CN111979127A - Method for separating lactic acid bacteria from yak triton - Google Patents
Method for separating lactic acid bacteria from yak triton Download PDFInfo
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- CN111979127A CN111979127A CN202010914206.0A CN202010914206A CN111979127A CN 111979127 A CN111979127 A CN 111979127A CN 202010914206 A CN202010914206 A CN 202010914206A CN 111979127 A CN111979127 A CN 111979127A
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- acid bacteria
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 100
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 title claims abstract description 59
- 241000894006 Bacteria Species 0.000 title claims abstract description 52
- 239000004310 lactic acid Substances 0.000 title claims abstract description 50
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000005406 washing Methods 0.000 claims abstract description 21
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 9
- 239000012154 double-distilled water Substances 0.000 claims description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 21
- 229920001817 Agar Polymers 0.000 claims description 20
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 20
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 239000008272 agar Substances 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 102000016938 Catalase Human genes 0.000 claims description 13
- 108010053835 Catalase Proteins 0.000 claims description 13
- 238000009630 liquid culture Methods 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 10
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 10
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 claims description 10
- 239000013078 crystal Substances 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 10
- 238000004043 dyeing Methods 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 235000013372 meat Nutrition 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 238000002791 soaking Methods 0.000 claims description 10
- 239000001632 sodium acetate Substances 0.000 claims description 10
- 235000017281 sodium acetate Nutrition 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000003794 Gram staining Methods 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 239000002250 absorbent Substances 0.000 claims description 5
- 230000002745 absorbent Effects 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 235000010216 calcium carbonate Nutrition 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 5
- 229910052740 iodine Inorganic materials 0.000 claims description 5
- 239000011630 iodine Substances 0.000 claims description 5
- 235000020191 long-life milk Nutrition 0.000 claims description 5
- 229940099596 manganese sulfate Drugs 0.000 claims description 5
- 239000011702 manganese sulphate Substances 0.000 claims description 5
- 235000007079 manganese sulphate Nutrition 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 230000002093 peripheral effect Effects 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 5
- 229920000136 polysorbate Polymers 0.000 claims description 5
- 229920000053 polysorbate 80 Polymers 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 238000010186 staining Methods 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 12
- 230000008901 benefit Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 235000013365 dairy product Nutrition 0.000 abstract description 2
- 238000002203 pretreatment Methods 0.000 abstract description 2
- 241000186660 Lactobacillus Species 0.000 abstract 2
- 229940039696 lactobacillus Drugs 0.000 abstract 2
- 239000002609 medium Substances 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000005238 degreasing Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000011109 contamination Methods 0.000 description 3
- 239000005862 Whey Substances 0.000 description 2
- 102000007544 Whey Proteins Human genes 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 235000020255 yak milk Nutrition 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 235000014048 cultured milk product Nutrition 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention relates to the technical field of bioengineering, and particularly relates to a method for separating lactic acid bacteria from yak triton. The method specifically comprises the seven steps of sample collection, surface disinfection, washing, pre-culture, lactobacillus separation, lactobacillus identification and preservation. According to the separation method of the lactic acid bacteria in the yak triton, the yak triton is treated by the pretreatment method, so that the separation of the lactic acid bacteria is facilitated, and the separation of the treated lactic acid bacteria has the advantages of high efficiency, less mixed bacteria pollution, simple steps, low cost and the like. The lactic acid bacteria separated by the method can be applied to dairy product production, and based on the advantages, the method can generate better social and economic benefits and has wide market prospect.
Description
Technical Field
The invention relates to the technical field of bioengineering, and particularly relates to a method for separating lactic acid bacteria from yak triton.
Background
The milk yeast is a fermented milk product prepared by degreasing yak milk, fermenting under natural conditions to coagulate casein and drying. The protein content of the Triton is higher than that of cheese made from common cow milk. Compared with fresh yak milk, the qula has the characteristics of higher protein content, easy storage and convenient transportation. The Qula is not only an important means for storing food protein for herdsmen in a Tibetan region, but also a starter for making yoghourt.
