CN104946566B - A kind of Lactococcus lactis subsp. lactis and its application in cheesemaking - Google Patents
A kind of Lactococcus lactis subsp. lactis and its application in cheesemaking Download PDFInfo
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Abstract
The invention discloses a kind of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis), preparation method and its applications in cheesemaking.The Lactococcus lactis subsp. lactis is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is：CGMCC No.10749.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 Lactococcus lactis subsp. lactis and its in cheesemaking
Using.
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, mesophilic, from China
The deficiency of the Lactococcus lactis subsp. lactis obtained in domestic microorganism resource provides a kind of Lactococcus lactis subsp. lactis
(Lactococcus lactis subsp.lactis) and its application in cheesemaking.The Lactococcus lactis
Subspecies can be used for preparing the mesophilic type lactic acid bacteria fermenting agent for cheesemaking.
One of technical scheme of the present invention is：A kind of Lactococcus lactis subsp. lactis (Lactococcus lactis
Subsp.lactis), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is：
CGMCC No.10749。
It is isolated from the milk pimple sample in China Tibet, obtains the Lactococcus lactis subsp. lactis CGMCC
No.10749.Lactococcus lactis subsp. lactis CGMCC No.10749 well-growns in breast, acid production speed is moderate, uses
Good in production cheesy flavor, yield is high.The bacterial strain is identified, result is Lactococcus lactis subsp. lactis
Lactococcus lactis subsp.lactis, are named as BD2263.The bacterial strain is on April 27th, 2015 is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, and receive collection and register on the books number CGMCC
No.10749, with Microbiological Characteristics below：
1, morphologic characteristic
After M17 agar medium cultures 24 hours, bacterium colony is creamy white, and bacterium colony is smaller, and circular protrusions are glossy, bacterium
Body is in oval.It mushrooms out later, bacterium colony is big and close after 2 days, and be creamy white yellowish.
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
Acid producing ability is strong, and acid production speed is very fast；Curdled milk speed is fast, curdled milk moderate viscosity；In terms of fermenting characteristic, breast can be precipitated
Clearly, curdled milk structural state is good, gives off a strong fragrance；Proteolytic activity is stronger, can by breaks down proteins be polypeptide, amino acid or its
Its inorganic, small molecule organic compound；And there is phage resistance.
Technical scheme of the present invention second is that：It is a kind of to prepare the Lactococcus lactis subsp. lactis CGMCC No.10749
Method comprising following step cultivates the Lactococcus lactis subsp. lactis CGMCC No.10749 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.10749, preferably MRS culture mediums, M17 agar culture mediums or degreasing milk medium.The MRS
Culture medium is the MRS culture mediums of this field routine, preferably comprising following constituent：8~12g/L of peptone, beef
9~12g/L of cream, 15~20g/L of glucose, 4~6g/L of yeast extract, 1.8~2g/L of ammonium citrate, dipotassium hydrogen phosphate 1.8~
2.0g/L, 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 comprising following constituent：Peptone 10g/L, beef extract 10g/L, glucose 20g/L, yeast extract 5g/L, lemon
Lemon acid ammonium 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.Institute
State the degreasing milk medium that degreasing milk medium is this field routine, preferably 10% degreasing milk medium.By skimmed milk powder
It is dissolved in the water and mixes up to the degreasing milk medium, the percentage accounts for the skimmed milk powder and institute for the skimmed milk powder
State the mass percent of water gross mass.The M17 agar culture mediums are the M17 agar culture mediums of this field routine, preferably
Ground comprising following constituent：4.5~5.5g/L of phytone, 4~5g/L of yeast extract, polyprotein peptone 4~
18~20g/L of 5g/L, β-phosphoglycerol disodium, 0.4~0.5g/L of ascorbic acid, 2.2~2.8g/L of beef extract, seven hydration sulphur
Sour 1~1.5g/L of magnesium and 2.5~3g/L of agar powder.More preferably comprising following constituent：Phytone 5g/L, ferment
Female 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 and agar powder 2.5g/L.
