CN108676742A - Lactobacillus casei (Lactobacillus casei）SITCC No.10009 - Google Patents

Lactobacillus casei (Lactobacillus casei）SITCC No.10009 Download PDF

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CN108676742A
CN108676742A CN201810414559.7A CN201810414559A CN108676742A CN 108676742 A CN108676742 A CN 108676742A CN 201810414559 A CN201810414559 A CN 201810414559A CN 108676742 A CN108676742 A CN 108676742A
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lactobacillus casei
sitcc
solution
sucrose
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CN108676742B (en
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田怀香
史雨桦
陈臣
于海燕
卢卓彦
俞本杰
刘晗
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Shanghai Institute of Technology
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    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
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    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/245—Lactobacillus casei

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Abstract

The invention discloses a kind of Lactobacillus caseis（Lactobacillus casei）SITCC No.10009, deposit number CCTCC No.M2017364, the ability with high yield diacetyl are applied in prepared by acidified milk or leben, are especially containing Lactobacillus casei（Lactobacillus casei）Diacetyl content is 56.84 mg/L in the acidified milk of SITCC No.10009, the addition of sodium citrate is so that the yield of diacetyl is up to 70.43mg/L in finally obtained acidified milk especially in preparation process, simultaneously when being compounded with normal fermentation agent, the type of the volatile flavor substance in acidified milk can be significantly improved, volatile flavor compound is up to 56 kinds, so as to improve the flavouring essence quality of acidified milk, promotes the raising of acidified milk quality, have broad prospects in fermented dairy product field.

Description

Lactobacillus casei (Lactobacillus casei) SITCC No.10009
Technical field
The present invention is related to a kind of Lactobacillus casei (Lactobacillus casei) SITCC No.10009 and its is sending out Ferment food, the especially application in fermented dairy product preparation process, belong to microorganisms technical field.
Background technology
Diacetyl, also known as biacetyl (2,3-butanedione, C4H6O2), appearance is yellow oily liquid, is had very strong Cream fragrance.In the flavor of fermented dairy product, diacetyl is considered as one of the important compound to work.In acidified milk Even if there are the flavors that Yoghourt can be substantially improved if micro diacetyl in, and when its concentration reaches l mg/L, people can feel By the peculiar fragrance of acidified milk.In production, endogenous generation is usually added or improved by external source to improve the wind of acidified milk Taste.
Diacetyl in acidified milk is mainly to be added by external source, mainly by being chemically synthesized, naturally carrying It follows the example of and the methods of microbe fermentation method preparation.
Diacetyl is chemically synthesized, mostly process is complicated, seriously polluted, and the safety of product is poor, and quality is not Stablize, and is extremely difficult to natural flavour mountaineous.Diacetyl is extracted from natural goods and then there are expensive starting materials, and production cost is high to be lacked Point, therefore limit large-scale production application.And microbial fermentation is then to synthesize diacetyl by a series of biochemical reactions, tool Have microbe species are more, fast, the raw materials used cheap, substance that generates of growth can biodegradable advantage, be the heat studied now Point.
Except external source is added, endogenous generate is also a kind of important producing method of diacetyl.Endogenous generation is to pass through leavening It is directly generated in acidified milk fermentation process, also more meets the current trend for advocating natural prodcuts.Currently, natural, no added Acidified milk has become dairy industry and develops irresistible trend, has the characteristics that mellow, soft, the another people's happiness of flavor receives. During the fermentation, there are many type that can generate the lactic acid bacteria of diacetyl, the fourth two in final acidified milk or leben Ketone content cannot all reach ideal effect, therefore, it is also desirable to which external source adds diacetyl.
Therefore, important function of the diacetyl in acidified milk can not be ignored, therefore screens the lactic acid bacteria of high yield diacetyl and carry Its high endogenous diacetyl that generates is one of the hot spot that current acidified milk is studied, and has highly important research significance and value.
Invention content
An object of the present invention is the lactic acid used in acidified milk in the prior art or leben for solution Bacterium produces the technical issues of diacetyl low output, and provides a kind of Lactobacillus casei (Lactobacillus casei) SITCC No.10009 finally produces the yield of diacetyl up to 56.84mg/L in acidified milk production.
The second object of the present invention is to provide above-mentioned a kind of Lactobacillus casei (Lactobacillus casei) SITCC NO.10009 are in fermented food, the application process especially in prepared by acidified milk and leben.
Technical scheme of the present invention
A kind of Lactobacillus casei (Lactobacillus casei) SITCC No.10009, belong to the bacterium of lactobacillus Pseudomonas Strain, which was the lactobacillus casei bacterial strain with high yield diacetyl screened in being fanned from breast, in preservation on June 23 in 2017 In China typical culture collection center (abbreviation CCTCC), preservation address：Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University is protected Tibetan center.Postcode：430072, deposit number CCTCC No.M2017364.
A kind of above-mentioned Lactobacillus casei (Lactobacillus casei) SITCC No.10009 have following microorganism It is as follows to learn feature：
(1), colony characteristics
Bacterial strain is detached in the flat lining outs of MRS, 37 DEG C of Anaerobic culturel 48h, and strain growth is good.Bacterium colony is circle, raised, Neat in edge, color milky is somewhat partially yellow, and opaque, surface wettability is smooth, picking energy wire drawing；
(2), thalline feature
Thalline is in rod-shaped, and multiple rows of row also have single dispersed arrangement, thalline size to be generally 0.6 μ at chain different in size Gemma, Gram-positive are not produced in m × 1.5 μm；
(3), feature is learned in culture
The minimum growth temperature of Lactobacillus casei (Lactobacillus casei) SITCC No.10009 is 15 DEG C, most Seedling height temperature is 40 DEG C, best in 30-40 DEG C of growth temperature；Highest and lowest initial growth pH is 9.0 and 4.0, most adaptability Long initial pH is 6.0；The period of delay of bacterial strain (Lactobacillus casei) SITCC No.10009 is relatively short, and 3h enters Exponential phase, 12h
Reach stationary phase.
Above-mentioned Lactobacillus casei (Lactobacillus casei) SITCC No.10009 derive from traditional fermented food In breast fan, belongs to generally recognized as safe (Generally Recognized As Safe, GRAS) strain, can be used in lactobacillus food.
