 |
| Figure 1: Total phenol content of unfermented and fermented citrus peels |
| Livestock Research for Rural Development 24 (1) 2012 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
Recently, the world production of citrus fruit is about 80 million tones per year; however, citrus fruits have a small edible portion and large amounts of waste such as peels, unlike other fruits. This study sought to integrate citrus peels to nutrient and nutraceutical sources through Saccharomyces cerevisae solid substrate fermentation techniques. Ground peels from orange (Citrus sinensis), grape fruit (Citrus maxima) and shaddock (Citrus maxima) were fermented with pure strain of Saccharomyces cerevisae for 14days, the products obtained were subsequently analyzed for their proximate composition, phytate and total phenol content, and antioxidant activity as typified by their reducing power, DPPH free radical scavenging ability and Fe (II) chelating ability.
This study revealed that S. cerevisae fermentation of the peels caused a significant increase (P<0.05) in the protein [unfermented (4.3 – 6.7g/100g), fermented (12.8 – 19.7g/100g)], fat [unfermented (8.4 – 12.6g/100g), fermented (13.6 – 14.8g/100g)], total phenol content [unfermented (1.0 – 2.7mg/g), fermented (3.7 – 5.4mg/g)] and antioxidant activities (reducing power, DPPH free radical scavenging and Fe (II) chelating ability). Conversely, there was a significant (P<0.05) decrease in the carbohydrate [unfermented (65.5 – 73.8g/100g), fermented (57.2 – 64.2g/100g)], crude fibre [unfermented (9.6 – 12.2g/100g), fermented (1.1 – 3.1g/100g)] and phytate [unfermented (310.3 – 394.9mg/100g), fermented (282.1 – 366.7mg/100g)] content. Therefore, cheap, non-pathogenic and aerobic Saccahromyces cerevisae could be used to enhance the nutritive value and antioxidant properties of citrus peels; however, fermented shaddock and orange peels show the highest nutritive and antioxidant potentials, while grape fruit peel showed the least potential. The fermented peels could be a source of nutrient and nutraceuticals for livestock.
Keywords: fruit waste, protein, phenol
The use of plant wastes, underutilized fruit and vegetables as livestock feed may be a practical and economic way of disposal of such materials. A number of residue materials are produced when fresh citrus is processed into juice, and canned fruit. These include peel, rag, and seeds. In contrast with other types of fruits, citrus fruits have small edible portion and large amounts of waste material such as peels and seeds. Therefore, citrus processing produces a considerable amount of by-products, which constitute problems since the plant material is usually prone to microbial spoilage, decay and contribute to environmental pollution (Famyima and Ough 1982; Nikolic et al 1986).
Residues of citrus juice production are a source of dried pulp and molasses, fiber-pectin, cold pressed oils, essences, d-limonene, juice pulps and pulp wash, ethanol, seed oil, pectin, ascorbic acid, limonoids and flanonoids (Braddock, 1995; Ozaki et al 2000). Most of these materials from citrus by-products could be used as functional ingredients when designing healthy foods (functional foods), especially non-digestible carbohydrates (dietary fiber) and bioactive compounds (ascorbic acid and flavonoids) (Marin et al 2002; Puupponen-Pimia et al 2002; Oboh and Ademosun 2006).
Citrus fruit peels, a by-product of the citrus industry, represent about 65% of the total weight of the processed fruits. Dried citrus waste (pulp) is usually a valuable feed commodity. The energy value is high and is used extensively in rations for dairy cattle and have a fairly high protein content (4 – 7%), however, micro-fungi solid substrate fermentation of this pulp could increase the protein content, antioxidant activity and digestibility of the pulp (Oboh and Akindahunsi, 2003; Oboh 2006).
Fermentation is one of the oldest applied biotechnologies, having been used in food processing and preservation as well as beverages production for over 6000 years ago (Motarjemi, 2002; Oboh 2006). Fermentation enhances the nutrient content of foods through the biosynthesis of vitamins, essential amino acids and proteins, by improving protein quality and fibre digestibility. It also enhances micronutrient bioavailability and aids in degrading antinutritional factors (Oboh and Akindahunsi, 2003; Oboh et al 2003; Oboh 2006). It also enhances the medicinal potentials of fermented foods through the proliferation of microorganism (single cell protein), secretion of extracellular enzymes; breakdown of food macromolecules by microbial enzymes to produce bioactive substances such as free soluble phenols and bioactive peptides with antioxidant activity is responsible for its nutraceutical effects (Oboh et al 2008; Oboh et al 2009). This study therefore sought to integrate citrus peels into useful nutritional and nutraceutical use through Saccharomyces cerevisae solid substrate fermentation techniques.
