Optimization of Solid-state Fermentation Conditions of Sophora japonica cv.jinhuai by Response Surface Methodology

[Objectives]To optimize the solid-state fermentation process of Flos Sophorae Immaturus by Penicillium with Sophora japonica cv.jinhuai as raw material.[Methods]The fermentation conditions were optimized by single factor experiment and response surface methodology with quercetin content as the dependent variable.[Results]According to the established model,the optimal fermentation process of Flos Sophorae Immaturus was obtained as follows:temperature 29.97℃,time 6.88 d,rotation speed 180.86 rpm,inoculation amount 3.93 mL,and the expected content of quercetin was 34.8053 mg/g.Based on this,the fermentation parameters were adjusted,and the actual content was 33.67 mg/g,which was close to the predicted value.[Conclusions]The optimization of fermentation process of Flos Sophorae Immaturus by response surface methodology provides a reference for the development and utilization of this medicinal material.

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.

Spore production in the solid-state fermentation of stevia residue by Trichoderma guizhouense and its effects on corn growth

Trichoderma is an important and widely used plant growth-promoting fungus(PGPF).In this study,stevia residue amended with amino acids hydrolyzed from animal carcasses was used for the production of Trichoderma guizhouense NJAU 4742 by solid-state fermentation,and then its potential to promote corn plant growth was evaluated in combination with chemical fertilizer(CF)or organic fertilizer(OF).The highest spore number of 7×10^(9) CFU g^(–1) fresh weight was obtained under the following optimal parameters:material ratio of 50%(stevia residue:rice bran=1:1),pH value of 3.0(amended with 6.67%amino acids),initial moisture content of 60%,inoculum size of 10%,material thickness of 3 cm and an incubation time of 4 days.The aboveground corn plant biomass obtained with T.guizhouense applied alone and with CF treatments were slightly higher than those of no fertilizer control and CF treatments,respectively.However,T.guizhouense applied with OF significantly(P<0.05)increased aboveground biomass compared to OF and yielded the highest aboveground biomass among all the treatments.Moreover,T.guizhouense applications primarily influenced the fungal bulk soil community composition,among which three OTUs(OTU_(2) and OTU_(9) classified as Chaetomium,and OTU_(4)classified as Trichoderma)were stimulated in both bulk and rhizosphere soil.Notably,a specific OTU_(3)(Phymatotrichopsis)was only stimulated by T.guizhouense applied with OF,possibly leading to high soil productivity.These results show that it is feasible to employ stevia residue in the eco-friendly fermentation of T.guizhouense,which is strongly suggested for enhancing OF applications.

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.

Optimization of the Chitinase Production by Different Metarhizium anisopliae Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD

Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control various insect pests. These fungi attack the integument of the host using an enzymatic complex. Among the enzymes found in this complex, chitinase is an important component. However, the relation between the chitinase production and the virulence from different M. anisopliae strains has not been analyzed. In this manuscript it is presented the chitinase production by four M. anisopliae strains with different potential of virulence in Solid-State Fermentation using silkworm chrysalis as substrate. The higher chitinase level was obtained with the strain IBCB 360 (7.14 U/g of substrate) with potential virulence of 68% on Diatrea saccharalis. The enzyme production was optimized for all strains using a factorial planning (CCRD) considering the cultivation time and medium humidity as independent variables. The maximal production of chitinase was obtained at a range from 8 to 12-days old cultures and from 45% to 62% of moisture according to the surface response plot, with high R2 value. The enzyme production by the strain IBCB 167 was increased two-folds under optimized conditions, while for the strains IBCB 360 and 425 the chitinase production was increased four-folds and nine-folds for the strain IBCB 384.

Solid-state fermentation of corn-soybean meal mixed feed with Bacillus subtilis and Enterococcus faecium for degrading antinutritional factors and enhancing nutritional value

