混菌固态发酵菜籽粕工艺优化

doi:10.3864/j.issn.0578-1752.2019.24.014

摘要/Abstract

摘要：

【目的】通过优化菜籽粕混菌固态发酵工艺以提高菜籽粕营养价值,为发酵菜籽粕在我国畜牧养殖上的应用提供理论参考。【方法】以普通菜籽粕为原料,采用嗜酸乳杆菌、枯草芽孢杆菌、酿酒酵母3种菌株,以硫甙降解率（X%)、总酸增加率(Y%)、多肽增加率(Z%)为评价指标,并采用加权法以M为综合评价指标（其中M = 0.7×X + 0.15×Y + 0.15×Z）评价发酵效果。其中,试验一以3株菌的不同添加量为试验因素,设计L₉（3 ⁴）三因素三水平正交试验,探究各菌株添加量及不同的菌株组合方式对发酵效果的影响;在试验一的基础上配置混菌发酵液,以发酵温度、料水比、发酵时间、接种量为试验因素,设计L₁₆（4 ⁵）四因素四水平正交试验,探究混菌固态发酵菜籽粕的最佳工艺,并结合最佳工艺进行中试验证。【结果】(1) 试验一对以总变化率M为指标的正交试验结果进行极差分析,结果显示混菌固态发酵菜籽粕最佳混菌组合为嗜酸乳杆菌:酿酒酵母:枯草芽孢杆菌为=1﹕3﹕2,在该条件下,硫甙降解率为23.5%,总酸增加率为179.2%,多肽增加率为375.0%。(2) 在试验一的基础上设计试验二并对结果进行极差分析,结果表明混菌固态发酵菜籽粕的最佳工艺条件为温度：33℃、料水比：1﹕1、时间：84h、接种量：6%,在该工艺条件下进行中试验证测得硫甙降解率为48.8%,总酸增加率为499.7%,多肽增加率为148.4%,总变化率为131.4%,符合试验预期;对各单因素的方差分析结果显示1水平温度（31℃）总变化率显著低于其他水平（P ＜ 0.05),2水平温度（33℃）总变化率高于3水平（35℃）和4水平（37℃）,但差异不显著（P ＞ 0.05);2水平（1﹕1）的料水比总变化率高于其他水平,差异未达到显著水平（P ＞0.05);3水平（84 h）的发酵时间总变化率高于其他水平,但差异未达到显著水平（P ＞0.05);接种量各水平之间对总变化率的影响差异不显著（P ＞ 0.05)。(3)在该最佳工艺条件下,微生物固态发酵增加了菜籽粕粗蛋白含量（从37.05％到40.90％)并降低了粗纤维浓度（从17.47%到16.72%）;发酵菜籽粕氨基酸组成分析表明发酵增加了菜籽粕中各种氨基酸的含量,尤其是Asp,Thr,Ser,Glu,Pro,Ala和Lys;发酵后菜籽粕硫甙含量从36.08 μmol·g ^-1降至18.48 μmol·g ^-1;发酵增加了菜籽粕中多肽含量（从0.84%到2.09%）和总酸含量（从1.01%到6.05%）;菜籽粕发酵前后粗脂肪含量相似（4.31%和4.39%）。【结论】通过本发酵工艺对菜籽粕进行混菌固态发酵可有效降解菜籽粕中的硫甙,并提高菜籽粕中多肽和总酸的含量,菜籽粕营养价值得到有效改善。

关键词: 菜籽粕, 混菌, 固态发酵, 正交设计

Abstract:

