Effect of temperature on chlorophyll extracted from

‫ت َو ْ‬ ‫ما ِء َما ًء َفأَ ْنبَ ْت َنا‬ ‫ن‬ ‫ق‬ ‫ما َوا ِ‬ ‫خل َ َ‬ ‫ن َ‬ ‫الس َ‬ ‫ُم ِم َ‬ ‫ض َوأَ ْن َز َ‬ ‫اْل َ ْر َ‬ ‫الس َ‬ ‫ل لَك ْ‬ ‫أَ َّم ْ‬ ‫َّ‬ ‫َّ‬ ‫ه‬ ‫ة َما َك َ‬ ‫ع اللَّ ِ‬ ‫بِ ِ‬ ‫ج ٍ‬ ‫ها ۗ أَإِ َٰلَه َم َ‬ ‫ج َر َ‬ ‫ق َذ َ‬ ‫ح َدائِ َ‬ ‫ش َ‬ ‫ُم أَ ْن ُت ْن ِب ُتوا َ‬ ‫ات بَ ْه َ‬ ‫ه َ‬ ‫ان لَك ْ‬ ‫ُون)‬ ‫م َق ْوم يَ ْع ِدل َ‬ ‫ه ْ‬ ‫ۚ بَ ْ‬ ‫ل ُ‬ ‫[سورة النمل ‪]60‬‬

Prof. Ashraf Mahdy Shroubeh Eng. Manal Mohamed Abbas Prof. Hassan Hassan El Tanahy Prof. Mostafa El Gharably 1 Prof. Effat Abdo Afifi

1

2

1

2

1 DEPARTMENT OF FOOD INDUSTRIES - FACULTY OF AGRICULTURE IN MOSHTOHOR - BENHA UNIVERSITY 2 NATIONAL NUTRITION INSTITUTE - GENERAL ORGANIZATION FOR TEACHING HOSPITAL AND INSTITUTES - EGYPTIAN MINISTRY OF HEALTH

The Impact of Conditions and Methods of Extraction on the Proportion of Some Plant Pigments in the Alfalfa Plant and the Pumpkin Fruits

content • introduction • Natural pigments

• The importance of using natural pigment • Aim of the study • structure of chlorophyll and carotenoids

• Color changes and chlorophyll modification • Color changes and carotenoids modification

• Recommendations

Introduction • Foods are complex systems; caution must be exercised when tracking color changes of molecular compounds during industrial applications. Moreover, the degradation of nutritional compounds involves a network of reactions that include aggregation, chemical transformation and decomposition reactions as well as a combination of these reactions. Furthermore, it is important to point out that foods may respond differently to the different techniques and conditions employed in different parts of the world. Consequently, there is not a standard rule to link the food characteristics. These issues bring about a complex scenario for large scale industrialization processes. It is essential to apply rigorous and periodical analysis by chemically measuring the presence of target bioactive compounds and monitoring their association with the color parameters.

Natural pigments • Color is one of the major attributes that affect the consumer perception of food quality. • Natural colorants are generally regarded as Safe (GRAS) substances. • Therefore, they are more desirable than the synthetic ones for industrial or commercial applications as food additives. • However, they are more expensive to obtain and usually they have lower stability, restricting sometimes their practical use as colorants.

The importance of using natural pigment: • Regarding food color industry trends, the use of natural food pigments has increased in foods and beverages as substitutes for their synthetic counterparts. This is mainly due to the growing awareness of the environmental hazards and the potential side-effect impacts of the chemicals used in the synthesis of food colorants Carocho et al. (2014).

• Commonly used natural food pigments include anthocyanins , carotenoids , betalains , and chlorophylls, among others. In addition to their food application, the consumption of these natural-colored compounds has been associated with a reduction of noncommunicable diseases such as cancer, diabetes, and obesity (Li et al. (2016a); Cooperstone and Schwartz (2016); Rodriguez et al.(2016)

• Color is one of the major attributes that affect the consumer perception of food quality. • Natural colorants are generally regarded as Safe (GRAS) substances. • Therefore, they are more desirable than the synthetic ones for industrial or commercial applications as food additives. • However, they are more expensive to obtain and usually they have lower stability, restricting sometimes their practical use as colorants. • There for this study was done for obtaining plant pigments with important characteristics in food processing (antiseptics, antimicrobials and enhancers for sensory properties) of waste or some non-traditional sources such as clover and pumpkin.

