高产乳酸菌种的选育及其发酵棉粕营养特性的研究-毕业论文.doc

分类号： 密 级：公开

学 号：2010108013 单位代码：10759

石河子大学

硕 士 学 位 论 文

高产乳酸菌种的选育及其发酵

棉粕营养特性的研究

学位申请人
吴 妍 妍

指 导 教 师
张 文 举 教授

申请学位门类级别
农学硕士

学科、专业名称
动物营养与饲料科学

向方究研
饲料资源的开发与利用

院学在所
动物科技学院

中国新疆石河子

2013 年 5 月

A Study on Screening and Breeding of High-yield Lactic Acid

Bacteria and the nutritional characteristics of Fermented

Cottonseed Meal

A Dissertation Submitted to

Shihezi University

In Partial Fulfillment of the Requirements

for the Degree of

Master of Agriculture

By

Yan-Yan Wu

(Animal Nutrition and Feed Science)

Dissertation Supervisor: Prof. Wen-Ju Zhang

May，2013

Shihezi, Xinjiang, China

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0 要

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目的：本研究以嗜酸乳杆菌（Lactobacillus acidophilus）为出发菌种，筛选出高产酸性能的嗜酸乳酸 菌，以棉粕和玉米为固体发酵底物，采用所选育菌种进行发酵，研究发酵条件参数、底料组成、接 种量的优化以及发酵产物代谢组分和营养价值 。并就发酵棉粕对肉仔鸡生长性能、血液理化指标和 免疫指标的影响进行了研究。为嗜酸乳杆菌发酵棉粕的综合评定和合理利用提供理论支撑。

方法：本研究由四个部分组成。试验一: 从S1、W2、J2三株嗜酸乳杆菌中选取一株产乳酸较高 、生长 较好的菌株，并对该菌株进行自然选育出一株高产乳酸的菌株 ，并鉴定其为嗜酸乳杆菌。试验二:旨

在优化嗜酸乳杆菌发酵棉粕的发酵工艺，采用 4 因子5 水平的正交设计法和二因子有重复观察值相结 合的设计方法，通过发酵底物中乳酸含量，确定发酵参数、发酵底料组成和接种比例。 试验三：分 别采用嗜酸乳杆菌 S5、W2、J2按照试验二的优化条件发酵棉粕，分 别为对照组、S5组、W2组、J2

组共4 组，研究发酵产物中有机酸、B族维生素、游离氨基酸含量的变化。 试验四：选取1 日龄AA肉

仔鸡100羽，随机分成2 组，每组5 个重复，每个重复 10羽，对照组添加6%的未发酵棉粕，试验组添 加6%的发酵棉粕，研究嗜酸乳杆菌发酵棉粕对 AA肉仔鸡生长性能、血液理化指标及免疫指标的影 响。

结果：试验一：菌株S5在培养36 h 后其产酸量可达2.14 g/L，通过生理生化鉴定和 16SDNA全序列分 析法确定其种属，然后经过 Gene Bank基因比对确定同源基因，用 DNAMAN软件绘制系统发育树， 鉴定为嗜酸乳杆菌（Lactobacillus acidophilus）。试验二：嗜酸乳杆菌发酵棉粕的优化结果为 ：温度 为37℃、时间为48 h、底物初水分为30%、初始pH为6 、底料组成为棉粕：玉米为9：1；接种量为50 mL/kg。试验三：S5组较对照组，戊酸含量极显著提高了49.32%，琥珀酸、柠檬酸显著降低了77.57%、 1.93%，VB1 、VB2 、VB5的含量极显著提高了28.53%、50.91%、89.47%（P<0.01），亮氨酸、赖氨酸 含量分别极显著提高了19.84%、33.63%（P <0.01）；W2组较对照组，戊酸、柠檬酸显著提高了79.82%、

10.77%（P <0.01），VB1、VB3 、VB5极显著降低了19.43%、91.35%、23.52%（P<0.01），亮氨酸、苯

丙氨酸含量分别极显著降低了 16.64%、4.43%（P <0.01）；J2组较对照组，戊酸、琥珀酸的相对含量

极显著提高了127.19%、14.95%（P <0.01），VB1、VB2、VB3、VB5分别极显著降低了44.54%、23.50%、

91.47%、23.52%（P <0.01）。试验四：添加嗜酸乳杆菌发酵棉粕后，（1 ）在0 ～42日龄，肉鸡平均日

采食量、平均日增重分别显著提高了10.20%和15.41%（P<0.05），料重比下降了4.63%（P>0.05）；（2 ）

在0 ～21日龄，肉鸡血清中白蛋白、总蛋白分别显著提高了 10.64%、5.68%（P<0.05），甘油三酯显

著提高了23.73%（P<0.05），总胆固醇显著降低了 6.72%（P<0.05）；（3 ）在21～42日龄，肉鸡血清

白蛋白提高了2.86%，但差异不显著（P>0.05），总蛋白显著提高了8.14%（P<0.05）；甘油三酯显著

提高了9.68%，总胆固醇极显著降低了 25.49%（P<0.01）；血清免疫球蛋白IgG、IgA、IgM含量显著

提高（P<0.05）。

结论：试验一：经过选育，确定了一株产酸性能较好的嗜酸乳杆菌 S5。试验二：确定发酵产物中乳 酸含量最优的发酵参数为，温度：37℃、时间：48 h 、底物初水分：30%、初始pH：6 、底料组成： 棉粕：玉米 = 9 ：1 ；接种量：50 mL/kg。试验三：发酵产物中的有机酸、 B 族维生素和游离氨基酸