One kind containing the working stock culture of Lactobacillus casei (Lactobacillus casei) SITCC No.10009, wherein Lactobacillus casei (Lactobacillus casei) SITCC No.10009 viable counts be at least 109(CFU refers to list to cfu/mL Bacterial community sum in the volume of position, and only calculate viable count), it is prepared by a method comprising the following steps:：
(1), the preparation of sterile absorbent breast
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g： 20-60g：1L, preferably For skimmed milk powder：Sucrose：Pure water is 120g：60g：The ratio of 1L carries out mixed dissolution, and then sterilize at 95 DEG C 20min, so After be cooled back to 37-40 DEG C, be preferably cooled to 37 DEG C to get to sterile absorbent milk solution；
(2), actication of culture
The Lactobacillus casei (Lactobacillus casei) that the freeze-drying pipe of sterile water dissolution preserves is taken with oese One ring of SITCC No.10009 strains cultivates 24-48h in the flat lining out of MRS agar mediums in 37 DEG C of incubators, until long Go out until single bacterium colony to get to tablet activated spawn；
(3), the preparation of working stock culture
With oese take step (2) obtained by one ring of tablet activated spawn access be equipped with 50mL MRS meat soup Liquid Cultures In the triangular flask of the 250mL specifications of base, it is placed in constant temperature incubation 10-15h in 37 DEG C of incubator, obtains culture solution；
It is 4000-6000r/min by the culture solution control rotating speed of above-mentioned gained, preferably 5000r/min carries out centrifugation 15- 30min, the precipitation for centrifuging gained are cleaned 2-3 times with sterile phosphate buffer solution, are then added in precipitation after cleaning Sterile absorbent milk solution obtained by 50mL steps (1), then control rotating speed be 4000-6000r/min, preferably 5000r/min into Thalline is resuspended to get to the work containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009 in the row concussion that is vortexed Make leavening；
The sterile phosphate buffer solution calculates, phosphoric acid potassium dihydrogen 0.27g, disodium hydrogen phosphate by every liter 1.42g, sodium chloride 8g, potassium chloride 0.2g, surplus are distilled water, and 121 DEG C of 20min that sterilize are cooling simultaneously to be preserved at 4 DEG C.
A kind of above-mentioned Lactobacillus casei (Lactobacillus casei) SITCC No.10009 are prepared in fermented food In application, the especially application in prepared by acidified milk or leben.
A kind of above-mentioned Lactobacillus casei (Lactobacillus casei) SITCC No.10009 are in acidified milk preparation Application, specifically comprise the following steps：
(1), the preparation of sterile absorbent breast
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g： 20-60g：1L, preferably For skimmed milk powder：Sucrose：Pure water is 120g：60g：The ratio of 1L carries out mixed dissolution, obtained mixed solution control temperature For 95 DEG C of sterilizing 20min, it is then cooled back to 37-40 DEG C, is preferably cooled to 37 DEG C to get to sterile absorbent milk solution；
Or by skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g： 20-60g：The ratio of 1L Example carries out mixed dissolution, and the lemon of quality of pure water 0.1-0.6% used in mixed solution is incorporated as in obtained mixed solution Then sour sodium is cooled back to 37-40 DEG C to get newborn to sterile absorbent after mixing controlled at 95 DEG C of sterilizing 20min Solution；
It is preferably added to as the sodium citrate of quality of pure water 0.5% used in mixed solution；
(2), fermented and cultured
It is calculated by percent by volume, in the ratio of 1-5%, preferably will contain Lactobacillus casei in 5% ratio The working stock culture of (Lactobacillus casei) SITCC No.10009 is inoculated into the breast of the sterile absorbent obtained by step (1) In solution, 37 DEG C of temperature of control is fermented to 70-80 ° of T of titratable acidity (about fermentation carries out 9-12h), then at 4 DEG C 12-24h is refrigerated to get to the acidified milk containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009；
A kind of above-mentioned Lactobacillus casei (Lactobacillus casei) SITCC No.10009 are in leben system Application in standby, specifically comprises the following steps：
(1), the preparation of sterile absorbent milk solution
By skimmed milk powder, glucose and pure water, by skimmed milk powder：Glucose：Pure water is 120g： 60-100g：1L, Preferably skimmed milk powder：Glucose：Pure water is 120g：80g：The ratio of 1L carries out mixed dissolution, then controlled at 95 DEG C sterilizing 120min, is then cooled back to 37-40 DEG C, is preferably cooled to 37 DEG C to get to sterile absorbent milk solution；
(2), the preparation of fermentation base-material
It is calculated by percent by volume, in the ratio of 1-5%, preferably will contain Lactobacillus casei in 5% ratio The working stock culture of (Lactobacillus casei) SITCC No.10009 is inoculated into the breast of the sterile absorbent obtained by step (1) In solution, 37 DEG C of temperature of control is fermented to 110-120 ° of T of titratable acidity (about fermentation carries out 58-64h), is then cooled down To 4-10 DEG C, preferably 4 DEG C refrigerations are to get to containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009 Fermentation base-material；
(3), the preparation of sucrose solution
Sucrose is added in 70-80 DEG C of water, control rotating speed, which is 1000-1200r/min stirrings 20-30min, makes sucrose Dissolving, then controlled at 95 DEG C of sterilizing 5-10min postcoolings to 20-30 DEG C, the sucrose solution sterilized；It is preferred that will Sucrose is added in 70 DEG C of water, and control rotating speed, which is 1000r/min stirrings 25min, makes sucrose dissolve, then controlled at 95 DEG C sterilizing 5min postcoolings are to 25 DEG C, the sucrose solution sterilized；
The dosage of above-mentioned sucrose and water, by sucrose：Water is 10-15g:100ml, preferably 12.5g:The ratio meter of 100ml It calculates；
(4), the allotment of acidified milk
It calculates by percent by volume, in the ratio of 10-25%, preferably does containing obtained by step (2) in 25% ratio The fermentation base-material of Lactobacillus paracasei (Lactobacillus casei) SITCC No.10009 is added to the sterilization obtained by step (3) Sucrose solution in, stir and adjust pH value to 3.6-3.7 with the aqueous citric acid solution of 5g/L after 10-15min, obtain deployed Acidified milk；
(5), homogeneous, sterilization
By the deployed acidified milk obtained by step (4) controlled at 20-30 DEG C, pressure 20-30Mpa, preferably 25 DEG C, carry out homogeneous under 20Mpa, then sterilize 5-10min, preferably 5min under the conditions of 95 DEG C, be then cooled to 4 DEG C of refrigerations, Obtain the leben containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009.
Beneficial effects of the present invention
A kind of Lactobacillus casei (Lactobacillus casei) SITCC No.10009 of the present invention are answered in acidified milk Used time, there is higher diacetyl to generate ability, and in acidified milk diacetyl content up to 56.84mg/L, therefore, this is dry Lactobacillus paracasei (Lactobacillus casei) SITCC No.10009 are in acidified milk in application, the acidified milk of final gained Its requirement to diacetyl content can be met by not needing external source addition diacetyl.
Further, a kind of Lactobacillus casei of the invention (Lactobacillus casei) SITCC No.10009 are being sent out In application, the type of the volatile flavor compound in acidified milk can also be significantly improved in kefir milk, so as to improve product Flavouring essence quality promotes the raising of product quality, has broad prospects in fermented dairy product field.
