棉籽粕的发酵脱毒及其在肉仔鸡中的应用研究
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摘要
本论文首先通过微生物发酵试验筛选出了能够有效降解棉粕中有害成分游离棉酚(Free Gossypol,FG)的微生物菌株,并且优化出了该微生物脱毒棉粕的最佳发酵工艺条件,结果表明,料水比1:0.94、发酵时间62.33h、装料量30.96g/150mL、接种量10%、麸皮量为20%时脱毒率最大(接近86%);随后进行了发酵棉粕在肉仔鸡日粮中的应用试验研究,结果表明,与对照组相比,发酵棉粕未对肉仔鸡生产性能、肉品质、免疫器官指数及表观能量和蛋白代谢产生显著影响,当添加量为20%时,肠道乳酸菌显著增加(P<0.05),大肠杆菌显著降低(P<0.05),且最节省成本。因此,发酵脱毒棉粕的最佳添加量为20%。
试验一发酵菌种的筛选
试验筛选了能够高效降解棉酚的微生物菌种。首先以白地霉、扣囊、热带假丝酵母、产朊假丝酵母、3株黑曲霉和2株米曲霉9种菌株为候选菌种,使用无碳源液体培养基各30ml,另加棉粕2g进行液态发酵,25~28℃,250 r/min,摇床48h后取出各菌种发酵液测定游离棉酚含量,结果表明,产朊假丝酵母对棉酚的降解率在70%左右,其他菌种未见显著效果;接着再用产朊假丝酵母固态发酵棉粕和麸皮混合物,150ml三角瓶装料30g,28℃静置培养48h,每12h进行一次搅拌,48h后取出发酵物烘干、粉碎、测定棉酚含量。结果表明,产朊假丝酵母固态发酵棉粕脱毒率在65%~70%之间。这表明产朊假丝酵母具有有效降解棉酚的作用。
试验二棉籽粕发酵工艺条件的优化
本试验优化了产朊假丝酵母降解棉粕中游离棉酚的最佳工艺参数。首先利用SAS软件的Plackett-Burman设计法对产朊假丝酵母固态发酵棉粕的发酵参数进行了筛选,得出3个影响较大的重要因素,分别为:料水比、发酵时间和装料量,然后再用SAS软件二次响应面分析法进行回归分析,得到了各因素的最佳水平值。结果表明,料水比1:0.94、发酵时间62.33h、装料量30.96g/150mL时脱毒率(85.99%)最高,与优化前相比提高近20%;随后在响应面优化基础上对麸皮和接种量进行了单因素优化。结果表明,接种量10%、麸皮量为20%时脱毒率相对最高。棉粕发酵后粗蛋白含量未有变化,可溶性蛋白从6.96%提高到10.2%,必需氨基酸——赖氨酸、苏氨酸、亮氨酸、异亮氨酸等的含量增加。发酵工艺参数优化后,棉粕的脱毒率有所提高,且发酵后棉粕的营养价值有所改善。
试验三发酵棉籽粕对肉仔鸡生产性能及相关指标的影响
本试验探讨了发酵棉粕对肉仔鸡生产性能、肉品质、免疫器官指数和蛋白能量代谢的影响。选用192只1日龄AA肉仔鸡,随机分为4个处理组,每处理6个重复,每重复8只鸡,对照组饲喂玉米豆粕基础日粮,处理组1-3分别添加发酵棉粕15%、20%和30%替代部分豆粕。试验期6周。试验结果显示,发酵棉粕处理组的肉仔鸡生产性能、肉品质、免疫器官指数和表观蛋白代谢值与对照组相比未有显著差异(p>0.05)。综合考虑经济成本认为,发酵棉粕的最佳添加量为20%。
试验四发酵棉籽粕对肉仔鸡肠道微生物区系的影响
本试验采用传统微生物培养和PCR-DGGE两种方法研究了发酵棉粕对肉仔鸡盲肠几种主要微生物的数量及区系的影响。选用192只1日龄AA肉仔鸡,随机分为4个处理组,每处理6个重复,每重复8只鸡,对照组饲喂玉米豆粕基础日粮,处理组1-3分别添加发酵棉粕15%、20%和30%替代部分豆粕。试验期6周。试验最后一天从每个重复中随机抽取1只肉仔鸡进行屠宰,无菌采集盲肠食糜,分析盲肠微生物数量和区系。结果表明,当发酵棉粕添加量为15%和20%时,盲肠乳酸杆菌的数量显著增加(P<0.05),同时大肠杆菌数量显著减少(P<0.05),双歧杆菌的数量增加但未达到显著水平(P>0.05);PCR-DGGE图谱表明,饲喂发酵棉粕后,肠道内微生物种群结构有轻微变化,发酵棉粕增加了微生物种群数量,各处理组中微生物种群类型既有相似又有差异,但未出现显著差异。试验表明,发酵棉粕可调节肉仔鸡肠道的微生态平衡,使有益菌尤其是乳杆菌的数量增加而大肠杆菌数量减少。
The microbe which can degrade free gossypol(FG) in cotton seed meal(CSM) was screened in this study, and optimal fermentation parameters for bio-degradation of FG was studied.The result showed substrate:water (moisture)1:0.94, fermentation time62.33h, material adding 30.96g/150mL, inoculum size 10%, wheat bran 20% where the elimination rate of FG was maximized to 86%.Animal experiment was also conducted to study effects of fermented cottonseed meal(FCSM) in diets on broilers. It showed that growth performance, meat quality, immune organ indices, apparent protein and enegy digestibility were not affected(P>0.05) in FCSM groups compared to the control, and viable count of Lactobacillus was enriched and E.coli was reduced significantly when birds fed a diet containing 20% FCSM. In conclusion, the optimum added proportion of FCSM was 20%.
