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.

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.

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.

不同发酵期浓香型青稞酒糟醅理化指标及基酒香气组分差异

基于前期优化的浓香型青稞酒发酵工艺,探究不同发酵期(40 d,50 d和60 d)对浓香型青稞酒糟醅理化指标和基酒挥发性香气组分的影响。结果表明,不同发酵期浓香型青稞酒出窖糟醅的水分、酸度、还原糖和淀粉差异显著(p<0.05)。随着发酵期的增加,出窖糟醅的水分增加,还原糖、酸度和淀粉呈现下降趋势。延长发酵期有利于延长微生物分解代谢,提高淀粉的利用率,提升出酒率。不同发酵期青稞酒的挥发性香气组分含量差异较大。随着发酵期增加,挥发性香气组分的种类及总含量呈现增加趋势。酸类、醇类和酯类为主要的挥发性香气组分,酸类和醇类组分的含量呈现先增加后下降的趋势,可能与醇酸酯化合成酯类有关,与酯类组分的含量增加趋势相一致。长发酵期有利于提高青稞酒中酯类组分的含量,进而增加青稞酒的香气浓郁度和丰富度。

酱香型白酒下沙到四轮次酒醅堆积终点特征研究

为探究酱香型白酒酒醅不同阶段堆积终点的特征,对贵州省某酒庄的53口窖池从下沙到四轮次酒醅进行理化及微生物指标的检测,并分析各轮次酒醅堆积终点时各指标对基酒产量与基酒品质的相关性。研究表明,随着轮次的增加,酒醅的温度、酸度、水分、发酵力呈明显的上升趋势;淀粉含量呈显著下降趋势(P<0.05);L-乳酸和葡萄糖含量整体呈上升趋势;乙醇含量、拜尔接合酵母(Zygosaccharomyces bailii)数量、酵母菌活菌率表现为前期快速上升,二轮次后趋于水平的状态。糖化力呈现先降低后水平波动的趋势;酿酒酵母和酵母总数总体变化不大。相关性分析结果表明,与基酒产量、基酒品质显著相关的指标有10个(P<0.05),分别是为水分、乙醇、拜尔接合酵母、乳酸、酿酒酵母、酸度、淀粉、葡萄糖、温度、发酵力。其中,水分、乙醇含量、葡萄糖含量对基酒的影响最大,且水分、乙醇含量对基酒的影响主要是正相关,葡萄糖含量对基酒的影响主要是负相关。

基于热量平衡模型的大曲发酵升温机理研究

中国白酒产业正面临产业转型升级,包括制曲在内的关键工艺采用基于经验式的直接改造,关键工程学机理的缺乏导致无法进行理性设计。该研究从大曲升温机理角度,对曲房固态发酵热量的产生与流动进行了定量分析,跟踪记录大曲发酵过程参数,简化大曲发酵过程生化反应,构建大曲热量平衡模型。通过模型计算,整个曲房发酵过程产生的热量,经水分蒸发途径散热占比54.22%,墙壁和微弱气体流动散热占比42.00%,由室内空气升温引起的热量变化可以忽略,剩余3.77%为累积热量部分,其与大曲升温呈正相关(R^(2)=0.85)。经验证,大曲温度变化模型的平均误差为5.05%,R^(2)=0.94。基于模型探讨了调控水分参数对大曲温度产生的影响,表明了水分控制对于调控大曲温度的重要性。初步探讨从数学建模的角度为未来大曲生产的调控与优化的可行性,研究结果可为智能曲房的构建提供理论支撑。

