Solid state fermentation of rapeseed cake with Aspergillus niger for degrading glucosinolates and upgrading nutritional value

Background： Rapeseed cake is a good source of protein for animal feed but its utilization is limited due to the presence of anti-nutritional substances, such as glucosinolates （GIs）, phytic acid, tannins etc. In the present study, a solid state fermentation （SSF） using Aspergillus niger was carried out with the purpose of degrading glucosinolates and improving the nutritional quality of rapeseed cake （RSC）. The effects of medium composition and incubation conditions on the GIs content in fermented rapeseed cake （FRSC） were investigated, and chemical composition and amino acid in vitro digestibility of RSC substrate fermented under optimal conditions were determined. Results： After 72 h of incubation at 34℃, a 76.89% decrease in GIs of RSC was obtained in solid medium containing 70% RSC, 30% wheat bran at optimal moisture content 60% （w/w）. Compared to unfermented RSC, trichloroacetic acid soluble protein （TCA-SP）, crude protein and ether extract contents of the FRSC were increased （P〈 0.05） 103.71, 23.02 and 23.54%, respectively. As expected, the contents of NDF and phytic acid declined （P〈 0.05） by 9.12 and 44.60%, respectively. Total amino acids （TAA） and essential amino acids （EAA） contents as well as AA in vitro digestibility of FRSC were improved significantly （P 〈 0.05）. Moreover, the enzyme activity of endoglucanase, xylanase, acid protease and phytase were increased （P 〈 0.05） during SSF. Conclusions： Our results indicate that the solid state fermentation offers an effective approach to improving the quality of proteins sources such as rapeseed cake.

Utilization of winery wastes for Trichoderma viride biocontrol agent production by solid state fermentation

Biocontrol agents are safe and environmental friendly alternatives for pesticides in agriculture application. Trichoderma viride WEBL0703 performed a high level of antagonistic activity toward a broad spectrum of phytopathogens and was determined as a biocontrol agent, which was produced by solid state fermentation using grape marc and wine lees. The maximum yield of T. viride conidia was up to 6.65 × 10^9 CFU/g initial dry substrate （IDS） after 10 d fermentation. As important enzymes for protecting plants from disease, chitinase, β-glucanase, and pectinase yields were 47.8 U/g IDS, 8.32 U/g IDS and 9.83 U/g IDS, respectively. These results show that it is feasible to convert winery wastes to a value-added and environmental friendly biocontrol agent.

Paddy Husk as Support for Solid State Fermentation to Produce Xylanase from Bacillus pumilus

To optimize culture conditions for xylanase production by solid state fermentation （SSF） using Bacillus pumilus, with paddy husk as support, solid medium contained 200 g of paddy husk with 800 mL of liquid fermentation medium [xylan, 20.0 g/L; peptone, 2.0 g/L; yeast extract, 2.5 g/L; K2HPO4, 2.5 g/L; KH2PO4, 1.0 g/L; NaCl, 0.1 g/L; （NH4）2SO4, 2.0 g/L, CaCl2-2H2O, 0.005 g/L; MgCl2.6H2O, 0.005 g/L; and FeCI3, 0.005 g/L] at pH 9.0 was applied. The highest xylanase activity （142.0 ±0.47 U/g DM] was obtained on the 6th day at 30℃ The optimized paddy husk to liquid fermentation medium ratio was 2：9, and the optimized culture temperature was 40℃. When commercial Birchwood xylan was replaced with different concentrations of corncob, xylanase production was maximized （224.2 U/g DM） in the medium with 150 g/L corncob. Xylanase production was increased by sucrose, fructose and arabinose, whereas reduced by glucose, galactose, lactose and amylose. When organic nitrogen sources were replaced with locally available nitrogen sources such as groundnut powder or sesame seedcake powder or coconut seedcake powder or soy meal powder, the highest xylanase production （290.7 U/g DM） was obtained in the medium with soy meal powder and 16.0 g/L of soy meal powder was the optimum （326.5±0.34 U/g DM）. Based on the optimization studies, B. pumilus produced 2.3 times higher xylanase activity. The medium cost was reduced from 2 458.3 to 178.3 SLR/kg and the total activity which could be obtained from 1 kg of the medium was increased from 48 624 to 220 253 Units.

Cellulase production by Aspergillus unguis in solid state fermentation

Lignocellulosic substrates are a good carbon source and provide rich growth media for a variety of microorganisms which prodLuce industrially important enzymes. Cellulases are a group of hydrolytic enzymes such as filter paperase (FPase), carboxymethyl cellulase(CMCase) andβ-glucosidase-responsible for release of sugars in the bioconversion of the lignocellulosic biomass into a variety of value-added products. This study examined cellulase production by a newly isolated Aspergillus unguis on individual lignocellulosic substrates in solid state fermentation (SSF). The maximum peak production of enzymes varied from one substrate to another, however,based on the next best solid support and local availability of groundnut fodder supported maximum enzyme yields compared with other solid supports used in this study.Groundnut fodder supported significant production of FPase (5.9 FPU/g of substrate), CMCase (1.1 U/g of substrate) andβ-glucosidase activity (6.5 U/g of substrate) in SSF. Considerable secretion of protein (27.0 mg/g of substrate) on groundnut fodder was recorded. Constant increment of protein content in groundnut fodder due to cultivation of A. unguis is an interesting observation and it has implications for the improvement of nutritive value of groundnut fodder for cattle.