Aim of the study: • Extract natural pigments from plants. • Study the effects of temperature and pH values on the pigment stability of alfalfa and pumpkin under different temperatures and pH. • The study helps to improve the extraction of pigments from alfalfa and pumpkin and promote the better use of pigments extracted chlorophyll and carotenoids as food.

ALFALFA • The dry weight of alfalfa includes not only the nutrients, minerals and vitamins, but also phytochemical substances: carotene, chlorophyll, coumarin, betasitosterol, fumaric acid, isoflavones, alkaloids, saponins, cryptoxanthin, daidzein, genistein, limonene, lutein, and zeaxanthin

PUMPKIN • Pumpkins, which are the fruits of different species of the genus Cucurbita, are cultivated worldwide for their pulp and seeds for human nutrition, either for direct consumption or for preparation of other foods such as syrups, jellies, jams, and purees. According to estimations by the Food and Agriculture Organization of the United Nations (FAO), world production of pumpkins in 2007 was over 20 million tons, especially in China, India, Russia, United States, and Egypt.

structure of chlorophyll and carotenoids: • Chlorophyll has a porphyrin ring similar to that of heme in hemoglobin , although the central atom in chlorophyll is magnesium instead of iron. Spinach, alfalfa, broccoli and etc.

• Carotenoids are titraterpenoid organic yellow pigments that are found naturally in vegetables and fruits and are abundant in some, such as carrots , pumpkin , tomatoes , pineapples and etc.

Color changes and chlorophyll modification: • Chlorophylls, which are pigments responsible for the characteristic green color of vegetables, are highly susceptible to degradation during processing conditions resulting in food color changes Schwartz and Elbe (1983) The simple reaction mechanism that occurs with the chlorophyll molecule is shown in fig.(1 ) • The chlorophyll degradation in foods may occur via chemical and biochemical reactions. Chemical reactions involve the formation of pheophytin from the chlorophyll by the replacement of magnesium ions from the porphyrin ring. The magnesium replacement can occur by an acidic substitution, heat treatment, or after the action of Mg dechelatase. Decarbomethoxylation may occur during a strong heat treatment, leading to the conversion of pheophytin to pyropheophytin Schwartz et al.(1981) Diop Ndiaye (2011).

Color changes and carotenoids modification: • The structural characteristics that are responsible for its antioxidant properties make this pigment susceptible to degradation by external agents, such as heat, low pH, dissolved oxygen, and light exposure. The degradation of carotenoids leads to colour changes as a result of a rearrangement or a formation of cis -isomers, epoxides, short chain products and, in some cases, volatile compounds Mercadante(2007).

reported that development of food products with attractive colors has been a major goal in the food industry. Carotenoids, especially β-carotene have been identified as the pigment responsible for the yellowish color in pumpkin PUMPKIN

EXTRACTION OF PIGMENTS Organic solvents extraction remains the widely used method. Indeed, carotenoids are mostly extracted by hexane for both food and pharmaceutical purposes. Petrochemical solvents are very efficient in the extraction of these molecules because they dissolve solutes easily and their volatility facilitates their removal at the end of the process. A green solvent is a environmentally friendly solvent from natural sources such as wood, fruits and vegetable oils. Ethanol is a green solvent widely used in industry because of its low cost, easy availability, safety and biodegradability. However, this solvent is less effective for the extraction of a polar compound such as lutein and chlorophyll to obtain high yields