The manufacturing methods of the triton in different regions are very different, and different herdsmen families in the same region have different manufacturing habits. The quality and flavor of the qula are closely related to the preparation method, and are mainly controlled by a degreasing method, a fermenting agent used for preparation and environmental conditions, and the quality and flavor of the qula are directly determined by the factors. With the gradual improvement of the degreasing technology of the families of herdsmen in the Tibetan region, the adoption of an electric degreasing machine can meet the degreasing requirement; so far, the traditional way of adding whey liquid is still selected as a leaven when the herdsman makes the qula, and because the traditional way of adding whey liquid contains abnormally complex microorganisms, a plurality of microorganisms are involved in the fermentation process besides lactic acid bacteria in the process of making the qula.
The fermentation process of the qula belongs to natural fermentation, and the fermentation temperature cannot be accurately controlled, so that the qula is suitable for the growth and the propagation of various microorganisms in the open environment, and therefore, the qula has a complex microecological system; in addition, the manufacture of the triton adopts a rough production mode, particularly, in the later airing, the herdsman directly spreads the triton on the ground and dries the triton through insolation, so that the triton is easily polluted by some impurities in the environment, such as dust and the like, and meanwhile, microorganisms in the environment can be gathered on the surface of the triton during the airing, thereby causing certain microbial pollution. In addition, because the moisture content of the triton is low, the protein content is high, microorganisms in the triton are wrapped in the protein, and a sample is difficult to dissolve when a common method is adopted for separation, the separation efficiency of internal lactic acid bacteria can be influenced.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for separating lactic acid bacteria from yak triton.
The invention relates to a technical scheme of a separation method of lactic acid bacteria in yak triton, which comprises the following steps: the method specifically comprises the following steps:
step one, collecting a sample: collecting yak triton samples from a pasturing area or a peripheral market, and checking whether the yak triton is mildewed or damaged by worms during collection;
step two, surface disinfection: weighing a yak triton sample, adding the yak triton sample into a sterile centrifugal tube filled with alcohol with a volume fraction of 75% according to a material-liquid ratio of 1:10, soaking for 30-50 s, centrifuging at 4000rpm, removing supernatant, adding hydrogen peroxide with the same amount as the alcohol and a mass fraction of 15%, sterilizing for 15-20 min, and centrifuging at 4000rpm to obtain precipitate;
step three, washing: washing the yak triton with sterilized surface with sterile water for 4-5 times, then soaking with 0.9% sterile normal saline for 5-8 min, and absorbing the water on the surface of the yak triton with sterile absorbent paper;
step four, pre-culturing: putting the washed yak triton into sterilized milk with the volume 20-40 times of the original weight of the yak triton, and culturing at the temperature of 37-40 ℃ until the yak triton is curd;
step five, separating lactic acid bacteria: diluting 10g of pre-cultured curd in 90mL of physiological saline according to 10-fold gradient, and coating 0.1mL of diluent with proper dilution on MRS agar medium for culture at 37 ℃;
step six, identifying lactic acid bacteria: selecting colonies for catalase test and gram staining and microscopic examination, and primarily determining gram-positive and catalase test-negative pure strains as lactic acid bacteria;
and seventhly, preserving: inoculating the lactic acid bacteria preliminarily determined in the step six into a preservation medium, and then preserving at-80 ℃.
