Enhancements of isoflavone aglycones, total phenolic content, and antioxidant activity of black soybean by solid-state fermentation with Rhizopus spp.

In the present study, a solid-state fermentation process of black soybean was developed. Three filamentous fungi including Rhizopus oligosporus (BCRC 31996), R. oligosporus (NTU-5), and Rhizopus oryzae (BCRC 30894) were cultivated with steam-cooked intact and dehulled black soybeans. It was found that fermentation enhanced the total phenolic and isoflavone aglycone content as well as antioxidant activity of the black soybean methanol [80 % (v/v)] extracts. Among the three candidates, R. oligosporus BCRC 31996 was chosen as the working strain, and dehulled Tainan NO.3 black soybeans were used as the substrate based on the relatively high total phenolic content, isoflavone aglycone accumulation, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effect of the fermented extracts. In a 6-day fermentation, extracts of the fermented dehulled black soybeans yielded higher total phenolic content (2,876.2 μg/g) which was 1.16 times of the result from the intact soybean extracts. The DPPH scavenging effect of the fermented dehulled black soybean extract reached its maximum on the third day. The activity of β-glucosidase of dehulled black soybean samples increased with time and resulted in accumulation of isoflavone aglycones. The product of solid-state black soybean fermentation serves as a functional ingredient in the products of nutritional supplements and health foods.     

Role of Carbohydrate-Cleaving Enzymes in Phenolic Antioxidant Mobilization from Whole Soybean Fermented with Rhizopus oligosporus

Previous research has suggested a relationship between free phenolic content and β-glucosidase activity in solid-state fermented food substrates and to amylase activity in germinating soybeans. This study was undertaken to examine the role of a number of carbohydrate-cleaving enzymes in phenolic antioxidant mobilization from whole soybean during solid-state fermentation. In addition to total soluble phenolic content, α- and β-glucosidase, α-amylase, and β-glucuronidase activities were measured in extracts of soybean fermented with a food-grade fungus, Rhizopus oligosporus. Antioxidant activity of the extracts was determined as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability. Our results demonstrate that while total soluble phenolic content increased 120–135% in the extracts, increased antioxidant activity (+61%) was limited to the early fermentation period, with activity decreasing with increased culture time. Higher antioxidant activity was linked to increased glucosidase and glucuronidase activities, while high total phenolic content partly linked to increased amylase activity. The overall results (enzymatic activities and phenolic antioxidant contents) suggest the possible involvement of lignin remobilization and/or degradation activities, as well as phenolic detoxification activities, by Rhizopus oligosporus in phenolic antioxidant mobilization from fermented whole soybean.     

Microbial Changes in Fermented Peanut and Soybean Pastes Containing Kojis Prepared using Aspergillus oryzae and Rhizopus oligosporus

Microbiological profiles of peanut and soybean misos containing 6 and 12% NaCl and kojis prepared with Aspergillus oryzae and Rhizopus oligosporus were monitored over a 90-day period of fermentation. Although total plate counts in misos containing R. oligosporus koji fluctuated while counts in misos containing A. oryzae koji remained constant during the fermentation period, the final microbial populations in sixteen test formulae were similar to the initial counts. Mold populations in mists containing two types of kojis declined similarly during fermentation. There was little difference in populations of Saccharomyces rouxii in misos containing the two types of mold kojis during the first month of fermentation; however, higher numbers of S. rouxii were noted in mitis containing R. oligosporus koji compared to those containing A. oryzae koji during later stages of fermentation. The final pH values of misos were similar, even though misos containing A. oryzae koji had a larger magnitude of drop in pH than did misos containing R. oligosporus koji over the 90-day test period.     

Bioavailability of Zinc in Fermented Soybeans

The bioavailability of zinc in soybean meal fermented by lactic acid-producing bacteria (LAPB) and by Rhizopus oligosporus in tempeh fermentation was evaluated with the rat bioassay, using the slope ratio method and log total femur zinc or weight gain versus zinc intake as response parameters. Fermentation significantly increased (P < 0.05) the RBV of zinc from 73.0% in boiled non-fermented soybeans (pH 6.7) to 84.5% in boiled LAPB fermented soybeans. The highest RBV (95.3%) was found in the autoclaved and LAPB fermented product (pH 6.5). Rhizopus oligosporus fermentation resulted in a RBV of 89.0%. Thus, fermentation in the processing of soybeans will improve zinc bioavailability in this food.     

