Biochemistry of Food Crops From Omics Studies to Nutrient Analysis
Shelly Praveen Ranjeet Ranjan Kumar Suneha Goswami
DIVISION OF BIOCHEMISTRY ICAR- INDIAN AGRICULTURAL RESEARCH INSTITUTE, NEW DELHI-110012
Center of Advanced Facility Training (CAFT)
Biochemistry of Food Crops From Omics Studies to Nutrient Analysis th
th
(September 25 – October 15 , 2018)
Indian Council of Agricultural Research Division of Biochemistry ICAR-Indian Agricultural Research Institute New Delhi-110012
Index Chapters Introduction
Chapter-1 Chapter-2 Chapter-3 Chapter-4 Chapter-5
Chapter-6 Chapter-7
Title
Page No.
Nutrition Sensitive Agriculture: A link between Agriculture, Nutrition and Health Module-I Omics Approaches for Characterizing Nutritional Pathway
1
Transcriptomic Approaches for Elucidating the Genes Network Associated with Starch Biosynthesis pathway Proteomics Approaches for the Identification of Novel/Conserved Proteins Linked with Different Metabolic Pathways Nutriepigenomics - a Bridge between Nutrition and Health Lipidomics in Nutrition Nutrigenomics ---- DNA >< Diet Module-II Natural Antioxidants – their Role in Human Health
7
Colorful Truth about Anthocyanins – Role in Human Nutrition Vitamin E- A Powerful Antioxidant: Chemistry and Analysis in Foods
16 23 29 34
39 44
Module-III Nutritional Biodiversity Chapter-8 Chapter-9
Coconut: A Treasure Trove of Nutrition and Health Benefits Omega-3 fatty acids: An Essential Contribution in Human Diet
50 58
Module-IV Fortification/ Biofortification – Approaches to Improve Nutritional Quality Chapter-10
62
Chapter-11 Chapter-12
Poor Shelf-life of Pearl Millet Flour - Approaches to Improve the Flour Quality Starch Hydrolyzation Kinetics of Pigmented Rice Bioavailability of Phytonutrients: A Key Determinant of their Bioefficacy
Chapter-13
Resistant Starch as Prebiotic: A Promise for Improving Human Health
78
67 72
Module-IV Food Safety Chapter-14
Street Food: The Food Safety Dimension
84
Practicals ĤĚ Ġ LŒ ŒL -I
Title
Page No.
Ċ -gel Activity Assay using Native-Gel Electrophoresis Ranjeet R. Kumar, SumedhaHasija, MohdTasleem, SunehaGoswami and Shelly Praveen
89
Practical-II
Differential Protein Profiling using SDS - Polyacrylamide Gel Electrophoresis Ranjeet R. Kumar, SumedhaHasija, MohdTasleem, SunehaGoswami and Shelly Praveen
92
Practical -III
Two-Dimensional Gel-Electrophoresis of Proteins SunehaGoswami, RR Kumar Sumedha Ahuja, Mohd. Tasleem and Shelly Praveen
96
Practical- IV
Expression Analysis of Specific Plant Protein by Immunoblotting Ranjeet R. Kumar, Sumedha Ahuja, SunehaGoswami and Shelly Praveen
103
Practical-V
Determination of Anthocyanin content in black rice/black soybean Veda Krishnan and Archana Singh
105
Practical-VI
Estimation of amylose and amylolytic activity in rice Veda Krishnan and Archana Singh
110
Practical-VII
Estimation of Enzymatic Lipid Hydrolysis in Pearl Millet SunehaGoswami, RR Kumar, Ansheef Ali TP, DV Bhargav, Abhishek Chitranshi and Shelly Praveen
118
Practical-VIII
Estimation of Resistant Starch (RS) Content Archana Singh, Monika Awana, Ms.Veda Krishnan, and Shelly Praveen
127
Practical-IX
Estimation of Phytic Acid Content in Soybean Seeds Archana Sachdev and Veda Krishnan
135
Practical-X
Content and Composition Analysis of Isoflavones in Soybean Seeds Sandeep Kumar, A.P. Rajarani and Anil Dahuja
141
Practical-XI
Extraction, Purification and Quantification of Gamma Oryzanol from Rice Bran Kishwar Ali and ArunaTyagi
144
Practical-XII
Estimation of DNA Methylation in a Gene Associated with Isoflavone Biosynthesis in Soybean Suresh Kumar, Archana Singh and Monika Awana
147
Nutrition Sensitive Agriculture A link between Agriculture, Nutrition and Health . Shelly Praveen
. Ranjeet Ranjan Kumar
. Suneha Goswami
Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
INTRODUCTION Nutri on-sensi ve agriculture is a foodbased approach to make the food system be er equipped to produce good nutri onal outcomes. Linking Agriculture with nutri on serve two purposes; it promotes agricultural development, with be er remunera on to farmer along with addressing malnutri on and over coming micro nutrient deficiencies. Collec vely, it addresses nutri onally rich foods, diversity of food and food for fica on / biofor fica on. Food system involves various stakeholders between farmers and consumers; hence nutri on sensi ve agriculture is not a term but a concept. This concept addresses the mul ple benefits involving variety of foods, nutri onal value of food, and the importance and social relevance of linking food sector with agricultural sector for suppor ng rural livelihoods. Agriculture and food are assumed to be cri cal determinants of malnutri on. However, agriculture research and development has primarily focused on improvement in yield and not much translated as expected into be er nutri on outcomes. Malnutri on is a global challenge having more adverse effects on developing world. Malnutri on results from a poor diet or a lack of food.
It happens when the intake of nutrients or energy is too high, too low, or poorly balanced. We argue that to do so, agriculture research needs to be fundamentally changed, from the current emphasis just on produc vity goals to understanding dynamics of malnourished popula on and addressing factors that can improve diet quality and health. In detail, malnutri on refers to deficiencies, excesses or imbalances in a person's intake of energy and/or nutrients. The term malnutri on broadly covers two types of groups: One is 'under-nutri on' —which includes stun ng (low height for age), was ng (low weight for height), underweight (low weight for age) and micro nutrient deficiencies or insufficiencies (a lack of important vitamins and minerals). The other is 'overweight or obese', which leads to obesity and diet-related noncommunicable diseases (such as heart disease, stroke, diabetes and cancer).
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How to develop link between Agriculture and Nutrition There is an urgent need to have a mental shi in the way we currently view agriculture. Currently it is le to farmers, who grow the crops for their livelihood. Farmers prefer to grow those crops, which requires less intensive care and fetch be er price. That is how farmers are inclined to wheat-rice system as it is covered under minimum support price. Although agriculture encompasses more than just cereal crop produc on-from hor culture to forestry and fisheries, agriculture should be seen not only as a means to an end, but as an essen al process for improving the nutri onal quality of foods available to the society. This process should also ensure healthy soils and ecosystems for farming in the future. Since agriculture is not considered as a mean to nutri on, it is therefore urgent to place the promo on of healthy diets and nutri on at the heart of agriculture research and extension policies and programs.
This is not only affec ng the current popula on but will translate many folds in the future genera ons. Programs should focus more on preven on of all forms of malnutri on, and nutri on should be incorporated into agricultural investment plans to ensure that there is a dedicated budget for nutri on-sensi ve ini a ves. Nutri on must be incorporated into all aspects of the value chain-star ng with nutrient-rich soils that will improve the quality of the crops, and extending across the food system to other elements like food safety, food processing, food for fica on and proper food prepara on and consump on,hygiene and sanita on in households . Nutri on educa on ini a ves that explain which food combina ons will provide essen al vitamins and minerals can have a big impact as well. Some of the research will be to improve the efficiency of supply for more nutri ous foods.
Nutrition to Health : a vital link Nutri onal security highlights the need of appropriate nutri ous diet with all essen al nutrients and water, adequate sanita on, healthy environment and proper health services for all the household members. It emphasizes the health component and nutri onal status of the individual or society. Further linking nutri on with health will percolate be er in rela ng it with dietrelated chronic disease such as diabetes, strokes and cancers. Deficiencies in vitamins and minerals remain unacceptably high in majority of popula on, which is one of the major cause of poor physical and mental growth.
To understand the dynamics of links between agriculture to nutri on, and nutri on to health, various factors should be analyzed to achieve sustainable nutri onal security. .'Nutri onal biodiversity' should be explored to develop dietary matrix capable of catering the nutri onal needs. This can be supplemented by the efforts of 'For fica on/ Biofor fica on', a process to for fy the micronutrients and vitamins in various food/food crops. Availability of nutrients in diet does not necessarily results in improved health, as gut health is crucial for nutrient bioavailability. 2
Hence it is important to flag the issue of 'Probio cs and Nutrient availability'. Since environmental and lifestyle stresses are increasing day by day, involvement of an oxidants in diet prevents several diseases. As colourful nutrients like anthocyanin, found abundant in many fruits and vegetables is considered as bliss in evading the development of various non-communicable diseases. This is being discussed in 'Colourful Nutrients and their Health benefits'. 'Food Safety' is another important issue to be discussed. If it not properly applied, the beneficial effects of availability of nutrients in food crops and bioavailability due to a health gut can be easily negated.
high in energy. The micro nutrient deficiencies and undernourishment has increased many fold in poor areas of the developing world .Biodiversity plays a key role in ensuring balanced diet with adequate nutrients in different varie es. Biodiversity are cri cally impaired by intensified agriculture for enhanced food produc on with increasing the load of fer lizer, pes cides and other chemicals and by frequently changing the cropping pa ern and varie es. The effect is evident from the change in the global nutri onal status and human health.
Fortification/ Biofortification
Nutritional Biodiversity
Food for fica on or enrichment is the process of adding micronutrients (essen al trace elements and vitamins) to food. This is one of the means to ensure healthy diet to the society reducing the dietary deficiencies within a popula on. Most of the staple foods in a region are lacking in one or more than one essen al nutrients, as evident from the specific diseases. The reason may be due to the soil of the region or from inherent inadequacy of a normal diet. World Health Organiza on (WHO) defines For fica on as "the prac ce of deliberately increasing the content of an essen al micro nutrient, ie., vitamins and minerals (including trace elements) in a food irrespec ve of whether the nutrients were originally in the food before processing or not, so as to improve the nutri onal quality of the food supply and to provide a public health benefit with minimal risk to health". Enrichment is defined as "the addi on of micronutrients to a food which are lost during processing". Biofor fica on is the concept of breeding crops to increase their nutri onal value. This can be done either through conven onal selec ve breeding, or through gene c engineering.
Nutri on and biodiversity are the most important and deciding factors for the food security and sustainable development. It has also been incorporated in the Millennium Development Goals to ensure hunger free world with sustainable environment. Our food systems and diet has severely affected the ecosystem and causes loss in the food biodiversity. The ecosystem and human diets has changed dras cally due to the increase in the popula on, urbaniza on, and change in the agriculture pa ern, as evident from the food produc on and consump on pa ern. Presently, one billion people suffer from hunger and another two billion suffer from micro nutrient deficiencies. The problems of obesity and chronic disease escalated over the last decade due to simplifica on of diets - low in variety but
3
B i o fo r fi c a o n d i ffe rs f ro m o rd i n a r y for fica on because it focuses on making plant foods more nutri ous as the plants are growing, rather than having nutrients added to the foods when they are being processed. Biofor fica on is seen as an upcoming strategy for dealing with deficiencies of micronutrients in the developing world.
Probiotics and Nutrient Bioavailability
Colourful Nutrients and their Health benefits
Probio cs are live bacteria and yeasts that are good for our diges ve system. Probio cs are o en called "good" or "helpful" bacteria because they help keep our gut healthy. Nutrient bioavailability refers to the propor on of a nutrient that is absorbed from the diet and is used for the normal body func ons. Different steps of the metabolic pathway where changes in nutrient bioavailability occur are ·Release of the nutrient from physicochemical dietary matrix ·Effects of diges ve enzymes in intes ne ·Binding and uptake by the intes mucosa · Transfer across the gut wall to blood or lympha c circula on · Systemic distribu on · Systemic deposi on (stores) ·Metabolic and func onal use ·Excre on
Bioavailability of a nutrient is governed by external and internal factors. External factors include the food matrix and the chemical form of the nutrient, whereas gender, age, nutrient status and life stage are among the internal factors. Bioavailability also refers to the frac on of a nutrient that is absorbed. The bioavailability of macronutrients – carbohydrates, proteins, fats – is usually very high (> 90% of the amount ingested), whereas micronutrients – vitamins, minerals, and bioac ve phytochemicals can vary widely in the extent they are absorbed and u lised.
Anthocyanins are natural plant pigments that have beneficial effects for the plant as well as for humans and animals. Dietary sources of anthocyanins are generally easy to iden fy due to their red, blue, or purple color. Examples include berries and redskinned grapes, apples, and pears and various vegetables such as radishes & red/purple cabbage. Anthocyanins may also be ingested through their use as a food addi ve and as dietary supplements, procured as anthocyanin-rich fruit extracts, powders, and purified compounds. The bioavailability of anthocyanins is considered to be limited; however, recent advances in targeted & non-targeted instrumenta on have enhanced our detec on capability, indica ng that anthocyanin metabolism can be extensive, is complex, and that the full por olio of anthocyanin metabolites are probably yet to be measured or characterized.
the the nal the
4
Nonetheless, it is clear that anthocyanin intake is associated with various health benefits as demonstrated in a number of study designs ranging from human epidemiology and clinical trial interven on to screening and mechanis c studies in animals and cell culture models. Anthocyanin molecular targets include transporters & receptors, second messenger signaling molecules and kinase enzymes, transcrip on factors, promoters, & growth factors, and a host of oxidant defense enzymes.Despite the poten al broadspanning biological ac vi es of anthocyanins, safety & toxicological concerns are rela vely low.
Access to sufficient amounts of safe and nutri ous food is key to sustaining life and promo ng good health. Unsafe food containing harmful bacteria, viruses, parasites or chemical substances, causes more than 200 diseases – ranging from diarrhoea to cancers. An es mated 600 million – almost 1 in 10 people in the world – fall ill a er ea ng contaminated food and 4,20,000 die every year, resul ng in the loss of 33 million healthy life years. Diarrhoeal diseases are the most common illnesses re s u l n g f ro m t h e co n s u m p o n o f contaminated food, causing 550 million people to fall ill and 2,30,000 deaths every year. Food safety, nutri on and food security are inter-linked. Unsafe food creates a vicious cycle of disease and malnutri on, par cularly affec ng infants, young children, elderly and the sick. Food borne diseases impede socioeconomic development by straining health care systems, and harming na onal economies, tourism and trade. Good collabora on between governments, producers and consumers helps ensure food safety. In many countries, street foods make an important contribu on to the employment, household revenue and food security, and help to meet the challenge of feeding urban popula ons, par cularly in developing countries. Dietary habits and tradi onal meal pa erns change when people move from rural to an urban environments, and ci es offer access to a variety of foods outside the home, including from street foods, restaurants and kiosks. As an 'informal' sector of food business street foods o en escape formal inspec on and control. They can therefore both be the source of food safety problems and co nt r i b u te to t h e d ete r i o ra o n o f environmental hygiene. Street foods require a comprehensive policy to ensure that food is safe for human consump on.
Food Safety Food Safety refers to handling, preparing and storing food in a way to best reduce the risk of individuals becoming sick from foodborne illnesses. Food safety is a global concern now-a-days that covers a variety of different areas of everyday life. Food Safety aims to prevent the food from becoming contaminated and causing food poisoning. This is achieved through a variety of different avenues, some of which are· Properly cleaning and sani sing all surfaces · Maintaining a high level of personal hygiene · Storing, chilling and hea ng food correctly · Effec ve pest control
5
Module-I
6
Chapter-1
Transcriptomic Approaches for Elucidating the Genes Network Associated with Starch Biosynthesis Pathway Ranjeet R. Kumar, Suneha Goswami and Shelly Praveen Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
Plant omics and new biotechnologies such as massively parallel sequencing and microarray analysis were preferred tools to identify and characterize the differentially expressed genes (DEGs) under stress in different modal species (Gong et al. 2014), but Next-Generation Sequencing (NGS) is now the most preferred technique
used
for
transcriptome
study.
