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.

Succession of bacteria communities during production and application of dairy bedding by membrane-covered aerobic fermentation

The cost of dairy manure treatment and bedding material purchase increases the operating cost of the dairy farm.Membrane-covered aerobic fermentation system has been widely used for dealing with dairy manure and recycling the final product as bedding material.However,the microbial safety in each processing step is still uncertain.To better understand the bacterial community dynamics during the whole bedding conversion process,a full-chain and large-scale experiment including 16-day membrane-covered aerobic fermentation and 11-day bedding material application was conducted.The results showed that the pile temperatures in the fermentation stage rapidly increased to 80°C and maintained>50°C for more than 11 days and the use of fermentation product as bedding material provided cows with a stable and comfortable bedding environment.The Chao1 and Shannon index decreased at the end of the fermentation stage and remained stable in the application stage,indicating that membrane-covered aerobic fermentation effectively killed some pathogenic bacteria and guaranteed both the maturity and stability of the final product.The dominant bacteria in the fermentation stage were Acinetobacter,Thermus,and Rhodothermus at genus level.Seven common potential pathogens of mastitis(Staphylococcus,Enterococcus,Serratia,Pseudomonas,Corynebacterium,Mycobacterium,and Bacillus)were found at the end of fermentation stage but the relative abundance was low(0.0025%-0.2727%).The dominant bacteria in the application stage mainly included Acinetobacter,Pseudomonas,and Flavobacterium at the genus level.The relative abundance of Pseudomonas increased in the application stage,which was a reminder to the dairy farm to pay attention to the disinfection and timely replacement of bedding material to prevent the occurrence of dairy mastitis.The results of this study contributed deep understanding of the microorganism-driven bedding conversion process and provide practical guidance and cautions for the bedding materials application.

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, aer- obic stability and in vitro gas production kinetics and digestibility of whole-crop corn based totalmixed ration （TMR） silage. Total mixed ration was ensiled with four treatments： （1） no additives （control）; （2） an inoculant （Lactobacillus plantarum） （L）; （3） propionicacid （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 for12 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 （NH3-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 NH3-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.

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 vacuum negative pressure aerobic hydrolysis pretreatment on corn stover anaerobic fermentation

Lignin degradation restricts corn stover anaerobic fermentation efficiency.The vacuum negative pressure aerobic hydrolysis pretreatment of corn stover was tested,and the optimal combined pretreatment conditions were presented in this paper.Because of the physical characteristics of light weight and large specific porosity of stover,it led to the formation of a scum layer during the fermentation process and thus reduced the gas production rate.In the pretreatment design,the vacuum conditions(0.02-0.08 MPa)and dwell time(5-20 min)were selected to see the changes of volumetric weight,swelling and specific porosity of corn stover,resulting in an increase of the volumetric weight by 7.18%-28.72%,an increase of the swelling by 3.18%-58.59%,and a decrease of the specific porosity by 9.34%-38.59%,as compared with the CK group.Continuous vacuum negative pressure treatment could discharge the air inside the stover destroy the microstructure,and cause the stover to settle more easily during the aerobic hydrolysis process.The optimal aerobic hydrolysis temperature and time were determined to be 39°C and 12.65 h,respectively.With the optimal pretreatment,the corn stover anaerobic fermentation test realized a cumulative methane yield of 260.44 mL/g VS,22.71%higher than CK group;meanwhile,the hydraulic retention time was shortened by 32.39%.

不同原料配比对芦笋秸秆堆肥发酵效果的影响

为促进芦笋秸秆资源化利用,通过加入不同比例的鸭粪和稻壳于芦笋秸秆中,进行资源化堆肥效果的试验研究。以发酵温度、pH、发芽指数、有机质、氮、磷、钾等为评价指标,研究鸭粪和稻壳添加对堆肥发酵过程的影响。结果表明:单一芦笋秸秆堆肥发酵过程,无高温阶段,未能达到发酵堆肥要求。添加不同比例的鸭粪和稻壳均能促进芦笋秸秆堆肥发酵,高温维持时间12~13 d,高温阶段平均温度为56.3~60.4℃,发酵周期21~29 d,发酵30 d结束时,发芽指数达到了92%~118%,pH>8.0,有机质含量50.70%~57.02%,氮磷钾总养分含量4.64%~5.20%。综合各项指标,在本试验条件下,70%芦笋秸秆+10%鸭粪+20%稻壳粉的配比发酵效果优于其他配比,更适合芦笋秸秆发酵。

生物质好氧发酵热生产与回收利用研究进展

好氧发酵是目前有机固体废弃物处理的一种有效手段。人们对于好氧发酵的研究主要集中在高效有机肥的获取上,但发酵过程产生的热能不容忽视。发酵热作为一种“零碳”能源,可代替传统化石能源应用于加温供暖、生物干化等领域,助力实现“碳达峰、碳中和”。为将生物质能高效转化为热能利用,人们对发酵热回收利用进行了研究,但是没有将热生产、热回收和热利用三个阶段进行系统联系,导致热回收工艺效率不高。该文主要阐述了好氧发酵产热原理,并从菌剂、原料理化性质和发酵工艺三个方面对发酵热生产的影响进行了探讨,总结了现有热回收利用系统,最后对生物质好氧发酵热生产与回收利用系统的发展方向进行展望,以期为生物质发酵热能利用提供支持。

