PREPARATION OF DIETARY FIBER FROM RICE ...

Original Research Paper ISSN: 1955-2068

Full length Research Article: PREPARATION OF DIETARY FIBER FROM RICE MILLED BY-PRODUCTS BY FERMENTATION OF EDIBLE FUNGI ASHFAQ AHMED1, MUHAMMAD KAMRAN TAJ2*, ZHUANG YONGLIANG3, IMRAN TAJ4, TAJ MUHAMMAD HASSANI5, MOHAMMAD SAJID6, ZAHOOR AHMED7, AJAZ-UL-HAQ8, AZIZULLAH9, MUHAMMAD AZAM MENGAl10, AND SUN LIPING11 1,3,11

Research Center of Food Engineering, College of Chemistry and Engineering, Kunming University of Science and Technology, Kunming, Yunnan 650224, China. 2,4,7,8,9 Centre for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan. 5 Tameer-e-Khalaq Foundation Balochistan 6 Department of Epidemiology and Public Health UVAS Lahore 10 Bolan Medical Collage Balochistan

* Corresponding Author: Name: Muhammad Kamran Taj E-mail: [email protected]

ABSTRACT The budding dilemma of food crisis, arising due to lower crop yields and escalating population particularly in the developing countries, needs to utilize each pent of available resources. For providing enough food to all people, there is great need for utilizing the by-products generated during food processing and preparations. Rice is the 2nd leading cereal crop and staple food of more than half of the world‟s population. Its harvest is milled to produce rice, bran and husk. The rice is consumed by human beings while its by-products (bran and husk) are used in animal feed or discarded as waste. Both by-products are reported to be rich in protein, fat and dietary fiber thus could have great potential to be converted into human food to improve food security in the world. For increased nutrient availability both by-products were studied for changes arising from fungal metabolic activity under liquid state fermentation process. The aim of this study was to prepare high quality dietary fiber from both by-products and to evaluate total dietary fiber, protein, fat, ash, functional properties and total phenolic contents of fermented by-products. The study results showed that rice milled by-product‟s total dietary fiber (SDF and IDF), protein and ash are increased after 8 days of fermentation with Ganoderma Lucidum, Agrocybe Aegerita, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes mushrooms with a decrease in fat contents which may be due to fat used by the fungi, possibly in the synthesis of phospholipids constituents of the cell membrane of fungal tissue. Key words: Rice milled by-products, Mushroom, Fermentation, Dietary fiber, Functional properties

HFSP Journal - HFSP Publishing Vol. 9, No. 10, October 2015, http://www.hfsp-journal.org

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INTRODUCTION Rice (Oryza sativa) is one of the main cereal crops, as well as staple food for most of the world‟s population, especially Asian countries (Bird et al., 2000). Approximately 600 million tons are harvested worldwide annually (Chen et al., 2012). Frequently, rice is eaten in cooked form by humans to obtain various nutrients, as well as to supplement their caloric intake (Kim et al., 2011). The milling of paddy rice has nearly a 70% yield of rice (endosperm) as its major product, although there are some unconsumed portions of the rice produced, such as rice husk (20%), rice bran (8%) and rice germ (2%) (Van Hoed et al., 2006). Rice bran, rice husks, rice germ and broken rice are the main rice by-products in the rice industry. The most important rice by-products are rice bran and rice husks. These both are used as animal feeds and human food to improve food security in the world. In recent years, rice by-products have received increased attention as functional foods due to their phenolic base compounds, in addition to having high amounts of vitamins, minerals and fiber, which can help to lower cholesterol and anti-atherogenic activity (Wilson et al., 2002). In recent years , dietary fiber has received increasing attention from researchers and industry due to the likely beneficial effects on the reduction of cardiovascular (Prosky, 2001) and diverticulitis diseases , blood cholesterol (Borderias et al., 2005), diabetes and colon cancer (Rodrı-guez et al., 2006). In addition to nutritional effects, dietary fiber has functional properties such as water binding capacity (WBC), oil holding capacity (OHC), swelling capacity (SC) and glucose dialysis retardation index (GDRI). So, addition of dietary fiber to a wide range of products will contribute to the development of value-added foods or functional foods that currently are in high demand (Day et al., 2009); also, it can give these functional properties to the foods. There are many definitions for dietary fiber. The American Association of Cereal Chemists (2001) adopted this definition for dietary fiber: the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. Gravimetric enzymatic (AOAC Official method 985.29) methods (Rodrı-guez et al., 2006) are currently used in the world (Cui and Roberts, 2009). In this research work we used liquid state fermentation technique to enhance the nutritive value of rice milled by-products. Many studies have shown that nutritional improvement of rice milled by-products is possible by fermentation (Aderolu et al., 2007) and different cultures around the world have independently discovered fermentation as a way to greatly improve the digestibility and nutritive value of grains. Recently fermentation became a trend for production of healthy foods from whole grain cereals (Ilowefah et al., 2014) and many researchers used fungus to improve the nutritional status of rice milled by-products (Aderolu et al., 2007). The objective of the present research was to explore the effect of mushroom fermentation on the soluble dietary fiber and other nutritional values of rice milled by-products bran/husks and to compare the values analyzed before fermentation. This is the first ever report on the narrated above condition.