Fermentation of Corn Gluten Meal with Aspergillus oryzae and Rhizopus oligosporus

PER values determined for corn gluten meal (CGM) and CGM fermented with Aspergillus oryzae NRRL 1988 were not significantly different (P > 0.05) and both diets failed to meet maintenance requirements of rats. In order to characterize some of the changes that occur during fungal fermentation, CGM was also fermented for 4 days at 28°C with A. oryzae NRRL 1988 and NRRL 506 and Rhizopus oligosporus NRRL 2710 and NRRL 2549, respectively. Proteolytic activity, pH, and nitrogen content increased rapidly between 20 and 70 hr for all the fungi. Decreases in some amino acids were observed, possibly due to their catabolism by the molds. Lysine as a proportion of total essential amino acids released by pepsin and pancreatin in vitro was increased as a result of these fungal fermentations.     

Degradation of anti-nutritional factors and reduction of immunoreactivity of tempeh by co-fermentation with Rhizopus oligosporus RT-3 and Actinomucor elegans DCY-1

The effects of solid-state fermentation with four filamentous fungi (Rhizopus oligosporus, Actinomucor elegans, Neurospora crassa and Rhizopus oryzae) and their combination on the degradation of protein allergens and anti-nutritional factors (ANFs) of tempeh were evaluated. Glycinin, β-conglycinin, trypsin inhibitor and flatulence-causing oligosaccharides were significantly hydrolysed by all assayed strains but the level of reduction of ANFs depended on the strain. ELISA was conducted to evaluate IgE immunoreactivity using sera from soy sensitive individuals. Results revealed that co-fermentation of R. oligosporus and A. elegans resulted in the highest reduction in IgE immunoreactivity for all sera used and showed greatest degradations on α’, α, β, acidic subunits (100% hydrolysed) and extensive hydrolysis of flatulence-causing oligosaccharides. Tempeh fermented with R. oligosporus and A. elegans displayed high level of soluble protein and peptide (<10 kD) content. Co-fermentation of R. oligosporus and A. elegans was proved to be effective in producing hypoallergenic tempeh.     

Isoflavone Conversion of Black Soybean by Immobilized Rhizopus spp.

Isoflavones exhibit many bioactive benefits, such as antioxidation, antimutagenic effects, and reduction of symptoms of postmenopause conditions. Aglycone forms of isoflavone have shown more biological effects over their glycosylated ones. Most isoflavones in nature, however, exist as glycosylated forms and cannot easily be absorbed in the intestines. The purpose of this study was to develop a fermentation process of black soybean to produce functional food using a Luffa cylindrical fiber biofilm reactor. Three filamentous-fungi, including Rhizopus oligosporus (BCRC 31996), Rhizopus oligosporus (NTU-5), and Rhizopus oryzae (BCRC 30894), were cultivated in black soybean medium. The assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effect of fermented broth and isoflavone aglycosylation rate of genistin and daidzin were evaluated as indicators to determine the most suitable strain. After a 2-day cultivation in flasks, DPPH radical scavenging effect of R. oligosporus NTU-5 reached 83%, which is the maximum value among three strains. R. oligosporus NTU-5 was subsequently chosen as target strain and used for the scale-up of fungal-based aerobic growth. The optimal cultivation condition obtained in a 5-L bioreactor with Luffa cylindrical fiber was 30°C, 0.1 vvm aeration, 100 rpm agitation, 10% inoculum volume, and 6% (w/v) black soybean as medium. After a 6-day cultivation, R. oligosporus NTU-5 converted isoflavones to their aglycones, daidzein and genistein, and these were 59.7 and 23.6 μM, respectively. Isoflavone aglycosylation rate of daidzin and genistin were 77.6 and 31.8%, respectively. The DPPH scavenging effect was 62%.     

Physical and Chemical Changes in Fermented Peanut and Soybean Pastes Containing Kojis Prepared Using Aspergillus oryzae and Rhizopus oligosporus

Studies were made to determine and compare physical and chemical changes occurring during 90 days of fermentation of miso-like products containing peanuts and soybeans as well as a combination of these oilseeds. Two koji molds (Aspergillus oryzae and Rhizopus oligosporus) and two levels of NaCl (6 and 12%) were evaluated. The L color values decreased more rapidly in misos containing A. oryzae koji compared to misos containing R. oligosporus koji and changes occurred earlier in low-salt formulae than in high-salt formulae. The type of mold koji had no apparent effect on changes in viscosity. The free fatty acid content of misos increased dramatically during the first 4 days of fermentation. Misos containing A. oryzae koji had higher soluble nitrogen and free amino acid content in the final product than did those containing R. oligosporus kojis. Peanut miso products had higher soluble nitrogen contents than did respective soybean products; however, the type of oilseed had little effect on accumulation of free amino acids.     