Citrus by-products (the remaining part after the extraction of the juice) from the following citrus; orange (Citrus sinensis), grape fruits (Citrus maxima) and shaddock (Citrus maxima) were collected from Oja-Oba Market, within Akure metropolis, Akure, Nigeria. Pure strain Saccharomyces cerevisae was collected from Biology Department, Federal University of Technology Akure, Nigeria.
The peels were washed, dried and ground to powdery form. Thereafter, 100g of the processed peels was spread on a 50cm diameter tray to an average thickness of 2cm. A 2.5g sub-cultured pure strain of Saccharomyces cerevisae in 370ml nutrient solution [containing urea (8.0g), MgSO4.7H2O (7.0g), KH2PO4 (1.3g) and citric acid (2.0g)] was carefully mixed with the peels to produce a solid matrix. This was allowed to ferment for 14 days; the temperature and the relative humidity of the air were kept constant at 300C and 90-93% respectively (Oboh 2006).
The nutrient composition (ash, fat, carbohydrate, and crude fiber) of the fermented and unfermented peels was determined using the standard AOAC (1990) methods and the protein content was determined using the micro-Kjeldahl method. The phytate content was determined by the method of Wheeler and Ferrel (1971) based on the ability of standard ferric chloride to precipitate phytate in dilute HCl extracts of the unfermented and fermented peels.
The aqueous extract of the unfermented and fermented peels were prepared using the procedure describe by Oboh et al (2009). Briefly, about 2 g of each of peel was soaked in 40 mL sterile water for 5 min. Thereafter the mixtures were centrifuged at 2000 rpm for 10 min. The supernatant was used for the determination of total phenolic content and antioxidant activity (reducing power, Fe (II) chelating ability and DPPH free radical scavenging ability).
The total phenol content was determined on the extracts using the method reported by Singleton et al (1999). Appropriate dilutions of the extracts were oxidized with 2.5ml 10% Folin-Ciocalteau’s reagent (v/v) and neutralized by 2.0ml of 7.5% sodium carbonate. The reaction mixture was incubated for 40minute at 45oC and the absorbance was measured at 765nm in the spectrophotometer. The total phenol content was subsequently calculated using tannic acid as standard.
The free radical scavenging ability of the extracts against DPPH (1,1-diphenyl–2 picrylhydrazyl) free radical was evaluated as described by Gyamfi et al (1999). Briefly, appropriate dilution of the extracts (1 ml) was mixed with 1 ml, 0.4 mM methanolic solution containing DPPH radicals, the mixture was left in the dark for 30 min and the absorbance was taken at 516 nm. The DPPH free radical scavenging ability was subsequently calculated.
The reducing properties of the extracts were determined by assessing the ability of the extract to reduce FeCl3 solution as described by Oyaizu (1986). 2.5ml aliquot was mixed with 2.5 ml 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml 1% potassium ferricyanide. The mixture was incubated at 50 oC for 20 min. and then 2.5 ml 10 % trichloroacetic acid was added. This mixture was centrifuged at 650 rpm for 10 min. 5 ml of the supernatant was mixed with an equal volume of water and 1 ml 0.1 % ferric chloride. The absorbance was measured at 700 nm; reading with higher absorbance was taken as having higher reducing power.
The Fe2+ chelating ability of the extracts was determined using a modified method of Minotti and Aust (1987). Freshly prepared 500µM FeSO4 (150 µl) was added to a reaction mixture containing 168 µl 0.1M Tris-HCl (pH 7.4), 218 µl saline and the extracts (0 - 25µl). The reaction mixture was incubated for 5min, before the addition of 13 µl 0.25% 1, 10-phenanthroline (w/v). The absorbance was subsequently measured at 510 nm in a spectrophotometer. The Fe (II) chelating ability was subsequently calculated.
The results of the triplicate were pooled and expressed as mean ± standard error (SE) (Zar 1984); student t test and one-way analysis of variance (ANOVA) were carried out using Graphpad Prism version 5. Significance was accepted at P < 0.05.