Experiment 1. The Screening of Microbe for Detoxification of Free Gossypol in Cootonseed Meal
This experiment was conducted to screen a microbe which can degrade FG in CSM.The liquid-state CSM culture without other carbon source was used to screen the microbe needed from 9 strains, 25~28℃, 250 r/min, and FG level was determined after 48h shaking culture, the result showed Candida utilis can degrade 70% FG in CSM, and no difference was observed in other 8 strains, then Candida utilis was used to ferment CSM in solid state substrate, 28℃, FG level was determined in fermented substrate after 48h static-culture. The result showed that elimination rate was around 65%~70%. The tested Candida utilis in this trial could largely degrade FG in CSM.
Experiment 2. The Optimization for Bio-detoxification of Free Gossypol in Cottonseed Meal
The optimal condition for biodegradation of FG was studied. Plackett-burman and response surface analysis method were applied to optimize fermentation parameters. The results indicated the elimination rate was mainly influenced by moisture, material adding,and fermentation time by Plackett-burman.In the second step, the levels of those three main factors were further optimized by using Box-Behnken design, the optimal levels of the variables were as follows: moisture 48% (water:substrate=0.94:1), material adding 30.96g, fermentation time 62.33h. Under the optimal conditions, the elimination rate was increased by almost 20%, up to 85.99%. At three fermentation batches under optimization, the experiment values were almost close to the predictive values. After fermentation, the crude protein level was not affected, and soluble protein was enhanced from 6.96% to 10.2%, some essential amino acids levels were also increased, but arginine was decreased. After optimization, FG was reduced largely, and the quality of cottonseed meal was improved remarkably.
Experiment 3. Studies on Application of Fermented Cottonseed Meal in Broiler Diet
This experiment was conducted to study the effects of fermented cottonseed meal (FCSM) in broiler diets on growth performance, meat quality, immune organ and digestibility of gross energy and crude protein.A total of one hundred and ninty-two day-old broiler chickens were alloted to 4 treatments with 6 replicates of 8 chickens each. The experiment lasted 6 weeks. The dietary treatments were: (1) 15% FCSM; (2) 20% FCSM;(3) 30% FCSM ; (4) the control (corn-soybean diet without FCSM) . The result showed no significant differences in growth performance were found between FCSM and the control group (P>0.05). Compared with the control, meat quality, immune organ and metabolism of gross energy and crude protein in broilers of FCSM groups were not affected (P>0.05). Taking economic cost into consideration, the optimum added amount of FCSM was 20%.
Experiment 4. Effect of Fermented Cottonseed Meal on Intestinal Microbial Flora in Broilers
The effect of FCSM on intestinal microbial community in broilers was studied by traditional method and denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA. A total of one hundred and ninty-two day-old broiler chickens were alloted into 4 treatments with 6 replicates of 8 chickens each.The experiment lasted 6 weeks. The dietary treatments were: (1) 15% FCSM; (2) 20% FCSM; (3) 30% FCSM ; (4) the control (corn-soybean diet without FCSM). At the end of the experiment, 24 randomly selected chickens from each replicate were sacrified to study microflora of caecum contents. The traditional method showed that compared with the control, FCSM groups did not affect Bifidobacterium enumeration (P>0.05), whereas, viable count of Lactobacillus was enriched and E. coli was reduced significantly when birds were fed diets containing 15% and 20% FCSM(P<0.05). DGGE analysis showed there were no abnormity of the microflora in the cecum. In conclusion, FCSM can improve intestinal micro-ecosystem, increased beneficial bacteria and reduced harmful bacteria.The optimum added amount of FCSM was 20%.