Enhancement of the antioxidant and starch hydrolase inhibitory activities of king coconut water (Cocos nucifera var. aurantiaca) by fermentation with kombucha ‘tea fungus’

Enhancement of antioxidant and starch hydrolase inhibitory properties of king coconut water when subjected to fermentation with the ‘tea fungus’ was evaluated. pH, colour, viscosity, ethanol content, antioxidant activity, total phenolic content, ferulic and p‐coumaric acid contents and starch hydrolase inhibitory activities were analysed for 7 days in fermented and unfermented king coconut water samples. The fermented sample had a statistically significant decrease (P < 0.05) in pH. Colour values indicated the fermented beverage to increase in ‘yellowness’ over time. A statistically significant increase in the total phenolic content and the antioxidant activity (P < 0.05) was observed from day 1 onwards in the fermented sample. The starch hydrolase inhibitory activities in the fermented beverage had statistically significantly increased (P < 0.05) by day 7. Overall, the study highlights an enhancement of the antioxidant and starch hydrolase inhibitory properties of king coconut water as a result of the kombucha‐based fermentation.     

Single‐cell protein production through microbial conversion of lignocellulosic residue (wheat bran) for animal feed

Agricultural residue (wheat bran) rich in carbohydrates was utilized in the fermentation process to produce microbial biomass. Single‐cell biomass consists of the dried cells of microorganisms, which are used as protein supplements in human food and in animal feed. In the present study, two different microorganisms (Candida utilis and Rhizopus oligosporus) were studied for biomass production. To enhance the nutritional contents of wheat bran, a number of different fermentation parameters (effect of inoculum size, age of inoculum, incubation period, moisture to substrate ratio and incubation temperature) were optimized. Maximum yield was obtained at an inoculum size of 10% (v/w), with the age of the inoculum being a 48 h old culture. A fermentation period of 48 h was found to give the maximum protein yield and viable counts of yeast cells and mould hyphae. The microorganisms showed good growth at 30 °C. After complete optimization of the fermentation parameters, a batch of wheat bran was fermented with C. utilis and R. oligosporus under the optimized conditions, resulting in a maximum crude protein yield of 41.02% compared with the 4.21% crude protein of the non‐fermented wheat bran. Copyright © 2015 The Institute of Brewing & Distilling     

AMYLASE AND HELICOBACTER PYLORI INHIBITION BY PHENOLIC EXTRACTS OF PINEAPPLE WASTES BIOPROCESSED BY RHIZOPUS OLIGOSPORUS

This study focused on the potential functional activity of phenolic‐enriched pineapple wastes bioprocessed by Rhizopus oligosporus. Using the phenolic‐enriched bioprocessed extracts, amylase inhibition relevant to Type 2 diabetes and Helicobacter pylori inhibition linked to stomach ulcer were investigated. Two treatments were studied: 9 g of pineapple residue plus 1 g of soy flour (P9) and 5 g of pineapple residue plus 5 g of soy flour (P5). Extracts obtained after 2 and 10 days of growth were selected to represent early and late stages of bioprocessing based on phenolic content. Potent α‐amylase inhibition was found in P9 treatment at 2 days of fungal growth and was maintained after subsequent boiling, freeze‐drying and/or autoclave treatment. Amylase inhibitory activity did not correlate with the DPPH radical scavenging antioxidant activity of the extracts and therefore it is likely linked to the structure of the phenolic compounds. H. pylori inhibitory activity was found in P5 treatment after 10 days of R. oligosporus growth and may be linked to phenolic compounds present at this stage.     

Solid-state fermentation of Lathyrus sativus seeds using Aspergillus oryzae and Rhizopus oligosporus sp T-3 to eliminate the neurotoxin β-ODAP without loss of nutritional value

The presence of a neurotoxic amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (β-ODAP), in the seeds of grass pea (Lathyrus sativus) is the cause of an irreversible spastic paraparesis, neurolathyrism, after over-consumption. This is a negative factor in an otherwise very tasty, nutritious, easily cultivated and hardy food crop in Asia and Africa. The reduction of β-ODAP to less than 10% of the original content has been reached by fermenting L sativas Jamalpur seeds with Aspergillus oryzae NRRL 1988 for 48 h, followed by fermentation with Rhizopus oligosporus sp T-3 for 48 h. Other nutritional qualities were also improved in the fermented seed meal: increased content of protein, higher amino acid scores for sulphur-containing and aromatic amino acids, better resistance to high temperature and to oxidation, and drastic reduction of the flatulence factors.     

