CN104560793A - Lactobacillus kefiri KL22 as well as screening method and application thereof - Google Patents

Lactobacillus kefiri KL22 as well as screening method and application thereof Download PDF

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CN104560793A
CN104560793A CN201410780337.9A CN201410780337A CN104560793A CN 104560793 A CN104560793 A CN 104560793A CN 201410780337 A CN201410780337 A CN 201410780337A CN 104560793 A CN104560793 A CN 104560793A
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lactobacillus
kefir
kefir grains
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朱宏
王�华
王世杰
冯丽莉
薛玉玲
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Shijiazhuang Junlebao Dairy Co Ltd
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    • C12R2001/225—Lactobacillus

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Abstract

The invention discloses a lactobacillus kefiri KL22 as well as a screening method and application thereof. The strain is collected in China General Microbiological Culture Collection Center (CGMCC) on September 16, 2014 and has a collection number of CGMCC No. 9669. Screened kefiri grains are sequentially subjected to separation, purification, a sugar fermentation characteristic experiment, a molecular biology identification, acid and bile salt tolerance characteristic experiment, an intestinal cell adhesion characteristic experiment, a harmful bacteria inhibition experiment and a strain fermentation characteristic experiment to finally screen lactobacillus kefiri KL22. The screened KL22 can be used for preparing yogurt, lactic bacteria drinks and bacterial powder preparations containing lactobacillus kefir KL22. The method is suitable for screening the lactobacillus kefiri KL22 which is used for preparing functional dairy products and probiotics powder containing lactobacillus kefiri KL22.

Description

Kefir grains lactobacillus KL22, its screening technique and application
Technical field
The invention belongs to bioengineering field, relate to a kind of lactobacillus and screening technique thereof, be specifically related to a kind of Kefir grains lactobacillus KL22, its screening technique and application.
Background technology
Current China specifies that the probio that can be used for food only has Bifidobacterium, lactobacillus bulgaricus, lactobacillus acidophilus, L. casei casei, streptococcus thermophilus.Kefir fermented milk has numerous prebiotic functions, and Kefir granule is containing multiple physiological beneficial bacterium, and stick to be colonizated in enteron aisle to compete with pathogen and stick site, nutriment, product lactic acid, the acetic acid of its metabolism can reduce gut pH; The antibacterial substance hydrogen peroxide, bacteriocin etc. that produce, effectively can suppress the field planting of invasive organism in enteron aisle.Therefore, from Kefir granule, screen the lactic acid bacteria with manifest function characteristic and there is very important realistic meaning.
Chinese invention patent CN102559561A discloses " a kind of food source property lactic acid bacteria and application thereof ", disclose a strain food source property lactic acid bacteria, the preserving number of lactic acid bacteria is CCTCC NO:M2011381, for a strain isolated from bean curd with odor saltwater brine has the lactic acid bacteria of bacteriostasis, can add in food and be used as food additives.And domestic and international report shows at present, the acidified milk of Kefir granule fermentation is adopted to have the effect suppressing harmful bacteria, but adopt Kefir granule prepare acidified milk institute with particular tastes by China consumer be difficult to acceptance, and directly adopt Kefir granule to carry out large-scale industrial production, complex operation step, easily pollute and batch between differ greatly.Therefore, from Kefir granule, screening has the lactic acid bacteria of bacteriostasis and is applied to have great importance in acidified milk preparation.
Summary of the invention
The technical problem to be solved in the present invention, is to provide a kind of Kefir grains lactobacillus KL22( lactobacilus kefiri strain) it be from the Kefir granule that Polish Ka Lishi takes back separation screening out, this probio Kefir grains lactobacillus KL22 be a strain there is fine acid and bile salt tolerance characteristic, fermentation character and stick enterocyte, suppress harmful bacteria growth bacterial strain.
Kefir grains lactobacillus of the present invention ( lactobacilus kefiri) KL22, on September 16th, 2014, be stored in Chinese microorganism strain preservationmanagement the committeegeneral Microbiological Culture preservationcenter, preserving number is CGMCC9669, and depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Another goal of the invention of the present invention is the Kefir granule filtered out, successively by separation, purifying, candy ferments characteristic test, molecular biology identification, acid and bile salt tolerance characteristic test, the experiment of enterocyte adhesion characteristics, suppression harmful bacteria characteristic test and strain fermentation characteristic test, finishing screen selects Kefir grains lactobacillus KL22.
