Screening and Identification of Lactic Acid bacteria in Traditionally Fermented and Acid Coagulated Hard Cheese and Analysis of Its in Vitro Probiotic Properties

doi:10.6048/j.issn.1001-4330.2022.06.021

Abstract

Abstract:

【Objective】 Using traditional separation and purification methods, the traditionally fermented cheese made by farmers in Zhaosu County, Yili, Xinjiang was used as the experimental raw material to try to obtain strains with relatively high acid-producing properties, and carry out in vitro probiotic properties test to screen out excellent probiotics.【Methods】 Thirty-six strains were isolated from spontaneously fermented cheese by using the method of dilution coating, scribing plate and re-screening purification, and through their colony and strain morphological features and physiological and biochemical identification, the targeted strains were selected and the molecular biology identification was conducted by analyzing 16S rDNA homologous sequence.【Results】 Six of them were lactic acid bacteria, five of which were Lactobacillus helveticus, the other one was Lactobacillus plantarum. The bile salt tolerance, acid tolerance, simulated gastrointestinal fluid tolerance and surface characteristics of these strains were verified through in vitro probiotic properties experiments, and it was known that Lactobacillus plantarum E₁₁ had the best performance in these aspects.【Conclusion】 Its viability reached 46.42% in 0.5% bile salt medium, and its survival rate remained above 20% under the condition of pH 2, and its self-aggregation rate reached 63.6% after standing still for 5 hours and the hydrophobic rate of para-xylene is 44.26%.

Key words: cheese; lactic acid bacteria; acidity; molecular biological identification; probiotic properties

摘要：

【目的】 研究传统发酵酸凝硬质奶酪中乳酸菌的分离鉴定及其体外益生特性,得到产酸性能较高的菌株,测试体外益生特性,筛选出优良益生菌。【方法】 采用传统分离纯化方法,以新疆伊犁地区昭苏县农家自制自然发酵酸凝硬质奶酪为原材料,运用稀释涂布、平板划线以及复筛纯化的方法,从自然发酵奶酪中分离得36株菌株,观察其菌落与菌株形态特征并检测生理生化,选取符合条件的菌株运用16S rDNA同源序列分析方法鉴定其分子生物学。【结果】 鉴定6株为乳酸菌,5株是瑞士乳杆菌(Lactobacillus helveticus),另外1株为植物乳杆菌(Lactobacillus plantarum)。这些菌株的耐胆盐、耐酸、耐模拟胃液以及表面特性得知植物乳杆菌E₁₁在这些方面的性能最佳。【结论】 该菌株在浓度为0.5%的胆盐培养基中存活率达到46.42%,pH 2的条件下存活率仍然保持在20%以上,静置5 h后其自凝集率达到63.6%,对二甲苯的疏水率为44.26%。

关键词: 奶酪, 乳酸菌, 酸度, 分子生物学鉴定, 益生特性

CLC Number:

S872

YilixiatiAireti , LI Wei, XU Yanglin, YAN Hongmeng, DAI Zhiwei, ZHOU Jianzhong. Screening and Identification of Lactic Acid bacteria in Traditionally Fermented and Acid Coagulated Hard Cheese and Analysis of Its in Vitro Probiotic Properties[J]. Xinjiang Agricultural Sciences, 2022, 59(6): 1491-1501.

伊力夏提·艾热提, 李伟, 徐杨林, 严宏孟, 戴志伟, 周建中. 传统发酵酸凝硬质奶酪中乳酸菌的分离鉴定及其体外益生特性[J]. 新疆农业科学, 2022, 59(6): 1491-1501.

Figures/Tables 14

References 37

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编号 Number	菌落特点 Characteristics of the colonies	菌体形态 Mycelia morphology
A₄	奶白色,扁圆形,周围整齐且不透明,中间凸起	细长杆状,单个或成连
A₇₅	奶白色,直径偏小,周围基本整齐及带有透明圈,中间凸起	细长杆状,单个或成连
B₂₅	奶白色,直径偏大,呈圆形,周围整齐及带有透明圈,中间凸起	细长杆菌,单个、成连或成团
B₃₄	奶白色,呈圆形,周边整齐,不透明,中间凸起	细长杆菌,单个
B₈	奶白色,呈圆形,周围整齐,不透明,微扁平	细长杆菌,单个
E₁₁	奶白色,直径偏小,周边整齐,不透明	短杆状,单独或成对

编号 Number	菌落特点 Characteristics of the colonies	菌体形态 Mycelia morphology
A₄	奶白色,扁圆形,周围整齐且不透明,中间凸起	细长杆状,单个或成连
A₇₅	奶白色,直径偏小,周围基本整齐及带有透明圈,中间凸起	细长杆状,单个或成连
B₂₅	奶白色,直径偏大,呈圆形,周围整齐及带有透明圈,中间凸起	细长杆菌,单个、成连或成团
B₃₄	奶白色,呈圆形,周边整齐,不透明,中间凸起	细长杆菌,单个
B₈	奶白色,呈圆形,周围整齐,不透明,微扁平	细长杆菌,单个
E₁₁	奶白色,直径偏小,周边整齐,不透明	短杆状,单独或成对

编号 Number	过氧化氢酶 Catalase	H₂S实验 H2S test	葡萄糖 Glucose	革兰氏染色 Gram stain
A₄	-	-	+	+
A₇₅	-	-	+	+
B₂₅	-	-	+	+
B₃₄	-	-	+	+
B₈	-	-	+	+
E₁₁	-	-	+	+

编号 Number	过氧化氢酶 Catalase	H₂S实验 H2S test	葡萄糖 Glucose	革兰氏染色 Gram stain
A₄	-	-	+	+
A₇₅	-	-	+	+
B₂₅	-	-	+	+
B₃₄	-	-	+	+
B₈	-	-	+	+
E₁₁	-	-	+	+

菌株编号 Strain No.	亲缘相似菌株 Related strains	Accession	相似度 Similarity(%)
A₄	Lactobacillus helveticusstrain 4538	MT459351.1	100
A₇₅	Lactobacillus helveticusstrainNWAFU1447	MG551111.1	99.93
B₈	Lactobacillus helveticusMG585	MN055698.1	100
B₂₅	Lactobacillus helveticusNWAFU1331	MG550993.1	100
B₃₄	Lactobacillus helveticusMGB67-2	HM058202.2	100
E₁₁	Lactobacillus plantarum strain 3334	MT613627.1	100