Response surface methodology for optimizing the fermentation medium of alpha-galactosidase in solid-state fermentation

AIMS: Alpha-galactosidase is applied in food and feed industries for hydrolysing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to develop an optimal culture medium for the production of alpha-galactosidase in solid-state fermentation (SSF) by a mutant strain Aspergillus foetidus. METHODS AND RESULTS: Response surface methodology (RSM) was applied to evaluate the effects of variables, namely the concentrations of wheat bran, soybean meal, KH(2)PO(4), MnSO(4).H(2)O and CuSO(4).5H(2)O on alpha-galactosidase production in the solid substrate. A fractional factorial design (FFD) was firstly used to isolate the main factors that affected the production of alpha-galactosidase and the central composite experimental design (CCD) was then adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 8.2137 g wheat bran, 1.7843 g soybean meal, 0.001 g MnSO(4).H(2)O and 0.001 g CuSO(4).5H(2)O in 10 g dry matter fermentation medium. CONCLUSIONS: After incubating 96 h in the optimum fermentation medium, alpha-galactosidase activity was predicted to be 2210.76 U g(-1) dry matter in 250 ml shake flask. In the present study, alpha-galactosidase activity reached 2207.19 U g(-1) dry matter. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimization of the solid substrate was a very important measure to increase enzyme activity and realize industrial production of alpha-galactosidase. The process of alpha-galactosidase production in laboratory scale may have the potential to scale-up.     

Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.     

Influence of cultivating conditions on the alpha-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation

AIMS: The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Penicillium sp. in solid-state fermentation (SSF). METHODS AND RESULTS: Certain fermentation parameters involving incubation temperature, moisture content, initial pH value, inoculum and load size of medium, and incubation time were investigated separately. The optimal temperature and moisture level for alpha-galactosidase biosynthesis was found to be 30 degrees C and 50%, respectively. The range of pH 5.5-6.5 was favourable. About 40-50 g of medium in 250-ml flask and inoculum over 1.0 x 10(6) spores were suitable for enzyme production. Seventy-five hours of incubation was enough for maximum alpha-galactosidase production. Substrate as wheat bran supplemented with soyabean meal and beet pulp markedly improved the enzyme yield in trays. CONCLUSIONS: Under optimum culture conditions, the alpha-galactosidase activity from Penicillium sp. MAFIC-6 indicated 185.2 U g(-1) in tray of SSF. SIGNIFICANT AND IMPACT OF THE STUDY: The process on alpha-galactosidase production in laboratory scale may have a potentiality of scaling-up.     

Influence of inocula and grains on sclerotia biomass and carotenoid yield of<Emphasis Type="Italic"> Penicillium</Emphasis> sp. PT95 during solid-state fermentation

Various inocula and grains were evaluated for carotenoid production by solid-state fermentation using Penicillium sp. PT95. Millet medium was more effective in both sclerotia growth and carotenoid production than other grain media. An inoculum in the form of sclerotia yielded higher sclerotia biomass compared to either a spore inoculum or a mycelial pellet inoculum. Adding wheat bran to grain medium favored the formation of sclerotia. However, neither the inoculum type nor addition of wheat bran resulted in a significant change in the carotenoid content of sclerotia. Among grain media supplemented with wheat bran (wheat bran:grain =1:4 w/w, dry basis), a medium consisting of rice and wheat bran gave the highest sclerotia biomass (15.10 g/100 g grain), a medium consisting of buckwheat and wheat bran gave the highest content of carotenoid in sclerotia (0.826 mg/g dry sclerotia), and a medium consisting of millet and wheat bran gave the highest carotenoid yield (11.457 mg/100 g grain).     

Enzyme formation during solid-substrate fermentation in rotating vessels

Aspergillus awamori NRRL 4869 was cultured on the solid substrate, wheat bran, in a modified Rollacell apparatus to produce alpha-galactosidase and invertase. The swivel cap on the elongated bottle permits the introduction of air while the bottle rotates. Parameters of air flow rate (0.05-0.2 liter/kg/min), rpm (0.15-15 rpm), and weight of solids (150 and 300 g) were varied. At low air flow rates (0.05 liter/kg solids/min), alpha-galactosidase production was minimal independent of the rotation rate. At 0.15 rpm and 0.2 liter/kg solids/min air flow rate, invertase production ceased after five days; whereas alpha-galactosidase production continued. The modified Rollacell can be a useful apparatus for studying solid-substrate cultures.     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

Use of evolutionary operation (EVOP) factorial design technique to develop a bioprocess using grease waste as a substrate for lipase production

The aim of the present work was to develop a bioprocess using EVOP-factorial design technique employing grease waste as a substrate for the production of lipase. A newly isolated fungal strain of Penicillium chrysogenum was explored for the fermentation process. Solid-state fermentation (SSF) was carried out using grease waste and Czapek-dox medium, supplemented with wheat bran. The yield of lipase was 38 U/ml when SSF was carried out at 32 °C for 8 days and grease:wheat bran:Czapek-dox media in 1:1:2 (w/w/v). Different physicochemical parameters affecting the production of lipase were optimized through evolutionary operation (EVOP) factorial design technique and after optimization yield was enhanced up to 46 U/ml at 30 °C, pH 7.0 with 1:1:2 (w/w/v) grease waste:wheat bran:Czapek-dox media. Industrial grease waste has never been reported before for the production of industrially important lipase enzyme.     