的含量因菌种而异，其中菌种 S5发酵产物中的有机酸、 B 族维生素和游离氨基酸含量得到了明显提

高。试验四：肉仔鸡的日粮中添加嗜酸乳杆菌发酵棉粕能提高肉鸡的生长性能、血液理化指标及其

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免疫性能。

关键词：嗜酸乳杆菌；固体发酵；发酵棉粕；鸡血清；代谢产物

论文类型： B（应用研究）

Abstract

Objective: Lactobacillus acidophilus was selected as starting strain in this study, screening out

Lactobacillus acidophilus with properties of high-yield acid, through the optimization of fermentation

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condition parameters, bottom material composition, inoculation amount of the selected breeding strains with cottonseed meal and corn as solid fermentation substrate to explore the metabolic components and nutritional value of fermentation products of the breeding Lactobacillus acidophilus strains, and the effects on growth performance, blood physical and chemical indicators and immune indexes of broiler chicken, therefore providing theoretical support for the comprehensive assessment and rational utilization of fermentation cottonseed meal of Lactobacillus acidophilus.

Method: This research is composed by four parts. Experiment 1: Choose a good strain with higher acid produce and better growth through S1, W2 and J2 Lactobacillus acidophilus, and identify it as Lactobacillus acidophilus. Experiment 2: In order to optimize biology forage fermentation parameters of Lactobacillus acidophilus, 4 factors 5 levels orthogonal and two factors with repeated observed value design method were combined through the lactic acid content of ferment substrate to determine fermentation condition parameters, bottom material composition and inoculation proportion. Experiment 3: Lactobacillus acidophilus S5 , W2 and J2 were selected according to the optimized conditions for ferment cottonseed meal, set four groups: CK、S5、W2、J2, respectively, to study the variation of organic acid, Vitamin B and free amino acid content. Experiment 4: To study the effects of Lactobacillus acidophilus fermentation cottonseed meal on growth performance, blood physical and chemical indicators and immune indexes of broiler chicken, 100 1-day-old AA broiler chicken were selected to separate randomly into 2 groups in this experiment, 5 repeats each group, and 10 in each repeat. CK add 6% no-fermentation cottonseed meal, the others add 6% Lactobacillus acidophilus fermentation cottonseed meal.

Results: Experiment 1: Acid product amount of S5 strain reached 2.14g/L after fostering for 36h, through physiology and biochemistry identification and 16SDNA complete sequence analytical method to ascertain its species, and then confirm homologous gene by Gene Bank comparison, use DNAMAN software to draw phylogenetic tree, and identify it as Lactobacillus acidophilus. Experiment 2: The optimized fermentation conditions parameters of Lactobacillus acidophilus biology forage are as follows: fermentation temperature is 37℃, fermentation time cycle is 48h, initial moisture of the substrate is 30% and pH 6; the optimized results of bottom material composition and inoculation amount are as follows: bottom material composition is 90% cottonseed meal and 10% corn, inoculation amount is 50ml/kg. Experiment 3: Compared to CK, S5 group pentanoic acid content significantly improved 49.32%, succinic acid and citric acid significantly decreased 77.57% and 1.93%, VB1, VB2 and VB5 content highly significantly increased 28.53%, 50.91% and 89.47% (P <0.01), leucine and lysine highly significantly increased 19.84% and 33.63% (P <0.01) respectively; Ⅲ group pentanoic acid and citric acid significantly improved 79.82% and 10.77% ( P<0.01), VB1, VB3 and VB5 content highly significantly reduced 19.43%, 91.35% and 23.52% (P <0.01), leucine and phenylalanine significantly decreased 16.64% and 4.43% (P <0.01); Ⅳ group pentanoic acid and succinic acid content highly significantly increased 127.19% and 14.95% (P <0.01), VB1, VB2 , VB3 and VB5 highly significantly decreased 44.54%, 23.50%, 91.47% and 23.52% (P <0.01) respectively. Experiment 4: After adding Lactobacillus acidophilus fermentation cottonseed meal, (1) in 0~42-day-old, compared to CK,

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average daily food consumption and average daily gain significantly increased 10.20%and 15.41%( P<0.05), forage weight proportion decreased 4.63% (P>0.05); (2) in 0~21-day-old, albumin and total protein significantly increased 10.64% and 5.68% (P <0.05), Ca and P improved 7.31% and 11.62%, but the difference is not significant (P>0.05), triglyceride significantly increased 23.73% (P<0.05), total cholesterol significantly decreased 6.72% ( P<0.05); (3) in 21~42-day-old, albumin improved2.86%, but the difference is not significant (P>0.05), total protein significantly increased 8.14% (P <0.05), triglyceride significantly increased 9.68% (P <0.05), total cholesterol decreased 25.49% (P <0.01); serum immunoglobulins（IgG、IgA、IgM）content is significantly improved (P <0.05).

