荔枝内生乳酸菌的筛选、鉴定及发酵效果分析
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摘要
【目的】筛选用于果汁发酵的荔枝内生乳酸菌,为解决发酵果汁专用乳酸菌少的问题提供新思路。【方法】以荔枝果肉为试验材料,通过MRS-溴甲酚紫平板法分离筛选荔枝内生乳酸菌,测定初筛菌株的发酵产酸和生长能力,筛选出发酵性能最优菌株,并进行形态学观察、生理生化试验和16S rDNA鉴定;以常用果蔬汁发酵菌种植物乳杆菌为对照菌株,进行荔枝内生菌发酵荔枝果汁的感官评价、微生物、理化指标和营养物质含量研究。【结果】从荔枝果肉中筛选出1株发酵性能优良的荔枝内生菌(编号LZ1),该菌菌落呈乳白色圆形凸起,边缘整齐,革兰氏阳性细菌,菌体细胞呈球形或卵圆形,单个、成对或链状排列;厌氧生长,能发酵纤维二糖、葡萄糖、果糖等8种碳水化合物;16S rDNA鉴定结果表明该菌与肠膜明串珠菌葡聚糖亚种(Leuconostoc mesenteroides subsp. dextranicum)的同源性达99%以上,结合形态特征及生理生化试验结果,鉴定该菌株为肠膜明串珠菌葡聚糖亚种。该荔枝内生肠膜明串珠菌能在荔枝果汁中生长良好,发酵果汁无分层现象,有浓郁的荔枝清香味,感官评分显著高于对照菌植物乳杆菌(P<0.05,下同),活菌数达4.85×109CFU/mL,高于植物乳杆菌,但差异不显著(P>0.05);肠膜明串珠菌和植物乳杆菌发酵果汁的乳酸含量(9.63和13.83 g/kg)显著高于不接菌果汁(0.44 g/kg),pH(3.73和3.54)显著低于不接菌果汁(4.68);荔枝内生肠膜明串珠菌发酵果汁中的维生素(Vc)含量高于植物乳杆菌和不接菌果汁,对果汁中果糖和葡萄糖的利用能力也强于植物乳杆菌,两株菌发酵果汁的葡萄糖含量分别较不接菌果汁下降31.4%和26.7%,果糖含量降幅分别为43.4%和21.7%;荔枝内生肠膜明串珠菌发酵果汁中游离氨基酸保留率为87.3%,略高于植物乳杆菌(84.4%),两株菌在发酵过程中各种氨基酸的种类和含量变化有所不同。【结论】筛选的荔枝内生肠膜明串珠菌在荔枝果汁中生长良好,发酵的荔枝果汁既保存和优化果汁的营养和口感,又增添益生菌的保健功能。
【Objective】The study screened the endogenous lactobacillus strains from litchi,which was used in juice fermentation. It provided a new way for solving the problem that the special bacteria used in fermented juice was few.【Method】Litchi endogenous lactobacillus strain was isolated from litchi pulp by using MRS-bromocresol plate. The strain with excellent fermentation performance was selected by measuring its fermentation acid production capacity and growth capacity,and the strain was subjected to morphological observation,physiological and biochemical tests and 16 S rDNA identification. The sensory evaluation,microbial and physicochemical indices,nutrient content of fermented litchi juice were studied with Lactobacillus plantarum as control strain.【Result】One endophytic strain(LZ1)of litchi with excellent fermentation performance was selected from the pulp. The results of morphological observation showed that the bacterial colony was milky white,round and convex,with neat edges,gram-positive bacteria,and the cells were spherical or ovoid,single,pair or chain arranged. The results of physiological and biochemical experiments showed that the strain was anaerobic and could ferment eight kinds of carbohydrates,such as fiber disaccharide,glucose and fructose. The results of 16 S rDNA identification showed that the strain had more than 99% homology with Leuconostoc mesenteroides subsp. dextranicum. Combining morphological characteristics and physiological and biochemical experimental results,the strain was identified as L. mesenteroides subsp. dextranicum. The litchi endophytic L. mesenteroides could grow well in litchi juice.The fermented juice presented no layering and had strong litchi fragrance. Sensory evaluation was significantly better thancontrol bacteria L. plantarum(P<0.05,the same below). The living bacterial number was 4.85×109CFU/mL,which was higher than that in L. plantarum,but the difference was not significant(P>0.05). Lactic acids contents of fermentation from L. mesenteroides(9.63 g/kg)and L. plantarum(13.83 g/kg)were significantly higher than uninoculated juice(0.44g/kg). But p H values of the two fermented juices(3.73 and 3.54)were significantly lower than uninoculated juice(4.68).The vitamin C(Vc)content of the fermented juice by L. mesenteroides was higher than those of control bacteria and uninoculated juice. The utilization of fructose and glucose in the juice were stronger than that of the control bacteria. The juices fermented by L. mesenteroides and L. plantarum declined by 31.4% and 26.7% respectively,and fructose content dropped by 43.4% and 21.7% compared with uninoculated juice.The retention rate of free amino acid in fermented juice L.mesenteroides was 87.3%,slightly higher than 84.4% of the control bacteria. But types and contents of free amino acids were different in two kinds of fermentation juice.【Conclusion】The selected litchi endogenous L. mesenteroides has good adaptability with litchi juice.The fermented litchi juice not only can be preserved and be optimized the nutrition and flavor of litchi juice,but also increases the health function of probiotics.