The protein content of the unfermented citrus peels ranged from 4.31 g/100g (orange) to 6.71 g/100g (shaddock) as shown in Table 1.
|
Table 1: Proximate composition of Saccharomyces cerevisae fermented citrus peels (g/100g dry weight) |
|||||
|
Sample |
Ash |
Protein |
Fat |
Crude fibre |
Carbohydrate |
|
Grape peels Unfermented Fermented Orange peels Unfermented Fermented Shaddock peels Unfermented Fermented SEM P-values |
4.69b 6.43a
1.28d 2.51c
5.22b 6.12 a 0.22 0.01 |
6.03cd 12.8b
4.31d 18.1a
6.71c 19.7a 1.25 0.03 |
12.1b 13.6a
8.42 c 14.1a
12.6b 14.8a 0.62 0.01 |
9.61b 3.13c
12.2a 1.12d
9.98b 2.21c 0.78 0.01 |
67.6b 64.1c
73.8a 64.2bc
65.5b 57.2d 2.17 0.03 |
|
Values represent means of triplicate readings. abc values with the same alphabet in a column are not significantly different (P>0.05). SEM = Standard error of the means. |
|||||
The protein content is within the range of protein content earlier reported for citrus peels (Figuerola 2009), however, this protein content is lower than that of cassava peels (Oboh 2006). This relatively low protein contents of this citrus limit its use as feed for monogastric animals. Our recent work revealed that a cheap non-pathogenic aerobe Saccharomyces cerevisae fermentation of citrus peels brought about a significant (P<0.05) increase in the protein content of the peels, ranging from 12.8 (Grape fruit) to 19.7 g/100g (Shaddock). This increase in the protein content in the citrus peels during the fermentation with S. cerevisae agrees with earlier report on S. cerevisae fermentation of cassava products (gari and flour) (Oboh and Akindahunsi 2003) and cassava peels (Oboh 2006) in that there was an increase in the protein content.
The increase in the protein content of the fermented citrus peels could be attributed to the possible secretion of some extracellular enzymes (proteins) such as amylases, linamarase and cellulase (Oboh and Akindahunsi, 2003) into the citrus peels by the fermenting organisms in an attempt to make use of the citrus peels starch as a source of carbon (Raimbault 1998). Apart from this, the increase in the growth and proliferation of the fungus in the form of single cell proteins may possibly account for the apparent increase in the protein content of the fermented peels (Antai and Mbongo 1994). In view of this significantly enhanced protein content in the fermented peels, the citrus peels, regarded as having no economic value, could be integrated into animal nutrition provided the peel is acceptable and highly digestible in farm animals.
Furthermore, fermentation of the peels also caused a significant (P<0.05) increase in the fat content [unfermented (8.42 – 12.6g/100g), fermented (13.6 – 14.8g/100g)] and the ash content [unfermented (1.28 – 5.22g/100g), fermented (2.51 – 6.43g/100g)], the increase in the fat content could be attributed to the possibility that the fungus could secrete microbial oil. This assertion agrees with earlier findings of Akindumila and Glatz (1998) that some microbes could produce microbial oil during fermentation. The increase in the ash content may not be a product of fermentation (Oboh 2006); however, it could be attributed to the inorganic content of nutrient solution, which contains both the salt of magnesium and potassium. However, fermented shaddock peel has the highest protein and fat content. Conversely, there was a significant (P<0.05) decrease in the crude fibre [unfermented (9.61 – 12.2g/100g), fermented (1.12 – 3.13g/100g)] and carbohydrate [unfermented (65.5 – 73.8g/100g), fermented (57.2 – 64.2g/100g)] content of the peels (Table 1). The decrease in the crude fibre and carbohydrate content could be attributed to the possibility that the fungus could have hydrolyze the complex carbohydrate into simple glucose and ultimately used it as carbon source to synthesize fungi biomass rich in protein (Oboh and Akindahunsi 2003).