Further, a kind of Lactobacillus casei of the invention (Lactobacillus casei) SITCC No.10009 are being sent out In application, adding suitable sodium citrate in the preparation process of sterile absorbent breast in kefir milk, finally obtained hair can be improved When the additive amount of the yield of the diacetyl of kefir milk, especially sodium citrate is the 0.5% of quality of pure water, finally obtained fermentation The yield of diacetyl in breast is up to 70.43mg/L, than do not add sodium citrate under equal conditions gained acidified milk in Diacetyl output increased 23.91%.
Description of the drawings
The cellular morphology (× 1600) of Fig. 1, SITCC No.10009 bacterial strains；
The growth curve of Fig. 2, Lactobacillus casei (Lactobacillus casei) SITCC No.10009 bacterial strains；
The dense pass with cultivation temperature of bacterium of Fig. 3, Lactobacillus casei (Lactobacillus casei) SITCC No.10009 System's figure；
Contain Lactobacillus casei (Lactobacillus casei) SITCC obtained by Fig. 4, Application Example 1 The content curve graph of the acidified milk of No.10009 different fermentations time corresponding diacetyl in preparation process；
Contain Lactobacillus casei (Lactobacillus casei) SITCC obtained by Fig. 5, Application Example 2 The GC-MS total ion current figures of volatile flavor compound in the acidified milk of No.10009 and customary commercial leavening；
Lactobacillus casei (Lactobacillus casei) SITCC is not contained obtained by Fig. 6, comparative example 1 No.10009 contains only the GC-MS total ion current figures of volatile flavor compound in the acidified milk of customary commercial leavening.
Specific implementation mode
It is illustrated below by specific embodiment and in conjunction with attached drawing to the present invention, but is not intended to limit the present invention.
Lactobacillus casei used in various embodiments of the present invention (Lactobacillus casei) SITCC No.10009, Its biolvgical name is known as：Lactobacillus casei are preserved on June 23rd, 2017 in China typical culture collection The heart, preservation address：Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University collection.Postcode：430072, deposit number is CCTCC No.M2017364。
The present invention filters out a strains of lactic acid bacteria L.casei TCS in being fanned from breast, is named as SITCC No.10009, utilizes shape The Microbiological Characteristics such as state feature, cultural colony and physiological and biochemical property and its hereditary capacity 16s rDNA are by the lactic acid bacteria L.casei TCS are accredited as Lactobacillus casei (Lactobacillus casei), and the bacterial strain is in preservation on June 23 in 2017 In China typical culture collection center (abbreviation CCTCC), deposit number is CCTCC No.M2017364.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to manufacturer Proposed condition." room temperature " described in embodiment refers to the temperature for the operation room tested, generally 25 DEG C.
Raw material used in various embodiments of the present invention or reagent are commercially available in addition to special instruction.
Sterile phosphate buffer solution used in various embodiments of the present invention calculates, phosphoric acid potassium dihydrogen by every liter 0.27g, disodium hydrogen phosphate 1.42g, sodium chloride 8g, potassium chloride 0.2g, surplus are distilled water, and 121 DEG C of sterilizing 20min, cooling is simultaneously It is preserved for use at 4 DEG C, preparation method is as follows：
Potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 1.42g, sodium chloride 8g, potassium chloride 0.2g are weighed respectively, use distilled water Constant volume is to 1L, 121 DEG C of sterilizing 20min, cooling and preserved at 4 DEG C to get to sterile phosphate buffer solution.
Embodiment 1
A kind of acquisition of Lactobacillus casei (Lactobacillus casei) SITCC No.10009, separation, step is such as Under：
(1), sample collection
From spontaneous fermentation fruit and vegetable product, traditional zymotic dairy product (breast fan, newborn cake, Yoghourt, Koumiss etc.), raw milk, It is sampled in dough/pasta, Kefir granule (Kefir grain), ensilage etc..The sample of collection is put into ice chest and is refrigerated, be maintained at compared with Laboratory is taken back under low temperature and is positioned in 4 DEG C of refrigerators, as early as possible detaches lactic acid bacteria；
(2), sample pretreatment
Solid sample 10g (fluid sample 10mL) is taken to be put into the 250mL triangular flasks (containing bead) equipped with 90mL sterile waters In, 20min is stood after oscillation, it is spare；
(3), lactic acid bacteria strains detach
It is 1 by volume：10 ratio calculates, and serial dilution is carried out to above-mentioned pretreated sample with sterile water, Each dilution takes 0.1mL dilute samples, MRS agar plates and M17 agar plates is respectively coated, in 37 DEG C of amphimicrobian conditions Lower constant temperature incubation 24-48h, micro white of different sizes, raised with sterile toothpick picking, moistening, neat in edge, the bacterium colony back side is The single bacterium colony of yellow；
Then scribing line point is pure on corresponding agar plate, obtains pure single bacterium colony and purifies bacterial strain, carries out gram dye Color, catalase experiment；
Purifying bacterial strain is deposited in corresponding isolation medium, adds 30% glycerine as protective agent, -20 DEG C freeze；
The M17 agar mediums used in MRS agar mediums, M17 agar plates used in above-mentioned MRS agar plates are equal For Bifidobacterium selective culture medium, Beijing overpass company buys；MRS fluid nutrient mediums (galactococcus selective medium, Beijing Overpass company reaches).
Different samples isolate 665 plants of bacterium on MRS agar mediums and M17 agar mediums.These bacterial strains are dividing From showing haircuts shape, thick and mucus shape on tablet.
The production diacetyl ability of 665 plants of bacterium of above-mentioned gained is measured, is as follows：
(1), the glycerine cryopreservation tube of 665 plants of separation strains is inoculated into MRS respectively by the inoculum concentration that percent by volume is 2% In fluid nutrient medium, for 24 hours controlled at 37 DEG C of cultures, corresponding 665 plants of separation strains bacterium solutions are obtained；
(2), the ratio that 665 plants of separation strains bacterium solutions of correspondence obtained by step (1) are again 2% in percent by volume is connect respectively In kind to MRS fluid nutrient mediums, 12h is cultivated at 37 DEG C, respectively obtains the zymotic fluid of corresponding 665 plants of separation strains；
(3), by 665 plants of separation strains zymotic fluids of correspondence obtained by step (2) at 4 DEG C, 5000rpm is centrifuged 20min, the precipitation for centrifuging gained are cleaned 3 times with sterile phosphate buffer solution, and 50mL then is added in precipitation after cleaning Thalline is resuspended through the concussion that is vortexed under 5000rpm in sterile phosphate buffer solution, spare；
(4), by skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120mg：60mg：1L's Ratio carries out mixed dissolution, then controlled at 95 DEG C of sterilizing 20min, is then cooled back to 37 DEG C to get to sterile absorbent Milk solution；
(5), the resuspension thalline obtained by step (3) is linked into the inoculum concentration of 5% (v/v) sterile obtained by step (4) In degreasing milk solution, ferment to curdled milk to get to the acidified milk of corresponding 665 plants of bacterium at 37 DEG C after mixing, 4 DEG C refrigerate It preserves.