The present invention also has a goal of the invention to be the application of Kefir grains lactobacillus KL22, has prepared yoghurt, sour milk beverage, bacterium powder preparation containing Kefir grains lactobacillus KL22.Compared with existing lactobacillus, Kefir grains Yoghourt lactobacillus KL22 has stronger resistance to low ph value and high bile salt, has the ability of better tolerance people alimentary canal poor environment, can play its prebiotic effect better.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
A kind of Kefir grains lactobacillus KL22, this bacteria strain on September 16th, 2014, is preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, deposit number: CGMCC No.9669.
Limit as one of the present invention, its 16SrDNA sequence results is as follows:
TTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTGGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCG
Present invention also offers the screening technique of above-mentioned Kefir grains lactobacillus KL22, it carries out according to following steps order:
(1) Kefir granule source
It is assorted that selected Kefir granule is derived from Polish Cali;
(2) separation of bacterial strain, purifying
A, Kefir granule was activated for 3 generations in defatted milk, get Kefir granule 1g, after sterile saline washing, in the mortar of sterilizing, grinding pulping, add 9ml physiological saline and dilute, get 1ml dilution and dilute, is 10 by dilution factor 4, 10 5with 10 6dilution is coated on bromocresol purple-MRS agar plate;
B, by Kefir granule by 5% inoculum concentration access sterilizing defatted milk in, cultivate 24h for 30 DEG C, draw Kefir granule acidified milk 1ml, suitably dilute with physiological saline, getting dilution factor is 10 6, 10 7, 10 8dilution coating bromocresol purple-MRS agar plate;
C, by step a and b coating bromocresol purple-MRS agar plate be placed in 37 DEG C at Anaerobic culturel 72h; Get above-mentioned plated growth good, there is colonies typical form and occur that the bacterium colony that color change carries out line separation, under aerobic conditions purifying 3 times repeatedly; Again through Gram's staining and catalase reaction, choose Gram-positive, the bacterial strain of catalase feminine gender saves backup, pick out the strain of doubtful lactic acid bacteria 50 altogether, comprising 38 strain bacillus and 12 strain coccuses;
(3) candy ferments characteristic test
Above-mentioned separation and purification 38 strain Gram-positive, the negative doubtful lactobacillus strain picking list bacterium colony of catalase are carried out plate streaking, cultivate 48h for 37 DEG C, at 37 DEG C, 48h is cultivated respectively in picking one oese bacterial strain access candy ferments pipe, observation color changes, and finally according to the change of candy ferments experiment color is different, 38 strain bacterial strains is divided into 8 classes;
(4) molecular biology identification
Above-mentioned 38 strain bacterial strains are carried out molecular biology identification, by DNA extraction, pcr amplification, 16SrDNA checks order, and NCBI website blast finally determines that 38 strain bacterial strains comprise 14 strain Kefir grains lactobacillus, 10 strain lactobacillus bulgaricus, 8 strain Lactobacillus brevis, 6 strain Lactobacillus helveticus;
(5) acid and bile salt tolerance characteristic test
By above-mentioned 14 strain Kefir grains lactobacillus, picking 1 oese purifying inoculation is in MRS culture medium respectively, and in 37 DEG C of cultivation 48h, taking-up counts; Meanwhile, respectively get this culture medium of 1mL, join respectively pH be 2.5 phosphate buffer and 3 ‰ containing bovine bile MRS culture solution in 37 DEG C hatch, carry out the mensuration of bacterium number in 0h, 3h, and calculate its survival rate; Finishing screen selects 6 strain bacterium, is respectively KL02, KL03, KL10, KL18, KL22 and KL36;
(6) enterocyte adhesion characteristics experiment
