固态发酵对棉籽粕棉酚脱毒及蛋白质降解的影响
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摘要
本文研究了固态发酵中高温高压灭菌处理对棉籽粕的脱毒及假丝酵母菌与黑曲霉单独或复合固态发酵对棉籽粕中棉酚的脱毒及蛋白质降解的影响,探讨微生物固态发酵脱除棉籽粕中游离棉酚(FG)的机理及发酵前后棉籽蛋白质和肽分子量分布范围的变化规律。
一、假丝酵母菌和黑曲霉生长曲线的测定
分别通过光电比浊法和菌丝干重法测定了假丝酵母菌和黑曲霉的生长曲线。结果表明:5株假丝酵母菌在麦芽汁培养液中28℃培养24 h达到对数生长末期,2株黑曲霉在马铃薯葡萄糖培养液中28℃培养24 h达到对数生长末期。在对数生长末期将其种子液接入发酵底物中发酵效果最佳。
二、微生物固态发酵对棉籽粕棉酚脱毒及蛋白质降解的影响
以棉籽粕、玉米面及麦麸按质量比7:2:1均匀混合后粉碎、过60目筛所得样品为试验的对照组。试验一设5个处理组:加水拌湿不灭菌组和4个湿热灭菌20 min组(105℃、112℃、115℃、121℃);试验二设6个处理组:加水拌湿不灭菌组、2个100℃蒸汽灭菌组(30 min、60 min)及3个湿热灭菌组(115℃20 min、115℃40 min、121℃20 min);试验三研究115℃灭菌40 min条件下,假丝酵母菌和黑曲霉固态发酵脱毒及蛋白质降解规律,试验设9个处理组:灭菌不接种组、5个接种假丝酵母菌组(C1、C2、C3、C4、C5)、2个接种黑曲霉组(A6、A7)及接种复合菌组(C1和A6)。处理结束后立即放入干燥箱中,80℃烘干48 h,粉碎,过60目筛,-20℃低温密封保存待测。苯胺法测定灭菌或发酵处理前后棉籽粕底物中FG与总棉酚(TG)含量变化,SDS-PAGE凝胶电泳法分析发酵前后棉籽蛋白质的分子量分布范围变化,并通过透析袋法测定发酵产物的蛋白质体外消化率。试验一和试验二结果表明:加水拌湿不灭菌组(底物含水47.37%)、其棉籽粕底物混匀拌湿后立即80℃烘干48 h脱毒率为54.57%~68.21%,各灭菌组脱毒率均在85%以上,不同灭菌温度及灭菌时间对棉籽粕底物脱毒率的差异不显著(P>0.05)。试验三结果表明:1)在高温高压灭菌处理对棉籽粕底物中FG已大部分脱毒的情况下,接种假丝酵母菌未见显著降低FG含量(P>0.05),接种黑曲霉A6和A7发酵组反而使FG含量从灭菌处理组的24.739 mg/kg分别增加到211.451 mg/kg和176.135 mg/kg(P<0.01),假丝酵母菌和黑曲霉复合发酵组FG含量为112.244 mg/kg(P<0.01);2)在灭菌处理已极显著(P<0.01)降低棉籽粕底物结合棉酚(BG)和TG含量的情况下,各菌株发酵对BG和TG的含量影响差异不显著(P>0.05);3)假丝酵母及黑曲霉固态发酵均能将棉籽粕底物中分子量在20~100 KDa之间的大分子蛋白质降解为小分子蛋白质和肽,其中黑曲霉可将蛋白质降解为14 KDa以下的小分子蛋白质和肽;4)黑曲霉发酵组的棉籽粕底物蛋白质体外消化率比对照组提高19.25%(P<0.05),复合发酵组提高8.18%(P>0.05);5)棉籽粕假丝酵母菌、黑曲霉单菌种及复合固态发酵的产率分别为96.34%、88.42%和89.69%,粗蛋白(CP)含量分别提高4.31%、13.76%和13.09%,发酵产物的CP含量与发酵产率呈极显著的负相关:y=97.52352-0.52748x(R2=0.9942,P<0.0001)。
综合本研究结果:1)微生物固态发酵对棉籽粕棉酚脱毒主要是高温高压灭菌作用;2)假丝酵母对棉籽粕中FG有一定的脱毒作用,而黑曲霉增加了FG含量,二者对棉酚均没有降解作用;3)黑曲霉固态发酵棉籽粕具有较强的蛋白降解能力,可将高分子量的棉籽蛋白降解为14 KDa以下的小分子蛋白和肽;4)黑曲霉固态发酵可显著提高棉籽粕CP体外消化率;5)棉籽粕发酵产物中的CP含量与发酵产率之间存在显著的线性负相关。
This article were conducted to study the effect of treatment sterilized by high temperature and high pressure in the solid-state fermentation on free gossypol detoxification of cottonseed meal (CSM) and solid-state fermentation on free gossypol detoxification and protein degradation on CSM by five strains of Candida spp. and two strains of Aspergillus niger solid-state fermentation, respectively.
