CN108265019A - Leuconostoc mesenteroides subsp mesenteroides, preparation method and application - Google Patents
Leuconostoc mesenteroides subsp mesenteroides, preparation method and application Download PDFInfo
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Abstract
Present invention is disclosed a kind of leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides), preparation method and applications, which is CCTCC NO：M 2018129.Leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides) the CCTCC NO：M 2018129 has high acid ability and degrading nitrite ability, and salt tolerant and acid and alkali-resistance, can extend product storage period again while improving product nutritional quality, develop Resource of lactic bacteria database, be conducive to the development of pickles industry, have a vast market prospect.
Description
Technical field
The present invention relates to microorganism fields, and in particular to a kind of leuconostoc mesenteroides subsp mesenteroides, preparation method and should
With.
Background technology
Leukonid is a category for lactic acid bacteria, is the gram-positive bacterium of a kind of heterolactic fermentation, is present in plant
The surface and root of object are the predominant bacterias in newborn or vegetable material spontaneous fermentation food.It is organic that fermentation can be metabolized generation
A variety of aromatic compounds such as acid, alcohols and biacetyl improve product special flavour；Blood thickener can be used as, relax by generating glucan
Spreader and stabilizer；Generation mannitol can be used as low calorie sweetener；Biological preservative can be used as by generating bacteriocin；It generates oligomeric
Sugar can maintain the ecological balance of human body intestinal canal normal flora, have probiotic effects, and leukonid is expected to become new Tiny ecosystem system
Agent.
Leuconostoc mesenteroides is the important strain of Leuconostoc, be one kind without the total of gemma gram-positive bacterium
Claim, Leuconostoc mesenteroides is non-toxic to humans and animals and pathogenic effects, especially leuconostoc mesenteroides subsp mesenteroides are by me
The Ministry of Public Health of state (No. 8 bulletin in 2012) is classified as one of edible strain with U.S. Food and Drug Administration (FDA).Goldbeater's skin
Leukonid goldbeater's skin subspecies, which have, adjusts intestinal flora balance, and nutriment is promoted to inhale, improves product special flavour, while is also had anti-
Oxidability and antagonism pathogenic bacteria ability.Because it can generate flavour substance, extensively should in the field of food such as breast and pickles
With.
Pickles are a kind of traditional foods for having long history, and the pickles of prevalence are main by the place of production and taste in the world at present
It is divided into Chinese pickle, Pickles and Japanese pickles, Japanese pickles belong to non-fermented type, and Chinese pickle and Pickles are fermentations
Type.Korean nationality's pickles are one of Chinese pickles of great national characters, and various in style, sorting is extensive, according to raw material, preparation method, usage
With the difference of time, taste is different, has unique dense flavor and rich in lactic bacteria activity and lactic acid.Cherry dish is the Koreans
The Specialty vegetable of plantation, growth period is short and with seasonality, and full of nutrition, taste is suitble to popular taste.At present from Henan Linzhou City
The in the majority of Leuconostoc is isolated in pickles, Pickles, Sichuan Style, Tian jin cabbage pickled in sweet and sour, but is not yet found in Korean traditional fermented cherry dish
Detach Leuconostoc mesenteroides.Same species of microorganism is because bacterium source, separation and cultural method difference, characteristic and functionality are in the presence of certain poor
It is different.In traditional fermented food, such as pickles, during the fermentation due to the slow-growing or degrading nitrite of lactic acid bacteria
Ability weaker cause to generate the shelf-life that many miscellaneous bacterias affect pickles in certain fermented foods, and Asia can be produced in pickles
Nitrate, nitrite are a kind of substances very harmful to human body, and excessive or long-term consumption is easily carcinogenic.Therefore one kind is needed
It can solve the bacterium of problems.
Invention content
The defects of it is an object of the invention to overcome the prior art, provides a kind of leuconostoc mesenteroides subsp mesenteroides and its system
Preparation Method and application, the leuconostoc mesenteroides subsp mesenteroides have high acid ability and degrading nitrite ability, and salt tolerant and
Acid and alkali-resistance, by the leuconostoc mesenteroides subsp mesenteroides can in the fermentation of pickles degrading nitrite, and high acid ability
It can inhibit the breeding of harmful bacteria, extend the shelf-life of pickles.
To achieve the above object, the present invention proposes the technical solution of first aspect：A kind of leuconostoc mesenteroides subsp mesenteroides
(Leuconostoc mesenteroides subsp.mesenteroides), is deposited in China typical culture collection
The heart, preserving number are：CCTCC NO：M 2018129.
