CN104946566A

CN104946566A - Lactococcus lactis subsp.lactis strain and application thereof in cheese production

Info

Publication number: CN104946566A
Application number: CN201510378583.6A
Authority: CN
Inventors: 莫蓓红; 黄宜; 刘振民; 孙颜君; 吴正钧; 石春权
Original assignee: Shanghai Bright Dairy and Food Co Ltd
Current assignee: Shanghai Bright Dairy and Food Co Ltd; Bright Dairy and Food Co Ltd
Priority date: 2015-07-01
Filing date: 2015-07-01
Publication date: 2015-09-30
Anticipated expiration: 2035-07-01
Also published as: CN104946566B

Abstract

The invention discloses a Lactococcus lactis subsp.lactis strain and a preparation method thereof, and application of the strain in cheese production. The Lactococcus lactis subsp.lactis is collected by China General Microbiological Culture Collection Center, and the collection number is CGMCC No.10749. The Lactococcus lactis subsp.lactis strain has the advantages of favorable growth performance in milk, favorable bacteriophage resistance and moderate acid production speed. The cheese produced from the Lactococcus lactis subsp.lactis strain has favorable flavor and high yield. The Lactococcus lactis subsp.lactis strain can be used for preparing a cheese ferment, and can also used in processing techniques of cheese or other fermented foods.

Description

A kind of Lactococcus lactis subsp.lactis and its application in cheesemaking

Technical field

The present invention relates to microorganism field, be specifically related to a kind of Lactococcus lactis subsp.lactis and its application in cheesemaking.

Background technology

Cheese, has another name called cheese, has taste miscellaneous, mouthfeel and form.Cheese take milk as raw material, containing rich in protein and lipid, has very high nutritive value.Along with our people's growth in the living standard, the demand of cheese is got more and more.In cheesemaking and ripening process, microorganism species plays an important role, and facilitates the matter structure of product and the formation of local flavor.The formation of Primary Fermentation agent to cheese matter structure and typical flavor of cheese plays extremely important effect.Acid is produced in Primary Fermentation agent in cheese manufacturing process, can improve the activity of rennin, contributes to getting rid of whey, suppressing harmful bacterial growth, and produces the formation that various enzyme participates in cheese typical flavor and matter structure.

The need of production of most of cheese adds different types of milk-acid bacteria (Lactic acidbacteria before curdled milk, LAB), their Main Function produces acid in cheese making process, in cheese manufacturing process, a key point is lactobacillary milk carbohydrate metabolism is lactic acid, and the speed of its acidifying and degree affect the effect of curdled milk and cheese yield and affect the activity of rennin.LAB also can produce the flavour substancess such as acetic acid, acetaldehyde and di-acetyl sometimes.Starter generally can be divided into two classes, addicted to warm nature starter (namely the suitableeest culture temperature is 30 DEG C) and thermophilic starter (namely the suitableeest culture temperature is 42 DEG C).Addicted to usually comprising Lactococcus lactis subsp.lactis (Lactococcus lactis) and leukonid (Leuconostoc spp.) etc. in warm nature starter, thermophilic starter generally includes thermophilus streptococcus (Streptococcus thermophilus) and lactobacillus bulgaricus (Lactobacillus bulgaricus) etc.The most of starter used now is directly again cultivated by curdled milk and obtains, and these curdled milks itself are through in the production practice of long-term extensive high-quality cheese select, usually containing 2 ~ 6 strain bacterium, so to a certain extent, current most starter can be called as mixed culture fermentation agent.Domestic research in starter mainly concentrates on the thermophilic starter being applicable to Yoghourt.

At present, domestic little to the starter research addicted to Wen Xin, mainly hanker after the screening of ferment agent for sour milk (mainly thermophilic milk-acid bacteria) and the research of preparation, and less to the screening study addicted to warm nature milk-acid bacteria that can be used for cheesemaking, the screening criteria aspect for cheese thalline system is blank especially.Therefore, the abundant microorganism resource of China is utilized to develop the fermentation strain being applicable to brand-new cheese starter significant.

Summary of the invention

Technical problem to be solved by this invention is, for lack at present be used for cheesemaking, the deficiency of Lactococcus lactis subsp.lactis that is mesophilic, that obtain from the domestic microorganism resource of China, a kind of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) and its application in cheesemaking are provided.Described Lactococcus lactis subsp.lactis may be used for for the preparation of cheesemaking addicted to warm type lactobacillus starter.

One of technical scheme of the present invention is: a kind of Lactococcus lactis subsp.lactis (Lactococcus lactissubsp.lactis), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is: CGMCC No.10749.

