乳酸菌的分离鉴定及其抗菌肽与发酵性能研究
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摘要
乳酸菌种质的性能直接关系到酸乳质量的好坏。在菌种保存与传代过程中,会发生菌种污染和变异退化等问题,及时进行菌种鉴定、复壮和优良菌种筛选对酸乳发酵剂和酸乳生产具有重要意义。本研究对本单位乳品厂1990-2006年间保存的3份乳酸菌菌种样本进行分离鉴定,获得7株乳酸菌,对其酸乳发酵性能、抗菌肽产生及生物学特性等进行了研究,主要结果如下：
(1)乳酸菌的分离鉴定：将3份乳酸菌样本接种MRS琼脂平板,根据菌落形态、细菌染色及生化特性初步鉴定结果,分离得7株乳酸菌,分别命名为IV-1、IV-2、IV-3、 ZK、G、ST和393。然后用PCR扩增其16S rDNA序列,利用测得的序列建立遗传进化树,结果显示Ⅳ-1株为嗜酸乳杆菌,Ⅳ-3株为保加利亚乳杆菌,393株为乳酸乳球菌,IV-2、ZK、G和ST株为嗜热链球菌。分离鉴定结果与原菌种标记不完全一致,说明原乳酸菌菌种标记有误、细菌不纯或在保存与传代过程中发生了菌种污染或变异退化。
(2)纯化乳酸菌的发酵特性研究：对不同种乳酸菌的代表菌株进行常规酸乳发酵试验,结果显示：不仅不同种乳酸菌的凝乳时间、产酸速度、粘度、酸乳风味和质地等存在差异,而且同种不同株嗜热链球菌也有显著差异。其中,嗜热链球菌株Ⅳ-2产酸特性与保加利亚杆菌Ⅳ-3株类似,而与同为嗜热链球菌的G、ST、ZK株区别较大；在所试的6株乳酸菌中,ST和G株嗜热链球菌发酵酸乳的综合性能指标较好,表现为产酸速度快、凝乳时间短、粘度值及感官评价高；保加利亚杆菌Ⅳ-3株发酵乳在风味及质地等指标方面有较好表现,综合评定也较好；其他菌株具有一定的单方面优势。这些研究结果表明,基于16SrDNA序列的遗传进化树分析能用于乳酸菌种属的准确鉴定,但不能取代常规方法用于优良酸乳发酵剂菌株的筛选。
(3)乳酸菌抗菌肽及其活性研究：从已经分离鉴定的乳酸菌株中,选择酸乳发酵性能综合表现较好的嗜链球菌ST株和保加利亚杆菌Ⅳ-3株,对其抗菌肽产生及抑菌活性进行了研究。结果显示：两株乳酸菌产生的抗菌肽不仅能抑制革兰氏阳性菌,也能抑制革兰氏阴性菌,具有较广谱抑菌效果；以金黄色葡萄球菌为指示菌,两个菌株产生的抗菌肽作用时间10h表现抑菌活性最高；两株乳酸菌在液体培养基中培养22-24h产生的抗菌肽活性最强,Ⅳ-3株产生的抗菌肽的抑菌作用强于ST株产生的抗菌肽；抗菌肽均具有较好的热稳定性,经100℃处理20min仍有较强的抑菌活性,而且在偏酸性及中性条件下稳定；抗菌肽容易被蛋白酶水解,作为食品添加剂,具有较好的食品安全性。
(4)乳酸菌酸乳发酵剂的研究：将单菌株发酵性能综合表现较好的嗜热链球菌ST株和保加利亚杆菌IV-3株,按照1：10、1：1、10：1、20：1比例制成酸乳发酵剂,分别标记为zh1、zh2、zh3和zh4。研究表明嗜热链球菌ST株和保加利亚杆菌IV-3株具有较好的互配共生特性,是可利用的酸乳发酵剂组合菌株,4个配比的发酵剂在酸乳凝乳时间、产酸速度、粘度、发酵乳活菌数和感官评价等方面均好于单菌株；在4℃储存期间后酸化较弱,粘度较稳定且略有增强,活菌数含量较高；存储24h香味物质乙醛与双乙酰含量达最大值,香味物质两者含量比值在合适的范围；发酵酸乳在比较长的货架期内品质基本保持稳定。总体来看,发酵剂zh3综合表现最好,发酵酸乳凝乳时间短、产酸速度快,冷藏期间粘度好,活菌数含量多,香味物质比例最合理,感官评定最好,品质稳定,具有较好的应用价值。
The quality of lactic acid bacteria (LAB) is directly related to the quality of yogurt. During preservation and passage of LAB, seed strains contamination, variation and/or degeneration are common problems. Therefore, regular identification, rejuvenation and selection of excellent strains are of great importance for high quality yogurt production. In this study, seven LAB strains were isolated and identified from the three bacterial seed stocks in the Experimental Dairy Farm of Yangzhou University from1990to2006. Their performaces in yogurt fermentation, antimicrobial peptide production and suitability for preparation of yogurt starters were studies with the following results.