Being added with every hole 1 mL cell suspension by enterocyte is added with in 24 orifice plates of cover glass, in 37 DEG C of incubators, hatch 48h; Treat that cell attachment is complete, wash 2 times with aseptic PBS, it is 10 that every hole adds 1 mL concentration 8the lactic acid bacteria suspension of individual/mL, hatches 1 h; Then cover glass is taken out, with aseptic PBS rinsing 3 times, then be that 4% paraformaldehyde fixes 30 min with mass fraction, dry rear violet staining, in basis of microscopic observation, random choose 20 visuals field, 50, each visual field cell, calculate the lactic acid bacterium number of its surface adhesion and the mean value getting 3 experiments represents the adhesive capacity of cell, finishing screen selects 4 strain bacterium, is respectively KL02, KL10, KL18 and KL22;
(7) harmful bacteria characteristic test is suppressed
The bacterial strain above-mentioned 4 strains to better enterocyte adhesion properties carries out shigella dysenteriae Antagonism effect experiment, adds in 24 orifice plates, in 37 DEG C of incubators, hatch 48h by enterocyte with every hole 1 mL cell suspension; Treat that cell attachment is complete, wash 2 times with aseptic PBS, every hole adds 10 8individual/ml lactic acid bacteria, hatches 30min, removes the lactic acid bacteria of not sticking, then adds 10 7the shigella dysenteriae of individual/ml, hatches 30min; Then cover glass is taken out, with aseptic PBS rinsing 3 times, then be that 4% paraformaldehyde fixes 30 min with mass fraction, dry rear violet staining, in basis of microscopic observation, random choose 20 visuals field, 50, each visual field cell, calculate the lactic acid bacterium number of its surface adhesion and the mean value getting 3 experiments represents the adhesive capacity of cell, finishing screen selects KL02 and KL22 two strain bacterium;
(8) strain fermentation characteristic test
Get fresh milk 200 parts, white granulated sugar 18 parts, after allotment evenly, in 65 DEG C, homogeneous under 15MPa, after 95 DEG C of sterilization 300s, be cooled to 35-40 DEG C, according to 10 6cfu/ml inoculates KL02 and KL22 respectively, in 37 DEG C of fermentation 24h, carries out sensory evaluation, select the Kefir grains lactobacillus KL22 suppressing harmful intestinal tract bacteria effect according to sensory evaluation result finishing screen after fermentation ends to fermented sample.
Limit as one of the present invention, the candy ferments pipe described in step (3) comprises: salicin, ribose, arabinose, maltose, sweet mellow wine, fiber two pool, sucrose, gossypose and sorbierite.
Present invention also offers the application of the Kefir grains lactobacillus KL22 of above-mentioned suppression harmful intestinal tract bacteria effect, this Kefir grains lactobacillus is applied to prepares functional dairy product and probiotics bacterial powder.
Owing to have employed above-mentioned technical scheme, compared with prior art, acquired technological progress is in the present invention:
1, the bacterial strain that the present invention adopts is by the strain Kefir grains lactobacillus KL22 filtered out in Kefir granule, this bacterial strain has good acid and bile salt tolerance characteristic, namely by can be good at survival when human stomach and enteron aisle, thus prebiotic effect can well be played in human body;
2, the bacterial strain that the present invention adopts has the effect of well sticking enteron aisle and suppressing pathogen, and the effect therefore having good regulating intestinal canal and suppress pathogen to attack, can be used for the preparation of probiotics bacterial powder.
3, the bacterial strain that the present invention adopts has good fermentation character, therefore, can be applied in the exploitation of functional dairy product.
The present invention is applicable to the screening of Kefir grains lactobacillus KL22, and contains the preparation of Kefir grains lactobacillus KL22 functional dairy product and probiotics bacterial powder.
The present invention is described in further detail below in conjunction with Figure of description and specific embodiment.
Accompanying drawing explanation
Fig. 1 is the embodiment of the present invention 1 Kefir grains lactobacillus KL22 and other lactic acid bacterias bacterial strain evolution tree graph based on 16SrDNA.
Detailed description of the invention
embodiment 1 Kefir grains lactobacillus KL22 and screening technique thereof
Kefir grains lactobacillus KL22, this bacterial strain on September 16th, 2014, is preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, deposit number: CGMCC No.9669.