【Objective】The study screened the endogenous lactobacillus strains from litchi,which was used in juice fermentation. It provided a new way for solving the problem that the special bacteria used in fermented juice was few.【Method】Litchi endogenous lactobacillus strain was isolated from litchi pulp by using MRS-bromocresol plate. The strain with excellent fermentation performance was selected by measuring its fermentation acid production capacity and growth capacity,and the strain was subjected to morphological observation,physiological and biochemical tests and 16 S rDNA identification. The sensory evaluation,microbial and physicochemical indices,nutrient content of fermented litchi juice were studied with Lactobacillus plantarum as control strain.【Result】One endophytic strain(LZ1)of litchi with excellent fermentation performance was selected from the pulp. The results of morphological observation showed that the bacterial colony was milky white,round and convex,with neat edges,gram-positive bacteria,and the cells were spherical or ovoid,single,pair or chain arranged. The results of physiological and biochemical experiments showed that the strain was anaerobic and could ferment eight kinds of carbohydrates,such as fiber disaccharide,glucose and fructose. The results of 16 S rDNA identification showed that the strain had more than 99% homology with Leuconostoc mesenteroides subsp. dextranicum. Combining morphological characteristics and physiological and biochemical experimental results,the strain was identified as L. mesenteroides subsp. dextranicum. The litchi endophytic L. mesenteroides could grow well in litchi juice.The fermented juice presented no layering and had strong litchi fragrance. Sensory evaluation was significantly better thancontrol bacteria L. plantarum(P<0.05,the same below). The living bacterial number was 4.85×109CFU/mL,which was higher than that in L. plantarum,but the difference was not significant(P>0.05). Lactic acids contents of fermentation from L. mesenteroides(9.63 g/kg)and L. plantarum(13.83 g/kg)were significantly higher than uninoculated juice(0.44g/kg). But p H values of the two fermented juices(3.73 and 3.54)were significantly lower than uninoculated juice(4.68).The vitamin C(Vc)content of the fermented juice by L. mesenteroides was higher than those of control bacteria and uninoculated juice. The utilization of fructose and glucose in the juice were stronger than that of the control bacteria. The juices fermented by L. mesenteroides and L. plantarum declined by 31.4% and 26.7% respectively,and fructose content dropped by 43.4% and 21.7% compared with uninoculated juice.The retention rate of free amino acid in fermented juice L.mesenteroides was 87.3%,slightly higher than 84.4% of the control bacteria. But types and contents of free amino acids were different in two kinds of fermentation juice.【Conclusion】The selected litchi endogenous L. mesenteroides has good adaptability with litchi juice.The fermented litchi juice not only can be preserved and be optimized the nutrition and flavor of litchi juice,but also increases the health function of probiotics.