Growth curves of Candida spp. and Aspergillus niger were determined by optoelectronic turbidimetry and dry weight of mycelium method, respectively. The results showed that the terminal period of log phase of Candida spp. was occurred at 24 h of incubation at 28℃in the malt extraction liquid, and that of Aspergillus niger was happened at 24 h of incubation at 28℃in potato dextrose liquid. In order to achieve the optimal fermentation product, the seed culture fluid of Candida spp. and Aspergillus niger should be inoculated into the fermentation substrate at the terminal period of log phase of them.
The control group was the material mixed with CSM, corn flour and wheat bran in the quality ratio of 7:2:1 and grinded by 60 mash screen. Exp.1 and Exp.2 were conducted to study sterilization of solid-state fermentation on free gossypol detoxification of CSM. Exp. 1 had treatment moistened in the ratio 1:0.9 of mixture to water but no sterilization, treatment autoclaved at 105℃for 20 min, treatment autoclaved at 112℃for 20 min, treatment autoclaved at 115℃for 20 min, treatment autoclaved at 121℃for 20 min. Exp. 2 had treatment moistened in the ratio 1:0.9 of mixture to water but no sterilization, treatment steam sterilized at 100℃for 30 min, treatment steam sterilized at 100℃for 60 min, treatment autoclaved at 115℃for 20 min, treatment autoclaved at 115℃for 40 min, treatment autoclaved at 121℃for 20 min. Exp. 3 was conducted to study Candida spp. and Aspergillus niger solid-state fermentation on free gossypol detoxification and protein degradation of CSM in the condition of CSM substrate autoclaved at 115℃for 40 min. Exp.3 had treatment sterilized but no inoculation, treatment inoculated C1, treatment inoculated C2, treatment inoculated C3, treatment inoculated C4, treatment inoculated C5, treatment inoculated A6, treatment inoculated A7, and treatment inoculated C1 and A6. Every sample after treated placed at the drying oven to dry at 80℃for 48 h, and grinded by 60-mash screen, then stored for determined at -20℃. The contents of free gossypol, bound gossypol and total gossypol on the CSM substrate before and after sterilization or fermentation were determined to investigate the mechanism of CSM free gossypol detoxification by microbe solid-state fermentation. The protein and peptide molecular weight distribution of CSM substrate before and after fermentation was analyzed by SDS-PAGE gel electrophoresis to investigate the degradation mechanism of CSM by solid-state fermentation, at the same time, the protein in vitro digestibility of fermented products was analyzed by dialysis tube method. Exp.1 and exp.2 showed that the detoxification