RNA
sequencing
(RNA-Seq)
is
revolutionizing the study of the transcriptome. A highly sensitive and accurate tool for measuring expression across the transcriptome, it is providing visibility to previously undetected changes occurring in disease states, in response to therapeutics, under different environmental conditions and across a broad range of other study designs. RNA-Seq allows researchers to detect both known and novel features in a single assay, enabling the detection of transcript isoforms, gene fusions, single nucleotide variants, allele-specific gene expression and other features without the limitation of prior knowledge. The transcriptome data generated through NGS provide very useful information on the regulatory pathway networks operating in plants under different conditions (Rensink and Buell, 2005). NGS data help to identify DEGs, along with their functional annotation under different stresses, which has been used in the past to elucidate the different mechanisms as well as pathways associated with stresstolerance (Mochida et al., 2006). RNA-Sequencing (RNA-Seq) is now the preferred technology used for global genome identification and de novo transcriptome of various organisms (Mousavi et al., 2014). In this technology, small stretches of cDNAs are sequenced at a very high coverage and assembled using different programs to reconstruct the contigs (Fig. 1). It has been successfully used for the relative expression study, editing 5’ and 3’ ends of annotated genes and functional gene identification with their respective exons and introns (Nagalakshmi et al., 2010). 7
In agriculturally important crops, RNA-Seq has been mostly used for the identification of novel and conserved stress-responsive and pathway-associated genes involve in tolerance and nutrient responsive regulation (Kugler et al., 2013). Other than this, RNA-Seq offers numerous advantages over gene expression arrays like more sensitive and accurate measurement of gene expression, helps in identification of both known and novel genes, can be used in any crop plant species. Various crops such as chickpea (Molina et al., 2011), rice (Mizuno et al., 2010), sorghum (Dugas et al., 2011), soybean (Hao et al., 2011), and parsley (Li et al., 2014) have been subjected to high-throughput NGS for identification of abiotic stress-related genes. RNA-Seq is virtually changing the area of sequencing and transcriptome profiling (Wang et al., 2009). Recently, de novo assembly has been used in some of the agriculturally important crops like chilli pepper (Liu et al., 2013), coconut (Fan et al., 2013), rice (Zhang et al., 2013), and sugarcane (Cardoso-Silva et al., 2014) for identification of novel genes. Earlier, Roche GS-FLX 454 was the most widely used platform for de novo transcriptome sequencing, due to its long read length in different organisms, for example, ginseng (Sun et al., 2010), A. thaliana (Wall et al., 2009), and maize (Vega-Arreguin et al., 2009). The Illumina transcriptome was mainly used for the sequencing of organisms whose genome was sequenced (Li et al., 2010). It was confirmed in due course of time that the relatively short reads can be effectively assembled by the Illumina transcriptome, or whole genome de novo sequencing and assembly with the advantage of paired-end sequencing (Maher et al., 2009). Starch Starch is the most significant form of carbon reserve in plants in terms of the amount made, the universality of its distribution among different plant species, and its commercial importance. It consists of different glucose polymers arranged into a three dimensional, semi-crystalline structure-the starch granule. The biosynthesis of starch involves not only the production of the composite glucans but also their arrangement into an organized form within the starch granule. The formation of the starch granule can be viewed as a simple model for the formation of ordered threedimensional polysaccharide structures in plants. Understanding the biochemical basis for the assembly of the granule could provide a conceptual basis for understanding other higher order biosynthetic systems such as cellulose 8
biosynthesis. For example, one emerging concept is that structure within the granule itself may determine or influence the way in which starch polymers are synthesized. Starch is synthesized in leaves during the day from photosynthetically fixed carbon and is mobilized at night. It is also synthesized transiently in other organs, such as meristems and root cap cells, but its major site of accumulation is in storage organs, including seeds, fruits, tubers, and storage roots. Almost all structural studies have used starch from storage organs because it is readily available and commercially important; we therefore focus on starch biosynthesis in storage organs. However, where aspects of transient biosynthesis are clearly different from long-term reserve synthesis, reference is made to biosynthesis in non-storage tissues. Starch is synthesized in plastids, which in storage organscommitted primarily to starch production are called amyloplasts (Fig. 2).These develop directly from proplastids and have littleinterna1 lamellar structure. Starch may also be synthesized inplastids that have other specialized functions, such as chloroplasts(photosynthetic carbon fixation), plastids of oilseed(fatty acid biosynthesis), and chromoplasts of roots such ascarrot (carotenoid biosynthesis). The Biochemistry of Starch Biosynthesis The biosynthetic steps required for starch biosynthesis arerelatively simple, involving three committed enzymes: ADPglucosepyrophosphorylase (ADPGPPase; EC 2.7.7.23), starchsynthase (SS; EC 2.4.1.21), and starch branching enzyme (SBE;EC 2.4.1.28). Amylose and amylopectin are synthesized from ADPglucose,which is synthesized from glucose-1-phosphate andATP in a reaction that is catalyzed by ADPGPPase and thatliberates pyrophosphate. This enzyme is active within theplastid, which means that its substrates, glucose-1-phosphateand ATP, must also be present in the plastid. In chloroplasts,ATP may be derived from photosynthesis, but in non-photosyntheticplastids, it must be specifically imported from the cytosol,probably by an ADP/ATP translocator. The glucose-1-phosphate canbe supplied
by the
reductive
pentose
phosphate
pathway
inchloroplasts
via
phosphoglucoisomerase and phosphoglucomutase. In non-photosynthetictissues, it may be imported directly from the cytosol or synthesized in the plastid from glucose6-phosphatevia
the
action
of
a
plastidial
phosphoglucomutase.The
pyrophosphate produced by ADPGPPase is removedby inorganic alkaline pyrophosphatase, which is probably confinedto plastids in both photosynthetic and 9
nonphotosynthetictissues.
The
removal
of
this
plastidial
pyrophosphate
effectivelydisplaces the equilibrium of the ADPGPPase reactionin favor of ADPglucose synthesis (Fig. 3). Further, SS catalyzes the synthesisof α (1-4) linkage between the non-reducing end ofa preexisting glucan chain and the glucosyl moiety of ADPglucose,causing the release of ADP. SSs can use both amyloseand amylopectin as substrates in vitro. How the initial primersfor the synthesis of glucan chains are produced in vivo is notknown. The α (1-6) branches in starch polymers are made by SBE,which hydrolyzes an (l-4) linkage within a chain and then catalyzesthe formation of an α (1-6) linkage between the reducingend of the “cut” glucan chain and another glucose residue,probably one from the hydrolyzed chain. SBEs show somespecificity for the length of the α (1-4) glucan chain that theywill use as a substrate. Remodelling Starch Biosynthesis Pathway The genes associated with starch biosynthesis pathway has not been fully identified and characterised. With the advent of technology like NGS and gel-free proteomics, now it becomes easy to identify the respective transcripts and their proteins associated with SBP. Even, the transcriptional regulation of the genes coding for these enzymes has not yet been fully explored. Transcriptional regulation may be a more important mechanism for long-term control of genes expression especially during
caryopsis
development.
Posttranslational
regulation
including
phosphorylation, interaction with 14-3-3 regulatory proteins and posttranslational redox activation, appear to be essential regulatory mechanisms controlling starch biosynthesis by providing a rapid response to short-term environmental changes. The pathway in terms of synthesis and regulation has not been extensively studied. In our lab, we have executed whole transcriptome sequencing of contrasting wheat cvs. HD2985, HD2329, Raj3765 and BT-Schomburgkin a tissue specific manner at different stages of growth and development and under differential stress treatment. The transcriptome data generated from the developing endosperm tissue of contrasting wheat cvs. was analysed and characterised using different bioinformatics software’s. We identified ~87, 100, and 46 novel transcripts associated with fructose, starch and sucrose metabolism pathway which was further validated in our lab. We identified 12 putative soluble starch synthase genes using de novo transcriptomic approach and cloned five of them for further functional validation (Table 1). Similarly, 10
we identified 4 AGPase, 2 SBE and 2 GBSS genes from contrasting wheat cvs. based on the information generated using transcriptomic approach. We also identified ~100000 SNIPs lying in differentially expressed genes associated with starch metabolism (Table 1). These are the potential resources to be utilized for the breeding program in order to develop a ‘climate-smart’ crop. References 1. Kumar RR, Goswami S, Sharma SK, et al (2015) Harnessing Next Generation Sequencing in Climate Change: RNA-Seq Analysis of Heat StressResponsive Genes in Wheat (Triticum aestivum L.). Omi A J Integr Biol 19:632–647. 2. Dugas D V, Monaco MK, Olson A, et al (2011) Functional annotation of the transcriptome of Sorghum bicolor in response to osmotic stress and abscisic acid. BMC Genomics 12:514. doi: 10.1186/1471-2164-12-514 3. Maher C a, Palanisamy N, Brenner JC, et al (2009) Chimeric transcript discovery by paired-end transcriptome sequencing. Proc Natl Acad Sci U S A 106:12353–8. 4. Nagalakshmi U, Waern K, Snyder M (2010) RNA-seq: A method for comprehensive transcriptome analysis. Curr. Protoc. Mol. Biol. 5. Kumar RR, Pathak H, Sharma SK, et al (2014) Novel and conserved heatresponsive microRNAs in wheat (Triticum aestivum L.). Funct Integr Genomics. doi: 10.1007/s10142-014-0421-0
11
Figure1. Schematic representation of protocol used for the Next-Generation Sequencing using Illumina HiSeq.
12
Figure 2.Schematic representation of starch biosynthesis pathway operating in plants.
13
Table 1.Identification of novel transcripts and SNIPs based marker associated with fructose, starch and sucrose metabolism pathways in wheat using de novo transcriptomic approach.
14
Figure 3.Schematic of the metabolic flux and expression of genes involved in sucrose to starch pathway in developing wheat seed. Upper box legend indicates level of gene expression and lower box legend indicates the time or developmental stage as days post-anthesis.
15
Chapter-2
Proteomics Approaches for the Identification of Novel/Conserved Proteins Linked with Different Metabolic Pathways Ranjeet Ranjan Kumar, Suneha Goswami and Shelly Praveen Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
Proteomics involve the large-scale study of proteins, their structure and physiological role or functions. Proteins are quintessential cellular components or biomolecules in any living organism. The term proteomics first appeared in 1997. It was very similar to the word genome. The word proteome is actually a combination of protein and genome and was coined by Mark Wilkins in 1994. To be precise and specific, proteome is the entire complement or database or set of proteins produced by a living organism. The proteome is a broad term that also encompasses the alterations or modifications produced in native protein when organisms are subjected to a plethora of changes. Proteomics is the large-scale study of proteomes. A proteome is a set of proteins produced in an organism, system, or biological context. The proteome is not constant; it differs from cell to cell and changes over time. To some degree, the proteome reflects the underlying transcriptome. However, protein activity (often assessed by the reaction rate of the processes in which the protein is involved) is also modulated by many factors in addition to the expression level of the relevant gene. Proteomics is used to investigate • • • • • •
when and where proteins are expressed rates of protein production, degradation, and steady-state abundance how proteins are modified (for example, post-translational modifications (PTMs) such as phosphorylation) the movement of proteins between subcellular compartments the involvement of proteins in metabolic pathways how proteins interact with one another
The conventional techniques for purification of proteins are chromatography based such as ion exchange chromatography (IEC), size exclusion chromatography (SEC) and affinity chromatography. For analysis of selective proteins, enzyme-linked immunosorbent assay (ELISA) and western blotting can be used. These techniques 16
may be restricted to analysis of few individual proteins but also incapable to define protein expression level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE) and two-dimensional differential gel electrophoresis (2D-DIGE) techniques are used for separation of complex protein samples (Fig. 1). Protein microarrays or chips have been established for high-throughput and rapid expression analysis; however, progress of a protein microarray enough to explore the function of a complete genome is challenging. The diverse proteomics approaches such as mass spectrometry (MS) have developed to analyze the complex protein mixtures with higher sensitivity (Fig. 2). Additionally, Edman degradation has been developed to determine the amino-acid sequence of a particular protein. Isotope-coded affinity tag (ICAT) labeling, stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tag for relative and absolute quantitation (iTRAQ) techniques have recently developed for quantitative proteomic. X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are two major high-throughput techniques that provide three-dimensional (3D) structure of protein that might be helpful to understand its biological function. Proteome analysis provides the complete depiction of structural and functional information of cell as well as the response mechanism of cell against various types of stress and drugs using single or multiple proteomics techniques. Gel-based approaches Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis SDS-PAGE is a high resolving technique for the separation of proteins according to their size, thus facilitates the approximation of molecular weight. Proteins are capable of moving with electric field in a medium having a pH dissimilar from their isoelectric point. Different proteins in mixture migrate with different velocities according to the ratio between its charge and mass. However, addition of sodium dodecyl sulfate denatures the proteins, therefore separate them absolutely according to molecular weight. Two-dimensional gel electrophoresis The two-dimensional polyacrylamide gel electrophoreses (2D-PAGE) is an efficient and reliable method for separation of proteins on the basis of their mass and charge. 2D-PAGE is capable of resolving ~5,000 different proteins successively, depending on the size of gel. The proteins are separated by charge in the first dimension while in second dimension separated on the basis of differences between their mass. The 2-DE is successfully applied for the characterization of post-translational modifications, mutant proteins and evaluation of metabolic pathways.
17
Two-dimensional differential gel electrophoresis 2D-DIGE utilizes the proteins labelled with CyDye that can be easily visualized by exciting the dye at a specific wavelength. Quantitative Techniques ICAT labelling The ICAT is an isotopic labelling method in which chemical labelling reagents are used for quantification of proteins. The ICAT has also expanded the range of proteins that can be analysed and permits the accurate quantification and sequence identification of proteins from complex mixtures. The ICAT reagents comprise affinity tag for isolation of labeled peptides, isotopically coded linker and reactive group.
Stable Isotopic Labeling with Amino Acids in Cell Culture SILAC is an MS-based approach for quantitative proteomics that depends on metabolic labeling of whole cellular proteome. The proteomes of different cells grown in cell culture are labeled with “light” or “heavy” form of amino acids and differentiated through MS. The SILAC has been developed as an expedient technique to study the regulation of gene expression, cell signaling, posttranslational modifications. Additionally, SILAC is a vital technique for secreted pathways and secreted proteins in cell culture. Isobaric tag for relative and absolute quantitation (iTRAQ) iTRAQ is multiplex protein labeling technique for protein quantification based on tandem mass spectrometry. This technique relies on labeling the protein with isobaric tags (8-plex and 4-plex) for relative and absolute quantitation. The technique comprises labeling of the N-terminus and side chain amine groups of proteins, fractionated through liquid chromatography and finally analyzed through MS. It is essential to find the gene regulation to understand the disease mechanism, therefore protein quantitation using iTRAQ is an appropriate method that helps to identify and quantify the protein simultaneously (Fig. 3). High-throughput techniques Mass spectrometry MS is used to measure the mass to charge ratio (m/z), therefore helpful to determine the molecular weight of proteins. The overall process comprises three steps. The molecules must be transformed to gas-phase ions in the first step, which poses a challenge for biomolecules in a liquid or solid phase. The second step involves the separation of ions on the basis of m/z values in the presence of electric or magnetic fields in a compartment known as mass analyzer. Finally, the separated ions and the 18
amount of each species with a particular m/z value are measured. Commonly used ionization method comprises matrix-assisted laser desorption ionization (MALDI), surface enhanced laser desorption/Ionization (SELDI) and electrospray ionization (ESI). NMR spectroscopy The NMR is a leading tool for the investigation of molecular structure, folding and behaviour of proteins. Structure determination through NMR spectroscopy typically involves various phases, each using a discrete set of extremely specific techniques. The samples are prepared and measurements are made followed by interpretive approaches to confirm the structure. The protein structure is fundamental in several research areas such as structure-based drug design, homology modelling and functional genomics. References 1. Kumar RR, Singh GP, Goswami S, Pathak H, Rai RD (2014) Proteome analysis of wheat (Triticum aestivum) for the identification of differentially expressed heatresponsive proteins.Australian Journal of Crop Science. 8(6):973. 2. Wilkins M (2009). "Proteomics data mining".Expert Review of Proteomics. England. 6 (6): 599–603. 3. Washinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humphery-Smith I (1995). "Progress with geneproduct mapping of the Mollicutes: Mycoplasma genitalium". Electrophoresis. 16(1): 1090–94. 4. Altelaar AF, Munoz J, Heck AJ (2013). "Next-generation proteomics: towards an integrative view of proteome dynamics.".Nature Reviews Genetics. 14(1): 35–48. 5. Kim, Min-Sik, et al. (2014)."A draft map of the human proteome".Nature. 509 (7502): 575–81. 6. Towbin, et al. (1979) Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. PNAS 76:4350–4354.