It is isolated from the cherry dish pickles of Yanbian, Jilin Province Korean nationality, obtains the leuconostoc mesenteroides subsp mesenteroides
(Leuconostoc mesenteroides subsp.Mesenteroides).The bacterial strain is identified, result is bright for goldbeater's skin
Beading bacterium goldbeater's skin subspecies (Leuconostoc mesenteroides subsp. mesenteroides).The bacterial strain is in 2018
On March 14, in is preserved in China typical culture collection center（Address：Wuhan, China Wuhan University）, and receive collection and step on
Charge to volume, number CCTCC NO：M 2018129, and on March 28th, 2018 determining testing result for survival.
The present invention proposes the technical solution of second aspect：A kind of leuconostoc mesenteroides subsp mesenteroides Leuconostoc mesenteroides goldbeater's skin
Subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO：The preparation side of M 2018129
Method includes the following steps：Using pickles as bacterium source, it is preferable that pickles are cherry dish.Goldbeater's skin is cultivated in culture medium under aerobic conditions
Leukonid goldbeater's skin subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO：M
2018129。
The culture medium of the present invention is the culture medium of this field routine, can grow the leuconostoc mesenteroides subsp mesenteroides
(Leuconostoc mesenteroides subsp.Mesenteroides)CCTCC NO：M 2018129.Preferably,
Culture medium includes MRS fluid nutrient mediums, LMM solid mediums and in the MRS solid medium containing vancomycin, it is preferable that ten thousand
Ancient a concentration of 30ug/ml of mycin.Using the concentration vancomycin when can either ensure to obtain best Leuconostoc mesenteroides intestines
Film subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO：M 2018129, and
Other bacterium can not grow in this concentration, be more advantageous to obtaining purebred leuconostoc mesenteroides subsp mesenteroides JNZB003.Specifically
Culture of isolated step is followed successively by, first, the Multiplying culture leuconostoc mesenteroides subsp mesenteroides in MRS fluid nutrient mediums
(Leuconostoc mesenteroides subsp. Mesenteroides)CCTCC NO：M 2018129, secondly, in LMM
Leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp. are separately cultured on solid medium
Mesenteroides)CCTCC NO：M 2018129 again, training is isolated and purified in the MRS solid mediums containing vancomycin
Support leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC
NO：M 2018129, wherein, it is separately cultured on solid medium using plate streak.
The temperature of the culture is the temperature and Conventional Time of this field routine, can grow Leuconostoc mesenteroides goldbeater's skin
Subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO：M 2018129, it is preferable that
Cultivation temperature is 25~36 DEG C in MRS fluid nutrient mediums, and incubation time is 24~72h, further, in MRS Liquid Cultures
Cultivation temperature is 30 DEG C in base, and condition of culture is 24~72h of 120-160r/min isothermal vibration cultures.Best, in MRS liquid
Cultivation temperature is 30 DEG C in culture medium, and condition of culture is 140r/min isothermal vibration culture 48h, can obtain performance under this condition
More excellent leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides)
CCTCC NO：M 2018129.
It is preferably, described that cultivation temperature is in LMM solid mediums and in the MRS solid mediums containing vancomycin
25~36 DEG C, incubation time is 24~72h, further, in LMM solid mediums and in the MRS solids containing vancomycin
Cultivation temperature is 30 DEG C in culture medium, and condition of culture is to be inverted constant temperature quiescent culture to obtain the Leuconostoc mesenteroides of best performance
Goldbeater's skin subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO：M 2018129.
The present invention proposes the technical solution of the third aspect：Leuconostoc mesenteroides subsp mesenteroides (Leuconostoc
mesenteroides subsp.Mesenteroides)CCTCC NO：Applications of the M 2018129 in pickle production.
The beneficial effects of the invention are as follows：The leuconostoc mesenteroides subsp mesenteroides intestines detached from Korean traditional fermented cherry dish
Film subspecies have high acid ability and degrading nitrite ability, and salt tolerant and acid and alkali-resistance.The same of product nutritional quality can be improved
When can extend product storage period again, develop Resource of lactic bacteria database.Be conducive to the development of pickles industry, before having a vast market
Scape.