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MATERIALS AND METHODS Rice Milled by-products: Rice bran, rice husk, rice germ and broken rice are the main rice milled by-products in the rice industry. But in this work we only used rice bran and rice husks together, which were purchased from local market of Kunming city, Yunnan, China. Mushroom Strains: Ganoderma Lucidum, Agrocybe Aegerita, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes were purchased from Guangdong microbiology culture center, Guangdong, China and were maintained on potato dextrose agar (PDA) containing Potato 200 g/l, Agar 20 g/l, Dextrose 20 g/, pH 5.5 at 4 0C by every month sub culturing. Chemicals and Reagents: Heat-stable α-amylase, Papain, Amyloglucosidase, Diatomaceous earth, were purchased from SigmaAldrich Co. (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade and ultrapure. Preparation of Liquid Culture Medium: Grounded to 40 mesh screen of rice milled by-products 50 g, potassium phosphate monoba KH2PO4 100 mg, magnesium sulphate heptahydrate MgSO4-7H2O 100 mg and vitamin B 40 mg were all mixed together in 1000 ml distilled water and heated in water bath at 40 0C/30 min with continuous stirring. After that 60 ml of liquid media was poured in 250 ml Erlenmeyer flasks and autoclaved at 1210C/30 min. Inoculation: The Erlenmeyer flasks containing liquid culture media were inoculated with 6 mm diameter portions of the unexposed mother mushroom cultures previously grown on potato dextrose agar (PDA). Fermentation Technique: The inoculated Erlenmeyer flasks were agitated in a shaker at a speed of 160 rpm at 28 0C for eight days. After eight days of fermentation the samples were withdrawn from shaker and lyophilized. The dried samples were then grounded to 40 mesh screen for analysis of total dietary fiber (insoluble and soluble), protein, fat and ash. Total (TDF), Insoluble (IDF) and Soluble (SDF) Dietary Fiber Determination: TDF, IDF, and SDF were determined by enzymatic-gravimetric standard (AOAC Method 991.43) method. The samples were defatted and desugared by petroleum ether and ethanol respectively. Then the experiment was