19
Figure 1.Overview of techniques used in proteomics.
20
Figure 2.Flow chart for the characterization of samples using different proteomic tools.
21
Figure 3.Identification of proteins using the iTRAQ labeling and sequencing.
22
Chapter-3
Nutriepigenomics - a Bridge between Nutrition and Health Suresh Kumar
Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
Accumulating evidence suggest that nutrients can initiate epigenetic changes like DNA methylation and histone modifications, thus alter the expression of the genes vital for normal physiological and biochemical processes inside the cell. From recent studies, it has emerged that food components, nutrients and xenobiotic contents influence epigenetic changes either by inhibiting the enzymes involved in catalyzing DNA methylation/histone modification reactions, or by affecting the availability of substrates necessary for their enzymatic activity (Kumar, 2018b). To deal with this emerging area of biology, a novel amalgamation of nutritional science and epigenomics has been conceived as a smarter tool to cope with the deleterious effects of environmental stresses and lethal pathological conditions such as cancer. In the recent years, epigenetic variations have emerged as a crucial factor involved in a range of human diseases like cancer, diabetes, inflammation, neurocognitive disorders and obesity. Although the possible epigenetic treatment/preventative measures of these diseases would be exciting, our current understanding of nutritional epigenetics (nutriepigenomics) is inadequate. Further investigations are needed to better understand the use of nutrients/bioactive/xenobiotic compounds for maintaining good health and preventing diseases by avoiding undesirable epigenetic changes. Epigenomics Epigenome is defined as the sum total of all the biochemical changes in DNA, histoneproteins andnon-coding RNAs (ncRNAs) biogenesis in a cell. Studies on the
epigenetic changes in and around DNA that regulate genome activity have been 23
defined as epigenetics, and the branch of genomics which deals with epigenomic studies is called epigenomics. The area of epigenomics is expanding continuously because of the identification of more and more epigenetic marks. With the identification of additional epigenetic DNA modifications [5-hydroxymethylcytosine (5-hmC) and N6-methyladenine (6-mA)] having the known epigenetic regulatory functions in animal system, the significance of epigenomic studies has increased considerably. Epigenomic changes are continuously being reported to be involved in gene regulation during the developmental processes, tissue differentiation, and suppression of transposable elements (TEs) in both animals and plants (Li et al., 2018). Unlike the genome, which is largely invariable within an individual throughout its life, the epigenome is dynamically altered by the developmental stage and environmental factors (Kumar, 2018a). Advanced studies in epigenetics, particularly in the area of cancer research, are being reported in the animal system (Kumar, 2018b), while the basicepigenomic study on the plant is still in the infancy and only little is known about the functional consequences of epigenetic/epigenomic changes in plants [Kumar et al., 2017b]. Epigenetic changes may also cause variation in the structure of chromatin and function of the genome. The epigenetic mechanisms instigate variation in gene expression with no change in the underlying DNA sequence and the same may be inherited through mitosis or meiosis (Kumar, 2018c; Kumar et al., 2017a). Epigenetic regulation of gene expression is mediated by a complex interplay among different molecular factors involved in DNA (de)methylation, the enzymes involved in posttranslational modifications of histone proteins (Fig 1), chromatin remodelers and ncRNAs (Feinberg et al., 2016; Kumar, 2017). The components that regulate targeting as well as enzymatic activation of DNA methyltransferase/glycosylases have been discovered, and DNA (de)methylation has been recognized to play crucial roles in developmental processes (Kumar and Singh, 2016). However, interaction between DNA (de)methylation and other epigenetic or chromatin features in controlling gene expression/genome activity remains unclear. DNA methylation Chemical modification of nitrogenous bases of DNA plays very important roles in epigenetic regulation of gene expression. DNA base modification is a tissue-specific, dynamic,
sequence-context
dependent 24
process.
Unraveling
these
complex
processes of DNA modifications may answer several biological questions. Methylcytosine (5-mC), also known as the 5th base, has long been known well before the DNA was accepted as genetic material in living organisms (Kumar, 2018a). About 4% of the cytosines present in the human genome are methylated, which reflects its abundance. However, the 5-mC level may vary greatly among the animal and plant genomes. Therefore, the significance of 5-mC cannot be delineated by its abundance. Rather, the importance of 5-mC lies in its positioning (in CG, CHG symmetric; CHH, asymmetric contexts; where H= A, T, or C) and presence in different parts of the gene (Wang et al., 2016; Kumar et al., 2017a). In the human genome, more than 80% of the cytosine present in CG context is methylated, which demonstrates a scenario of ubiquitous methylation. However, local gaps are common at regulatory elements like promoters and enhancers of the actively transcribed genes. Methylation at non-CG sites plays key roles in plants by silencing the activity of the foreign DNA via an RdDM pathway (Law and Jacobsen, 2010). Therefore, it would be reasonable to assume that the default state of the plant genomes
is
“methylated” and that
specific mechanisms
are
required
to
make/maintain the specific regions free from methylation by DNA demethylation processes, which may take place by the active or passive method (Li et al., 2018). Epigenetic DNA modifications affect the accessibility of genomic regions to the regulatory proteins or protein complexes, which influence chromatin structure and/or regulate transcriptional activity.
Figure 1. Environment-mediated epigenetic variations showing the connections between epigenetics, environmentand gene expression. Environmental factors
25
cause epigenetic modifications including DNA methylation and histone modifications (acetylation, methylation, glutathionylation etc.). Nutriepigenomics In the field of nutrition, epigenetics is becoming exceptionally important becausenutrients and bioactive components of food can cause epigenetic changes and alter expression of genes at the transcriptionallevel. Betaine, choline, folate, methionine and vitamin B-12 canaffect DNA methylation and histone methylation through alteringcarbon metabolism. S-adenosylmethionine serves as a methyl donor for methylation reactions, andS-adenosylhomocysteine serves as inhibitorof methyltransferases. Thus, nutrient, bioactivecomponent, or environmental condition can alter methylation of DNA and histones. Other components of vitamin-B like biotin, niacin, and pantothenic acidalso play important roles in histone modifications. Niacin is involved in histone acetylation as a substrate of Sirt1. Similarly,pantothenic acid is a part ofCoA to form acetyl-CoA, which is the source of acetyl group in histoneacetylation. Bioactive food components like genistein and tea-catechin affects DNA methyltransferases. Resveratrol, butyrate, sulforaphane, and diallylsulfide inhibit histone deacetylase, and curcumininhibits histone acetyltransferases. Alteration in enzyme activity due to these compounds may affect physiological and pathologic status by altering gene expression. Environmental stressescause accumulation of reactive oxygen species (ROS) inside the cell which result in several chemical changes in biomolecules like nucleic acids, proteins and lipids. Antioxidant compounds (ascorbic acid,α-tocopherols, glutathione, proline, flavonoids and carotenoids) consumed by an organism serve as non-enzymatic ROS-scavenging machinery to combat the deleterious effects of ROS accumulated during the stress.It is well-known that many biological processes like responses to biotic and environmental stresses, growth, development, and programmed cell death are regulated by ROS scavenging mechanisms.ROS affect biochemistry, physiology, genetics and epigenetics of the organism which may result into critical health issues like cancer. Glutathione (GSH) is a powerful reducing agent, in addition to post-translational modifier of histone proteins, which modulates chromatin structure (Fig 1). Thus, nutrients play a significant role not only in affecting epigenome of the organism but also in determining physiological and pathological status. 26
Effects of nutrients on DNA methylation In a study on femalesheep, restricted supply of folate, vitamin B-12, and methionine in the diet during periconceptional period induced obesity in adult offspring as wellas altered immune responses to antigenic challenge. In the adultoffspring, methylation status of 4% CG islands (out of 1400) was found to be altered.The study indicates that
dietary
methyl
group
containing
nutrients
consumed
during
periconceptionalperiod mayaffect DNA methylation status in the progeny, and it may alter adult’s health-related phenotypes (Sinclair et al., 2007). Growing body of evidence suggests that certain bioactive food components like
tea-polyphenols,
genistein
from
soybean,
and
isothiocyanates
inhibit
development ofcancer by reducing DNA hypermethylation of the genesassociated with cancer. Thus, individual nutrient and food component and the diet can affect DNA methylation which alters gene expression. These epigenetic changes may affect physiological and pathologic processes in the body. Therefore, to maintain healthy life style we need to consume food/nutrients as per our personal requirements dictated by the genome, environment and their combination. This is where, the importance of nutriepigenomics comes in the picture. Conclusion Chromatin structure is quite dynamic, and it is more than a naturalsystem for packaging and condensing genomic DNA. It is a criticalplayer in controlling the accessibility of DNA for transcription process. Due to the reversible nature, epigenetics is considered an attractive field of nutritional intervention. Nutrients can modify physiological and pathologicprocesses through epigenetic mechanisms that are critical forgene expression.However, it is still very difficult today to delineate the preciseeffect of nutrients on epigeneticmodulation and their associations with physiological and/or pathological processes. Our current understanding about nutritional epigenetics is still limited, particularly the effects of nutrients/bioactive food componentson histone modification or chromatin remodeling complexes. In the near future, we must investigate the effects of different nutrientsor bioactive compounds to maintain our good health through nutritional modulationthat could be more natural than any other pharmacotherapies. While nutriepigenomics is a promising branch of
27
science, research in this area is still in the preliminary stages and it may take a few more years to suggest an accurate and effective diet for an individual. References 1. Feinberg, A.P., Koldobskiy, M.A., Göndör, A. (2016). Epigenetic modulators, modifiersand mediators in cancer aetiology and progression. Nature Review Genetics 17: 284–299. doi: 10.1038/nrg.2016.13 2. Kumar, S. (2017) Epigenetic control of apomixis: a new perspective of an old enigma. Advances Plants Agriculture Research 7: e243. 3. Kumar, S. (2018) Epigenomics of plant responses to environmental stress. Epigenomes 2: 6. doi: 10.3390/epigenomes2010006 4. Kumar, S. (2018a) Environmental stress, food safety, and global health: biochemical, genetic and epigenetic perspectives. Medical Safety Global Health 7: e145. doi: 10.4172/2574-0407.1000145. 5. Kumar, S. (2018c) Epigenetic memory of stress responses in plants. Journal of Phytochemistry and Biochemistry 2: e102. 6. Kumar, S., Beena, A.S., Awana, M., Singh, A. (2017a) Salt-induced tissuespecific cytosine methylation downregulates expression of HKT genes in contrasting wheat (Triticum aestivum L.) genotypes.DNA Cell Biology 36: 283−294.
28
Chapter-4
Lipidomics in Nutrition Aruna Tyagi Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
Lipids, the fundamental components of biological membranes, play multiple important roles in biological systems. First, lipids make the cell a sub-system in the context of the whole and relatively independent of the exterior environment through lipid bilayer structures. Second, lipids can provide an appropriate hydrophobic medium for the functional implementations of membrane proteins and their interactions. Third, a variety of lipid molecular species can produce second messengers by enzyme reactions. Furthermore, the aberrant lipid metabolism observed in numerous human diseases such as diabetes, obesity, atherosclerosis and Alzheimer's disease has attracted increased attention from lipid researchers. In summary, all these characteristics make lipids a focal point in cutting-edge biosciences. Current research on lipids tends to shift from determining the individual molecular structures of single lipids in biological samples to characterizing global changes of lipid metabolites in a systems-integrated context in order to understand the crucial role of lipids in physiopathology. Lipidomics, the large-scale study of the structures and functions of a wide range of lipids is of increasing importance in this research field. In the recent decade, many significant efforts have been made to promote research activities in this new-emerging field of lipidomics. Lipids are among the major components of food and constitute the principal structural biomolecules of human body together with proteins and carbohydrates. Lipidomics encompasses the investigation of the lipidome, defined as the entire spectrum of lipids in a biological system at a given time. Among metabolomics technologies, lipidomics has evolved due to the relevance of lipids in nutrition and their well-recognized roles in health. Lipidomics or lipid-profiling is a lipid-targeted metabolomics approach aiming at comprehensive analysis of lipids in biological systems. The extent of information in the genomic and proteomic fields is greater than that in the lipidomics field, because 29
of the complex nature of lipids and the limitations of tools for analysis. Modern technological advances in mass spectrometry and chromatography have greatly improved the developments and applications of metabolic profiling of diverse lipids in complex biological samples. Mass spectrometry advances have greatly facilitated lipidomics, but owing to the complexity and diversity of the lipids, lipidome purification and analysis are still challenging. Lipids are a diverse class of metabolites that play several key roles in the maintenance of human health. Lipidomics, which focuses on the global study of molecular lipids in cells, tissues, and biofluids, has been advancing rapidly over the past decade. Recent developments in MS and computational methods enable the lipid analysis with high throughput, resolution, sensitivity, and ability for structural identification of several hundreds of lipids. In nutrition research, lipidomics can be effectively used to elucidate the interactions between diet, nutrients, and human metabolism. Lipidomics can also be applied to optimize the effects of food processing on the dietary value, and in the evaluation of food-related health effects. Lipidomics will not only provide insights into the specific functions of lipid species in health and disease, but will also identify potential biomarkers for establishing preventive or therapeutic programs for human diseases and their applications in disease biomarker discovery and clinical application. The application of lipidomics in clinical studies may provide new insights into lipid profiling and pathophysiological mechanisms. Lipidomics is a lipid-targeted metabolomics approach focusing on comprehensive analysis of all lipids with which they interact in biology systems. Lipidomics will not only provide insights into the specific functions of lipid species in health and disease, but will also identify potential biomarkers for establishing preventive or therapeutic programs for human disease. Recent applications of lipidomics extend to
animal models of
disease such as metabolic syndromes, neurodegenerative diseases, cancer and infectious diseases.