rate of treat drying the CSM substrate containing water 47.37% at 80℃for 48 h was 54.57%~68.21%, and that of every treat sterilizing was above 85%, at the same time, the temperature and time of sterilization had no significant difference in the detoxification rate of CSM substrate(P>0.05). Exp. 3 showed as follow: 1)Compared with the free gossypol content of treat sterilizing that have better detoxified free gossypol, that of treats Candida spp. fermenting had no significant difference (P>0.05), and that of treats Aspergillus niger A6 and A7 fermenting increased to 211.451 mg/kg and 176.135 mg/kg from 24.739 mg/kg, respectively(P<0.01),and that of treat of mixed cultured fermentation by Candida spp. C1 and Aspergillus niger A6 was 112.244 mg/kg(P<0.01). 2)Compared with bound and total gossypol content of treat sterilizing, that of treat of microbe fermentation had no significant difference (P>0.05). 3)The solid state fermentation of three kinds of fungi all can degrade the large molecular weight(MW) protein to low molecule proteins and peptides, and especially, Aspergillus niger can degrade protein(MW=20~100 KDa) into low molecule mass(MW<14 KDa). 4)Compared with the protein in vitro digestibility of control, that of CSM substrate fermented by Aspergillus niger had a significant increase by 19.25%(P<0.05), and that of CSM substrate mixingly fermented increased by 8.18%, but they had no significant difference(P>0.05). 5)The productivity of the products fermented by Candida spp., Aspergillus niger or mixingly were 96.34±0.58%, 88.42±1.31% or 89.69±0.62%, respectively. Their crude protein contents had a very significant increase by 4.31%, 13.76% or 13.09%(P<0.01) with that of control. The crude protein contents of fermentation products had a very significant negative correlation with the fermentation productivity of products: y=97.52352-0.52748x(R2=0.9942,P<0.0001).
The conclusion in this study was that: 1)The treatment by sterilization is the main function that led to the detoxification of free gossypol in CSM. 2)Aspergillus niger increased the content of free gossypol of CSM detoxified by sterilization, however, Candida spp. can detoxify the free gossypol of CSM, but both cann’t degrade the gossypol. 3)Solid-state fermentation of CSM by Aspergillus niger possessed a stronger capability of protein degradation as a result is that the large molecular weight can be degraded to proteins or peptides with MW less than 14 KDa. 4)Aspergillus niger solid-state fermentation can significantly increase the protein in vitro digestibility of CSM substrate. 5)The fermentation could decrease the content of dry matter and increase the content of crude protein of fermented product, and the crude protein contents of fermented products and fermentation productivity had significantly a negative correlation.
