Extraction and characterization of dietary fiber from coconut residue

WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland e-mail: info@world-food.net Journal of Food, Agriculture & Environment Vol.8 (2): 172-177. 2010 www.world-food.net Extraction and characterization of dietary fiber from coconut residue S. P. Ng 1, C. P. Tan 1 *, O. M. Lai 2, K. Long 3 and H. Mirhosseini 1 1 Department of Food Technology, Faculty of Food Science and Technology, University Putra Malaysia, 43400, Serdang, Selangor, Malaysia. 2 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, UPM, 43400, Serdang, Selangor, Malaysia. 3 Malaysian Agricultural Research & Development Institute (MARDI), P.O. Box 12301, 50774 Kuala Lumpur, Malaysia. *e-mail: tancp@putra.upm.edu.my Received 13 February 2010, accepted 8 April 2010. Abstract The present work was conducted to investigate the coconut residue (CR) left after the extraction of coconut milk by subjecting it to physical treatments. Water-washed samples as well as the original samples were analyzed for their chemical composition, soluble dietary fiber (SDF), insoluble dietary fiber (IDF) and total dietary fiber (TDF), and measurements were based on dry matter. Fractionated IDF was further treated into four fractions: cellulose, lignin, hemicelluloses A and B. IDF, TDF, moisture, crude fat, protein and CHO (carbohydrate) values were significantly (p < 0.05) different between the samples. The IDF values ranged from 27.32 to 35.08 in both CR samples. The TDF of the CR with treatment differed significantly (p < 0.05) between two different analytical methods. Cellulose contents were 72.67 and 72.33 for TDF in original and waterwashed CR samples, respectively. Key words:, dietary fiber, fractionation, cellulose, physical treatment. Introduction An emerging product of importance from coconuts, both in the local and foreign markets, is the virgin coconut oil. The coconut residue obtained after the extraction of milk is an inedible byproduct of the coconut industry, and only a small part of it is being utilized as fertilizer or feed for cows. Large quantities of the coconut residue by-product are left to rot on the fields as waste material. This defatted coconut residue, after milk extraction and drying, can be utilized as dietary fiber 1, 2. Only one report, by Trinidad et al. 2 is available in the literature regarding the utilization of spent coconut fiber as dietary fiber, in which only the physicochemical and nutritional properties of coconut fiber were emphasized. Besides, fruits and vegetables form the important part in human diet 3. Dietary fiber (DF) consists of a heterogeneous mixture of nonstarch polysaccharides, including cellulose, hemicelluloses, pectin, hydrocolloids and lignin, which cannot be degraded by enzymes in the human gastrointestinal tract 4, 5. Recent studies have indicated that dietary fiber may prevent some health condition and diseases such as constipation, hemorrhoids, hypercholesterolemia and colorectal cancer 6. The by-products of plant food processing represent a major disposal problem for the industry 7,and their transformation into value-added products, which fibers are, may diminish the problem and recover valuable biomass and nutrients. The use of fibers from new origins that are currently not fully exploited and the possibility of modifying the fibers by chemical, enzymatic or physical treatments will probably widen the fields of application for dietary fibers 8. Techniques for the fractionation of dietary fiber into its individual components are limited in number. Furda 9, 10 proposed a fiber extraction technique that included the isolation of water soluble fiber fractions from various food sources. Southgate 11 outlined and updated an extraction and fractionation procedure for lignocelluloses, crude lignin and cellulose fractions. In addition, numerous researchers have determined the cellulose, hemicelluloses and lignin contents of dietary fiber from food sources, while few studies have attempted to isolate and fractionate them into their major components. The objective of this work was to determine the content of insoluble and soluble DF fractions in CR as well as the constituents of each of these fractions. The proximate composition was also studied. In addition, this study aimed to provide an in-depth study on coconut-based dietary fiber. Materials and Methods Material and preparation of sample: Coconuts (Cocos nucifera) were purchased fresh from a local grocery store. After removal of the shell and parings, the coconut endosperm was passed through a Krauss-Maffei (Rotary wedge type) with a sieve plate (3-mm hole) through which shredded coconut meat was forced out 12. The resulting coconut gratings were pressed in a screw press (Model CME-100) to extract coconut milk. Besides, oils are easily removed by using extrusion and screw pressing method 13.CR with treatment was washed with running tap water at ambient temperature and CR without treatment was simply collected. The residual coconut fiber for both groups was subjected to an 8-hour Soxhlet extraction for fat removal at 40 to 60 C. Finally, the sample was kept in a hot air oven at 55 C for 3 h to remove the petroleum ether 1. 