CN104911134B - A kind of Leuconostoc mesenteroides and its application in cheesemaking - Google Patents
A kind of Leuconostoc mesenteroides and its application in cheesemaking Download PDFInfo
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Abstract
The invention discloses a kind of Leuconostoc mesenteroides (Leuconostoc mesenteroides), preparation method and its applications in cheesemaking.The Leuconostoc mesenteroides is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is：CGMCC No.10750.Its growth performance in breast is good, and phage resistance is good, and acid production speed is moderate, good for producing cheesy flavor, and yield is high.It can be used for preparing the leavening of cheese, and applied in cheese or other fermented food processing technologys.
Description
Technical field
The present invention relates to microorganism fields, and in particular to a kind of Leuconostoc mesenteroides and its application in cheesemaking.
Background technology
Cheese also known as cheese have miscellaneous taste, mouthfeel and form.Cheese is using milk as raw material, containing abundant
Protein and lipid have very high nutritive value.With the improvement of people's living standard in our country, more next to the demand of cheese
It is more.In cheesemaking and maturation microorganism species play an important role, and promote the texture and flavor of product
It is formed.The main fermentation agent of cheese has played the formation of cheese texture and characteristic flavor on basis extremely important.Main fermentation agent is dry
Acid is produced in junket production process, can improve the activity of renin, is helped to exclude whey, is inhibited harmful bacterial growth, and generates
Various enzymes participate in the formation of cheese characteristic flavor on basis and texture.
The production needs of most of cheese add different types of lactic acid bacteria (Lactic acid before curdled milk
Bacteria, LAB), their main function is that acid is produced in cheese making process, and a key point is in cheese manufacturing process
It is lactic acid for lactobacillary milk glycometabolism, the effect and cheese yield of speed and degree the influence curdled milk of acidification simultaneously influence renin
Activity.LAB can also generate the flavor substances such as acetic acid, acetaldehyde and biacetyl sometimes.Leavening can be generally divided into two classes, thermophilic
Warm nature leavening (i.e. most suitable cultivation temperature is 30 DEG C) and thermophilic leavening (i.e. most suitable cultivation temperature is 42 DEG C).Mesophilic property hair
Lactococcus lactis subsp. lactis (Lactococcus lactis) and leukonid (Leuconostoc are generally comprised in ferment agent
Spp.) etc., thermophilic leavening generally includes streptococcus thermophilus (Streptococcus thermophilus) and Bulgaria
Lactobacillus (Lactobacillus bulgaricus) etc..It is used today major part leavening directly cultivated again by curdled milk and
, and these curdled milks itself are select in production practices by long-term extensive high-quality cheese, usually contain 2~
6 plants of bacterium, so to a certain extent, current most leavening is referred to alternatively as mixed culture fermentation agent.The country is in terms of leavening
Research focus primarily upon the thermophilic leavening suitable for Yoghourt.
Currently, domestic study seldom mesophilic new leavening, it is mainly keen to ferment agent for sour milk (mainly thermophilic breast
Sour bacterium) screening and preparation research, and the screening study of the mesophilic property lactic acid bacteria to can be used for cheesemaking is less, for dry
It is even more blank in terms of screening criteria of the junket with thalline system.Therefore, it is suitable for completely newly using the abundant microorganism resource exploitation of China
The fermentation strain of cheese starter is of great significance.
Invention content
The technical problem to be solved by the present invention is to, for it is current lack it is for cheesemaking, from the country of China strain
The deficiency of the Leuconostoc mesenteroides obtained in resource provides a kind of Leuconostoc mesenteroides (Leuconostoc
Mesenteroides the application) and its in cheesemaking.The Leuconostoc mesenteroides can be used for preparing gives birth to for cheese
The mesophilic type lactic acid bacteria fermenting agent of production.
One of technical scheme of the present invention is：A kind of Leuconostoc mesenteroides (Leuconostoc mesenteroides),
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is：CGMCC No.10750.
It is isolated from the milk sample product in China Tibet, obtains the Leuconostoc mesenteroides CGMCC No.10750.Institute
The Leuconostoc mesenteroides CGMCC No.10750 well-growns in breast stated, acid production speed is moderate, good for producing cheesy flavor
Good, yield is high.The bacterial strain is identified, result is Leuconostoc mesenteroides (Leuconostoc mesenteroides), name
For LM79.The bacterial strain is preserved on April 27th, 2015 in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, and receive collection and register on the books number CGMCC No.10750, with Microbiological Characteristics below：
1, morphologic characteristic
After M17agar medium cultures 24 hours, bacterium colony is just transparent, and bacterium colony is smaller, mushrooms out later, after 2d
Bacterium colony is transparent greatly and has mobility.Leukonid cell spheroid, 0.9-1.2 microns of diameter, in pairs, at short chain or length
Chain, morphological feature meet the feature of Leuconostoc mesenteroides.