机械化模式下酱酒不同层次堆积糟醅的理化指标与风味物质的差异分析

为研究机械化模式下酱香型白酒不同层次堆积糟醅的差异,对酒质好且出酒率高的第4轮次酒堆积糟醅进行了跟踪检测。结果表明,各层次糟醅在温度、黏度、还原糖之间都存在显著性差异,除中层和内层糟醅之间硬度差异不显著外,其余层次间差异显著;在酸度、水分、淀粉指标上,只有外层糟醅与其他层次糟醅差异显著,其余层次之间不显著。通过GC-MS分析,发现外层挥发性风味物质含量最多,中层和内层含量相近,底层含量最少;酯类、酸类、醇类和芳香族物质占总挥发性物质含量的90%以上,其中酯类是堆积发酵过程中最丰富的挥发性物质;不同层次堆积糟醅的关键差异风味物质主要有24种,其中酯类有12种,酸类有4种,醇类有3种,其他类有5种。外层与其他层次的糟醅风味物质存在明显的差异,中层与底层糟醅风味物质存在一定的差异,在堆积前期,内层与中层风味物质相似,在堆积后期,内层与底层风味物质相似。造成糟醅差异的驱动因素主要是溶氧量及温度,底层由于溶氧量和温度较低,导致发酵进度较慢。研究发现,通过破堆移位可以提高发酵糟醅的均匀性和稳定性,有利于风味物质的产生和积累从而加快发酵进度。本研究为进一步剖析酱酒机械化模式下改进酿造工艺参数、提升堆积质量提供了参考依据。

高产乙酸乙酯酵母的筛选鉴定及发酵条件优化

该研究通过传统培养分离方法及测定乙酸乙酯产量从清香型白酒大曲中分离筛选高产乙酸乙酯的酵母菌株,通过形态观察及分子生物学技术对其进行菌种鉴定,并对其耐受性进行分析。以乙酸乙酯产量为响应值,采用单因素试验及响应面试验对筛选菌株的发酵条件进行优化。结果表明,分离筛选得到1株高产乙酸乙酯的酵母菌株,编号为YM001,经鉴定,其为异常威克汉姆酵母(Wickerhamomyces anomalus)。该菌株具有较强的耐受性,在乙醇体积分数10%,乙酸含量0.08%、温度50℃、pH 2、乙酸乙酯含量30 g/L、NaCl含量150 g/L、葡萄糖含量50 g/L的条件下仍能生长,能够较好地适应白酒酿造环境。异常威克汉姆酵母YM001产乙酸乙酯的最优发酵条件为:接种量4%、乙醇体积分数4%、乙酸添加量0.02%、初始糖度9°Bx、初始pH值6、转速210 r/min、发酵温度22℃、第2阶段发酵时间36 h。在此发酵条件下,异常威克汉姆酵母YM001的乙酸乙酯产量最高,达(17.39±0.362)g/L。

窖池中酸性脲酶产生菌的分离鉴定及其对模拟窖泥pH值和风味物质的影响

为改善窖池老化现象,该文从水井坊窖泥中分离酸性脲酶产生菌,将分离株与产香克氏梭菌S008复配后固态发酵,探究酸性脲酶产生菌对模拟窖泥pH值及挥发性风味物质影响。结果表明,筛选出4株酸性脲酶产生菌,鉴定并命名为布氏乳杆菌S162、科氏葡萄球菌S163、鲁梅利芽孢杆菌S164和水原拉梅尔芽孢杆菌S165;4株菌的脲酶活性在0.45~1.51 U/mL;复配菌株的模拟窖泥pH值呈现:上部>中部>下部,其中S163的模拟窖泥pH值最高(P<0.05)。在风味物质检测中,协同发酵后S163乙醛含量(2.38 mg/100 mL)最高,而S162乙缩醛含量(2.50 mg/100 mL)最高;正丙醇、正丁醇、正己醇和异戊醇含量分别是S008单菌发酵的1.41~1.97倍、0.92~1.85倍、0.66~1.58倍、1.13~1.69倍;S164与S008协同发酵对己酸含量提升最显著(P<0.05);4株菌与S008协同发酵可以分别提高“四大酯”含量2.66~5.02倍、1.21~4.85倍、1.33~3.31倍、1.27~2.86倍。研究表明,酸性脲酶产生菌与克氏梭菌S008协同发酵可以不同程度提高模拟窖泥pH值及主要挥发性风味物质含量。