The temperature of the culture is the temperature of this field routine, can grow the Lactococcus lactis subsp. lactis
CGMCC No.10749, preferably 25~35 DEG C are more preferably 28~30 DEG C.The time of the culture is that this field is normal
The time of rule, preferably 24~72 hours are more preferably 24~48 hours.The pH of the culture is the pH of this field routine,
It can grow the described Lactococcus lactis subsp. lactis CGMCC No.10749, 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 Lactococcus lactis subsp. lactis CGMCC No.10749 are to freeze state, the seed culture packet
The step of including heating.The temperature of the heating is the temperature of this field routine, and preferably 10~34 DEG C, be more preferably 20~28
℃。
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 Lactococcus lactis subsp. lactis CGMCC No.10749 are dry
Application in junket production.
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 Lactococcus lactis subsp. lactis CGMCC
No.10749, growth performance is good in breast, and phage resistance is good, and acid production speed is moderate, good for producing cheesy flavor,
Yield is high.It can be used for preparing the leavening of cheese, and applied 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 BD164, and Classification And Nomenclature is Lactococcus lactis breast
Sour subspecies Lactococcus lactis 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 BD401, and Classification And Nomenclature is Lactococcus lactis breast
Sour subspecies Lactococcus lactis 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 BD2263, and Classification And Nomenclature is Lactococcus lactis breast
Sour subspecies Lactococcus lactis 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 Leuconostoc mesenteroides, 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 yogurt coccus lactic acid subspecies CGMCC No.10749 of embodiment
(1), it activates：The milk sample product for being derived from Tibet are taken out from cryopreservation tube, with the inoculum concentration of 2% (v/v) be inoculated in through
121 DEG C are crossed, (M17 broth bouillons are the M17 Agar culture without containing agar in the M17 broth bouillons of sterilizing in 15 minutes
Base is purchased from OXOID companies of Britain) 30 DEG C of cultures 36 hours, it is continuous to cultivate for 3 generations, obtain the strain of activation.
(2), cheese analogue maturation environmental selection：By the strain of the activation obtained by step (1) with the inoculum concentration of 2% (v/v)
It is inoculated in sodium chloride content 4%, (skimmed milk powder is purchased from New Zealand Westland cooperation dairy industries to 10% degreasing milk medium of pH5
Skimmed milk powder is dissolved in the water and mixes up to degreasing milk medium by Co., Ltd, and the percentage is that the skimmed milk powder accounts for
The mass percent of the skimmed milk powder and the water gross mass) in, 30 DEG C of cultures obtain culture solution in 16 hours.By culture solution according to
Total plate count assay method described in GB4789.2-2010 carries out plate count.It measures total viable count and reaches >=106cfu/mL。
If control group：Well-grown strain obtained by step (1) is inoculated in the inoculum concentration of 2% (v/v) and is not added with
Sodium chloride is not adjusted in 10% degreasing milk medium of pH, and 30 DEG C are cultivated 16 hours, and culture solution is obtained.By culture solution according to
After step method processing described in GB4789.2-2010,37 DEG C are cultivated 48 hours.
It after 48 hours, is measured by colony counting method, total viable count reaches >=107cfu/mL。
(3), temperature is pressed to select：
B) well-grown bacterial strain obtained by step (2) is inoculated in the inoculum concentration of 2% (v/v) be not added with sodium chloride,
In the triangular flask of 10% degreasing milk medium for not adjusting pH, 15 DEG C are cultivated 16 hours, and culture solution b is obtained；
C) well-grown bacterial strain obtained by step (2) is inoculated in the inoculum concentration of 2% (v/v) be not added with sodium chloride,
In the triangular flask of 10% degreasing milk medium for not adjusting pH, 30 DEG C are cultivated 16 hours, and culture solution c is obtained；
D) well-grown bacterial strain obtained by step (2) is inoculated in the inoculum concentration of 2% (v/v) be not added with sodium chloride,
In the triangular flask of 10% degreasing milk medium for not adjusting pH, after 30 DEG C of cultures 14 hours, in 40 DEG C of water-baths 2 hours, it must cultivate
Liquid d.