(1) According to their colony morphorlogy, staining property and biochemical reaction characteristics,7LAB strains were isolated by streaking3lactic acid bacterial stocks on MRS agar plates, which were named as strains Ⅳ-1, Ⅳ-2, Ⅳ-3, ZK, G, ST and393, respectively. To identify further the LAB strains,16S ribosomal DNA (rDNA) were amplified for sequence analysis. On the phylogenetic tree generated, the seven LAB strains belonged to Bacillus acidophilus (strain Ⅳ-1), Lactobacillus bulgaricus (strain Ⅳ-3), Lactococcus lactis (strain393) or Streptococcus thermophilus (strains Ⅳ-2, ZK, G and ST). Some isolated strains differed with the labelings of the bacterial seed stocks. These results suggest mis-labeling, impurity, contamination and/or degeration of the seed stocks during passage and/or storage, and thus regular rejuvenation, isolation, identification and selection of the bacterial stocks are needed for high quality yogurt production.
(2) Yogurt fermentation tests were carried out with the representative strains of different lactic acid bacteria. The result showed that differences in the clotting time, acid production speed, viscosity, yogurt flavor and/or quality existed not only between different species of LABs, but also between different strains of the same species. Among them, Streptococcus thermophilus strain Ⅳ-2has the same acid production characteristics as Lactobacillus bulgaricus strain Ⅳ-3, but different from Streptococcus thermophilus strains G, ST and ZK. Among the seven LAB strains tested, Streptococcus thermophilus strain G and ST strains had a better performance in yogurt fermentation, including higher acid production speed, shorter solidify time, higher viscosity and sensory evaluation. Strain IV-3performed better in flavor, quality and comprehensive evaluation. The other strains had some unilateral advantages. These findings suggest that the phylogenetic tree based on the16S rDNA sequences can be used for accurate identification of LABs, but can't replace the conventional methodsd for selection of excellent yogurt fermentation strains.
(3) Two representative strains with better performance in yogurt fermentation, Streptococcus thermophilus strain ST and Lactobacillus bulgaricus strain IV-3, were selected for further study on antibacterial peptide production. The result showed that the antibacterial peptides produced by the two strains had a brord inhibitory profile against Grams-positive and negative bacteria. By using Staphylococcus aureus as the indicator bacterium, the antibacterial peptides had the highest antibacterial activity at10h after incubation. The two strains had the highest peptide production levels after culturing for22-24h. The antibacterial peptide from strain IV-3was more active than that from strain ST. Both antibacterial peptides had a good thermostability without loss of antibacterial activity after boiling for20min. They were also stable under acidulous and neutral conditions, but can be easily hydrolyzed by proteases.
(4) Streptococcus thermophilus strain ST and Lactobacillus bulgaricus strain IV-3were mixed in1:10,1:1,10:1or20:1as the fermentation starter agent, which are called zhl, zh2, zh3and zh4, respectively. Yogurt fermentation experiments showed that the mixed strain had a better complementary performance than the single strains as yogurt fermentation agent, including shorter milk-clotting time, quicker lactic acid production speed, higher viscosity, more live bacterial number and better flavor of the product. When storage at4℃, the yogurt had a weaker postacidification, the viscosity was slightly increased with a relatively large number of viable lactic acid bacteria. After storage for24h, the contents of acetaldehyde and diacetyl reached to the highest levels but the ratio was within the proper concentration range. The fermented milk remained stable with a longer shelf life and thus the starter culture was suitable for yogurt production. The overall performance of the zh3starter agent was the best among the four starter agents tested.