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performed under sequential enzymatic digestion by heat stable α-amylase, papain and amyloglucosidase to remove starch and protein. For total dietary fiber (TDF) determination enzyme digestates were treated with ethanol to precipitate and were washed with ethanol and acetone by filtration. Then sample were overnight dried and weighed. For insoluble dietary fiber (IDF) analysis, the total dietary fiber residues were washed with 70 °C hot water by filtration and the filtrates (water washings) were used for soluble dietary fiber determination as total dietary fibers were determined, overnight dried and weighed. Kjeldahl Nitrogen Method for Protein: In the basic method for protein, nitrogen is determined by a method of chemical analysis known as the Kjeldahl procedure. The nitrogen found multiplied by a factor of 6.25 to determine the crude protein of rice milled by-products. The accurately weighed samples were placed in Kjeldahl flask, concentrated sulfuric acid, powdered potassium sulfate, copper sulfate, and catalyst tablets were added to samples to complete the oxidation and conversion of nitrogen to ammonium sulfate. After digestion Alkali-containing sodium thiosulfate was added to neutralized the sulfuric acid and when the ammonia formed was distilled into a boric acid solution containing the indicators methylene blue and methylene red (AOAC Method 955.04), and then borate anion was titrated with standardized HCL. Fat Extraction by Semi Continuous Solvent Extraction Method: Soxhlet Method: The Soxhlet method (AOAC Method 920.39C) was applied for the fat extraction. Accurately weighed predried samples into predried extraction thimble, covered the samples with glass wool. Put petroleum ether 3 rd half of flasks and assembled Soxhlet flasks, and condenser. Extracted in Soxhlet apparatus at a rate of four drops per second for 16 h by heating solvent in boiling flasks, after extraction the solvents were aspirated by aspirator and boiling flasks were dried with extracted fat in an air oven at 100 0C for 30 min, cooled in desiccator and weighed. Ashing: Ashing was done by dry ashing method (AOAC Methods 900.02), 1 g of predried sample was weighed into a tared crucible. The samples in tared crucibles were incinerated in muffle furnace for overnight at 550 0C. Physicochemical properties: Water-Holding Capacity (WHC) Twenty five milliliters of distilled water were added to 520 mg of dried powdered samples in a 50 mL centrifuge tube. The samples was stirred and left, at room temperature, for 1h. After centrifugation at 3000g for 15 min, supernatant was discarded and the residue was weighed. Results were expresses as g of water/ g dry base.

Oil-Holding Capacity (OHC)


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Twelve milliliters of olive oil was added to 125 mg of dried powder samples in a 50 mL centrifuge tube. Sample was stirred and left, at room temperature, for 1h. After centrifugation at 3000g for 15 min, supernatant was discarded and residue was weighed. Results were expressed as g of olive oil/ g dry base. Swelling Capacity (SC) The dried powder sample 500 mg was weighed in a 10 mL measuring cylinder (0.1 mL graduations), then was added 10 mL of distilled water. Total volume (mL) occupied by the sample was measured. After that, it was gently stirred to eliminate trapped air bubbles and left on a level surface, at room temperature, 18h to settle the sample. The volume (mL) occupied by sample was measured and SC expressed as mL of water/ g of dry base.

Glucose Dialysis Retardation Index (GDRI) The measurement of glucose retardation index was performed using the method described by Lecumberri et al. (2007) with slight modifications by loading dialysis bags (12 000 MWCO; Sigma Chemical Co., St Louis, MO, USA), with 400 mg of sugar free samples twice extracted with 80 % ethanol and 7.5 ml of distilled water and 7.5 ml of glucose (4 mg mL -1) was added. 7.5 ml of distilled water as the control and 7.5 ml of glucose as control. Each bag containing a mixture solution was transferred and suspended in a beaker containing 400 ml of distilled water at 37 0C for 1 h with constant stirring. The volume of 0.5 mL of glucose diffused from fiber and control samples was taken at 20, 40 and 60 min time intervals; the absorbance of each sample was measured using a spectrophotometer at 490 nm. The GDRI was calculated as follows: GDRI = 100 – (Total glucose diffused from sample / Total glucose diffused from control) × 100

Determination of total phenolic contents

The total phenolic content of rice milled by-products and the fermented rice milled by-products with different mushrooms extracts were determined, using the Folin–Ciocalteu reagent as followed by Abu Bakar et al. (2009).. The reaction mixture contained 0.5 mL extracts was mixed with 2.5 mL of the freshly prepared Folin– Ciocalteu 0.1 mol/mL reagent left at room temperature for 5 min and a further 2 mL of sodium carbonate (20% w/v) was added to get the mixture of 5 mL. The mixture was shaken vigorously for 30 min. After the mixture was left at room temperature for 1h, the absorbance was measured at 765 nm by using a spectrophotometer. Gallic acid was used as a standard, and results were calculated as mg Gallic acid equivalents in 0.5 g of dried sample (mg GAE/g).