Culture solution b, culture solution c and culture solution d are carried out according to the total plate count assay method described in GB4789.2-2010
Plate count.
Total viable count reaches >=10 during selection meets b)5Cfu/mL, c) in total viable count reach >=107Cfu/mL and
106Cfu/mL >=d) in total viable count reach >=104The bacterial strain of cfu/mL conditions, is named as BD2263.
By Lactococcus lactis subsp. lactis BD2263 on April 27th, 2015 in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center (CGMCC) preservation, obtaining deposit number is：CGMCC No.10749, culture title are BD2263,
Classification And Nomenclature is Lactococcus lactis subsp. lactis Lactococcus lactis subsp.lactis.
Feature is learned in the morphology of 2 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment and culture
Cultural characteristic：Using 30 DEG C of culture Lactococcus lactis subsp. lactis CGMCC No.107492 of M17 agar culture mediums
After it, cultural characteristic is observed.
Cultural characteristics of the 1 Lactococcus lactis subsp. lactis CGMCC No.10749 of table on culture medium
| Culture medium | Bacterium colony quality | Bacterium colony surface color | Bacterium colony back side color |
| M17 agar | Circular protrusions, it is glossy | Milky | Milky band yellow slightly |
The result of table 1 illustrates that Lactococcus lactis subsp. lactis CGMCC No.10749 are grown on M17 agar culture mediums
Well, after cultivating 24 hours, bacterium colony is creamy white, and bacterium colony is smaller, and circular protrusions are glossy, thalline oval.It is rapid later raw
Long, bacterium colony is big and close after 2 days, and be creamy white yellowish.
The physiological and biochemical property of 3 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
A), product acid activity
Lactococcus lactis subsp. lactis CGMCC No.10749 are inoculated in by the inoculum concentration of 1% (v/v) equipped with sterilizing
In the triangular flask of degreasing milk medium, 30 DEG C of constant incubator cultures are placed in, a triangular flask are taken out within every 3 hours, not to be inoculated with
The degreasing milk medium of Lactococcus lactis subsp. lactis CGMCC No.10749 as a contrast, surveys pH value, the results are shown in Table 2.
The rate of producing acid of 2 Lactococcus lactis subsp. lactis CGMCC No.10749 of table
| 0 hour | 3 hours | 6 hours | 9 hours | 12 hours | 15 hours | 18 hours | 21 hours | 24 hours | |
| pH | 6.33 | 6.30 | 5.69 | 4.78 | 4.53 | 4.5 | 4.42 | 4.36 | 4.30 |
As shown in Table 2, Lactococcus lactis subsp. lactis CGMCC No.10749 acid production speeds are very fast, and after 24 hours, pH is only
It is 4.30, the sour power of production is stronger, can be used for the pre-acidified speed of raw material milk, and the formation of acid has conducive to the function of renin is strengthened
The dissolving of calcium, is convenient for the formation of ziega in effect and breast.
B), the curdled milk time in skimmed milk and curdled milk viscosity measurement
Lactococcus lactis subsp. lactis CGMCC No.10749 are inoculated in by the inoculum concentration of 1% (v/v) equipped with sterilizing
In the triangular flask of degreasing milk medium, 30 DEG C of constant incubator cultures are placed in, are kept for 24 hours up under its curdled milk, 4 DEG C of environment.