30
Lipidome analysis by electrospray ionization triple quadrupole mass spectrometry Recently, lipid profiling, or so-called lipidomics research, has captured increased attention due to the well-recognized roles of lipids in numerous human diseases to which
lipid-associated
disorders
contribute,
such
as
diabetes,
obesity,
atherosclerosis and Alzheimer's disease. Investigating lipid biochemistry using a lipidomics approach will not only provide insights into the specific roles of lipid molecular species in health and disease, but will also assist in identifying potential biomarkers for establishing preventive or therapeutic approaches for human health. Plant membrane lipids undergo many changes when the plants are exposed to various stress conditions such as cold, mechanical wounding, and phosphorus deficiency. Lipidomics allows researchers to reveal the lipid changes induced by the stress conditions.Product‐ion analysis after collision‐induced dissociation (CID) is the essential approach for identification and characterization of plant lipids. Electrospray ionisation mass spectrometry (ESI‐MS)‐based methods have
advantages over
traditional lipid analytical approaches for analysis of plant lipids as there are a few lipid classes relatively specific to plant lipidomes in comparison to those of mammalian cellular lipidomes. Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), production of 31
fish oil-like proportions of DHA was observed
in Arabidopsis seed oil. Liquid
chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds. Challenges remaining in lipidomics include: •
Standardizing lipidomics protocols
•
Increasing availability of appropriate internal standards
•
Establishment of analyses for additional groups of lipids
•
Development of a standardized data processing system for interpreting mass spectral data to produce lipid profiles
•
Development of a web-accessible lipid profile database that facilitates integration with genomic, gene expression, proteomic, and other metabolomic data
•
Increasing access to lipidomics technologies
32
References 1. Christine ER, Marie H, Yurika O, Daisy Z, Jun Y, Bruce D H, Ameer YT (2017) Lipidomic Analysis of Oxidized Fatty Acids in Plant and Algae OilsJ. Agric. Food Chem. 65 (9): 1941–1951 2. Liu M-Y, Burgos A, Ma L, Zhang Q, Tang D, Ruan J (2017) Lipidomics analysis unravels the effect of nitrogen fertilization on lipid metabolism in tea plant (Camellia Sinensis L.) BMC Plant Biology DOI 10.1186/s12870.017-1111-6 3. Zhou X-R, Callahan D L, Shrestha P, Liu Q, Petrie J, Singh S P (2014)Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA. Front. Plant Sci.,doi.org/10.3389/fpls.2014.00419 4. Li-Beisson, Y., Shorrosh, B., Beisson, F., Andersson, M.X., Arondel, V., Bates, P.D., Baud, S., Bird, D., DeBono, A., Durrett, T.P., Franke, R. B., Graham, I.A., Katayama, K., Kelly, A.A., Larson, T., Markham, J.E., Miquel, M., Molina, I., Nishida, I., Rowland, O., Samuels, L., Schmid, K.M., Wada, H., Welti, R., Xu, C., Zallot, R. and Ohlrogge, J. Acyl lipid metabolism: June 11, 2010. The Arabidopsis Book. (American Society of Plant Biologists, Rockville, MD), doi: 10.1199/tab.0133, www.aspb.org/publications/arabidopsis 5. Pan X, Welti R, Wang X (2010) Quantitative analysis of major plant hormones in crude
plant
extracts
by
high-performance
spectrometry. Nat. Protoc., 5: 986-992
33
liquid
chromatography-mass
Chapter 5
Nutrigenomics ---- DNA >< Diet
Archana Sachdev Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi Nutrigenomics is the study of the effects of foods and food constituents on gene expression. It is an approach to nutrition and human health that studies the effect of genetic differences in human response to food and how food has an impact on gene expression, biochemistry, metabolism and promotion of health. It is based on two main observations: (1) the nutritional environment modifies the expression of genes, and (2) depending on the genotype of an individual, the metabolism of nutrients may vary and ultimately result in a different health status. Thus, nutrigenomics treats food as a major environmental factor in the gene-environment interaction, with the final aim to individualize or personalize food and nutrition, and ultimately individual strategies to preserve health, by tailoring the food to the individual genotype. It involves the application of the sciences of genomics, transcriptomics, proteomics, and metabolomics to human nutrition in order to understand the relationship between nutrition and health. Nutrigenomics has also been defined as the application of highthroughput genomic tools in nutrition research. The term "high throughput tools" in nutrigenomics refers to genetic tools that enable millions of genetic screening tests to be conducted at a single time. Such tools include those that measure the transcriptome - DNA microarray, Exon array, Tiling arrays, single nucleotide polymorphism arrays and genotyping. Tools that measure the proteome
include
methods based on gel electrophoresis, chromatography and mass spectrometry and the tools that measure the metabolome being less developed include methods based on nuclear magnetic resonance imaging and mass spectrometry often in combination with gas and liquid chromatography. When such high throughput screening is applied in nutrition research, it allows the examination of how nutrients affect the thousands of genes present in the human genome. Nutrigenomics involves the characterization of gene products and the physiological function and interactions 34
of these products. This includes how nutrients impact on the production and action of specific gene products and how these proteins in turn affect the response to nutrients. Nutrigenomic research also aims to address management and storage of different types of data derived from the various technological platforms used, development and application of new biostatistical algorithms, and inaccessibility of tissue samples from healthy volunteers. There are two strategies for conducting research in nutrigenomics. The first strategy would provide detailed molecular data on the interaction between nutrition and the genome, whereas the second strategy might be important for human nutrition, given the difficulty of collecting tissue samples from healthy individuals. The first strategy, typically applied by smaller research groups, would reveal the identification of transcription factors that function as nutrient sensors and the genes they target; elucidation of the signaling pathways involved, and characterization of the main dietary signals; measurement and validation of cell- and organ-specific gene expression signatures of the metabolic consequences of specific micronutrients and macronutrients; elucidation of interactions between nutrient-related regulatory pathways and proinflammatory stress pathways, to understand the process of metabolic dysregulation that leads to diet-related diseases; and identification of genotypes that are risk factors for development of diet-related human diseases (such as diabetes, hypertension, or atherosclerosis) and quantification of their impact. The second strategy would be the application of nutritional systems biology to develop biomarkers of early metabolic dysregulation and susceptibility (stress signatures) that are influenced by diet. This strategy would require large consortia, considerable research funding, and excellent multidisciplinary (and possible multinational) collaboration. Amongst the early research initiatives set up with the aim to fulfil the goal of nutrigenomics were, the Center of Excellence for Nutritional Genomics, established in 2003 with a $6.5 million grant at the University of California (UC), Davis, to coordinate nutrigenomics studies among participating institutes. 25 experts in nutrition, molecular biology, bioinformatics, and related fields from UC Davis, the Children's Hospital Oakland Research Institute, the U.S. Department of Agriculture Western Human Nutrition Research Center, and the Ethnic Health Institute at Alta Bates Summit Medical Center. Center members are involved in exploring how different foods interact with genes to increase the risk of type 2 diabetes mellitus, obesity, heart disease, and cancer. Across the Atlantic, the European Nutrigenomics Organisation (NuGO) was 35
launched in February 2004. This network of 22 scientists from 10 European countries
with a grant of 17.3 million [euro]
is involved in
developing new
technologies, improved model systems, and advance nutritional bioinformatics. It is hoped that by building up knowledge in this area, nutrigenomics will promote an increased understanding of how nutrition influences metabolic pathways and homeostatic control, which will then be used to prevent the development of chronic diet related diseases such as obesity and type two diabetes. Part of the approach of nutrigenomics involves finding markers of the early phase of diet related diseases; this is the phase at which intervention with nutrition can return the patient to health. As nutrigenomics seeks to understand the effect of different genetic predispositions in the development of such diseases, once a marker has been found and measured in an individual, the extent to which they are susceptible to the development of that disease will be quantified and personalized dietary recommendation can be given for that person. The aim of nutrigenomics also includes being able to demonstrate the effect of bioactive food compounds on health and the effect of health foods on health, which should lead to the development of functional foods that will keep people healthy according to their individual needs. Eventually it will lead to evidence-based dietary intervention strategies for restoring health and fitness and for preventing diet-related disease. Nutrigenomics-based signature analysis is a promising strategy for learning more about phenotypic responses to a nutrition intervention. Though a rapidly emerging science, nutrigenomics is still in its infancy but with a great potential in the near future where we can achieve within the scope of the expertise and techniques available now and in the coming years,
a thorough
understanding of how nutrients interact with the human genome at a molecular level. By identifying the mechanisms driving the connection between diet and the outward manifestation of our genes, our phenotype, we will thus be able to use genetic blueprints or genotypes for dietary prevention of diseases.
36
References 1. M. Muller, S. Kersten, (2003),
Nutrigenomics: goals and strategies,
Nat. Rev.
Genet., 4, 315-322 2. V.S. Neeha, Kinth P. Priyamvadah, (2013), Nutrigenomics research: A review, J. Food Sci. Technol., 50 (3) 415-428 3.A.Palou (2007) From nutrigenomics to personalised nutrition, Gen. Nutr., 2, 5-7 4.G.P. Patrinos, B. Prainsack, (2014) Working towards personalization of medicine: genomics in 2014, Pers. Med., 11 (7), 611-613
37
Module-II
38
Chapter-6
Colorful Truth about Anthocyanins – Role in Human Nutrition Veda Krishnan, Anil Dahuja and Shelly Praveen Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi Anthocyanins responsible for the colors - red, purple, and blue in fruits and vegetables which serves as attractants for pollination and seed dispersal; give protection against the harmful effects of UV irradiation; provide antiviral and antimicrobial activities. These are flavylium or phenyl-2-benzopyrylium salts (anthocyanidins) conjugated with free sugars and/or acylated sugars. The differences in glycosylation and acylation pattern result in the existing structural diversity in anthocyanins and exist in six major forms – Cyanidin, Delphinidin, Malvidin, Petunidin, Pelargonidin and Peonidin. Various naturally occurring sources are rich in anthocyanins like purple corn (~1642mg/100g), berries (~550mg/100g), eggplant (~750mg/100g), black rice (~116mg/100g), black soybean (~500mg/100g) etc. This purplish water soluble pigments which have emerged out as one of the most promising ingredient for functional food industry. The color and stability of these pigments are influenced by pH, light, temperature, and structure. Chromatography has been largely applied in extraction, separation, and quantification of anthocyanins. Besides the use of anthocyanidins and anthocyanins as natural dyes, these colored pigments are potential pharmaceutical ingredients that give various beneficial health effects. Scientific studies, such as cell culture studies, animal models, and human clinical trials, show that anthocyanidins and anthocyanins possess anti-oxidative and antimicrobial activities, improve visual and neurological health, and protect against various non-communicable diseases. These studies confer the health effects of anthocyanidins and anthocyanins, which are due to their potent antioxidant properties. Global anthocyanin market of US$ 300mn has been segmented into food, beverage, pharmaceuticals and personal care and Europe, mainly UK is the front runner. Therapeutic effects of anthocyanins has boomed the fortification industry where varied percentage of anthocyanin been fortified (2% 25%) in bread, confectionaries, desserts etc. Concentrates of anthocyanins have 39
also marketed as brands like ‘vita foods’ or ‘actiplants’ (>36% anthocyanins) where Asia-Pacific have a huge market as well as consumer demand for this. Thus anthocyanins are premium food supplements, which can value add the traditional farming community.
Figure1. Proposed mechanism for the ONOO- scavenging activity of pelargonidin (adapted from Tsuda et al., 2000). Nutraceutical properties Anthocyanins are absorbed in blood in an intact form, so they can reach various tissues and can modulate metabolic changes in the body. Anthocyanins present in some food and beverages has shown to play an important role in the prevention of diverse diseases such as cancer; cardiovascular diseases involving mechanisms of antioxidant activity, detoxification activity, anti-proliferation, induction of apoptosis, and anti-angiogenic activity; anti-inflammatory activity; inhibition of digestive enzymes (α-glucosidase, α-amylase, protease, and lipase), which is a clinical therapeutic target for controlling type II diabetes and obesity; improvement of the immune system; improvement of night vision as well as in the retardation of the aging process, reducing, for example, the risk of degenerative disorders, such as Alzheimer’s disease (Ames et al., 1993; Jing, 2006; Nikkhah et al., 2008). A detailed depiction of various nutraceutical attributes of anthocyanins have been included in Table 1.
40
Table.1. Biological activity of anthocyanins: based on in vitro studies Trait
Source
Anti-oxidant
Red
Concentration Effect wine 10µL
anthocyanin Anti-oxidant
Pigmented
0.2mg/L
rye
Reference
Decreased
Tedesco et al.,
ROS
2001
Decreased
Zykin et al., 2018
ROS
Cardio-
Bilberry
0.01
vascular
anthocyanin
mg/L
to
1.00 Decreased
Ziberna
et
al.,
et
al.,
rate of LDH, 2010
protection
increased post-ischemic coronary flow, decreased the incidence and
duration
of reperfusion arrhythmias Anti-obesity and
Cy-3-glc
100.00 μM
anti-
Regulation of Tsuda adipocytokine
diabetic
2005
secretion, total
633
genes glc)
(cy-3-
or
genes
427 (cy)
were upregulated Anti-
Red
wine 10.00 nmol/L
inflammatory anthocyanins
Increased
Garcia-Alonso et
oxidant status al., 2010 and decreased MCP-1, decreased TNF-α induced 41
inflammation Anticancer
Cy-3-glc
activity
12.50
to Increased
200.00 μg/mL
induction
Fimognari et al., 2004
differentiation, decreased cell proliferations , increased apoptosis Anti-
Bilberry (cy) 14.80 mg/L
Decreased
Lacombe et al.,
microbial
and
growth
2010
activity
blueberry (mv) extracts
Summary High level of antioxidant ability is reported for anthocyanins. Nevertheless, food and beverages in which phenolic compounds can be found may greatly change depending of several factor such as: (a) plant, agronomic and technological conditions (plant species, varieties, climatic and agronomical conditions in which plants grow, post-harvesting conditions of vegetable and fruits, technological treatment of rich-anthocyanin foods); (b) related to human beings (age, gender, health status, genetics and the presence of some pathologies which is determinant in the biotransformation, interaction between anthocyanins and other foods ingested at the same time or within a short interval of time) are only some examples which hampered studies concerning the effect of anthocyanins in human health. A step ahead, health care professionals can advise with certitude consumers which are looking for information about such anthocyanin rich foods/products for taking a decision about its inclusion in their dietary as supplement, nutraceutical, or other form to improve health, prevent or retard some diseases.
42
References 1. Ames BN, Shigenaga MK, Hagen TM (1993).Oxidants, antioxidants, and the degenerative diseases of aging.Proc. Natl. Acad. Sci. 90: 7915-7922 2. Fimognari C, Berti F, Nüsse M, Cantelli-Forti G, Hrelia P (2004). Induction of apoptosis in two human leukemia cell lines as well as differentiation in human promyelocytic
cells
by
cyanidin‐3‐O‐beta‐glucopyranoside. Biochem
Pharm 67(11):2047–56. 3. García‐Alonso M, Rimbach G, Rivas-Gonzalo JC, De Pascual-Teresa S (2004). Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A—studies in platelets, monocytes, and human endothelial cells. J Agric Food Chem 52(11):3378–84. 4. Jing P. (2006). Purple corn anthocyanins: chemical structure, chemopreventive activity and structure/function relationships. PhD thesis, 2006; The Ohio State University, U.S.A. p: 5-90 5. Lacombe A, Wu VCH, Tyler S, Edwards K(2010). Antimicrobial action of the American cranberry constituents; phenolics, anthocyanins, and organic acids, against Escherichia coli O157:H7. Intl J Food Microbiol 139(1–2):102–7. 6. Nikkhah E, Khayami M, Heidari R (2008).In vitro screening for antioxidant activity and cancer suppressive effect on blackberry (Morus nigra).Iran. J Canc. Prevent. 1: 167-172
43
Chapter-7
Vitamin E- a Powerful Antioxidant: Chemistry and Analysis in Foods Vinutha T, Navita Bansal and Shelly Praveen Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
Vitamin E (Vit-E) is a powerful, fat-soluble antioxidant, which protects cell membranes against damage caused by free radicals. It was one of the first two antioxidant compounds to be sold as dietary supplements, the second being vitamin C. Vitamin E is necessary for structural and functional maintenance of skeletal, cardiac, and smooth muscle. It also assists in the formation of red blood cells and helps to maintain stores of vitamins A and K, iron, and selenium. The term Vit- E encompasses a group of eight compounds, called tocopherols and tocotrienols, with various subsets of each, that comprise the vitamin complex as it is found in nature. Each of these different compounds has distinct chemistries and biological effects. Chemistry of Vitamin-E Vitamin-E is made of two compounds viz., tocopherols and tocotrienols together are also called as tocochromanol. Vitamin E exists in eight chemical forms; α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol. The tocopherols are characterized by the 6-chromanol ring structure methylated to varying degrees at the 5, 7, and 8 positions and the C16 saturated phytyl side chain is attached to chromanol ring at position 2 (Fig 1A). The tocotrienols are characterized by the presence of unsaturated double bonds at the 3', 7', and 11' positions of the phytyl side chain (Fig 1B). The different forms of tocopherols and tocotrienols are differing by the number and positions of the methyl groups on the 6-chromanol ring; α-tocopherol/ tocotrienol are tri-methylated; β –tocopherol/ tocotrienol, γ-tocopherol/tocotrienol are dimethylated; and δ-tocopherol/tocotrienol are mono-methylated.
