优良发酵性能和产抑菌肽乳酸菌筛选及在发酵蔬菜中的应用
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摘要
泡菜是一种传统而有特色的蔬菜发酵产品,主要由乳酸菌发酵而成,一直以来,泡菜以其独特的诱人风味和多种营养功效而受到广大消费者的青睐。本研究从四川不同地区采集分别具有百年历史的4户家庭自制泡菜的老谭子,从中分离存化出16株乳酸菌,对16株乳酸菌和实验室保藏的15株试验菌株进行发酵性能、亚硝酸盐降解率、感官评价研究,采用蔬菜汁模拟蔬菜环境进行试验,筛选出6株乳酸菌优势菌株,这6株菌分别为SJ4.4、SJ4.6、SJ1.1、IWQ、WT2和J21。对这6株菌进行Biolog鉴定,其结果为副干酪乳杆菌(Lactobacillus paracasei)3株,菌株编号分别为J21、SJ1.1和SJ4.6,徳氏乳杆菌（Lactobacillus delbrueckii)2株,菌株编号分别为IWQ和WT2,植物乳杆菌(Lactobacillus plantarum)1株为SJ4.4。采用手性柱的高效液相色谱法,分析确定这6株乳酸菌代谢产生的乳酸构型,其中菌株J21旋光度最高0.863,所产生的L-乳酸含量最高15.115g/L,D-乳酸含量最低0.878 g/L。
将筛选出的6株优势菌株两两复配以1:1的比例接入蔬菜汁中进行发酵性能、亚硝酸盐降解率、感官评价研究,以纯菌株发酵为对照,筛选出1组优良发酵剂组合IWQ:J21。同时将组合IWQ:J21接入白菜发酵中进行验证试验,以工业化应用菌株3(Lactobacillus brevis)、9(Streptococcus thermophilus)、1.1(Lactobacillus bulgaricus)为对照,试验结果表明,试验组IWQ:J21在整个发酵过程中其产酸性能、亚硝酸盐降解率和感官评价都要优于工业化发酵剂组合3:9:1.1,且在发酵过程中没有亚硝峰的出现。
从产细菌素的菌株J11、J23、M18、M22和X20中筛选可以做为辅助蔬菜发酵的菌株,通过发酵性能、亚硝酸盐降解率和细菌素效价试验,采用蔬菜汁模拟蔬菜环境进行试验,成功筛选出1株菌株J11与发酵剂组合IWQ:J21可以共同在蔬菜汁中进行发酵,并且不影响菌株J11产细菌素,经Biolog鉴定J11为短乳杆菌(Lactobacillus brevis J11)。对复配组合J11:IWQ:J21经行发酵工艺优化,采用蔬菜汁模拟蔬菜环境进行试验,确定发酵温度为34℃、盐浓度3%、接菌量3%、菜汁浓度20%。将复配组合J11:IWQ:J21做为蔬菜发酵剂接入白菜发酵中进行验证试验,对照组为IWQ:J21,在优化后的发酵条件下经行腌制,结果表明复配组合J11:IWQ:J21的发酵速率和产酸量与对照组IWQ:J21并没有明显区别(p﹤0.05),说明菌株J11并不影响发酵剂IWQ:J21的发酵性能,发酵蔬菜汁中细菌素的抑菌圈可以达到12.45mm。
将复配组合J11:IWQ:J21接入纯蔬菜汁中,采用高效液相色谱法分别测定发酵前后蔬菜汁中的糖、有机酸和游离氨基酸的组成及含量变化。并对白菜腌制后剩余的菜汁进行调配,使之成为适合大众消费者口味的乳酸菌饮料。
Chinese paocai is a traditional and distinctive vegetables fermented products, which is mainly formed by the fermentation of lactic acid bacteria. Because of its unique attractive flavor and nutrition, paocai has a large number of consumers of all ages. In this study, 16 strains of lactic acid bacteria collected from four hundred-year-old home-made paocai of different regions of SiChuan and 15 strains of lactic acid bacteria of laboratory preservation are investigated by fermentation performance, sub-nitrate degradation rate, and sensory evaluation in vegetable juice which is simulated environment. Six strains of lactic acid bacteria are selected, and they are SJ4.4, SJ4.6, SJ1.1, IWQ, WT2 and J21. Carryying out the Biolog identification, there are two strains of Lactobacillus paracasei marking by J21, SJ1.1 and SJ4.6, two strains of Lactobacillus delbrueckii marking by IWQ and WT2, and one strain of Lactobacillus plantarum marking by SJ4.4. The configuration of lactic acid produced by the six strains of lactic acid bacteria are analysed by chiral HPLC columns. The highest content of L-lactic acid produced by strain J21 is 15.115g / L, and the minimum content of D-lactic acid is 0.878g/L, and its specific rotation is 0.863.
