Microbiomics and metabolomics insights into the microbial regulation on the formation of flavor components in the traditional fermentation process of Chinese Hongqu aged vinegar

This study aimed to investigate microbial succession and metabolic dynamics during the traditional fermentation of Hongqu aged vinegar,and explore the core functional microbes closely related to the formation of flavor components.Microbiome analysis demonstrated that Lactobacillus,Acetobacter,Bacillus,Enterobacter,Lactococcus,Leuconostoc and Weissella were the predominant bacterial genera,while Aspergillus piperis,Aspergillus oryzae,Monascus purpureus,Candida athensensis,C.xylopsoci,Penicillium ochrosalmoneum and Simplicillium aogashimaense were the predominant fungal species.Correlation analysis revealed that Acetobacter was positively correlated with the production of tetramethylpyrazine,acetoin and acetic acid,Lactococcus showed positive correlation with the production of 2-nonanone,2-heptanone,ethyl caprylate,ethyl caprate,1-hexanol,1-octanol and 1-octen-3-ol,C.xylopsoci and C.rugosa were positively associated with the production of diethyl malonate,2,3-butanediyl diacetate,acetoin,benzaldehyde and tetramethylpyrazine.Correspondingly,non-volatile metabolites were also detected through ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.A variety of amino acids and functional dipeptides were identified during the traditional brewing of Hongqu aged vinegar.Correlation analysis revealed that Lactobacillus was significantly associated with DL-lactate,indolelactic acid,D-(+)-3-phenyllactic acid,pimelic acid,pregabalin and 3-aminobutanoic acid.This study is useful for understanding flavor formation mechanism and developing effective strategies for the suitable strains selection to improve the flavor quality of Hongqu aged vinegar.

Condition Optimization of Lipopeptide Production by Bacillus natto NT-6 in Solid-State Fermentation with Box-Behnken Design

Solid-state fermentation has certain advantages in improving the yield of lipopetide, Box-Behnken Design（BBD） was adopted to optimize the producing condition of the antibacterial lipopetide produced by Bacillus natto in this article. The optimal solid state fermentation conditions were obtained： 10 g solid medium（7 g of wheat bran, 3 g of soybean meal） with appropriate inorganic salt（glucose 0.67%,sodium glutamate 0.64%,（NH4）2SO40.15%, K2HPO40.10%）; moisture content 123.78%; inoculation amount 10%; cultivation temperature 36.75 ℃ and cultivation time 72.4 h. The maximum production of lipopetide is 61.76 mg/gds under such conditions. This is the first report on the optimization of lipopeptide fermentation conditions in solid-state fermentation by wheat bran and soybean meal with Bacillus natto NT-6 strain, and will contribute to the development of lipopetide production.

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.

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 ofA. 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 ℃.

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 ,Aspergillusfoetidus ZU-GI 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 ℃ 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 2037.51 U/g dry matter near the 144th hour of fermentation.

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 Lipase Using Cassava Peel and Sunflower Oil in Solid-State Fermentation： Preliminary Study

Full use of residues from industrial processes is a fundamental necessity of contemporary society, since it avoids impacts to the environment by using residues as inputs for other products of high economic and social importance. In this study, lipase production of the crude enzymatic extracts obtained by Aspergillus niger using cassava peel as substrate and sunflower oil as an inductor was investigated. The optimized cultivation temperature and concentration of inductor were determined using the response surface methodology. The two variables studied exercised influence in the production of lipase in the 95% level of confidence. The response surface obtained indicated that the conditions that maximize lipase activity production were 30.5 ~C and initial concentration of sunflower oil was 2.5% （w/w）. Through this analysis, it is evident that extremes in temperature and concentration of inductor tend to decrease lipase production, since low temperatures decrease metabolism and high temperatures may inactivate the lipase. Optimum lipase yield was 59.8 U/g of dry peel which was fermented for 60 h. Lipase production presents a peak of 61.3 U/g, at 72 h of fermentation. However, this value is statistically equal （p 〉 0.05） of the value of lipase activity obtained for 60 h and 84 h of fermentation.

