CN108265019B - 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 discloses a kind of leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides), preparation method and applications, the leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.mesenteroides) its in the deposit number of China typical culture collection center be CCTCC NO:M 2018129.The leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO:M 2018129 has high acid ability and degrading nitrite ability, and salt tolerant and acid and alkali-resistance, product storage period is able to extend while product nutritional quality can be improved again, develop Resource of lactic bacteria database, the development for being conducive to pickles industry, has a vast market foreground.
Description
Technical field
The present invention relates to microorganism fields, and in particular to a kind of leuconostoc mesenteroides subsp mesenteroides, preparation method and answers
With.
Background technique
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 the spontaneous fermentation food of cream or vegetable material.It is organic that fermentation can be metabolized generation
A variety of aromatic compounds such as acid, alcohols and biacetyl improve product special flavour；Generating glucan can be used as blood thickener, relaxes
Spreader and stabilizer；Generating mannitol can be used as low calorie sweetener；Generating bacteriocin can be used as biological preservative；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, is 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, 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, promotes nutriment to inhale, improves product special flavour, while also having anti-
Oxidability and antagonism pathogenic bacteria ability.Because it can generate flavour substance, answered extensively in the field of food such as cream 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 the 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 is rich in lactic bacteria activity and lactic acid.Cherry dish is the Koreans
The Specialty vegetable of plantation, growth period is short and has seasonality, and full of nutrition, taste is suitble to popular taste.At present from Henan Linzhou City
Pickles, Pickles, Sichuan Style are isolated the in the majority of Leuconostoc in Tian jin cabbage pickled in sweet and sour, but are not yet found in Korean traditional fermented cherry dish
Separate 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 growth of lactic acid bacteria or degrading nitrite
Ability it is 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 the very harmful substances of a kind of pair of human body, and excessive or long-term consumption is easy carcinogenic.Therefore one kind is needed
It is able to solve the bacterium of problems.
Summary of the invention
It is an object of the invention to overcome the deficiencies of existing technologies, a kind of leuconostoc mesenteroides subsp mesenteroides and its system are provided
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 is able to suppress the breeding of harmful bacteria, extends the shelf-life of pickles.
To achieve the above object, the present invention proposes a kind of technical solution of first aspect: leuconostoc mesenteroides subsp mesenteroides
(Leuconostoc mesenteroides subsp.mesenteroides), is deposited in China typical culture collection
The heart, deposit number are as follows: 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 that goldbeater's skin is bright
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 receives collection and step on
Volume, number CCTCC NO:M 2018129 are charged to, and is to survive in determining testing result on March 28th, 2018.
A kind of technical solution of present invention proposition second aspect: leuconostoc mesenteroides subsp mesenteroides Leuconostoc mesenteroides goldbeater's skin
The preparation side of subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO: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。
Culture medium of the 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 medium, LMM solid medium and in the MRS solid medium containing vancomycin, it is preferable that ten thousand
Ancient mycin concentration is 30ug/ml.Using the concentration vancomycin when can either guarantee to obtain optimal 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 and obtain purebred leuconostoc mesenteroides subsp mesenteroides JNZB003.Specifically
Culture of isolated step is followed successively by, firstly, the Multiplying culture leuconostoc mesenteroides subsp mesenteroides in MRS fluid nutrient medium
(Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO:M 2018129, secondly, in LMM
Leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides is separately cultured on solid medium
Subsp.Mesenteroides) CCTCC NO:M 2018129 is separated in the MRS solid medium containing vancomycin again
Purifying culture leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides)
CCTCC NO:M 2018129, wherein be 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 medium, and incubation time is 24~72h, further, in MRS Liquid Culture
Cultivation temperature is 30 DEG C in base, and condition of culture is 24~72h of 120-160r/min isothermal vibration culture.It is optimal, 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 medium and in the MRS solid medium containing vancomycin
25~36 DEG C, incubation time is 24~72h, further, in LMM solid medium and in the MRS solid containing vancomycin
Cultivation temperature is 30 DEG C in culture medium, and condition of culture is the Leuconostoc mesenteroides for being inverted constant temperature stationary culture to obtain best performance
Goldbeater's skin subspecies (Leuconostoc mesenteroides subsp.Mesenteroides) CCTCC NO:M 2018129.
The technical solution of the present invention proposition third aspect: leuconostoc mesenteroides subsp mesenteroides (Leuconostoc
Mesenteroides subsp.Mesenteroides) application of the CCTCC NO:M 2018129 in pickle production.
The beneficial effects of the present invention are: the leuconostoc mesenteroides subsp mesenteroides intestines separated 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 be able to extend product storage period again, develop Resource of lactic bacteria database.The development for being conducive to pickles industry, before having a vast market
Scape.