Be isolated from the milk pimple sample in China Tibet, the Lactococcus lactis subsp.lactis CGMCC No.10749 described in acquisition.Described Lactococcus lactis subsp.lactis CGMCC No.10749 is at Ruzhong well-grown, and acid production speed is moderate, and good for the production of cheesy flavour, yield is high.Identify this bacterial strain, result is Lactococcus lactis subsp.lactis Lactococcus lactis subsp.lactis, called after BD2263.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 27th, 2015, and receives preservation center and to register on the books numbering CGMCC No.10749, and it has following Microbiological Characteristics:

1, morphologic characteristic

In M17 agar culture medium culturing after 24 hours, bacterium colony is creamy white, and bacterium colony is less, and circular protrusions is glossy, and thalline is oval.Ramp afterwards, after 2 days, greatly and closely, be creamy white bacterium colony yellowish.

2, the characteristic learned is cultivated

Well-grown under acid (pH5.0), high salt concentration (4%NaCl, described per-cent is mass percent) environment; Well-grown at 15 ~ 30 DEG C; At 40 DEG C, under the cheese cutting heating process condition of 2 hours, viability is good.

3, physiological property

Acid producing ability is strong, and acid production speed is very fast; Curdled milk speed is fast, curdled milk moderate viscosity; Fermenting characteristic aspect, can separate out whey, and curdled milk structural state is good, give off a strong fragrance; Proteolytic activity is comparatively strong, can be polypeptide, amino acid or other inorganic, small molecule organic compound by breaks down proteins; And there is phage resistance.

Two of technical scheme of the present invention is: a kind of method preparing described Lactococcus lactis subsp.lactis CGMCC No.10749, and it comprises the following steps, cultivates described Lactococcus lactis subsp.lactis CGMCC No.10749 in the medium.

Wherein, described substratum is the substratum of this area routine, can grow described Lactococcus lactis subsp.lactis CGMCC No.10749, is preferably MRS substratum, M17 agar substratum or degreasing milk medium.Described MRS substratum is the MRS substratum of this area routine, preferably, it comprises following moiety: peptone 8 ~ 12g/L, extractum carnis 9 ~ 12g/L, glucose 15 ~ 20g/L, yeast extract paste 4 ~ 6g/L, ammonium citrate 1.8 ~ 2g/L, dipotassium hydrogen phosphate 1.8 ~ 2.0g/L, sodium acetate 4.8 ~ 5.2g/L, magnesium sulfate 0.5 ~ 0.6g/L, manganous sulfate 0.5 ~ 0.55g/L and tween 80 ~ 85g/L.More preferably, it comprises following moiety: peptone 10g/L, extractum carnis 10g/L, glucose 20g/L, yeast extract paste 5g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.5g/L, manganous sulfate 0.5g/L and tween 80 g/L.Described degreasing milk medium is the degreasing milk medium of this area routine, is preferably 10% degreasing milk medium.Be dissolved in the water by skimmed milk powder and mix and obtain described degreasing milk medium, described per-cent is the mass percent that described skimmed milk powder accounts for described skimmed milk powder and described water total mass.Described M17 agar substratum is the M17 agar substratum of this area routine, preferably, it comprises following moiety: phytone 4.5 ~ 5.5g/L, yeast extract 4 ~ 5g/L, polyprotein peptone 4 ~ 5g/L, β-Phosphoric acid glycerol esters disodium 18 ~ 20g/L, xitix 0.4 ~ 0.5g/L, beef extract 2.2 ~ 2.8g/L, bitter salt 1 ~ 1.5g/L and agar powder 2.5 ~ 3g/L.More preferably, it comprises following moiety: phytone 5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-Phosphoric acid glycerol esters disodium 19g/L, xitix 0.5g/L, beef extract 2.5g/L, bitter salt 1.2g/L and agar powder 2.5g/L.

The temperature of described cultivation is the temperature of this area routine, and can grow described Lactococcus lactis subsp.lactis CGMCC No.10749, be preferably 25 ~ 35 DEG C, be more preferably 28 ~ 30 DEG C.The time of described cultivation is the time of this area routine, is preferably 24 ~ 72 hours, is more preferably 24 ~ 48 hours.The pH of described cultivation is the pH of this area routine, can grow described Lactococcus lactis subsp.lactis CGMCC No.10749, is preferably 4 ~ 6, is more preferably 4 ~ 5.

Preferably, the step using seed culture medium to carry out seed culture is also comprised before described cultivation.Described seed culture is the seed culture of this area routine.Described seed culture medium is the seed culture medium of this area routine, is preferably degreasing milk medium, is more preferably 10 ~ 12% degreasing milk mediums, and described per-cent is mass percent.Described skimming milk cultivates bacterium through 115 DEG C, 15 ~ 20min sterilizing.The time of described seed culture is the time of this area routine, is preferably 16 ~ 32 hours, is more preferably 16 ~ 24 hours.The temperature of described seed culture is the temperature of this area routine, is preferably 28 ~ 32 DEG C.The inoculum size of described seed culture is the inoculum size of this area routine, and be preferably 1 ~ 2%, described per-cent is volume percent.The algebraically of the activation of described seed culture is the algebraically of this area routine, is preferably 2 ~ 4 generations, is more preferably 2 ~ 3 generations.Preferably, described Lactococcus lactis subsp.lactis CGMCCNo.10749 is frozen state, and described seed culture comprises the step of intensification.The temperature of described intensification is the temperature of this area routine, is preferably 10 ~ 34 DEG C, is more preferably 20 ~ 28 DEG C.

