CN106554925A - One plant of leuconostoc lactis with high-yield extracellular polysaccharide - Google Patents
One plant of leuconostoc lactis with high-yield extracellular polysaccharide Download PDFInfo
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Abstract
The invention discloses one plant of leuconostoc lactis with high-yield extracellular polysaccharide isolated from Qinghai-Tibet traditional zymotic yak yoghourt, preserving number is CGMCC No.11248, ability with high-yield extracellular polysaccharide, ability with certain hydrophobic ability and digestion resistant road environment, and with stronger bacteriostasis.Bacterial strain of the present invention has immune induction and adjustment effect in mice body, can be applied to the preparation of fermented dairy product and the preparation of health food and medicine.
Description
Technical field
The present invention relates to biological technical field, more particularly to extreme environment functional lactobacillus are developed and application, and separation, screening, identification and the prebiotic functional evaluation of the leuconostoc lactis with high-yield extracellular polysaccharide (EPS), and the application in food and pharmaceutical production.
Background technology
Microbial exopolysaccharides (exopolysaccharides, EPS) are that some peculiar microorganisms are secreted into during growth metabolism outside cell wall, are easy to the detached capsular polysaccharide of thalline or glue polysaccharide, belong to secondary metabolite.The generation of fermented-milk products and the prebiotic effect of lactic acid bacteria not only from lactic acid bacteria itself and cell wall constituent, some extracellular polysaccharide (EPS) produced in coming from the metabolite such as peptide and sweat of lactic acid bacteria.Lactic acid bacteria is acknowledged as safe microorganism (GRAS) as the microorganism of food stage, and its EPS for producing contributes to improving the quality and viscosity of natural fermentation dairy productss, and prevents fermented dairy product from shrinking dehydration.In addition, substantial amounts of research confirms that EPS has immunomodulating and antineoplastic physiologically active, the health of the mankind is had great significance.For antibacterial itself, EPS contributes to preventing being dried, toxic compounds and phage corrode, osmotic stress and promote its be attached to the surface of solids and biomembranous formation etc. (Vuyst et al., 1999).In addition, also research point out EPS have suppress pathogenic bacterium ability (Wu et al., 2010).Improvement results of the EPS to fermented dairy product quality, and prebiotic effect to human body itself, have had many research reports.And going deep into the prebiotic functional studies of EPS, the exploitation of prebioticses product also becomes health and extremely welcome prebiotic product new at present.But as the time of EPS researchs is shorter, the excavation of high yield EPS microorganisms shortcoming causes in fermented dairy product the industrial gel of addition to improve the scandal of product quality, and remains incessant after repeated prohibition, and the probiotic products containing EPS supply falls short of demand.This also illustrates that market is so urgent to the demand of high yield EPS lactic acid bacterias.
From from the perspective of ecology, Archimycetess or antibacterial all have a similitude, exactly using itself producing biopolymer protecting somatic cells, so as to from extreme environment infringement (Khan et al., 2011).Therefore; living in low temperature, High aititude, the extreme environment of strong ultraviolet, dilute oxygen for a long time may make lactic acid bacteria define a kind of special genetic mechanism; reach with more EPS are produced and resist external environment stress and protect the possibility of itself, and there is very high viscosity in the case of not adding any additive from the apparent observation of yak in Qinghai-tibet Yoghourt.
Thus, particularity of the probiotic lactobacillus with high-yield extracellular polysaccharide for screening in extreme circumstances based on its prebiotic function, is worth with larger development and application, to maintaining health tool to be of great significance.
The content of the invention
It is an object of the invention to provide a kind of have high-yield extracellular polysaccharide and maintain basic probiotic properties, the probiotic bacteria of special prebiotic function can be played in vivo, to further developing.
To achieve these goals, the inventive method step is as follows：
(1) microorganism in Qinghai-Tibet traditional zymotic yak yoghourt is selected feature bacterial strain purification, obtains pure detached bacterial strain by tetra- kinds of different culture medium culturings of MRS, M17, KFS and SL.
(2) with reference to hydrogen peroxide feminine gender, Gram-positive, thalli morphology and microscopy separating, selecting like lactobacilli strain, 16S rDNA sequencings are carried out, in NCBI, carries out Blast comparisons, identify and preserve 403 strains of lactic acid bacteria bacterial strains.
