Solid state fermentation of rapeseed cake with Aspergillus niger for degrading glucosinolates and upgrading nutritional value

Background： Rapeseed cake is a good source of protein for animal feed but its utilization is limited due to the presence of anti-nutritional substances, such as glucosinolates （GIs）, phytic acid, tannins etc. In the present study, a solid state fermentation （SSF） using Aspergillus niger was carried out with the purpose of degrading glucosinolates and improving the nutritional quality of rapeseed cake （RSC）. The effects of medium composition and incubation conditions on the GIs content in fermented rapeseed cake （FRSC） were investigated, and chemical composition and amino acid in vitro digestibility of RSC substrate fermented under optimal conditions were determined. Results： After 72 h of incubation at 34℃, a 76.89% decrease in GIs of RSC was obtained in solid medium containing 70% RSC, 30% wheat bran at optimal moisture content 60% （w/w）. Compared to unfermented RSC, trichloroacetic acid soluble protein （TCA-SP）, crude protein and ether extract contents of the FRSC were increased （P〈 0.05） 103.71, 23.02 and 23.54%, respectively. As expected, the contents of NDF and phytic acid declined （P〈 0.05） by 9.12 and 44.60%, respectively. Total amino acids （TAA） and essential amino acids （EAA） contents as well as AA in vitro digestibility of FRSC were improved significantly （P 〈 0.05）. Moreover, the enzyme activity of endoglucanase, xylanase, acid protease and phytase were increased （P 〈 0.05） during SSF. Conclusions： Our results indicate that the solid state fermentation offers an effective approach to improving the quality of proteins sources such as rapeseed cake.

Utilization of winery wastes for Trichoderma viride biocontrol agent production by solid state fermentation

Biocontrol agents are safe and environmental friendly alternatives for pesticides in agriculture application. Trichoderma viride WEBL0703 performed a high level of antagonistic activity toward a broad spectrum of phytopathogens and was determined as a biocontrol agent, which was produced by solid state fermentation using grape marc and wine lees. The maximum yield of T. viride conidia was up to 6.65 × 10^9 CFU/g initial dry substrate （IDS） after 10 d fermentation. As important enzymes for protecting plants from disease, chitinase, β-glucanase, and pectinase yields were 47.8 U/g IDS, 8.32 U/g IDS and 9.83 U/g IDS, respectively. These results show that it is feasible to convert winery wastes to a value-added and environmental friendly biocontrol agent.

Paddy Husk as Support for Solid State Fermentation to Produce Xylanase from Bacillus pumilus

To optimize culture conditions for xylanase production by solid state fermentation （SSF） using Bacillus pumilus, with paddy husk as support, solid medium contained 200 g of paddy husk with 800 mL of liquid fermentation medium [xylan, 20.0 g/L; peptone, 2.0 g/L; yeast extract, 2.5 g/L; K2HPO4, 2.5 g/L; KH2PO4, 1.0 g/L; NaCl, 0.1 g/L; （NH4）2SO4, 2.0 g/L, CaCl2-2H2O, 0.005 g/L; MgCl2.6H2O, 0.005 g/L; and FeCI3, 0.005 g/L] at pH 9.0 was applied. The highest xylanase activity （142.0 ±0.47 U/g DM] was obtained on the 6th day at 30℃ The optimized paddy husk to liquid fermentation medium ratio was 2：9, and the optimized culture temperature was 40℃. When commercial Birchwood xylan was replaced with different concentrations of corncob, xylanase production was maximized （224.2 U/g DM） in the medium with 150 g/L corncob. Xylanase production was increased by sucrose, fructose and arabinose, whereas reduced by glucose, galactose, lactose and amylose. When organic nitrogen sources were replaced with locally available nitrogen sources such as groundnut powder or sesame seedcake powder or coconut seedcake powder or soy meal powder, the highest xylanase production （290.7 U/g DM） was obtained in the medium with soy meal powder and 16.0 g/L of soy meal powder was the optimum （326.5±0.34 U/g DM）. Based on the optimization studies, B. pumilus produced 2.3 times higher xylanase activity. The medium cost was reduced from 2 458.3 to 178.3 SLR/kg and the total activity which could be obtained from 1 kg of the medium was increased from 48 624 to 220 253 Units.