44
Vit-E forms
R1
R2
Relative activity of Vit-E (tocopherolvstocotrienol
α - tocopherol/trienol
-CH3
-CH3
100% vs 30%
β - tocopherol/trienol
-CH3
-H
50% vs 5%
γ - tocopherol/trienol
-H
-CH3
10% vs 0%
δ - tocopherol/trienol
-H
-H
Figure 1:
3%
vs 0%
The general structure of tocopherol (A) and tocotrienol (B). The table
indicates the number/position of ring methyls in α, β, γ and δ tocopherol and tocotrienols and relative Vit-E activities are shown. Of the different tocopherol and tocotrienol species present in foods, α-tocopherol has the highest Vit-E activity. Due to the occurrence of α-tocopherols on the surface of the plasma membrane, it has highest antioxidant potential (KamalEldin and Appelqvist, 1996) where in, other forms of Vit-E are buried within the membrane and physically inaccessible to scavenge Reactive Oxygen Species (ROS). Vitamin E is believed to act as membrane stabilizer. The stabilization process is caused by the interaction between the chromanol hydroxyl group of α-tocopherol with the carboxyl group of the ester carbonyl bond of the phospholipid molecules which increases the rigidity of the membrane (Munne-Bosch, 2002).
45
Sources of vitamin E In plants, tocopherol composition differs between different species and between different tissues within one species. Usually, leaves commonly accumulate αtocopherol whereas seeds are rich in γ-tocopherol, β- and δ tocopherol are not very abundant in most plant species. The variations in tocopherol isoforms are detected in several plant species such as soybean, rapeseed and Arabidopsis with most of the tocopherols are present in the form of γ or δ-tocopherol in seeds. In case of sunflower and safflower seeds, α-tocopherol comprises more than 95% of the total tocopherol content. Wide variations in tocopherol content and concentration have been reported in different fats and oils (Fig 2).
Figure 2: A.Tocopherols and tocotrienols content in various fats and oils; Slover, H T.
(1971), B. α-tocopherol and total tocopherol content in various crops Potential applications of Vitamin E Vitamin E as animal feed: Vit-E supplements in animal feed and diet is increasing at an alarming rate and is due to its antioxidant activity, membrane integrity, immunity and better muscle function. Moreover, it enhances the shelf life of feed by imparting oxidative stability. Vitamin E as dietary supplements: Due to high incidence of lifestyle disorders contributed by diet as well as sedentary life style, there is a growing awareness on health concerns and hence great market for wellness products and supplements. As a health supplement, it improves the immune function and maintains vitamin A levels etc. Natural and synthetic forms of Vit-E are well known as anti-cancerous, especially RRR-α-tocopherol ether-linked acetic acid analog (α-TEA).
46
Vitamin E as sports blend: As known for muscle developing attributes, Vit-E is used in sports blend for the tired muscles. Vitamin E in cosmetic industry: As an antioxidant natural Vit-E is widely used in cosmetic industry in cosmetics and skin care; including lotions, creams, lipsticks, sunscreens etc. It protects skin from harmful radiations like UV and prevents deposition of melanin. It delays the signs of aging, heal scars and acnes, improves moisture content in skin and its texture. It also improves the stability of lipid-based cosmetics. Other industrial uses:Mixed tocopherols from soybean oil processing are generally used for stabilizing oxidatively-sensitive lipid supplements such as those from marine oils and other products. In addition, tocopherol-rich oils, such as germ oils from oat, barley and wheat may be mixed with other oils to stabilize them. Among the tocotrienol-rich oils, palm oil, annatto, and rice bran oil are important, and these are often used in product enrichment. Considerable amounts of tocopherols are removed during the refining process of edible oils. Commercially gamma-tocopherol is being used as an anti-oxidative protectant to preserve food emulsions like fish oil-enriched salad emulsions. References 1. Prieto, P; Pineda, M. Aguilar, M (1999). Spectrophotometric Quantitation of Antioxidant Capacity through the Formation of a Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E. Anal. Biochem. 269: 337341. 2. Sicaire AG , Vian M , Fine F, Joffre F , Carre P ,Tostain S and Chemat F (2015). Alternative Bio-Based Solvents for Extraction of Fat and Oils:Solubility Prediction, Global Yield, Extraction Kinetics,Chemical Composition and Cost of Manufacturing. Int. J. Mol. Sci.16, 8430-8453. 3. Vinutha T, ChiragMaheswari, NavitaBansal, Rama Prashat G, Veda Krishnan, SwetaKumari, Anil Dahuja, ArchanaSachdev and R.D. Rai (2015). Expression analysis of γ-tocopherol methyl transferase genes and alpha- tocopherol content in developing seeds of soybean (Glycine max). Indian J Biochem&Biophys. 52(3&4): 267-273. 47
4. Vinutha, T; Bansal, Navita; Kumari, Khushboo ; G, Rama; Sreevathsa, Rohini; Krishnan, Veda; Kumari, Sweta; Dahuja, Anil; Lal, Sanjay; Sachdev, Archana ; Praveen, Shelly Comparative analysis of tocopherol biosynthesis genes and its transcriptional regulation in soybean seeds (2017) Journal of Agricultural and Food Chemistry doi: 10.1021/acs.jafc.7b03448
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Module-III
49
Chapter-8
Coconut: A Treasure Trove of Nutrition and Health Benefits
S.V. Ramesha, M. Arivalagan a,b and K.B. Hebbara a
Division of Physiology, Biochemistry and Post-Harvest Technology, ICAR- Central Plantation Crops Research Institute, Kasaragod, Kerala b ICAR-Indian Institute of Horticultural Research (ICAR-IIHR), Bengaluru, Karnataka
Coconut [Cocosnucifera L.,] belongs to the family Arecaceae and is widely called as ‘Kalpaviriksha’ or ‘Tree of Heaven’. It is also one of the most important sources of vegetable oil in the world. Coconut and various values added products obtained from coconut have numerous health benefits. Coconut endosperm: Coconut endosperm is the edible part of the coconut and exists in two forms: Liquid form called nut water and solid form called kernel. At initial developmental stage, coconut endosperm is in the liquid form containing free nuclei. Endosperm nucleus undergoes several cycles of division without cytokinesis. The process of cytokinesis initially takes place in the periphery of the nut, which progress further towards the centre of the nut, forming a cellular endosperm layer. At initial stages of development, the cellular endosperm appear as translucent jelly like structure, which hardens and become white flesh (coconut meat) upon maturity. In coconut, cellularization process does not fill up the entire cavity of the embryo sac instead the cavity is filled with substantial amount of water, termed coconut water. Table 1. Biochemical composition of coconut endosperm/kernel Constituents
Values*
Constituents
Values*
Proximates Moisture (g)
46.99
Carbohydrates# (g)
15.23
Protein (g)
3.33
Total dietary fibre (g)
9.00
50
Total fat (g)
33.49
Total sugar (g)
6.23
14
Potassium (mg)
356
Sodium (mg)
20
Minerals Calcium (mg) Iron (mg)
2.43
Magnesium (mg)
32
Phosphorus (mg)
113
Zinc (mg)
1.10
Vitamins Vitamin C (mg)
3.3
Vitamin B6 (mg)
0.054
Thiamine (mg)
0.066
Folate, DEE (µg)
26
Riboflavin (mg)
0.020
Vitamin E, (mg)
0.24
Niacin (mg)
0.540
Vitamin K (µg)
0.2
Total SFA (g)
29.698
Total PUFA (g)
0.366
Total MUFA (g)
1.425
Lipids
(Source: USDA); * values are giver on fresh weight basis per 100 g coconut kernel; # carbohydrates by difference; SFA – Saturated fatty acids; MUFA – Monounsaturated fatty acids; PUFA – Poly unsaturated fatty acids. Coconut water and health benefits: Coconut water, a liquid endosperm, is a refreshing nutritional beverage. It is one of the richest sources of complex vitamins, macro and micro minerals, amino acids, etc. The electrolytes present in the coconut water mimics that of human plasma, and hence it is usedas an electrolyte supplement and in oral rehydration. It is extensively used for treating childhood diarrhea, cholera and gastroenteritis and to reduce the problem of intestinal disturbance in infants and prevent the nutritional rickets. Coconut water showed cardio protective effect in the rats induced with myocardial infarction, reduces the high blood pressure and improves the blood circulation. It improves the production of nitric oxide, which favors the vasorelaxation and thus cardioprotective effect. Folate present in the coconut water protects the mitochondrial toxicity. cytokinins, especially kinetin, has anti-aging property. It has potential therapeutic properties for arterial thrombosis due to its anti-platelet effect, and also possess anti-carcinogenic effects. Coconut water is used in treating various illnesses in urinary systems and functions more effectively in removing the kidney 51
stones and reduces the bladder infection. Coconut water also showed hypolipidemic effect in rats that are fed with cholesterol enriched diet. Value added products of coconut: Coconut oil/Virgin coconut oil (VCO): Coconut oil is extracted from the kernel has various applications in food, medicine, and industry. As it is relativelystable in atmospheric air, it get oxidized very slowly hence resistant to rancidity.Coconut oil contains high saturated fat content. Depending on the production methods used, coconut oil is called as traditional or RBD (refined, bleached and deodorized, refined) oil and Virgin Coconut Oil (VCO). VCO profoundly differs from the traditionally produced coconut oil in terms of quality attributes. The RBD coconut oil does not contain Vitamin E since it is degraded when the oil is subjected to high temperature and various physiochemical processes. Fatty acid composition of VCO is similar to that of traditional and refined coconut oil, but it is rich in antioxidants such as poly phenolic compounds, tocopherols and phytosterols. Major polyphenolic compounds identified in VCO were protocatechuic, vanillic, caffeic, syringic, ferulic and p-coumaric acids. Health benefits of coconut oil: •
Digestion of medium chain fatty acids (MCFAs) in coconut oil by the liver provides ketones to the brain which acts as an alternate source of energy to help repair brain function in cases of Alzheimer’s’ disease.
•
Coconut oil is high in natural saturated fats, which not only increases the healthy cholesterol (HDL) in our body, but also converts LDL into HDL and lower the risk of heart disease
•
The MCFAs in the coconut oil work as a natural antibiotic by disrupting the lipid coating on bacteria and killing them. Coconut oil directly protects the liver from damage “and cures” urinary and kidney infection.
52
•
Since tumor cells are not able to access the energy from ketones and are glucose dependent, the ketones produced during digestion of coconut oil can help to fight cancer
•
Coconut oil is very effective against a variety of infections due to its antifungal, antiviral, and antibacterial properties. When applied on infections, it forms a chemical layer which protects the infected body part from external dust, air, fungi, bacteria and virus.
•
Lauric acid in coconut oil reduces candida, fight bacteria, and create a hostile environment for viruses. When taken orally, the oil is found to be a useful adjunct therapy in children with community-acquired pneumonia.
•
Coconut oil also improves digestion as it helps the body to absorb fat-soluble vitamins, calcium, and magnesium and reduces stomach ulcers and ‘ulcerative colitis’.
•
The oil is remarkably effective against Streptococcus mutans b, a leading contributor to tooth decay. Additionally, coconut oil was shown to be effective against Candida albicans, the yeast that notoriously causes thrush, another common oral health concern. Calcium is an important element present in teeth. Since coconut oil facilitates absorption of calcium by the body, it helps in getting strong teeth.
•
The MCFA’s in coconut oil helps balance the insulin reactions in the cells and promote healthy digestive process. They take off the strain on the pancreas and give the body a consistent energy source which can prevent insulin resistance and Type II diabetes.
•
Coconut oil also helps to burn fat, decrease appetite and is especially helpful in losing belly fat. MCFA can be incorporated in the weight loss program without fear of metabolic risk factors.
•
Coconut oil, being a triglyceride of lauric acid, has a high affinity for hair proteins and, because of its low molecular weight and straight linear chain, is able to penetrate inside the hair shaft to reduce the protein loss compared to other oils.
•
Coconut oil improves antioxidant levels and can slow aging, by reducing stress on the liver and by lowering oxidative stress.
53
•
Numerous studies have suggested that the consumption of foods containing dietary phenolics might significantly contribute to the human health. Beneficial effects resulting from phenolic antioxidants have created a niche for specialty foods with phenolic compounds. The total phenolic content of VCO was nearly seven times higher than that of commercial coconut oil. The antioxidant activity in VCO was reported to be high compared to refined coconut oil.
Coconut milk residue and VCO cake: The two important co-products during VCO production are coconut milk residue and VCO cake flour. These are being used mainly in animal feed production at present. As a source of dietary fiber and other nutrients, these co-products provide a number of health benefits in preventing coronary heart diseases, colon cancer and diabetes. The dried coconut milk residue and VCO cake flour can be utilized in the preparation of extrudates and sweet snacks along with the broken rice, maize grits and pearl millet grits. Coconut inflorescence sap: Coconut inflorescence sap (neera, kalparasa) is a phloem sap traditionally tapped from unopened inflorescence of coconut and consumed largely by rural population. Sap is reported as a natural and non-alcoholic beverage, high in nutritional value and an instant thirst quencher. Sap contains a number of minerals and salts and is high in protein. It contains acids like ascorbic acid, nicotinic acid (vitamin B3) and riboflavin. Due to its high nutrient content, sap is prone to fermentation by yeast and bacteria. In order to collect fresh unfermented sap, a device has been developed at ICAR-Central Plantation Crops Research Institute (CPCRI), Kasaragod, Kerala, India. The device has been designed so as to conceal the cut end of spadix to the container in the ice box in order to avoid
the
contamination and arrest the process of fermentation. Fresh sap is rich in sugar, minerals and proteins. It is also a rich source of phenolics and ascorbic acid. Coconut sap contains high amounts of essential elements such as N, P, K, Mg and micronutrients (Zn, Fe, and Cu). Since, it is rich in minerals and vitamins it is considered as one of the best natural health drinks. It can be promoted as an instant energy provider, as a functional food, nutraceutical. It is good for post operative care 54
due to high content of electrolytes. Frequent consumption of kalparasa known to prevent diseases like jaundice and keeps one healthy. Coconut sugar: Coconut sugar is also known as coconut palm sugar, coco sugar or coco sap sugar which is one of the best natural sweeteners. It is a rich source of potassium, magnesium, zinc and iron and contains all essential amino acids and is rich in B complex Vitamins.Coconut sugar has a low glycemic index and hence is a promising product for diabetics. Characteristic features of coconut sugar •
It is completely natural, and no added chemicals.
•
Unlike cane sugar coconut sugar supplies calories and nutrients as well.
•
It contains considerable amount of minerals like calcium, magnesium, potassium, zinc, iron, copper, manganese, phosphorus and boron, they have important roles to play in alleviation of micronutrient deficiencies especially iron and zinc and such other public health problems
•
It contains about 2.5 to 3 % of dietary fibres, helps maintain bowel health, lowers cholesterol levels and helps control blood sugar levels.
•
It is a rich source of phenolics which are potent and important contributors in reducing oxidative stress. Natural antioxidants is associated with a lower risk of cardiovascular disease and cancer.
•
Coconut sugar has low glycemic index and is in the range of 35 to 54 Gi/ serving compared to the high glycemic index rating of cane sugar which falls in the range of 65-100 Gi/serving.
Coconut haustorium: Coconut haustorium is a spongy absorbent tissue formed from the basal part of coconut embryo during germination. Coconut haustorium contains about 85 to 88% moisture, and about 10 % of soluble nutrients, which include a considerable amount of sugars (mostly of reducing sugars) and rich in dietary fibre, small amount of aminoacids and proteins.