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RESULTS The effect of fermentation on TDF, IDF and SDF of rice milled by-products: As the Figure-1 shows that the fermentation technique enhanced the TDF of rice milled by-products after 8 days at 28 0C/160rpm. The TDF before fermentation was 69.8985% and after fermentation with Ganoderma Lucidum, Agrocybe Aegerita, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes were 87.33%, 91.04%, 87.59%, 90.23% and 90.37% respectively.

Figure-1: The total dietary fiber of rice milled by-product was increased after 8 days of fermentation with Ganoderma Lucidum (GL), among all Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE). AA and LE had a significant effect on TDF of rice milled by-products.

Dietary fiber consists of two distinct types of fiber, categorized into insoluble and soluble fiber based on their chemical nature, functionality and specific health benefits. In order to benefit from dietary fiber intake both the fibers in the proper ratio need to be consumed. Insoluble dietary fiber (IDF) is that component which is insoluble in water and includes cellulose hemicelluloses and lignin. Rice husk and rice bran are found to be rich in insoluble fiber. Insoluble fibers are considered gut-healthy fiber because they have a laxative effect and add bulk to the diet, helping prevent constipation (Knudsen, 2001). Our result revealed that fermentation technique increased the IDF because before fermentation it was 64.2445 % and after fermentation with Ganoderma Lucidum, Agrocybe Aegirite, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes the IDF were 65.59%, 65.33%, 71.34%, 75.37% and 62.58% respectively as shown in Figure-2:


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Figure-2: The IDF of rice by-product was increased after 8 days of fermentation with Ganoderma Lucidum (GL), Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE). PE and FV had a significant effect on IDF of rice milled by-products

than rest.

The effect of fungal treatment on rice bran and rice husks was significant and increase in the SDF was observed throughout the study period as compared to the control. Our trail results showed that soluble dietary fiber of rice milled by-products (rice barn and husks) before fermentation was 2.2938% and after fermentation with Ganoderma Lucidum, Agrocybe Aegerita, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes the SDF were 4.24%, 9.56%, 4.14%, 10.06% and 7.89% respectively as shown in Figure-3:

Figure-3: The SDF of rice by-product was increased after 8 days of fermentation with Ganoderma Lucidum (GL), Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE). AA and FV had a significant effect on SDF among rest.


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The effect of fermentation on protein contents of rice milled by-products: The study confirms that rice bran and rice husks were excellent source of protein and mineral, so it could be used in food industry especially developing functional foods. While in our experiments we observed that protein before fermentation was 6.2044% and after fermentation with Ganoderma Lucidum, Agrocybe Aegerita, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes mushrooms were 6.46%, 6.75%, 6.39%, 6.44% and 6.47% respectively. The result shown that protein contents found of rice milled by-products increased after fermentation with different types of fungus as shown in Figure-4:

Figure-4: The protein of rice by-product was increased after 8 days of fermentation with Ganoderma Lucidum (GL), Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE). AA and LE had a better effect on protein contents of rice milled by-

products.

The effect of fermentation on fat contents of rice milled by-products: Rice husk and rice bran are the largest by-product of rice milling industry. These by-products are rich source of fat. The fats of rice husk and rice bran are used for oil, protective coating and tocopherol (Esa et al., 2013). While our result shown that fat contents found of rice milled by-products reduced after fermentation with different types of fungi as shown in Figure-5. The fat content of rice by-product was decreased after fermentation; it may be the result of fat used by fungi, possibly in the synthesis of phospholipids constituents of the cell membrane of fungal tissue. The results showed that fat before fermentation was 5.6827 % and after fermentation with Ganoderma Lucidum, Agrocybe Aegerita, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes mushrooms were 5.23%,5.56%, 4.69%, 5.56% and 5.58% respectively as presented in Figure-5


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Figure-5: The fat of rice by-product was increased after 8 days of fermentation with Ganoderma Lucidum (GL), Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE). The fat contents of rice milled by-products were decreased with all

mushrooms.