Manganese peroxidase production from cassava residue by Phanerochaete chrysosporium in solid state fermentation and its decolorization of indigo carmine

Bioconversion of lignocellulosic wastes to higher value products through fungal fermentation has economic and ecological benefits. In this study, to develop an effective strategy for production of manganese peroxidase(Mn P)from cassava residue by Phanerochaete chrysosporium in solid state fermentation, the stimulators of Mn P production were screened and their concentrations were optimized by one-at-a-time experiment and Box–Behnken design. The maximum Mn P activity of 186.38 nkat·g-1dry mass of the sample was achieved after 6 days of fermentation with the supplement of 79.5 mmol·L-1·kg-1acetic acid, 3.21 ml·kg-1soybean oil, and 28.5 g·kg-1alkaline lignin, indicating that cassava residue is a promising substrate for Mn P production in solid state fermentation. Meanwhile, in vitro decolorization of indigo carmine by the crude Mn P was also carried out, attaining the ratio of 90.18% after 6 h of incubation. An oxidative mechanism of indigo carmine decolorization by Mn P was proposed based on the analysis of intermediate metabolites with ultra-high performance liquid chromatography and gas chromatography tandem mass spectrometry. Using the crude Mn P produced from cassava residue for indigo carmine decolorization gives an effective approach to treat dyeing effluents.

Mechanism of Solid State Fermentation in Reducing Free Gossypol in Cottonseed Meal and the Effects on the Growth of Broiler Chickens

[Objective] This study aims to reduce the free gossypol(FG) and improve utilization rate of cottonseed meal(CSM) by solid state fermentation(SSF). [Method]Bacillus subtilis GJ00141 and Saccharomyces cerevisiae GJ00079 were applied for the SSF and the optimal carbon source, nitrogen source, inorganic salt, moisture content, inoculum level, fermentation temperature, and fermentation time were investigated. The detoxifying effects of different products of GJ00141 were examined with gossypol as substrate. A total of 90 one-day-old broilers were randomized into group A [control, basal diet with 36% soybean meal(SM)], group B(basal diet with 18% SM and 18% CSM), and group C [basal diet with 18% SM and 18% fermented CSM(FCSM)] and thereby the influence of FCSM on the growth of broilers was explored. [Results] The maximum reduction rate(59%) of FG was achieved under the following fermentation conditions: solid medium composed of 96% CSM, 1%glucose, 1% ammonium sulfate, and 2% corn grits, 45% moisture content, 20%inoculum, fermentation at 30 °C for 60 h. Both the viable and inactivated cells of GJ00141 can reduce the content of gossypol, but the reduction rates were only about 20% after 72 h of incubation. Cellular contents and supernatant demonstrated strong detoxifying activity, which achieved the reduction rates of about 95% after 48 h, and the removal was free from the influence of proteinase K, heat, or EDTA. In the 42 d feeding experiment on broilers, the ratios of feed to gain were insignificantly different between the group C and group A. [Conclusion] This method achieved high rate of removing FG in CSM. The reason was the likelihood that the stable compounds in the cellular contents and supernatant of GJ00141 adsorb or bind to FG. Broilers grew well with the FCSM. Thus, it was an efficient detoxifying method for CSM.

Optimization of Solid State Fermentation Conditions Using a Mixture of Bean Curd Residue and Marc with Bacillus natto

[Objective] The aim was to optimize the appropriate solid state fermentation(SSF)conditions.[Method] The optimization of solid state fermentation using a mixture substrate of bean curd residue and the marc with Bacillus natto was developed.[Result] The best fermentation condition optimized by the test of single factor and the orthogonal design respectively was mixing ratio of bean curd residue to marc 2∶1,substrate pH value 6,fermentation temperature 39 ℃,inoculum volume 10% and fermentation time 48 h.Under this optimized fermentation condition,the content of crude fiber in the substrate decreased from 107.8 mg/g before SSF to 56.2 mg/g after SSF,and the degeneration rate of crude fiber was 47.87%.[Conclusion] The bean curd residue in its palatability was enormously improved by SSF with Bacillus natto strain,which could be expected to be widely used as raw material of health foodstuff.