2, the characteristic that culture is learned
The well-grown under acid (pH5.0), high salt concentration (4%NaCl, the percentage are mass percent) environment；
The well-grown at 15~30 DEG C；Viability is good under the conditions of 40 DEG C, 2 hours cheese cutting heating process.
3, physiological property
It gives off a strong fragrance；Structural state is good, and proteolytic activity is stronger, can by breaks down proteins be polypeptide, amino acid or
Other inorganic, small molecule organic compounds；Has phage resistance.
Technical scheme of the present invention second is that：A kind of side preparing the Leuconostoc mesenteroides CGMCC No.10750
Method comprising following step cultivates the Leuconostoc mesenteroides CGMCC No.10750 in the medium.
Wherein, the culture medium is the culture medium of this field routine, and it is sub- can to grow the Lactococcus lactis
Kind CGMCC No.10752, preferably the M17agar culture mediums of MRS culture mediums or improvement.The MRS culture mediums are
The MRS culture mediums of this field routine, preferably comprising following constituent：8~12g/L of peptone, beef extract 9~
12g/L, 15~20g/L of glucose, 4~6g/L of yeast extract, 1.8~2g/L of ammonium citrate, 1.8~2.0g/L of dipotassium hydrogen phosphate,
4.8~5.2g/L of sodium acetate, 0.5~0.6g/L of magnesium sulfate, 0.5~0.55g/L of manganese sulfate and Tween 80~85g/L.More preferably,
It includes following constituent：Peptone 10g/L, beef extract 10g/L, glucose 20g/L, yeast extract 5g/L, ammonium citrate
2g/L, dipotassium hydrogen phosphate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.5g/L and Tween 80 g/L.The degreasing
Newborn culture medium is the degreasing milk medium of this field routine, preferably 10% degreasing milk medium.Skimmed milk powder is dissolved in
It is mixed in water up to the degreasing milk medium, the percentage is that the skimmed milk powder accounts for the skimmed milk powder and the water is total
The mass percent of quality.The M17agar culture mediums of the improvement be this field routine M17agar culture mediums in add 4~
5% sucrose, the percentage are mass percent, preferably comprising following constituent：Phytone 4.5~
5.5g/L, 4~5g/L of yeast extract, 4~5g/L of polyprotein peptone, β -18~20g/L of phosphoglycerol disodium, ascorbic acid 0.4
~0.5g/L, 2.2~2.8g/L of beef extract, 1~1.5g/L of bitter salt, 2.5~3g/L of agar powder and 4~5% sugarcanes
Sugar, the percentage are mass percent.More preferably comprising following constituent：Phytone 5g/L, yeast extraction
Object 5g/L, polyprotein peptone 5g/L, β-phosphoglycerol disodium 19g/L, ascorbic acid 0.5g/L, beef extract 2.5g/L, seven hydrations
Magnesium sulfate 1.2g/L, agar powder 2.5g/L and 5% sucrose, the percentage are mass percent.
The temperature of the culture is the temperature of this field routine, can grow the Leuconostoc mesenteroides CGMCC
No.10750, preferably 25~35 DEG C are more preferably 28~30 DEG C.The time of the culture be this field it is conventional when
Between, preferably 24~72 hours, be more preferably 24~48 hours.The pH of the culture is the pH, Neng Gousheng of this field routine
Long described Leuconostoc mesenteroides CGMCC No.10750, preferably 4~6, it is more preferably 4~5.
Preferably, further including the steps that carrying out seed culture using seed culture medium before the culture.The seed
Culture is the seed culture of this field routine.The seed culture medium is the seed culture medium of this field routine, preferably
Degreasing milk medium is more preferably 10~12% degreasing milk mediums, and the percentage is mass percent.The degreasing
Breast culture bacterium sterilizes by 115 DEG C, 15~20min.The time of the seed culture is the time of this field routine, preferably
16~32 hours, be more preferably 16~24 hours.The temperature of the seed culture be this field routine temperature, preferably 28
~32 DEG C.The inoculum concentration of the seed culture be this field routine inoculum concentration, preferably 1~2%, the percentage be body
Product percentage.The algebraically of the activation of the seed culture is the algebraically of this field routine, and preferably 2~4 generations are more preferably 2
~3 generations.Preferably, the Leuconostoc mesenteroides CGMCC No.10750 are the state that freezes, the seed culture includes heating
The step of.The temperature of the heating is the temperature of this field routine, and preferably 10~34 DEG C, be more preferably 20~28 DEG C.