The acidified milk of 665 plants of bacterium of correspondence of above-mentioned gained is measured the content of diacetyl as follows respectively：
The acidified milk for weighing the above-mentioned gained of 7g is respectively placed in 15ml headspace solid-phase microextraction bottles, ml headspace bottle is placed in In 55 DEG C of thermostat water bath, it is inserted into the good extracting head of prior aging (50/30 μm of DVB/CAR/PDMS), headspace absorption 40min, extraction terminate, and extracting head is transferred at GC (Agilent 7890)-MS (Agilent 5975) injection port, parsing 5min, completes sample introduction, and GC-MS keeps 3min, then the rate of 5 DEG C/min is warming up to 230 DEG C, and keeps with 40 DEG C of initial temperature 10min completes the measurement of the content of diacetyl in the acidified milk of corresponding 665 plants of bacterium, finally obtain 19 plants of production diacetyl contents compared with High bacterial strain, specifically see the table below：
As can be seen from the above table, the diacetyl yield highest of bacterial strain CA-1 chooses this strain in conjunction with the wire-drawing performance of bacterium colony Bacterium is named as SITCC No.10009.
Embodiment 2
The SITCC No.10009 bacterial strains of embodiment 1 are subjected to microbiology identification
(1), colony characteristics
By SITCC No.10009 bacterial strains in the flat lining out separation of MRS, 37 DEG C of Anaerobic culturels 48h, SITCC No.10009 strain growths are good, and bacterium colony is circle, and protrusion, neat in edge, color milky is somewhat partially yellow, impermeable Bright, surface wettability is smooth, picking energy wire drawing；
(2), morphological features
Using Gram's stain (Zhou Deqing the study course of microbiologies [J] .2011.) to the SITCC of gained in step (1) No.10009 thalline carry out Gram's staining observation, shown in the result is shown in Figure 1, from figure 1 it appears that thalline is in no motion of bar Shape, multiple rows of row also have single dispersed arrangement, thalline size to be generally 1.5 μm of 0.6 μ m, do not produce bud at chain different in size Spore, Gram-positive；
(3), feature is learned in culture
The minimum growth temperature of SITCC No.10009 bacterial strains is 15 DEG C, and maximum growth temperature is 45 DEG C, in 30-40 DEG C of life Long temperature is best；
Highest growth pH is 9.0, minimum growth pH is 4.0, and the most suitable growth pH is 6.0；
The period of delay of SITCC No.10009 bacterial strains is relatively short, and 3h enters exponential phase, and 12h reaches stationary phase；
(4), Physiology and biochemistry is identified
SITCC No.10009 bacterial strains peroxidase is negative；
(5), genetics characteristic：
The 16S rDNA sequence analyses of SITCC No.10009 bacterial strains；
SITCC No.10009 strain gene group DNA extracting methods：
The SITCC No.10009 single bacterium colonies of picking purifying are inoculated into 10mL MRS fluid nutrient mediums, after 37 DEG C of culture 8h Thalline is collected into bacterium solution centrifugation (4000rpm, 15min).
Above-mentioned gained is extracted using genome DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd.) Thallus DNA.PCR amplification uses universal primer (the 16s 27F of two kinds of synthesis:GAGAGTTTGATCCTGGCTCAG is specifically shown in SEQ IDNO：1；16s 1492R：CGGCTACCTTGTTACGACTT is specifically shown in SEQ IDNO：2), PCR product uses pillar PCR Product Purification Kits (Sangon Biotech (Shanghai) Co., Ltd.) recycle, and send raw work bioengineering after purification (Shanghai) limited liability company is sequenced.
The 16s rDNA nucleotides sequences of gained SITCC No.10009 bacterial strains are classified as 1482bp, are specifically shown in SEQ IDNO：3, Sequencing result is compared with the correlated series in GenBank, comparison result shows homologous with SITCC No.10009 bacterial strains The highest bacterial strain of property is Lactobacillus casei NWAFU1550 (Sequence ID：MG551225.1), homology is 99%.The G+C's (mol%)≤10%~12% and 16S rRNA of DNA described in Goodfellow and O ' Donnell The kind of sequence homology >=95% can be classified as a category, and Embley and Stackebrangdt think the sequence as 16S rRNA One kind is may be considered when row homology >=97%.It can therefore be concluded that：SITCC No.10009 bacterial strains with Lactobacillus casei NWAFU1550 belong to same kind, and SITCC No.10009 bacterial strains are accredited as Lactobacillus casei (Lactobacillus casei)。
Embodiment 3
The growth characteristics of Lactobacillus casei (Lactobacillus casei) SITCC No.10009
(1), Lactobacillus casei (Lactobacillus casei) SITCC No.10009 strain growths Drawing of Curve will live Lactobacillus casei (Lactobacillus casei) the SITCC No.10009 changed access MRS liquid by 2% (v/v) inoculum concentration In body culture medium, 37 DEG C of constant temperature incubation 16h are plotted against time every the OD values that 1-2h measures culture solution in 600nm with OD values Growth curves of Lactobacillus casei (Lactobacillus casei) the SITCC No.10009 in MRS is obtained, result is such as Shown in Fig. 2, as can be seen from Figure 2：Lactobacillus casei (Lactobacillus casei) SITCC No.10009 are trained in MRS It supports and is grown in base rapidly, enter logarithmic phase in 3h or so, 12h or so enters stationary phase；
(2), Lactobacillus casei (Lactobacillus casei) SITCC No.10009 optimum growth temperatures measurement will live Lactobacillus casei (Lactobacillus casei) the SITCC No.10009 changed are connected to respectively by 2% (v/v) inoculum concentration In 10mL MRS fluid nutrient mediums, constant temperature incubation under the conditions of being respectively placed in 15 DEG C, 25 DEG C, 32 DEG C, 37 DEG C, 40 DEG C and 45 DEG C 10h is compared with nonvaccinated MRS fluid nutrient mediums, and the OD values for the culture solution cultivated under different temperatures are measured in 600nm, with Cultivation temperature is abscissa, OD600nmIt maps for ordinate, as shown in figure 3, from figure 3, it can be seen that Lactobacillus casei The growth temperature range of (Lactobacillus casei) SITCC No.10009 is wider, DEG C is all grown from 15 DEG C to 45,30 DEG C -40 DEG C of well-growns, optimum growth temperature are 37 DEG C.
