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.

The Advantages of Methane Production by Combined Fermentation of Lignite and Wheat Straw

Biogasification of coal is important for clean utilization of coal. Experiments on the fermentation of single lignite, single straw and their mixture were performed to explore the variation characteristics of gas production potential, microbial community and methanogenic metabolic pathways of mixture. Research has shown that mixed fermentation of lignite and straw significantly promoted biomethane production. The abundance of hydrolytic acidifying functional bacteria genera (Sphaerochaeta, Lentimicrobium) in mixed fermentation was higher than that in the fermentation of single lignite and single straw. The abundance of some key CAZy metabolic enzyme gene sequences in mixed fermentation group was increased, which was favorable to improve methane production. Aceticlastic methanogenesis was the most critical methanogenic pathway and acetic acid pathway was more competitive in methanogenic mode during peak fermentation. Macrogenomics provided theoretical support for the claim that mixed fermentation of coal and straw promoted biomethane metabolism, which was potentially valuable in expanding methanogenesis from mixed fermentation of lignite with different biomasses.

Effects of molasses on the fermentation characteristics of mixed silage prepared with rice straw, local vegetable by-products and alfalfa in Southeast China

This experiment was conducted to study the effect of molasses on the fermentation characteristics of mixed silage ensiled rice straw and vegetable by-products with alfalfa.Mixture(202 g kg^(–1) dry matter(DM))consisting of rice straw,broccoli residue and alfalfa at the ratio of 5:4:1 was ensiled with three experimental treatments:(1)no additives(control);(2)molasses at 2.5%(M1);(3)molasses at 5%(M2)on a fresh matter basis of mixture,respectively.All treatments were packed into laboratory-scale silos,and three silos per treatment were sampled on days 1,3,5,14 and 30.The result showed that the p H value of all mixed silages decreased gradually with the time of ensiling except for the control silage,in which a significant increase(P<0.05)on day 30 occurred.The lactic acid content increased gradually with the time of ensiling and reached the highest value on day 14,and a marked decrease(P<0.05)was found in the control silage on day 30.The changes of acetic acid content showed similar pattern with lactic acid content.A trace amount of propionic and butyric acid contents were found in the three mixed silages during the fermentation period.Comparing to the control,M1 and M2 treatments improved the fermentation quality of mixed silages as indicated by higher(P<0.05)lactic acid contents and lower(P<0.05)p H and ammonia-N contents.The Flieg points also showed that M1 and M2 silages were well preserved,whereas the control silage had a bad quality.Overall,the findings of this study suggested that adding molasses could improve fermentation quality of mixed silage,and M1 was more suitable for practical application.

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.

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.

Production of Protein Feedstuff by Mixed Fermentation of Waste Lees

[Objective] To produce protein feedstuff by mix fermentation of waste lees.[Method] Waste lees was fermented by mixture of Aspergillus niger,Trichoderma viride and Candida tropicalis to produce protein feedstuff.The fermentation conditions were optimized by single factor test and orthogonal test.[Result] The optimum fermentation conditions of waste lees are as following:raw materials with fineness of 20 meshes composed of waste lees of 750 g/kg,corn meal of 50 g/kg and wheat bran of 200 g/kg;pH value of inoculum culture,5.0;Aspergillus niger:Trichoderma viride:Candida tropicalis,1:1:1;inoculum size,12%(V/V);fermentation temperature,30 °C;and fermentation time,6 d.After fermentation,the crude protein content,pure protein content,and crude fiber content were increased by 78.97%,56.29% and 31.60%,respectively.In addition,the fermented waste lees was rich in beneficial live microorganisms and bioactive substances such as enzymes and vitamins.[Conclusion] Waste lees can be well fermented to produce protein feedstuff by mix fermentation.

ETHANOL FERMENTATION OF MIXED GLUCOSE AND XYLOSE BY PICHIA STIPITIS

Pichia stipitis CBS 5773 yeast cells were used to ferment the mixed substrates consisted of glucose andxylose to produce ethanol.The effects of aeration rate,initial substrate concentration and pH on substrateutilization and ethanol yield were evaluated.During batch fermentation,the oscillation phenomena in cell growthwere observed at low aeration rate,whereas the diauxic growth at high aeration rate.The substrate utilizationratio and ethanol yield reached 95％ and 0.46g/g respectively under appropriate operation conditions.Amodified unstructural model was proposed to simulate the diauxic cell growth,substrate consumption andproduct formation.