Optimization of Solid-State Fermentation with Lactobacillus brevis and Aspergillus oryzae for Trypsin Inhibitor Degradation in Soybean Meal

The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology （RSM） with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were： pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were： substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically （57.1 and 89.2% respectively）. L. brevis fermentation did not affect phytic acid （0.4%） and crude fat （5.2%） considerably, whereas A. oryzae fermentation degraded phytic acid （34.8%） and crude fat （22.0%） contents to a certain extent. Crude protein content was increased after both fermentation （6.4 and 12.9% for L. brevis and A. oryzae respectively）. Urease activity was reduced greatly （83.3 and 58.3% for L. brevis and A. oryzae respectively）. In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.

Production of iturin A through glass column reactor (GCR) from soybean curd residue (okara) by <i>Bacillus subtilis</i>RB14-CS under solid state fermentation (SSF)

The present study was conducted with an aim to scale up the production of iturin A using soybean curd residue (okara). Iturin A was produced by indigenous bacterial strain Bacillus subtilis RB14-CS through glass column reactor (GCR) under solid state fermentation (SSF) was characterized. The enhanced iturin A production was observed with respect to enhanced substrate bed height when SSF was conducted in Erlenmeyer flask. To check the effect of substrate bed height on iturin A production under SSF of okara, GCR was introduced. Substrate bed height of 15 cm was suitable for iturin A production which was about 2700 mg/kg wet substrate. The observed iturin A production by the aerobic bacteria Bacillus subtilis in nearly anaerobic condition in such high substrate bed for SSF is a wonderful finding for development of SSF system in future.

Lovastatin production by Aspergillus terreus in solid-state fermentation

Lovastatin production by Aspergillus terreus ATCC 20542 in solid-state fermentation (SSF) was studied. Various substrates were used to evaluate the ability of A. terreus to produce lovastatin. The results showed that either rice or wheat bran was suitable substrate for lovastatin production in SSF. The maximum yield of lovastatin (2.9 mg/g dry substrate) using rice as substrate was achieved after incubating for 11 d at the following optimized process parameters: 50%～60% initial moisture content,pH 5.5,incubation temperature 28 °C.

Effect of cultivating conditions on α-galactosidase production by a novel Aspergillus foetidus ZU-G1 strain in solid-state fermentation

The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 °C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0～6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2 037.51 U/g dry matter near the 144th hour of fermentation.

Optimization of Solid State Fermentation Conditions Using a Mixture of Bean Curd Residue and Marc with Bacillus natto

[Objective] The aim was to optimize the appropriate solid state fermentation(SSF)conditions.[Method] The optimization of solid state fermentation using a mixture substrate of bean curd residue and the marc with Bacillus natto was developed.[Result] The best fermentation condition optimized by the test of single factor and the orthogonal design respectively was mixing ratio of bean curd residue to marc 2∶1,substrate pH value 6,fermentation temperature 39 ℃,inoculum volume 10% and fermentation time 48 h.Under this optimized fermentation condition,the content of crude fiber in the substrate decreased from 107.8 mg/g before SSF to 56.2 mg/g after SSF,and the degeneration rate of crude fiber was 47.87%.[Conclusion] The bean curd residue in its palatability was enormously improved by SSF with Bacillus natto strain,which could be expected to be widely used as raw material of health foodstuff.

The variation of two extracellular enzymes and soybean meal bitterness during solid-state fermentation of Bacillus subtilis

The debittering effect of extracellular enzymes from Bacillus subtilis ACCC 01746 was studied using soybean meal as a substrate for solid-state fermentation(SSF).Results showed that B.subtilis produces proteases and carboxypeptidase in the early stage of SSF(0–8 h).Proteases are dominant and can hydrolyze the soybean protein into long-chain peptides with mild bitterness.Carboxypeptidase production is dominant at 8–16 h SSF,at which point soybean protein is further hydrolyzed and bitterness is enhanced.The strain then produces additional carboxypeptidase after 16 h,and bitterness is reduced.We compared the amino acid composition of the hydrolysates from soybean protein isolates to that of the fermented liquid of SSF.In the hydrolysates from soybean protein isolates that exhibit strong bitterness,62.81%of amino acids are hydrophobic and occur in the form of peptides.In the fermented liquid from soybean meal,16.22%of amino acids are hydrophobic and are mainly present in the form of free amino acids.The bitterness of fermented soybean hydrolysate is reduced from 5 to 0 when fermented for 24 h,suggesting that B.subtilis can effectively reduce bitterness,possibly due to the carboxypeptidase.Enzyme analysis shows that B.subtilis excretes carboxypeptidase during growth.The amino acids phenylalanine,alanine,tyrosine,and leucine at the C-terminal of the soybean bitter peptides in hydrolysates are cleaved in the presence of carboxypeptidase,resulting in complete debitterness.

