Screening And Breeding Of Highly Effective Strains For Degrading Gossypol And Biological Fermentation Of Cottonseed Meal

Cottonseed meal (CSM) with high concentration of protein is a very good resources of plant protein feedstuff. However, the potential of cottonseed meal for animal feed is limited by the presence of gossypol (C₃₀H₃₀O₈), a toxic polyphenolic pigment. Therefore, a number of methods have been developed for removing gossypol from cottonseed meal including solvent extraction of free gossypol (FG), chemical treatment with ferrous sulfate or calcium hydroxide, and microbes fermentation. These methods play an important role in detoxification of CSM, but the reduction of gossypol using solvent system suffers from the difficulty of totally removing residual solvents that may be potentially harmful or have detrimental effects on flavour. Ferrous sulfate causes feed turn black and feed intake depression, whereas calcium hydroxide often reduces the biological activity of vitamins and lower detoxification efficiency. Microbial fermentation may offer promise as a detoxification method, because fermented CSM not only contains lower free gossypol and higher protein, but also contains some kinds of exoenzyme (secreted by microorganism) such as cellulolytic enzyme, amylase, protease and lipolytic enzyme, some variety of vitamins, and some unknown active substance, therefore, it was paid more attention.In the present study, a strain with high efficiency of gossypol detoxification was screened out; the process parameters for fermentation detoxification was optimized; the factors of affecting fermentation detoxification, mixed cultures fermentation and its influence factors were also investigated.The Czapek's medium containing acetic gossypol instead of sucrose as carbon source and malt extract medium with acetic gossypol were used for plate screening. Cottonseed meal was employed for practically biological fermentation. Through these methods, a strain of Candida tropicalis ZD-3 with high efficiency of gossypol detoxification was screened out. The cell shape of Candida tropicalis ZD-3 was oval, with size of 3～4×4～5μm, and its reproduction was by budding. Cultured by malt extract medium, yeast body was usually sunk to the bottom of test tube. Cultured by malt extract solid medium, plate colony surface had wrinkle, and could form pseudomycelium. C. tropicalis ZD-3 could not fermented raffinose under the semi-anaerobic or aerobic conditions, therefore, fermentable carbohydrates such as starch need to be supplemented when CSM was fermented for detoxification. The log growth phase of C. tropicalis ZD-3 was at the range of 12～18 h, during this phase, the culture fit for fermentation or other research.To optimize the process parameters for detoxification of gossypol in CSM by C. tropicalis ZD-3 during solid substrate fermentation (SSF). The maximum detoxification efficiency of gossypol was achieved by employing the substrate which consists of 70% of CSM, 20% of corn flour and 10% of wheat bran. The optimum fermentation conditions for gossypol detoxification are incubation period of 48 h, incubation temperature at 30℃, inoculum level 5% v/w, moisture content of solid substrate 50% and pH in nature.Different treated CSM substrate was fermented by C. tropicalis ZD-3, then to assay free gossypol (FG), crude protein (CP) content in CSM substrate. The objective was to study the effect of carbohydrate sources, heat treatment, urea, ferrous sulfate, sodium carbonate and mixed minerals on the reduction of gossypol levels during solid substrate fermentation of CSM. The results showed that CSM substrate supplemented with selected carbohydrate sources was beneficial for fermentation detoxification, of which, starch was most effective, detoxification efficiency up to 91.91%; Heat treatment was instrumental in reducing gossypol levels during CSM fermentation, but CSM substrate supplemented with urea could decrease detoxification rate of FG, and was not beneficial for CSM fermentation; Adding ferrous sulfate, sodium carbonate and mixed minerals to CSM substrate could increase detoxification rate of FG significantly, C. tropicalis ZD-3 detoxification rate reached up to 94.70%, 93.96% and 96.67%, respectively.CSM substrate was added with 0, 0.5%, 1% and 2% of ethanolamine (or choline chloride) respectively, mixed evenly, then heat treatment and fermentation experiments were carried out. The results showed that CSM substrate supplemented with different percentage of ethanolamine or choline chloride was not beneficial for CSM heat treatment and fermentation detoxification.CSM substrate was treated with different concentration of ethanolamine, choline chloride, lecithin and heated whole soy, then fermented by C. tropicalis ZD-3. After fermentation was over, to assay the FG content and neutral, alkaline and acetic proteinase activity, as well as microbial numbers in fermented CSM substrate. The objective was to study the effect of ethanolamine, choline chloride, lecithin and heated whole soy on CSM fermentation.