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.

Selection of Relevant Variables to the Enzyme Production on Red Grape Pomace by Solid-State Fermentation

With the aim of to valorise red grape pomace and to reduce its environmental impact, the production of enzymatic preparations appear as an interesting choice. Statistical experimental Plackett-Burman designs were applied for the selection of relevant medium components and culture conditions for cellulase, xylanase, polygalacturonase and tannase production by Aspergillus awamori, in solid-state fermentation on red grape pomace. Ten variables were tested： initial moisture content （IMC）, particle size （PS）, temperature, initial pH, time of cultivation, mixing （Mx）, and additions of： fructose, tannic acid, sodium phosphate, and ammonium sulphate （ASA）. Results indicate that the production of each enzyme was affected in a distinct way by the different variables. Also, for each of the enzyme activities considered, IMC must be carefully controlled, and optimized above 65%; PS and Mx, must not be taken into account and ASA must be discarded. The other variables studied, must be selected according to the enzyme activity that will be favored.

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.

Effects of solid-state fermentation product of yeast supplementation on liver and intestinal health,and resistance of common carp(Cyprinus carpio)against spring viraemia carp virus

This study aimed to investigate the effects of solid-state fermentation products of yeast(SFPY)on liver and intestinal health and disease resistance of common carp(Cyprinus carpio).A total of 200 common carp with an initial average weight of 2.55±0.004 g were divided into 5 groups(4 replications per group and 10 fish per replication),and were fed with one of five diets,including a control diet and 4 diets supplemented with 2‰(Y2),3‰(Y3),4‰(Y4),or 5‰(Y5)SFPY,respectively,for 8 weeks.Results indicated that,the addition of SFPY to the diet of common carp did not affect the growth performance or survival rate of fish(P=0.253).Interestingly,with the addition of SFPY,the triacylglycerol(TAG)content of the liver presented a linear decreasing tendency(P=0.004),with significantly decreased in Y4 and Y5 groups(P=0.035)compared with control.Serum lipopolysaccharide(LPS)content and diamine oxidase(DAO)activity presented a negative linear relationship with the addition of SFPY(P=0.015,P=0.030),while serum lipopolysaccharide binding protein(LBP)content first decreased and then increased(P<0.001).The total antioxidant capacity(T-AOC)in the intestine of fish increased continuously with increasing SFPY supplementation(P=0.026),reaching the highest level in Y5 group.The villus height in all experimental groups were significantly higher than that in the control group(P<0.001).Furthermore,compared to the control,adding 3‰SFPY to the control diet of common carp significantly increased the relative abundance of Fusobacteria(P=0.018)and decreased that of Proteobacteria(P=0.039)at phylum level,and increased the relative abundance of Cetobacterium(P=0.018)and decreased that of Shewanella(P=0.013)at genus level.Compared with the control,the relative mRNA expression level of spring viraemia of carp virus N protein(SVCV-n)in the kidney was lower than that of the control group without significance and bottomed out in Y4 group(P=0.138).In conclusion,dietary SFPY enhanced the SVCV resistance capacity of common carp by improving liver and intestinal health and modulating the gut microbiota.Thus,SFPY is a potential feed additive to be used in aquaculture to reduce the huge economic loss of common carp due to SVCV disease.Based on liver TAG content and intestinal villus height,the optimal addition level of SFPY was 3.02‰and 2.72‰,respectively.

Fermentation characteristics of vinegar residue and some natural materials

Solid state fermentation is an attractive process to produce cellulase economically due to its lower financial investment and lower operating costs. Generally available natural materials in our study included vinegar residue, wheat bran, rice bran and wheat straw. Cellulase production was carried out by solid state fermentation using these materials as the substrate of Trichoderma koningii. The ingredients of natural materials, associated with the effects of water content, time and nitrogen sources on cellulase synthesis were studied. A comparatively high cellulase activity （〉4 IU·g^-1 SDM） was obtained in the fermentation batch. The optimum culture time of vinegar residue, wheat bran and wheat straw were 72 hours, but that office bran was 60 hours. Total water contents of wheat bran, rice bran and wheat straw should not exceed 50% and that of vinegar residue should not be more than 60%. It was also shown that nitrogen salts contributed much to fermentation. （NH4）3PO4 and urea achieved good results in promoting enzyme activity.