The six strains screened out are inoculated in vegetable juice by the ratio of 1:1, and the fermente performance, nitrite degradation rate and sensory evaluation are studied compared with the fermentation of pure strains. And the best combination of fermentation is IWQ: J21, which is verified in cabbage fermentation tests compared to the industrialized application strain 3 (Lactobacillus brevis), 9 (Streptococcus thermophilus) and 1.1 (Lactobacillus bulgaricus). The experimental group IWQ: J21 show better production of acid, nitrite and sensory evaluation of the degradation rate in the entire fermentation process than the combination of industrialization 3:9:1.1,and there is not the emergence of peak nitrosourea.in the fermentation process. Bacteria selected from bacteriocin producing strains J11, J23, M18, M22 and X20 can be used in vegetables fermented. The fermentation performance, growth, nitrite degradation rate and bacteriocin titer test are investigated in vegetable juice environment, and the selected strain J11 which is identified by the Biolog as Lactobacillus brevis are fermented with the combination of IWQ: J21 and does not affect the producing of bacteriocin. Complex combination of J11: IWQ: J21 are optimized in vegetable juice environment, and the optimal condition is that fermentation temperature is 34℃, concentration of salt is 3%, the inoculate volume is 3%, and the concentration of juices is 20%. The complex combination of J11: IWQ: J21 are inoculated to cabbage fermentation a to carry out verification tests comparing to group IWQ: J21 in the optimum fermentation conditions, and the results showed that there is not significant difference between the two compound in fermentation acid production rate and volume and the strain J11 does not affect the fermentation performance, fermented of IWQ: J21. The inhibition zone of bacteriocin in vegetable juice can be achieved 12.45mm.
The complex combination of J11: IWQ: J21 is inoculated to pure vegetable juice, and the sugars, organic acids and free amino acid composition and other content of fermentation vegetable juice are measured by high-performance liquid chromatography. The succus after fermention is remade and becomes lactic acid bacteria beverage being suitable for mass consumer tastes.
将筛选出的6株优势菌株两两复配以1:1的比例接入蔬菜汁中进行发酵性能、亚硝酸盐降解率、感官评价研究,以纯菌株发酵为对照,筛选出1组优良发酵剂组合IWQ:J21。同时将组合IWQ:J21接入白菜发酵中进行验证试验,以工业化应用菌株3(Lactobacillus brevis)、9(Streptococcus thermophilus)、1.1(Lactobacillus bulgaricus)为对照,试验结果表明,试验组IWQ:J21在整个发酵过程中其产酸性能、亚硝酸盐降解率和感官评价都要优于工业化发酵剂组合3:9:1.1,且在发酵过程中没有亚硝峰的出现。
从产细菌素的菌株J11、J23、M18、M22和X20中筛选可以做为辅助蔬菜发酵的菌株,通过发酵性能、亚硝酸盐降解率和细菌素效价试验,采用蔬菜汁模拟蔬菜环境进行试验,成功筛选出1株菌株J11与发酵剂组合IWQ:J21可以共同在蔬菜汁中进行发酵,并且不影响菌株J11产细菌素,经Biolog鉴定J11为短乳杆菌(Lactobacillus brevis J11)。对复配组合J11:IWQ:J21经行发酵工艺优化,采用蔬菜汁模拟蔬菜环境进行试验,确定发酵温度为34℃、盐浓度3%、接菌量3%、菜汁浓度20%。将复配组合J11:IWQ:J21做为蔬菜发酵剂接入白菜发酵中进行验证试验,对照组为IWQ:J21,在优化后的发酵条件下经行腌制,结果表明复配组合J11:IWQ:J21的发酵速率和产酸量与对照组IWQ:J21并没有明显区别(p﹤0.05),说明菌株J11并不影响发酵剂IWQ:J21的发酵性能,发酵蔬菜汁中细菌素的抑菌圈可以达到12.45mm。
将复配组合J11:IWQ:J21接入纯蔬菜汁中,采用高效液相色谱法分别测定发酵前后蔬菜汁中的糖、有机酸和游离氨基酸的组成及含量变化。并对白菜腌制后剩余的菜汁进行调配,使之成为适合大众消费者口味的乳酸菌饮料。