Its 16SrDNA sequence results is as follows:
TTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTGGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCG
This bacterial strain essential characteristic is as shown in the table:
The screening technique of above-mentioned Kefir grains lactobacillus KL22, it carries out according to following steps order:
(1) Kefir granule source
It is assorted that selected Kefir granule is derived from Polish Cali;
(2) separation of bacterial strain, purifying
A, Kefir granule was activated for 3 generations in defatted milk, get Kefir granule 1g, after sterile saline washing, in the mortar of sterilizing, grinding pulping, add 9ml physiological saline and dilute, get 1ml dilution and dilute, is 10 by dilution factor 4, 10 5with 10 6dilution is coated on bromocresol purple-MRS agar plate;
B, by Kefir granule by 5% inoculum concentration access sterilizing defatted milk in, cultivate 24h for 30 DEG C, draw Kefir granule acidified milk 1ml, suitably dilute with physiological saline, getting dilution factor is 10 6, 10 7, 10 8dilution coating bromocresol purple-MRS agar plate;
C, by step a and b coating bromocresol purple-MRS agar plate be placed in 37 DEG C of Anaerobic culturel 72h; Get above-mentioned plated growth good, there is colonies typical form and occur that the bacterium colony that color change carries out line separation, under aerobic conditions purifying 3 times repeatedly; Again through Gram's staining and catalase reaction, choose Gram-positive, the bacterial strain of catalase feminine gender saves backup, pick out the strain of doubtful lactic acid bacteria 50 altogether, comprising 38 strain bacillus and 12 strain coccuses;
(3) candy ferments characteristic test
Above-mentioned separation and purification 38 strain Gram-positive, the negative doubtful lactobacillus strain picking list bacterium colony of catalase are carried out plate streaking, cultivate 48h for 37 DEG C, carry out 37 DEG C in picking one oese bacterial strain access candy ferments pipe respectively and cultivate 48h, observation color changes, and finally according to the change of candy ferments experiment color is different, 38 strain bacterial strains is divided into 8 classes;
Wherein, candy ferments pipe comprises: salicin, ribose, arabinose, maltose, sweet mellow wine, fiber two pool, sucrose, gossypose, sorbierite.
Note: "+" represents that fermentation utilizes; "-" represents that azymic utilizes.
(4) molecular biology identification
Above-mentioned 38 strain bacterial strains are carried out molecular biology identification, by DNA extraction, pcr amplification, 16SrDNA checks order, and NCBI website blast finally determines that 38 strain bacterial strains comprise 14 strain Kefir grains lactobacillus, 10 strain lactobacillus bulgaricus, 8 strain Lactobacillus brevis, 6 strain Lactobacillus helveticus;
According to sequencing result, this bacterial strain chadogram result as shown in Figure 1.
The 16SrDNA sequencing result of 14 strain Kefir grains lactobacillus is as follows:
KL01 bacterial strain 16SrDNA sequence is:
TACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTTCGACTG
KL02 bacterial strain 16SrDNA sequence is:
TTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTTCGACTG
KL03 bacterial strain 16SrDNA sequence is:
TTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTTCGACTG
KL10 bacterial strain 16SrDNA sequence is:
TTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTCGACTG
KL11 bacterial strain 16SrDNA sequence is:
GTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTTCGACTG
KL12 bacterial strain 16SrDNA sequence is:
ACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTCGAC
KL18 bacterial strain 16SrDNA sequence is:
ACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTCGACTG
KL19 bacterial strain 16SrDNA sequence is:
TGGTCCCGAAGGTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACT
KL22 bacterial strain 16SrDNA sequence is:
TTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTGGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCG
KL24 bacterial strain 16SrDNA sequence is:
CTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCA
KL30 bacterial strain 16SrDNA sequence is:
GTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCA
KL31 bacterial strain 16SrDNA sequence is:
CTGGTCCCGAAGGTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTCACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTT
KL36 bacterial strain 16SrDNA sequence is:
TTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTCGACTG
KL40 bacterial strain 16SrDNA sequence is:
ACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCGTTCGACTG
(5) acid and bile salt tolerance characteristic test
By above-mentioned 14 strain Kefir grains lactobacillus, picking 1 oese purifying inoculation is in MRS culture medium respectively, and cultivate 48h for 37 DEG C, taking-up counts; Meanwhile, respectively get this culture medium of 1mL, join respectively pH be 2.5 phosphate buffer and 3 ‰ containing bovine bile MRS culture solution in 37 DEG C hatch, carry out the mensuration of bacterium number in 0h, 3h, and calculate its survival rate; Finishing screen selects 6 strain bacterium, is respectively KL02, KL03, KL10, KL18, KL22 and KL36; Different Kefir grains lactobacillus acid and bile salt tolerance experimental result is as shown in table 2:
Table 2 different Kefir grains lactobacillus acid and bile salt tolerance experimental result
As seen from the above table, bacterial strain KL02, KL03, KL10, KL18, KL22 and KL36 survival rate in acid solution and bile all higher than other bacterial classifications, all higher than 1 ‰.