Detailed description of the invention
Fig. 1 is the colonial morphology figure that bacterial strain JNJB003 of the invention is separated on LMM culture medium；
Fig. 2 is the colonial morphology figure that bacterial strain JNJB003 of the invention is purified on MRS culture medium；
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 is that bacterial strain growth test under the conditions of 37 DEG C identifies picture；
Fig. 5 b is that bacterial strain growth test in the culture medium containing 30 μ g/mL vancomycins identifies picture；
Fig. 5 c is the Salt tolerance identification picture of bacterial strain；
Fig. 5 d is the arginine hydrolysis experiment identification picture of bacterial strain；
Fig. 5 e is the sweet hydrolysis experiment identification picture of seven leaves of bacterial strain；
Fig. 5 f is the Metabolism of Citric Acid test for identification picture of bacterial strain；
Fig. 5 g is that the glucan of bacterial strain generates identification picture；
Fig. 5 h is the carbohydrate fermentation test for identification picture 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 value of leuconostoc mesenteroides subsp mesenteroides JNZB003 with incubation time change curve；
Figure 10 is that leuconostoc mesenteroides subsp mesenteroides JNZB003 changes over time the curve graph for producing acid；
Figure 11 is the leuconostoc mesenteroides subsp mesenteroides JNZB003 curve graph that optical density is grown at various ph values；
Figure 12 is curve graph 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 picture.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Control strain of the invention is selected as Leuconostoc mesenteroides bacterial strain (BNCC195309), by the comparison of the bacterial strain to obtain
Leuconostoc mesenteroides bacterial strain is obtained, which is purchased from Bei Na Chuan Lian Bioisystech Co., Ltd；The training of MRS solid
It supports base, MRS fluid nutrient medium, 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- xylose, gossypose,
L- sorbose, melibiose are purchased from SIGMA；Ezup pillar bacterial genomes DNA extraction agent box, SanPrep pillar DNA glue return
It receives kit and is purchased from Sangon Biotech (Shanghai) Co., Ltd..
Instrument and equipment of the invention: II autoclave Spain SELECTA of PRESOCLAVE-；2720thermal
Cycler PCR instrument Applied Biosystems；HC-2518R high-speed refrigerated centrifuge BBI；CX41-RF fluorescence microscope day
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, Korean Autonomous Prefecture of Yanbian, the market of farm produce and supermarket
System fermentation cherry dish filters out single Leuconostoc mesenteroides bacterial strain using separation, purification process, method particularly includes: aseptic condition
Under, Korea-Yanbian traditional zymotic cherry vegetable juice 1mL is taken, is placed in the test tube of sterilizing, LMM solid is 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 for generating drops toughness polysaccharide in culture dish containing LMM solid medium around picking is containing 30ug/mL ten thousand
Scribing line isolates and purifies 3 times in the culture dish of the MRS solid medium of ancient mycin, picking single bacterium colony, by microscopy, and and goldbeater's skin
Leukonid bacterial strain (BNCC195309) comparison, confirmation obtain single pure culture, JNJB003 are named as, such as Fig. 1 and 2 institute
Show.
The identification of embodiment 2, bacterial strain JNJB003
One, 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.As shown in figure 4,
Catalase test photo.It is Gram-positive, 17 plants of negative catalases, by 17 plants of peroxides through 20 plants of bacterium of microscopy
Change hydrogen enzyme negative bacterium further to identify.
Two, the Physiology and biochemistry identification of bacterial strain JNJB003
The physiological property of bacterial strain is identified
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 test, 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 vancomycin, 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 are close in sodium solution, and 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, as follows to the tolerance test method of 30ug/ml vancomycin:
(1) culture for taking activation, containing crossing on 30ug/mL vancomycin MRS solid medium, 37 DEG C are cultivated
The bacterium colony of growth is repeated primary inoculation, culture, if still there is bacterium colony, indicates that 37 DEG C can grow by 24-72h if growth.
(2) culture for taking activation, containing crossing on 30ug/mL vancomycin MRS solid medium, 1 DEG C is cultivated
The bacterium colony of growth is repeated primary inoculation, culture, if still there is bacterium colony, indicates that 1 DEG C can grow by 48h if growth.