Description of the drawings
Fig. 1 is the colonial morphology figure that the bacterial strain JNJB003 of the present invention is detached on LMM culture mediums；
Fig. 2 is the colonial morphology figure that the bacterial strain JNJB003 of the present invention is purified on MRS culture mediums；
Fig. 3 is part bacterial strain gram stain microscopy photo；
Fig. 4 is strain morphology figure of the bacterial strain after catalase is tested in Fig. 3；
Fig. 5 a are that bacterial strain growth test under the conditions of 37 DEG C identifies picture；
Fig. 5 b are that bacterial strain growth test in the culture medium containing 30 μ g/mL vancomycins identifies picture；
Fig. 5 c are the Salt tolerance identification pictures of bacterial strain；
Fig. 5 d are the arginine hydrolysis experiment identification pictures of bacterial strain；
Fig. 5 e are the sweet hydrolysis experiment identification pictures of seven leaves of bacterial strain；
Fig. 5 f are the Metabolism of Citric Acid test for identification pictures of bacterial strain；
Fig. 5 g are the glucan generation identification pictures of bacterial strain；
Fig. 5 h are the carbohydrate fermentation test for identification pictures of bacterial strain；
Fig. 6 is the strain bacterium pcr amplification product result figure filtered out；
Fig. 7 is that leuconostoc mesenteroides subsp mesenteroides JNZB003 grows bar chart at different temperatures；
Fig. 8 is the bar chart that leuconostoc mesenteroides subsp mesenteroides JNZB003 is grown under different salt concentration conditions；
Fig. 9 is the OD values of leuconostoc mesenteroides subsp mesenteroides JNZB003 with incubation time change curve；
Figure 10 is the curve graph that leuconostoc mesenteroides subsp mesenteroides JNZB003 changes over time production acid；
Figure 11 is the leuconostoc mesenteroides subsp mesenteroides JNZB003 curve graphs that optical density is grown at various ph values；
Figure 12 is curve graphs of the leuconostoc mesenteroides subsp mesenteroides JNZB003 to various concentration nitrite degradation ability；
Figure 13 is leuconostoc mesenteroides subsp mesenteroides JNZB003 degrading nitrite ability qualification test pictures.
Specific embodiment
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.Test method without specific conditions in the following example, according to conventional methods and conditions or according to quotient
Product specification selects.
The control strain of the present invention is selected as Leuconostoc mesenteroides bacterial strain (BNCC195309), by the comparison of the bacterial strain to obtain
Leuconostoc mesenteroides bacterial strain is obtained, the bacterial strain (BNCC195309) is purchased from Bei Na Chuan Lian Bioisystech Co., Ltd；MRS solids are trained
It supports base, MRS fluid nutrient mediums, 1% vancomycin and is purchased from Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd；Amygdalin, bear
Fructose, bigcatkin willow are sweet, erythrite is purchased from MACKLIN；Mannose is purchased from MERCK；Sugar is purchased from two factory of Shanghai reagent in L- mouse；
Galactolipin, sorbierite, cellobiose, lactose, maltose, sucrose, fructose, L-arabinose, trehalose, D- xyloses, gossypose,
L- sorboses, melibiose are purchased from SIGMA；Ezup pillar bacterial genomes DNA extraction agent boxes, SanPrep pillar DNA glue
QIAquick Gel Extraction Kit is purchased from Sangon Biotech (Shanghai) Co., Ltd..
The instrument and equipment of the present invention：II autoclave Spain SELECTA of PRESOCLAVE-；2720thermal
Cycler PCR instrument Applied Biosystems；HC-2518R high-speed refrigerated centrifuges BBI；CX41-RF fluorescence microscope days
This OLYMPUS.
The reagents and materials used in the present invention are commercially available.
Unmentioned method is conventional method in preparation method of the present invention.
The isolation and purification of embodiment 1, bacterial strain JNJB003
Strain source：It is taken respectively from the biography of Kumgang mountain pickles brand shop of Korean Autonomous Prefecture of Yanbian, the market of farm produce and supermarket
System fermentation cherry dish, using separation, purification process, filters out single Leuconostoc mesenteroides bacterial strain, specific method is：Aseptic condition
Under, Korea-Yanbian traditional zymotic cherry vegetable juice 1mL is taken, is placed in the test tube of sterilizing, LMM solids are being contained using plate streak
It crosses in the culture dish of culture medium, is inverted culture dish after scribing line, under 30 DEG C, aerobic conditions, constant temperature incubation 48h.From
The bacterium colony of drops toughness polysaccharide is generated in culture dish containing LMM solid mediums around picking, is containing 30ug/mL ten thousand
Scribing line isolates and purifies 3 times in the culture dish of the MRS solid mediums of ancient mycin, picking single bacterium colony, by microscopy, and and goldbeater's skin
Leukonid bacterial strain (BNCC195309) compares, and confirms and obtains single pure culture, JNJB003 is named as, such as Fig. 1 and 2 institutes
Show.