(6) enterocyte adhesion characteristics experiment
Being added with every hole 1 mL cell suspension by enterocyte is added with in 24 orifice plates of cover glass, in 37 DEG C of incubators, hatch 48h; Treat that cell attachment is complete, wash 2 times with aseptic PBS, it is 10 that every hole adds 1 mL concentration 8the lactic acid bacteria suspension of individual/mL, hatches 1 h; Then cover glass is taken out, with aseptic PBS rinsing 3 times, then be that 4% paraformaldehyde fixes 30 min with mass fraction, dry rear violet staining, in basis of microscopic observation, random choose 20 visuals field, 50, each visual field cell, calculate the lactic acid bacterium number of its surface adhesion and the mean value getting 3 experiments represents the adhesive capacity of cell, finishing screen selects 4 strain bacterium, is respectively KL02, KL10, KL18 and KL22; To enterocyte experimental result sticked for not homophyletic Kefir grains lactobacillus as shown in table 3:
Table 3 not homophyletic Kefir grains lactobacillus sticks experiment to enterocyte
As seen from the above table, bacterial strain KL02, KL10, KL18 and KL22 are all greater than 10/cell for the lactic acid bacteria lived to the adherence rate of cell, are all greater than 9/cell for the lactic acid bacteria for deactivation to the adherence rate of cell.
(7) harmful bacteria characteristic test is suppressed
The bacterial strain above-mentioned 4 strains to better enterocyte adhesion properties carries out shigella dysenteriae Antagonism effect experiment, adds in 24 orifice plates, in 37 DEG C of incubators, hatch 48h by enterocyte with every hole 1 mL cell suspension; Treat that cell attachment is complete, wash 2 times with aseptic PBS, every hole adds 10 8individual/ml lactic acid bacteria, hatches 30min, removes the lactic acid bacteria of not sticking, then adds 10 7the shigella dysenteriae of individual/ml, hatches 30min; Then cover glass is taken out, with aseptic PBS rinsing 3 times, then be that 4% paraformaldehyde fixes 30 min with mass fraction, dry rear violet staining, in basis of microscopic observation, random choose 20 visuals field, 50, each visual field cell, calculate the lactic acid bacterium number of its surface adhesion and the mean value getting 3 experiments represents the adhesive capacity of cell, finishing screen selects KL02 and KL22 two strain bacterium; As shown in table 4 to the result of shigella dysenteriae Antagonism effect:
Table 4 Kefir grains lactobacillus is to the effect of shigella dysenteriae Antagonism
As seen from the above table, no matter bacterial strain KL02 and KL22 is that viable bacteria state or inactivated bacteria state are all less than KL10 and KL18 to shigella dysenteriae to the adherence rate of cell.
(8) strain fermentation characteristic
Get fresh milk 200 parts, white granulated sugar 18 parts, after allotment evenly, in 65 DEG C, homogeneous under 15MPa, after 95 DEG C of sterilization 300s, be cooled to 35-40 DEG C, inoculate KL02 and KL22 10 respectively 6cfu/ml, in 37 DEG C of fermentation 24h, carry out sensory evaluation to fermented sample after fermentation ends, select the Kefir grains lactobacillus KL22 suppressing harmful intestinal tract bacteria effect according to sensory evaluation result finishing screen, result is as shown in table 5:
Table 5 Kefir grains lactobacillus ferment characteristic
Bacterial strain The mouthfeel of institute's yoghourt The fragrance of institute's yoghourt
KL02 acidified milk Astringent, meta-acid Fragrance is general
KL22 acidified milk Smooth, mild acidity Fragrance is abundant
embodiment 2 is containing the preparation of the sour milk beverage of Kefir grains lactic acid bacteria KL22
By plain chocolate 50 parts, 30 parts, water, sucrose 5 parts mixes sterilizing 15min at 110 DEG C and prepares liquid medium;
By 10 6the Kefir grains lactobacillus KL22,10 of cfu/ml 6the lactobacillus bulgaricus of cfu/ml and 10 6mix in the streptococcus thermophilus access liquid medium of cfu/ml, 37 DEG C of fermentations, to pH to 4.2-4.6, stop fermentation, cool filling.Stored refrigerated.