引文
杜慧娟,王伯初,米鹏程,冯薇.2008.药用植物内生菌的分离及抗菌活性的初步研究[J].氨基酸和生物资源,30(1):61-64.[Du H J,Wang B C,Mi P C,Feng W.2008.Isolation of endophytes in pharmaceutical plants and pilot study of its antimicrobial activity[J].Amino Acids&Biotic Resources,30(1):61-64.]
龚小洁.2015.荔枝果渣的乳酸菌发酵特性及其功能性果粉的加工工艺研究[D].南昌:江西农业大学.[Gong X J.2015.Study on fermentation characteristics of lactic acid bacteria in litchi pulp pomace and processing technology of its functional fruit powder[D].Nanchang:Jiangxi A-gricultural University.]
郭丽琼,郭心悦,储炫,林俊芳,刘英丽,叶志伟,李宏略,廖雪义.2015.海带益生菌发酵条件的优化和营养成分分析[J].现代食品科技,31(8):280-285.[Guo L Q,Guo XY,Chu X,Lin J F,Liu Y L,Ye Z W,Li H L,Liao X Y.2015.Optimization of kelp fermentation conditions using probiotics and the analysis of nutrient composition[J].Modern Food Science and Technology,31(8):280-285.]
赖婷,张名位,刘磊,张瑞芬,邓媛元,池建伟.2016.龙眼果浆复合乳酸菌发酵工艺优化[J].农业工程学报,32(S2):390-397.[Lai T,Zhang M W,Liu L,Zhang R F,Deng YY,Chi J W.2016.Optimization of process conditions of dry longan pulp fermented by complex lactic acid bacteria[J].Transactions of the Chinese Society of Agricultural Engineering,32(S2):390-397.]
廖雪义,郭丽琼,林俊芳,邱灵燕.2014.益生乳酸菌在发酵果蔬饮品开发上的应用[J].食品工业,35(7):223-229.[Liao X Y,Guo L Q,Lin J F,Qiu L Y.2014.Application of probiotic lactic acid bacteria in functional fruit and vegetable drink[J].The Food Industry,35(7):223-229.]
凌代文,东秀珠.1999.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社:54-57.[Ling D W,Dong XZ.1999.Identification and Characterization and Experiment Method of Lactic Acid Bacteria[M].Beijing:China Light Industry Press:54-57.]
马锞,徐匆,张长勇,黄应维,罗华建,罗诗,胡文锋.2014.一株荔枝生物保鲜乳酸菌的筛选及鉴定[J].现代食品科技,30(5):111-117.[Ma K,Xu C,Zhang C Y,Huang YW,Luo H J,Luo S,Hu W F.2014.Screening and identification of a lactic acid bacteria strain with lychee freshkeeping function[J].Modern Food Science and Technology,30(5):111-117.]
宋苏华.2012.发酵果蔬饮料益生乳酸菌的强化筛选及其发酵工艺技术研究[D].南昌:南昌大学.[Song S H.2012.Improved screening of probiotic lactic acid bacteria specific for fruit-vegetable beverages fermentation and its fermentation technology[D].Nanchang:Nanchang University.]
谢明勇,熊涛,关倩倩.2014.益生菌发酵果蔬关键技术研究进展[J].中国食品学报,14(10):1-9.[Xie M Y,Xiong T,Guan Q Q.2014.Research progress on the key techniques of the fruit and vegetable products fermented by probiotics[J].Journal of Chinese Institute of Food Science and Technology,14(10):1-9.]
杨月欣,王光亚.2002.实用食物营养成分分析手册[M].北京:中国轻工业出版社.[Yang Y X,Wang G Y.2002.Practical Food Nutrient Analysis Manual[M].Beijing:China Light Industry Press.]