The effect of fermentation on ash contents of rice milled by products:

Ash of rice husk and rice bran is a carbon neutral green product. Lots of ways are being thought of for disposing them by making commercial use of this ash. Ash is a good super-pozzolan and this super-pozzolan can be used in a big way to make special concrete mixes. There is a growing demand for fine amorphous silica in the production of special cement and concrete mixes, high performance concrete, high strength, low permeability concrete, for use in bridges, marine environments, nuclear power plants etc. The results showed that the ash contents were increased but the difference was not significant after 8 days of fermentation by different fungi as shown is Figure-6. Our experiments revealed that ash before fermentation was 13.6700 % and after fermentation with Ganoderma Lucidum, Agrocybe Aegerita, Pleurotus Eryngii, Flammulina Velutipes and Lentinus Edodes mushrooms were 16.3085%, 14.8872%, 16.2909%, 14.0706% and 15.0819% respectively

.


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Figure-6: The ash of rice by-product was increased after 8 days of fermentation with Ganoderma Lucidum (GL), Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE). The ash contents of rice milled by-products were increased with all

nushrooms but GL and PE had a better effect.

The effect of fermentation on physicochemical properties of rice milled by-products: The effect of fermentation on physicochemical properties of rice milled by-products Chemical and physical properties of tested dietary fibers which would provide a clue to its physiological function will be helpful for their selection in diets. Tested dietary fiber sources could be as a good source of dietary fiber with multiple functional tasks and significant technical advantages. The results of current study indicated that DF components would likely be the main determining factors for physic-chemical and functional properties assessed in this trial. Table 1 shows the effect of fermentation on the physicochemical properties including water holding capacity, oil holding capacity, swelling capacity and glucose retardation index of rice milled by-products with different mushrooms. The fermentation of rice milled by-products indicated that the fermented rice milled byproducts could be considered as a new good DF. Water-holding capacity of five series of rice milled by-product varied from 3.14 to 3.92 g/g, and PE had the highest WHC, being 3.92 g/g. The higher WHC might open the possibility of using PE as a functional ingredient for reducing calories, avoiding syneresis and modifying the viscosity of formulated foods. Swelling capacity of Gl, AA, PE, FV and LE were 0.75, 0.90, 0.50, 0.80, 0.20 and 0.40 mL/g, respectively (Table1). GL showed significantly higher SC than other four, which might be related to its higher content of SDF. The OHC is another important functional property of fibre and our result shown that (Table-1) OHC of GL was higher than that of AA, PE, FV and LE. Glucose dialysis retardation index is a useful in vitro index to predict the effect of a fibre on delaying glucose absorption in the gastrointestinal tract. As shown in Table 1, GDRI of PE was significant higher than that of


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Gl, AA, FV and LE, showing 27.89% for 20 min, 30.51% for 40 min and 29.70% for 60 min. The results demonstrated PE could be used to improve glucose tolerance in diabetes. Table 1: The effect of 8 days fermentation on physicochemical properties of rice milled by-products

WHC g/g OHC g/g SC ml/g GDRI % 20 min 40 min 60 min

RMBP 3.83±0.34 4.50±0.13 0.75±o.oo

GL AA 3.85±0.05 3.86±0.33 5.25±0.045 5.04±0.22 o.90±o.14 0.50±0.14

PE 3.92±0.14 4.98±0.13 0.80±o.oo

FV 3.14±0.03 4.64±0.28 0.20±0.00

LE 3.20±0.12 4.21±0.40 0.40±0.00

14.54±1.80 21.17±1.80 15.81±0.72 27.89±4.69 20.15±0.35 15.05±3.25 22.18±1.11 22.50±1.11 16.99±0.45 30.51±2.23 36.80±1.77 19.19±3.11 13.75±0.18 14.01±0.90 16.07±0.90 29.70±1.63 27.64±1.64 18.13±0.54

Rice milled by-products (RMBP) fermentation with Ganoderma Lucidum (GL), Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE).