Salt tolerance test method is as follows:
The picking colony on the solid medium containing 30ug/mL vancomycin, access is containing 3.0% and 6.5%NaCl's
It in MRS fluid nutrient medium, after being cultivated for 24 hours at 30 DEG C, shakes up, measures its OD600Value, using MRS fluid nutrient medium 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 medium, with
3% hydrogen peroxide mixes, if there is bubble to indicate positive reaction, bubble-free indicates 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 generate test, aesculin hydrolysis experiment, and qualification result is shown in Table 2.From
As can be seen that 17 plants of bacterium arginine hydrolysis experiments are positive reaction in table 2, described in this and " primary Jie Shi Bacteria Identification handbook "
Leuconostoc characteristic it is inconsistent；Citric acid can be metabolized for 7 plants in 17 plants of strains of screening, 10 plants of not energy metabolism lemons
Acid；6 plants can hydrolyze aesculin, and 11 plants cannot hydrolyze aesculin；3 plants can generate glucan using sucrose.In Leuconostoc
In only leuconostoc mesenteroides subsp mesenteroides can using sucrose generate glucan, thick lawn is formed on culture medium,
It, which is planted, does not have this feature, compares " primary Jie Shi Bacteria Identification handbook " and " lactic acid bacteria taxonomic identification and reality according to this characteristic
Proved recipe method ", identified in conjunction with physiological property, by bacterial strain JNZB003, JNZB005, JNZB009, JNZB011, JNZB015 and
This 6 plants of bacterium of JNZB018 are further cooked carbohydrate fermentation test and 16S rDNA Molecular Identification.
6 plants of bacterium may be by cellobiose, melibiose, sucrose, fructose, L-arabinose, black bearberry sugar, trehalose, sweet
Reveal sugar, D- xylose, salicin, amygdalin；6 plants of bacterium cannot all utilize erythrite, sorbierite, L- sorbose and L- rhamnose；This
Outer bacterial strain JNZB003 can use galactolipin, maltose and ribose, the results are shown in Table 3.According to " primary Jie Shi Bacteria Identification handbook " and
Description in " lactic acid bacteria taxonomic identification and experimental method " to Leuconostoc is reflected in conjunction with morphological feature, physiological property
Fixed, basic biochemistry identification and carbohydrate fermentation identification.Tentatively judge that bacterial strain JNZB003 is that Leuconostoc mesenteroides goldbeater's skin is sub-
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 for taking activation is inoculated into arginine hydrolyzing culture medium, above the 1 sterile paraffinic mineral oil of drop of drop, and 30 DEG C
Lower culture 24-72h.If culture medium is still purple, indicates that arginine is hydrolyzed, generate alkaline matter, positive reaction；If turning yellow
Color indicates to produce acid, negative reaction.
It is as follows that glucan generates test method:
Bacterium colony on picking MRS solid medium generates in glucan and crosses on culture medium, cultivates 24-72h at 30 DEG C.
Bacterium colony is observed, if generating drops, sticky lawn in periphery of bacterial colonies, indicates to generate glucan, positive reaction；No drops glues
Thick lawn is generated as negative reaction.
Citrate utilization test method is as follows:
The culture for taking activation cultivates 24-72h at 30 DEG C in citric acid using crossing on culture medium, if forming blue bacterium
It falls, indicates 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 for taking activation, is inoculated in esculin medium, after cultivating 72h at 30 DEG C, aesculin culture solution is taken to set
In colorimetric disc, a little ironic citrate is added dropwise, is positive reaction if aobvious black indicates 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 the fermentation medium containing different carbohydrate respectively, cultivates 24- at 30 DEG C
72h, culture solution turn yellow, and indicate that carbohydrate is metabolized production acid, are positive reaction；It otherwise is negative reaction.
2 bacterial strain basic biochemistry CHARACTERISTICS IDENTIFICATION result of table
Arginine hydrolysis: "+" represents arginine and is hydrolyzed, and showing purple is positive reaction；"-" 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 hydrolysis: "+" represents aesculin hydrolysis, and showing black is positive reaction；"-" representative does not hydrolyze aesculin, no
Colour developing is negative reaction.
3 bacterial strain carbohydrate fermentation biochemical identification result of table
"+", which is represented, produces acid using carbohydrate, and displaing yellow is positive reaction；"-" represents negative reaction.
Three, leuconostoc mesenteroides subsp mesenteroides 16S rDNA Molecular Identification
It is raw using molecule on traditional Physiology and biochemistry evaluation of foundation for more acurrate, more effective identification bacterial strain JNJB003
Object 16S rDNA gene sequence analysis is identified.It is filtered out with the extraction of Ezup pillar bacterial genomes DNA extraction agent box
6 plants of bacterial strain DNA, PCR amplification obtains 1400 ± 100bp band, the band of pcr amplification product recycled again, is connected to
The conversion of Takara pMD*18-T Vector carrier cloning carries out blue hickie screening, reuses M13+/- primer sequencing.Amplification produces
Object such as Fig. 6,16S rDNA gene order is 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 order of LC260035.1 and LC260034.1 reaches 99%, identifies that bacterial strain JNZB003 is intestines
Film leukonid goldbeater's skin subspecies JNZB003；And China typical culture collection center, preservation were preserved on 03 14th, 2018
Number is CCTCC NO:M 2018129, and is to survive in determining testing result on March 28th, 2018.