Study on Free Amino Acid and Short Peptide Fertilizer Production by Solid State Fermented Castor Bean Meal

Using microbial fermentation to increase the content of free amino acids and short peptides in the organic fertilizer for castor bean meal can effectively promote plant growth and improve fruit quality.Using free amino acid and short peptide content as an indicator,through single factor and response surface optimization experiments,the process parameters(moisture content,fermentation time and inoculum quantity)of castor meal solid-state fermentation were optimized.The best process parameters for the solid-state fermentation were:the moisture content 62%,the fermentation time 20 d,and the inoculum quantity 0.23%.The moisture content had the greatest impact on the conversion rate of free amino acids and short peptides,and the protein conversion rate reached 65.6%.The scale-up experiment under the optimal conditions showed that the solid-state fermentation using the inoculum had a significant beneficial effect compared with other fermentation methods.The fermentation of castor cake fertilizer provides a theoretical and practical basis for production feasibility,and has important guiding significance for the effective utilization of castor bean meal.

Multi-enzymes Production Studies in Single Tray Solid State Fermentation with Opened and Closed System

The robustness ofA. awamori and A. oryzae as enzyme producers is exploited in fungal fermentation on agricultural solid waste. High-level production of extracellular glucoamylase, protease, cellulase and xylanase has been achieved. Three different types of ＇waste＇ solids （wheat bran, soybean hulls and rapeseed meal） have been used in studies of solid state fermentation （SSF）. The enzymes could be produced in significant levels by continuously supplying oxygen （02） through the tray system known as ＂closed＂ and ＂opened＂ tray systems. A perforated tray system was developed in this study that permits direct access to 02. Testing the tray system with different perforated mesh aperture sizes in this study did not yield different results in growth performance of A. awamori and A. oryzae. A. awamori and A. oryzae can be very versatile in producing various enzymes with different substrates with different starch, protein, hemiceilulose and cellulose contents. These studies indicate that A. awamori is more suitable for the efficient production of multiple enzymes in the closed system including xylanase and cellulase, while the production of glucoamylase and protease is superior in the opened system. A. oryzae is more suitable for the efficient production of protease and cellulase in the closed system, while the production of protease is more favourable the opened system. A. awamori efficiently consumed starch in wheat bran medium and produced very high glucoamylase activity, and after that, the fungus efficiently produced other enzymes to degrade other complex nutrients such as protein, hemicellulose and cellulose. Meanwhile, A. oryzae efficiently consumed protein in rapeseed meal and produced very high protease activity. The ability of both filamentous fungi, to convert biomass through SSF bioconversion will have a great impact on food and agro-industry in every aspect of life from food and medicine to fuel.

Production of Amylase Enzyme through Solid State Fermentation by Aspergillus niger

Traditionally, coconut dregs will be used as animal feed after the extraction of coconut oil and coconut milk from the copra. This study was carried out to discover the commercial value of coconut dregs as a solid substrate in the production of amylase through solid state fermentation （SSF） since this agro-waste is fairly rich in nutrients, providing the necessary nutrients supplementation for better microbial activity to produce enzymes. In this study, amylase is to be produced from coconut dregs by Aspergillus niger through solid state fermentation （SSF）. Three parameters were covered, which are incubation time, initial moisture content of substrate and inoculum sizes. SSF was carried out by using incubator at 37 ~C to test for enzyme activity at these following parameters： incubation time： 24, 48, 72, 96 and 120 hours; substrate moisture content： 64, 66, 68, 70 and 72% （w/w）; inoculum sizes： 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL spore suspension （5.5 × 10^6 spores/mL）. Enzyme activities were measured through the estimation of liberated reducing sugars after the assay of amylase enzyme by using DNS （3, 5 dinitrosalicylic acid） method. Highest enzyme activities were obtained at these following parameters： incubation time： 72 hours （31.76 U/gds）; initial moisture content ofsubstrate： 66% （26.66 U / gds） and inoculum sizes： 2.0 mL （30.56 U/gds）.