浓香型白酒发酵体系中己酸菌的研究进展

浓香型白酒发酵体系中己酸生成菌(以下简称“己酸菌”)的己酸合成代谢对提高浓香型白酒的发酵质量非常重要。因此,有必要深入全面了解浓香型白酒发酵体系中己酸菌的种类及其己酸合成代谢特征。本综述介绍了目前浓香型白酒发酵体系中已经分离的己酸菌株的种类多样性、系统进化关系、生理代谢特征、己酸合成代谢机制以及其与己酸菌、非己酸菌之间的协同代谢关系。本文可为理解己酸菌群在浓香型白酒发酵体系中的原位己酸合成代谢规律提供参考,为将来靶向提高己酸菌群在浓香型白酒发酵和生物质转化高附加值己酸工艺中进行己酸合成培养工程的应用提供理论依据。

浓香型白酒窖泥己酸菌的研究进展

浓香型白酒是中国白酒的重要类型之一,己酸乙酯是浓香型白酒特征风味物质,该物质合成所需的前体己酸主要依靠发酵过程中的己酸菌代谢生成。己酸菌的分离、鉴定及代谢功能研究已成为白酒品质提升相关领域的研究热点之一。本文综述了窖泥中己酸菌的分离概况,介绍了浓香型白酒发酵系统中常见的3种己酸菌及己酸的合成途径,探讨了pH及底物组成对己酸产量的影响;此外,介绍了微生物群落中其他微生物对己酸菌的影响,以期通过探讨浓香型白酒发酵体系中微生物驱动己酸形成的机制及其影响因素,为提升浓香型白酒品质提供理论基础。

芦台春酱香型白酒酿造过程中真菌菌群多样性及代谢分析

该研究采用高通量测序技术对天津芦台春酱香型白酒发酵过程中不同轮次出窖酒醅的真菌群落结构进行研究,并对其与理化因子、挥发性风味成分相关性分别进行典范对应分析(CCA)及偏最小二乘判别分析(PLS-DA)。结果表明,随着轮次的增加,真菌菌群的多样性升高,而丰度先升高后降低;酵母菌是白酒发酵过程中的主要真菌,但相对丰度逐步降低,在属水平上以伊萨酵母属(Issatchenkia)为主,其相对丰度由第一轮次的97.48%逐渐下降至第七轮次的3.53%。相关性分析结果表明,总酸、还原糖和酒精度是窖池发酵微生物演替的重要理化因子;伊萨酵母属(Issatchenkia)与乙酸乙酯、乙酸异戊酯、异戊醇等相关性较大;哈萨克斯坦酵母属(Kazachstania)、丝衣霉属(Byssochlamys)、罗萨氏菌属(Rasamsonia)、复膜孢酵母属(Saccharomycopsis)等与正己酸乙酯、庚酸乙酯、3-羟基丁酸乙酯、丁二酸二乙酯、苯乙酸乙酯、油酸乙酯、乙醇等相关性较大;而Leiothecium、未分类的酵母目(unclassified_o__Saccharomycetales)、Apiotrichum、Cutaneotrichosporon等则对壬酸乙酯、乙酸苯乙酯、正己醇、2,3-丁二醇、糠醇、糠醛等的形成起到重要作用。

酵母菌种类及发酵条件对白酒正丙醇含量的影响

酵母菌是白酒发酵中酒精的主要产生菌种,具有合成正丙醇等高级醇的作用,发酵条件对酵母菌产酒精和正丙醇等的含量有重要影响。本研究采用液态发酵方法对酒醅中分离的不同酵母菌种类及不同糖度下产正丙醇产量的差异进行比较分析,结果表明:热带假丝酵母产正丙醇效果最好,东方伊萨酵母产正丙醇效果最弱,酿酒酵母产正丙醇效果适中。正丙醇的含量随着发酵培养基中葡萄糖含量的增加而增加。实验室模拟固态白酒发酵条件实验表明:小曲酒发酵温度28℃时正丙醇含量最高,38℃发酵时正丙醇含量最低;酒醅含水率为53%、pH为3.5时正丙醇含量最高。大曲酒实验室模拟发酵结果表明:堆积阶段补加葡萄糖或淀粉酶,可明显提高正丙醇的含量。