Embodiment 4
One kind containing the working stock culture of Lactobacillus casei (Lactobacillus casei) SITCC No.10009, passes through The method included the following steps is prepared：
(1), the preparation of sterile absorbent breast
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120mg： 60mg：The ratio of 1L Mixed dissolution is carried out, then sterilize at 95 DEG C 20min, is then cooled back to 37 DEG C to get to sterile absorbent milk solution；
(2), actication of culture
Lactobacillus casei (the Lactobacillus that the freeze-drying pipe of sterile water dissolution preserves is taken with oese Casei) one ring of SITCC No.10009 strains is cultivated for 24 hours in the flat lining out of MRS agar mediums in 37 DEG C of incubators, To get to tablet activated spawn until growing single bacterium colony；
(3), the preparation of working stock culture
With oese take step (2) obtained by one ring of tablet activated spawn access be equipped with 50mL MRS meat soup Liquid Cultures In the triangular flask of the 250mL specifications of base, it is placed in constant temperature incubation 10h in 37 DEG C of incubator, obtains culture solution；
It is that 5000r/min carries out centrifugation 20min by the culture solution control rotating speed of above-mentioned gained, centrifuges the precipitation nothing of gained Bacterium phosphate buffer solution is cleaned 3 times, and the sterile absorbent breast obtained by 50mL steps (1) is then added in precipitation after cleaning Solution, it is that 5000r/min be vortexed concussion resuspension thalline to get to containing Lactobacillus casei then to control rotating speed The working stock culture of (Lactobacillus casei) SITCC No.10009.
The working stock culture containing Lactobacillus casei (Lactobacillus casei) TCS of above-mentioned gained after measured, wherein It is 10 containing Lactobacillus casei (Lactobacillus casei) TCS viable counts9cfu/mL。
Application Example 1
The preparation of acidified milk of the one kind containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009, Specifically comprise the following steps：
(1), the preparation of sterile absorbent breast
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g：60g：The ratio of 1L into Row mixed dissolution obtains mixed solution, then controlled at 95 DEG C sterilizing 20min, be then cooled back to 37 DEG C to get to Sterile absorbent milk solution；
(2), fermented and cultured
It is calculated by percent by volume, 4 gained of embodiment is contained into Lactobacillus casei in 5% ratio The working stock culture of (Lactobacillus casei) SITCC No.10009 is inoculated into the breast of the sterile absorbent obtained by step (1) In solution, 37 DEG C of temperature of control carry out fermentation 10h to titratable acidity be 70-80 ° of T to get to containing Lactobacillus casei The acidified milk of (Lactobacillus casei) SITCC No.10009,4 DEG C stored refrigerated.
It in above-mentioned fermentation process, is sampled per 1h, until fermentation ends, are measured by above-mentioned method obtained by the different fermentations time Zymotic fluid in diacetyl content measurement, using fermentation time as abscissa, using the content of diacetyl in zymotic fluid as ordinate Mapping, the results are shown in Figure 4, and the dynamic change of diacetyl during as can be seen from Figure 4 fermenting is indicated above 3h fourths before fermentation The content of diketone is kept approximately constant, and Lactobacillus casei (Lactobacillus casei) SITCC No.10009 bacterium after 3h Strain starts to play a role, diacetyl content exponentially ascendant trend after 4h, until reaching peak 56.84mg/L when fermentation 10h.
Application Example 2
One kind containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009 and customary commercial leavening Acidified milk preparation, specifically comprise the following steps：
(1), the preparation of sterile absorbent breast
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g：60g：The ratio of 1L into Row mixed dissolution obtains mixed solution, then controlled at 95 DEG C sterilizing 20min, be then cooled back to 37 DEG C to get to Sterile absorbent milk solution；
(2), fermented and cultured
It is calculated by percent by volume, 4 gained of embodiment is contained into Lactobacillus casei in 5% ratio The working stock culture and customary commercial leavening of (Lactobacillus casei) SITCC No.10009 is inoculated into step (1) In the sterile absorbent milk solution of gained, 42 DEG C of temperature of control carry out fermentation 5h to titratable acidity be 70-80 ° of T to get to containing The acidified milk of Lactobacillus casei (Lactobacillus casei) SITCC No.10009 and customary commercial leavening, 4 DEG C cold It hides and preserves；
Customary commercial leavening is YF-L811, and manufacturer is Chr. Hansen A/S.
Weigh above-mentioned gained containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009d and often The acidified milk 7g for advising commodity leavening, is placed in headspace solid-phase microextraction bottle (15ml).Ml headspace bottle is placed in 55 DEG C of constant temperature In water-bath, it is inserted into the good extracting head of prior aging (50/30 μm of DVB/CAR/PDMS), headspace absorption 40min, extraction terminates, Extracting head is transferred at GC (Agilent 7890)-MS (Agilent 5975) injection port, 5min is parsed, completes sample introduction.GC- MS is kept 3min, is then warming up to 230 DEG C with the rate of 5 DEG C/min, and keep 10min with 40 DEG C of initial temperature, is completed to containing Volatility in the acidified milk of Lactobacillus casei (Lactobacillus casei) SITCC No.10009 and customary commercial leavening The detection of aroma compound, gained containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009 and often The GC-MS total ion current figures for advising volatile flavor compound in the acidified milk of commodity leavening are shown in Fig. 5, as can be drawn from Figure 5, In acidified milk containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009 and customary commercial leavening Detect 56 kinds of volatile flavor compounds altogether, wherein 9 kinds of alkanes, 7 kinds of olefines, 7 kinds of alcohols, 8 kinds of aldehydes, 9 kinds of ketone, 2 kinds of esters, 7 kinds of heterocyclic, other 7 kinds.
Application Example 3
A kind of above-mentioned Lactobacillus casei (Lactobacillus casei) SITCC No.10009 are in leben system Application in standby, specifically comprises the following steps：
(1), the preparation of sterile absorbent milk solution
By skimmed milk powder, glucose and pure water, by skimmed milk powder：Glucose：Pure water is 120g： 80g：The ratio of 1L Example carries out mixed dissolution, then controlled at 95 DEG C of sterilizing 120min, is then cooled back to 37 DEG C to get newborn to sterile absorbent Solution；
(2), the preparation of fermentation base-material
It is calculated by percent by volume, 4 gained of embodiment is contained into Lactobacillus casei in 5% ratio The working stock culture of (Lactobacillus casei) SITCC No.10009 is inoculated into the breast of the sterile absorbent obtained by step (1) In solution, 37 DEG C of temperature of control carries out fermentation 60h, is subsequently cooled to 4 DEG C of refrigerations to get to containing Lactobacillus casei The fermentation base-material of (Lactobacillus casei) SITCC No.10009；
(3), the preparation of sucrose solution
Sucrose is added in 70 DEG C of water, control rotating speed, which is 1000r/min stirrings 25min, makes sucrose dissolve, and then controls Temperature processed is 95 DEG C of sterilizing 5min postcoolings to 25 DEG C, the sucrose solution sterilized；
The dosage of above-mentioned sucrose and water, by sucrose：Water is 12.5g:The ratio of 100ml calculates；
(4), the allotment of acidified milk
It is calculated by percent by volume, in 25% ratio, Lactobacillus casei will be contained obtained by step (2) The sucrose that the fermentation base-material of (Lactobacillus casei) SITCC No.10009 is added to the sterilization obtained by step (3) is molten In liquid, uses the citric acid solution of 5g/L to adjust pH value to 3.6 after stirring 15min, then obtain deployed acidified milk；
(5), homogeneous, sterilization
The deployed fermented lactate-containing solution of step (3) is subjected to homogeneous for 20Mpa controlled at 25 DEG C, pressure, is then existed 5min is sterilized under the conditions of 95 DEG C, is then cooled to 4 DEG C of refrigerations to get to containing Lactobacillus casei (Lactobacillus Casei) the leben of SITCC No.10009.