周丽娟,郑琳,刘煜宇,王松峰,端李祥,陈彩和,普兴伟.2010.荔枝产香内生菌的挥发性成分分析及其在卷烟中的应用研究[J].精细化工,27(10):1013-1015.[Zhou LJ,Zheng L,Liu Y Y,Wang S F,Duan L X,Chen C H,Pu X W.2010.Analysis and study of volatile components and its cigarette utilization from lichee endophyte[J].Fine Chemicals,27(10):1013-1015.]
郑欣.2014.荔枝汁乳酸菌发酵饮料的工艺研究[D].南昌:江西农业大学.[Zheng X.2014.Study on fermentantation technology of lactic acid bacteria in litchi juice[D].Nanchang:Jiangxi Agricultural University.]
Abdel-Rahman M A,Tashiro Y,Sonomoto K.2011.Lactic acidproduction from lignocellulose-derived sugars using lacticacid bacteria:Overview and limits[J].Journal of biotechnology,156(4):286-301.
Di Cagno R,Cardinali G,Minervini G,Antonielli L,Rizzello C G,Ricciuti P,Gobbetti M.2010.Taxonomic structure of the yeasts and lactic acid bacteria microbiotaof pineapple(Anana scomosus L.Merr.)and use of autochthonous starters for minimally processing[J].Food Microbiology,27(3):381-389.
Di Cagno R,Surico R F,Minervini G,Rizzello C G,Lovino R,Servili M,Taticchi A,Urbani S,Gobbetti M.2011.Exploitation of sweet cherry(Prunus avium L.)puree added of stem infusion through fermentation by selected autochthonous lactic acid bacteria[J].Food Microbiology,28(5):900-909.
Liao X Y,Guo L Q,Ye Z W,Qiu L Y,Gu F W,Lin J F.2016.Use of autochthonous lactic acid bacteria startersto ferment mango juice for promoting its probioticroles[J].Preparative Biochemistry and Biotechnology,46(4):399-405.
Mousavi Z E,Mousavi S M,Razavi S H,Emam-Djomeh Z,Kiani H.2011.Fermentation of pomegranate juice by probiotic lactic acid bacteria[J].World Journal of Microbiology and Biotechnology,27(1):123-128.
Siti M M,Lee S C,Hesham A E,Fadzilah M A A,Roslinda AM.2016.A review on fruit juice probiotication:Pomegranate[J].Current Nutrition&Food Science,12(1):4-11.
龚小洁.2015.荔枝果渣的乳酸菌发酵特性及其功能性果粉的加工工艺研究[D].南昌:江西农业大学.[Gong X J.2015.Study on fermentation characteristics of lactic acid bacteria in litchi pulp pomace and processing technology of its functional fruit powder[D].Nanchang:Jiangxi A-gricultural University.]
郭丽琼,郭心悦,储炫,林俊芳,刘英丽,叶志伟,李宏略,廖雪义.2015.海带益生菌发酵条件的优化和营养成分分析[J].现代食品科技,31(8):280-285.[Guo L Q,Guo XY,Chu X,Lin J F,Liu Y L,Ye Z W,Li H L,Liao X Y.2015.Optimization of kelp fermentation conditions using probiotics and the analysis of nutrient composition[J].Modern Food Science and Technology,31(8):280-285.]
赖婷,张名位,刘磊,张瑞芬,邓媛元,池建伟.2016.龙眼果浆复合乳酸菌发酵工艺优化[J].农业工程学报,32(S2):390-397.[Lai T,Zhang M W,Liu L,Zhang R F,Deng YY,Chi J W.2016.Optimization of process conditions of dry longan pulp fermented by complex lactic acid bacteria[J].Transactions of the Chinese Society of Agricultural Engineering,32(S2):390-397.]
廖雪义,郭丽琼,林俊芳,邱灵燕.2014.益生乳酸菌在发酵果蔬饮品开发上的应用[J].食品工业,35(7):223-229.[Liao X Y,Guo L Q,Lin J F,Qiu L Y.2014.Application of probiotic lactic acid bacteria in functional fruit and vegetable drink[J].The Food Industry,35(7):223-229.]