The phytate content of the fermented and unfermented citrus peels is presented in Table 2.
|
Table 2: Phytate content of saccharomyces cerevisae fermented citrus peels (mg/100g dry weight) |
||||
|
Sample Grape Orange Shaddock |
Unfermented 395a 310a 339a |
Fermented 367a 299b 282b |
SEM 10.7 2.41 8.85 |
P-values 0.03 0.02 0.03 |
|
Values represent means of triplicate readings. abc values with the same alphabet in a row are not significantly different (P>0.05). SEM = Standard error of the means. |
||||
Phytate content of the unfermented peels ranged from 310.3 (orange) to 394.9 mg/100g (grape), these values are low when compared with the phytate content of cassava peels (Oboh, 2006). Phytic acid (1,2,3,4,5,6- hexakis dihydrogen phosphate myoinositol) is a common storage form of phosphorus in seeds and in a few tubers and fruits. However, it is also considered an antinutritional factor. The complexing of phytic acid with nutritionally essential elements and the possibility of interference with proteolytic digestion have been suggested as responsible for antinutritional activity (Aletor, 1993). However, the fermentation of these peels with S. cerevisae caused a significant (P<0.05) decrease in the phytate content of the fermented peels [282 (shaddock) – 367 mg/100g (grape)]. The decrease in the phytate content of the fungus fermented citrus peels could possibly be attributed to the secretion of the enzyme phytase by the fungus. This enzyme is capable of hydrolyzing phytate, thereby, decreasing the phytate content of the peels. This decrease in phytate agrees with our earlier report, where S. cerevisae solid substrate fermentation of cassava decreased the phytate content of cassava products and peels (Oboh and Akindahunsi, 2003; Oboh 2006).
The effect of S. cerevisae fermentation on the total phenol content and the antioxidant activities of the citrus peels are presented in Figures 1 – 4. Natural phenolics are capable of removing free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce ɑ-tocopherol radicals and inhibit oxidases (Oboh and Rocha 2007).
|
| Figure 1: Total phenol content of unfermented and fermented citrus peels |
The total phenol content of citrus peels ranged from 1.0 (grape) to 2.7mg/g (shaddock), this phenolic content are quite low when compared with the total phenol content of green and sour teas (Oboh and Rocha 2008). However, fermentation of the citrus peels with pure strain of Saccharomyces cerevisae caused a significant (P<0.05) increase in the total phenolic content [unfermented (3.69), fermented (5.44mg/g)] of the peels. This increase in free soluble phenolic content of the peels during fermentation agrees with the report of Ju et al. (2009) where Saccharomyces cerevisae fermentation of Bokbunja fruits (Rubus coreanus Miq) caused an increase in the protein content. Likewise, the fermentation of black bean with some micro-fungi like Aspergillus awamori, Aspergillus oryzae, Aspergillus sojae, Rhizopus azygosporus and Rhizopus sp. No. 2 caused an increase in the total phenol content of the bean (Lee et al 2008). The basis for the increase in the phenolic content cannot category stated, however, recent report as shown that the activity of β-glucosidase and esterase increases during fermentation (Cho et al 2009), this increased enzyme activities may have enhanced the biotransformation of the bound phenolics to free soluble phenolics by cleaving the β-glycosidic linkage in the bound phenols (Chu et al 2002). However, fermented orange peels have the highest total phenol content, closely followed by that of shaddock peels.
Antioxidants carry out their protective properties on cells either by preventing the production of free radicals or by neutralizing/scavenging free radicals produced in the body or reducing/ chelating the transition metal composition of foods. Prevention of the chain initiation step by scavenging various reactive species such as free radicals is considered to be an important antioxidant mode of action (Dastmalchi et al 2007); the DPPH free radical scavenging ability of the citrus peels are presented in Figure 2.
 |
| Figure 2: DPPH free radical scavenging ability of unfermented and fermented citrus peels |
The results revealed that the micro-fungus fermentation of the peels caused a significant increase in the free radical scavenging ability of the peels. This increase in free radical scavenging ability with fermentation agrees with earlier report, in that there was an increase in the DPPH free radical scavenging ability of the fruits of Bokbunja (Rubus coreanus Miq) when fermented with Saccharomyces cerevisiae (Ju et al 2009). The fermentation of black bean with some like Aspergillus awamori, Aspergillus oryzae, Aspergillus sojae, Rhizopus azygosporus and Rhizopus sp. No. 2 also caused an increase in the free radical scavenging ability of the beans (Lee et al 2008). It is worth noting that fermented orange peels, with the highest total phenol content had the highest free radical scavenging ability.