(3) the extracellular polysaccharide ability for determining the lactic acid bacteria thalline identified in (2) is screened, the ability that bacterial strain H52 of the present invention has high-yield extracellular polysaccharide is filtered out, leuconostoc lactis (Leuconostoc lactis) is accredited as in (2).
(4) the external prebiotic function of bacterial strain H52 of the present invention is evaluated, is determined including enzyme activity, bacteriostasis, Gl tract, bile tolerance, hydrophobic ability.
(5) by animal model test, vivo immunization characteristic research is carried out to bacterial strain of the present invention.
Leuconostoc lactis with high-yield extracellular polysaccharide of the present invention is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 17th in August in 2015, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.11248.
The present invention has the bacterial strain of combination property, the bacterial strain high-yield extracellular polysaccharide strains, with stronger peptidase and glycosidase hydrolysing activity, preferable probiotic properties are all showed in the in vitro tests of cholate tolerance, bacteriostasis, hydrophobic ability and Gl tract, there is stronger rejection ability to escherichia coli, staphylococcus aureuses, listeria spp, mouse typhuss shigella especially.
Jing animal experiments prove that bacterial strain of the present invention has no toxic side effect to internal organs, the increase of energy blood serum induced middle immune factor IL-10, IL-12 and IFN-γ, with immunoregulation effect.
Description of the drawings
Fig. 1 is impact of the 4 invention bacterial strain of the embodiment of the present invention to mice serum cytokine and SigA sIgA
Specific embodiment
To make the object, technical solutions and advantages of the present invention become more apparent, below in conjunction with specific embodiment, and referring to the drawings, the present invention is described in more detail.
Related culture medium prescription
Lactic acid bacteria culture medium (MRS) (Zhang Gang, 2007)：Peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, agar 15g, distilled water 1000mL, pH 6.2-6.6.Preparation method：Heating for dissolving will be added to the water by all the components in addition to agar, adjust pH 6.2～6.4, and add agar, 121 DEG C of sterilizing 15min to be down flat plate while hot.
M17 culture medium (Zhang Gang, 2007)：Plant matter peptone 5.0g, yeast extract 5.0g, polyprotein peptone 5.0g are weighed accurately, ascorbic acid 0.5g, beef extract 2.5g, β-phosphoglycerol disodium 19g measures 1.0mol/L MgSO4·7H2O 1.0mL, distilled water 1000mL, agar 15g.Preparation method：Heating for dissolving will be added to the water by all the components in addition to agar, adjust pH 7.0, and add agar, 121 DEG C of sterilizing 15min to be down flat plate while hot.
SL culture medium (Zhang Gang, 2007)：Weigh casein hydrolysate 10g, yeast extract 5g, diammonium hydrogen citrate 2g, sodium acetate 25g, Magnesium sulfate heptahydrate 0.58g, agar 15g, glucose 20g, Tween 80 1.0mL, the water manganese sulfate 0.15g of dipotassium hydrogen phosphate 6g, ferrous sulfate heptahydrate 0.03g, four, distilled water 1000mL.Preparation method：Heating for dissolving will be added to the water by all the components in addition to agar, adjust pH 5.4, and add the agar of heating for dissolving, mix homogeneously to boil 5min while stirring, be cooled to 50 DEG C, be down flat plate.
KFS streptococcus selective mediums：Proteose peptone 10g, yeast powder 10g, sodium glycerophosphate 10g, Sodium Chloride 5g, maltose 20g, Lactose 1g, sodium azide 0.4g, agar 13g, bromocresol purple 0.015g adjust pH 7.2.Preparation method：Heating for dissolving will be added to the water by all the components in addition to agar, adjust pH 7.2, and add agar, 121 DEG C of sterilizing 15min when being cooled to 50-60 DEG C, to add aseptic 1%TTC solution 10mL, mix, be down flat plate while hot.
Nutrient medium (NB)：Peptone 10g, Carnis Bovis seu Bubali cream 3g, Sodium Chloride 5g, distilled water 1000mL, pH 7.0.Preparation method：Mentioned component is mixed, pH, 121 DEG C of sterilizing 15min is adjusted after dissolving, is cooled down standby.When preparing Nutrient medium flat board, 0.8% agar is added.