Total Chlorophyll from Different Solvent M E T H A N O L W A S T H E M O S T E F F I C I E N T S O LV E N T I N E X T R A C T O F C H L O R O P H Y L L P I G M E N T S F R O M A L FA L FA L E AV E S A N D S T E M . I N L E AV E S , F O L L O W E D B Y E T H A N O L 7 7 % , H E X A N E A N D T O L U E N E , T H E N D I E T H Y L E T H E R , A C E T O N E A N D E T H A N O L T O TA L C H L O R O P H Y L L W A S 6 2 . 3 0 , 6 0 . 0 6 , 5 0 . 7 1 , 4 9 . 1 7 , 3 2 . 9 8 A N D 2 5 . 1 9 M G / L R E S P E C T I V E LY. W H I L E T H E S T E M M E T H A N O L I S T H E M O S T E F F I C I E N T S O LV E N T I N T H E E X T R A C T I O N O F C H L O R O P H Y L L F R O M A L FA L FA S T E M , F O L L O W E D B Y D I E T H Y L E T H E R , E T H A N O L 7 7 % , A C E T O N E , T H E N E T H A N O L A N D H E X A N E A N D T O L U E N E T H E T O TA L C H L O R O P H Y L L P I G M E N T S 0 2 3 . 7 4 , 1 3 . 7 3 , 1 3 . 0 1 , 1 2 . 4 3 , 1 2 . 3 1 A N D 1 1 . 7 5 M G / L , R E S P E C T I V E LY

Solvent Ethanol Ethanol 77% Methanol Acetone Di ethyl ether Hexane, toluene and ethanol

Total Chlorophyll mg/l Alfalfa Stem Alfalfa Leaves 12.319 25.196 13.011 60.063 23.744 62.302 12.439 32.898 13.737 49.179 11.759 50.715

Total carotenoids from Different Solvent T HE R ESULTS O BTA INED S HOWED T HAT P ET ROL EU M ET HER WAS T HE MO ST EFFI CI ENT SO LV ENT IN EXT R AC TIN G PU MP KIN CAROT ENO IDS PI GMENTS FOLLOWED BY ETHANO L AN D HEX ANE. T HE TOTAL CAROT ENO I D S P I GM ENT WA S 28 .68, 26.55 AN D 1 6 . 64 Μ G/ G SAMP L E, RESP EC T IV ELY

Solvent

Total carotenoids

Hexane

16.64

Ethanol

26.55

Petroleum ether

28.68

Green color losses in processed and minimally processed fruit and vegetable products are associated with decreases in the quality of such products. Chlorophyll, the pigment responsible for the characteristic green color of several fruit and vegetables, can degrade to undesirable grey-brown compounds such as pheophorbide and pheophytin. This degradation is mediated by acid and the enzyme chlorophyllase. Pheophorbide can be further metabolized to colourless compounds in metabolically active tissue

Effect of pH on Chlorophyll extracted from Alfalfa Stem and Leaves Using methanol and Ethanol 77% Solvent pH

pH pH pH pH pH pH pH

4 5 6 7 8.4 9.2 10

Stem Methanol Retention%

98.4 63.15 88.4 43.96 80.4 84.85 98.93

Leaves Methanol Retention %

55.73 60.21 64.44 87.72 99.59 61.009 70.98

Stem ethanol77% Retention%

40.33 -33.53 -59.56 69.27 -3.61 20.83 -13.01

Leaves ethanol77% Retention%

70.53 47.69 57.15 65.63 91.35 85.26 84.72

Carotenoids Carotenoids are responsible for the color of a wide variety of both vegetable and animal foods. Several studies published recently have shown that these compounds have a beneficial effect in human health, thus, from a nutritional point of view, it is important to know the factors related to their degradation, because carotenoids losses, not only produce changes in food color, but also decrease their nutritional value. The instability of carotenoids is due to the fact that they are highly unsaturated compounds, thus degradation is due mainly to oxidation. Other factors, such as temperature, light or pH can produce important qualitative changes in these compounds by means of isomerization reactions

Effect of pH from Carotenoids extracted PH pH 3 pH 4 pH 5 pH 6 pH 7 pH 8.4 pH 9.2 pH 10

Retention %pulp 93.96 85.09 95.37 96.47 98.9 98.23 93.95 94.35

Retention %peels 82.06 83.49 92.34 87.58 93.93 85.96 65.87 87.14

Effect of temperature on chlorophyll extracted from alfalfa Stem using methanol and ethanol 77% solvent

There is general agreement that the main cause of green vegetable discoloration during processing is the conversion of chlorophylls to pheophytins by the influence of pH. The green color of vegetables turns to an olive green when heated or placed in acidic conditions

Effect of Temperature on Chlorophyll extracted from Alfalfa Stem Using Methanol Solvent Temp.°C