Comparison of releasing bound phenolic acids from wheat bran by fermentation of three Aspergillus species

Wheat bran was fermented at 28 °C for 7 days under 70% humidity by Aspergillus niger, Aspergillus oryzae and Aspergillus awamori. Total phenolic content (TPC) of the unfermented sample was 1531.5 μg g^?1 wheat bran. After the fermentation of Aspergillus awamori, Aspergillus oryzae and Aspergillus niger, TPC reached 5362.1, 7462.6 and 10 707.5 μg g^?1, respectively. The antioxidant activity in the extractions of fermented wheat bran also increased significantly compared with the unfermented sample (P < 0.05). Aspergillus niger showed the greatest capacity to release bound ferulic acid (416.6 μg g^?1). Aspergillus oryzae and Aspergillus awamori had the advantages of releasing more chlorogenic acid (84.0 μg g^?1) and syringic acid (142.3 μg g^?1), respectively. The destructive effect of Aspergillus niger on wheat bran structure was the strongest, followed by that of Aspergillus oryzae. This effect of Aspergillus niger may be due to its higher cellulase, xylanase, arabinofuranosidase and β‐xylosidase activities. Besides, Aspergillus oryzae possessed higher β‐glucosidase activity, and Aspergillus awamori had higher α‐amylase and feruloyl esterase activities. Aspergillus niger may be the best to release bound phenolic acids in the three Aspergillus species. These will provide the helpful information for understanding mechanism of the fermentation by Aspergillus species releasing bound phenolic in wheat bran.     

The phytate and phytase of soybean tempeh

Soybeans were fermented into tempeh by Rhizopus oligosporus NRRL 2710. The phytic acid content of soybeans was reduced by about one-third as a result of this fermentation, while an equivalent amount of phosphate was released in the tempeh. The reduction of phytic acid was due to phytase elaborated by the mould of the fermentation. The pH optimum of this enzyme was 5.6 and the K_m of the phytase-phytate reaction was 0.28 × 10^?3 M.     

Myo-inositol phosphates profile of buckwheat and quinoa seeds: Effects of hydrothermal processing and solid-state fermentation with Rhizopus oligosporus

Phytate content, bioavailability and myo-inositol phosphates profile were analysed in pseudocereals (i.e., white and coloured quinoa, buckwheat) subjected to thermal processing and solid-state fermentation with Rhizopus oligosporus. Fair buckwheat groats were characterised by the highest level of phytate (18.3 µmol/g), with 54% share of InsP₆ fraction. Processing (cooking, roasting, tempe-type fermentation) resulted in the significant decrease in phytate content, its bioavailability and the share of higher phosphorylated forms of myo-inositol (InsP_6-5) in the profile of all the samples. Fermentation of cereals resulted in generation of high amounts of inositol triphosphates, particularly in the case of buckwheat (32% share in the whole profile). The path of dephosphorylation, identified after fermentation of quinoa seeds including intermediate products – Ins(1,2,4,5,6)P₅ and Ins(1,2,6)P₃ – confirmed that the dominant phytase activity could be attributed to the presence of microbial 3-phytases from R. oligosporous.     

Bioavailability of Iron in Fermented Soybeans

Hemoglobin depletion-repletion assay was used for determining iron bioavailability. The relative biological value (RBV) of iron in boiled nonfermented soybeans was the lowest among the products evaluated. The RBV of iron increased from 60.1% in boiled nonfermented soybeans to 86.7% by lactic acid producing microorganisms and to 87.5% by Rhizopus oligosporus fermentation (FeSO₄= 100%). Results of this research suggested that fermentation by lactic acid producing organisms and Rhizopus oligosporus increased the RBV of iron.     