Brain heart infusion broth (BHI)：Peptone 10g, is dehydrated little Medulla Bovis seu Bubali and soaks powder 12.5g, be dehydrated beef heart infusion 5g, Sodium Chloride 5g, glucose 2g, disodium hydrogen phosphate 2.5g, distilled water 1000mL, pH 7.4.Preparation method：Mentioned component is mixed, pH, 121 DEG C of sterilizing 15min is adjusted after dissolving, is cooled down standby.When preparing culture medium flat plate, 0.8% agar is added.
The reference strain adopted in test
Standard pathogenic bacterium：Salmonella typhimurium (Salmonella typhimurium, CICC10420), it is single to increase listeria spp (Listeria monocytogenes, CICC21583), staphylococcus aureuses (Staphylococcus aureus, CICC10384), colon bacillus (Escherichia coli, CICC20234).It is single to increase listeria spp brain-heart infusion medium culture, the Nutrient medium culture of remaining pathogenic bacterium.
Embodiment 1：Separate from Qinghai-Tibet traditional zymotic yak yoghourt and identify lactic acid bacteria
First, sample source
From China's Tianzhu Zang Autonomous County, Gansu Province traditional zymotic yak milk product.
2nd, from isolated lactic acid bacteria in traditional yak milk product
1mL fermentation yak milk product is taken by 1:10 ratios, are diluted with sterile saline, and this is 10-1Dilution factor.Then the above-mentioned diluents of 1mL, then 10 times of dilutions are drawn, is 10-2Dilution factor, by that analogy.Choose 10-5、10-6、10-7Three dilution factors, draw 100 μ L diluents respectively equably to be coated on MRS, M17, KFS and SL (adjusting pH5.4) plating medium respectively, respectively in 37 DEG C, 42 DEG C, 42 DEG C and 37 DEG C anaerobism constant temperature culture 48 hours, picking feature bacterium colony, and be further purified, obtain pure detached lactobacilli strain.
3rd, bacterial strain is identified
(1) Gram’s staining, catalase experiment
Gram’s staining：Pure bacterium colony on picking flat board carries out dye at the beginning of smear, fixation, crystal violet, iodine solution mordant dyeing, ethanol decolourizes, sarranine is redyed, is dried, oil mirror microscopy.Bluish violet is gram positive bacteria, and red is gram negative bacteria.
Catalase is tested：Bacterium to be measured is exposed into 30min in atmosphere, a small amount of 3% (volume fraction) hydrogen peroxide is drawn with dropper and is dripped the bacterium colony that grown in planar surface, after 2-3 minutes observation have that bubble produces for the positive, inaction is negative.
The bacterial strain Gram’s staining is the positive, and hydrogen peroxide experiment is feminine gender, can tentatively be considered as lactic acid bacteria.
(2) 16SrRNA identifications
After lactic acid bacteria after purification cultivates 8h in MRS culture medium, 10000rpm/min centrifugation 3-5min collects thallines extract DNA according to test kit using method.Then enter performing PCR amplification, (Monis et al, under amplification 2005), carry out the amplification of 16SrRNA in the universal primer 27F and 1492R of antibacterial.Amplification condition：95℃5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min 30s；72 DEG C of 5min, 30 circulations.PCR primer is served Hai Meijisheng sequencing companies and carries out sequence analysis.Lactobacilli strain 16S rRNA gene order BLAST (http will be measured：//wwwncbi.nlm.nih.gov/blast/) search in GenBank, it is more than 98% with test strains homology score, then it is believed that they belong to same kind.Totally 403 plants of appraisable lactic acid bacteria, is completely used for the screening of high-yield extracellular polysaccharide lactic acid bacteria.
Embodiment 2：The screening of high-yield extracellular polysaccharide lactic acid bacteria
(1) separation of extracellular polysaccharide
By isolated lactobacillus inoculum in sterilized MRS culture medium, 37 DEG C of cultures are activated 2-3 time.Bacterium solution after activation is adjusted into bacterium number to 2 × 108, then by 2% inoculum concentration be inoculated with respectively bacillus and coccus to 2mL MRS and M17 (containing 5% Lactose) culture medium in.37 DEG C of fermentation culture 24h, (4 DEG C) centrifugation 10min of 10000g, take supernatant.3 times of 95% cold ethanol of volume are added in supernatant, 4 DEG C of overnight precipitations in 12000g (4 DEG C), are centrifuged 6min, collect precipitation.Add the ultrapure water dissolution of 2mL heat, and load place in 8000-14000 bag filters 4 DEG C of refrigerators in dialyse 24h, change 3 water () daily per 8h once, constant volume determine sugared content (Guo Xinghua, 2008；Nuria et al.,2009).