The method of the culture is the method for the culture of this field routine, preferably shaking flask culture or fermentation tank culture.
The three of technical solution provided by the invention are：The Leuconostoc mesenteroides CGMCC No.10750 are in cheesemaking
In application.
The cheese is the cheese of this field routine, preferably Gouda cheese, Cheddar and Pa Masen cheese.More
It is Gouda cheese goodly.
Obtained cheese quality is close, and elasticity is moderate, and color and luster is micro- yellow and uniform, has aged cheese characteristic chicken flavor and taste
Taste, the cheese yield prepared with the strain is high, and no whey precipitation or rancid in 10~24 months can be stored under non-refrigerated conditions.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined arbitrarily to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that：The present invention provides a kind of Leuconostoc mesenteroides CGMCC No.10750,
Aromatic flavour, tissue morphology is good, good for producing cheesy flavor, and yield is high.It can be used for preparing the leavening of cheese, and apply
In cheese or other fermented food processing technologys.
Biomaterial preservation information
The Lactococcus lactis subsp. lactis BD164 of the present invention, is deposited in Chinese microorganism strain on April 27th, 2015
Preservation administration committee common micro-organisms center (CGMCC), preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postal
It compiles：100101, deposit number is：CGMCC No.10751, culture title are Lactococcus lactis subsp. lactis, and Classification And Nomenclature is
Lactococcuslactis subsp.lactis。
The Lactococcus lactis subsp. lactis BD2263 of the present invention, is deposited in Chinese microorganism strain April 27 in 2015
Preservation administration committee common micro-organisms center (CGMCC), preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postal
It compiles：100101, deposit number is：CGMCC No.10749, culture title are Lactococcus lactis subsp. lactis, and Classification And Nomenclature is
Lactococcuslactis subsp.lactis。
The Lactococcus lactis subsp. lactis BD401 of the present invention, is deposited in Chinese microorganism strain on April 27th, 2015
Preservation administration committee common micro-organisms center (CGMCC), preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postal
It compiles：100101, deposit number is：CGMCC No.10752, culture title are Lactococcus lactis subsp. lactis, and Classification And Nomenclature is
Lactococcuslactis subsp.lactis。
The Leuconostoc mesenteroides LM79 of the present invention, is deposited in Chinese microorganism strain preservation pipe on April 27th, 2015
Reason committee common micro-organisms center (CGMCC), preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：
100101, deposit number is：CGMCC No.10750, culture title are Lactococcus lactis subsp. lactis, and Classification And Nomenclature is
Leuconostocmesenteroides。
Specific implementation mode
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
Product specification selects.
The acquisition of 1 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
(1), it activates：The milk sample for being derived from Tibet is taken out from cryopreservation tube, with the inoculum concentration of 2% (v/v) be inoculated in by
121 DEG C, the 30 DEG C of cultures 36 hours that (are purchased from Britain OXOID companies) in the M17 broth bouillons of sterilizing in 15 minutes, it is continuous to cultivate
Three generations obtains the strain of activation.
(2), the strain of the activation obtained by step (1) is inoculated in the inoculum concentration of 2% (v/v) in degreasing milk medium,
After 15 DEG C are cultivated 16 hours, the bacterial strain of aromatic flavour is selected, you can.Selection meets the bacterial strain of conditions above, is named as LM79.
Leuconostoc mesenteroides LM79 is deposited in China Committee for Culture Collection of Microorganisms on April 27th, 2015
Common micro-organisms center (CGMCC), preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101, preservation
Number is：CGMCC No.10750, culture title are Lactococcus lactis subsp. lactis, and Classification And Nomenclature is
Leuconostocmesenteroides。
Feature is learned in the morphology of 2 Leuconostoc mesenteroides CGMCC No.10750 of embodiment and culture
Cultural characteristic：Using 30 DEG C of culture Leuconostoc mesenteroides CGMCC No.107502 days of M17agar culture mediums of improvement
Afterwards, cultural characteristic is observed.