embodiment 3 is containing the preparation of the probiotics bacterial powder of Kefir grains lactic acid bacteria KL22
Raw material form: Kefir grains lactobacillus KL22 3%, Lactobacillus plantarum 3%, lactobacillus acidophilus 1%,
Bifidobacterium lactis 1%, Lactobacillus casei 2%, FOS (prebiotics) 80%, Fragaia ananassa Duchesne extract 10%;
This probiotics bacterial powder preparation technique is: take bacterium powder by formula rate, FOS, Fragaia ananassa Duchesne extract mix, vacuum racking machine carries out packing, 2g/ bag (wherein biodiasmin sum>=4 × 10 10cfu/ bag), labeling is put in storage, stored refrigerated.
comparative example
Carry out enterocyte to Kefir grains lactobacillus KL22, lactobacillus bulgaricus, Lactobacillus plantarum, Bacillus bifidus and Lactobacillus casei (viable bacteria, inactivated bacteria) and stick experiment and enterocyte shigella dysenteriae Antagonism effect experiment, experimental result is distinguished as shown in table 6 and table 7:
The different lactobacillus cell of table 6 sticks experiment
The different lactic acid bacteria of table 7 is tested pathogenic bacteria enterocyte Antagonism
Shown in table 6 and table 7, Kefir grains lactobacillus KL22 has good Adhesion To Intestinal Epithelium and suppresses harmful bacteria to the adhesion of enterocyte compared with lactobacillus bulgaricus, Bifidobacterium, Lactobacillus plantarum, Lactobacillus casei.Point out Kefir grains lactobacillus KL22 may have better effect at suppression harmful bacteria in human body intestinal canal invasion and attack.
The above is only preferred embodiment of the present invention, is not restriction the present invention being made to other form, and any those skilled in the art may utilize above-mentioned technology contents to be changed or be modified as the Equivalent embodiments of equivalent variations as enlightenment.But everyly do not depart from technical solution of the present invention content, according to technical spirit of the present invention to the simple modification done by above embodiment, equivalent variations and remodeling, still belong to the protection domain of the claims in the present invention.
Organization Applicant
----------------------
Street: No. 36, Shi Tong road, Luquan district
City: Hebei province
State: Shijiazhuang City
Country: China
PostalCode :
PhoneNumber : 0311-80813256
FaxNumber :
EmailAddress :
<110> OrganizationName: Shijiazhuang Junlebao Dairy Co., Ltd.