Methanol Stem Retention %

metanol Leaves Retention %

50 °C 60 °C 70 °C 80 °C 90 °C 100 °C

98.96 98.78 97.87 95.26 92.65 91.6

97.3 96.6 96.57 95.26 95.02 94.58

ethanol 77% Stem Leaves etathanol Retention % 77% Retention % 92.094 96.81 91.563 94.35 87.401 92.41 84.591 90.00 83.74 89.89 80.252 84.69

Effect of Temperature on Carotenoids extracted from pumpkin pulp and peel CAROTENOIDS ARE SENSITIVE ORGANIC COMPOUNDS THAT CAN BE DENATURED BY WATER, OXYGEN, TRACE MINERALS, HEAT AND OTHER FACTORS Temp. °C/30 min. 50 60 70 80 90 100

Retention %pulp 98.77 98.56 95.21 93.86 93.71 93.34

Retention% peel 86.57 80.65 79.3 71.95 66.43 58.02

Effect of thermal stability on chlorophyll extracted from alfalfa stem using methanol solvent • Since color is a major sensory characteristic in determining product acceptability, it is important to prevent or at least minimize chlorophyll degradation during thermal processing in food industry. Thermal processing induces structural and chemical variations to the tissue of vegetables that often result in color change

Time (min) Temp 30 60 90 .°C min. min. min. 70°C 96.46 96.28 95.02 80 °C 92.91 86.36 85.45 82. 90 °C 92.42 95 81.29 100 82. °C 85.78 32 80.9

120m 150m 180m in. in. in. 93.22 91.26 91.08 84.3 82.23 78.18 80.18 79.18

78.1

79.18 78.17 77.63

Effect of thermal stability on chlorophyll extracted from alfalfa leaves using methanol solvent Temp.°C 70 °C 80 °C 90 °C 100 °C

30min 99.17 96.5 95.46 88.83

60 min 98.86 95.71 92.43 86.91

Time (min) 90 min 120min 150min 180min 98.64 98.58 97.84 94.58 93.04 90.26 89.46 87.58 87.68 87.08 86.27 85.58 85.9 84.26 83.37 81.36

Effect of Thermal Stability on Chlorophyll extracted from Alfalfa Leaves Using Methanol and Ethanol 77% Solvent Time (min) stem Tem p.°C 70°C 80 °C 90 °C 100 °C

30mi 60 90 120m 150m 180m n min min in in in 93.54 88.10 87.34 87.32 84.86 80.76 86.45 82.83 81.09 79.84 79.04 78.15

83.64 82.02 80.04 77.65 77.16 76.98 82.83 80.62 78.71 77.49 73.37 70.85

Time (min) leaves Tem p.°C 70°C 80 °C 90 °C 100 °C

30mi 60 90 120m 150m 180m n min min in in in 92.52 91.92 90.61 86.51 83.75 82.48 89.98 86.79 84.73 83.09 81.41 81.01

85.12 82.37 80.27 75.75 75.41 75.36 82.87 77.93 77.58 74.45 72.27 72.15

Effect of thermal stability on carotenoids extracted from pumpkin pulp Time (min) 30mi 60 90 120m 150m n min min in in 99.99 99.7 99.46 99.3 98.67 99.91 99.83 99.4 98.55 98.54 97.03 96.43 95.99 95.94 95.9

Temp .°C 70 °C 80 °C 90 °C 100 °C 96.82 95.67 95.39 95.38 94.9

180m in 98.42 97.92 94.38

92.8

• Color degradation is common for natural pigments, and is therefore a major concern in coloring foods, frequently compensated by the proper dosage of the pigment

Effect of thermal stability on carotenoids extracted from pumpkin peel • Thermal treatment is one of the most important methods of food preservation. However, excessive heating produces considerable loss in the quality and particularly in the sensory properties of foods

Temp .°C 70 °C 80 °C 90 °C 100 °C

30mi 60 n min 99.94 99.82 99.91 99.65 98.49 94.55

Time (min) 90 120m min in 97.31 96.4 97.16 95.99 93.47 92.17

150m in 95.98 95.77 91.25

180m in 95.78 95.72 90.76

91.08 86.59 84.86 81.58 81.55 81.3