Cultural characteristics of the 1 Leuconostoc mesenteroides CGMCC No.10750 of table on culture medium
| Culture medium | Bacterium colony quality | Colonial morphology | Colony colour |
| The M17agar of improvement | With mobility | In pairs, at short chain or long-chain | It is transparent |
The result of table 1 illustrates that Leuconostoc mesenteroides CGMCC No.10750 grow good on the M17agar culture mediums of improvement
Good, bacterium colony is just transparent, and bacterium colony is smaller, mushrooms out later, and bacterium colony is transparent greatly and has mobility after 2d.A bright beading
Bacterium cell spheroid, 0.9~1.2 μm of diameter, in pairs, at short chain or long-chain, morphological feature meets the feature of Leuconostoc mesenteroides.
The physiological and biochemical property of 3 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
A), fermentation character is tested
Leuconostoc mesenteroides CGMCC No.10750 are inoculated in by the inoculum concentration of 1% (v/v) and are taken off equipped with 10% to sterilize
In the triangular flask of fat breast culture medium, 30 DEG C of constant incubator cultures are placed in, until its curdled milk, investigates its curd taste, whey analysis
The quality having troubles with grumeleuse, the results are shown in Table 2.
The fermentation character of 2 Leuconostoc mesenteroides CGMCC No.10750 of table
| Bacterial strain | Curdled milk situation | It judges |
| Leuconostoc mesenteroides CGMCC No.10750. | Whey is precipitated less, and structural state is fine | Fragrance is dense |
B), proteolytic activity is tested
Forint (Folin)-phenol reagent process measures the proteolytic activity of bacterial strain：1mL sample solutions are taken, 2.5mL is added
0.28M NaOH solutions, mixing place 10min in 25 DEG C or so.0.75mL forint phenol reagent (being purchased from traditional Chinese medicines reagent) is added,
It shakes up immediately, reacts 15min in 35 DEG C or so, using ultraviolet specrophotometer (purchased from the U.S. scientific instrument in Shanghai day at 660nm
Co., Ltd) measure its light absorption value.The sample solution is：Leuconostoc mesenteroides CGMCC No.10750 are pressed into 1% (v/v)
Inoculum concentration be inoculated in the triangular flask of the degreasing milk medium equipped with sterilizing, after being placed in 30 DEG C of constant incubator cultures 24 hours
Suspension.As a result, it has been found that the OD660 of Leuconostoc mesenteroides CGMCC No.10750 is 0.82.Illustrate Leuconostoc mesenteroides
CGMCC No.10750 proteolytic activities are stronger, and in Cheese during Ripening, leavening bacterial strain can be by breaks down proteins
For polypeptide, amino acid or other inorganic, small molecule organic compounds, play an important role to the formation of cheesy flavor.
C), phage resistance
By the cheese starter fermentation exception containing Leuconostoc mesenteroides, (cheese does not produce acid with bacterium or production acid is extremely slow and solidifying
It is newborn abnormal be ferment it is abnormal) cheese raw milk (being obtained from bright milk industry research institute pilot plant) be used as sample, by
Leuconostoc mesenteroides CGMCC No.10750 are inoculated in by the inoculum concentration of 2% (v/v) in the triangular flask equipped with sample, are placed in 30 DEG C
Constant incubator fermented and cultured 12 hours.The pH value of the zymotic fluid of fermented and cultured is measured, Δ pH >=0.3 is found, illustrates that goldbeater's skin is bright
Beading bacterium CGMCC No.10750 have phage resistance.
Illustrate that the Leuconostoc mesenteroides CGMCC No.10750 have microorganism below by the data of embodiment 2~3
Learn characteristic：
1, morphologic characteristic
After M17agar medium cultures 24 hours, bacterium colony is just transparent, and bacterium colony is smaller, mushrooms out later, after 2d
Bacterium colony is transparent greatly and has mobility.Leukonid cell spheroid, 0.9-1.2 microns of diameter, in pairs, at short chain or length
Chain, morphological feature meet the feature of Leuconostoc mesenteroides.
2, the characteristic that culture is learned
The well-grown under acid (pH5.0), high salt concentration (4%NaCl, the percentage are mass percent) environment；
The well-grown at 15~30 DEG C；Viability is good under the conditions of 40 DEG C, 2 hours cheese cutting heating process.