【Objective】 The aim of this study was to optimize the solid state fermentation conditions for rapeseed meal with mixed strains and to improve the feeding value of rapeseed meal. It would provide a reference for the application of fermented rapeseed meal in animal husbandry in China.【Method】The rapeseed meal used in this study was conventional rapeseed meal. Three strains of Lactobacillus acidophilus, Bacillus subtilis and Saccharomyces cerevisiae were used, and the glucosinolates degradation rate (X%), total acid increase rate (Y%), polypeptide increase rate (Z%) were used as evaluation indexes. Method M was used as the comprehensive evaluation index (M=0.7*X+0.15*Y+0.15*Z). In the experiment one, the effects of different strains addition levels and the best mixed ratio of the three strains in fermented rapeseed meal were investigated through L₉ (3 ⁴) three-factors and three-levels orthogonal array design methods. Based on the result of experiment one, an L₁₆ (4 ⁵) four-factors and four-levels of orthogonal array design experiment was designed to explore the optimal conditions of mixed strains solid state fermentation of rapeseed meal, and the fermentation temperature, feed water ratio, time, and inoculation quantity were selected as experimental factors. 【Result】The results showed as follow: (1) L₉ (3 ⁴) orthogonal array design showed that the optimal mixed strains ratio of fermented rapeseed meal were Lactobacillus acidophilus : Bacillus subtilis : Saccharomyces cerevisiae = 1:3:2. Under these condition, the contents of glucosinolates was degraded by 23.5%, the total acid and polypeptide increase rate were 179.2% and 375.0%, respectively. (2) Based on the result of experiment one, the range analysis of experiment two showed that the optimal conditions of mixed strains solid state fermentation of rapeseed meal were as follows: fermentation temperature was 33 °C, feed water ratio was 1:1, time was 84h and inoculation quantity was 6%. The pilot test of this conditions showed that the contents of glucosinolates was degraded by 48.8%, the total acid increase rate was 499.7%, the polypeptide increase rate was 148.4%, and the total change rate M was 131.4%. The results were in line with our expectation. The lowest total change rate M was noted when the temperature was 31 °C, and the total change rate M in temperature 33 °C was higher than 35 °C and 37 °C, but the difference between groups was not significant (P ＞ 0.05). The higher M was also noted when feed water ratio was 1:1.0 and fermentation time was 84h. No significant differences in M were observed between conditions in different levels of inoculation quantity. (3) The fermentation of rapeseed meal by mixed strains also increased the content of CP (37.05% vs. 40.90%) and decreased the concentration of crude fiber (17.47% vs. 16.72%). The amino acid composition of rapeseed meal and fermented rapeseed meal showed that fermentation increased the content of several amino acids in rapeseed meal, especially Asp, Thr, Ser, Glu, Pro, Ala, and Lys. The greatest change caused by fermentation was for glucosinolates, which decreased from 36.08 μmol·g ^-1 to 18.48 μmol·g ^-1. The fermentation of rapeseed meal increased the content of polypeptides (from 0.84% to 2.09%) and increased the content of total acid (from 1.01% to 6.05%) compared to unfermented rapeseed meal. The concentration of crude fat was similar in rapeseed meal and FRSM (4.31% and 4.39%).【Conclusion】The optimal fermentation conditions could effectively degrade the glucosinolates and increase the content of polypeptide and total acid in rapeseed meal, so the nutritional value of rapeseed meal was significantly improved.

Key words: rapeseed meal, mixed strains, solid state fermentation, orthogonal design

吴正可,刘国华,李阳,郑爱娟,常文环,陈志敏,蔡辉益. 混菌固态发酵菜籽粕工艺优化[J]. 中国农业科学, 2019, 52(24): 4603-4612.

ZhengKe WU,GuoHua LIU,Yang LI,AiJuan ZHENG,WenHuan CHANG,ZhiMin CHEN,HuiYi CAI. Optimization of Solid State Fermentation for Rapeseed Meal with Mixed Strains[J]. Scientia Agricultura Sinica, 2019, 52(24): 4603-4612.

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水平 Level	因素Factors
水平 Level	嗜酸乳杆菌Lactobacillus acidophilus	枯草芽孢杆菌 Bacillus subtilis	酿酒酵母 Saccharomyces cerevisiae
1	5%	5%	5%
2	10%	10%	10%
3	15%	15%	15%