Conclusions: Experiment 1: Selected and breed a strain Lactobacillus acidophilus S5 with better acid production. Experiment 2: Ascertained the optimal biological fermentation forage parameters for acid production of fermentation metabolite, temperature is 37℃, fermentation time cycle is 48h, initial moisture of the substrate is 30% and pH 6; The optimized bottom material composition is 90% cottonseed meal and 10% corn, inoculation amount is 50 mL /kg. Experiment 3: Organic acid, Vitamin B and free amino acid content of fermentation products differ because of strain type. After fermentation of cottonseed meal, organic acid, Vitamin B and free amino acid content of strain S5 were significantly improved. Experiment 4: Add Lactobacillus acidophilus fermentation cottonseed meal in daily forage of broiler chicken can improve its growth performance, blood physicochemical indexes and immune properties.

Key words: Lactobacillus acidophilus; solid fermentation; fermentation cottonseed meal; chicken serum;

metabolites

主要缩略词

Main Abbreviation

英文缩写 英文全称 中文全称

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LAB
Lacticacid bacteria
乳酸菌

Lactic acid

Lac

乳
酸

FG
Free gossypol
游离棉酚

EAA
Essential Amino Acid
必需氨基酸

AA
Amino Acid
氨基酸

FAA
Free Amino Acid
游离氨基酸

DM
Dry matter
干物质

CP
Crude protein
粗蛋白

EE
Ether extract
乙醚浸出物

目 录
摘 要..............................................................................................................................................................
I
Abstract..........................................................................................................................................................
III
主要缩略词.....................................................................................................................................................
V
目 录
.............................................................................................................................................................
VI
前 言..........................................................................................................................................................

VIII
1..............................................................................................................................
研究目的及意义
VIII
2 ............................................................................................................................................
立题依据
IX

VII

3
论文总体研究思路............................................................................................................................
IX
第一章 文献综述.......................................................................................................................................
1
乳酸菌的研究进展及微生物发酵棉粕在畜禽中的应用 ..............................................................................
1
1
乳酸菌的分类、培养及其保存..........................................................................................................
1

1.1
乳酸菌的分类.........................................................................................................................
..2

1.2
乳酸菌的培养...........................................................................................................................
2

1.3
乳酸菌的保存...........................................................................................................................
2
2
乳酸菌的生理功能..............................................................................................................................
2

2.1
维持动物肠道菌群平衡...........................................................................................................
2

2.2
促进动物对营养物质的吸收 ..................................................................................................
2

2.3
增强动物机体免疫力...............................................................................................................
3

2.4 产生具有抑菌活性的代谢物...... ...............................................................................................
3
3
乳酸菌在动物生产中的应用..............................................................................................................
3

3.1
在鸡生产中的应用....................................................................................................................
3

3.2
在猪生产中的应用....................................................................................................................
4

3.3
在反刍动物生产中的应用.......................................................................................................
4

3.4
在水产中的应用.......................................................................................................................
4
4
微生物育种方法及微生物发酵棉粕的研究进展 ...............................................................................
4

4.1
微生物育种方法的研究进展....................................................................................................
4

4.2
微生物发酵棉粕的研究进展...................................................................................................
5

4.3
微生物发酵饲料在畜禽生产中的应用 ....................................................................................
6
第二章 试验研究.........................................................................................................................................
7
试验一
嗜酸乳杆菌的产酸性研究及鉴定........................................................................................
7
1
材料与方法..........................................................................................................................................
8

1.1
试验材料与仪器........................................................................................................................
8

1.2
菌种的筛选................................................................................................................................
8

1.3
菌种的鉴定部分........................................................................................................................
9

1.4
数据分析...................................................................................................................................
9
2
结果与分析.........................................................................................................................................
9

2.1
嗜酸乳杆菌培养液 pH、产酸量..............................................................................................
9

2.2
菌种的生理生化鉴定及形态学鉴定......................................................................................
11

2.3
嗜酸乳杆菌的 RCR 鉴定结果和测序分析...........................................................................
11
3
结论...................................................................................................................................................

12
4
小结....................................................................................................................................................

12
试验二 嗜酸乳杆菌生物饲料发酵参数的优化 .............