Physicochemical Properties and Bioactivities of Rice Beans Fermented by Bacillus amyloliquefaciens

The purpose of this study was to investigate the physicochemical properties and bioactivities of rice beans(Vigna umbellata)fermented by Bacillus amyloliquefaciens.The fermentation conditions were optimized on the basis of the fibrinolytic activity.Under the optimal fermentation conditions,the fibrinolytic activity reached a maximum of 78.0 FU·g^(-1)(4890 IU·g^(-1),fibrin plate method,FU:fibrin degradation unit).The contents of peptides(which increased from 2.1 to 10.9 g per 100 g),total phenolics(from 116.7 to 388.5 mg gallic acid per 100 g),total flavonoids(from 235.5 to 354.3 mg rutin per 100 g),and anthocyanin(from 20.1 to 47.1 mg per 100 g),as well as the superoxide dismutase activity(from 55.3 to 263.6 U·g^(-1))in rice beans were significantly increased after fermentation.The 2,2-diphenyl-1-picrylhydrazyl(DPPH)and 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt(ABTS)free radical scavenging activities and ferric reducing antioxidant power(FRAP)of fermented rice beans were 1.9–4.8 times higher than those of unfermented rice beans.Moreover,fermentation induced an increase in the dipeptidyl peptidase IV(DPP-IV)inhibition,a-glucosidase inhibition,and anticoagulant activities of rice beans.Rice beans fermented by Bacillus amyloliquefaciens may serve as a functional food with potential benefits for the prevention of thrombotic diseases.

Upgrading the Fermentation Process of Zhejiang Rosy Vinegar by Purebred Microorganisms

Purebred microorganisms were employed to upgrade the fermentation process of Zhejiang rosy vinegar. The fermentation cycle was greatly shorten from 5 months to 72 d. The transformation rate of raw materials was increased from 1:4.5 in the traditional fermentation to 1:5 or more in the upgraded fermentation. The content of organic acids in the traditional vinegar (TRV), the upgraded vinegar (UPV) and the submerged fermentation vinegar (SFV) were also investigated by HPLC. No significant difference was found regarding the proportion of phenylethanol to the total volatile components in UPV (7.47% ± 0.00324%) and TRV (7.23% ± 0.00329%), but it was significantly higher than that in SFV (2.26% ± 0.00143%). This study provides deep insight into upgrading the fermentation process of Zhejiang rosy vinegar by purebred microorganisms.

Optimization of Acetic Acid Fermentation Process of Apple Cider Vinegar

This paper studied the fermentation rules of apple cider vinegar from fruit juice,to provide a theoretical guidance for the production of apple cider vinegar.Using Fuji apples as raw materials,the process parameters(fermentation temperature,fermentation time,stirring speed,and inoculation amount)of apple cider vinegar fermentation were optimized through single-factor experiments and response surface analysis.The results indicated that the fermentation temperature had no significant effect on the total acid content of apple cider vinegar fermentation,the fermentation time had an extremely significant effect on the total acid content of apple cider vinegar fermentation,and the stirring speed and inoculation amount had a significant effect on the total acid content of apple cider vinegar fermentation.Through process optimization,the optimal process parameters for apple cider vinegar fermentation are fermentation temperature of 33℃,fermentation time of 39 h,stirring speed of 1500 r/min,and acetic acid bacteria inoculation amount of 7%.Under such conditions,the total acid content of fermented apple cider vinegar is 62.22 g/L,very close to the predicted value of the model,indicating that the process parameters of acetic acid fermentation obtained by response surface methodology(RSM)optimization are reliable and can be used for actual production prediction.