 
Application Project
-------------------
<120> Title: Kefir grains lactobacillus KL22, its screening technique and application
 
<160> 1
<170> PatentIn version 3.3
Sequence
--------
<213> OrganismName : Lactobacilus kefiri strain
<400> PreSequenceString :
ttacctcacc ggctttgggt gttacaaact ctcatggtgt gacgggcggt gtgtacaagg 60
cccgggaacg tattcaccgt ggcatgctga tccacgatta ctagcgattc caacttcatg 120
caggcgagtt gcagcctgca atccgaactg agaacggctt taagagatta gcttgacctc 180
gcggtttcgc gactcgttgt accgtccatt gtagcacgtg tgtagcccag gtcataaggg 240
gcatgatgat ttgacgtcat ccccaccttc ctccggtttg tcaccggcag tcttgctaga 300
gtgcccaact gaatgctggc aactaacaat aagggttgcg ctcgttgcgg gacttaaccc 360
aacatctcac gacacgagct gacgacaacc atgcaccacc tgtcattctg tccccgaagg 420
gaacgcctaa tctcttaggt tggcagaaga tgtcaagacc tggtaaggtt cttcgcgtag 480
catcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt 540
caaccttgcg gtcgtactcc ccaggcggag tgcttaatgc gttagctgca gcactgaagg 600
gcggaaaccc tccaacactt agcactcatc gtttacggca tggactacca gggtatctaa 660
tcctgttcgc tacccatgct ttcgagcctc agcgtcagtt acagaccaga cagccgcctt 720
cgccactggt gttcttccat atatctacgc atttcaccgc tacacatgga gttccactgt 780
cctcttctgc actcaagtct cctggtttcc gatgcacttc tccggttaag ccgaaggctt 840
tcacatcaga cctaagaaac cgcctgcgct cgctttacgc ccaataaatc cggacaacgc 900
ttgccaccta cgtattaccg cggctgctgg cacgtagtta gccgtggctt tctggttgga 960
taccgtcaag atgtcaacag ttactctgac acctgttctt ctccaacaac agagttttac 1020
gagccgaaac ccttcatcac tcacgcggcg ttgctccatc agactttcgt ccattgtgga 1080
agattcccta ctgctgcctc ccgtaggagt ttgggccgtg tctcagtccc aatgtggccg 1140
attaccctct caggtcggct acgtatcatt gccttggtag gccattacct taccaacaag 1200
ctaatacgcc gcgggtccat cctaaagtga tagccgaagc catcttttaa accaaaacca 1260
tgtggttttg gttgttatac ggtattagca cctgtttcca agtgttatcc cctacttcaa 1320
gggcaggtta cccacgtgtt actcaccagt tcgccactcg tttggtgtta aatcattcaa 1380
atgcaagcac ctaaaatcaa taacggaaac gc 1412
<212> Type : RNA
<211> Length : 1412
SequenceName : KL22
SequenceDescription :
 

Claims (5)

1. Kefir grains lactobacillus KL22, its screening technique and application, it is characterized in that:this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, deposit number: CGMCC No.9669, and preservation date is on September 16th, 2014.
2. Kefir grains lactobacillus KL22 according to claim 1, it is characterized in that:its 16SrDNA sequence results is as follows:
TTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTGGTGTTAAATCATTCAAATGCAAGCACCTAAAATCAATAACGGAAACGCG。
3. a screening technique of Kefir grains lactobacillus KL22 as claimed in claim 1 or 2, it is characterized in thatit carries out according to following steps order:
(1) Kefir granule source
Selected Kefir granule is derived from Polish Ka Lishi;
(2) separation of bacterial strain, purifying
A, Kefir granule was activated for 3 generations in defatted milk, get the Kefir granule 1g after activation, after sterile saline washing, in the mortar of sterilizing, grinding pulping, add 9ml physiological saline and dilute, get 1ml dilution and dilute, is 10 by dilution factor 4, 10 5with 10 6dilution is coated on bromocresol purple-MRS agar plate;
B, by Kefir granule by 5% inoculum concentration access sterilizing defatted milk in, cultivate 24h for 30 DEG C, draw Kefir granule acidified milk 1ml, suitably dilute with physiological saline, getting dilution factor is 10 6, 10 7, 10 8dilution factor coating bromocresol purple-MRS agar plate;
C, by step a and b coating bromocresol purple-MRS agar plate be placed in 37 DEG C at Anaerobic culturel 72h; Get above-mentioned plated growth good, there is colonies typical form and occur that the bacterium colony that color change carries out line separation, under aerobic conditions purifying 3 times repeatedly; Again through Gram's staining and catalase reaction, choose Gram-positive, the bacterial strain of catalase feminine gender saves backup, pick out lactic acid bacteria 50 strain altogether, comprising 38 strain bacillus and 12 strain coccuses;
(3) candy ferments characteristic test
38 strain Gram-positives of above-mentioned separation and purification, catalase negative lactobacillus strain picking list bacterium colony are carried out plate streaking, cultivate 48h for 37 DEG C, at 37 DEG C, 48h is cultivated respectively in picking one oese bacterial strain access candy ferments pipe, observation color changes, and finally according to the change of candy ferments experiment color is different, 38 strain bacterial strains is divided into 8 classes;