Background: Corn and soybean meal(SBM) are two of the most common feed ingredients used in pig feeds.However, a variety of antinutritional factors(ANFs) present in corn and SBM can interfere with the bioavailability of nutrients and have negative health effects on the pigs. In the present study, two-stage fermentation using Bacillus subtilis followed by Enterococcus faecium was carried out to degrade ANFs and improve the nutritional quality of corn and SBM mixed feed. Furthermore, the microbial composition and in vitro nutrient digestibility of inoculated mixed feed were determined and compared those of the uninoculated controls.Results: During the fermentation process, B. subtilis and lactic acid bacteria(LAB) were the main dominant bacteria in the solid-state fermented inoculated feed, and fermentation produced a large amount of lactic acid(170 mmo L/kg),which resulted in a lower pH(5.0 vs. 6.4) than the fermented uninoculated feed. The amounts of soybean antigenic proteins(β-conglycinin and glycinin) in mixed feed were significantly decreased after first-stage fermentation with B. subtilis. Inoculated mixed feed following two-stage fermentation contained greater concentratioin of crude protein(CP), ash and total phosphorus(P) compared to uninoculated feed, whereas the concentrations of neutral detergent fiber(NDF), hemicellulose and phytate P in fermendted inoculated feed declined(P < 0.05) by 38%, 53%, and 46%,respectively. Notably, the content of trichloroacetic acid soluble protein(TCA-SP), particularly that of small peptides and free amino acids(AA), increased 6.5 fold following two-stage fermentation. There was no difference in the total AA content between fermented inoculated and uninoculated feed. However, aromatic AAs(Phe and Tyr) and Lys in inoculated feed increased, and some polar AAs, including Arg, Asp, and Glu, decreased compared with the uninoculated feed. In vitro dry matter and CP digestibility of inoculated feed improved(P < 0.05) compared with the uninoculated feed.Conclusions: Our results suggest that two-stage fermentation using B. subtilis followed by E. faecium is an effective approach to improve the quality of corn-soybean meal mixed feed.

Antioxidant and Anti-inflammatory Capacity of Ferulic Acid Released from Wheat Bran by Solid-state Fermentation of Aspergillus niger

Objective A strain of Aspergillus niger(A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain(FA-WB) were evaluated. Methods Molecular identification techniques based on PCR analysis of specific genomic sequences were conducted; antioxidant ability was examined using oxygen radical absorbance capacity(ORAC), cellular antioxidant activity(CAA) assays, and erythrocyte hemolysis assays. RAW264.7 cells were used as a model to detect anti-inflammatory activity. Results The filamentous fungal isolate was identified to be A. niger. ORAC and CAA assay showed that FA-WB had better antioxidant activity than that of the ferulic acid standard. The erythrocyte hemolysis assay results suggested that FA-WB could attenuate AAPH-induced oxidative stress through inhibition of reactive oxy gen species(ROS) generation. FA-WB could significantly restore the AAPH-induced increase in intracellular antioxidant enzyme activities to normal levels as well as inhibit the intracellular malondialdehyde formation. TNF-?, IL-6, and NO levels indicated that FA-WB can inhibit the inflammation induced by lipopolysaccharide(LPS). Conclusion Ferulic acid released from wheat bran by a new strain of A. niger had good anti-inflammatory activity and better antioxidant ability than standard ferulic acid.

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.

Direct Solid-State Fermentation of Soybean Processing Residues for the Production of Fungal Chitosan by Mucor rouxii

The feasibility of utilizing soybean-processing residues such as soybean meal and hulls as substrates for chitosan production by the fungus Mucor rouxii ATCC 24905 via solid-state fermentation (SSF) was investigated. The effects of the type of soybean-based substrate, length of cultivation period, substrate moisture content, substrate pH, incubation temperature and extraction conditions on chitosan yield were determined. The results showed that a maximum fungal chitosan yield of up to 3.44% by dry substrate weight (34.4 g/kg) could be achieved using a pure soybean meal substrate with an initial moisture content of 50% (w/w) and pH of 5 - 6 incubated for six days at 25&degC. A more severe heat treatment (autoclaving vs. refluxing) resulted in higher chitosan extraction yields regardless of the strength of extraction reagents. Fourier transform infrared (FTIR) analysis of the fungal chitosan revealed its degree of deacetylation (DDA) to be between 55% and 60%.

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.

Study on Temperature Gradients and Protein Enrichment by <i>Aspergillus oryzae</i>in Solid-State Fermentation on Packed Bed Bioreactor Using Jowar (Sorghum) Straw as Substrate

The packed bed solid state bioreactor designated as PBSSB is constructed in the present study. The experiments are carried out in packed bed bioreactor with jowar straw and inoculated with Aspergillus oryzae. Temperature gradient has been measured at different axial positions. It is found that the organisms grew rapidly during the period from 20 to 30 h during which heat generation is more. These results are in agreement with other researchers. The fermented jowar straw shows threefold increase in protein content. This can be utilized as high value nutritional feed to animals.

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.