3, physiological property
It gives off a strong fragrance；Structural state is good, and proteolytic activity is stronger, can by breaks down proteins be polypeptide, amino acid or
Other inorganic, small molecule organic compounds；Has phage resistance.
The 16S rDNA sequence analyses of 4 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
16S rDNA sequence analyses：Utilize 27F, 1492R primer amplification Lactococcus lactis subsp. lactis CGMCC
The 16S rDNA segments of No.10752 purify, recycling, and then using sequenator ABI3730-XL to carry out DNA sequencing, (the upper Shanghai's style is gloomy
Promise bio tech ltd).As a result, it has been found that Leuconostoc mesenteroides CGMCC No.10750 and Leuconostoc mesenteroides type strain
The homology of the 16S rDNA sequences of Leuconostoc mesenteroides strain ATCC8293 is 100%.Due to working as
When 16S rDNA sequence homologies are higher than 97%, it is believed that be of the same race, therefore Leuconostoc mesenteroides CGMCC in belonging to
No.10750 belongs to Leuconostoc mesenteroides (Leuconostoc mesenteroides).
Wherein, primer pair sequence as described above is：27F：agagtttgat cctggctcag；1492R：
Ctacggctac cttgttacga (its nucleotide sequence is respectively as shown in sequence table SEQ ID NO.2 and SEQ ID NO.3).
The result of the 16S rRNA gene sequencing of Leuconostoc mesenteroides CGMCC No.10750 is as shown in SEQ ID NO.1.
The fermentation of 5 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
The Leuconostoc mesenteroides CGMCC No.10750 frozen are to slowly warm up to 10 DEG C.
Seed culture medium：(skimmed milk powder is purchased from the limited public affairs of New Zealand Westland cooperation dairy industries to 10% degreasing milk medium
Skimmed milk powder is dissolved in the water and mixes up to degreasing milk medium by department, and the percentage is that the skimmed milk powder accounts for described take off
The mass percent of fat milk powder and the water gross mass), the percentage is mass percent.The skimmed milk culture bacterium passes through
115 DEG C, 15min sterilizings.
28 DEG C, cultivation cycle 32h of the cultivation temperature of seed culture medium, inoculum concentration 1%, the percentage in seed bottle
To account for the percent by volume of fermentation medium.Activated for 2 generations in seed culture medium.
Fermentation medium：Peptone 8g/L, beef extract 9g/L, glucose 15g/L, yeast extract 4g/L, ammonium citrate 1.8g/
L, dipotassium hydrogen phosphate 1.8g/L, sodium acetate 4.8g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.5g/L and Tween 80 g/L.
Cultivation temperature is 25 DEG C, cultivation cycle 72h, and culture pH is 4.
The fermentation of 6 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
The Leuconostoc mesenteroides CGMCC No.10750 frozen are to slowly warm up to 20 DEG C.
Seed culture medium：12% degreasing milk medium, the percentage are mass percent.The skimmed milk culture bacterium passes through
Cross 115 DEG C, 20min sterilizings.
32 DEG C, cultivation cycle 16h of the cultivation temperature of seed culture medium, inoculum concentration 2%, the percentage in seed bottle
To account for the percent by volume of fermentation medium.Activated for 4 generations in seed culture medium.
Fermentation medium：Peptone 12g/L, beef extract 12g/L, glucose 20g/L, yeast extract 6g/L, ammonium citrate 2g/
L, dipotassium hydrogen phosphate 2.0g/L, sodium acetate 5.2g/L, magnesium sulfate 0.6g/L, manganese sulfate 0.55g/L and polysorbate85 g/L.
Cultivation temperature is 35 DEG C, and for 24 hours, culture pH is 6 to cultivation cycle.
The fermentation of 7 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
The Leuconostoc mesenteroides CGMCC No.10750 frozen are to slowly warm up to 28 DEG C.
Seed culture medium：12% degreasing milk medium, the percentage are mass percent.The skimmed milk culture bacterium passes through
Cross 115 DEG C, 20min sterilizings.
32 DEG C, cultivation cycle 16h of the cultivation temperature of seed culture medium, inoculum concentration 2%, the percentage in seed bottle
To account for the percent by volume of fermentation medium.Activated for 3 generations in seed culture medium.
Fermentation medium：Peptone 10g/L, beef extract 10g/L, glucose 20g/L, yeast extract 5g/L, ammonium citrate 2g/
L, dipotassium hydrogen phosphate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.5g/L and Tween 80 g/L.
Cultivation temperature is 28 DEG C, cultivation cycle 48h, and culture pH is 5.