55
Haustorium contains considerable amount of dietary fibrecomprising both soluble and insoluble fibres, between 4.96 to 6.20 % and 17.1 to 23.2 %, respectively. As it is rich source of simple sugars (except lactose), coconut haustorium could be used as primary energy source for the children who are suffering lactose intolerance. Aspartic acid was found maximum in the haustorium (29.9%), followed by alanine (11.4%), proline (9.32 %) and glutamic acid (6.35%). The amino acid score value of histidine (170%), valine (160%) and lysine (135%) was higher than the recommended value of FAO, whereas the threonine score was on par with the FAO reference value. The aromatic amino acids (phenylalanine and tyrosine), leucine, sulphur containing amino acids and isoleucine scores were very less (32.5, 45.7, 57.6 and 68%, respectively) indicating the deficit state. Thus, in order to make coconut haustorium as nutritionally superior, fortification with essential amino acid rich food matrices is essential. Presence of high amount of acidic amino acids viz., aspartic and glutamic acids will help in improving the flavor of the haustorium. coconut haustorium is rich source of magnesium, manganese and especially iron, and it can certainly be a high potential diet in many developing countries, where the average diet is deficient in iron. The fortification of haustorium with calcium and phosphorous is essential for to transform it into a nutritionally balanced food as it is low in calcium and phosphorus. References 1. Hebbar, K.B and Arivalagan, M. 2015. Health benefits of coconut oil. Indian Coconut Journal. LVII (9), Pp27-30 2. Hebbar, K.B., M. Arivalagan., A.C. Mathew and P. Chowdappa. 2015. Kalparasa Collection and value addition. Technical Bulletin No. 92. ICARCPCRI, Kasaragod, Kerala, India. 28 p. 3. Jean WH, Yong, LiyaGe, Yan Fei Ng and SweeNgin Tan, The chemical composition
and
biological
properties
of
coconut
(CocosnuciferaL.)
Water.Molecules, 14 (2009) 5144. 4. Manikantan, M.R., Mathew, A.C., Madhavan, K., Arumuganathan, T., Arivalagan, M., ShameenaBeegum, P.P., Hebbar, K.B. 2016. Coconut Chips Entrepreneurship driven ICAR-CPCRI technology for healthy alternative nonfried snack food. Technical bulletin No. 107 ICAR- Central Plantation Crops Research Institute, Kasaragod, Kerala, India. 28 p. 56
5. Manikantan, M.R., Mathew, A.C., Madhavan, K., Arumuganathan, T., Arivalagan, M., ShameenaBeegum, P.P., Hebbar, K.B. 20162016. Virgin coconut oil- Hot and fermentation process. Technical bulletin No. 108. ICARCentral Plantation Crops Research Institute, Kasaragod, Kerala, India. 37 p.
57
Chapter 9
Omega-3 Fatty Acids: An Essential Contribution in Human Diet G.Venkateshwarlu Education Division, ICAR-HQ Indian Council of Agricultural Research, New Delhi
Consumption pattern of fats and fatty acids Human diet and lifestyle issues are closely associated with a myriad of cardiovascular risk factors including abnormal plasma lipid, hypertension, insulin resistance, diabetes, and obesity, suggesting that diet based approaches may be of immense benefit. Substantial evidence from epidemiological and clinical trial studies indicates consumption of long chain omega-fatty acids reduce risk of cardiovascular mortality. Low fat intake and associated chronic energy deficiency have been the major nutritional problem of developing countries. The consumption of fat has been found to be lower in developing countries, that is, 49 g/person/day in comparison to 128 g/person/day in the developed countries. It has been observed that the supply of fat and omega -3 fatty acids decreases significantly with decreasing gross domestic product (GDP). Therefore, it is imperative to look for sources of PUFA, particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and other fatty acids for steady supply for health and nutrition of millions of people in the developing countries. Health and nutritional advisories on the use of highly unsaturated fatty acids date back to 1975 when the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) recommended that infant formula should mimic breast milk. With particular reference to PUFAs, human breast milk is rich in arachidonic acid and DHA. General recommendation for daily intakes of DHA/EPA is 0.5 g for infants and 1 g/day for adult and patients with heart disease. Beneficial role of Omega-3 fatty acids Fatty acids play crucial role inmaintaining health and cellular functions. The preventive effect of omega-3 fatty acids on coronary heart disease is based upon hundreds of experiments in animals, humans, tissue culture studies, and even clinical trials which first became apparent in the investigation on the health status ofGreenland Eskimos who consumed a very high fat diet from seal, whale, and fish and yet had a low incidence of coronary heart disease. Further studies have shown that the kind of fat the Eskimos consumed contained large quantities of omega-3 58
fatty acids namely EPA(20:5) andDHA(22:6). Among the long chain omega-3 fatty acids (LC-PUFA), DHA is the principal PUFA constituent of brain neurons, retinal cells, and primary structural component of skin, sperm, and testicles. Apart from being an important structural component of cellular membranes, it performs varieties of functions in a number of cellular processes like transport of neurotransmitters and amino acids and modulates the functioning of ion channels and responses of retinal pigments. DHA has been shown to be particularly important for brain development, optimal development of motor skills and visual acuity in infants, lipid metabolism in children and adults, and cognitive support in the elderly. DHA along with EPA play important role in preventing atherosclerosis, dementia, rheumatoid arthritis, diseases of old age like Alzheimer’s disease, and age related macular degeneration. DHA is an essential nutrient as it is synthesized in very less quantity in human body and is obtained mainly through diet. Biosynthetic pathways The pathway for biosynthesis of Arachidonic acid [20:4(n-6)] and DHA [22:6(n-3)] involves desaturation and elongation of the dietary essential fatty acids, linoleic acid [18:2(n-6)] and linolenic acid [18:3(n-3)], respectively. Both the (n6/omega-6) and (n-3/omega-3) fatty acid pathways are believed to use the same desaturase enzymes, and desaturation is subject to competitive substrate and product inhibition. Thus, desaturation of 18:2(n-6) to 20:4(n-6) can be inhibited by 18:3(n-3), 20:5(n-3) and 22:6(n-3).
No vertebrate species can synthesize polyunsaturated fatty acids (PUFA) de novo, as they lack the fatty acid desaturase enzymes required for the production of linoleate (18:2 n-6) and linolenate (18:3 n-3) from oleic acid (18:1n-9). However, many vertebrates can convert dietary 18:2n-6 and 18:3 n-3 to long chain highly unsaturated fatty acids (HUFA) such as arachidonic acid (20:4 n-6, AA), eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (22:6 n-3, DHA) via a pathway involving a series of microsomal fatty acid desaturation and elongation steps. The production of EPA requires ∆6and ∆5 desaturases, and the production of DHA from EPA requires a further desaturation originally thought to be effected by a ∆4 desaturase acting on a C22 fatty acid intermediate, although evidence now 59
suggests it may actually be affected by a ∆6 desaturase acting on a C24 intermediate. Significance of Omega-3/Omega-6Ratio Improving the dietary ratio by decreasing omega-6 fatty acids and increasing omega-3 fatty acids is essential for brainfunction, prevention of cardiovascular diseases,arthritis and cancer. Omega-3 and omega-6 fatty acids competefor the same metabolic enzymes in human body and the ratio of omega-3/omega-6 will significantly influence the ratio of ensuing eicosanoids, which can altermetabolic functions and it is essential to maintain a favourable omega-3/omega-6 ratio in the diet.
References 1. M. Y. Abeywardena and G. S. Patten, “Role of 3 longchain polyunsaturated fatty acids in reducing cardio-metabolic risk factors,” Endocrine, Metabolic & Immune Disorders-Drug Targets, vol. 11, no. 3, pp. 232–246, 2011. 2. H. O. Bang, J. Dyerberg, and N. Hjøorne, “The composition of food consumed by Greenland Eskimos,” Acta Medica Scandinavica, vol. 200, no. 1-2, pp. 69– 73, 1976. 3. P. M. Kidd, “Omega-3 DHA and EPA for cognition, behavior, and mood: clinical findings and structural-functional synergies with cell membrane phospholipids,” Alternative Medicine Review, vol. 12, no. 3, pp. 207–227, 2007. 4. W. E. Conor, “Importance of n-3 fatty acids in health and diseases,” American Journal of Clinical Nutrition, vol. 71, no. 1, supplement, pp. 171S–175S, 2000. 5. P. C. Calder, “The role of marine omega-3 (n-3) fatty acids in inflammatory processes, atherosclerosis and plaque stability,”Molecular Nutrition and Food Research, vol. 56, no. 7, pp. 1073–1080, 2012. 6. J. Beare-Rogers, A. Ghafoorunissa, O. Korver, G. Rocquelin, K. Sundram, and R. Uauy, “Dietary fat in developing countries,” Food and Nutrition Bulletin, vol. 19, supplement 1, pp. 251–266, 1998.
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Module-IV
61
Chapter-10
Poor Shelf-life of Pearl Millet Flour - Approaches to Improve the Flour Quality Suneha Goswami1, RR Kumar1, SP Singh2, Tara C. Satyavathi3 and Shelly Praveen1 1
Division of Biochemistry,2Division of Genetics, ICAR- Indian Agricultural Research Institute (IARI) 3All India Coordinated Research Project-Pearl Millet, Jodhpur Pearl millet (Pennisetum glaucumL.) is one of the most important drought-tolerant crops cultivated mostly in semi-arid parts of Africa and Asia and is a major source of energy and proteins for about 500 million people. India is the largest producer of pearl millet in the world. It is a highly tillering, cross-pollinating millet crop with a short life cycle and a large genome size (1.79 Gb). It is the third most widely cultivated food crop after rice and wheat in India. It is usually grown in marginal environment of arid and semi-arid regions of Sub-Saharan Africa and the Indian subcontinent, characterized by scanty and erratic rainfall, poor soil conditions and high temperature, where staple cereals such as rice, wheat, maize, and even sorghum are likely to fail. Pearl millet (Bajra) is a multipurpose crop, which is grown for food, feed and forage. Pearl millet has an excellent nutritional composition and is a rich source of energy (361 Kcal/100 g) as compared to staple cereals like rice (345 Kcal/100 g) and wheat (346 Kcal/100 g). Further with regard to nutritional quality, it is generally equivalent to maize and superior to sorghum in terms of protein content, quality, and metabolizable energy. Although it is deficient in essential amino acids, it contains 35% more lysine as compared to sorghum. Additionally, it also does not contain condensed polyphenols which are a major cause of decreased digestibility, for example, tannins in sorghum. Pearl millet grains contain 5–6% oil and are also rich in important micronutrients like iron and zinc (Palande et al., 1996). Hence, pearl millet significantly contributes toward protein, iron, and zinc uptake as well as serves as the cheapest source of energy to low-income group consumers of semi-arid tropics including India. Pearl Millet has an immense potential toward medicinal uses like, it is a potential component in the diets of patients suffering from constipation, 62
obesity, and gallstones, beneficial for persons with celiac disease and/or diabetes and it can be safely incorporated in the diets of pregnant women, infants, lactating mothers, elderly and convalescents.
Chemistry behind poor shelf life Despite nutritionally very rich and high medicinal value, the full potential of pearl millet flour is limited due to rancidity and off odour during storage. High fat content along with highly active lipases causes hydrolysis of fat into fatty acids. It contains 74% unsaturated fatty acids like oleic (C18:1), linoleic (C18:2), and linolenic (C18:3). The lipase (triacylglycerol acyl hydrolase, E.C.3.1.1.3) enzyme is accumulated in the pericarp, aleurone layer and germ. This enzyme is responsible for stepwise hydrolysis of the triacyl glycerol into diacyl glycerol, monoacylglycerol, glycerol and short chain free fatty acids. Unsaturated fatty acids undergoes to oxidation in the presence of oxygen and moisture, resulting in development of undesired off odour. Pearl millet is also reported to contain oxidative enzyme such as peroxidase (Reddy et al., 1986) and enzymatic browning, catalysed by enzyme such as polyphenol oxidase (PPO), both of which play important role in pearl millet flour quality deterioration. Hence, there are two type of rancidity mechanisms contribute to the development of off odour in pearl millet flour. First is enzymatic rancidity caused by lipase enzyme which results the release of free fatty acids, which further converted into phenolic aglycones, causes the generation of mousy odour. Poly phenol oxidase causes enzymatic browning, which results the generation of bitter compounds. Second mechanism is oxidative rancidity, caused by the lipoxygenase activity on free fatty acids and converted into hydro-peroxides and further through peroxidase activity, into aldehyde, ketones, polymers of peroxides etc, which is responsible for off odour development. Due to above reason, pearl millet flour can only be stored for short period of span and it quickly get rancid and off odour (Fig. 1). Technological interventions to improve the flour shelf life Nutritional composition and health benefits attracted present day health conscious consumer segment emphasizing commercial viability of the crop. Pearl millet can be stored for longer period without substantial quality alteration if the kernel remains 63
intact but once the grain is decorticated and ground the quality of meal deteriorates rapidly. Pearl millet is used in preparation of conventional foods such as porridge, roti or bhakhri (stiff roti), made from either coarsely or finely milled flour. But the major constraint is that the flour becomes rancid within a few days after milling. Rancidity of pearl millet flour during storage is one of the long-lasting unresolved problems for food process engineers. Now various studies and technological interventions trying to improve the pearl millet flour quality and storability as mentioned in Table 1. Method of milling and type of packaging are crucial unit operations in pearl millet processing as these determine the quality, shelf life and also influences market value of the product. This is the phase with maximum opportunity for rancidity development in the flour.The pearling of pearl millet grains brings changes in its chemical composition. During the pearling operation, nutrients present in the grain are distributed due to the reason that certain nutrients are concentrated into certain part of grain. The pearl millet grains are processed by friction and abrasion operation in a simple pearler to peel and strip various layers of bran from grains. Beside this, other methods like pre-milling processing such as blanching, malting, popping and dry heat treatment etc. are given to the grains before product development, which produces flour with longer shelf life (Akinola et al., 2017). Some studies reports that microwave heating of pearl millet grains decreased lipase activity significantly (p < 0.05) with an increase in moisture level from 12 to 18 % and maximum reduction of lipase activity (92.9 %) was observed at 18 % moisture level for 100 s microwave treatment (Yadav et al., 2012). Progressive demand for pearl millet flour and its storability complications emphasizes need for innovative interventions for enhancing storability of pearl millet flour. References 1. Akinola SA, Badejo AA,Osundahunsi OF,
Edema MO (2017) Effect of
preprocessing techniques on pearl millet flour andchanges in technological properties. International Journal of Food Science and Technology.52(4):992999.
64
2. Yadav DN, Anand T, Kaur J, Singh AK. (2012) Improved Storage Stability of Pearl Millet Flour Through Microwave Treatment. Agric Res (2012) 1: 399. 3. Palande KB, Kadlag RY, Kachare DP, Chavan JK (1996) Effect of blanching of pearl millet seeds on nutritional composition and shelf life of its meal. J Food SciTechnol 33(2):153–155. 4. Arora P, Sehgal S, Kawatra A (2002) The role of dry heat treatment in improving the shelf life of pearl millet flour. J Food SciTechnol 16:331–336.
Figure 1. Role of different enzymes in off odour development in pearl millet flour
65
Table 1. Technology used to mitigate the poor shelf life and improve the flour storability
Methodology
Result/findings
References
Storage in cotton and
Fat acidity increased in cotton
Kaced etal.,(1984)
polyethylene
bags compared to polyethylene bags
Defatted pearl
Fat acidity and peroxide valuesdidn’t Kapoor and
millet flour using n-hexane change during one month storage
Kapoor (1990)
Hot water blanching
Palade etal.,(1996)
Enzymes are inactivated. Phenolics are reduced. Reduced minerals, antioxidants, vitamins
Use of antioxidants
Inhibit the radical propagation
Eskin and
(BHA, BHT, Citric acid,
stages in the triglycerideoxidation
Przybylski, (2001)
tocopherols and a
process
range of phenolics) Oven heating
Enzyme inactivation; reduced
Arora etal., (2002)
antioxidant vitamins Toasting and Boiling
Reduced rate of rancidity.
Mohamed etal., (2011)
Enzymes are inactivated
Yadavetal.,(2012)
Followed by refrigerated storage Microwave treatment
(Tempered grains treated at 18% moisture) Germination and Malting
Improves digestibility; changes in functional properties of flour.