The identification of embodiment 2, bacterial strain JNJB003
First, the Preliminary Identification of bacterial strain JNJB003
Gram's stain is widely used a kind of discrimination method in bacteriology, and catalase experiment is that identification leather is blue
The important experiment of family name's positive bacteria, it is doubtful to isolating and purifying 20 plants from Korean traditional fermented cherry dish further to identify bacterial strain
The bacterial strain of leukonid carries out Gram's staining and catalase experiment, as shown in figure 3, part bacterial strain gram stain microscopy
Photo, wherein, a is bacterial strain JNZB003；B is bacterial strain JNZB005；C is bacterial strain JNZB011, d JNZB020.Such as Fig. 4 institutes
Show, catalase test photo.Through 20 plants of bacterium of microscopy be Gram-positive, 17 plants of negative catalases, by 17 plants
Catalase negative is further identified.
2nd, the Physiology and biochemistry identification of bacterial strain JNJB003
The physiological property identification of bacterial strain
17 plants of bacterial strains of screening have been subjected to physiological property identification, including 37 DEG C of growth tests, 1 DEG C of growth test,
Catalase test, vancomycin resistance experiment, 3% sodium chloride growth test and 6.5% sodium chloride growth test, qualification result
It is shown in Table 1.From table 1 it follows that the 17 plants of bacterial strains screened have tolerance to 30ug/ml vancomycins, and can be
It is grown in 3% sodium chloride and 6.5% sodium chloride solution, but the extent of growth between each bacterial strain is presented with very big difference,
This 6 plants of bacterium of JNZB003, JNZB009, JNZB011, JNZB012, JNZB015 and NZB019 are in 3% sodium chloride and 6.5% chlorination
Well-grown and degree approaches in sodium solution, JNZB005 and JNZB017 are in 3% sodium chloride well-grown, but in 6.5% chlorination
Faint growth in sodium solution；17 plants of bacterial strains are not grown at 1 DEG C, and 11 plants of strain growths at 37 DEG C, 6 plants of bacterial strains are not grown.
Wherein, it is as follows to the tolerance test method of 30ug/ml vancomycins：
(1) culture of activation is taken, is crossed on containing 30ug/mL vancomycin MRS solid mediums, 37 DEG C of cultures
The bacterium colony of growth if growth, is repeated primary inoculation, culture, if still there is bacterium colony, representing 37 DEG C can grow by 24-72h.
(2) culture of activation is taken, is crossed on containing 30ug/mL vancomycin MRS solid mediums, 1 DEG C of culture
The bacterium colony of growth if growth, is repeated primary inoculation, culture, if still there is bacterium colony, representing 1 DEG C can grow by 48h.
Salt tolerance test method is as follows：
The picking colony on the solid medium containing 30ug/mL vancomycins, access contain 3.0% and 6.5%NaCl's
It in MRS fluid nutrient mediums, after being cultivated for 24 hours at 30 DEG C, shakes up, measures its OD600Value, using MRS fluid nutrient mediums as blank control.
Catalase test method is as follows：
In sterilizing, on clean glass slide, the hydrogen peroxide of drop upper 3%, the bacterium colony on picking MRS solid mediums, with
3% hydrogen peroxide mixing, if there is bubble to represent positive reaction, bubble-free represents negative reaction.
1 bacterial strain physiological property qualification result of table
"+" represents growth；"-" representative is not grown
The biochemical identification result of bacterial strain JNJB003
On the basis of the identification of above-mentioned physiological property, basic biochemistry identification is carried out to 17 plants of bacterial strains of screening, is specifically included
Arginine hydrolysis experiment, citrate utilization test, glucan generation experiment, aesculin hydrolysis experiment, qualification result are shown in Table 2.From
In table 2 as can be seen that 17 plants of bacterium arginine hydrolysis experiments be positive reaction, this with《Primary Jie Shi Bacteria Identifications handbook》Described in
Leuconostoc characteristic it is inconsistent；Citric acid can be metabolized in 17 plants of strains of screening for 7 plants, 10 plants of not energy metabolism lemons
Acid；6 plants can hydrolyze aesculin, and 11 plants cannot hydrolyze aesculin；3 plants can utilize sucrose to generate glucan.In Leuconostoc
In only leuconostoc mesenteroides subsp mesenteroides can utilize sucrose generate glucan, thick lawn is formed on culture medium,
It, which is planted, does not have this feature, is compareed according to this characteristic《Primary Jie Shi Bacteria Identifications handbook》With《Lactic acid bacteria taxonomic identification and reality
Proved recipe method》, identified with reference to physiological property, by bacterial strain JNZB003, JNZB005, JNZB009, JNZB011, JNZB015 and
This 6 plants of bacterium of JNZB018 are further cooked carbohydrate fermentation experiment and 16S rDNA Molecular Identifications.