(2) measure of sugared content
Sugared content is determined using phend-sulphuric acid.Standard glucose 20mg is weighed accurately in 500mL volumetric flasks, add water constant volume.After various reagents are added in test tube according to the amount shown in table 1,10min being stood, being shaken up, room temperature is placed 20min and absorbance detected under 490nm wavelength.2mL distilled water is pressed after equally processing as blank.With OD values and polyoses content do standard curve (Guo Xinghua, 2008).
1 glucose standard curve of table makes
Mensuration program of the isolated sugar juice from streptococcus acidi lactici fermented solution according to standard curve by more than, detects the light absorption value at 490nm.The amount of EPS is calculated according to standard curve, and filters out the lactobacilli strain of high yield EPS.
Table 2 is bacterial strain EPS yield of the present invention that filters out in test, and under no optimal conditions, EPS yield is 304.86mg/L.
2 lactic acid bacteria EPS yield of the present invention of table
Embodiment 3：External probiotic properties research
First, invention bacterial strain enzyme activity determination (API-ZYM)
Detected using French Mei Liai companies API-zym reagents, the technology is a semiquantitative micro method system, aimed at designed by research enzyme activity.Bacterium solution is collected by centrifugation thalline using aseptic MRS culture fluid Secondary Culture 3 times, prepares 1 × 10 with physiological saline solution7The bacteria suspension of CFU/mL is tested.
Table 3 shows that lactic acid bacteria H52 has stronger peptidase activity, can hydrolysis amino acid be effectively volatile molecules, and the local flavor generation to fermented product carries very important effect.And H52 strong glycosidase activity can effectively utilizes sugar source, the offer of lactic acid bacteria fermentation energy is provided, galactosidase activity is higher, can effectively alleviate human body intestinal canal lactose intolerance.
The enzyme activity of 3 invention lactic acid bacteria H52 of table
Note：" 1 " is equivalent to 5 micro- millimole concentration (5nM) of release；" 2 " are equivalent to 10nM；" 3 " are 20nM；" 4 " are 30nM；" 5 " are 40nM or higher.
2nd, invention bacterial strain bacteriostatic test
Using agar diffusion method, (Papamaloni et al., 2003) carry out bacteriostatic test.
Lactic acid bacteria fermentation supernatant prepares：By lactic acid bacteria activated overnight twice, bacterium number is adjusted to 1 × 108CFU/mL, 2% is inoculated in 3 different 10mL MRS fluid mediums, respectively numbering 1,2,3,37 DEG C, cultivates 24h.Streptococcus acidi lactici fermented solution 9500g is centrifuged into 10min, supernatant is taken.(Aslim et al.,2005).
45 DEG C will be cooled to after 20mL Nutrient medium or brain-heart infusion medium (containing 0.75% agar) sterilizing, be respectively connected to the different pathogenic bacterium of incubated overnight so as to final concentration of 1 × 106CFU/mL, mixes, is poured in sterile petri dish.After cooled and solidified, the hole of 8mm is beaten on flat board with aseptic card punch, 100 μ L difference 1,2, No. 3 fermentation liquids of lactic acid bacteria are separately added in hole, 37 DEG C of culture 24h after 4 DEG C of diffusion 5h, are placed.Antibacterial circle diameter is finally measured, the size in 8mm apertures is deducted.
Table 4 shows that the 24h fermentation liquids of invention bacterial strain H52 all show stronger rejection ability to staphylococcus aureuses, listeria spp, escherichia coli and mouse typhuss shigella, with relatively broad bacteriostasis.
The bacteriostasis of 4 invention bacterial strain H52 of table
Note:“–”,≤0mm；“±”,0–4mm；“+”,4–8mm；“++”,8–12mm；“+++”,>12mm.
3rd, invention bacterial strain digestive tract simulation test and hydrophobic ability are determined
(1) hydrophobic ability
Tested according to the method for Collado etc. (2008), thalline is inoculated in 37 DEG C of aseptic MRS culture medium Secondary Culture 3 times by the inoculum concentration of 1% (v/v) before experiment.Thalline is collected by centrifugation, PBS is washed twice, is resuspended in PBS, adjust bacterium number 1 × 108CFU/mL.Bacteria suspension is placed at room temperature, the absorbance under 600nm is detected in different time (0,2,4,6,8,10,20h).