â‘ The results showed that, when substrate was heated, ethanolamine, choline chloride, lecithin and heated whole soy had no efficiency for fermentation detoxification, decreased detoxification rate significantly except for choline chloride; neutral proteinase activity was increased markedly by choline chloride, ethanolamine and lecithin; alkaline proteinase activity was improved greatly by choline chloride and lecithin; and acidic proteinase activity was elevated significantly by all treatment factors; microbial numbers in fermented substrates were increased markedly by all treatment factors, of which, the effect of choline chloride was best.â‘¡The results demonstrated that, when substrate was not heated, ethanolamine, lecithin and heated whole soy had no efficiency for fermentation detoxification, and decreased detoxification rate significantly, but choline chloride had the trend to increased detoxification rate; the neutral proteinase activity was increased greatly by lecithin and whole soy; the alkaline proteinase activity was elevated markedly by all treatment factors, but acidic proteinase activity was decreased significantly by all treatment factors; microbial numbers in substrate treated with choline chloride was increased greatly, whereas treated with others, microbial numbers were decreased significantly.â‘¢The results showed that choline chloride had important implication for fermentation detoxification of CSM, because its substrate proteinase activity was higher and microbial number was larger than other factors. When substrate was not heated, lower concentration of choline chloride could facilitate fermentation detoxification.Cottonseed meal was fermented by Candida tropicalis ZD-3 or (with) Aspergillus niger ZD-8, then to assay free gossypol, crude fiber (CF), crude fat (ether extract, EE), crude protein (CP) and amino acids content in CSM substrate, analyzing their in vitro digestibility of crude protein and amino acids, and investigating morphology of fermented CSM under the environmental scanning electron microscope (ESEM). The objective was to investigate the effect of mixed culture solid substrate fermentation of C. tropicalis ZD-3 with A. niger ZD-8 on nutritional value and detoxification of CSM. The results showed that the mixed culture fermentation could decrease significantly FG content in fermented CSM, detoxification rate up to 91.64%. Crude protein, total amino acids and essential amino acids content in fermented CSM were increased by 27.83%, 18.15% and 19.18%, respectively; and their in vitro digestibilities were also increased by 20.90%, 26.16% and 24.47%, respectively. Lysine and methionine levels in fermented CSM were improved by 20.24% and 66.29%. CF and EE content in fermented substrate were also decreased significantly. The changes of morphology of A. niger ZD-8 under ESEM were as follows: mycelium became thin, and sporangium was not observed but sporangiophore could be seen. The morphology of C. tropicalis ZD-3 was not found transformed. The study indicated that the fermentation efficiency of the mixed cultures was superior to that of single strain fermentation, which not only could detoxify gossypol in CSM, but also increase the nutritional value of fermented CSM. And found that morphology of A. niger ZD-8 had being transformed significantly by C. tropicalis ZD-3 during mixed culture solid substrate fermentation.Different treated CSM substrate was fermented by C. tropicalis ZD-3 with A. niger ZD-8, then assaying FG, crude protein (CP) content in fermented CSM substrate, and analyzing in vitro CP digestibility. Objective was to investigate the effect of fermentation period, proportion of CSM in substrate, sodium carbonate, heat treatment, ferrous sulfate and minerals on the reduction of FG levels during mixed culture fermentation of CSM.. The results showed that the proper fermentation period was 48h, and the proper substrate composition consisted of 70% CSM, 20% corn flour and 10% wheat bran. Addition of 0.5%～1.0% sodium carbonate to CSM substrate was beneficial for fermentation detoxification. Heat treatment could facilitate fermentation detoxification, and supplementation with ferrous sulfate or minerals was instrumental in reducing gossypol levels during mixed culture fermentation of CSM. These results were consistent with the experimental effects of C. tropicalis ZD-3 fermentation.