The fermentation of 8 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
The Leuconostoc mesenteroides CGMCC No.10750 frozen are to slowly warm up to 34 DEG C.
Seed culture medium：10% degreasing milk medium, the percentage are mass percent.The skimmed milk culture bacterium passes through
Cross 115 DEG C, 15min sterilizings.
28 DEG C, cultivation cycle 32h of the cultivation temperature of seed culture medium, inoculum concentration 1%, the percentage in seed bottle
To account for the percent by volume of fermentation medium.Activated for 2 generations in seed culture medium.
Fermentation medium：Peptone 10g/L, beef extract 10g/L, glucose 20g/L, yeast extract 5g/L, ammonium citrate 2g/
L, dipotassium hydrogen phosphate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.5g/L and Tween 80 g/L.
Cultivation temperature is 30 DEG C, cultivation cycle 48h, and culture pH is 5.
The fermentation of 9 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
The Leuconostoc mesenteroides CGMCC No.10750 frozen are to slowly warm up to 20 DEG C.
Seed culture medium：10% degreasing milk medium, the percentage are mass percent.The skimmed milk culture bacterium passes through
Cross 115 DEG C, 15min sterilizings.
28 DEG C, cultivation cycle 32h of the cultivation temperature of seed culture medium, inoculum concentration 1%, the percentage in seed bottle
To account for the percent by volume of fermentation medium.Activated for 4 generations in seed culture medium.
Fermentation medium：Phytone 4.5g/L, yeast extract 4g/L, polyprotein peptone 4g/L, β-phosphoglycerol two
Sodium 18g/L, ascorbic acid 0.4g/L, beef extract 2.2g/L and bitter salt 1g/L, agar powder 2.5g/L and 4% sugarcane
Sugar, the percentage are mass percent.
Cultivation temperature is 35 DEG C, and for 24 hours, culture pH is 4 to cultivation cycle.
The fermentation of 10 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
The Leuconostoc mesenteroides CGMCC No.10750 frozen are to slowly warm up to 28 DEG C.
Seed culture medium：12% degreasing milk medium, the percentage are mass percent.The skimmed milk culture bacterium passes through
Cross 115 DEG C, 15min sterilizings.
28 DEG C, cultivation cycle 32h of the cultivation temperature of seed culture medium, inoculum concentration 1%, the percentage in seed bottle
To account for the percent by volume of fermentation medium.Activated for 2 generations in seed culture medium.
Fermentation medium：Phytone 5.5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-phosphoglycerol two
Sodium 20g/L, ascorbic acid 0.5g/L, beef extract 2.8g/L and bitter salt 1.5g/L agar powders 3g/L and 5% sugarcane
Sugar, the percentage are mass percent.
Cultivation temperature is 30 DEG C, cultivation cycle 48h, and culture pH is 4.
The fermentation of 11 Leuconostoc mesenteroides CGMCC No.10750 of embodiment
The Leuconostoc mesenteroides CGMCC No.10750 frozen are to slowly warm up to 34 DEG C.
Seed culture medium：10% degreasing milk medium, the percentage are mass percent.The skimmed milk culture bacterium passes through
Cross 115 DEG C, 20min sterilizings.
28 DEG C, cultivation cycle 32h of the cultivation temperature of seed culture medium, inoculum concentration 1%, the percentage in seed bottle
To account for the percent by volume of fermentation medium.Activated for 3 generations in seed culture medium.
Fermentation medium phytone 5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-phosphoglycerol disodium
19g/L, ascorbic acid 0.5g/L, beef extract 2.5g/L, bitter salt 1.2g/L agar powders 2.5g/L and 5% sugarcane
Sugar, the percentage are mass percent.
Cultivation temperature is 28 DEG C, and for 24 hours, culture pH is 5 to cultivation cycle.
Effect example 1
(1) by Lactococcus lactis subsp. lactis BD164, Lactococcus lactis subsp. lactis BD2263, Lactococcus lactis
Subspecies BD401 and Leuconostoc mesenteroides LM79 is inoculated in the skimmed milk culture of 10% (w/v) sterilizings by the inoculum concentration of 2% (v/v)
In base (be purchased from New Zealand Westland cooperation dairy industries Co., Ltd), it is placed in 30 DEG C of constant incubator cultures 24 hours, activation 2
In generation, obtains the bacterial strain of activation.The bacterial strain of activation is inoculated in the skimmed milk culture of 10% (w/v) sterilizings by the inoculum concentration of 2% (v/v)
It in base, is placed in 30 DEG C of constant incubator cultures and is enlarged culture in 24 hours, be repeated 3 times, obtain culture.