Antimutagenic effects of wheat bran diet through modification of xenobiotic metabolising enzymes

Diets containing wheat bran (WB) protect against cancers of the colon or breast in rats, and may be beneficial in humans. In a previous study of rats treated with the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), inclusion of 10% wheat bran in the diet led to an apparent reduction in IQ metabolites but not of intact IQ in plasma. In the present study, male Wistar rats were fed diets containing 0, 10 or 20% wheat bran, and effects on xenobiotic metabolising enzymes compared. Wheat bran-supplementation showed differential effects on phase I enzymes, significantly increasing the activity of hepatic cytochrome P450 isozyme CYP3A2, but slightly reducing the activity of CYP1A1/2. The activities of both hepatic phase II detoxification enzymes glutathione-S-transferase and glucuronosyl transferase were also reduced. Western blotting revealed similar effects on expression of the proteins. Interestingly, the expression of xenobiotic metabolising enzymes (XME) in the colon appeared to be modulated independently of hepatic XME. Although the wheat bran-supplemented diet still led to an increased expression of CYP3A, it now slightly increased CYP1A in the colon. However, 20% wheat bran significantly increased the expression of both glutathione transferase isozymes, GST A1 & A2, in the colon. Natures Gold (NG) is a commercial wheat bran derivative which is lower than wheat bran in dietary fibre, but enriched in vitamins, minerals and various phytochemicals. Dietary supplementation with 20% Natures Gold led to similar trends as seen in wheat bran-fed rats, but more potent effects in both hepatic and colonic enzymes. The significance of these changes for activation of carcinogens to mutagenic metabolites was investigated using the Salmonella/mammalian microsome mutagenicity test. The activation of IQ and benzo[a]pyrene, but not cyclophosphamide, to a mutagen by hepatic S9 from wheat bran-fed or Natures Gold-fed rats was significantly reduced compared with S9 from animals on a diet lacking wheat bran. We suggest that modulation of xenobiotic metabolising enzymes may be an important component of cancer protection by wheat bran, and this effect may relate to micronutrients or cancer-protective non-nutrient phytochemicals rather more than to dietary fibre.     

Use of wheat bran as a nutritive supplement for the production of ethanol by Zymomonas mobilis

A strain of Zymomonas mobilis (ZYM-TS 1) was isolated from fermenting palm wine (toddy). In addition to glucose, sucrose, and fructose, the organism utilized hydrolysates of corn ( Zea mays ) flour, corn starch and ragi ( Eleusine coracana ) flour. Amounts of ethanol produced in media (adjusted to 10% (w/v) total carbohydrates) fortified with wheat bran extract only, were comparable with those obtained from the defined media containing yeast extract, (NH₄)₂SO₄, KH₂PO₄, and MgSO₄.7H₂O. The results indicate that wheat bran extract can supply all the necessary nutrients required for ethanol production by Zymomonas mobilis .     

海洋真菌Fusariumsp.LD8岩藻多糖酶的液态发酵条件研究

通过对海洋真菌Fusariumsp.LD8产岩藻多糖酶发酵培养基组成及工艺条件的研究,得到较优培养基配方为:麸皮5%,海带粉2%,(NH4)2SO40.8%。以人工海水代替蒸馏水,接种量为3%,在恒温振荡器中以180r/min的速度振荡培养,岩藻多糖酶活可达2.10IU/mL。     

Use of chitin to improve a Beauveria bassiana alginate-pellet formulation

A study was undertaken to evaluate optimum concentrations of chitin in sodium alginate pellet formulations to enhance conidia production. Chitin concentrations tested were 0, 0.5, 1, 2, 3 and 4% (w/v), with (2%, w/v) or without wheat bran. The different chitin-wheat bran pellet combinations were prepared with Beauveria bassiana isolate Qu-B306 at 10⁸ conidia mL^-1. After 21 days of incubation in a humid chamber at 28°C, conidia production was assessed. Improvements up to three times the initial conidia number were achieved using 2% chitin and 2% wheat bran. Higher levels of chitin decreased the number of conidia per pellet. For all chitin concentrations, conidia number increased with the addition of wheat bran (P≤0.05). Contamination by saprophytic fungi was reduced by the incorporation of chitin in the pellet formulation.     