Further, the MRS agar culture medium in the fifth step is 10.5g of peptone, 10.5g of meat extract, 4.5g of yeast extract powder, 23.0g of glucose, 1.8g of dipotassium phosphate, 1.7g of diammonium hydrogen citrate, 21.5g of calcium carbonate, 4.5g of sodium acetate, and 7H of magnesium sulfate2O0.25 g, manganesemaganese 4H2O0.06 g, Tween 801.2 mL, agar 8.0g and ddH2O 1000mL。
Further, the gram stain in the sixth step is: carrying out primary dyeing on crystal violet for 50s, and mordanting the crystal violet for 50s by using iodine solution after washing by using sterile double distilled water; washing with sterile double distilled water, and decolorizing with 95% ethanol for 40 s; rinsing with sterile double distilled water, and re-staining with alkaline eosin for 40 s; observing the shape under an oil lens, and recording the shape, the size and the gram dyeing result under the oil lens.
Further, the preservation medium in the seventh step is a mixed solution of MRS liquid culture bacterial suspension and 15% of glycerol, wherein the MRS liquid culture bacterial suspension contains peptone, meat extract, yeast extract powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, calcium carbonate, sodium acetate, magnesium sulfate 7H2O, manganese sulfate 4H2O, Tween 80, agar and ddH2O。
Compared with the prior art, the invention has the following beneficial effects: after the yak triton is treated by the pretreatment method, the separation of lactic acid bacteria from the yak triton is more facilitated, and the separation of the treated lactic acid bacteria has the advantages of high efficiency, less mixed bacteria pollution, simple steps, low cost and the like. The lactic acid bacteria separated by the method can be applied to dairy product production, and based on the advantages, the method can generate better social and economic benefits and has wide market prospect.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for separating lactic acid bacteria from yak triton specifically comprises the following steps:
step one, collecting a sample: collecting yak triton samples from a pasturing area or a peripheral market, and checking whether the yak triton is mildewed or damaged by worms during collection;
step two, surface disinfection: weighing 5g of yak triton sample, adding the yak triton sample into a sterile centrifuge tube filled with 50mL of alcohol with volume fraction of 75% for soaking for 30s, centrifuging at 4000rpm, removing supernatant, adding 50mL of hydrogen peroxide with mass fraction of 15%, sterilizing for 15min, and centrifuging at 4000rpm to obtain precipitate;
step three, washing: washing yak Qula with sterilized surface with sterile water for 4 times, soaking with 0.9% sterile normal saline for 5min, and drying yak Qula surface water with sterile absorbent paper;
step four, pre-culturing: putting the washed yak triton into 100mL of sterilized milk, and culturing at 37 ℃ until curd;
step five, separating lactic acid bacteria: diluting 10g of pre-cultured curd in 90mL of physiological saline according to 10-fold gradient, and coating 0.1mL of diluent with proper dilution on MRS agar medium for culture at 37 ℃; wherein the MRS agar culture medium comprises peptone 10.5g, meat extract 10.5g, yeast extract 4.5g, glucose 23.0g, dipotassium hydrogen phosphate 1.8g, diammonium hydrogen citrate 1.7g, calcium carbonate 21.5g, sodium acetate 4.5g, magnesium sulfate 7H2O0.25 g, manganesemaganese 4H2O0.06 g, Tween 801.2 mL, agar 8.0g and ddH2O 1000mL；
Step six, identifying lactic acid bacteria: selecting colonies for catalase test and gram staining and microscopic examination, and primarily determining gram-positive and catalase test-negative pure strains as lactic acid bacteria; wherein, the gram stain is: carrying out primary dyeing on crystal violet for 50s, and mordanting the crystal violet for 50s by using iodine solution after washing by using sterile double distilled water; washing with sterile double distilled water, and decolorizing with 95% ethanol for 40 s; rinsing with sterile double distilled water, and re-staining with alkaline eosin for 40 s; observing the form under an oil lens, and recording the form, the size and the gram dyeing result under the oil lens;
and seventhly, preserving: inoculating the lactic acid bacteria preliminarily determined in the step six into a preservation medium, and then preserving at-80 ℃. Wherein the preservation medium is mixed solution of MRS liquid culture bacterial suspension and 15% glycerol, wherein the MRS liquid culture bacterial suspension contains peptone, meat extract, yeast extract powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, calcium carbonate, sodium acetate, magnesium sulfate 7H2O, manganese sulfate 4H2O, Tween 80, agar and ddH2And O. The strains obtained by identification and separation are all lactic acid bacteria, and the pollution rate is 0%.