试验一发酵菌种的筛选
试验筛选了能够高效降解棉酚的微生物菌种。首先以白地霉、扣囊、热带假丝酵母、产朊假丝酵母、3株黑曲霉和2株米曲霉9种菌株为候选菌种,使用无碳源液体培养基各30ml,另加棉粕2g进行液态发酵,25~28℃,250 r/min,摇床48h后取出各菌种发酵液测定游离棉酚含量,结果表明,产朊假丝酵母对棉酚的降解率在70%左右,其他菌种未见显著效果;接着再用产朊假丝酵母固态发酵棉粕和麸皮混合物,150ml三角瓶装料30g,28℃静置培养48h,每12h进行一次搅拌,48h后取出发酵物烘干、粉碎、测定棉酚含量。结果表明,产朊假丝酵母固态发酵棉粕脱毒率在65%~70%之间。这表明产朊假丝酵母具有有效降解棉酚的作用。
试验二棉籽粕发酵工艺条件的优化
本试验优化了产朊假丝酵母降解棉粕中游离棉酚的最佳工艺参数。首先利用SAS软件的Plackett-Burman设计法对产朊假丝酵母固态发酵棉粕的发酵参数进行了筛选,得出3个影响较大的重要因素,分别为:料水比、发酵时间和装料量,然后再用SAS软件二次响应面分析法进行回归分析,得到了各因素的最佳水平值。结果表明,料水比1:0.94、发酵时间62.33h、装料量30.96g/150mL时脱毒率(85.99%)最高,与优化前相比提高近20%;随后在响应面优化基础上对麸皮和接种量进行了单因素优化。结果表明,接种量10%、麸皮量为20%时脱毒率相对最高。棉粕发酵后粗蛋白含量未有变化,可溶性蛋白从6.96%提高到10.2%,必需氨基酸——赖氨酸、苏氨酸、亮氨酸、异亮氨酸等的含量增加。发酵工艺参数优化后,棉粕的脱毒率有所提高,且发酵后棉粕的营养价值有所改善。
试验三发酵棉籽粕对肉仔鸡生产性能及相关指标的影响
本试验探讨了发酵棉粕对肉仔鸡生产性能、肉品质、免疫器官指数和蛋白能量代谢的影响。选用192只1日龄AA肉仔鸡,随机分为4个处理组,每处理6个重复,每重复8只鸡,对照组饲喂玉米豆粕基础日粮,处理组1-3分别添加发酵棉粕15%、20%和30%替代部分豆粕。试验期6周。试验结果显示,发酵棉粕处理组的肉仔鸡生产性能、肉品质、免疫器官指数和表观蛋白代谢值与对照组相比未有显著差异(p>0.05)。综合考虑经济成本认为,发酵棉粕的最佳添加量为20%。
试验四发酵棉籽粕对肉仔鸡肠道微生物区系的影响
本试验采用传统微生物培养和PCR-DGGE两种方法研究了发酵棉粕对肉仔鸡盲肠几种主要微生物的数量及区系的影响。选用192只1日龄AA肉仔鸡,随机分为4个处理组,每处理6个重复,每重复8只鸡,对照组饲喂玉米豆粕基础日粮,处理组1-3分别添加发酵棉粕15%、20%和30%替代部分豆粕。试验期6周。试验最后一天从每个重复中随机抽取1只肉仔鸡进行屠宰,无菌采集盲肠食糜,分析盲肠微生物数量和区系。结果表明,当发酵棉粕添加量为15%和20%时,盲肠乳酸杆菌的数量显著增加(P<0.05),同时大肠杆菌数量显著减少(P<0.05),双歧杆菌的数量增加但未达到显著水平(P>0.05);PCR-DGGE图谱表明,饲喂发酵棉粕后,肠道内微生物种群结构有轻微变化,发酵棉粕增加了微生物种群数量,各处理组中微生物种群类型既有相似又有差异,但未出现显著差异。试验表明,发酵棉粕可调节肉仔鸡肠道的微生态平衡,使有益菌尤其是乳杆菌的数量增加而大肠杆菌数量减少。
The microbe which can degrade free gossypol(FG) in cotton seed meal(CSM) was screened in this study, and optimal fermentation parameters for bio-degradation of FG was studied.The result showed substrate:water (moisture)1:0.94, fermentation time62.33h, material adding 30.96g/150mL, inoculum size 10%, wheat bran 20% where the elimination rate of FG was maximized to 86%.Animal experiment was also conducted to study effects of fermented cottonseed meal(FCSM) in diets on broilers. It showed that growth performance, meat quality, immune organ indices, apparent protein and enegy digestibility were not affected(P>0.05) in FCSM groups compared to the control, and viable count of Lactobacillus was enriched and E.coli was reduced significantly when birds fed a diet containing 20% FCSM. In conclusion, the optimum added proportion of FCSM was 20%.