Hypoallergenic buckwheat flour preparation by Rhizopus oligosporus and its application to soba noodle

The effect of Rhizopus oligosporus on the structure and functional properties of buckwheat grain during fermentation was investigated and the production of buckwheat hypoallergenic flour was studied. The spores of R. oligosporus were mixed with steamed buckwheat grains, and they were incubated at 30 °C and 85% relative humidity (RH) for 0, 24, 48 and 72 h. The changes of structure, free amino acids, minerals, phytic acid and allergenic proteins in the grains were determined during the time course of incubation. The 48-h-fermented buckwheat (FeB) was found to be involved in formation of amino acids with higher amounts of total amino acids, and some of them increased 50 times more; including isoleucine, leucine, lysine, valine, glycine, histidine, tyrosine and γ-amino butyric acid (GABA). SDS–PAGE and Western Blot analyses showed that R. oligosporus was very effective to reduce allergenic proteins of buckwheat and could produce the hypoallergenic buckwheat flour. In its application to noodle making, the FeB-substituted wheat flour noodle was softer and had more elasticity than the control without FeB flours (1CW), except for the 10% substitution of FeB. Uncooked and cooked noodles were brownish compared to those of the 1CW. The R. oligosporus could possibly be used to produce hypoallergenic buckwheat with higher functional and good rheological properties, and thus produced FeB flour was considered to become a big prospect for development of new food in the future.     

Degradation of Phytic Acid in Oncom (Fermented Peanut Press Cake)

Peanut press cake was fermented with Neurospora sitophila, Rhizopus oligosporus, a Neurospora strain isolated from Indonesian oncom, and mixed cultures of these molds. The phytic acid content of the uninoculated cake was 1.36% on a dry basis, but decreased rapidly during fermentation. Oncom prepared with R. oligosporus contained the least amount of phytic acid (0.05% after 72 hr of fermentation), while oncom prepared with the Indonesian strain of Neurospora contained the greatest amount of it (0.70%). Penta-, tetra-, tri-, di- and mono-phosphates of inositol, as well as inorganic phosphate and inositol, were found in the fermenting cake.     

In vitro digestibility of processed and fermented soya bean,cowpea and maize

Tropical legumes, ie soya bean and cowpea, were pre‐treated and subsequently fermented using pure cultures of Rhizopus spp. Impact of soaking, cooking and fermentation of the legumes on their digestibility was determined using an in vitro digestion method. Processing of white maize included, amongst others, natural lactic acid fermentation, cooking and saccharification using barley malt. An in vitro method was standardised to carry out comparative determinations of the dry matter digestibility of cereal and legume food samples as a function of processing conditions, without attempting to exactly mimic gastrointestinal digestion. Using this method based on upper digestive tract digestion, it was observed that digestibility of the legumes increased during cooking and fermentation. Cooking improved the total digestibility of both soya bean and cowpea from 36.5 to 44.8% and from 15.4 to 40.9% respectively. Subsequent fungal fermentation increased total digestibility only by about 3% for both soya bean and cowpea. Digestibility was also influenced by fungal strain and fermentation time. Cooking and subsequent saccharification using malt almost tripled total digestibility of white maize from 25.5 to 63.6%, whereas lactic fermentation of maize had no effect on in vitro dry matter digestibility. Although total digestibility of cooked legumes was only slightly improved by mould fermentation (3% for both soya bean and cowpea), the level of water‐soluble dry matter of food samples increased during fermentation with Rhizopus oryzae from 7.0 up to 27.3% for soya bean and from 4.3 up to 24.1% for cowpea. These fermented products could therefore play a role as sources of easily available nutrients for individuals suffering from digestive disorders. © 2000 Society of Chemical Industry     

Enhanced Antioxidative Activity of Chinese Traditionally Fermented Okara (Meitauza) Prepared with Various Microorganism

In order to investigate the antioxidative activity of Meitauza koji (Meitauza, a Chinese traditionally fermented okara), the okara was fermented with Aspergillus oryzae 3.951, Rhizopus oligosporus 3.912, Actinomucor elegans 3.118 and Bacillus subtilis B2 as kojis. All the methanol extract yields and peptide content of the kojis were higher than that of non-fermented okara. Moreover, the Meitauza kojis displayed enhanced antioxidative activities in comparison with the non-fermented okara. Among the four kinds of kojis tested, those fermented with B. subtilis B2 exhibited the highest levels of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals scavenging activities and reducing power. The ABTS and DPPH radicals scavenging activities of Meitauza koji fermented with B. subtilis B2 were ca. 12.53 and 7.92 folds that of the non-fermented okara. Analysis of the dose-response effect also revealed that before reaching a threshold point, there is a linear relationship between increases in antioxidative activity and increases in the concentration of koji extract. In addition, the peptide content of the Meitauza koji prepared with B. subtilis B2 increased from 8.6 mg to 34 mg per 100 mg extract. These results suggest the potential for developing traditional functional food with okara fermented by B. Subtilis B2.