Chinese paocai is a traditional and distinctive vegetables fermented products, which is mainly formed by the fermentation of lactic acid bacteria. Because of its unique attractive flavor and nutrition, paocai has a large number of consumers of all ages. In this study, 16 strains of lactic acid bacteria collected from four hundred-year-old home-made paocai of different regions of SiChuan and 15 strains of lactic acid bacteria of laboratory preservation are investigated by fermentation performance, sub-nitrate degradation rate, and sensory evaluation in vegetable juice which is simulated environment. Six strains of lactic acid bacteria are selected, and they are SJ4.4, SJ4.6, SJ1.1, IWQ, WT2 and J21. Carryying out the Biolog identification, there are two strains of Lactobacillus paracasei marking by J21, SJ1.1 and SJ4.6, two strains of Lactobacillus delbrueckii marking by IWQ and WT2, and one strain of Lactobacillus plantarum marking by SJ4.4. The configuration of lactic acid produced by the six strains of lactic acid bacteria are analysed by chiral HPLC columns. The highest content of L-lactic acid produced by strain J21 is 15.115g / L, and the minimum content of D-lactic acid is 0.878g/L, and its specific rotation is 0.863.
The six strains screened out are inoculated in vegetable juice by the ratio of 1:1, and the fermente performance, nitrite degradation rate and sensory evaluation are studied compared with the fermentation of pure strains. And the best combination of fermentation is IWQ: J21, which is verified in cabbage fermentation tests compared to the industrialized application strain 3 (Lactobacillus brevis), 9 (Streptococcus thermophilus) and 1.1 (Lactobacillus bulgaricus). The experimental group IWQ: J21 show better production of acid, nitrite and sensory evaluation of the degradation rate in the entire fermentation process than the combination of industrialization 3:9:1.1,and there is not the emergence of peak nitrosourea.in the fermentation process. Bacteria selected from bacteriocin producing strains J11, J23, M18, M22 and X20 can be used in vegetables fermented. The fermentation performance, growth, nitrite degradation rate and bacteriocin titer test are investigated in vegetable juice environment, and the selected strain J11 which is identified by the Biolog as Lactobacillus brevis are fermented with the combination of IWQ: J21 and does not affect the producing of bacteriocin. Complex combination of J11: IWQ: J21 are optimized in vegetable juice environment, and the optimal condition is that fermentation temperature is 34℃, concentration of salt is 3%, the inoculate volume is 3%, and the concentration of juices is 20%. The complex combination of J11: IWQ: J21 are inoculated to cabbage fermentation a to carry out verification tests comparing to group IWQ: J21 in the optimum fermentation conditions, and the results showed that there is not significant difference between the two compound in fermentation acid production rate and volume and the strain J11 does not affect the fermentation performance, fermented of IWQ: J21. The inhibition zone of bacteriocin in vegetable juice can be achieved 12.45mm.
The complex combination of J11: IWQ: J21 is inoculated to pure vegetable juice, and the sugars, organic acids and free amino acid composition and other content of fermentation vegetable juice are measured by high-performance liquid chromatography. The succus after fermention is remade and becomes lactic acid bacteria beverage being suitable for mass consumer tastes.