The comparative example was set to compare with example 1 above, the steps of surface sterilization and rinsing were omitted, the remaining steps were identical to example 1, and 40% of the identified and isolated strains were lactic acid bacteria, with a contamination rate of 60%.
Example 2
A method for separating lactic acid bacteria from yak triton specifically comprises the following steps:
step one, collecting a sample: collecting yak triton samples from a pasturing area or a peripheral market, and checking whether the yak triton is mildewed or damaged by worms during collection;
step two, surface disinfection: weighing 15g of yak triton sample, adding the yak triton sample into a sterile centrifuge tube filled with 150mL of alcohol with volume fraction of 75% for soaking for 40s, centrifuging at 4000rpm, removing supernatant, adding 150mL of hydrogen peroxide with mass fraction of 15%, sterilizing for 18min, and centrifuging at 4000rpm to obtain precipitate;
step three, washing: washing yak Qula with sterilized surface with sterile water for 4 times, soaking in 0.9% sterile normal saline for 6min, and drying with sterile absorbent paper;
step four, pre-culturing: putting the washed yak triton into 450mL of sterilized milk, and culturing at 39 ℃ until curd;
step five, separating lactic acid bacteria: diluting 10g of pre-cultured curd in 90mL of physiological saline according to 10-fold gradient, and coating 0.1mL of diluent with proper dilution on MRS agar medium for culture at 37 ℃; wherein the MRS agar culture medium comprises peptone 10.5g, meat extract 10.5g, yeast extract 4.5g, glucose 23.0g, dipotassium hydrogen phosphate 1.8g, diammonium hydrogen citrate 1.7g, calcium carbonate 21.5g, sodium acetate 4.5g, magnesium sulfate 7H2O0.25 g, manganesemaganese 4H2O0.06 g, Tween 801.2 mL, agar 8.0g and ddH2O 1000mL；
Step six, identifying lactic acid bacteria: selecting colonies for catalase test and gram staining and microscopic examination, and primarily determining gram-positive and catalase test-negative pure strains as lactic acid bacteria; wherein, the gram stain is: carrying out primary dyeing on crystal violet for 50s, and mordanting the crystal violet for 50s by using iodine solution after washing by using sterile double distilled water; washing with sterile double distilled water, and decolorizing with 95% ethanol for 40 s; rinsing with sterile double distilled water, and re-staining with alkaline eosin for 40 s; observing the form under an oil lens, and recording the form, the size and the gram dyeing result under the oil lens;
and seventhly, preserving: gram-positive and catalase test-negative strains are selected to be inoculated in a preservation medium formed by mixing MRS liquid culture bacterial suspension and 15% of glycerol, and then the strain is preserved at the temperature of minus 80 ℃. Inoculating the lactic acid bacteria preliminarily determined in the step six into a preservation medium, and then preserving at-80 ℃. Wherein the preservation medium is mixed solution of MRS liquid culture bacterial suspension and 15% glycerol, wherein the MRS liquid culture bacterial suspension contains peptone, meat extract, yeast extract powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, calcium carbonate, sodium acetate, magnesium sulfate 7H2O, manganese sulfate 4H2O, Tween 80, agar and ddH2And O. The strains obtained by identification and separation are all lactic acid bacteria, and the pollution rate is 0%.
The comparative example was set to compare with example 2 above, the steps of surface sterilization and rinsing were omitted, the remaining steps were identical to example 2, and the identified and isolated strains were 50% lactic acid bacteria and the contamination rate was 50%.