6 plants of bacterium may be by cellobiose, melibiose, sucrose, fructose, L-arabinose, black bearberry sugar, trehalose, sweet
Reveal sugar, D- xyloses, salicin, amygdalin；6 plants of bacterium cannot all utilize erythrite, sorbierite, L- sorboses and L- rhamnoses；This
Outer bacterial strain JNZB003 can utilize galactolipin, maltose and ribose, the results are shown in Table 3.According to《Primary Jie Shi Bacteria Identifications handbook》With
《Lactic acid bacteria taxonomic identification and experimental method》In description to Leuconostoc, reflect in conjunction with morphological feature, physiological property
Fixed, basic biochemistry identification and carbohydrate fermentation identification.It is preliminary to judge that bacterial strain JNZB003 is Leuconostoc mesenteroides goldbeater's skin Asia
Kind, JNZB005, JNZB009 and JNZB011 are cold leukonids, and JNZB015 and JNZB018 are meat leukonids.
Wherein, arginine hydrolysis experiment method is as follows：
The culture of activation is taken, is inoculated into arginine hydrolyzing culture medium, above the 1 sterile paraffinic mineral oil of drop of drop, 30 DEG C
Lower culture 24-72h.If culture medium is still purple, represents that arginine is hydrolyzed, generate alkaline matter, positive reaction；If turn yellow
Color represents production acid, negative reaction.
Glucan generation test method is as follows：
Bacterium colony on picking MRS solid mediums crosses on glucan generation culture medium, 24-72h is cultivated at 30 DEG C.
Bacterium colony is observed, if generating drops, sticky lawn in periphery of bacterial colonies, represents generation glucan, positive reaction；No drops glues
Thick lawn is generated as negative reaction.
Citrate utilization test method is as follows：
The culture of activation is taken, in citric acid using crossing on culture medium, 24-72h is cultivated at 30 DEG C, if forming blue bacterium
It falls, represents that there is Metabolism of Citric Acid ability；If not developing the color, expression is not metabolized citric acid.
Aesculin hydrolysis experiment method is as follows：
The culture of activation is taken, is inoculated in esculin medium, after cultivating 72h at 30 DEG C, aesculin culture solution is taken to put
In colorimetric disc, a little ironic citrate is added dropwise, is positive reaction if aobvious black represents aesculin hydrolysis；It does not develop the color for negative reaction.
It is compared with nonvaccinated culture solution.
Carbohydrate fermentation test method is as follows：
The culture of activation is taken, is inoculated in respectively in the fermentation medium containing different carbohydrate, 24- is cultivated at 30 DEG C
72h, culture solution turn yellow, and represent that carbohydrate is metabolized production acid, are positive reaction；Otherwise it is negative reaction.
2 bacterial strain basic biochemistry CHARACTERISTICS IDENTIFICATION result of table
Arginine hydrolyzes："+" represents arginine and is hydrolyzed, and it is positive reaction to show purple；"-" representative does not hydrolyze arginine,
Displaing yellow is negative reaction.
Citric acid utilizes："+" represents metabolism citric acid, forms blue colonies；"-" representative is not metabolized citric acid, is formed white
Color bacterium colony.
Glucan generates："+", which represents, generates thick lawn；"-" representative does not generate thick lawn.
Aesculin hydrolyzes："+" represents aesculin hydrolysis, and it is positive reaction to show black；"-" representative does not hydrolyze aesculin, no
It develops the color for negative reaction.
3 bacterial strain carbohydrate fermentation biochemical identification result of table
"+" is represented using carbohydrate production acid, and displaing yellow is positive reaction；"-" represents negative reaction.