水平 Level	因子Factors			结果Result
水平 Level	嗜酸乳杆菌 Lactobacillus acidophilus	枯草芽孢杆菌 Bacillus subtilis	酿酒酵母 Saccharomyces cerevisiae	硫甙降解率X Degradation rate of GS (%)	总酸增加率Y Increase rate of total acid (%)	多肽增加率Z Increase rate of polypeptide (%)	总变化率M Total rate of change (%)
1	3	3	3	24.19	287.13	197.62	89.65
2	1	3	2	23.45	179.21	375.00	99.55
3	3	1	2	23.22	204.95	176.19	73.43
4	2	1	3	22.89	140.59	238.10	72.83
5	2	3	1	21.33	107.92	330.95	80.76
6	2	2	2	19.39	184.16	328.57	90.48
7	1	1	1	20.45	147.52	282.14	78.76
8	1	2	3	21.04	90.10	342.86	79.67
9	3	2	1	22.80	206.93	171.43	72.71
标准误SEM				1.83	18.32	21.37	2.25
P值 P value				0.68	0.03	0.04	0.06
K1	85.99	75.01	77.41
K2	81.36	80.96	87.82
K3	78.59	89.98	80.71
优水平Optimal levels	A1	K3	N2
极差(RJ)	7.40	14.97	10.41
排序 Rank	枯草芽孢杆菌（Bacillus subtilis）＞酿酒酵母（Saccharomyces cerevisiae）＞嗜酸乳杆菌（Lactobacillus acidophilus）

编号 Number	因子Factors				结果Result (%)
编号 Number	温度 Temperature (℃)	料水比 Feed water ratio	发酵时间 Time (h)	接种量 Inoculation quantity (%)	硫甙降解率X Degradation rate of GS	总酸增加率Y Increase rate of total acid	多肽增加率Z Increase rate of polypeptide	总变化率M Total rate of change
1	1	1	1	1	31.15	220.64	184.85	82.63
2	1	2	2	2	31.55	318.98	148.96	92.28
3	1	3	3	3	36.16	363.23	117.67	97.45
4	1	4	4	4	41.82	214.42	122.50	79.81
5	2	1	2	3	29.75	483.75	148.16	115.61
6	2	2	1	4	53.58	508.91	134.70	134.04
7	2	3	4	1	53.92	471.88	114.88	125.76
8	2	4	3	2	56.49	541.77	79.19	132.69
9	3	1	3	4	48.21	451.77	154.93	124.76
10	3	2	4	3	48.77	488.55	131.40	127.13
11	3	3	1	2	46.37	468.02	118.30	120.40
12	3	4	2	1	47.26	452.96	56.60	109.51
13	4	1	4	2	41.69	432.11	120.58	112.08
14	4	2	3	1	42.06	529.86	148.41	131.19
15	4	3	2	4	43.23	494.08	114.10	121.49
16	4	4	1	3	41.66	447.40	62.62	105.66
标准误SEM					1.15	19.94	5.67	6.35
P值 P value					0.01	0.04	0.07	0.06
K1	88.04	108.77	110.68	112.27
K2	130.78	121.16	109.72	118.11
K3	120.45	116.27	125.27	111.46
K4	117.60	110.67	111.20	115.02
优水平 Optimal levels	2	2	3	2
极差 RJ	42.73	12.39	15.55	6.65
排序 Rank	温度 Temperature＞时间 Time＞料水比 Feed water ratio＞接种量 Inoculation quantity

水平 Level	结果Result
水平 Level	温度 Temperature(℃)	料水比 Feed water ratio	发酵时间 Time(h)	接种量 Inoculation quantity（%）
1	88.04 a	108.77	110.68	112.27
2	130.78b	121.16	109.72	118.11
3	120.45b	116.27	125.27	111.46
4	117.60 b	110.67	111.20	115.02
标准误SEM	4.35	4.35	4.35	4.35
P值 P value	0.02	0.67	0.79	0.99

水平 Level	结果Result
水平 Level	温度 Temperature (℃)	料水比 Feed water ratio	发酵时间 Time (h)	接种量 Inoculation quantity (%)
1	35.17a	37.70	43.19	43.59
2	48.43b	43.99	37.94	44.02
3	47.65b	44.92	45.73	39.08
4	42.16ab	46.80	46.55	46.71
标准误SEM	2.04	2.04	2.04	2.04
P值 P value	0.06	0.46	0.47	0.66