Study on the Saccharification Process of Citrus Vinegar by Fruit-Grain Mixed Fermentation

[Objective] This study aimed to enhance acid production rate and yield of citrus vinegar,and improve the quality of liquid fermented with fruit juice.[Method]The saccharification process of citrus vinegar was investigated preliminarily with fruit-grain mixed fermentation method using liquor starters as sacchariferous agents. [Result] The results indicated that the optimal glutinous rice saccharification conditions were as follows: fruit-grain ratio 3 ∶ 1,Aspergillus nigerMonascus purpureus ratio 3∶ 1,saccharification temperature 60 ℃,and saccharification time 2. 5 h. Under the optimized saccharification conditions,reducing sugar content in saccharified glutinous rice reached 146. 43 mg / ml; after fermentation,alcohol content in fermentation broth was 6%,amino nitrogen content was 0. 44 mg / ml,and acid yield was 3. 1 g / L. [Conclusion] Citrus vinegar brewed based on the optimized technological conditions exhibited better sensory and physicochemical properties than that brewed with pure juice.

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.

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.

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.

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.

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.

Effect of lactic acid bacteria and propionic acid on conservation characteristics,aerobic stability and in vitro gas production kinetics and digestibility of whole-crop corn based total mixed ration silage

This study was conducted to evaluate the effect of lactic acid bacteria and propionic acid on the fermentation quality, aerobic stability and in vitro gas production kinetics and digestibility of whole-crop corn based total mixed ration(TMR) silage. Total mixed ration was ensiled with four treatments:(1) no additives(control);(2) an inoculant(Lactobacillus plantarum)(L);(3) propionic acid(P);(4) propionic acid+lactic acid bacteria(PL). All treatments were ensiled in laboratory-scale silos for 45 days, and then subjected to an aerobic stability test for 12 days. Further, four TMR silages were incubated in vitro with buffered rumen fluid to study in vitro gas production kinetics and digestibility. The results indicated that all TMR silages had good fermentation characteristics with low pH(<3.80) and ammonia nitrogen(NH_3-N) contents, and high lactic acid contents as well as Flieg points. Addition of L further improved TMR silage quality with more lactic acid production. Addition of P and PL decreased lactic acid and NH_3-N contents of TMR silage compared to the control(P<0.05). After 12 days aerobic exposure, P and PL silages remained stable, but L and the control silages deteriorated as indicated by a reduction in lactic acid and an increase in pH, and numbers of yeast. Compared to the control, addition of L had no effects on TMR silage in terms of 72 h cumulative gas production, in vitro dry matter digestibility, metabolizable energy, net energy for lactation and short chain fatty acids, whereas addition of PL significantly(P<0.05) increased them. L silage had higher(P<0.05) in vitro neutral detergent fiber digestibility than the control silage. The results of our study suggested that TMR silage prepared with whole-crop corn can be well preserved with or without additives. Furthermore, the findings of this study suggested that propionic acid is compatible with lactic acid bacteria inoculants, and when used together, although they reduced lactic acid production of TMR silage, they improved aerobic stability and in vitro nutrients digestibility of TMR silage.

Research on Fermentation Technology of Raspberry-mulberry Leaven

The mixed fermentation is based on the raw material of raspberry and mulberry. The research is to explore the sticking point for the fermentation progress. To attain the optimized fermentation condition which gained by integrating fermentation data,the concentration of pectinase should be 80 mg/L,the mass fraction of Lactobacillus,Acetobacter and yeast should be 3%,1% and 3%,respectively,the fermentation temperature should be 28 ℃,the initial sugar content should be 24°,and the duration of fermentation should be 16 d. After the fermentation,sugar content should be adjusted to 25°,and the products should be stored for 3 d in the condition of 15 ℃. The Raspberry-mulberry leaven containing 8. 5 × 10~8 probiotics per milliliter and 61. 2 g total acid per liter should be diluted with various proportions of water to drink according to different habits of customers[1]. The volume ratio of leaven and water should be at least 1∶ 1 and at most 1∶ 3.