Optimization of Parameters for the Production of Lipase from Pseudomonas sp. BUP6 by Solid State Fermentation

Solid-state fermentation (SSF) holds tremendous potentials for the production of industrially significant enzymes. The present study describes the production of lipase by a novel rumen bacterium, Pseudomonas sp. strain BUP6 on agro-industrial residues. Pseudomonas sp. strain BUP6 showed higher lipase production when grown in Basal salt medium (BSM) supplemented with oil cakes. Initially, five different oil cakes (obtained after extracting oil from coconut, groundnut, cotton seed, gingelly or soybean) were screened to find out the most suitable substrate-cum-inducer for the production of lipase. Among them, groundnut cake supported the maximum production of lipase (107.44 U/gds). Box-Behnken Design (BBD), followed by response surface methodology (RSM) was employed to optimize the culture parameters for maximizing the production of lipase. Using the software Minitab 14, four different parameters like temperature, pH, moisture content and incubation time were selected for the statistical optimization, which resulted in 0.7 fold increase (i.e., 180.75 U/gds) in production of lipase under the optimum culture conditions (temperature 28&#176C, pH 5.9, moisture 33% and incubation 2 d). Thus, this study signifies the importance of SSF for the production of industrially-significant lipase using agro-industrial residues as solid support.

Solid-State Fermentation Production of Chitosanase by <i>Streptomyces</i>with Waste Mycelia of <i>Aspergillus niger</i>

Solid-state fermentation was carried out using mycelium powder of Aspergillus niger as substrate for the production of chitosanase of Streptomyces. Results of the experiments indicated that the optimal medium consisted of wheat bran and mycelium powder of Aspergillus niger with initial moisture content of 60% - 70%. The enzyme activity reached 41.33 U per gram dry medium after cultured for 5 days at 28°C - 30°C and an initial pH 6.5. Chitosanase was detected on the second day of incubation and had maximal activity at 5 days and decreased gradually within a 1 month period. Solid-state fermentation is maybe an economic alternative in the production.

Production of Xylanase and <i>β</i>-Xylosidase Enzymes by <i>Pseudozyma hubeiensis</i>in Solid State Fermentation

Xylanase is an important enzyme with potential application in the degradation of xylan component in the lignocellulosic biomass. There are very few reports on the production of cellulase free xylanases especially by yeast strains which have great potential in paper and pulp industry in removing the hemicellulose from the treated or untreated pulp. In this study, P. hubeiensis NCIM 3574 isolated in our laboratory produced significant levels of extracellular cellulase free xylanase (2480 IU/g DSS) in solid state fermentation (SSF) using wheat bran and xylan. It also produced high levels of β-xylosidase (198 IU/g DSS) when grown in SSF using ground nut oil cake and xylan. These highest activities were obtained when fermented Koji was extracted with 1% NaCl supplemented with 0.5% of Triton X-100. These are the highest activities reported so far from yeast strains in the available literature. The crude xylanase preparation of P. hubeiensis produced xylooligosaccharides (XOS) without xylose proving its potential for XOS production with no further requirement of downstream processing. The XOS as prebiotic show beneficial effect on gut microflora such as Lactobacilli and Bifidobacteria which suppress the activity of pathogenic organisms. This xylanase also has a potential application as a bio-bleaching agent in paper and pulp industry.

Pool of Biological Resources for Potential Applications in Solid State Fermentation Obtained from a Forest Plantation of Pinus pseudostrobus Lindl, Mexico

A forest plantation, product of the reforestation of pine trees, represented a pool of biological resources for the implementation of a solid state fermentation process. The trees were identified as Pinus pseudostrobus Lindl from which lignocellulosic material in the form of pine needles was collected. Soil fungi, responsible for plant litter decomposition, were cultured at laboratory conditions and tested for their ability to grow on cellulose and hemicellulose as the sole carbon sources. A fungal strain, belonging to the genus Penicillium, was selected for growing it on pine needles as the substrate in a solid state culture. After following the culture for six days, the newly isolated strain exhibited a much higher capacity for spore production and holocellulose degradation, compared to a purchased strain of Penicillium chrysogenum and two control conditions. This work marks the beginning of future studies focused on commercial applications and represents the first report of a biotechnological process based on pine needles and their degradation by an ascomycetes species belonging to the genus Penicillium.

Novel Protease from Aspergillus tamarii URM4634: Production and Characterization Using Inexpensive Agroindustrial Substrates by Solid-State Fermentation

This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited KM = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40°C. The proteolytic activity was inhibited by Cu2+ (33.98%) and Hg2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.

Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from <i>Bacillus thuringiensis</i>subsp. <i>kurstaki</i>

Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt);and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h;while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores;malachite green-saffranin stained both δ-crystals and endospores;and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.