Reducing power is a novel antioxidation defense mechanism; the two mechanisms that are available to affect this property are electron transfer and hydrogen atom transfer (Dastmalchi et al 2007). The reducing power of the unfermented and fermented citrus peels was assessed based on their ability to reduce Fe (III) to Fe (II) and the results are presented in figure 3. The results of the study revealed that fermentation significantly increased the reducing power of the peels. This increase in reducing power agrees with our earlier report on the increased reducing power of fermented underutilized legumes (Oboh et al 2008; Oboh et al 2009), and what Lee et al (2008) reported on solid-state fermentation of black bean with various GRAS filamentous fungi including Aspergillus awamori, Aspergillus oryzae, Aspergillus sojae, Rhizopus azygosporus and Rhizopus sp. No. 2
 |
| Figure 3: Reducing power of unfermented and fermented citrus peels |
Furthermore, the ability of antioxidant substances to chelate and deactivate transition metals, prevent such metals from participating in the initiation of lipid peroxidation and oxidative stress through metal catalysed reaction. The Fe (II) chelating ability of the fermented and unfermented peels are presented in Figure 4.
 |
| Figure 4: Fe (II) chelating ability of unfermented and fermented citrus peels |
The results also revealed that S. cerevisiae solid substrate fermentation of the citrus peels caused a significant increase in the Fe (II) chelating ability of the peels. This increase in the Fe (II) chelating ability of the peels agree with earlier report by Lee et al (2008), in that solid-state fermentation of black bean with various filamentous fungi including Aspergillus awamori, Aspergillus oryzae, Aspergillus sojae, Rhizopus azygosporus and Rhizopus sp. No. 2 brings about an increase in the Fe (II) chelating ability of the beans. Therefore, this increase in the antioxidant activity as typified by Fe (II) chelating ability, reducing power and free radical scavenging ability of the peels during fermentation may have been due to the increase in the total phenol content. However, this increased total phenol content and antioxidant activities makes this fermented peels good sources of antioxidant for both nutritional and industrial use.
Cheap, non-pathogenic and aerobic Saccahromyces cerevisae could be used to enhance the nutritive value and antioxidant properties of citrus peels; however, fermented shaddock and orange peels show the highest nutritive and antioxidant potentials, while grape peel showed the least potential. The fermented peels could be a source of nutrient and nutraceuticals for livestock and industrial use.
The authors acknowledge the research grant provided by Federal University of Technology Akure, Nigeria for the execution of the project.
Akindumila F and Glatz B A 1998 Growth and oil production of Apiotrichum curvatum in tomato juice. Journal of Food Protection 61: 1515–1517.
Aletor V A 1993 Allelochemicals in plant food and feedingstuffs: Nutritional, Biochemical and physiopathological aspects in animal production. Veterinary and Human Toxixology 35: 57–67.
Antai S P and Mbongo P M 1994 Utilization of cassava peels as substrate for crude protein formation. Plant Foods for Human Nutrition 46 (4) : 345-351.
AOAC 1990 Official methods of analysis of the Association of Official Analytical Chemists. 15th ed. Association of Official Analytical Chemists. Washington D C
Cho K M, Hong S, Math R K, Lee J H, Kambiranda D M, Kim J M, Islam S M A, Yun MG, Cho J J, Lim W J and Yun H D 2009 Biotransformation of phenolics (isoflavones, flavanols and phenolic acids) during the fermentation of cheonggukjang by Bacillus pumilus HY1. Food Chemistry 114: 413-419
Chu Y, Sun J, Wu X and Liu R H 2002 Antioxidant and antiproliferative activity of common vegetables. Journal of Agricultural and Food Chemistry 50: 6910–6916.
Dastmalchi K, Dorman H J D and Kosar Mand Hiltunen R 2007 Chemical composition and in vitro antioxidant evaluation of a water solubleMoldavian balm (Dracocephalum moldavica L.) extract. Lebensmittel-Wissenschaft and Technologie 40:239–248
Famyima O and Ough C 1982 Grape pomace: possibilities as animal feed. American Journal of Enology and Viticulture 33: 44–46.