Example 3
A method for separating lactic acid bacteria from yak triton specifically comprises the following steps:
step one, collecting a sample: collecting yak triton samples from a pasturing area or a peripheral market, and checking whether the yak triton is mildewed or damaged by worms during collection;
step two, surface disinfection: weighing 20g of yak triton sample, adding the yak triton sample into a sterile centrifuge tube filled with 200mL of alcohol with volume fraction of 75% for soaking for 50s, centrifuging at 4000rpm, removing supernatant, adding 200mL of hydrogen peroxide with mass fraction of 15%, sterilizing for 20min, and centrifuging at 4000rpm to obtain precipitate;
step three, washing: washing yak Qula with sterilized surface with sterile water for 5 times, soaking with 0.9% sterile normal saline for 8min, and drying with sterile absorbent paper;
step four, pre-culturing: putting the washed yak triton into 800mL of sterilized milk, and culturing at 40 ℃ until curd;
step five, separating lactic acid bacteria: diluting 10g of pre-cultured curd in 90mL of physiological saline according to 10-fold gradientCoating 0.1mL of diluent with proper dilution on MRS agar medium at 37 ℃ for culture; wherein the MRS agar culture medium comprises peptone 10.5g, meat extract 10.5g, yeast extract 4.5g, glucose 23.0g, dipotassium hydrogen phosphate 1.8g, diammonium hydrogen citrate 1.7g, calcium carbonate 21.5g, sodium acetate 4.5g, magnesium sulfate 7H2O0.25 g, manganesemaganese 4H2O0.06 g, Tween 801.2 mL, agar 8.0g and ddH2O 1000mL；
Step six, identifying lactic acid bacteria: selecting colonies for catalase test and gram staining and microscopic examination, and primarily determining gram-positive and catalase test-negative pure strains as lactic acid bacteria; wherein, the gram stain is: carrying out primary dyeing on crystal violet for 50s, and mordanting the crystal violet for 50s by using iodine solution after washing by using sterile double distilled water; washing with sterile double distilled water, and decolorizing with 95% ethanol for 40 s; rinsing with sterile double distilled water, and re-staining with alkaline eosin for 40 s; observing the form under an oil lens, and recording the form, the size and the gram dyeing result under the oil lens;
and seventhly, preserving: gram-positive and catalase test-negative strains are selected to be inoculated in a preservation medium formed by mixing MRS liquid culture bacterial suspension and 15% of glycerol, and then the strain is preserved at the temperature of minus 80 ℃. Inoculating the lactic acid bacteria preliminarily determined in the step six into a preservation medium, and then preserving at-80 ℃. Wherein the preservation medium is mixed solution of MRS liquid culture bacterial suspension and 15% glycerol, wherein the MRS liquid culture bacterial suspension contains peptone, meat extract, yeast extract powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, calcium carbonate, sodium acetate, magnesium sulfate 7H2O, manganese sulfate 4H2O, Tween 80, agar and ddH2And O. The strains obtained by identification and separation are all lactic acid bacteria, and the pollution rate is 0%.
The comparative example was set to compare with example 3 above, the steps of surface sterilization and rinsing were omitted in the comparative example, the remaining steps were completely the same as example 3, and 60% of the identified and isolated strains were lactic acid bacteria, and the contamination rate was 40%.
Example 4
The method disclosed by the invention is adopted to separate 97 gram-positive and catalase test-negative strains from a yak triton sample, and the strains are determined to be lactic acid bacteria. After the separated strains are continuously passaged for 20 times, determining the genetic stability of the strains, and selecting the strains from which the curd time is within 8h and the titer acidity is greater than 70 DEG T after the genetically unstable strains are removed. And 6 strains with better fermentation performance are obtained by primary screening, and the results are shown in table 1.
TABLE 1 screening results of lactic acid bacteria
Note: different letters in the same column indicate significant difference (p < 0.05).