3rd, leuconostoc mesenteroides subsp mesenteroides 16S rDNA Molecular Identifications
For more accurate, more effective identification bacterial strain JNJB003, on traditional Physiology and biochemistry evaluation of foundation, given birth to using molecule
Object 16S rDNA gene sequence analysis is identified.It is filtered out with the extraction of Ezup pillar bacterial genomes DNA extraction agents box
6 plants of bacterial strain DNA, PCR amplification obtains 1400 ± 100bp bands, the band of pcr amplification product recycled again, is connected to
The conversion of Takara pMD*18-T Vector carrier clonings carries out blue hickie screening, reuses M13+/- primer sequencing.Amplification production
Object such as Fig. 6,16S rDNA gene orders are as shown in sequence 1 in sequence table.
Sequence homology comparison is carried out to blast program of the sequencing result in ncbi database, comparison result is shown in Table 4.
From table 4, it can be seen that bacterial strain JNZB003 and bright string strain bacterium goldbeater's skin subspecies (the Leuconostoc mesenteroides of goldbeater's skin
subsp.Mesenteroides)CP020731.1、 LC119136.1、LC119132.1、CP015442.1、LC260037.1、
The similitude of the 16s rDNA gene orders of LC260035.1 and LC260034.1 reaches 99%, identifies that bacterial strain JNZB003 is
Leuconostoc mesenteroides subsp mesenteroides JNZB003；And China typical culture collection center was preserved on 03 14th, 2018, it protects
It is CCTCC NO to hide number：M 2018129, and on March 28th, 2018 determining testing result for survival.
4 bacterial strain 16S rDNA gene orders of table compare
The feature of embodiment 3, leuconostoc mesenteroides subsp mesenteroides JNZB003
Same species of microorganism is because of source difference, and there are certain differences in certain characteristics.In order to grasp the goldbeater's skin of the present invention
Leukonid goldbeater's skin subspecies JNJB003CCTCC NO：The characteristic of M 2018129, by the leuconostoc mesenteroides subsp mesenteroides of screening
JNJB003CCTCC NO：After M 2018129 is activated, culture solution is inoculated in MRS fluid nutrient mediums using volume ratio as 2% and 3%
In.
Bacterial strain optimum growth temperature is investigated by being cultivated under condition of different temperatures, as a result sees Fig. 7, Leuconostoc mesenteroides intestines
Film subspecies JNJB003CCTCC NO：2018129 growth temperature ranges of M are very wide, can well be grown at 28 DEG C and 30 DEG C, most suitable
Growth temperature is 30 DEG C.
Salt tolerance observes different salinity to leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：M 2018129
The influence of growth, from figure 8, it is seen that with the increase of salinity, leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC
NO：2018129 growth ability of M gradually weakens, and when salinity is in 4% and 6%, can well grow, when salinity increases to
When 8%, leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：M 2018129 is slow-growing, when salinity reaches 10%
When, leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：2018129 faint growths of M.
It is grown by measuring bacterial strain growth course OD600 values and pH value dynamic change in MRS fluid nutrient mediums to evaluate
Situation and acid producing ability, from fig. 9, it can be seen that leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：M 2018129 has
There are good growth characteristics, enter stationary phase after cultivating 14h, at this time OD600Value reaches 1.52；When cultivating 48h, OD600It is worth highest,
Up to 1.909, OD later600Value starts slowly to reduce, into degradation period；Leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC
NO：M 2018129 produces sour very fast, culture 12h, and pH value drops to 4.60, and for 24 hours, pH value drops to 4.16 for culture, cultivates 28h, pH value drop
It is no longer reduced after to 4.0 or so, refers to Figure 10.
Observe leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：M 2018129 is raw under different pH condition
Long situation investigates leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：The resistance to acid and alkali of M 2018129, is as a result shown in figure
11, leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：2018129 the most suitable growth pH value of M is 6.When 4 Hes of pH value ＜
During pH value ＞ 10, leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：M 2018129 is slow-growing；The bright beading of goldbeater's skin
Bacterium goldbeater's skin subspecies JNJB003CCTCC NO：M 2018129 is compared with acid and alkali-resistance, when pH value is 2 and 11, Leuconostoc mesenteroides goldbeater's skin
Subspecies JNJB003CCTCC NO：M 2018129 faint can also be grown.
Screen leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：M 2018129 to nitrite degradation ability,
See Figure 12 and 13, there it can be seen that with the increase of nitrite concentration, leuconostoc mesenteroides subsp mesenteroides
JNJB003CCTCC NO：M 2018129 is first to increase to reduce again to nitrite degradation rate.Leuconostoc mesenteroides subsp mesenteroides
JNJB003CCTCC NO：Degradation rate highests of the M 2018129 to 150ug/ml nitrite for 24 hours, reaches 95.92%, 75ug/
Ml takes second place, and reaches 94.18%.