Solid-State Fermentation Production of Chitosanase by <i>Streptomyces</i>with Waste Mycelia of <i>Aspergillus niger</i>

Solid-state fermentation was carried out using mycelium powder of Aspergillus niger as substrate for the production of chitosanase of Streptomyces. Results of the experiments indicated that the optimal medium consisted of wheat bran and mycelium powder of Aspergillus niger with initial moisture content of 60% - 70%. The enzyme activity reached 41.33 U per gram dry medium after cultured for 5 days at 28°C - 30°C and an initial pH 6.5. Chitosanase was detected on the second day of incubation and had maximal activity at 5 days and decreased gradually within a 1 month period. Solid-state fermentation is maybe an economic alternative in the production.

Pool of Biological Resources for Potential Applications in Solid State Fermentation Obtained from a Forest Plantation of Pinus pseudostrobus Lindl, Mexico

A forest plantation, product of the reforestation of pine trees, represented a pool of biological resources for the implementation of a solid state fermentation process. The trees were identified as Pinus pseudostrobus Lindl from which lignocellulosic material in the form of pine needles was collected. Soil fungi, responsible for plant litter decomposition, were cultured at laboratory conditions and tested for their ability to grow on cellulose and hemicellulose as the sole carbon sources. A fungal strain, belonging to the genus Penicillium, was selected for growing it on pine needles as the substrate in a solid state culture. After following the culture for six days, the newly isolated strain exhibited a much higher capacity for spore production and holocellulose degradation, compared to a purchased strain of Penicillium chrysogenum and two control conditions. This work marks the beginning of future studies focused on commercial applications and represents the first report of a biotechnological process based on pine needles and their degradation by an ascomycetes species belonging to the genus Penicillium.

Water Retention Value： A Study Model-based by Asperglillus awamori and Aspergillus oryzae Embrace Three Models of Solid Substrate

The goal of this study was to evaluate the water retention value （WRV） of a test solid substrate and a fungal cell in solid state fermentation （SSF）. WRV is the ratio of the weight of water retained after centrifugation under specific conditions by a wet sample to the oven dry weight of the same sample. SSF refers to the microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment. Many factors are involved in a successful SSF process. In addition to biological parameters, the SSF process is also dependent on physical factors such as WRV. A centrifugal technique has been modified and applied to the evaluation of WRV. Wheat bran, soybean hulls and rapeseed meal were used as model substrate. Aspergillus awamori and Aspergillus oryzae were used as model microorganism. Results revealed that the ability of wheat bran to retain water in the solid substrate is 56% higher than that of soybean hulls and rapeseed meal. In the term of fungal cell, the ability of A. oryzae to retain water in the cells was higher （73% higher） than that ofA. awamori. In addition, through oven method moisture content loss from A. awamori is 46% higher than that from A. oryzae during drying process. Nevertheless, it can be seen that A. oryzae is able to retain water content about 5 times higher than A. awamori. Through this results, we found that WRV varies depending on solid substrates and microorganisms. This initial information can be beneficial in the SSF process to be carried out.

Biotransformation of Shrimp Wastes by Bacillus subtilis OKF04 and Evaluation of Growth Promoting Effect in Crop Planting