Application Example 4
A kind of preparation of the acidified milk for adding different the content of sodium citrate corresponding in the preparation process of sterile absorbent breast, tool Body includes the following steps：
(1), the preparation of sterile absorbent breast
By 120g skimmed milk powders, 60g sucrose and 1L pure water, mixed dissolution is carried out, mixed solution is obtained；
Above-mentioned mixed solution prepares 6 parts, be separately added into for above-mentioned quality of pure water used 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% sodium citrate obtains 6 parts and corresponds to the different citric acids of addition in the preparation process of sterile absorbent breast The mixed solution of sodium content, then carries out sterilizing 20min controlled at 95 DEG C, is then cooled back to 37 DEG C to get in nothing The corresponding sterile absorbent milk solution for adding different the content of sodium citrate in the preparation process of bacterium skimmed milk；
(2), fermented and cultured
It is calculated by percent by volume, will contain Lactobacillus casei (Lactobacillus casei) SITCC in 5% ratio The working stock culture of No.10009 is inoculated into being corresponded in the preparation process of sterile absorbent breast obtained by step (1) and adds different lemons In the sterile absorbent milk solution of lemon acid sodium content, 37 DEG C of temperature of control carry out fermentation 10h to titratable acidity be 70-80 ° of T to get To the corresponding acidified milk for adding different the content of sodium citrate in the preparation process in sterile absorbent breast, 4 DEG C stored refrigerated.
Comparative example 1
One kind not containing Lactobacillus casei (Lactobacillus casei) SITCC No.10009, contains only customary commercial The preparation of the acidified milk of leavening, specifically comprises the following steps：
(1), the preparation of sterile absorbent breast
By 120g skimmed milk powders, 60g sucrose and 1L pure water, mixed dissolution is carried out, mixed solution is obtained；
Above-mentioned mixed solution carries out sterilizing 20min controlled at 95 DEG C, is then cooled back to 37 DEG C and is taken off to get to sterile Fat milk solution；
(2), fermented and cultured
It calculates, is inoculated into routine business bacterium powder in 5% ratio sterile de- obtained by step (1) by percent by volume In fat milk solution, 42 DEG C of temperature of control carries out fermentation 5h to curdled milk, and refrigeration is to 4 DEG C and stored refrigerated, obtains a kind of without containing dry Lactobacillus paracasei (Lactobacillus casei) SITCC No.10009, the acidified milk for containing only customary commercial leavening, it is as empty White hair kefir milk；
Customary commercial leavening is YF-L811, and manufacturer is Chr. Hansen A/S.
The blank run breast of above-mentioned gained is weighed into 7g, is placed in headspace solid-phase microextraction bottle (15ml).By ml headspace bottle It is placed in 55 DEG C of thermostat water bath, is inserted into the good extracting head of prior aging (50/30 μm of DVB/CAR/PDMS), headspace absorption 40min, extraction terminate, and extracting head is transferred at GC (Agilent 7890)-MS (Agilent 5975) injection port, parsing 5min completes sample introduction.GC-MS is kept 3min, is then warming up to 230 DEG C with the rate of 5 DEG C/min, and keep with 40 DEG C of initial temperature 10min completes the detection to blank run volatile flavor compound.Volatile flavor compound in the blank run breast of gained GC-MS total ion current figures as shown in Figure 6, as can be seen from Figure 6 do not contain Lactobacillus casei (Lactobacillus Casei) 35 kinds of volatile flavor compounds are detected altogether in the acidified milk of SITCC No.10009, that is, blank run breast, wherein 5 kinds of alkanes, a kind of olefines, 4 kinds of alcohols, 4 kinds of aldehydes, 9 kinds of ketone, a kind of esters, 5 kinds of heterocyclic, other 6 kinds.
It is compared by Fig. 5 and Fig. 6, it can be clearly seen that contain Lactobacillus casei (Lactobacillus casei) Volatile flavor compound type showed increased in the acidified milk of SITCC No.10009 and customary commercial leavening.Thus table Bright Lactobacillus casei (Lactobacillus casei) SITCC No.10009 can significantly improve the volatility in acidified milk The type of aroma compound, therefore Lactobacillus casei (Lactobacillus casei) SITCC No.10009 are in acidified milk It is with a wide range of applications.
Effect example 1
Acidified milk containing Lactobacillus casei and the acidified milk diacetyl of sodium citrate containing various concentration generate ability comparison
1 gained of above application embodiment is contained into Lactobacillus casei (Lactobacillus casei) SITCC The different sodium citrates of addition are corresponded in the acidified milk and Application Example 4 of No.10009 in the preparation process of sterile absorbent breast to contain The acidified milk of amount gained carries out the measurement that diacetyl generates ability respectively, the results showed that 1 gained of embodiment contains cheese breast bar Bacterium (Lactobacillus casei) SITCC be No.10009's and the life of diacetyl that is not added in the acidified milk of sodium citrate Yield is only 56.84mg/L, and adds the sodium citrate of different content in Application Example 4 in the preparation process of sterile absorbent breast The yield of diacetyl in finally obtained acidified milk is all improved, and especially sodium citrate is quality of pure water used When 0.5%, the yield of the diacetyl in finally obtained acidified milk is up to 70.43mg/L.
In conclusion a kind of Lactobacillus casei (Lactobacillus casei) SITCC No.10009 of the present invention exist In application, generating ability with higher diacetyl in acidified milk, while flavor substance in acidified milk can be significantly improved Type, and suitable sodium citrate is added in the preparation process of sterile absorbent breast, finally obtained acidified milk can be improved When the additive amount of the yield of diacetyl, especially sodium citrate is the 0.5% of skimmed milk powder, sucrose and pure water gross mass, most The yield of diacetyl in the acidified milk obtained eventually is up to 70.43mg/L, than not adding sodium citrate under equal conditions institute Acidified milk in diacetyl output increased 23.91%.
The above is only the citing of embodiments of the present invention, it is noted that for the ordinary skill of the art For personnel, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improve and become Type also should be regarded as protection scope of the present invention.