一、假丝酵母菌和黑曲霉生长曲线的测定
分别通过光电比浊法和菌丝干重法测定了假丝酵母菌和黑曲霉的生长曲线。结果表明:5株假丝酵母菌在麦芽汁培养液中28℃培养24 h达到对数生长末期,2株黑曲霉在马铃薯葡萄糖培养液中28℃培养24 h达到对数生长末期。在对数生长末期将其种子液接入发酵底物中发酵效果最佳。
二、微生物固态发酵对棉籽粕棉酚脱毒及蛋白质降解的影响
以棉籽粕、玉米面及麦麸按质量比7:2:1均匀混合后粉碎、过60目筛所得样品为试验的对照组。试验一设5个处理组:加水拌湿不灭菌组和4个湿热灭菌20 min组(105℃、112℃、115℃、121℃);试验二设6个处理组:加水拌湿不灭菌组、2个100℃蒸汽灭菌组(30 min、60 min)及3个湿热灭菌组(115℃20 min、115℃40 min、121℃20 min);试验三研究115℃灭菌40 min条件下,假丝酵母菌和黑曲霉固态发酵脱毒及蛋白质降解规律,试验设9个处理组:灭菌不接种组、5个接种假丝酵母菌组(C1、C2、C3、C4、C5)、2个接种黑曲霉组(A6、A7)及接种复合菌组(C1和A6)。处理结束后立即放入干燥箱中,80℃烘干48 h,粉碎,过60目筛,-20℃低温密封保存待测。苯胺法测定灭菌或发酵处理前后棉籽粕底物中FG与总棉酚(TG)含量变化,SDS-PAGE凝胶电泳法分析发酵前后棉籽蛋白质的分子量分布范围变化,并通过透析袋法测定发酵产物的蛋白质体外消化率。试验一和试验二结果表明:加水拌湿不灭菌组(底物含水47.37%)、其棉籽粕底物混匀拌湿后立即80℃烘干48 h脱毒率为54.57%~68.21%,各灭菌组脱毒率均在85%以上,不同灭菌温度及灭菌时间对棉籽粕底物脱毒率的差异不显著(P>0.05)。试验三结果表明:1)在高温高压灭菌处理对棉籽粕底物中FG已大部分脱毒的情况下,接种假丝酵母菌未见显著降低FG含量(P>0.05),接种黑曲霉A6和A7发酵组反而使FG含量从灭菌处理组的24.739 mg/kg分别增加到211.451 mg/kg和176.135 mg/kg(P<0.01),假丝酵母菌和黑曲霉复合发酵组FG含量为112.244 mg/kg(P<0.01);2)在灭菌处理已极显著(P<0.01)降低棉籽粕底物结合棉酚(BG)和TG含量的情况下,各菌株发酵对BG和TG的含量影响差异不显著(P>0.05);3)假丝酵母及黑曲霉固态发酵均能将棉籽粕底物中分子量在20~100 KDa之间的大分子蛋白质降解为小分子蛋白质和肽,其中黑曲霉可将蛋白质降解为14 KDa以下的小分子蛋白质和肽;4)黑曲霉发酵组的棉籽粕底物蛋白质体外消化率比对照组提高19.25%(P<0.05),复合发酵组提高8.18%(P>0.05);5)棉籽粕假丝酵母菌、黑曲霉单菌种及复合固态发酵的产率分别为96.34%、88.42%和89.69%,粗蛋白(CP)含量分别提高4.31%、13.76%和13.09%,发酵产物的CP含量与发酵产率呈极显著的负相关:y=97.52352-0.52748x(R2=0.9942,P<0.0001)。
综合本研究结果:1)微生物固态发酵对棉籽粕棉酚脱毒主要是高温高压灭菌作用;2)假丝酵母对棉籽粕中FG有一定的脱毒作用,而黑曲霉增加了FG含量,二者对棉酚均没有降解作用;3)黑曲霉固态发酵棉籽粕具有较强的蛋白降解能力,可将高分子量的棉籽蛋白降解为14 KDa以下的小分子蛋白和肽;4)黑曲霉固态发酵可显著提高棉籽粕CP体外消化率;5)棉籽粕发酵产物中的CP含量与发酵产率之间存在显著的线性负相关。
This article were conducted to study the effect of treatment sterilized by high temperature and high pressure in the solid-state fermentation on free gossypol detoxification of cottonseed meal (CSM) and solid-state fermentation on free gossypol detoxification and protein degradation on CSM by five strains of Candida spp. and two strains of Aspergillus niger solid-state fermentation, respectively.
Growth curves of Candida spp. and Aspergillus niger were determined by optoelectronic turbidimetry and dry weight of mycelium method, respectively. The results showed that the terminal period of log phase of Candida spp. was occurred at 24 h of incubation at 28℃in the malt extraction liquid, and that of Aspergillus niger was happened at 24 h of incubation at 28℃in potato dextrose liquid. In order to achieve the optimal fermentation product, the seed culture fluid of Candida spp. and Aspergillus niger should be inoculated into the fermentation substrate at the terminal period of log phase of them.
