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。

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。

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。

固态法白酒糟醅添加黄原胶保水发酵研究

[目的]提升糟醅的保水能力,增加糟醅的液相含量。[方法]以特香型白酒为研究对象,分别进行糟醅添加黄原胶的模拟酿造试验(1.5%添加量、稻壳用量减少10%)与车间窖池试验(0.7%添加量),分析保水发酵对白酒酿造的影响。[结果]黄原胶具有良好的保水作用,上层/粮糟层水分升高,上下层/粮糟层与底糟层水分差异变小,水分含量与糟醅酒精度正相关,与酸度无明显相关性。模拟酿造试验发现基酒总酸总酯含量升高,表现出“增乙抑乳”与降低高级醇含量的作用。车间试验面糟酒、粮糟酒出酒数与总产量均增加,粮糟层基酒总酯含量更高,而底糟酒出酒数下降,己酸、己酸乙酯含量更低;粮糟酒感官评价优于对照组。[结论]添加一定比例的黄原胶可增强糟醅的保水能力,提升基酒总酯含量,本研究为固态法白酒糟醅改良、降低稻壳用量、提升基酒质量提供了新的思路。

酱酒1、2轮次窖池不同醅层微生态结构与酸性化合物组成解析及其相关性预测

以酱香白酒酿造过程中1、2轮次上、中、底层窖池酒醅为研究对象,采用高通量测序技术偶联超高效液相色谱等方法解析酒醅中微生物群系结构、酸类化合物结构及其酸类化合物分布与微生物群系相关性,进一步揭示其内在机制。结果表明,2轮次酒醅总酸含量大于1轮次;7种酸类化合物在窖池不同时空径向的分布规律不同;1、2轮次酒醅中乳酸和乙酸含量占比较大。1、2轮次绝对优势细菌属为Limosilactobacillus,优势真菌属为Saccharomyces和Candida,且随发酵时间延长,微生物群系结构差异显著。总酸和酸类化合物含量与Limosilactobacillus、Schizosaccharomyces、Zygosaccharomyces、Candida和Kazachstania相对丰度呈正相关,与Lactobacillus、Saccharomyces、Paecilomyces和Torulaspora相对丰度呈负相关。本研究揭示了酱香白酒1、2轮次窖池酒醅不同时空径向的酸度及酸类化合物与微生物群落间的相关性,可为进一步阐明酱香白酒窖池发酵机理提供理论参考。

洞酿酱香型白酒第三轮次酒醅发酵过程中微生物与挥发性风味物质相关性分析

该研究应用Illumina MiSeq高通量测序技术和顶空固相微萃取-气相色谱质谱联用(HS-SPME-GC-MS)技术解析洞酿酱香型白酒第三轮次酒醅发酵过程中微生物多样性与挥发性风味物质变化。结合偏最小二乘法-判别分析(PLS-DA)筛出关键差异风味物质,并揭示优势菌属与挥发性风味物质的相关性。结果表明,所有酒醅样品共检测出4个优势菌门,16个优势菌属(包含8个细菌属和8个真菌属)。第三轮次酒醅发酵过程样品中共检出68种挥发性风味物质,其中醇类15种,酯类27种,醛类10种,酸类4种,酚类4种,其他类8种。PLS-DA模型共筛出关键差异风味物质16种。相关性结果表明,曲霉属(Aspergillus)与大多数酯类物质呈极显著正相关(P<0.01),Priceomyces和乳杆菌属(Lactobacillus)与十七烷酸乙酯呈极显著正相关(P<0.01)。

外源氨基酸对液态发酵法白酒风味品质的影响

在液态法发酵白酒的基础上,添加不同质量浓度的甘氨酸(Gly)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe),探究外源氨基酸对液态法发酵白酒产生的酒精度、总酸、总酯和主要风味物质的影响。结果表明,甘氨酸对白酒酒精度、辛酸乙酯含量的影响最大,最大值比空白组分别增加了7.14%、69.75%;亮氨酸对总酸、总酯、异戊醇的影响最大,最大值比空白组分别增加了8.10%、12.07%和92.94%;缬氨酸对异丁醇的影响最大,最大值比空白组增加了404.28%;Phe对苯乙醇、乙酸乙酯的影响最大,最大值比空白组分别增加了81.54%、32.66%;随着四种氨基酸添加量的增加,酒精度和总酸产量先升高后降低,其中甘氨酸和亮氨酸添加量分别为400 mg/L时,酒精度和总酸最大分别为12%vol和0.227 g/L;四种氨基酸对白酒总酯的生成影响明显,其中亮氨酸添加量为600 mg/L时总酯产量最大,为0.43 g/L;由挥发性风味物质聚类结果可知,亮氨酸、缬氨酸和苯丙氨酸对液态发酵法白酒风味物质生成影响较大。在液态发酵法白酒制备中合理添加氨基酸,可明显提高液态发酵法白酒酒精、总酸、总酯的含量,有效调节高级醇产生。

浓香型白酒在新、老窖池发酵过程中酒醅微生物群落结构差异分析

为了分析新、老窖池浓香型白酒发酵过程中微生物群落演替特征及差异性,采用高通量测序技术分析酒醅的微生物群落演替与组成结构,并通过相关性网络分析阐明新、老窖池酒醅中微生物群落的相关性与稳定性。结果表明,老窖池酒醅样品中细菌和真菌菌群的多样性均低于新窖池,细菌菌群多样性的增长趋势也要滞后于新窖池,而真菌菌群多样性则相反。新、老窖池酒醅样品中的真菌群落组成及演替规律存在明显差异,而细菌群落较为相似。在老窖池酒醅样品中,曲霉属(Aspergillus)与哈萨克斯坦酵母属(Kazachstania)的相对丰度更高,而在新窖池酒醅样品中酿酒酵母(Saccharomyces)更具优势。另外,相关性网络分析显示,老窖池酒醅的微生物互作网络具有更高的聚集程度,微生物群落的共存关系也更加复杂,稳定性更高。

不同层次堆积粮醅对复合香型白酒发酵特性及香气品质的影响

堆积是白酒生产过程中重要的环节,通过研究不同层次醅对复合香白酒发酵的影响。结果表明:(1)堆积后内层、外层和混合中淀粉含量消耗分别下降5.23%、5.85%、7.2%。还原糖消耗分别上升1.56%、0.63%、1%;不同堆积醅所产原酒经检测,内层、外层及混合醅的总酯含量分别为1.94 g/L、2.57 g/L、5.86 g/L,总酸含量分别为0.33g/L、0.54 g/L、1.23 g/L。其中乙酸乙酯含量最高分别为1.17 g/L、1.67 g/L、4.74 g/L。(2)对所产原酒进行GC-MS分析,样品中共检测到挥发性物质121种,其中酯类物质种类最多有60种,占比49.6%。内层、外层及混合层原酒中分别检测到103种、95种、95种,从挥发性物质含量来看:混合(157.63μg/g)>外层(150.05μg/g)>内层>(107.76μg/g)。PCA和热力图结果显示内、外和混合之间的相关性较大,其中混合样得分最高。

清香型大曲白酒酿造中发酵微生物的分析

从清香型大曲白酒酿造过程中分离到的不同种类的微生物中,筛选出具有代表性的优势微生物,并对之进行检测鉴定,以能了解发酵工艺及影响白酒酿造口感、口味的影响因素。在实际进行酿酒时,细菌和真菌以固态发酵为主,而曲酒的糖化效果和酒化效果比液态发酵好,在酿造过程中若控制好细菌或真菌含量就可以提高酒醅的糖化率、提升酒醅酸度。