(2) culture obtained by step (1) is obtained into Lactococcus lactis subsp. lactis BD164, Lactococcus lactis Asia
Kind BD2263, Lactococcus lactis subsp. lactis BD401 and Leuconostoc mesenteroides LM79, then according to the Lactococcus lactis
Subspecies BD401, the Lactococcus lactis subsp. lactis BD2263, the Lactococcus lactis subsp. lactis BD164 and the goldbeater's skin
The viable count of leukonid LM79 is 1:1:1:1 ratio mixing, obtains cheese starter.
2 cheese starter of effect example produces Gouda cheese
1) 100kg fresh cow milks (being purchased from Kingsoft pasture) add 0.01% calcium chloride, stir evenly through filtering, at 72 DEG C,
For 15s pasteurizes postcooling to 30 DEG C, the percentage is mass percent.By cheese starter made from effect example 1
It pours into the fresh cow milk after pasteurize, the viable count of the cheese starter in fresh cow milk is made to reach 107Cfu/mL is slowly stirred
15min.It stands 32 DEG C of fermentations to pH reductions by 0.2 and 20mg/100L renins (being purchased from Danisco A/S BJ Rep Office) is added later (wherein,
Renin makes it dissolve uniformly using the preceding sterile water dissolution with 10 times of quality, stirring 2min), curdled milk is obtained after 30min.
2) curd cutting made from step 1) is discharged at the cubical grumeleuse of length of side 5mm after being slowly stirred rear 15min
The whey of total volume 35%；Divide 2 50 DEG C of additions, the water that volume is fresh cow milk total volume 25%, stirring 20min to curd temperature
Reach 35 DEG C.It is 6.1 to continue stirring to the whey pH of discharge, exhausts whole wheys.Grumeleuse is entered into mould later, squeezing forming, is taken off
Mould takes out.Using a concentration of 20% 12 DEG C of salt marsh 20h of brine, the percentage is mass percent.It is vacuum-packed later, 10
DEG C, humidity 90%RH, ripe 5 weeks to get young Gouda cheese.
The sensory evaluation of 3 cheese of effect example
This subjective appreciation personnel include 12 personnel for being engaged in food research, are familiar with subjective appreciation points for attention and comment
Minute mark is accurate.Total score is 50 points, and score is evaluated in accordance with table 3.Each assessment officer independently evaluates, and clear water is used when replacing sample every time
It gargles, the results are shown in Table 4.Wherein, control group is (to be purchased from Dan Nike using direct putting type cheese starter CHOOZIT RM 32
This (China) Co., Ltd), according to effect example 2 the step of obtained cheese.
3 cheese subjective appreciation method of table
4 cheese results of sensory evaluation of table
The result of table 4 illustrates, the Gouda cheese (control group) that commercial fermentation agent makes in terms of quality with effect example 2
Difference between the cheese of gained is not notable；In growing smell scoring, the cheese of 2 gained of effect example is grown in cheese feature
Scoring and total score in terms of smell, tart flavour, bitter taste are all higher than control group.Therefore, the cheese of 2 gained of effect example has good
Good quality and preferably grow smell.
It should be understood that after the above for having read the present invention, those skilled in the art can make the present invention various
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (11)
1. a kind of Leuconostoc mesenteroides（Leuconostoc mesenteroides）, which is characterized in that it is deposited in the micro- life of China
Object culture presevation administration committee common micro-organisms center, preserving number are：CGMCC No.10750.
2. a kind of method preparing Leuconostoc mesenteroides CGMCC No.10750, which is characterized in that it includes the following steps,
Leuconostoc mesenteroides CGMCC No.10750 are cultivated in culture medium.