Production of cellulase-free endoxylanase from novel alkalophilic thermotolerent Bacillus pumilus by solid-state fermentation and its application in wastepaper recycling

The present study aimed at optimization of culture condition for the enhanced production of extra cellular thermostable cellulase-free xylanase from Bacillus pumilus by solid-state fermentation. Batch studies were carried out to evaluate various agro-industrial residues such as rice bran, rice husk, rice straw, sawdust, coconut pith, sugarcane bagasse and wheat bran for enzyme production by the bacterial culture. The endoxylanase production was highest on wheat bran media (5582 U/gds), which was enhanced 3.8-fold (21,431 U/gds) by optimization of cultivation conditions. The enzymatic extracts was used in mixed wastepaper recycling, which resulted in a considerable improvement of the paper strength with high drainage and easy drying up. The results of enzyme application with recycled paper clearly indicated that the effective use of enzymes in fiber separation could reduce the cost of carton paper production.     

Optimization of some additives to improve protease production under SSF

In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation.     

Production of haloduracin by <Emphasis Type="Italic">Bacillus halodurans</Emphasis> using solid-state fermentation

Bacillus halodurans was cultivated on wheat bran as a solid-state substrate and produced haloduracin, a bacteriocin, at about 245 AU per wheat bran. Supplementation of the bran with Lauria-Bertani broth decreased haloduracin production. However, production was stimulated by addition of Mg₂SO₄ and K₂HPO₄. The highest production was achieved at a wheat bran/moisture ratio of 1:1.8 and in the presence of 10% (w/w) Na₂CO₃. Under optimum conditions, the organism produced about 3,000 AU per gram dry bran.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides.

Hydrolysis of wheat bran and wheat straw by a 20.7 kDa thermostable endoxylanase released 35 and 18% of the cell-wall xylan content, respectively. Separation of the cinnamoyl-oligosaccharides (accounting for 6%) from the bulk of total oligosaccharides was achieved by specific anion-exchange chromatography. The cinnamoyl-oligosaccharides were further purified by preparative paper chromatography (PPC) and their molecular weight was determined by MALDI-TOF mass spectrometry. The partially purified hydrolysis end-products contained from 4 to 16 and from 4 to 12 pentose residues for wheat bran and straw, respectively, and only one cinnamic acid per molecule. The primary structure of the new feruloyl arabinoxylopentasaccharide from wheat bran hydrolysis, which has been determined using 2D NMR spectroscopy, is O-beta-D-xylopyranosyl-(1-->4)-O-[5-O- (feruloyl)-alpha-L-arabinofuranosyl-(1-->3)]-O-beta-D-xylopyranosy l-(1-->4) -O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

Treatment of germinated wheat to increase levels of GABA and IP6 catalyzed by endogenous enzymes

We found that the levels of bioactive products from wheat can be increased dramatically by manipulating germination conditions and taking advantage of the activity of endogenous enzymes. The yield of phytic acid (IP(6)) from wheat germinated in the presence of high, controlled levels of dissolved oxygen (188 +/- 28 mg/100 g wheat) was almost three times greater than that from wheat germinated with no supplemental oxygen (74 +/- 10 mg/100 g wheat). The yield of gamma-aminobutyric acid (GABA) from wheat germinated in the presence of uncontrolled levels of dissolved oxygen was 18 +/- 3 times greater than that from nonsupplemented wheat (1 mg/100 g wheat). The concentration of GABA was much greater in wheat germ than in whole wheat, and the yield of GABA from wheat germ processed with supplemental water (163 +/- 7 mg/100 g wheat germ) was notably greater than that from wheat germ processed with no supplemental water (100 +/- 2 mg/100 g wheat germ). In contrast, IP(6) was more concentrated in wheat bran, and the yield of IP(6) from wheat bran processed with supplemental water (3100 +/- 12 mg/100 g wheat bran) was notably higher than that from wheat bran processed with no supplemental water (2420 +/- 13 mg/100 g wheat bran). We conclude that the large amount of GABA extracted from wheat germ is likely due to high glutamate decarboxylase activity and low aminotransferase activity and that the large amount of IP(6) extracted from wheat bran is likely due to high levels of tyrosinase activity. Our findings indicate that bioactive molecules such as GABA and IP(6) can be successfully mass-produced by taking advantage of endogenous enzymatic activities.     

In situ starch degradation of different feeds in the rumen

The in situ starch degradation of 5 feeds (barley, maize, pea, oats and wheat bran) has been measured (trial 1), and the influence of particle size on starch degradation investigated with 3 feeds (barley, maize, pea) (trial 2). The starch degradability of barley, oats and wheat bran was found to be higher than that of pea, and higher again than that of maize: 98, 97, 96, 90 and 58% respectively. For barley, oats and wheat bran, starch was degraded more rapidly than the other dry matter (pm) components. Maize and pea starches were degraded at the same rate as non-starchy components. The particle size variations between feeds ground on the same screen may partly explain variations in starch degradability. When the particle size increased from 0.8 to 6.0 mm screen grinding, in situ starch degradability decreased; the decrease was higher for maize (13.8 points) than for barley (7.4 points) or pea (10.4 points).     

Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068.

Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.