The application of embodiment 4, leuconostoc mesenteroides subsp mesenteroides JNJB003 in pickles
The pickles of the present invention can select conventional production method, and the raw material of pickles can select conventional raw material, and
Not as the limitation of the present invention, it is preferable that pickles raw material is maturity period cherry dish, and whole, following steps may be used in production method.
(1) leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：The preparation of 2018129 bacteria suspensions of M, the preparation side
Method is identical with the method for above-mentioned leuconostoc mesenteroides subsp mesenteroides separating-purifying, under aseptic condition, takes Leuconostoc mesenteroides goldbeater's skin
Subspecies JNJB003CCTCC NO：M 2018129 is inoculated in 30 DEG C of culture 48h in MRS fluid nutrient mediums, obtains the bright beading of goldbeater's skin
Bacterium goldbeater's skin subspecies JNJB003CCTCC NO：2018129 bacteria suspensions of M, leuconostoc mesenteroides subsp mesenteroides in bacteria suspension
JNJB003CCTCC NO：The content of M 2018129 is 118cfu/ml.
Wherein, the solvent of MRS fluid nutrient mediums is water, and solute and its concentration are as follows：Peptone 10g/L, beef extract 10g/
L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate
2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L；pH6.8.
(2) whole maturity period cherry dish is taken out with 6% saline sook after 12 hours, the moisture control in cherry dish is gone out, is taken
500g is put into jar, the leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO that step (1) is obtained：M 2018129
300 μ l of every bag of bacteria suspension are added in jar, mixing, sealing, are put into 4 DEG C of refrigerator-freezers and are fermented to obtain traditional cherry dish pickles.
Since leuconostoc mesenteroides subsp mesenteroides are mainly enriched in pickle fermentation early period, ferment rapidly and generate a large amount of breast
Acid, makes the pH value of yeasting reduce, and inhibits harmful microbe growth and inhibits the formation of nitrite, long-term so as to reach
It preserves, improve nutritional quality and ensures edible safety purpose.And leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO：M
2018129 there is higher acid producing ability and degrading nitrite ability to cause the quality of pickles and edible more healthy and peace
Entirely.
The technology contents and technical characteristic of the present invention have revealed that as above, however those skilled in the art still may base
Make various replacements and modification without departing substantially from spirit of the present invention, therefore, the scope of the present invention in teachings of the present invention and announcement
The revealed content of embodiment should be not limited to, and various replacements and modification without departing substantially from the present invention should be included, and is this patent Shen
Please claim covered.
<110>Jilin Academy of Agricultural Science
<120>Leuconostoc mesenteroides subsp mesenteroides, preparation method and application
<160> 1
<210> 1
<211> 1385
<212> DNA
<213>Leuconostoc mesenteroides subsp mesenteroides（Leuconostoc mesenteroides subsp.
Mesenteroides）
<400> 1
cacgtcgaac gcacagcgaa aggtgcttgc acctttcaag tgagtggcga acgggtgagt 60
aacacgtgga caacctgcct caaggctggg gataacattt ggaaacagat gctaataccg 120
aataaaactt agtgtcgcat gacacaaagt taaaaggcgc ttcggcgtca cctagagatg 180
gatccgcggt gcattagtta gttggtgggg taaaggccta ccaagacaat gatgcatagc 240
cgagttgaga gactgatcgg ccacattggg actgagacac ggcccaaact cctacgggag 300
gctgcagtag ggaatcttcc acaatgggcg aaagcctgat ggagcaacgc cgcgtgtgtg 360
atgaaggctt tcgggtcgta aagcactgtt gtatgggaag aacagctaga ataggaaatg 420
attttagttt gacggtacca taccagaaag ggacggctaa atacgtgcca gcagccgcgg 480
taatacgtat gtcccgagcg ttatccggat ttattgggcg taaagcgagc gcagacggtt 540
tattaagtct gatgtgaaag cccggagctc aactccggaa tggcattgga aactggttaa 600
cttgagtgca gtagaggtaa gtggaactcc atgtgtagcg gtggaatgcg tagatatatg 660
gaagaacacc agtggcgaag gcggcttact ggactgcaac tgacgttgag gctcgaaagt 720
gtgggtagca aacaggatta gataccctgg tagtccacac cgtaaacgat gaacactagg 780
tgttaggagg tttccgcctc ttagtgccga agctaacgca ttaagtgttc cgcctgggga 840
gtacgaccgc aaggttgaaa ctcaaaggaa ttgacgggga cccgcacaag cggtggagca 900
tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tttgaagctt 960
ttagagatag aagtgttctc ttcggagaca aagtgacagg tggtgcatgg tcgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat tgttagttgc 1080
cagcattcag atgggcactc tagcgagact gccggtgaca aaccggagga aggcggggac 1140
gacgtcagat catcatgccc cttatgacct gggctacaca cgtgctacaa tggcgtatac 1200
aacgagttgc caacccgcga gggtgagcta atctcttaaa gtacgtctca gttcggattg 1260
tagtctgcaa ctcgactaca tgaagtcgga atcgctagta atcgcggatc agcacgccgc 1320
ggtgaatacg ttcccgggtc ttgtacacac cgcccgtcac accatgggag tttgtaatgc 1380
Tccaaag 1385
Claims (10)
1. a kind of leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides
Subsp.Mesenteroides), which is characterized in that it is deposited in China typical culture collection center, and preserving number is：CCTCC
NO：M 2018129.