3. method as claimed in claim 2, which is characterized in that the culture medium is MRS culture mediums or the M17 of improvement
Agar culture mediums or degreasing milk medium；The MRS culture mediums include following constituent：8 ~ 12g/L of peptone, beef
9 ~ 12g/L of cream, 15 ~ 20g/L of glucose, 4 ~ 6 g/L of yeast extract, 1.8 ~ 2g/L of ammonium citrate, 1.8 ~ 2.0g/L of dipotassium hydrogen phosphate,
4.8 ~ 5.2g/L of sodium acetate, 0.5 ~ 0.6g/L of magnesium sulfate, 0.5 ~ 0.55g/L of manganese sulfate and Tween 80 ~ 85g/L；The skimmed milk
Culture medium is 10% degreasing milk medium, and the percentage is mass percent；And/or the M17 agar cultures of the improvement
Base includes following constituent：4.5 ~ 5.5g/L of phytone, 4 ~ 5g/L of yeast extract, 4 ~ 5g/L of polyprotein peptone, β-
18 ~ 20 g/L of phosphoglycerol disodium, 0.4 ~ 0.5g/L of ascorbic acid, 2.2 ~ 2.8g/L of beef extract, bitter salt 1 ~
1.5g/L, 2.5 ~ 3g/L of agar powder and 4 ~ 5% sucrose, the percentage are mass percent.
4. method as claimed in claim 3, which is characterized in that the MRS culture mediums include following constituent：Albumen
Peptone 10g/L, beef extract 10g/L, glucose 20g/L, yeast extract 5g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 2g/L, sodium acetate
5g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.5g/L and Tween 80 g/L；And/or the M17 agar culture medium packets of the improvement
Include following constituent：Phytone 5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-phosphoglycerol disodium
19g/L, ascorbic acid 0.5g/L, beef extract 2.5g/L, bitter salt 1.2g/L, agar powder 2.5g/L and 5% sucrose,
The percentage is mass percent.
5. method as claimed in claim 2, which is characterized in that the temperature of the culture is 25 ~ 35 DEG C；The time of the culture
It is 24 ~ 72 hours；And/or the pH of the culture is 4 ~ 6.
6. method as claimed in claim 5, which is characterized in that the temperature of the culture is 28 ~ 30 DEG C；The time of the culture
It is 24 ~ 48 hours；And/or the pH of the culture is 4 ~ 5.
7. method as claimed in claim 2, which is characterized in that further include being planted using seed culture medium before the culture
The step of son culture, the seed culture medium are degreasing milk medium；The degreasing milk medium by 115 DEG C, 15 ~
20min sterilizes；The time of the seed culture is 16 ~ 32 hours；The temperature of the seed culture is 28 ~ 32 DEG C；The seed
The inoculum concentration of culture is 1 ~ 2%, and the percentage is percent by volume；And/or the algebraically of the activation of the seed culture is 2 ~ 4
Generation.
8. the method for claim 7, which is characterized in that the seed culture medium is 10 ~ 12% degreasing milk mediums,
The percentage is mass percent；The time of the seed culture is 16 ~ 24 hours；And/or the work of the seed culture
The algebraically of change was 2 ~ 3 generations.
9. the method for claim 7, which is characterized in that the seed culture includes the steps that heating；The heating
Temperature is 10 ~ 34 DEG C.
10. method as claimed in claim 9, which is characterized in that the temperature of the heating is 20 ~ 28 DEG C.
11. applications of the Leuconostoc mesenteroides CGMCC No.10750 as described in claim 1 in cheesemaking.
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| KR101930166B1 (en) * | 2016-07-15 | 2018-12-17 | 씨제이제일제당 (주) | Leuconostoc mesenteroides CJLM181 producing decreased amounts of gas and methods for preparing kimchi using the same |
| JP6875041B2 (en) * | 2016-07-15 | 2021-05-19 | シージェイ チェイルジェダン コーポレーションＣｊ Ｃｈｅｉｌｊｅｄａｎｇ Ｃｏｒｐｏｒａｔｉｏｎ | Lyconostock mesenteroides CJLM627 strain with low gas generation and kimchi production method using this strain |
| CN107080173A (en) * | 2017-05-04 | 2017-08-22 | 光明乳业股份有限公司 | A kind of style milk pudding and preparation method thereof |
| CN108060099B (en) * | 2017-12-26 | 2021-09-07 | 光明乳业股份有限公司 | Auxiliary leavening agent, preparation method thereof and application thereof in fermented products |
| CN109097297B (en) * | 2018-08-07 | 2022-10-04 | 宁夏大学 | Leuconostoc mesenteroides strain and application thereof |
| CN113662164A (en) * | 2021-08-16 | 2021-11-19 | 宁夏春升源生物科技有限公司 | Biological coupling oil extracted onion oil and preparation method thereof |
| CN115843848B (en) * | 2022-12-12 | 2024-03-01 | 江南大学 | Method for improving bread quality by fermenting dough in situ and producing high-molecular polysaccharide |
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