172 Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010

Chemical composition of coconut residue: Moisture, ash, fat, protein, determinations of coconut residue were determined according to AOAC procedures 14. The acid detergent fiber of defatted fiber was analyzed using the method of Robertson and Van Soest 15.The moisture content of the original sample was calculated based on weight loss after the sample was heated in oven at 105 C for overnight 14. Fat content was determined by extraction with petroleum ether in a Soxhlet apparatus 14.The total protein content from defatted and dried fiber sources was determined by micro-kjeldahl method, using 6.25 as a conversion factor 14. The ash content of dry fiber was determined by incineration in the muffle furnace at 550 C for 5 h. Dietary fiber - AOAC 991.43 method: Soluble (SDF), insoluble (IDF) and total (TDF) dietary fiber were determined by an enzymatic-gravimetric method according to Lee et al. 16 Duplicate test samples were sequentially treated for starch gelatinization and enzymatic starch and protein digestion in three incubation steps: heat stable alpha-amylase (or termamyl) (1500-3000 units/ mg protein; Sigma Chemical Co.) at 95 to 100 C for 15 min; amyloglucosidase (5000-8000 units/ml; Sigma Chemical Co.) at 60 C for 30 min, ph 4.0-4.7; and protease (7-15 units/mg protein; Sigma Chemical Co.), ph 7.5. In this experiment, sample was suspended in MES/ TRIS buffer. The enzyme digestate was then filtered using acid-washed celite on a Fibertec system E1023 filtration unit (Tecator, Sweden). Following filtration, the remaining residue was the IDF and the filtrate was the SDF. The IDF was washed with two portions of 15 ml 78 ethanol, 15 ml 95 ethanol and 15 ml acetone. For SDF, the filtrate was precipitated with 95 ethanol at 60 C before filtering. The SDF was then washed with two portions of 15 ml 78 ethanol, 15 ml 95 ethanol and 15 ml acetone. TDF, IDF and SDF residue values were all corrected for undigested protein, ash and blank. Dietary fiber - ASP method: The TDF (total dietary fiber), a measure of the sum of insoluble and soluble dietary fibers that is based on the digestion of food samples (1 g) with enzymes, was determined as described by Asp et al. 17. Dry sample was suspended in sodium phosphate buffer (ph 6.0) and treated with different enzymes, including termamyl (ph 1.5), pepsin (ph 6.8) and pancreatin (ph 4.5), for 1 h at specific ph. The enzyme digestate was then filtered using acid-washed celite on a Fibertec system E1023 filtration unit (Tecator, Sweden). Following the filtration of the enzyme digestate, the remaining residue was the IDF and the filtrate was the SDF. The IDF was washed with ethanol and acetone and finally incinerated and weighed. Soluble dietary fiber was estimated by precipitating the filtrate using ethanol. The precipitate was washed with ethanol and acetone, dried, incinerated and finally weighed. Fractionation procedure: The fiber extraction and fractionation was conducted as outlined by Southgate 11 with some modifications. The generalized fractionation procedure may be seen in Fig. 1. To optimize the component yield, cold and hot water extraction of the fibers was used to remove partially soluble polysaccharides and proteins before enzyme treatment. Extraction with cold and hot water: The defatted fiber samples were extracted with 25 ml of cold, slightly alkaline water (ph 7.0-7.5) for 2 h at 20 C. The samples were then centrifuged at 1500 g or 3000 rpm for 10 min. The supernatants were removed and collected, and the procedure was repeated twice. Residues were extracted with 0.01 M EDTA solution for 2 h to bind cations and solubilize the more pectic substances 9. The mixtures were filtered and the extraction repeated twice. After extraction, the residue was washed twice with 15 ml 80 ethanol and three times with 20 ml distilled deionized water. The washed