菌酶添加对喀斯特地区全株青贮玉米发酵品质和有氧稳定性的影响

试验旨在研究添加菌酶对喀斯特地区全株青贮玉米发酵品质和有氧稳定性的影响,为新型青贮菌剂的应用提供参考。以真空袋法青贮全株玉米,设5个试验组和1个对照组,分别为添加等量纯化水(对照组)、植物乳杆菌(试验A组)、纤维素分解菌(试验B组)、纤维素酶(试验C组)、植物乳杆菌+纤维素分解菌(试验AB组)、植物乳杆菌+纤维素酶(试验AC组)。其中植物乳杆菌添加1×10^(5) CFU/g;纤维素分解菌为黑曲霉∶枯草芽孢杆菌=2∶1,添加量为3.0×10^(5)、1.5×10^(5) CFU/g,纤维素酶按0.3%添加。发酵60 d后同时采样并测定全株玉米的发酵特性、乳酸菌数量、营养成分、有氧稳定性指标。结果表明:试验组均有微弱香味,对照组则气味较平淡,综合评分均到达一级;试验组pH均低于4.2,达到优质青贮料标准,C、AC组pH显著低于A、B、AB组(P<0.05);A、B、C组乳酸(LA)含量显著高于对照组(P<0.05);试验组乙酸(AA)含量高于对照组,但差异不显著(P>0.05),丙酸(PA)和丁酸(BA)均未检测出;A、B、C、AB组氨态氮(NH3-N)含量显著高于对照组(P<0.05);试验组有氧稳定性(AS)时间均显著高于对照组(P<0.05);AB组粗蛋白(CP)显著高于其他试验组(P<0.05);试验组干物质(DM)含量显著高于对照组(P<0.05);B、AB组粗灰分(Ash)含量显著低于对照组(P<0.05)。采用隶属函数法综合各项指标进行评价,各组优劣排序为AC组>C组>AB组>B组>A组>对照组。说明AC、AB组可有效改善全株青贮玉米的青贮品质,提高有氧稳定性,以AC组效果最好;因此,纤维素分解菌可在一定程度上替代纤维素酶用于全株青贮玉米发酵。

餐厨垃圾生物减量技术研究进展

餐厨垃圾占城市生活垃圾比重日益增高,其成分特性非常适合微生物生长繁殖,若处理不当则极易腐败污染环境。目前市面上存在多种针对餐厨垃圾的资源化处理技术,各有优劣。通过介绍生物减量技术原理并将其与同为好氧处理中的好氧堆肥与生物干化对比,体现出生物减量技术能更好实现减量化的优势,更适用于餐厨垃圾前端处理。重点从减量菌、餐厨垃圾预处理、填充剂选择与复配三角度介绍了餐厨垃圾生物减量的强化策略,并列举部分生物减量技术规模化的应用,为进一步提高生物减量效率提供参考。

超嗜热菌对乡村有机固体废弃物发酵过程减量化的影响研究

传统发酵在废弃物资源化处理中已经广泛应用,但是存在发酵周期长、发酵温度低、容量大、水分高、减量化效果不佳等缺陷。采用超高温好氧发酵技术对乡村有机固体废弃物进行处理,将超高温环境下培养出的以芽孢杆菌(Bacillus)为核心的超嗜热菌剂添加到有机固体废弃物中,采用高通量测序技术,对堆体不同阶段的微生物种群进行分析,同时,对堆体温度、含水率、碳氮比(C/N,质量比)、pH、种子发芽指数(GI)和电导率(EC)等指标进行分析。结果表明:堆体的最高温度为86℃,以80℃为节点,发酵超高温期占比达到2/3,含水率从44.57%下降到29.09%,总减重率和总减容率分别为85.50%、50.51%,发酵结束时C/N、pH分别为15.56、7.55,发酵结束时物料的GI和EC分别为110.24%和3.72 mS/cm,发酵过程中芽孢杆菌目(Bacillales)和放线菌目(Actinomycetales)是达到堆体减量化效果的主要菌目。综上所述,超嗜热菌对好氧堆肥有一定的促进作用,是乡村固体废弃物减量化处理较为理想的发酵菌剂。

大豆秸和稻草对甜玉米秸秆和湿啤酒糟混合青贮的影响

甜玉米(Zea mays var.rugosa)秸秆和湿啤酒糟含水量较高,青贮可有效解决其不易贮藏的问题。通过设置甜玉米秸秆单贮、湿啤酒糟单贮、两者不同比例(3∶1和4∶1)混合青贮以及分别添加大豆(Glycine max)秸和稻(Oryza sativa)草将混合青贮的含水量调节为60%和70%,共12个处理,探究混合比例及含水量对其混合青贮发酵品质及有氧稳定性的影响。结果表明:甜玉米秸秆单贮发酵品质良好,湿啤酒糟单贮发酵品质较差,二者按3∶1混贮后显著提高了乳酸及乙酸含量(P<0.05),4∶1混贮后显著提高了乳酸含量(P<0.05),但4∶1混贮后好氧细菌及酵母数量较3∶1高;与甜玉米秸秆单贮相比,添加大豆秸显著提高了混合青贮料的有氧稳定性(P<0.05),有氧暴露7 d后的pH值仍接近4.2,添加大豆秸将含水量调节为60%显著降低了NH3-N含量(P<0.05);添加稻草的所有处理均显著降低了NH3-N含量(P<0.05)。综上,甜玉米秸秆与湿啤酒糟按3︰1的比例混合,并添加大豆秸将含水量调节为60%,其发酵品质好,有氧暴露7 d有氧稳定性也较好。