66
Akinola et al., (2017)
Chapter-11
Starch Hydrolyzation Kinetics of Pigmented Rice Veda Krishnan, Archana Singh and Shelly Praveen Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi Starch or amylum is a polymeric carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. This polysaccharide is the main energy source and rice is its major source in our diet. Rice grain is largely composed of starch (approximately 80%-90%) and hence slowly vanished from our diet due to increasing incidence of type II diabetes. But other than the commonly consumed white rice, there are different variants by their husk and endosperm colour into red, green, violet, black, and brown varieties.Even though white rice rules in terms of consumption worldwide, some Asian countries also consume pigmented cultivars like black, purple, red, and reddish brown rice varieties as part of tradition or due to their medicinal attributes.Pigmented rice belongs to two species - Oryza sativa L, which originated from South-East Asia and Oryza glaberrima Steud, which is native to West Africa.In India, the total cultivated area has been recordedas 43.77 M ha (29.4% of the global rice area) with aproduction of 90 million tons. Among which ‘Njavara’, ‘jyothi’, ‘Chakhao’, ‘Kavuni’ etc are commonly known for its medicinal properties owing to its rich cocktail of bio-actives. Nutraceutical properties A varied list of nutraceutical attributes has been deciphered including anti-oxidant activity, hypo-glycemic activity, anti-inflammatory activity etc (Table 1). A study conducted in Thai pigmented rice varieties showed anti-glycation capacity and proanthocyanidins in red rice bran also exhibited moderate chelating activity. Chen et al. (2016) reported that the pigmented rice extracts are very strong intracellular candidates inside the cell-based systems. Various researchers mentioned that the lightness (L*), redness (a*) and yellowness (b*) values of pigmented rice are strong indicators of its bioactive components. The major bioactive components responsible for such nutraceutical properties are gallic, protocatechuic, hydroxybenzoic, 67
pcoumaric,ferulic, glucoside,flavan-3-ol
sinapic (+)
acid,
catechin
cyanidin-3-O-glucoside, and
epicatechin,flavanols
peonidin-3-O(flavan-
3-ols),
isoflavones, c-oryzanol contents, compositions of steryl, triterpene alcohol ferulates proportions, and tocopherols, etc. Exploring these nutraceutical matrices will value add and can include this pigmented rice into the therapeutic regimen in future.
Starch quality and hydrolyzation dynamics Physiological quality of starch is contributed by its structure and composition which impacts its digestibility. Starch is mainly classified into three based on its amylolytic activities – rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). The comparative change in digestibility over a period of time i.e. from 0-240min is commonly termed as ‘starch hydrolyzation kinetics’ (Fig.1). Enzymatic digestion and classification of starch based on such kinetics can act as potential tool for screening varieties with good starch quality. Other than the inherent biochemical nature, the major factor that affect digestibility is particle size of starch granule. An inverse relation has been observed between particle size and digestibility. This particle size dependent reduction in the extent of starch digestion is indicative of reduced starch bio-accessibility where the food matrix imposes a physical barrier to enzyme ingress. Alternatively, it has been hypothesized that in high amylose starches anindigestible fraction is formed as a result of α-amylase activity, due tothe action of the enzyme releasing linear glucan chains which are ableto recrystallize during digestion.
Figure 1. Starch hydrolyzation curves obtained for milled particle sizes of hydrothermally processed WT (A) and r-mutant rice (B) samples digested by porcine 68
pancreatic α-amylase. Values are means of triplicates ± SEM. Thus understanding the starch hydrolysis kinetics will be a new direction in starch quality analysis and varieties with less hydrolysis index can be included to diversify the diet of diabetics, which will assist in management of it.
Table1. Nutraceutical properties of pigmented rice Type of
Bioactivities
Method
Concentration/amount
Reference
pigmented rice Black
rice Antioxidant
bran
and activity
red bran
Oxygen
Mean ORAC values for Chen et al.,
Anti- radical
the accessions with red 2016
atherosclerosis absorbance
and purple bran were
activity
426.40
capacity
and
725.56±
69.32A µM TE g-1 bran Blackish
Inhibitory
Xanthine
Suwon 415 (blackish– Nam et al.,
purple rice
activities
oxidase
purple), inhibition of
inhibition
Xanthine
(%)
2005
oxidase
activity at different concentration (0.1–5 µg mL-1) by rice bran extracts were ranged from 12.12 - 73.09 µg
Black rice
Hypoglycemic
In
activities
method
vivo High-fructose diet from Guo et al., black
rice
pigment 2007
fraction showed insulin sensitivity 5.00±0.3
reduction to
4.4±0.4
(mmol/L) from 4 to 8 weeks of feeding. Black rice
Reduction the
of In
vivo Anthocyanin-rich
method
Xia et al.,
extract from aleurone 2003 69
atherosclerotic
layer
of
black
rice
plague
caused a marked (60%)
formation
reduction in Serum total cholesterol
(TC)
and
non-HDL
cholesterol
(HDL-C) levels Red rice
Antioxidant
In
capacity
method
vitro The highest activity was Oki et al., observed in rice with a 2002 red hull 2.77 µmol of Trolox equivalents mL-1 ),
followed
by black
(0.92) µmol of Trolox equivalents mL-1 ). Red rice
Anti-
In
inflammatory
method
vivo Red
rice
extract Niu et al.,
achieved the strongest
2013
inhibition against IL-1b at 100 mg mL-1 Red rice
ric
varities Finocchiaro
Antioxidant
Trolox
Red
capacity
equivalent
observed to have anti- et al., 2007
antioxidant
oxidant activity ranged
capacity
from
(TEAC)
Trolox/kg to
method
10.2 mmol Trolox/kg
35.9
mmol
Summary The pigmented rice has been consumed for a long time, and is widely known as enriched rice for taste and health improvements. The nutraceutical attributes have been well characterized till date, but the quality of starch has been neglected till date. Fine tuning of starch for consistent glucose release being the need of the hour for tackling type II diabetes, starch hydrolyzation dynamics will be a vital tool in screening the available pigmented/non-pigmented germ plasm for better quality starch. 70
References 1. Chen M, McClungAM and Bergman CJ (2016).Concentrations of oligomers and polymers of pro-anthocyanidins in red and purple rice bran and their relationships to total phenolics, flavonoids, antioxidant capacity and whole grain color.Food Chemistry 208: 279–287 2. Finocchiaro F, Ferrari B, Gianinetti A, Dall'asta C, Galaverna G, Scazzina F, Pellegrini N (2007). Characterization of antioxidant compounds of red and white rice and changes in total antioxidant capacity during processing.Mol Nutr Food Res 51(8):1006-19 3. Guo H, Ling W, Wang Q, Liu C, Hu Y and Xia M (2007). Effect ofanthocyaninrich extract from black rice (Oryza sativaL. indica) onhyperlipidemia and insulin resistance in fructose-fed rats. Plant Foods for Human Nutrition, 62: 1– 6. 4. Nam SH, Choi SP, Kang MY, Koh HJ, Kozukue Nand Friedman M (2005). Bran extracts from pigmented rice seeds inhibit tumor promotion in lymphoblastoid B cells by phorbol ester. Food Chemical Toxicology, 43: 741– 745 5. Niu Y, Gao B, Slavin M. et al (2013).Phytochemical compositions and antioxidant and anti-inflammatory properties of twenty-two red rice samples grown in Zhejiang.LWT- Food Science and Technology 54: 521–527 6. Oki T, Masuda M, Kobayashi M. et al (2002). Polymeric procyanidins as radical-scavenging components in red-hulled rice.Journal of Agricultural and Food Chemistry 50: 7524–7529
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Chapter-12
Bioavailability of Phytonutrients: a Key Determinant of their Bioefficacy Sandeep Kumar, Minnu Sasi, Monika Awana, Khushboo Kumari, A.P. Rajarani and Anil Dahuja Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
Plants contain plethora of nutrients which are required for the sustenance and nourishment of human beings. Besides, during the last decade or so, it is being increasingly recognized that they are also rich source of many non-nutrient, bioactive components having immense potential for providing additional health benefits beyond the basic nutrition. These phytochemicals not only play pivotal role in health promotion but have also been reported to be useful for disease prevention. However, in order to exert a health benefit these phytochemicals need to withstand food processing, be released from the food matrix after the ingestion, undergo metabolism in the gastrointestinal tract, and reach the target tissue of action. In other words, bioactive phytochemical needs to be bioavailable before it can have a physiological effect. It is, therefore, of utmost importance to fully elucidate bioavailability of dietary bioactive compounds before any specific recommendations are made w.r.t. their daily intake and for deriving maximal health benefits without any side effects from their consumption. From a nutritional perspective, bioavailability is the fraction of a given food component that the body can utilize and is therefore a matter of nutritional efficacy. Bioavailability addresses several processes such as Liberation from a food matrix, Absorption, Distribution, Metabolism and Elimination (LADME) [Holst and Williamson, 2008]. The first step of bioavailability of any compound is its bio-accessibility, which is defined as the fraction of the compound released from the food matrix in the gastro-intestinal lumen and therefore made available for the intestinal absorption. Bio-accessibility is influenced by the composition of the food matrix in which bioactive component is localized and also by the pH, temperature and texture of the food matrix. The bio-accessibility of a component can also be influenced to a great extent by processing of the plant foods, which brings about changes in the structure and properties of the plant cell wall. There are sufficient numbers of examples in the literature where it has been shown that processing of grain into flour and fermentation of grains significantly enhances the bioavailability of bioactive components as both the processes help in breaking their strong linkages with fibre 72
and thereby facilitating their release. Hence, researchers are really optimistic about the prospects of improving the bio-efficacy of bioactives via their greater liberation from the food matrices. Indeed, in the future, we may realize that the dietary fibre in different plant-based foods are simply the delivery vehicle for the more precious phytochemical cargo carried within, which must be unpacked by the gut microbiota before their contents can be enjoyed by the fortunate human host (Biernat et al, 2018). After overpowering the first challenge of becoming bioaccessible, the bioactive compound needs to be absorbed in the gastro-intestinal tract. The absorption of these compounds can be influenced by 1) solubility, 2) interaction with other dietary ingredients, 3) biotransformation, 4) different cellular transporters, 5) metabolism and 6) interaction with the gut microbiota. The absorption mechanisms are different for the hydrophilic and lipophilic bioactive compounds; the mechanism being simple for hydrophilic compounds such as polyphenols. The absorbed metabolites enter next into hepatocytes via the hepatic portal vein and then these can either be excreted into systemic circulation or into to the bile. The metabolites produced from bioactives' metabolism are finally excreted from systemic circulation into urine. The larger bioactive molecules which are not absorbed in the small intestine reach the large intestine where they can be metabolized by microbiota into smaller molecules. Some of these could be responsible for the health benefits ascribed to the parent phytochemical, such as equol, which is formed by gut microflora by their action on isoflavones such as daidzein. Increasing evidence shows that clinical effectiveness of soy isoflavones depends partly on the microbial transformation of isoflavones into stronger oestrogen, equol.
Factors affecting bioavailability Structure of bioactive molecules The molecular structure of a bioactive compound affects its absorption considerably. The bioavailability of anthocyanins is modulated by the nature of the sugar conjugate and the aglycones. Anthocyanins with more hydrophilic groups exhibit superior bioavailability compared with anthocyanins with fewer hydroxyl groups and low hydrophilicity (Fang, 2014). Further, most of the plant bioactives are secondary metabolites, which are predominantly stored in their β-glucosidic forms to acquire enhanced solubility and stability. Besides, these conjugated forms are invariably poorly absorbed in the small intestine and need to travel to the large intestine to have their sugar moiety cleaved off by the intestinal microbiota before absorption. The type of sugar moiety linked to phenolics like anthocyanins play an important role in this process. Hexoside anthocyanins are degraded by β-glucosidases provided by the gut microbiota but anthocyanins linked to pentose and acids are more resistant to degradation.
73
In addition to chemical structure, the isomeric configuration of a bioactive also affects its absorption significantly as the flavonoids with different stereochemistry have been shown to exhibit different bioavailability and bioefficacy. Some of the notable examples are given below:
1. The lycopene, which is present as 95% all-trans isomer in tomatoes yet the cis isomer represent around 50% of lycopene in human plasma indicating the fact that cis-isomer is more bioavailable than transand thus there is trans to cis isomerization in the GI tract. 2. Equol, a metabolite produced from daidzein-a prominent isoflavone in soybean by the gut microflora, is known to be estrogenic just like its precursor. It is a chiral molecule and can exist in two enantiomeric forms but the enantiomer produced by metabolic reduction of Daidzein is known to be S (-)-equol. Further, it has been found that the estrogenic activity of S-equol exceeds that of daidzein. 3. The hesperidin-7-glucoside has been found to have an R;S ratio of 39:69 in human plasma and urine samples, suggesting that the S-configuration could be more bioavailable.
Transport of bioactive molecules Like many drugs, bioactive food components do not have the optimal physicochemical properties necessary for the passive diffusion. The trans-membrane transporters are, therefore, needed for enhancing their permeability. These transporters are involved in two mechanisms related to the permeability of compounds- uptake and efflux. The uptake transporters like OAT1 enhance the bioavailability of bioactives while the efflux transporters (ABC-family of transporters) hinder their bioavailability. It has been reported that one of the major reason for the poor bioavailability of flavonoids could be their strong affinity for ABC family of efflux transporters. Furthermore, it has been hypothesized that if different bioactives are transported by a common membrane transporter, then they may cause competitive inhibition of one another. For example, plant sterols have been shown to reduce the absorption of cholesterol, carotenoids and alpha-tocopherol, most likely by competing with the lipid membrane transporters.
Metabolism of bioactive molecules After entering into enterocytes, the bioactive food molecules are subjected to PhaseI (oxidation and reduction) and Phase-II (Conjugation with methyl, sulfate and glucouronyl groups) metabolism resulting in molecular forms, which are different from the original constituents of the digested food (Braga et al., 2018). Strategies for improving the bioavailability of bioactives 74
Improving the bioavailability of bioactive food components is fundamental to improving their bioefficacy. Several strategies have been explored for enhancing the bioaccessibility and bioavailability of bioactive components (Rein et al., 2012). •
Chemical modifications of the bioactive molecules Quercetin, the aglycone form of rutin is more bioavailable than the parent flavonol
•
Processing of food matrix For example, by processing raw tomatoes into tomato paste, the bioavailability of lycopene is increased due to its release from the food matrix and also due to isomerization of trans lycopene into more bioavailable cis form.
•
Inhibition of intestinal cell transporters In vitro experiments suggests that the bioavailability of the flavonoid hesperidin may be enhanced by inhibiting the ABC transporters by competitive exposure to other flavonoids, such as quercetin resulting in a decrease of the efflux of hesperidin Design of colloidal systems It has been found that crystallization retardation, emulsification and synthesis of colloidal phytosterols leads to enhanced solubilization of phytosterols thereby improving the bioaccessibility and consequently the bioavailability of phytosterols Use of nanotechnology The curcumin is well known for many potential health benefits. However, due to its insolubility in water, this compound has a very low bio-availability. However, a 5.6 fold increase in its bioavailability has been achieved by using PLGA [Poly (lactic-co-glycolic acid)] nano-particles.
•
•
Role of microbes in enhancing the bioavailability of bioactives The rate and extent of absorption of health associated compounds vary widely between the individuals. The inter-individual variability in bioavailability may be due to several factors including genetic background but the one factor which has received the most attention is the composition and activity of gut microbiota. The microbiota maintains an important role in human metabolism and provides a number of protective, immune and metabolic functions, which altogether have an enormous imact on the nutritional and health status of the host. It is now being considered as a "Metabolizing Organ". The human gastro-intestinal (GI) tract contains a huge collection of micro-organisms. Although microbiota has not been fully described, but it is clear that human gut is home for an ecosystem of around 1013-1014 bacterial cells. As a whole, the micro-organisms that live inside humans are estimated to outnumber human cells by a factor of 10, and the microbiome represents overall 75
>100 times the human genome. The gut colonization process starts immediately after birth and development and establishment of infant's microbiota is highly dependent on the environmental factors. The infant's microbiota initially shows low diversity and instability, but evolves into a more stable adult-type microbiota over the first 24 months of life. The gene set of microbiota (the gut microbiome) is estimated to be about 3 million genes-150 times larger than that of the human genome (Rowland et al., 2017). The microbiota also has extensive capacity to metabolize phytochemicals. For example, the catabolism of anthocyanins by microbiota results in the production of new phenolic compounds, which are absorbed easily and exhibit different bioactivity from that of parents. The wealth of metabolic functionality encoded within the gut microbiome extends the biochemical flexibility of the host to process a wide range of dietary substances (Laparra and Sanz , 2010). The changes in the gut microbiota are being accepted as important elements in the development of many diseases. Many nutraceuticals including prebiotics, anthocyanins etc. may impart their health benefits by restoring microbial homeostasis. Conclusion The determination of the bioavailability of bioactive food components is essential when evaluating their potential health benefits. The current analytical methods for estimating the bioavailability are mainly based on the identification of main metabolites in urine. However, since metabolism of most of the bioactive components has not been fully characterized, the researchers more often than not end up in the underestimation of the actual bioavailability of a bioactive. Hence, there is an urgent need for the development of expansive methods for the comprehensive evaluation of the circulating metabolites in order to have a better understanding of the fate of bioactive molecules in human body. This would not only provide a complete picture about the bioavailability of a bioactive compound but would also help in understanding the relationship between the bioavailabitity and bioefficacy of bioactive.