residue was freeze-dried and kept for further analysis. This residue was called non-purified insoluble residue (NPIR). Enzymatic treatment of non-purified insoluble residue: The NPIR of each fiber was enzymatically treated using the method of Southgate 11. NPIR (25 g) was weighed into beaker and 0.1 M acetate buffer ph 4.8 was added (50 ml/g fiber). Duplicate Sample Grind Lipid Extraction Cold Water Extraction Hot Water Extraction (1) NONPURIFIED INSOLUBLE RESIDUE Amyloglucosidase Trypsin (2) PURIFIED INSOLUBLE RESIDUE Extraction (5 Ammonium Oxalate, 85 C) (3) PECTIN FREE INSOLUBLE RESIDUE Extraction (5 KOH, N 2 Atmosphere) (4) LIGNOCELLULOSE Filtrate KMNO 4 Extraction 72 H 2 SO 4 Acetic Acid (7) CRUDE CELLULOSE (8) CRUDE LIGNIN (5) HEMICELLULOSE A 4 Volumes Ethanol (6) HEMICELLULOSES B Figure 1. Extraction and fractionation of fiber sources. Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010 173

treatments were conducted. An amyloglucosidase solution was added (0.15 ml/g fiber), the beaker covered with aluminium foil and incubated for 3 h at 55 C, with continuous agitation. After cooling, the ph was adjusted to ph 8 ± 0.1 by adding 0.275 natrium hydroxide solution. Trypsin was added (5 mg/g fiber), and incubated and stirred for 18 h at 37 C. Finally, the mixture was filtered and washed three times with 15 ml 80 ethanol, once with 15 m1 95 ethanol and three times with 20 ml distilled deionized water, then freeze-dried. The residue obtained was considered as pure insoluble residue (PIR). Removal of insoluble pectic substances: Duplicate (1 g) of purified insoluble residue samples were extracted three times using 10 m1 of 0.5 (w/v) ammonium oxalate solution at 85 C for 2 h 18. Finally, the mixture was filtered and washed three times with 15 ml 80 ethanol, once with 15 ml 95 ethanol and three times with 20 ml distilled deionized water, then freeze-dried. Hemicelluloses A and B extraction: The method of Monte and Maga 18 was used to extract hemicelluloses A and B. Depectinated insoluble fiber (5 g) was weighed in duplicate into 250 ml plastic stoppered centrifuge bottles and 100 ml of 5 potassium hydroxide solution was added. The bottles were flushed with nitrogen gas and shaken for 24 hours, then centrifuged at 1500 g for 10 minutes. The supernatant was decanted and kept while the residue was further extracted for two times using the same conditions. The residue described as lignocelluloses was washed, dried and kept for further analysis. Filtrates were combined with 50 acetic acid adjusted to ph 5.0-5.5 and centrifuged. The hemicellulose A (HCA) fraction was washed and freeze-dried while the supernatant was diluted further with 4 volumes of 95 ethanol to produce second precipitate, hemicellulose B (HCB). Crude cellulose extraction: The method of Robertson and Van Soest 15 was used for the extraction of crude cellulose. Duplicate (2 g) portions of lignocelluloses were extracted using about 20 ml of combined reagent (KMNO 4 + lignin buffer, 2:1 ratio) in sintered glass crucibles and allowed to stand for 90 ± 15 min at 22 C with periodic stirring. The reagent in the crucibles was made to remain purple by changing frequently for the duration of the extraction process. The combined reagent was drawn out by suction and crucibles were transferred to a clean pan. Demineralizing solution (20 ml) was added to each crucible, allowed to stand for 5 min, refilling as necessary and then removed by suction. Lignin extraction: In this study, the Klason lignin method 15 was used. Duplicate samples of lignocelluloses (5 g) were extracted with cold 72 sulfuric acid solution (1 g/ml, w/v) at 4 C for 30 h. Cold distilled deionized water was added (150 ml) and the residue was allowed to precipitate before further treatment. The residue was then washed with warm distilled deionized water until no acid was detectable. The crude lignin residue (CLR) was then air-dried and kept in a freezer for later studies. Statistical analysis: Results were expressed as mean ± standard deviation (SD) either duplicate or triplicate for each determination. The dietary fiber for both samples treatment were performed through one-way analysis of ANOVA and followed by pair-wise multiple comparisons evaluated by Tukey s significant difference test. The statistical significance paired t-test analysis was carried out for chemical composition and fractionation analysis by using MINITAB 13.2 (Minitab Inc., Pennsylvania, USA). Differences at p < 0.05 were considered to be significant. Results and Discussion Table 1 shows the chemical compositions