Hydrophobic ability (%)=(A0-At)/A0×100
A0Represent the absorbance under 0h, AtRepresent the absorbance under different time (0,2,4,6,8,10,20h).
(2) Gl tract survival rate
Using the method for Carteris etc. (1998).Comprise the following steps that：
The preparation of simulated gastric fluid：0.35g pepsin is dissolved in the physiological saline solution of 100mL 0.2%, adjusts pH to 3.0 with concentrated hydrochloric acid, crosses 0.45 μm of filter membrane degerming.
The preparation of simulated intestinal fluid：0.1g trypsin, 1.8g cholate are dissolved in sterile vehicle NaHCO containing 1.1g3, 0.2g NaCl and 100mL distilled water, with the NaOH of 0.5M adjustment pH to 8.0.It is degerming that solution crosses 0.45 μm of filter membrane.
The bacterium solution for having been activated 3 times is inoculated in simulated gastric fluid (pH3) by 10% inoculum concentration, is mixed, 37 DEG C of Anaerobic culturels, samples plate count in 0,3h respectively.After 3h is cultivated in simulated gastric fluid, draw 1mL culture fluid and be inoculated in 9mL simulated intestinal fluids (pH 8), 37 DEG C of Anaerobic culturels, respectively 0,3,6,11,24h samplings are counted.
Counting MRS culture plates, normal saline gradient dilution, sampling coating, 37 DEG C of Anaerobic culturel 48h count the plate of 30～300 bacterium numbers.
Survival rate (%)=(log CFU N1/log CFU N0) × 100%
N1Represent the lactic acid bacterium number after Gl tract culture；N0Lactic acid bacterium number before representative simulation gastrointestinal tract culture.
(3) bile tolerance ability
In aseptic MRS-THIO culture medium (containing 0.2% sodium thioglycolate), the Fel Bovis seu Bubali of 0.3% (W/V) of addition, is then inoculated with 1% lactic acid bacteria (having activated three times), and matched group is the MRS-THIO culture fluid without Fel Bovis seu Bubali.37 DEG C of cultures of water-bath, every two hours sample mensuration absorbance under 620nm, with not lactobacteria-containing corresponding blank cultures zeroing.Record OD620nmThe time of 0.3 unit of change.Retardation time (LT) lactobacter growth is calculated as to OD620nmThe time postponed during 0.3 unit of change.The less bile tolerance abilities of LT it is stronger (Walker and Gilliland, 1993).
Table 5 shows that bacterial strain of the present invention has digestive tract tolerance and hydrophobic ability, can keep higher survival rate, and hydrophobicity increased the chance for being attached to intestinal walls, meet the primary condition as probiotic bacteria in digestive tract environment.
5 invention bacterial strain H52 of table simulates digestive tract tolerance and hydrophobic ability
1Lactic acid bacteria is survived after 3h in pH3 simulated gastric fluids, then the survival rate after 24h is digested in simulated intestinal fluid.
2In absorbance OD620nmWhen, in two kinds of culture medium of MRS-THIO and MRS-THIO (containing 0.3% cholate), strain growth increases the time difference required for 0.3 unit.
Embodiment 4：Animal experiment
(1) mice is fed
18 male BALB mices, body weight 25g or so, free choice feeding and drinking-water, Jing after the preliminary trial period of 7 days, empty stomach 12h, water restriction, does not weigh.2 groups are randomly divided into according to body weight, 9 per group, each treatment group feeds basal diet.Mice group process and dosage are shown in Table 6, daily gavage once, 2 weeks experimental periods.Mice natural lighting, free choice feeding and drinking-water.
6 laboratory animal of table is grouped and processes
After last day gavage, stop feed 8h, not water restriction.Weigh, pluck eyeball and take blood, room temperature is placed, and centrifugation (4000g, 10min) separates serum, and -80 DEG C save backup.The vertebra dislocation of blood sampling reserve strength is put to death, and is positioned on ice, separates liver, the heart, kidney, spleen, thymus.With the normal saline flushing surface bloodstain of pre-cooling, hospital gauze is wiped dry, is weighed respectively.It is calculated as follows internal organs proportion：
(2) measure of cytokine IL-12, IL-10 and IFN-γ
Blood serum sample room temperature is melted, and carries out (purchased from Shanghai Yan Ji bio tech ltd) according to the workbook of IL-12, IL-10 and IFN-γ ELISA kit.