(4) molecular biology identification
Above-mentioned 38 strain bacterial strains are carried out molecular biology identification, by DNA extraction, pcr amplification, 16SrDNA checks order, and NCBI website blast finally determines that 38 strain bacterial strains comprise 14 strain Kefir grains lactobacillus, 10 strain lactobacillus bulgaricus, 8 strain Lactobacillus brevis, 6 strain Lactobacillus helveticus;
(5) acid and bile salt tolerance characteristic test
By above-mentioned 14 strain Kefir grains lactobacillus difference picking 1 oese purifying inoculation in MRS culture medium, at 37 DEG C, cultivate 48h, take out counting; Meanwhile, respectively get the postvaccinal culture medium of 1mL, join respectively pH be 2.5 phosphate buffer and 3 ‰ containing bovine bile MRS culture solution in, hatch for 37 DEG C, carry out the mensuration of bacterium number in 0h, 3h, and calculate its survival rate; Finishing screen selects 6 strain bacterium, is respectively KL02, KL03, KL10, KL18, KL22 and KL36;
(6) enterocyte adhesion characteristics experiment
Being added with every hole 1 mL cell suspension by enterocyte is added with in 24 orifice plates of cover glass, in 37 DEG C of incubators, hatch 48h; Treat that cell attachment is complete, wash 2 times with aseptic PBS, it is 10 that every hole adds 1 mL concentration 8the lactic acid bacteria suspension of individual/mL, hatches 1 h; Then cover glass is taken out, with aseptic PBS rinsing 3 times, then be that 4% paraformaldehyde fixes 30 min with mass fraction, dry rear violet staining, in basis of microscopic observation, random choose 20 visuals field, 50, each visual field cell, calculate the lactic acid bacterium number of its surface adhesion and the mean value getting 3 experiments represents the adhesive capacity of cell, finishing screen selects 4 strain bacterium, is respectively KL02, KL10, KL18 and KL22;
(7) harmful bacteria characteristic test is suppressed
The bacterial strain above-mentioned 4 strains to better enterocyte adhesion properties carries out shigella dysenteriae Antagonism effect experiment, adds in 24 orifice plates, in 37 DEG C of incubators, hatch 48h by enterocyte with every hole 1 mL cell suspension; Treat that cell attachment is complete, wash 2 times with aseptic PBS, every hole adds 10 8individual/ml lactic acid bacteria, hatches 30min, removes the lactic acid bacteria of not sticking, then adds 10 7the shigella dysenteriae of individual/ml, hatches 30min; Then cover glass is taken out, with aseptic PBS rinsing 3 times, then be that 4% paraformaldehyde fixes 30 min with mass fraction, dry rear violet staining, in basis of microscopic observation, random choose 20 visuals field, 50, each visual field cell, calculate the lactic acid bacterium number of its surface adhesion and the mean value getting 3 experiments represents the adhesive capacity of cell, finishing screen selects KL02 and KL22 two strain bacterium;
(8) strain fermentation characteristic
Get fresh milk 200 parts, white granulated sugar 18 parts, after allotment evenly, in 65 DEG C, homogeneous under 15MPa, after 95 DEG C of sterilization 300s, be cooled to 35-40 DEG C, according to 10 6cfu/ml inoculates KL02 and KL22 respectively, in 37 DEG C of fermentation 24h, carries out sensory evaluation, select the Kefir grains lactobacillus KL22 suppressing harmful intestinal tract bacteria effect according to sensory evaluation result finishing screen after fermentation ends to fermented sample.
4. the screening technique of Kefir grains lactobacillus KL22 according to claim 3, it is characterized in thatcandy ferments pipe described in step (3) comprises: salicin, ribose, arabinose, maltose, sweet mellow wine, fiber two pool, sucrose, gossypose and sorbierite.
5. the application of the Kefir grains lactobacillus KL22 described in claim 1, it is characterized in that:this Kefir grains Yoghourt lactobacillus is applied to prepares functional dairy product and probiotics bacterial powder.
CN201410780337.9A 2014-12-17 2014-12-17 Lactobacillus kefiri KL22 as well as screening method and application thereof Pending CN104560793A (en)

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