Figuerola F, Hurtado M L, Estevez A M, Chiffelle I and Asenjo F 2009 Fibre concentrates from apple pomace and citrus peel as potential fibre sources for food enrichment. Retreived January 22 2010 from http://captura.uchile.cl/dspace/bitstream/2250/2304/1/Figuerola_F.pdf
Gyamfi M A, Yonamine M and Aniya Y 1999 Free-radical scavenging action of Medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced liver injuries. General Pharmacology 32: 661-667
Ju H K, Cho E J, Jang M E, Lee Y Y, Hong S S, Park J H and Kwon S W 2009 Characterization of increased phenolic compounds from fermented Bokbunja (Rubus coreanus Miq.) and related antioxidant activity. Journal of and Biomedical Analysis 49: 820 – 827
Lee I H, Hung Y H and Chou CC 2008 Solid-state fermentation with fungi to enhance the antioxidative activity, total phenolic and anthocyanin contents of black bean. International Journal of Food Microbiology 121: 150-156
Minotti G and Aust S D 1987 An investigation into the mechanism of citrate-Fe2+-dependent lipid peroxidation. Free Radicals in Biology and Medicine 3: 379–387.
Miyake Y, Fukumoto S, Osawa T and Sakaida K 2004 Antioxidative activity of citrus peels fermented by three species of Aspergillus. Journal of the Japanese Society for Food Science and Technology 51: 181-184
Motarjemi Y 2002 Impact of small scale fermentation technology on food safety indeveloping countries. International Journal of Food Microbiology 75: 213-229.
Nikolic J A, Cuperlovic M, Milijic C, Djordjevic D and Krsmanovic J 1986 The potential nutritive value for ruminants of some fibrous residues from the food processing industry. Acta Veterinaria 36: 13–22.
Oboh G 2006 Nutrient enrichment of cassava peels using Saccharomyces cerevisae and Lactobacillus spp solid media fermentation techniques. Electronic Journal of Biotechnology 9:46–49.
Oboh G, Ademiluyi A O and Akindahunsi A A 2009 Change in the polyphenol distribution and antioxidant activity during fermentation of some underutilized legumes. Food Science and Technology International 15: 41 – 46
Oboh G and Ademosun A O 2006 Comparative studies on the ability of crude polyphenols from some Nigerian citrus peels to prevent lipid peroxidation—in vitro. Asian Journal of Biochemistry 1: 169–177
Oboh G and Akindahunsi A A 2003 Biochemical changes in cassava products (flour and gari) subjected to Saccharomyces cerevisae solid media fermentation. Food Chemistry 82:599–602.
Oboh G, Akindahunsi A A and Oshodi A A 2003 Dynamics of Phytate-Zn balance of Fungi Fermented Cassava products (Flour and Gari). Plant Foods forHuman Nutrition 58: 1-7.
Oboh G, Alabi K B and Akindahunsi A A 2008 Fermentation changes the nutritive value, polyphenol distribution and antioxidant properties of Parkia biglobosa seeds (African locust beans). Food Biotechnology 22: 363 – 376
Oboh G and Rocha J B T 2007 Polyphenols in Red pepper [Capsicum annuum var. aviculare (Tepin)] and their protective effect on some pro-oxidants induced lipid peroxidation in Brain and Liver. European Food Research and Technology 225: 239-247.
Oboh G and Rocha J B T 2008 Antioxidant and Neuroprotective Properties of Sour tea (Hibiscus sabdariffa, calyx) and Green tea (Camellia sinensis) on some Pro-oxidants induced Lipid Peroxidation in Brain - In vitro. Food Biophysics 3: 382-389
Oyaizu M 1986 Studies on products of browning reaction: antioxidative activity of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition 44:307–315
Puupponen-Pimia R, Aura A M, Oskman-Caldentey KM, Myllarinen P, Saarela M, Mattila-Sandholm T and Poutanen K 2002 Development of functional ingredients for gut health. Trends in Food Science and Technology 13: 3 – 11.
Raimbault M 1998 General and
microbiological aspects of solid substrate fermentation, Electronic Journal of
Biotechnology [online] 1 (3). Retreived February 4, 2010 from
http://www.ejbiotechnology.info/content/vol1/issue3/full/9/9.PDF
Singleton V L, Orthofer R and Lamuela-Raventos R M 1999 Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteau’s reagent. Methods in Enzymology 299:152–178
Wheeler E L and Ferrel R E 1971 A method for phytic acid determination in wheat fractions. Cereal Chemistry 48: 312 – 316.
Received 23 June 2011; Accepted 25 October 2011; Published 4 January 2012