(3) measure of S-IgA sIgA
Blood serum sample room temperature is melted, and carries out according to the workbook of sIgA radioimmunoassay kitss.
It is shown in Table 7 result of the tests to show, invention bacterial strain has no toxic side effect internal organs.And Fig. 1 shows, the increase of invention bacterial strain energy blood serum induced middle immune factor IL-10, IL-12 and IFN-γ is induced with vivo immunization and adjustment effect.
Impact of the 7 invention bacterial strain of table to mice organs
The bacterial strain leuconostoc lactis H52 tests that the present invention is provided prove which has higher extracellular polysaccharide ability.The bacterial strain meets the primary condition as probiotic bacteria, the ability with stronger suppression common causative bacteria growing, to maintaining intestinal normal microbial flora balance tool to have certain effect, can keep passing through digestive tract environment compared with high viability.The bacterial strain preferably hydrophobic ability increased which and be attached to intestinal walls and play the chance of prebiotic effect.The product has no toxic side effect, and can improve the effect such as immunity of organisms, regulating intestinal canal flora as health food or medicine.The invention bacterial strain is used in Dairy fermentation the quality that can improve fermented product.
Those of ordinary skill in the art should be understood：The specific embodiment of the present invention is the foregoing is only, the present invention, all any modification, equivalent substitution and improvements within the spirit and principles in the present invention, done etc. is not limited to, be should be included within the scope of the present invention.
Claims (5)
1. a kind of leuconostoc lactis with high-yield extracellular polysaccharide, deposit number are CGMCC No.11248.
2. the process for screening and identifying of the leuconostoc lactis described in a kind of claim 1, it is characterised in that comprise the following steps：
(1) from separating lactic acid bacteria strain in Qinghai-Tibet traditional zymotic yak yoghourt, and purification, obtain pure detached bacterial strain.
(2) lactobacilli strain filtered out by catalase experiment, Gram’s staining, thalli morphology and 16SrRNA sequence analysis, authentication step (1), including bacterial strain H52 leuconostoc lactises of the present invention.
(3) sugared content in bacterial strain 24h fermentation liquids of the present invention is determined by phend-sulphuric acid, filters out the bacterial strain of the present invention with high-yield extracellular polysaccharide ability.
3. the probiotic properties assessment method of the leuconostoc lactis described in a kind of claim 1, it is characterised in that including herein below：
(1) verify that bacterial strain of the present invention has stronger bacteriostasis by bacteriostatic test.
(2) verify that bacterial strain of the present invention has basic probiotic properties and immunoregulation effect by internal, in vitro tests.
4. application of the leuconostoc lactis with high-yield extracellular polysaccharide described in claim 1 in fermented dairy product is prepared.
5. application of the leuconostoc lactis with high-yield extracellular polysaccharide described in claim 1 in health food and medicine is prepared.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107557303A (en) * | 2017-09-08 | 2018-01-09 | 天津大学 | A kind of ocean thraustochytriale Isolation and screening of bacterial strain method of high-yield extracellular polysaccharide |
| CN108441434A (en) * | 2017-09-19 | 2018-08-24 | 西藏农牧学院 | A kind of method of the separation of probiotic yak source lactic acid bacteria, identification and preliminary screening |
| CN110024849A (en) * | 2019-04-23 | 2019-07-19 | 兰州大学 | A kind of application of leuconostoc lactis |
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| CN1695469A (en) * | 2005-05-17 | 2005-11-16 | 田星 | Flavor rancid milk beneficial for health and preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107557303A (en) * | 2017-09-08 | 2018-01-09 | 天津大学 | A kind of ocean thraustochytriale Isolation and screening of bacterial strain method of high-yield extracellular polysaccharide |
| CN108441434A (en) * | 2017-09-19 | 2018-08-24 | 西藏农牧学院 | A kind of method of the separation of probiotic yak source lactic acid bacteria, identification and preliminary screening |
| CN110024849A (en) * | 2019-04-23 | 2019-07-19 | 兰州大学 | A kind of application of leuconostoc lactis |
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