Experiment 1. The Screening of Microbe for Detoxification of Free Gossypol in Cootonseed Meal
This experiment was conducted to screen a microbe which can degrade FG in CSM.The liquid-state CSM culture without other carbon source was used to screen the microbe needed from 9 strains, 25~28℃, 250 r/min, and FG level was determined after 48h shaking culture, the result showed Candida utilis can degrade 70% FG in CSM, and no difference was observed in other 8 strains, then Candida utilis was used to ferment CSM in solid state substrate, 28℃, FG level was determined in fermented substrate after 48h static-culture. The result showed that elimination rate was around 65%~70%. The tested Candida utilis in this trial could largely degrade FG in CSM.
Experiment 2. The Optimization for Bio-detoxification of Free Gossypol in Cottonseed Meal
The optimal condition for biodegradation of FG was studied. Plackett-burman and response surface analysis method were applied to optimize fermentation parameters. The results indicated the elimination rate was mainly influenced by moisture, material adding,and fermentation time by Plackett-burman.In the second step, the levels of those three main factors were further optimized by using Box-Behnken design, the optimal levels of the variables were as follows: moisture 48% (water:substrate=0.94:1), material adding 30.96g, fermentation time 62.33h. Under the optimal conditions, the elimination rate was increased by almost 20%, up to 85.99%. At three fermentation batches under optimization, the experiment values were almost close to the predictive values. After fermentation, the crude protein level was not affected, and soluble protein was enhanced from 6.96% to 10.2%, some essential amino acids levels were also increased, but arginine was decreased. After optimization, FG was reduced largely, and the quality of cottonseed meal was improved remarkably.
Experiment 3. Studies on Application of Fermented Cottonseed Meal in Broiler Diet
This experiment was conducted to study the effects of fermented cottonseed meal (FCSM) in broiler diets on growth performance, meat quality, immune organ and digestibility of gross energy and crude protein.A total of one hundred and ninty-two day-old broiler chickens were alloted to 4 treatments with 6 replicates of 8 chickens each. The experiment lasted 6 weeks. The dietary treatments were: (1) 15% FCSM; (2) 20% FCSM;(3) 30% FCSM ; (4) the control (corn-soybean diet without FCSM) . The result showed no significant differences in growth performance were found between FCSM and the control group (P>0.05). Compared with the control, meat quality, immune organ and metabolism of gross energy and crude protein in broilers of FCSM groups were not affected (P>0.05). Taking economic cost into consideration, the optimum added amount of FCSM was 20%.
Experiment 4. Effect of Fermented Cottonseed Meal on Intestinal Microbial Flora in Broilers
The effect of FCSM on intestinal microbial community in broilers was studied by traditional method and denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA. A total of one hundred and ninty-two day-old broiler chickens were alloted into 4 treatments with 6 replicates of 8 chickens each.The experiment lasted 6 weeks. The dietary treatments were: (1) 15% FCSM; (2) 20% FCSM; (3) 30% FCSM ; (4) the control (corn-soybean diet without FCSM). At the end of the experiment, 24 randomly selected chickens from each replicate were sacrified to study microflora of caecum contents. The traditional method showed that compared with the control, FCSM groups did not affect Bifidobacterium enumeration (P>0.05), whereas, viable count of Lactobacillus was enriched and E. coli was reduced significantly when birds were fed diets containing 15% and 20% FCSM(P<0.05). DGGE analysis showed there were no abnormity of the microflora in the cecum. In conclusion, FCSM can improve intestinal micro-ecosystem, increased beneficial bacteria and reduced harmful bacteria.The optimum added amount of FCSM was 20%.
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