2. a kind of leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides)
CCTCC NO：The preparation method of M 2018129, which is characterized in that include the following steps：Using pickles as bacterium source, in aerobic item
Leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides are cultivated under part and in culture medium
subsp.Mesenteroides)CCTCC NO：M 2018129.
3. preparation method according to claim 2, which is characterized in that the pickles are cherry dish.
4. preparation method according to claim 2, which is characterized in that Leuconostoc mesenteroides goldbeater's skin is cultivated in the culture medium
Subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO：M 2018129 is included in
Multiplying culture leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides in MRS fluid nutrient mediums
subsp.Mesenteroides)CCTCC NO：M 2018129 is separately cultured Leuconostoc mesenteroides on LMM solid mediums
Goldbeater's skin subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO：M 2018129 and
Isolation and purification culture leuconostoc mesenteroides subsp mesenteroides (Leuconostoc in MRS solid mediums containing vancomycin
mesenteroides subsp.Mesenteroides)CCTCC NO：M 2018129.
5. preparation method according to claim 4, which is characterized in that a concentration of 30ug/ml of vancomycin.
6. preparation method according to claim 4, which is characterized in that the cultivation temperature in MRS fluid nutrient mediums is
25~36 DEG C, incubation time is 24~72h.
7. preparation method according to claim 6, which is characterized in that the cultivation temperature in MRS fluid nutrient mediums is
30 DEG C, condition of culture is 120-160r/min isothermal vibration cultures.
8. preparation method according to claim 4, which is characterized in that described in LMM solid mediums and containing through the ages
Cultivation temperature is 25~36 DEG C in the MRS solid mediums of mycin, and incubation time is 24~72h.
9. preparation method according to claim 8, which is characterized in that described in LMM solid mediums and containing through the ages
Cultivation temperature is 30 DEG C in the MRS solid mediums of mycin, and condition of culture is is inverted constant temperature quiescent culture.
10. leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides according to claim 1
subsp.Mesenteroides)CCTCC NO：Applications of the M 2018129 in pickle production.
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|---|---|---|---|---|
| CN110004090A (en) * | 2019-04-15 | 2019-07-12 | 东北农业大学 | A kind of Leuconostoc mesenteroides and its application in fermented pickled Chinese cabbage |
| CN113913342A (en) * | 2021-11-15 | 2022-01-11 | 广西壮族自治区农业科学院 | Leuconostoc mesenteroides and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105231369A (en) * | 2015-11-18 | 2016-01-13 | 四川东坡中国泡菜产业技术研究院 | Method for quickly reducing nitrate and nitrite in pickled vegetables |
-
2018
- 2018-04-17 CN CN201810344233.1A patent/CN108265019B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105231369A (en) * | 2015-11-18 | 2016-01-13 | 四川东坡中国泡菜产业技术研究院 | Method for quickly reducing nitrate and nitrite in pickled vegetables |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004090A (en) * | 2019-04-15 | 2019-07-12 | 东北农业大学 | A kind of Leuconostoc mesenteroides and its application in fermented pickled Chinese cabbage |
| CN113913342A (en) * | 2021-11-15 | 2022-01-11 | 广西壮族自治区农业科学院 | Leuconostoc mesenteroides and application thereof |
| CN113913342B (en) * | 2021-11-15 | 2023-06-20 | 广西壮族自治区农业科学院 | Leuconostoc mesenteroides and application thereof |
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