76
References 1. Biernat KA, Li B and Redinbo MR (2018) Microbial unmasking of plant glycosides. Am. Soc. Microbiol 9(1) e02433-17. 2. Braga ARC, Murador DG, Mesquita LMD and de Rosso VV (2018) Bioavailability of anthocyanins: Gaps in knowledge, challenges and future research. J. Food Comp. & Analysis 68: 31-40 3. Fang J. (2014) Bioavailability of anthocyanins. Drug Metab. Rev. 46(4): 508520. 4. Holst B and Williamson G (2008) Nutrients and phytochemicals: from bioavailability to bioefficacy beyond antioxidants. Curr. Opinion Biotechnol.19: 73-82. 5. Laparra JM, and Sanz Y (2010) Interactions of gut microbiota with functionl food components and nutraceuticals. Pharmac Res. 61: 219-225. 6. Rein MJ, Renouf M, Hernandez CC, Goretta LA, Thakkar SK, Pinto MS (2012) Bioavailability of bioactive food compounds: a challenging journey to bioefficacy. Br. J. Clin. Pharmacol. 75(3): 588-602.
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Chapter-13
Resistant Starch as Prebiotic: A Promise for Improving Human Health Archana Singh, Veda Krishnan, Shelly Praveen Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi
Prebiotics are utilized to promote the survival of probiotics. Prebiotics are nondigestible carbohydrates that are not absorbed in the intestine, such as resistant starch (RS). RS is a type of starch got recent recognition due to its slow /incomplete digestion which doesn’t contribute to blood sugar spike and act as a matrix for fermentation by helping microbiome of human gut to grow. This non-digestible starch fraction known as RS occurs basically in all starchy foods and has a long history as food source for humans. RS includes the portion of starch that can resist digestion by human pancreatic amylase in the small intestine and thus, reach the colon. It is of great nutritional interest associated with it's physiological effects, similar to those of dietary fibres. The regular consumption of certain subclasses of highly fermentable dietary fibre sources result in gut associated immune and microbiota modulation as well as a significant production of small chain fatty acids (SCFAs). Recently, molecular assembly of starch granules as an interplay of metabolizing enzymes in rice has been elaborated to correlate the integration of starch biosynthesis pathway genes to starch packaging and RS involved in distinct roles in different steps of starch synthesis (Praveen et al.,2018). Among the different physiological roles of RS, its prebiotic effect is of great interest. RS can be considered prebiotic as it is not absorbed in the intestine. The best approach of prebiotic–probiotic symbiosis is achieved by encapsulation. But other food bio-compounds can be encapsulated too using RS. Changes in human lifestyle and food consumption have resulted in a large increase in the incidence of type-2 diabetes, obesity, and colon disease, especially in Asia. These conditions are a growing threat to human health, but consumption of foods high in RS can potentially reduce their incidence (Zhou et al., 2016). Dietary starches are important sources of energy for human kind and it is clear that they can also make quite specific contributions to health. It is normally hydrolyzed by enzymes in 78
the digestive tract to be converted into glucose that cells directly use to produce energy for their metabolic functions. The concept of resistant starch arose from research in the 1970s and is currently considered to be one of three starch types: rapidly digested starch (RDS), slowly digested starch (SDS) and resistant starch (RS), each of which may affect levels of blood glucose (Sharma et al., 2008). RS is a fraction of starch that resists hydrolysis by the digestive enzymes α-amylase and amyloglucosidase. It is a recently recognized source of fibre and is classified as a fibre component with partial or complete fermentation in the colon, producing various beneficial effects on health (Birt et al.,2013). RS also offers an exciting new potential as a food ingredient. As a functional fibre, its fine particles and bland taste make the formulation of a number of food products possible with better consumer acceptability and greater palatability than those made with traditional fibres (Fuentes-Zaragoza et al.,2010). The concept regarding digestion of RS being a source of dietary fiber has evoked new interest in the bioavailability of starch with immense health benefits but its mechanism of synthesis as well as structure has not been explored yet. Based on structural modifications, RS has been categorized into five types (Fig. 1).
Figure 1.Resistant starch types and their putative structures(Praveen et al., 2018). 79
Resistant starch is starch, including its degradation products, that escapes from digestion in the small intestine of healthy individuals. Resistant starch occurs naturally in foodsbut is also added to foods by the addition of isolated or manufactured types of resistant starch.Isolated and extracted resistant starch and foods rich in resistant starch have been used to fortify foods to increase their dietary fiber content. Typically, food fortification utilizes RS2 from high amylose corn, RS3 from cassava and RS4 from wheat and potato, as these sources can survive varying degrees of food processing without losing their RS content. RS has a small particle size, white appearance, bland flavor and low water-holding capacity. RS typically replaces flour in foods such as bread and other baked goods, pasta, cereal and batters because it can produce foods with similar color and texture of the original food. It has also been used for its textural properties in imitation of cheese. RS2 from potato starch and green banana starch maintain their resistance as long as they are consumed raw and unheated. If they are heated or baked, these types of starch become rapidly digestible. RS2 from high amylose corn can be consumed raw or baked into foods. Technically, it is possible to increase the RS content in foods by modifying the processing conditions such as pH, heating temperature and time, the number of heating and cooling cycles, freezing, and drying. There are varied processing conditions which increase the RS content like high pressure, refrigeration, enzymatic treatment, extrusion etc., as well parallel certain household strategies (Fig. 2) are also instrumental to increase the RS content. RS shows improved crispness and expansion in certain products, which have better mouthfeel, color and flavor than products produced with traditional insoluble fibers. RS does not release glucose within the small intestine, but rather reaches the large intestine where it is consumed or fermented by colonic bacteria (gut microbiota). The fermentation of RS produces short-chain fatty acids, including acetate, propionate, and butyrate and increased bacterial cell mass. The short-chain fatty acids are produced in the large intestine where they are rapidly absorbed from the colon, then are metabolized in colonic epithelial cells, liver or other tissues(Pryde et al., 2002; Andoh et al., 2003).
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Figure 2. House-hold strategies to increase RS content. Studies have shown that high amylose content, short chains of amylopectins, and amylose-lipid complexes contribute to resistance towards amylolytic degradation. It has received much attention for both its potential health benefits (similar to soluble fibre) and functional properties. RS positively influences the functioning of the digestive tract, microbial flora, the blood cholesterol level, the glycemic index (GI) and assists in the control of diabetes. In 2016, the U.S. FDA approved a qualified health claim stating that RS might reduce the risk of type 2 diabetes, but with qualifying language for product labels that limited scientific evidence exists to support this claim. Some types of resistant starch (RS1, RS2 and RS3) are fermented by the large intestinal microbiota, conferring benefits to human health through the production of short-chain fatty acids, increased bacterial mass, and promotion of butyrate-producing bacteria (Brouns et al., 2002). RS in various ways has similar physiologic effect as dietary fiber, which is why it functions as a mild laxative.Apart from the potential health benefits of RS, another positive advantage is its lower impact on the sensory properties of food compared with traditional sources of fibre, as whole grains, fruits or bran. The studies revealed consistent evidence that consumption of RS can aid blood sugar control bypassing the digestion and reaching, where bacteria ferment to releases SCFA thus support gut health. Here, RS acts as a prebiotic as well nutraceutical and its consumption leads to many health benefits. Being so important, RS is a major area of interest from more than a decade. Biochemists are unraveling the various types and pathways involved; biotechnologist keen to explore on how to engineer the pathways; food technologists playing a role 81
in improving RS content in food stuffs by various processing methods and nutritionists are the bridging factor which connects these research community to consumers. More than a decade research which explored the RS content and glycemic index in various food stuffs and the potential mechanisms underpinned in them has gained great attention of the nutritional world. It is considered both as a dietary and functional fiber, depending on whether it is naturally present in foods or can be added. In particular, considering the tremendous diversity of RS in plants, very few of these starches have been studied for their effects on animal or human health.A great source of variation has been observed in RS content among due to genetic, environmental and mutation effects in the plants. Deciphering the molecular switches, which turn normal starch to digestion- resistant phenotype, is a potential for future genetic engineers. Thus, integrative research that addresses all of these fronts will help to expand thepromising uses of RS as a "prebiotic" in human health promotion. References 1. Andoh A, Tsujikawa T, Fujiyama Y(2003). Role of Dietary Fiber and ShortChain Fatty Acids in the Colon. Cur. Pharma Design. 9:347-358. 2. Fuentes-Zaragoza E, Riquelme-Navarrete MJ, Sánchez-Zapata E, PérezÁlvarez JA (2010). Resistant starch as functional ingredient. Food Res Int. 43: 931-942. 3. Pryde SE, Duncan SH, Hold GL, Stewart CS,
Flint HJ (2002). The
microbiology of butyrate formation in the human colon (PDF). FEMS Microbiol Letters. 217: 133-139. 4. Sharma A, Yadav B, Singh R (2008). Resistant Starch: Physiological Roles and Food Applications. Food Rev Int. 24:193-234. 5. Shelly P, Singh A, Krishnan V (2018).Molecular Assembly of Starch Granules: Interplay of Metabolizing Enzymes in Rice.J. Rice Res. 10:1-10. 6. Zhou H, Wang L, Liu G, Meng X, Jing Y, Shu X, Kong X, Sun J, Yu H, Smith SM, Wu D, Li J
(2016). Critical roles of soluble starch synthase SSIIIa and
granule-bound starch synthase Waxy in synthesizing resistant starch in rice. PNAS. 113: 12844-12849.
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Module V
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Chapter 14
Street Food: The Food Safety Dimension Ms. Vinod Kotwal DDG (FIPP), Department of Telecommunications, GoI
Street foods are ready-to-eat foods and beverages prepared and/or sold by vendors and hawkers especially in streets and other similar public places (FAO, 1989). This definition of street foods was agreed at a Food and Agriculture (FAO) Regional Workshop on Street Foods in Asia, held in Jogjakarta, Indonesia in 1986. Examined carefully, the emphasis is on the retail location of vending, which in this case is ‘on the street' and therefore, pushcarts, bicycles, baskets carried on the head are used to sell and carry their wares. Street foods are recognized by academics, FAO and World Health Organisation (WHO) inter-alia as an essential instrument to achieve food security in urban areas because of their easy availability, accessibility and economics. Street vended foods are also appreciated for their unique flavours, convenience and the role they play in the cultural and social heritage of societies. The sale of street foods also provides a source of livelihood to millions of individuals with limited access to financial sources as the investment for starting street food vending are low, and skills required are generally available with them. Thus, street food vendors play an important role in not only generating self-employment but also meeting the nutritional requirement of many low-income people. Despite their importance, due to informal nature of the street food vending, the activities of the vendors are difficult to regulate, and this leads to practices that may pose a risk to human health. Food Safety concerns The primary concern related to street food vending is of food safety and other matters related to pollution and sanitation problems, traffic congestion, illegal occupation of public/private spaces and various social issues. But, food safety of 84
street food takes precedence as these are prepared and sold under unhygienic conditions, with limited access to safe water, sanitary services, or garbage disposal facilities. There are threat and health risk to the consumers due to crosscontamination of food, ignorance of food safety measures by the vendors and noncompliance of guidelines on food safety. According to WHO, one of the leading cause of foodborne diseases (FBD) is lack of adequate food hygiene, and 1 in 10 people in the world fall ill every year due to eating contaminated food (WHO,2016) and therefore, food safety has to be an essential aspect of public health. Also, there may be a need to design intervention programs involving street food consumers, aiming to protect the health of population. The location of vending carts, which are mostly located on the roadsides with high traffic also poses a health risk.. Street food vendors are often poor, uneducated and may lack the appreciation of hygienic practices. It is borne out from different studies across the world. Apart from safety and hygiene practices, there are issues with the nutritional content and the quality of street-vended food based on the type of fats and oils used. Majority of the SFVs use “karahi” for frying and cooking, and food items are high in Trans Fatty Acids (TFA). There are increasing demands for restricting certain foods usually considered energy-dense and nutrient poor. Regulatory interventions It is recognized that regulatory interventions help in addressing the need for basic and hygienic food safety and sanitary measures. Food Safety and Standards Act, 2006 (FSSA,2006) led to the creation of the food safety regulator, Food Safety and Standards Authority of India (FSSAI) in 2008. It is responsible for ensuring food safety in the country. FSSA,2006 has been operationalised w.e.f 5th August 2011 with the notification of various regulations including the Food Safety and Standards (Licensing and Registration of Food Businesses) Regulation 2011. It lays down the criteria for licensing and registration of the food business operators (FBOs) including the sanitary and hygiene practices to be followed by them as licensing and registration requirements. Petty manufacturers including street food vendors are to be registered and comply with Schedule IV Part I (A), which lays down the sanitary and hygiene requirements to be followed by the SFVs. Meeting the regulatory requirements involve multiple stakeholders with different responsibilities. Some of these requirements to be fulfilled are under the control of street food vendors and others which are under the control of other stakeholders. It 85
is easy to address controllable components through training and awareness generation, and numerous studies have been conducted addressing this aspect. Steps for improving the safety of street foods Non-controllable components include location of points-of-sale that usually has limited infrastructure, with restricted access to drinking water, toilets, water disinfecting methods, refrigeration or ice, as well as hand washing and waste disposal facilities. A logical step towards reducing the risks of foodborne illness from street foods should focus on (a) educating the food handlers on the safety and hygiene (b) improving the environmental conditions under which the trade is carried out (c) providing essential services to the vendors to ensure the safety of their commodities. Training and capacity building has been widely used to address the safety and hygiene aspects of street-vended food following a hygienistic approach aimed at ensuring food safety through SFVs sterilization only, neglecting the role of consumers’ awareness and importance of provision of services and infrastructure at the vending site. It needs to be supplemented with focus on other areas of equal importance viz., design of the food cart; meeting the energy requirements of lighting sustainably; developing interventions to reduce unhealthy fats in street food, sensitizing about the harmful effects of air pollution on the health of SFV etc. This would require the involvement of various government agencies, important ones being the municipal, and police authorities, the food safety organizations; nongovernment organizations; consumers; educational and research institutions.
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1. Facilitating regulatory compliance (use of technology, census /surveys, simplification of processes, single-window approach, infrastructural facilities)
2.Training/capacity building/awareness generation (Training of the vendors at location nearest to their vending area, repitition of training modules at regular intervals)
3. Inter-sectoral involvement (consumers, civil society, research institutes)
Figure 1 : Based on the strategy "Triangle that moves the Mountain Strategy" for street vended food
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Chapter 15
Biofortification of Maize for Nutritional Security: Challenges and Opportunities Firoz Hossain*, Rajkumar U. Zunjare and Vignesh Muthusamy Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi *Corresponding author:
[email protected] Micronutrient malnutrition has emerged as one of the major health problems across the globe (Bouis 2018). Two billion people worldwide suffer from malnutrition of which 815 million people are under-nourished (Global Nutrition Report 2017). Malnutrition is predominant in African and Asian countries where cereals are consumed as staple. In India too, around 194 million people are undernourished, where 42% of children (