The control group was the material mixed with CSM, corn flour and wheat bran in the quality ratio of 7:2:1 and grinded by 60 mash screen. Exp.1 and Exp.2 were conducted to study sterilization of solid-state fermentation on free gossypol detoxification of CSM. Exp. 1 had treatment moistened in the ratio 1:0.9 of mixture to water but no sterilization, treatment autoclaved at 105℃for 20 min, treatment autoclaved at 112℃for 20 min, treatment autoclaved at 115℃for 20 min, treatment autoclaved at 121℃for 20 min. Exp. 2 had treatment moistened in the ratio 1:0.9 of mixture to water but no sterilization, treatment steam sterilized at 100℃for 30 min, treatment steam sterilized at 100℃for 60 min, treatment autoclaved at 115℃for 20 min, treatment autoclaved at 115℃for 40 min, treatment autoclaved at 121℃for 20 min. Exp. 3 was conducted to study Candida spp. and Aspergillus niger solid-state fermentation on free gossypol detoxification and protein degradation of CSM in the condition of CSM substrate autoclaved at 115℃for 40 min. Exp.3 had treatment sterilized but no inoculation, treatment inoculated C1, treatment inoculated C2, treatment inoculated C3, treatment inoculated C4, treatment inoculated C5, treatment inoculated A6, treatment inoculated A7, and treatment inoculated C1 and A6. Every sample after treated placed at the drying oven to dry at 80℃for 48 h, and grinded by 60-mash screen, then stored for determined at -20℃. The contents of free gossypol, bound gossypol and total gossypol on the CSM substrate before and after sterilization or fermentation were determined to investigate the mechanism of CSM free gossypol detoxification by microbe solid-state fermentation. The protein and peptide molecular weight distribution of CSM substrate before and after fermentation was analyzed by SDS-PAGE gel electrophoresis to investigate the degradation mechanism of CSM by solid-state fermentation, at the same time, the protein in vitro digestibility of fermented products was analyzed by dialysis tube method. Exp.1 and exp.2 showed that the detoxification rate of treat drying the CSM substrate containing water 47.37% at 80℃for 48 h was 54.57%~68.21%, and that of every treat sterilizing was above 85%, at the same time, the temperature and time of sterilization had no significant difference in the detoxification rate of CSM substrate(P>0.05). Exp. 3 showed as follow: 1)Compared with the free gossypol content of treat sterilizing that have better detoxified free gossypol, that of treats Candida spp. fermenting had no significant difference (P>0.05), and that of treats Aspergillus niger A6 and A7 fermenting increased to 211.451 mg/kg and 176.135 mg/kg from 24.739 mg/kg, respectively(P<0.01),and that of treat of mixed cultured fermentation by Candida spp. C1 and Aspergillus niger A6 was 112.244 mg/kg(P<0.01). 2)Compared with bound and total gossypol content of treat sterilizing, that of treat of microbe fermentation had no significant difference (P>0.05). 3)The solid state fermentation of three kinds of fungi all can degrade the large molecular weight(MW) protein to low molecule proteins and peptides, and especially, Aspergillus niger can degrade protein(MW=20~100 KDa) into low molecule mass(MW<14 KDa). 4)Compared with the protein in vitro digestibility of control, that of CSM substrate fermented by Aspergillus niger had a significant increase by 19.25%(P<0.05), and that of CSM substrate mixingly fermented increased by 8.18%, but they had no significant difference(P>0.05). 5)The productivity of the products fermented by Candida spp., Aspergillus niger or mixingly were 96.34±0.58%, 88.42±1.31% or 89.69±0.62%, respectively. Their crude protein contents had a very significant increase by 4.31%, 13.76% or 13.09%(P<0.01) with that of control. The crude protein contents of fermentation products had a very significant negative correlation with the fermentation productivity of products: y=97.52352-0.52748x(R2=0.9942,P<0.0001).
The conclusion in this study was that: 1)The treatment by sterilization is the main function that led to the detoxification of free gossypol in CSM. 2)Aspergillus niger increased the content of free gossypol of CSM detoxified by sterilization, however, Candida spp. can detoxify the free gossypol of CSM, but both cann’t degrade the gossypol. 3)Solid-state fermentation of CSM by Aspergillus niger possessed a stronger capability of protein degradation as a result is that the large molecular weight can be degraded to proteins or peptides with MW less than 14 KDa. 4)Aspergillus niger solid-state fermentation can significantly increase the protein in vitro digestibility of CSM substrate. 5)The fermentation could decrease the content of dry matter and increase the content of crude protein of fermented product, and the crude protein contents of fermented products and fermentation productivity had significantly a negative correlation.
引文
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