In this study,we proposed a reliable and sustainable technique for the clean utilization of shrimp wastes,which can yield a solid inoculant of Bacillus subtilis OKF04 containing micronutrients at low cost without the risk of contamination.Study of the culture conditions revealed that the head of shrimp Litopenaus vannamei and the wheat bran acted as suitable substrates for the growth of B.subtilis OKF04.With 60%initial moisture content,30℃culture temperature,and 5%inoculation amount,followed by 48 hours of fermentation and 0.5%soluble starch added during the drying process(50℃for 6h),a solid B.subtilis OKF04 inoculant with a spore amount of 2.4×10^(10)CFU g^(-1)and a high amino acid content was obtained.The solid B.subtilis OKF04 inoculant was applied to cultivate pakchoi under pot experiment.As the result,of adding to,the size of stems and leaves,nutritional composition,and physiological activity of pakchoi were significantly(P<0.05)enhanced by solid B.subtilis OKF04 inoculant.B.subtilis OKF04 also significantly(P<0.05)increased the soil’s nutrient content and improved its microbial composition.Furthermore,pakchoi cultivated with a low dose of solid B.subtilis OKF04 inoculant(0.05 g kg^(-1)soil)resulted in the best results.This study provides a new method for the preparation of microbial inoculants with solid waste shrimp heads.

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.

Biological pretreatment of corn stover by solid state fermentation of Phanerochaete chrysosporium

Biological pretreatment is a promising way to overcome the biorecalcitrance of cleaving the super- molecular structure of lignocellulose by lignin degrading enzymes from microorganisms. Solid state fermentation of corn stover with the white-rot fungus Phanerochaete chrysosporium was carried out and the efficiency of this pretreatment was evaluated. The enzymatic hydrolysis yield reached a maximum when the corn stover was biologically pretreated for nine days, and the hydrolysis yield decreased sharply if the solid state fermentation was carried out for more than nine days. A possible explanation for this sharp decrease is that not only the lignin degrading enzymes （LiP and MnP） were secreted, but also other metabolites, which were toxic or fatal to the hydrolysis enzymes resulting in the lower hydrolysis yield were generated during the prolonged period of biopretreatment. These results are usefuI to help determine the optimal timing and to understand the lignin structure and degradation mechanism in biological pretreatment processes.

Purification and Characterisation of a Coffee Pulp Tannase Produced by Penicillium verrucosum

Tannase was extracted from a new fungal strain Penicillium verrucosum using coffee pulp as a substrate by solid state fermentation. The extracellular tannase was purified using Sephadex G-100 to 86.02 folds with 34.3% yield and a single band corresponding to 81 kDa was observed in SDS-PAGE. Tannase was immobilized on alginate by entrapment method. The graphical analysis of the effect of the substrate concentration on the tannase activity yielded a Km of 48.88 mM and a Vmax of 169.49 U/mL. The optimum temperature and pH of the enzyme were 30 ℃ and pH 5. The thermal stability, as well as stability over a wide range ofpH, was significantly improved by the immobilization process. The activity was enhanced by Mg^2＋ （23.5%） and K^＋ （5.3%）, whereas Zn^2＋, Cu^2＋, Ca^2＋, NH^4＋ and Hg^2＋ inhibited tannase activity. Maximum substrate bioconversion （58.94%） from tannic acid to gallic acid and tea cream solubilisation （32.82%） was achieved using immobilized tannase. The extracellular tannase produced by P. verrucosum showed distinctive characteristics such as monomeric structure and activation by Mg^2＋, suggesting a new kind of fungal tannase with biotechnological potential.

Cellulase Production from Species of Fungi and Bacteria from Agricultural Wastes and Its Utilization in Industry: A Review

In energy deficient world, cellulases play a major role for the production of alternative energy resources utilizing lignocellulosic waste materials for bioethanol and biogas production. This study highlights fungal and bacterial strains for the production of cellulases and its industrial applications. Solid State Fermentation (SSF) is more suitable process for cellulase production as compared to submerge fermentation techniques. Fungal cellulosomes system for the production of cellulases is more desirable and resistant to harsh environmental conditions. Trichoderma species are considered as most suitable candidate for cellulase production and utilization in industry as compared to Aspergillus and Humicola species. However, genetically modified strains of Aspergillus have capability to produce cellulase in relatively higher amount. Bacterial cellulase are more resistant to alkaline and thermophile conditions and good candidate in laundries. Cellulases are used in variety of industries such as textile, detergents and laundries, food industry, paper and pulp industry and biofuel production. Thermally stable modified strains of fungi and bacteria are good future prospect for cellulase production.