Sequence table
<110>Shanghai application technology university
<120>Lactobacillus casei（Lactobacillus casei）SITCC No.10009
<130> 2017
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Primer (16s 27f)
<400> 1
gagagtttga tcctggctca g 21
<210> 2
<211> 20
<212> DNA
<213>Primer (16s 1492r)
<400> 2
cggctacctt gttacgactt 20
<210> 3
<211> 1482
<212> DNA
<213>Lactobacillus casei (lactobacillus casei)
<400> 3
ggcctggcgg gtgctataca tgcagtcgaa cgagttctcg ttgatgatcg gtgcttgcac 60
cgagattcaa catggaacga gtggcggacg ggtgagtaac acgtgggtaa cctgccctta 120
agtgggggat aacatttgga aacagatgct aataccgcat agatccaaga accgcatggt 180
tcttggctga aagatggcgt aagctatcgc ttttggatgg acccgcggcg tattagctag 240
ttggtgaggt aatggctcac caaggcgatg atacgtaggc cgaacttgaa gaggttgatc 300
ggccacattg ggactggaga acacggccca aactcctacg gaaggcagca gtagggaatc 360
ttccacaatg gacgcaagtc tgatggagca acgccgcgtg agtgaagaag gctttcgggt 420
cgtaaaactc tgttgttgga gaagaatggt cggcagagta actgttgtcg gcgtgacggt 480
atccaaccca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540
aagcgttatc cggatttatt gggcgtaaag cgagcgcagg cggtttttta agtctgatgt 600
gaaagccctc ggcttaaccg aggaagcgca tcggaaactg ggaaacttga gtgcagaaga 660
ggacagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg 720
cgaaggcggc tgtctggtct gtaactgacg ctgaggctcg aaagcatggg tagcgaacag 780
gattagatac cctggtagtc catgccgtaa acgatgaatg ctaggtgttg gagggtttcc 840
gcccttcagt gccgcagcta acgcattaag cattccgcct ggggagtacg accgcaaggt 900
tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 960
agcaacgcga agaaccttac caggtcttga catcttttga tcacctgaga gatcaggttt 1020
ccccttcggg ggcaaaatga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1080
ttgggttaag tcctgcatcg agcgcaaccc ttatgactag ttgccagcat ttagttgggc 1140
actctagtag gactgccggt gacaaacccg gacgaaggtg gggatggacg gtcaaatcat 1200
catgcccctt atgacctggg gcgtacacac acgctacaat ggatggtaca acgagttgcg 1260
agaccgcgag gtcaagctaa tctcttaaag ccattctcag ttcggactgt aggctgcaac 1320
tcgcctacac gaagtcggaa tcgctagtaa tcgcggatca gcacgccgcg gtgaatacgt 1380
tcccgggcct tgtacacacc gcccgtcaca ccatgagagt ttgtaacacc cgaagccggt 1440
ggcgtaaccc ttttagggag cgagccgtct aagtgacaat gt 1482

Claims (10)

1. a kind of Lactobacillus casei（Lactobacillus casei）SITCC No.10009, the energy with high yield diacetyl Power is deposited in China typical culture collection center, deposit number CCTCC No. M2017364.
2. one kind containing Lactobacillus casei（Lactobacillus casei）The working stock culture of SITCC No.10009, feature It is to be prepared by a method comprising the following steps：
（1）, sterile absorbent breast preparation
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g：20-60g：The ratio of 1L carries out Mixed dissolution, then sterilize at 95 DEG C 20min, is then cooled back to 37-40 DEG C to get to sterile absorbent milk solution；
（2）, actication of culture
The Lactobacillus casei that the freeze-drying pipe of sterile water dissolution preserves is taken with oese（Lactobacillus casei） One ring of SITCC No.10009 strains cultivates 24-48h in the flat lining out of MRS agar mediums in 37 DEG C of incubators, until long Go out until single bacterium colony to get to tablet activated spawn；
（3）, working stock culture preparation
Step is taken with oese（2）One ring of the tablet activated spawn access of gained is equipped with 50mL MRS meat soup fluid nutrient mediums In the triangular flask of 250mL specifications, it is placed in constant temperature incubation 10-15h in 37 DEG C of incubator, obtains culture solution；
It is that 4000-6000r/min carries out centrifugation 15-30min by the culture solution control rotating speed of above-mentioned gained, centrifuges the precipitation of gained It is cleaned 2-3 times with sterile phosphate buffer solution, 50mL steps is then added in precipitation after cleaning（1）The sterile of gained takes off Fat milk solution, it is that 4000-6000r/min be vortexed concussion resuspension thalline to get to containing Lactobacillus casei then to control rotating speed （Lactobacillus casei）The working stock culture of SITCC No.10009.
3. containing Lactobacillus casei as claimed in claim 2（Lactobacillus casei）The work of SITCC No.10009 Make leavening, it is characterised in that preparation process step（1）In：Skimmed milk powder：Sucrose：Pure water is 120g：60g：1L；
Step（3）In：The culture solution control rotating speed of gained is that 5000r/min is centrifuged, and control rotating speed carries out for 5000r/min Thalline is resuspended in the concussion that is vortexed；
The sterile phosphate buffer solution calculates, phosphoric acid potassium dihydrogen 0.27g, disodium hydrogen phosphate 1.42g, chlorine by every liter Change sodium 8g, potassium chloride 0.2g, surplus is distilled water, and 121 DEG C of 20min that sterilize are cooling simultaneously to be preserved at 4 DEG C.
4. containing Lactobacillus casei as claimed in claim 2（Lactobacillus casei）The work of SITCC No.10009 Make leavening, it is characterised in that calculated by per mL, contain Lactobacillus casei（Lactobacillus casei）SITCC No.10009 viable counts are at least 109cfu/mL。
5. a kind of Lactobacillus casei as described in claim 1（Lactobacillus casei）SITCC No.10009 are being sent out Application in the preparation of ferment food.
6. a kind of Lactobacillus casei as claimed in claim 5（Lactobacillus casei）SITCC No.10009 are being sent out Application in the preparation of ferment food, it is characterised in that the fermented food is acidified milk or leben.