凌代文,东秀珠.1999.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社:54-57.[Ling D W,Dong XZ.1999.Identification and Characterization and Experiment Method of Lactic Acid Bacteria[M].Beijing:China Light Industry Press:54-57.]
马锞,徐匆,张长勇,黄应维,罗华建,罗诗,胡文锋.2014.一株荔枝生物保鲜乳酸菌的筛选及鉴定[J].现代食品科技,30(5):111-117.[Ma K,Xu C,Zhang C Y,Huang YW,Luo H J,Luo S,Hu W F.2014.Screening and identification of a lactic acid bacteria strain with lychee freshkeeping function[J].Modern Food Science and Technology,30(5):111-117.]
宋苏华.2012.发酵果蔬饮料益生乳酸菌的强化筛选及其发酵工艺技术研究[D].南昌:南昌大学.[Song S H.2012.Improved screening of probiotic lactic acid bacteria specific for fruit-vegetable beverages fermentation and its fermentation technology[D].Nanchang:Nanchang University.]
谢明勇,熊涛,关倩倩.2014.益生菌发酵果蔬关键技术研究进展[J].中国食品学报,14(10):1-9.[Xie M Y,Xiong T,Guan Q Q.2014.Research progress on the key techniques of the fruit and vegetable products fermented by probiotics[J].Journal of Chinese Institute of Food Science and Technology,14(10):1-9.]
杨月欣,王光亚.2002.实用食物营养成分分析手册[M].北京:中国轻工业出版社.[Yang Y X,Wang G Y.2002.Practical Food Nutrient Analysis Manual[M].Beijing:China Light Industry Press.]
周丽娟,郑琳,刘煜宇,王松峰,端李祥,陈彩和,普兴伟.2010.荔枝产香内生菌的挥发性成分分析及其在卷烟中的应用研究[J].精细化工,27(10):1013-1015.[Zhou LJ,Zheng L,Liu Y Y,Wang S F,Duan L X,Chen C H,Pu X W.2010.Analysis and study of volatile components and its cigarette utilization from lichee endophyte[J].Fine Chemicals,27(10):1013-1015.]
郑欣.2014.荔枝汁乳酸菌发酵饮料的工艺研究[D].南昌:江西农业大学.[Zheng X.2014.Study on fermentantation technology of lactic acid bacteria in litchi juice[D].Nanchang:Jiangxi Agricultural University.]
Abdel-Rahman M A,Tashiro Y,Sonomoto K.2011.Lactic acidproduction from lignocellulose-derived sugars using lacticacid bacteria:Overview and limits[J].Journal of biotechnology,156(4):286-301.
Di Cagno R,Cardinali G,Minervini G,Antonielli L,Rizzello C G,Ricciuti P,Gobbetti M.2010.Taxonomic structure of the yeasts and lactic acid bacteria microbiotaof pineapple(Anana scomosus L.Merr.)and use of autochthonous starters for minimally processing[J].Food Microbiology,27(3):381-389.
Di Cagno R,Surico R F,Minervini G,Rizzello C G,Lovino R,Servili M,Taticchi A,Urbani S,Gobbetti M.2011.Exploitation of sweet cherry(Prunus avium L.)puree added of stem infusion through fermentation by selected autochthonous lactic acid bacteria[J].Food Microbiology,28(5):900-909.
Liao X Y,Guo L Q,Ye Z W,Qiu L Y,Gu F W,Lin J F.2016.Use of autochthonous lactic acid bacteria startersto ferment mango juice for promoting its probioticroles[J].Preparative Biochemistry and Biotechnology,46(4):399-405.
Mousavi Z E,Mousavi S M,Razavi S H,Emam-Djomeh Z,Kiani H.2011.Fermentation of pomegranate juice by probiotic lactic acid bacteria[J].World Journal of Microbiology and Biotechnology,27(1):123-128.
Siti M M,Lee S C,Hesham A E,Fadzilah M A A,Roslinda AM.2016.A review on fruit juice probiotication:Pomegranate[J].Current Nutrition&Food Science,12(1):4-11.