Measure the viscosity (mPa/s) of each fermentation yogurt after curdled milk respectively with rotational viscometer (being purchased from proRheo companies of Germany).It measures
No. 2 rotors of Shi Xuanyong, speed 64r/s.Primary per 15s values when measurement, minute 3min, the results are shown in Table 3.By
For watch 3 it is found that Lactococcus lactis subsp. lactis CGMCC No.10749 curdled milk speed is fast, curdled milk viscosity is higher.
The curdled milk time of 3 Lactococcus lactis subsp. lactis CGMCC No.10749 of table and curdled milk viscosity
| Bacterial strain | The curdled milk time/h | Viscosity mPa/s |
| Lactococcus lactis subsp. lactis CGMCC No.10749 | 10 | 213 |
C), fermentation character is tested
Lactococcus lactis subsp. lactis CGMCC No.10749 are inoculated in by the inoculum concentration of 1% (v/v) equipped with sterilizing
In the triangular flask of degreasing milk medium, 30 DEG C of constant incubator cultures are placed in, until its curdled milk, investigates its curd taste, whey
The quality of precipitation situation and grumeleuse, the results are shown in Table 4.
The fermentation character of 4 Lactococcus lactis subsp. lactis CGMCC No.10749 of table
| Bacterial strain | Curdled milk situation | It judges |
| Lactococcus lactis subsp. lactis CGMCC No.10749 | Whey is precipitated very much, and structural state is partially hard | Fragrance is very dense |
D), 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：Lactococcus lactis subsp. lactis CGMCC No.10749 are pressed
The inoculum concentration of 1% (v/v) is inoculated in the triangular flask of the degreasing milk medium equipped with sterilizing, is placed in 30 DEG C of constant incubator cultures
Suspension after 24 hours.As a result, it has been found that the OD of Lactococcus lactis subsp. lactis CGMCC No.10749660It is 0.93.Illustrate breast
Yogurt coccus lactic acid subspecies CGMCC No.10749 proteolytic activities are stronger, and in Cheese during Ripening, leavening bacterial strain
It can be polypeptide, amino acid or other inorganic, small molecule organic compounds by breaks down proteins, the formation of cheesy flavor is risen emphatically
It acts on.
E), phage resistance
(cheese does not produce acid with bacterium or production is sour extremely slow and curdled milk exception is exception of fermenting for the exception that will ferment.) cheese
Use raw milk (being obtained from bright milk industry research institute pilot plant) as sample, by Lactococcus lactis subsp. lactis CGMCC
No.10749 is inoculated in by the inoculum concentration of 2% (v/v) in the triangular flask equipped with sample, is placed in 30 DEG C of constant incubator fermented and cultureds
12 hours.The pH value of the zymotic fluid of fermented and cultured is measured, Δ pH >=0.5 is found, illustrates Lactococcus lactis subsp. lactis CGMCC
No.10749 has phage resistance.
Illustrate that the Lactococcus lactis subsp. lactis CGMCC No.10749 have below by the data of embodiment 2~3
Microbiological Characteristics：
1, morphologic characteristic
After M17 agar medium cultures 24 hours, bacterium colony is creamy white, and bacterium colony is smaller, and circular protrusions are glossy, bacterium
Body oval.It mushrooms out later, bacterium colony is big and close after 2 days, and be creamy white yellowish.
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
Acid producing ability is strong, and acid production speed is very fast；Curdled milk speed, curdled milk viscosity are high；In terms of fermenting characteristic, breast can be precipitated
Clearly, curdled milk structural state is good, gives off a strong fragrance；Proteolytic activity is stronger, can by breaks down proteins be polypeptide, amino acid or its
Its inorganic, small molecule organic compound；And there is phage resistance.
The 16S rDNA sequence analyses of 4 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
16S rDNA sequence analyses：Utilize 27F, 1492R primer amplification Lactococcus lactis subsp. lactis CGMCC
The 16S rDNA segments of No.10749 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 Lactococcus lactis subsp. lactis CGMCC No.10749 and Lactococcus lactis
The 16S rDNA sequences of subspecies type strain Lactococcus lactis subsp.lactis Il1403 strain IL1403
Homology be 99%.Due to when 16S rDNA sequence homologies are higher than 97%, it is believed that be of the same race, therefore yogurt in belonging to
Coccus CGMCC No.10749 belong to Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis).