of the CR byproducts. The production of CR from raw coconut meat is illustrated in Fig. 2. The composition (moisture, crude fat, protein and CHO) of the two samples differed significantly. The higher percentages of moisture for both samples were found in the CR as compared to other, and this is likely due to the high water-holding capacity, water retention and swelling capacity of the CR compared to other dietary fibers 1.The protein contents in CR without treatment and with treatment were 5.32 and 1.43, respectively, while the range of protein contents in fruits and vegetables was 2.70-24.9. The low protein content in the CR with treatment is most likely due to the physical treatment; water washing may influence the result. The crude fat content of CR with treatment (2.91) falls in the range of fruits and vegetables (0.5-10.9) 19. The carbohydrate contents of CR without treatment and with treatment on a dry basis were 56.70 and 71.77, respectively, and they were lower than the carbohydrate contents of other fruits and vegetables. Most of the fruits showed higher carbohydrate contents (>72.3) than vegetables, with the exception of apples, which were only 25.8 carbohydrate 19. Therefore, increased carbohydrates were expected due to fat removal 20. Shelled Coconut (3.6 kg) Pared coconuts (3.0 kg) Coconut Gratings Disintegration Screw pressing Parings (1.2 kg) Coconut Residue Coconut milk (400 g) (650 g) Figure 2. Flow chart for the production of coconut residue from raw coconut meat. The total dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) in the CR of both groups are shown in Table 2. The SDF values varied significantly (p < 0.05) and ranged from 2.22 for CR with the Lee et al. 16 treatment to 3.41 for CR without treatment following Asp et al. 17. The IDF values ranged from 27.32 for CR with treatment to 35.08 for CR without treatment by the method of Lee et al. 16. The TDF in the Asp et al. 17 and Lee et al. 16 CRs without treatment were not significantly different, while the Asp et al. 17 CR with treatment was significantly (p < 0.05) higher than the Lee et al. 16 CR method. The high value of TDF by the Asp et al. 17 method indicates that it is suitable for further fractionation of the IDF content. The TDF of CRs with and without treatment using the Asp et al. 17 method 174 Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010

were compared to other sources of dietary fiber, such as pears, oranges, peaches, artichokes and asparagus 21 ; wheat bran 22 ; barley bagasse 23 and oat bran 24. CR without treatment and with treatment gave 37.4 and 31.1 TDF, respectively. According to Lund and Smoot 25, fiber contents of tropical fruits and vegetables differ from those of non-tropical fruits and vegetables. The IDF contents of both CR samples using the Asp et al. 17 method were found to be the highest among most types of fruit sources but they had lower values than vegetable and cereal sources (Table 3). Insoluble pectin removed from the CR fractions varied from 3.60 to 4.32 for both treatments (Table 4). CR without treatment had significantly higher pectin levels than CR with treatment (p < 0.05). The residue obtained after insoluble pectin extraction was regarded as pure insoluble fiber (PIF). The flow chart for the extraction and fractionation of fiber sources is given in (Fig. 1). The amounts of hemicelluloses A and B extracted are indicated in Table 4. The hemicelluloses A content was significantly different between the fibers and ranged from 1.13 for CR without treatment to 3.25 for CR with treatment (p < 0.05). Hemicelluloses B values did not differ between the samples, with 0.72 for the CR without treatment and 1.07 for the CR with treatment. The ratio of hemicelluloses A to hemicelluloses B was low in both samples. The reason the values were lower may be due to the procedures Table 1. Chemical composition of fiber sources x. Total carbohydrate Fiber source Moisture (w.b. ) Crude fat Ash Protein ADF y CHO z 50.26±0.24a 14.62±0.38a 0.23±0.15a 5.32±0.23a 23.13±0.38a 56.70±0.89a (without treatment) (with treatment) 68.13±0.39b 2.91±0.24b 0.22±0.08a 1.43±0.20b 23.66±0.31a 71.77±0.38b x Each value in the table represents the mean ± standard deviations of six determination from two replicate experiments. y ADF = Acid Detergent Fiber. z CHO = Carbohydrate (calculated by difference): 100 (protein + fat + ash + acid detergent fiber). Means within each column with different letters (a, b) are significantly different (p* < 0.05). Table 2. Soluble, insoluble and total dietary fiber values of