7. a kind of Lactobacillus casei as claimed in claim 6（Lactobacillus casei）SITCC No.10009 are being sent out Application in kefir milk preparation, specifically comprises the following steps：
（1）, sterile absorbent breast preparation
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g：20-60g：The ratio of 1L carries out Then mixed dissolution, obtained mixed solution are cooled back to 37-40 DEG C controlled at 95 DEG C of sterilizing 20min；
Or by skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g：20-60g：The ratio of 1L into Row mixed dissolution is incorporated as the sodium citrate of quality of pure water 0.1-0.6% used in mixed solution in obtained mixed solution, After mixing controlled at 95 DEG C of sterilizing 20min, it is then cooled back to 37-40 DEG C, obtains sterile absorbent milk solution；
（2）, fermented and cultured
It is calculated by percent by volume, will contain Lactobacillus casei in the ratio of 1-5%（Lactobacillus casei）SITCC The working stock culture of No.10009 is inoculated into step（1）In the sterile absorbent milk solution of gained, 37 DEG C of temperature of control is fermented To titratable acidity 70-80oT, 12-24h is then refrigerated at 4 DEG C to get to containing Lactobacillus casei（Lactobacillus casei）The acidified milk of SITCC No.10009;
Described contains Lactobacillus casei（Lactobacillus casei）The working stock culture of SITCC No.10009 passes through packet The method for including following steps is prepared：
1., the preparation of sterile absorbent breast
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g： 60g：The ratio of 1L is mixed Dissolving is closed, then sterilize at 95 DEG C 20min, is then cooled back to 37-40 DEG C to get to sterile absorbent milk solution；
2., actication of culture
The Lactobacillus casei that the freeze-drying pipe of sterile water dissolution preserves is taken with oese（Lactobacillus casei） One ring of SITCC No.10009 strains cultivates 24-48h in the flat lining out of MRS agar mediums in 37 DEG C of incubators, until long Go out until single bacterium colony to get to tablet activated spawn；
3., the preparation of working stock culture
Taking step with oese, 2. one ring of tablet activated spawn of gained access 50mL MRS meat soup fluid nutrient mediums is housed In the triangular flask of 250mL specifications, it is placed in constant temperature incubation 10-15h in 37 DEG C of incubator, obtains culture solution；
It is that 5000r/min carries out centrifugation 15-30min by the culture solution control rotating speed of above-mentioned gained, centrifuges the precipitation nothing of gained Bacterium phosphate buffer solution is cleaned 2-3 time, and the sterile absorbent that 50mL steps 1. gained is then added in precipitation after cleaning is newborn Solution, it is that 5000r/min be vortexed concussion resuspension thalline to get to containing Lactobacillus casei then to control rotating speed （Lactobacillus casei）The working stock culture of SITCC No.10009.
8. a kind of Lactobacillus casei as claimed in claim 7（Lactobacillus casei）SITCC No.10009 are being sent out Application in kefir milk preparation, it is characterised in that step（1）Middle skimmed milk powder：Sucrose：Pure water is 120g：60g：1L, after sterilizing It is cooled to 37 DEG C；Or skimmed milk powder：Sucrose：Pure water is 120g：20-60g：The ratio of 1L carries out mixed dissolution, and what is obtained is mixed The sodium citrate that 0.5 % of quality of pure water used in mixed solution is incorporated as in solution is closed, sterilizing postcooling is to 37 DEG C；
Step（2）In by percent by volume calculate, be inoculated in 5% ratio, control 37 DEG C of temperature ferment.
9. a kind of Lactobacillus casei as claimed in claim 6（Lactobacillus casei）SITCC No.10009 are being sent out Application in the preparation of kefir milk beverage, it is characterised in that specifically comprise the following steps：
（1）, sterile absorbent milk solution preparation
By skimmed milk powder, glucose and pure water, by skimmed milk powder：Glucose：Pure water is 120g：60-100g：The ratio of 1L Mixed dissolution is carried out, then controlled at 95 DEG C of sterilizing 120min, is then cooled back to 37-40 DEG C to get to sterile absorbent Milk solution；
（2）, fermentation base-material preparation
It is calculated by percent by volume, will contain Lactobacillus casei in the ratio of 1-5%（Lactobacillus casei）SITCC The working stock culture of No.10009 is inoculated into step（1）In the sterile absorbent milk solution of gained, 37 DEG C of temperature of control is fermented To titratable acidity 110-120oT, 4-10 DEG C of refrigeration is subsequently cooled to get to containing Lactobacillus casei（Lactobacillus casei）The fermentation base-material of SITCC No.10009；
Described contains Lactobacillus casei（Lactobacillus casei）The working stock culture of SITCC No.10009 passes through packet The method for including following steps is prepared：
1., the preparation of sterile absorbent breast
By skimmed milk powder, sucrose and pure water, by skimmed milk powder：Sucrose：Pure water is 120g： 60g：The ratio of 1L is mixed Dissolving is closed, then controlled at 95 DEG C of sterilizing 20min, is then cooled back to 37-40 DEG C to get to sterile absorbent milk solution；
2., actication of culture
The Lactobacillus casei that the freeze-drying pipe of sterile water dissolution preserves is taken with oese（Lactobacillus casei） One ring of SITCC No.10009 strains cultivates 24-48h in the flat lining out of MRS agar mediums in 37 DEG C of incubators, until long Go out until single bacterium colony to get to tablet activated spawn；
3., the preparation of working stock culture
Taking step with oese, 2. one ring of tablet activated spawn of gained access 50mL MRS meat soup fluid nutrient mediums is housed In the triangular flask of 250mL specifications, it is placed in constant temperature incubation 10-15h in 37 DEG C of incubator, obtains culture solution；
It is that 5000r/min carries out centrifugation 15-30min by the culture solution control rotating speed of above-mentioned gained, centrifuges the precipitation nothing of gained Bacterium phosphate buffer solution is cleaned 2-3 time, and the sterile absorbent that 50mL steps 1. gained is then added in precipitation after cleaning is newborn Solution, it is that 5000r/min be vortexed concussion resuspension thalline to get to containing Lactobacillus casei then to control rotating speed （Lactobacillus casei）The working stock culture of SITCC No.10009；
（3）, sucrose solution preparation
Sucrose is added in 70-80 DEG C of water, control rotating speed, which is 1000-1200r/min stirrings 20-30min, makes sucrose dissolve, Then controlled at 95 DEG C of sterilizing 5-10min postcoolings to 20-30 DEG C, the sucrose solution sterilized；
The dosage of above-mentioned sucrose and water, by sucrose：Water is 10-15g:The ratio of 100ml calculates；
（4）, acidified milk allotment
It is calculated by percent by volume, in the ratio of 10-25% by step（2）Gained contains Lactobacillus casei（Lactobacillus casei）The fermentation base-material of SITCC No.10009 is added to step（3）In the sucrose solution of the sterilization of gained, 10- is stirred PH value is adjusted to 3.6-3.7 with the aqueous citric acid solution of 5g/L after 15min, obtains deployed acidified milk；
（5）, homogeneous, sterilization
By step（3）Deployed acidified milk is that 20-30Mpa carries out homogeneous controlled at 20-30 DEG C, pressure, then 95 5-10min is sterilized under the conditions of DEG C, is then cooled to 4 DEG C of refrigerations to get to containing Lactobacillus casei（Lactobacillus casei）The leben of SITCC No.10009.
10. a kind of Lactobacillus casei as claimed in claim 9（Lactobacillus casei）SITCC No.10009 exist Application in leben preparation, it is characterised in that：
Step（1）Middle skimmed milk powder：Glucose：Pure water is 120g：80g：1L, sterilizing postcooling is to 37 DEG C；
Step（2）In by percent by volume calculate, by 5% ratio be inoculated with, fermentation postcooling to 4 DEG C refrigerations；
Step（3）The dosage of middle sucrose and water, by sucrose：Water is 12.5g:The ratio of 100ml calculates；
Step（4）In by percent by volume calculate, will contain Lactobacillus casei in 25% ratio（Lactobacillus casei） The fermentation base-material of SITCC No.10009 is added to step（3）The sucrose solution of the sterilization of gained；
Step（5）In controlled at 25 DEG C, pressure be 20Mpa carry out homogeneous.
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