Saccharification of Sugarcane Bagasse Using an Enzymatic Extract Produced by Aspergillus fumigatus

This study investigates the efficiency of a crude enzymatic extract produced by Aspergillus fumigatus SCBM6 by solid state fermentation(SSF)in the hydrolysis of alkali pretreated sugarcane bagasse(PTB).After SSF using in natura sugarcane bagasse(SCB),the enzymatic extract presented 21.33 U.g^-1 of β-glucosidase and 544.46 U.g^-1 of xylanase.The alkaline pretreatment with sodium hydroxide 2%NaOH(w/v)removed 43% of the lignin from PTB and the cellulosic fraction increased to 75%.The hydrolysis was optimized as a function of time,temperature,and concentration of PTB.After hydrolysis,the maximum yield(30.05%)of total released reducing sugars(TRS)was obtained under the following conditions:24 h,55℃,2% of PTB and 3 U.g^-1 of β-glucosidase(CBU).Furthermore,an approximate TRS value(26.4%)was also obtained after saccharification carried out during 6 h,55℃,4% of PTB and 1 CBU.These results indicate that hydrolysis can be performed in a short incubation period and with low enzymatic load for reasonable TRS release.

Inclusion Levels of Fermented Apple Bagasse on in Vitro Rumen Fermentation of Alfalfa Hay

The aim of this study was to assess the effect of inclusion of fermented apple bagasse （FAB） obtained through solid state fermentation on pH, ammonia nitrogen （N-NH3）, volatile fatty acids （VFA） content, in vitro dry matter digestibility （IVDMD）, lactic acid and microbial counting of alfalfa hay under in vitro rumen environment; four levels of FAB were evaluated （0, 0.25, 0.50 and 0. 75 g/dry matter of FAB） replacing 1.5 g dry matter （DM） of alfalfa hay and incubated at different fermentation times （0, 4, 8, 12 and 24 h） using a complete random design with repeated measures on time. Counts of live yeast colonies （6.08, 6.33, 6.24 and 6.51 CFU/mL expressed as log 10） was higher when FAB was included in the different levels up to the 12 h of fermentation （P 〈 0.0001）; lactic acid content also increased as FAB was included in the different levels （10.61, 13.86, 16.84 and 14.57μg/mL） up to the 12 h of incubation （P 〈 0.001）. Nevertheless, the other variables measured as pH, N-NH3, VFA, IVDMD, total bacteria and fungi counts, were not affected by the treatments. It is concluded that substitution of FAB by alfalfa hay in an in vitro rumen ecosystem positively modified live yeast colonies and lactic acid concentration, without effect on the other fermentative and microbial parameters of the in vitro rumen environment, but considering mixes of FAB and alfalfa hay as a quality ingredient for the feeding of ruminants.

Partial purification,immobilization and preliminary biochemical characterization of lipases from Rhizomucor pusillus

Lipases have important applications in biotechnological processes, motivating us to produce, purify, immobilize and perform a biochemical characterization of the lipase from Rhizomucor pusillus. The fungus was cultivated by solid state fermentation producing lipolytic activity of about 0.5 U/mL(4U/g). A partial purification by gel filtration chromatography in Se-phacryl S-100 allowed obtaining a yield of about 85% and a purification factor of 5.7. Our results revealed that the purified enzyme is very stable with some significant differences in its properties when compared to crude extract. The crude enzyme extract has an optimum pH and temperature of 7.5 ° C and 40 ° C, respectively. After purification, a shift of the optimum pH from 7 to 8 was observed, as well as a rise in optimumtemperature to 60 ° C and an increase in stability. The enzyme was immobilized on CNBr-Agarose and Octyl-Agarose supports, having the highest immobilization yield of 94% in the second resin. The major advantage of immobilization in hydrophobic media such as Octyl is in its hyper activation, which in this case was over 200%, a very interesting finding. Another advantage of this type of immobilization is the possibility of using the derivatives in biotechnological applications, such as in oil enriched with omega-3 as the results obtained in this study display the hydrolysis of 40% EPA and 7% DHA from sardine oil, promising results compared to the literature.