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 Lactococcus lactis subsp. lactis CGMCC No.10749 is as shown in SEQ ID NO.1.
The fermentation of 5 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
The Lactococcus lactis subsp. lactis CGMCC No.10749 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 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
The Lactococcus lactis subsp. lactis CGMCC No.10749 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 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
The Lactococcus lactis subsp. lactis CGMCC No.10749 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 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
The Lactococcus lactis subsp. lactis CGMCC No.10749 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 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
The Lactococcus lactis subsp. lactis CGMCC No.10749 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 and agar powder 2.5g/L.
Cultivation temperature is 35 DEG C, and for 24 hours, culture pH is 4 to cultivation cycle.
The fermentation of 10 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
The Lactococcus lactis subsp. lactis CGMCC No.10749 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 and agar powder 3g/L.
Cultivation temperature is 30 DEG C, cultivation cycle 48h, and culture pH is 4.
The fermentation of 11 Lactococcus lactis subsp. lactis CGMCC No.10749 of embodiment
The Lactococcus lactis subsp. lactis CGMCC No.10749 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 and agar powder 2.5g/L.
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 5.Each assessment officer independently evaluates, and clear water is used when replacing sample every time
It gargles, the results are shown in Table 6.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.
5 cheese subjective appreciation method of table
6 cheese results of sensory evaluation of table
The result of table 6 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 distinctive in cheese
Scoring and total score in terms of cheese feature taste smell and bitter taste are all higher than control group.Therefore, the cheese of 2 gained of effect example
Grow with good quality and preferably 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 Lactococcus lactis subsp. lactis（Lactococcus lactis subsp. lactis）, which is characterized in that its
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is：CGMCC No.10749.
2. a kind of method preparing Lactococcus lactis subsp. lactis CGMCC No.10749, which is characterized in that it includes below
Step cultivates Lactococcus lactis subsp. lactis CGMCC No.10749 in the medium.
3. method as claimed in claim 2, which is characterized in that the culture medium is MRS culture mediums, M17 agar culture mediums
Or degreasing milk medium；The MRS culture mediums include following constituent：8 ~ 12g/L of peptone, 9 ~ 12g/L of beef extract,
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, sodium acetate 4.8 ~
5.2g/L, 0.5 ~ 0.6g/L of magnesium sulfate, 0.5 ~ 0.55g/L of manganese sulfate and Tween 80 ~ 85g/L；The degreasing milk medium is 10%
Degreasing milk medium, the percentage are mass percent；And/or the M17 agar culture mediums include following composition
Ingredient：4.5 ~ 5.5g/L of phytone, 4 ~ 5g/L of yeast extract, 4 ~ 5g/L of polyprotein peptone, β-phosphoglycerol disodium 18 ~ 20
G/L, 0.4 ~ 0.5g/L of ascorbic acid, 2.5 ~ 3g/ of 2.2 ~ 2.8g/L of beef extract, 1 ~ 1.5g/L of bitter salt and agar powder
L。
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 mediums include as follows
Constituent：Phytone 5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-phosphoglycerol disodium 19g/L, resist
Bad hematic acid 0.5g/L, beef extract 2.5g/L, bitter salt 1.2g/L and agar powder 2.5g/L.
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 the culture further includes carrying out seed using seed culture medium
The step of 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 Lactococcus lactis subsp. lactis CGMCC No.10749 as described in claim 1 in cheesemaking.
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| CN106434441B (en) * | 2016-09-21 | 2019-07-26 | 光明乳业股份有限公司 | A kind of lactococcus lactis subsp and its application in cheesemaking |
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