Continuous Ethanol Production Through Fermentation with Glucose and Xylose by Immobilized Yeasts in Two Cascade Fixed Beds

Glucose and xylose were used as mixed carbon sources for ethanol production by a cascade immobi-lized-cell reactor.In the first fixed-bed reactor,immobilized S.cerevisiae was used to metabolize the glucoseanaerobically,and in the second fixed-bed column,the immobilized Pichia stipitis was applied to metabolize xy-lose aerobically.The effect of xylose on the glucose uptake by S.cerevisiae and the effect of ethanol on the xylosemetabolization by P.stipitis were studied.A mathematical model taking both tbe substrate and xylose inhibitioninto consideration was suggested for the description of the two-substrate fermentation process,and a total effec-tiveness factor η_s in the model was derived to take into account the mass transfer resistances of intra-particlesand liquid films.Calculated results are in fair agreement with the experimental data.

Effects of Facultative Anaerobic Cellulolytic Bacteria and Nitrogen-fixing Bacteria Isolated from Cow Rumen Fluid on Rumen Fermentation and Dry Matter Degradation in Vitro

The purpose of the paper was to study the effects of cellulolytic bacteria(CB)mixed with nitrogen-fixing bacteria(NFB)on fermentation in vitro.Nine CB strains and seven NFB strains were isolated from numen fluid of three Holstein cows.Based onhigher activity of cellulose or nitrogenase,three CB types[CB-2(KT725624),CB-5(KT725623)and CB-6(KT725622)]and one NFB type[NFB-3(KT735054)]were screened out,respectively.Two parts were included in this study.The first part explored the optimal mixed ratio of CB to NFB through inoculating filter paper medium with the bacteria of 2×10^5 cfu·mL^-1.According to CMCase and FPase activities in the medium,the ratio of 4 to 1 was proven to be more effective.In the second part,rumen fermentation in vitro was conducted at 4:1 of CB to NFB,aiming at studying the effects of mixed bacteria.Six groups were classified,namely,control group(no bacteria),Group 1(CB-2+NFB-3),Group 2(CB-5+NFB-3),Group 3(CB-6+NFB-3),Group 4(NFB-3)and Group 5(CB-6).All the experimental groups had the same amount of bacteria(4×10^6 cfu·mL^-1)in the fermentation liquid.Samples were collected at 2,4,8,12 and 24 h of incubation.Compared with the groups with CB or NFB alone,gas production,dry matter degradability and bacterial protein expressions in the mixed groups increased.However,NH3-N concentration decreased and p H was stable.Meanwhile,related values among three mixed groups were significantly different;values in Group 2 were the best.

Cyanides Reduction and Pasting Properties of Cassava (Manihot Esculenta Crantz) Flour as Affected by Fermentation Process

BACKGROUND: The effect of fermentation process on total cyanides content reduction and pasting properties of cassava flour was evaluated. The fermentation process studied was spontaneous fermentation (SF) and the fermentation with the addition of mixed-starter at the 0 hour of fermentation (FAS). Unfermented cassava flour (UCF) was also produced to serve as a control. Total cyanides and pasting properties of flours from the fermented cassava were compared with unfermented flour. RESULTS: The results showed that the addition of mixed-starter effected a significant reduction in total cyanides level (79.6% to 99%) depending on the fermentation period. Pasting properties of cassava flour were also affected by fermentation process. SF flour paste (stability ratio = 0.6) was more resistant to shear thinning than FAS and UCF (stability ratio = 0.5) indicating that, SF flour has the ability to withstand severe processing conditions better than FAS and UCF pastes. Fermentation up to 48 h had resulted in cassava flour SF having a higher peak viscosity value (6615 cP), but did not affect the tendency to retrograde. CONCLUSION: The use of mixed-starter in fermentation greatly reduced cyanide intake of the population in Africa and has potential to eliminate Konzo without significant modifications of flour properties.