(1)乳酸菌的分离鉴定：将3份乳酸菌样本接种MRS琼脂平板,根据菌落形态、细菌染色及生化特性初步鉴定结果,分离得7株乳酸菌,分别命名为IV-1、IV-2、IV-3、 ZK、G、ST和393。然后用PCR扩增其16S rDNA序列,利用测得的序列建立遗传进化树,结果显示Ⅳ-1株为嗜酸乳杆菌,Ⅳ-3株为保加利亚乳杆菌,393株为乳酸乳球菌,IV-2、ZK、G和ST株为嗜热链球菌。分离鉴定结果与原菌种标记不完全一致,说明原乳酸菌菌种标记有误、细菌不纯或在保存与传代过程中发生了菌种污染或变异退化。
(2)纯化乳酸菌的发酵特性研究：对不同种乳酸菌的代表菌株进行常规酸乳发酵试验,结果显示：不仅不同种乳酸菌的凝乳时间、产酸速度、粘度、酸乳风味和质地等存在差异,而且同种不同株嗜热链球菌也有显著差异。其中,嗜热链球菌株Ⅳ-2产酸特性与保加利亚杆菌Ⅳ-3株类似,而与同为嗜热链球菌的G、ST、ZK株区别较大；在所试的6株乳酸菌中,ST和G株嗜热链球菌发酵酸乳的综合性能指标较好,表现为产酸速度快、凝乳时间短、粘度值及感官评价高；保加利亚杆菌Ⅳ-3株发酵乳在风味及质地等指标方面有较好表现,综合评定也较好；其他菌株具有一定的单方面优势。这些研究结果表明,基于16SrDNA序列的遗传进化树分析能用于乳酸菌种属的准确鉴定,但不能取代常规方法用于优良酸乳发酵剂菌株的筛选。
(3)乳酸菌抗菌肽及其活性研究：从已经分离鉴定的乳酸菌株中,选择酸乳发酵性能综合表现较好的嗜链球菌ST株和保加利亚杆菌Ⅳ-3株,对其抗菌肽产生及抑菌活性进行了研究。结果显示：两株乳酸菌产生的抗菌肽不仅能抑制革兰氏阳性菌,也能抑制革兰氏阴性菌,具有较广谱抑菌效果；以金黄色葡萄球菌为指示菌,两个菌株产生的抗菌肽作用时间10h表现抑菌活性最高；两株乳酸菌在液体培养基中培养22-24h产生的抗菌肽活性最强,Ⅳ-3株产生的抗菌肽的抑菌作用强于ST株产生的抗菌肽；抗菌肽均具有较好的热稳定性,经100℃处理20min仍有较强的抑菌活性,而且在偏酸性及中性条件下稳定；抗菌肽容易被蛋白酶水解,作为食品添加剂,具有较好的食品安全性。
(4)乳酸菌酸乳发酵剂的研究：将单菌株发酵性能综合表现较好的嗜热链球菌ST株和保加利亚杆菌IV-3株,按照1：10、1：1、10：1、20：1比例制成酸乳发酵剂,分别标记为zh1、zh2、zh3和zh4。研究表明嗜热链球菌ST株和保加利亚杆菌IV-3株具有较好的互配共生特性,是可利用的酸乳发酵剂组合菌株,4个配比的发酵剂在酸乳凝乳时间、产酸速度、粘度、发酵乳活菌数和感官评价等方面均好于单菌株；在4℃储存期间后酸化较弱,粘度较稳定且略有增强,活菌数含量较高；存储24h香味物质乙醛与双乙酰含量达最大值,香味物质两者含量比值在合适的范围；发酵酸乳在比较长的货架期内品质基本保持稳定。总体来看,发酵剂zh3综合表现最好,发酵酸乳凝乳时间短、产酸速度快,冷藏期间粘度好,活菌数含量多,香味物质比例最合理,感官评定最好,品质稳定,具有较好的应用价值。
The quality of lactic acid bacteria (LAB) is directly related to the quality of yogurt. During preservation and passage of LAB, seed strains contamination, variation and/or degeneration are common problems. Therefore, regular identification, rejuvenation and selection of excellent strains are of great importance for high quality yogurt production. In this study, seven LAB strains were isolated and identified from the three bacterial seed stocks in the Experimental Dairy Farm of Yangzhou University from1990to2006. Their performaces in yogurt fermentation, antimicrobial peptide production and suitability for preparation of yogurt starters were studies with the following results.