Protein Enrichment of Potato Peels Using Saccharomyces cerevisiae via Solid-State Fermentation Process

In order to add value to potato peels and also curb their environmental pollution problems, this study investigated the protein enrichment of potato peels with Saccharomyces cerevisiae via Solid-State Fermentation (SSF). SSF is a fermentation process which involves solid matrix and is carried out in absence or near absence of free water. SSF of potato peel mashed was carried out with S. cerevisiae at 30°C, pH of 5.5, moisture adjustment between 40 and 90%, addition of ammonium sulphate and urea salts as nitrogen supplements for the microorganisms for 3 days. The results showed that the percentage crude protein content of all the fermented samples increased significantly when compared with the unfermented sample. 40% moisture content adjustment and ammonium sulphate as nitrogen source gave the best result. The crude protein increased from 12.5% to 21.86%, which is 74.88% increment for ammonium sulphate supplementation, and 12.5% to 18.42%, which is 47% increment for urea supplementation. Therefore, the fermented peels could serve as good source of cheap protein enriched feed for livestock.

Feasibility Study of Ethanol Production from Food Wastes by Consolidated Continuous Solid-State Fermentation

To save the cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation (CCSSF) system composed of a rotating drum reactor, a humidifier and a condenser has been developed. In this research, the feasibility of using this system for production of ethanol from food wastes was carried out. The ethanol conversion of bread crust and rice grain (uncooked rice) as substrates reached up to 100.9% ± 5.1% and 108.0% ± 7.9% (against theoretical yield), respectively. Even for bread crust, a processed starchy material which contained lower carbohydrate content than rice grain, the amount of ethanol obtained in a unit of CCSSF per year was higher due to easy saccharification and fermentation. The salt contained in potato chips directly affected yeast activity resulting to low ethanol conversion (80.7% ± 4.7% against theoretical yield).

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.

Protein enrichment of cassava pulp by solid-state fermentation using Aspergillus niger

This study aimed to assess protein enrichment of sterile and non-sterile cassava pulp using four strains of Aspergillus niger-AN1,AN2,AN3,AN4.First,studies were conducted to evaluate nutritional requirements of A.niger strains and their suitability for protein enrichment of cassava pulp.Second,sterile and non-sterile cassava pulps were inoculated with spores of A.niger strains and incubated under conditions of solid-state fermentation for 8 days using standard methods.Protein contents of sterile and non-sterile cassava pulps were determined by the Kjeldahl method.Initial nutritional requirements studies showed that A.niger strains grew at rates between 2.0 cm and 8.5 cm over 10 days on potato dextrose agar medium of different pH.The dry weight of mycelia of A.niger strains varied in liquid media separately supplemented with different concentrations of thiamine,ammonium nitrate and sodium chloride.A.niger strains also showed different levels of sporulation when cultured in growth media supplemented with different concentrations of sodium chloride.Increases in protein contents of sterile cassava pulp by AN1,AN2,AN3 and AN4 were 15.65%,22.61%,18.30%and 19.13%,respectively.On non-sterile cassava pulp,the increases in protein content by AN1,AN2,AN3 and AN4 were 15.40%,21.54%,10.80%and 3.85%,respectively.A.niger strain AN2 was the most suitable strain for the enrichment of protein content of cassava because it sparsely produced spores,it had the highest mycelial growth and resulted in the greatest increase in protein content of both sterile and non-sterile cassava pulp.

Biotechnological potential of agro-industrial wastes for protein enrichment by solid-state fermentation using Aspergillus niger

This study is to assess the biotechnological potential of agro-industrial wastes of pineapple(Ananas comosus),sweet potato(Ipomoea batatas)and watermelon(Citrullus lanatus)for protein enrichment by solid-state fermentation using Aspergillus niger.Spore suspensions of A.niger were prepared with potato dextrose broth that had been supplemented with sodium chloride,ammonium nitrate and thiamine,and adjusted to pH of 5.Initial protein contents of sterile and non-sterile wastes of the pineapple,sweet potato,and watermelon were determined by the Kjeldahl method.The remaining sterile and non-sterile wastes was inoculated with spore suspensions of A.niger and incubated at temperature of 24℃ under conditions of solid-state fermentation for 14 days.Protein contents of the inoculated sterile and non-sterile wastes were determined after 7 and 14 days.The sterile wastes of pineapple,sweet potato and watermelon recorded initial percentage protein contents of 4.37,4.39,and 10.89,respectively,whereas their corresponding non-sterile wastes recorded initial percentage protein contents of 3.76,4.00,and 10.16,respectively.The results further show that percentage increase in protein content of sterile wastes after 14 days of fermentation were pineapple,35.01%;sweet potato,27.60%;and watermelon,64.40%.Percentage increase in protein content of non-sterile wastes after 14 days of fermentation were pineapple,72.34%;sweet potato,85.25%;and watermelon,80.51%.These findings affirm the biotechnological potential of pineapple,sweet potato and watermelon wastes and the importance of fungi as agents for protein enrichment of agro-industrial wastes.