4 bacterial strain 16S rDNA gene order of table compares
The feature of embodiment 3, leuconostoc mesenteroides subsp mesenteroides JNZB003
Same species of microorganism has a certain difference in certain characteristics because of source difference.In order to grasp goldbeater's skin of the invention
The characteristic of leukonid goldbeater's skin subspecies JNJB003CCTCC NO:M 2018129, by the leuconostoc mesenteroides subsp mesenteroides of screening
After JNJB003CCTCC NO:M 2018129 is activated, culture solution is inoculated in for 2% and 3% by MRS fluid nutrient medium with volume ratio
In.
Bacterial strain optimum growth temperature is investigated by cultivating under condition of different temperatures, as a result sees Fig. 7, Leuconostoc mesenteroides intestines
Film subspecies JNJB003CCTCC 2018129 growth temperature ranges of NO:M are very wide, can well grow 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
2018129 growth ability of NO:M gradually weakens, and when salinity is in 4% and 6%, can well grow, when salinity increases to
When 8%, 2018129 slow growth of leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M, when salinity reaches 10%
When, the faint growth of leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M 2018129.
By measurement bacterial strain, growth course OD600 value and pH value dynamic change evaluate growth in MRS fluid nutrient medium
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, enters 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, is detailed in Figure 10.
Observation leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M 2018129 is grown under different ph values
Situation, the resistance to acid and alkali of investigation leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M 2018129, the result is shown in Figure 11,
2018129 the most suitable growth pH value of leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M is 6.When pH value < 4 and pH value
When > 10,2018129 slow growth of leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M；Leuconostoc mesenteroides goldbeater's skin
Subspecies JNJB003CCTCC NO:M 2018129 is compared with acid and alkali-resistance, when pH value is 2 and 11, leuconostoc mesenteroides subsp mesenteroides
JNJB003CCTCC NO:M 2018129 faint can also be grown.
Leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M 2018129 is screened 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
Degradation rate highest of the JNJB003CCTCC NO: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
Pickles of the invention can choose conventional production method, and the raw material of pickles can choose conventional raw material, and
Not as limitation of the invention, it is preferable that pickles raw material is maturity period cherry dish, and whole, production method can use following steps.
(1) preparation of 2018129 bacteria suspension of leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO: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 medium, obtains the bright beading of goldbeater's skin
2018129 bacteria suspension of bacterium goldbeater's skin subspecies JNJB003CCTCC NO:M, leuconostoc mesenteroides subsp mesenteroides in bacteria suspension
The content of JNJB003CCTCC NO:M 2018129 is 118cfu/ml.
Wherein, the solvent of MRS fluid nutrient medium 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) it is taken out after impregnating whole maturity period cherry dish 12 hours with 6% salt water, the moisture control in cherry dish is gone out, is taken
500g is put into jar, 2018129 bacterium of leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M that step (1) is obtained
300 μ l of every bag of suspension is added in jar, mixes, and sealing is put into 4 DEG C of refrigerator-freezers and ferments 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 cream
Acid reduces the pH value of yeasting, inhibits harmful microbe growth and inhibits the formation of nitrite, to reach long-term
It saves, improve nutritional quality and guarantees edible safety purpose.And leuconostoc mesenteroides subsp mesenteroides JNJB003CCTCC NO:M
2018129 acid producing ability and degrading nitrite abilities with higher make the quality and edible more healthy and peace of pickles
Entirely.
Technology contents and technical characteristic of the 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 that invention, therefore, the scope of the present invention in teachings of the present invention and announcement
It should be not limited to the revealed content of embodiment, and should include various without departing substantially from replacement and modification of the invention, and be 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 (3)
1. a kind of leuconostoc mesenteroides subsp mesenteroides (Leuconostoc mesenteroides subsp.Mesenteroides)
Application in pickle production, the leuconostoc mesenteroides subsp mesenteroides are deposited in China typical culture collection center, preservation
Number are as follows: CCTCC NO:M 2018129, the ferment making pickles under 4 DEG C, 6% salinity.
2. application as described in claim 1, it is characterised in that the leuconostoc mesenteroides subsp mesenteroides are used in pickle production
In production acid, degrading nitrite.
3. application as described in claim 1, it is characterised in that the bacteria suspension for preparing the leuconostoc mesenteroides subsp mesenteroides is used
In production pickles.
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| 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 |
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| 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 |
Non-Patent Citations (2)
| Title |
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| 熊涛等.直投式菌种发酵泡菜过程中亚硝酸盐的变化规律.《南昌大学学报（理科版）》.2013,第37卷(第1期),第47-50页，参见摘要、第48页左栏第5-9段，第49页右栏第2段及表1. * |
| 王昌禄等.亚硝酸盐降解菌的分离及其降解特性.《中国酿造》.2008,(第9期),第33-35页，参见摘要及第35页图4. * |
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