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A method for separating lactic acid bacteria from yak triton is characterized by comprising the following steps:
step one, collecting a sample: collecting yak triton samples from a pasturing area or a peripheral market, and checking whether the yak triton is mildewed or damaged by worms during collection;
step two, surface disinfection: weighing a yak triton sample, adding the yak triton sample into a sterile centrifugal tube filled with alcohol with a volume fraction of 75% according to a material-liquid ratio of 1:10, soaking for 30-50 s, centrifuging at 4000rpm, removing supernatant, adding hydrogen peroxide with the same amount as the alcohol and a mass fraction of 15%, sterilizing for 15-20 min, and centrifuging at 4000rpm to obtain precipitate;
step three, washing: washing the yak triton with sterilized surface with sterile water for 4-5 times, then soaking with 0.9% sterile normal saline for 5-8 min, and absorbing the water on the surface of the yak triton with sterile absorbent paper;
step four, pre-culturing: putting the washed yak triton into sterilized milk with the volume 20-40 times of the original weight of the yak triton, and culturing at the temperature of 37-40 ℃ until the yak triton is curd;
step five, separating lactic acid bacteria: diluting 10g of pre-cultured curd in 90mL of physiological saline according to 10-fold gradient, and coating 0.1mL of diluent with proper dilution on MRS agar medium for culture at 37 ℃;
step six, identifying lactic acid bacteria: selecting colonies for catalase test and gram staining and microscopic examination, and primarily determining gram-positive and catalase test-negative pure strains as lactic acid bacteria;
and seventhly, preserving: inoculating the lactic acid bacteria preliminarily determined in the step six into a preservation medium, and then preserving at-80 ℃.
2. The method for separating lactic acid bacteria from yak triton as claimed in claim 1, wherein MRS agar medium in the fifth step is peptone 10.5g, meat extract 10.5g, yeast extract powder 4.5g, glucose 23.0g, dipotassium hydrogen phosphate 1.8g, diammonium hydrogen citrate 1.7g, calcium carbonate 21.5g, sodium acetate 4.5g, magnesium sulfate 7H2O0.25 g, manganesemaganese 4H2O0.06 g, Tween 801.2 mL, agar 8.0g and ddH2O 1000mL。
3. The method for separating the lactic acid bacteria in the yak trita according to claim 1, wherein the gram staining in the sixth step is as follows: carrying out primary dyeing on crystal violet for 50s, and mordanting the crystal violet for 50s by using iodine solution after washing by using sterile double distilled water; washing with sterile double distilled water, and decolorizing with 95% ethanol for 40 s; rinsing with sterile double distilled water, and re-staining with alkaline eosin for 40 s; observing the shape under an oil lens, and recording the shape, the size and the gram dyeing result under the oil lens.
4. The method for separating lactic acid bacteria from yak triton as claimed in claim 1, wherein the preservation medium in step seven is a mixture of MRS liquid culture suspension and 15% glycerol, wherein the MRS liquid culture suspension contains peptone, meat extract, yeast extract powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, calcium carbonate, sodium acetate, magnesium sulfate 7H2O, manganese sulfate 4H2O, Tween 80,Agar and ddH2O。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20140322273A1 (en) * | 2011-12-06 | 2014-10-30 | Bright Dairy & Food Co., Ltd. | Strain of exopolysaccharide-secreting lactobacillus plantarum and application thereof |
| CN107488604A (en) * | 2016-06-12 | 2017-12-19 | 张家港市山牧新材料技术开发有限公司 | MRS culture mediums, its preparation method and Isolation and purification method are used in lactic acid bacteria separation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20140322273A1 (en) * | 2011-12-06 | 2014-10-30 | Bright Dairy & Food Co., Ltd. | Strain of exopolysaccharide-secreting lactobacillus plantarum and application thereof |
| CN107488604A (en) * | 2016-06-12 | 2017-12-19 | 张家港市山牧新材料技术开发有限公司 | MRS culture mediums, its preparation method and Isolation and purification method are used in lactic acid bacteria separation |
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