引文
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66 K. J. Park, S. J. Woo. Effect of Na-acetate, Na-malate and K-sorbate on the pH Acidity and Sourness During Kimchi Fermentation. Korean J. Food Science and Technology. 1988,63(20):40~44
67 C. L. Randazzo, D. Romano, C. Caggia, et al. Lactobacillus casei, Dominant Species in Naturally Fermented Sicilian Green Olives. International Journal of Food Microbiology . 2004,90:9~14
68 J. Y. Leea, C. J. Kimb, B. Kunzc. Identification of Lactic Acid Bacteria Isolated from Kimchi and Studies on Their Suitability for Application as Starter Culture in the Production of Fermented Sausages. Meat Science. 2005,72(3):437~445
69食品中亚硝酸盐与硝酸盐的测定. GB/T 5009.3 3~1996
70 C. O. Kyung, C. O. Myung, S. H. Kim. The Depletion of Sodium Nitrite by Lactic Acid Bacteria Isolated from Kimchi. Medicinal Food. 2004,7(1):38~44
71 M. A. Leea, D. J. Hana, J. Y. Jeong. Effect of kimchi Powder Level and Drying Methods on Quality Characteristics of Breakfast Sausage. Meat Science. 2008,80(3):708~714
72 H. P. Song, M. W. Byun, J. Cheorun. Effects of Gamma Irradiation on the Microbiological, Nutritional, and Sensory Properties of Fresh Vegetable Juice. Food Control. 2005,18(1):5~10
73 Y. M. Choi, J. H. Whang, J. M. Kim. The Effect of Oyster Shell Powder on the Extension of the Shelf-life of Kimchi. Food Control. 2005,17(1):695~699
74 Y. S. Kim, Y. S. Kim, S.Y. Kim, et al. Application of Omija (Schiandra chinensis) and Plum (Prunus mume) Extracts for the Improvement of Kimchi Quality. Food Control. 2007,19(7):662~669
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76程池,杨梅,李金霞. Biolog微生物自动分析系统—细菌鉴定操作规程的研究.食品与发酵工业. 2006,4(2):41~44
77尹玉英,刘春蕴.乳酸及其某些衍生物的旋光性与结构的关系.北京石油化工学院学报. 2002,42(9):99~103
78 E. Giraud, A. Brauman, S. Keleke. Isolation and Physiological Study of anAmylolytic Strain of Lactobacillus plantarum. Applied Microbiology and Biotechnology. 2004,36(3):379~383
79 P. M. Yana, W. T. Xuea, S. S. Tan. Effect of Inoculating Lactic Acid Bacteria Starter Cultures on the Nitrite Concentration of Fermenting Chinese paocai. Food Control. 2007,19(1):50~55
80食品中还原糖的测定方法. GB 5009.7~85
81 J. G. Park, J. H. Kim, J. N. Park. The Effect of Irradiation Temperature on the Quality Improvement of Kimchi, Korean Fermented Vegetables, for its Shelf Stability. Radiation Physics and Chemistry. 2008,77(4):497~502
82 A. Kapley, S. Prasada, H. J. Purohit. Changes in Microbial Diversity in Fed-batch Reactor Operation with Wastewater Containing Nitroaromatic Residues. Bioresource Technology. 2007,98(13):2479~2484
83蔡永峰,熊涛,岳国海.直投式生物法快速生产泡菜工艺条件的研究.食品与发酵工业. 2006,12(3):123~126
84 L. Settanni, A. Corsetti. Application of Bacteriocins in Vegetable Food Biopreservation. International Journal of Food Microbiology. 2008,121(2):123~138
85 M. Cocchi, C. Durante, M. Grandi. Simultaneous Determination of Sugars and Organic Acids in Aged Vinegars and Chemometric Data Analysis. Talanta. 2006,69(5):1166~1175
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41 K. Y. Yoon, E. E. Woodams, Y. D. Hang . Production of Probiotic Cabbage Juice by Lactic Acid Bacteria. Bioresource technology. 2006,97:1427~1430
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56叶淑红.植物乳杆菌的筛选及其在泡菜中的应用.中国酿造. 2004, 41(2): 53~56