The effect of fermentation on total phenolic contents of rice milled-by products (TPC): As table:2 shows that the total phenolic contents of rice milled-by products before fermentation was 2.90 and the highest TPC effect after fermentation was shown by FV increasing by 1.56 there was also a significant effect by AA increasing 1.13. The lowest effect was also significant for PE lowering the TPC by 1.14. while the rest didn‟t have a great influence. The increase in the total phenolic compound content can be explained by the ability of fungi to degrade lignocellulosic materials due to their highly efficient enzymatic system.

Table 2: The effect of 8 days fermentation on total phenolic contents of rice milled by-products

RMBP(mg/g) 2.90±0.16

GL(mg/g) 2.18±0.06

AA(mg/g) 4.03±0.11

PE(mg/g) 1.76±0.06

FV(mg/g) 4.46±0.14

LE(mg/g) 2.53±0.12

Rice milled by-products (RMBP) fermentation with Ganoderma Lucidum (GL), Agrocybe Aegerita (AA), Pleurotus Eryngii (PE), Flammulina Velutipes (FV) and Lentinus Edodes (LE).

DISCUSSION Rice is a staple food of over half the world's population and about one-fifth of the world‟s population is engaged in rice cultivation (Reidy, 2011). Rice is the world‟s second largest cereal crop and produces the largest amount of crop residues (Soest, 2006). Rice husk and rice straw are the main by-products of rice cultivation and processing (Binod et al., 2010). The average ratio of rice grain: rice husk: rice straw is 1:0.25:1.25 (Haefele et al., 2011). Rice husk and bran are by-products of rice and can be used as an energy source. Proper understanding of the physical properties of rice residues is necessary for utilizing them in different field.


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Our results revealed that fermentation technique enhanced the nutritive value of rice husks and rice bran total dietary fiber as Xie et al, (2012) reported that fermentation of rice by-product with fungi improved its nutritive value. The study confirms that rice bran and rice husks were excellent source of protein and mineral, so it could be used in food industry especially developing functional foods. Our results revealed that fermentation technique enhanced the nutritive value of rice bran and rice husks protein as Oduguwa et al, (2008) reported that fermentation of rice by-product with fungi improved its nutritive valve. In this research we found that the fat contents of rice milled by-product were decreased and same condition was observed by Oliveira et al, (2010) and by Silveira and Badiale-Furlong (2007). The variation in the contents of ashes in unfermented and fermented biomass was increased insignificantly as Feddern et al, (2007) also reported that fermentation cannot effect ash contents. The effect of fermentation on water holding capacity of PE was high, which was agreement with the studies of Sangnark & Noomhorm (2003), suggesting that the smaller particle size of DF was associated with the higher WHC. The swelling capacity of GL showed significantly higher than other four as Chantaro et al, (2008) reported that SC is related to its higher content of SDF. While the OHC of GL was also higher as Elleuch et al, (2011) reported that OHC have good effects on cholesterol absorption.

Glucose dialysis retardation index is a useful in vitro index to predict the effect of a fibre on delaying glucose absorption in the gastrointestinal tract. As shown in Table 1, GDRI of PE was significant higher than that of Gl, AA, FV and LE, showing 27.89% for 20 min, 30.51% for 40 min and 29.70% for 60 min. The results demonstrated PE could be used to improve glucose tolerance in diabetes as Hassan et al, (2011) reported.

Fungi have two types of extracellular enzymatic system: the hydrolytic system, which produces hydrolases that are responsible for polysaccharide degradation and a unique oxidative and extracellular ligninolytic system, which degrades and opens phenyl rings Sanchez, (2009). During fermentation, several enzymes are produced, such as a-amylase, b-glycosidase and xylanase, directly in the substrate with a consequent release of phenols Bhanja et al., (2009) evidenced by the gradual increase observed for 72 hours. The decrease in the TPC contents after that period probably resulted from degradation, as shown by other authors Hegde et al., (2006)

ACKNOWLEDGMENTS We are highly thankful to faculty of chemical engineering for providing financial support for this study. I would also like to express my very great appreciation to Professor Zhuang Yong Liang and Sun Liping, for their valuable and constructive suggestions during the planning and development of this research work.


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