fiber sources. Fiber source Soluble fiber a g/100 g Insoluble fiber a g/100 g Total dietary fiber a g/100 g Reference method 3.41 ± 0.24b 33.97 ± 0.67a 37.38 ± 0.91b Asp et al. 17 Fiber (without treatment) 2.43 ± 0.12ac 35.08 ± 0.60c 37.51 ± 0.72b Lee et al. 16 Fiber (without treatment ) 2.68 ± 0.39a 28.42 ± 0.59b 31.10 ± 0.98a Asp et al. 17 Fiber (with treatment) Fiber (with treatment) 2.22 ± 0.11c 27.32 ± 0.39d 29.54 ± 0.50c Lee et al. 16 a Each value in the table represents mean ± standard deviations of six determination from two replicate experiments. Means within each column with different letters (a, b, c) are significantly different (p* < 0.05). Table 3. Dietary fiber of some cereal derivatives, fruits and vegetables ( dry matter). Dietary source IDF SDF TDF Reference Fruits DF coconut residue 28.4 2.7 31.1 From this study (with treatment) DF coconut residue 34.0 3.4 37.4 From this study (without treatment) DF pear 22.0 14.1 36.1 Grigelmo-Miguel and Martin-Belloso 19 DF orange 24.3 13.6 37.8 Grigelmo-Miguel and Martin-Belloso 19 DF peach 26.1 9.7 35.8 Grigelmo-Miguel and Martin-Belloso 19 Vegetables DF asparagus 38.6 10.4 49.0 Grigelmo-Miguel and Martin-Belloso 19 DF artichoke 44.5 14.3 58.8 Grigelmo-Miguel and Martin-Belloso 19 Cereal derivatives Wheat bran 41.6 2.9 44.5 Prosky et al. 21 Barley bagasse 41.4 1.7 43.1 Molla et al. 23 Oat bran 20.2 3.6 23.8 Grigelmo-Miguel and Martin-Belloso 19 DF = Dietary fiber. Table 4. Cellulose, hemicelluloses, and lignin content of fiber sources c. Fiber source Fiber (without treatment) Fiber (with treatment) Hemicelluloses c Cellulose c Lignin c Insoluble pectin c A B 1.13±0.22a 0.72±0.45a 72.67±1.75a 1.88±0.19a 4.32±0.36a 3.25±0.24b 1.07±0.23a 72.33±2.58a 2.08±0.24a 3.60±0.41b c Each value in the table represents mean ± standard deviations of six determination from two replicate experiments. Means within each column with different letters (a, b) are significantly different (P* < 0.05). Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010 175

used to isolate the fiber fractions. The recovery of hemicelluloses might be important since hemicelluloses can affect the quality of baked products and can be used as a thickening and gelling agent. Jeltema et al. 26, 27 demonstrated that insoluble hemicelluloses had the greatest effects on the baking quality of cakes. The cellulose values for CR without treatment and with treatment were 72.67 and 72.33, respectively (NS) (Table 4). Using the permanganate procedure, Southgate 11 reported cellulose values of 33 for oat fiber, 26 for rice, 21 for apple and 20 for tomato fiber. In the present work, the fiber fraction of cellulose from the CR of both samples had the highest yield component compared to other fiber sources. This might be explained by differences in the methods used for determination. The results of this study suggested that some dietary fibers, notably cellulose, can be added to or partially substituted for cryoprotectants in surimi or other textured products for their texture-modifying and freeze-thaw-stabilizing properties 28. Crude lignin contents were not significantly different between the samples; CR without treatment yielded 1.88, while CR with treatment yielded 2.08 (Table 4).This was mainly due to the appropriate length of time for acid hydrolysis and as well as the methodology. Therefore, the lignin values obtained in this study were lower than other sources of fibers reported by Dreher 29.The fractionation procedure provides a guideline for determining the components of various food fiber sources and for further studies on the physiological effects and physicochemical properties of food. Previous works have indicated that various fiber sources have different physical properties that are associated with physiological responses and food applications 30, 31. The fractionation of dietary fiber to cellulose, hemicelluloses and lignin may elucidate which fractions affect mineral availability. Further studies are needed to more thoroughly examine the fractionation component. Conclusions The IDF components of the samples studied were different from each other. Cellulose was found to be the major component in all samples and was followed by hemicelluloses A and lignin. The results presented in this study of CR were different than other fruit sample contents. This would therefore suggest that the methods provide reliable fractions for further characterization. Standard procedures for fractionation are needed to study various sources of fiber. 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