57沈国华.纯菌接种发酵技术在腌渍蔬菜加工上的应用研究.中国调味品. 2002,41(3):101~104
58毕金峰.自然发酵酸菜汁中乳酸菌的分离、鉴定及发酵剂的筛选.沈阳农业大学学报. 2000,41(1):90~93
59 J. P. Tamang, B. Tamang, U. Schillinger. Identification of Predominant Lactic Acid Bacteria Isolated from Traditionally Fermented Vegetable Products of the Eastern Himalayas. International Journal of Food Microbiology, 2005,105(3):347~356
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65 F. Devlieghere, L. Vermeiren, J. Debevere, et al. New Preservation Technologies: Possibilities and Limitations. International Dairy Journal. 2003,14(4):273~285
66 K. J. Park, S. J. Woo. Effect of Na-acetate, Na-malate and K-sorbate on the pH Acidity and Sourness During Kimchi Fermentation. Korean J. Food Science and Technology. 1988,63(20):40~44
67 C. L. Randazzo, D. Romano, C. Caggia, et al. Lactobacillus casei, Dominant Species in Naturally Fermented Sicilian Green Olives. International Journal of Food Microbiology . 2004,90:9~14
68 J. Y. Leea, C. J. Kimb, B. Kunzc. Identification of Lactic Acid Bacteria Isolated from Kimchi and Studies on Their Suitability for Application as Starter Culture in the Production of Fermented Sausages. Meat Science. 2005,72(3):437~445
69食品中亚硝酸盐与硝酸盐的测定. GB/T 5009.3 3~1996
70 C. O. Kyung, C. O. Myung, S. H. Kim. The Depletion of Sodium Nitrite by Lactic Acid Bacteria Isolated from Kimchi. Medicinal Food. 2004,7(1):38~44
71 M. A. Leea, D. J. Hana, J. Y. Jeong. Effect of kimchi Powder Level and Drying Methods on Quality Characteristics of Breakfast Sausage. Meat Science. 2008,80(3):708~714
72 H. P. Song, M. W. Byun, J. Cheorun. Effects of Gamma Irradiation on the Microbiological, Nutritional, and Sensory Properties of Fresh Vegetable Juice. Food Control. 2005,18(1):5~10
73 Y. M. Choi, J. H. Whang, J. M. Kim. The Effect of Oyster Shell Powder on the Extension of the Shelf-life of Kimchi. Food Control. 2005,17(1):695~699
74 Y. S. Kim, Y. S. Kim, S.Y. Kim, et al. Application of Omija (Schiandra chinensis) and Plum (Prunus mume) Extracts for the Improvement of Kimchi Quality. Food Control. 2007,19(7):662~669
75李运,盛慧,赵荣华. Biolog微生物鉴定系统在菌种鉴定中的应用.酿酒科技. 2005,12(3):25~28
76程池,杨梅,李金霞. Biolog微生物自动分析系统—细菌鉴定操作规程的研究.食品与发酵工业. 2006,4(2):41~44
77尹玉英,刘春蕴.乳酸及其某些衍生物的旋光性与结构的关系.北京石油化工学院学报. 2002,42(9):99~103
78 E. Giraud, A. Brauman, S. Keleke. Isolation and Physiological Study of anAmylolytic Strain of Lactobacillus plantarum. Applied Microbiology and Biotechnology. 2004,36(3):379~383
79 P. M. Yana, W. T. Xuea, S. S. Tan. Effect of Inoculating Lactic Acid Bacteria Starter Cultures on the Nitrite Concentration of Fermenting Chinese paocai. Food Control. 2007,19(1):50~55
80食品中还原糖的测定方法. GB 5009.7~85
81 J. G. Park, J. H. Kim, J. N. Park. The Effect of Irradiation Temperature on the Quality Improvement of Kimchi, Korean Fermented Vegetables, for its Shelf Stability. Radiation Physics and Chemistry. 2008,77(4):497~502
82 A. Kapley, S. Prasada, H. J. Purohit. Changes in Microbial Diversity in Fed-batch Reactor Operation with Wastewater Containing Nitroaromatic Residues. Bioresource Technology. 2007,98(13):2479~2484
83蔡永峰,熊涛,岳国海.直投式生物法快速生产泡菜工艺条件的研究.食品与发酵工业. 2006,12(3):123~126
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