(1) According to their colony morphorlogy, staining property and biochemical reaction characteristics,7LAB strains were isolated by streaking3lactic acid bacterial stocks on MRS agar plates, which were named as strains Ⅳ-1, Ⅳ-2, Ⅳ-3, ZK, G, ST and393, respectively. To identify further the LAB strains,16S ribosomal DNA (rDNA) were amplified for sequence analysis. On the phylogenetic tree generated, the seven LAB strains belonged to Bacillus acidophilus (strain Ⅳ-1), Lactobacillus bulgaricus (strain Ⅳ-3), Lactococcus lactis (strain393) or Streptococcus thermophilus (strains Ⅳ-2, ZK, G and ST). Some isolated strains differed with the labelings of the bacterial seed stocks. These results suggest mis-labeling, impurity, contamination and/or degeration of the seed stocks during passage and/or storage, and thus regular rejuvenation, isolation, identification and selection of the bacterial stocks are needed for high quality yogurt production.
(2) Yogurt fermentation tests were carried out with the representative strains of different lactic acid bacteria. The result showed that differences in the clotting time, acid production speed, viscosity, yogurt flavor and/or quality existed not only between different species of LABs, but also between different strains of the same species. Among them, Streptococcus thermophilus strain Ⅳ-2has the same acid production characteristics as Lactobacillus bulgaricus strain Ⅳ-3, but different from Streptococcus thermophilus strains G, ST and ZK. Among the seven LAB strains tested, Streptococcus thermophilus strain G and ST strains had a better performance in yogurt fermentation, including higher acid production speed, shorter solidify time, higher viscosity and sensory evaluation. Strain IV-3performed better in flavor, quality and comprehensive evaluation. The other strains had some unilateral advantages. These findings suggest that the phylogenetic tree based on the16S rDNA sequences can be used for accurate identification of LABs, but can't replace the conventional methodsd for selection of excellent yogurt fermentation strains.
(3) Two representative strains with better performance in yogurt fermentation, Streptococcus thermophilus strain ST and Lactobacillus bulgaricus strain IV-3, were selected for further study on antibacterial peptide production. The result showed that the antibacterial peptides produced by the two strains had a brord inhibitory profile against Grams-positive and negative bacteria. By using Staphylococcus aureus as the indicator bacterium, the antibacterial peptides had the highest antibacterial activity at10h after incubation. The two strains had the highest peptide production levels after culturing for22-24h. The antibacterial peptide from strain IV-3was more active than that from strain ST. Both antibacterial peptides had a good thermostability without loss of antibacterial activity after boiling for20min. They were also stable under acidulous and neutral conditions, but can be easily hydrolyzed by proteases.
(4) Streptococcus thermophilus strain ST and Lactobacillus bulgaricus strain IV-3were mixed in1:10,1:1,10:1or20:1as the fermentation starter agent, which are called zhl, zh2, zh3and zh4, respectively. Yogurt fermentation experiments showed that the mixed strain had a better complementary performance than the single strains as yogurt fermentation agent, including shorter milk-clotting time, quicker lactic acid production speed, higher viscosity, more live bacterial number and better flavor of the product. When storage at4℃, the yogurt had a weaker postacidification, the viscosity was slightly increased with a relatively large number of viable lactic acid bacteria. After storage for24h, the contents of acetaldehyde and diacetyl reached to the highest levels but the ratio was within the proper concentration range. The fermented milk remained stable with a longer shelf life and thus the starter culture was suitable for yogurt production. The overall performance of the zh3starter agent was the best among the four starter agents tested.
引文
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