The method of described cultivation is the method for the cultivation of this area routine, is preferably shake-flask culture or fermentor cultivation.

Three of technical scheme provided by the invention is: the described application of Lactococcus lactis subsp.lactis CGMCCNo.10749 in cheesemaking.

Described cheese is the cheese of this area routine, is preferably Gouda cheese, cheddar cheese-and Pa Masen cheese.It is more preferably Gouda cheese.

Obtained cheese quality is tight, and elasticity is moderate, the micro-Huang of color and luster and evenly, have ripened cheese characteristic chicken flavor and flavour, the cheese yield prepared with this bacterial classification is high, can deposit 10 ~ 24 months no whey and separate out or become sour under non-refrigerated conditions.

On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.

Agents useful for same of the present invention and raw material are all commercially.

Positive progressive effect of the present invention is: the invention provides a kind of Lactococcus lactis subsp.lactis CGMCC No.10749, and it is good at Ruzhong growth performance, and phage resistance is good, and acid production speed is moderate, and good for the production of cheesy flavour, yield is high.Can be used for the starter preparing cheese, and be applied in cheese or other leavened food complete processing.

biomaterial preservation information

Lactococcus lactis subsp.lactis BD164 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on April 27th, 2015, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, deposit number is: CGMCC No.10751, culture title is BD164, and Classification And Nomenclature is Lactococcus lactis subsp.lactis Lactococcus lactissubsp.lactis.

Lactococcus lactis subsp.lactis BD401 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on April 27th, 2015, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, deposit number is: CGMCC No.10752, culture title is BD401, and Classification And Nomenclature is Lactococcus lactis subsp.lactis Lactococcus lactissubsp.lactis.

Lactococcus lactis subsp.lactis BD2263 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) April 27 in 2015, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, deposit number is: CGMCC No.10749, culture title is BD2263, and Classification And Nomenclature is Lactococcus lactis subsp.lactis Lactococcus lactissubsp.lactis.

Leuconostoc mesenteroides LM79 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on April 27th, 2015, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, deposit number is: CGMCC No.10750, culture title is Leuconostoc mesenteroides, and Classification And Nomenclature is Leuconostocmesenteroides.

Embodiment

Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.

The acquisition of embodiment 1 yogurt coccus lactic acid subspecies CGMCC No.10749

(1), activate: the milk sample product taking from Tibet are taken out from cryopreservation tube, to be inoculated in the inoculum size of 2% (v/v) in the M17 broth culture that have passed through sterilizing in 121 DEG C, 15 minutes that (M17 broth culture is the M17 Agar substratum not containing agar, purchased from OXOID company of Britain) 30 DEG C cultivate 36 hours, in cultured continuously 3 generation, obtain the bacterial classification of activation.

(2), the ripe environmental selection of cheese analogue: the bacterial classification of the activation of step (1) gained is inoculated in sodium chloride content 4% with the inoculum size of 2% (v/v), (skimmed milk powder is purchased from New Zealand Westland cooperation dairy industry company limited for 10% degreasing milk medium of pH5, skimmed milk powder is dissolved in the water and mixes and obtain degreasing milk medium, described per-cent is the mass percent that described skimmed milk powder accounts for described skimmed milk powder and described water total mass) in, cultivate 16 hours to obtain nutrient solution for 30 DEG C.Nutrient solution is carried out plate count according to the total number of bacterial colony measuring method described in GB4789.2-2010.Record total viable count to reach>=10 ⁶cfu/mL.

If control group: well-grown bacterial classification of step (1) gained is inoculated in the inoculum size of 2% (v/v) and does not add sodium-chlor, do not regulate in 10% degreasing milk medium of pH, cultivate 16 hours, obtain nutrient solution for 30 DEG C.By nutrient solution according to after the step method process described in GB4789.2-2010, cultivate 48 hours for 37 DEG C.

After 48 hours, being measured by colony counting method, total viable count reaches>=and 10 ⁷cfu/mL.

(3), by thermal creep stress:

B) well-grown bacterial strain of step (2) gained is inoculated in the inoculum size of 2% (v/v) does not add sodium-chlor, do not regulate in the triangular flask of 10% degreasing milk medium of pH, cultivate 16 hours, obtain nutrient solution b for 15 DEG C;

C) well-grown bacterial strain of step (2) gained is inoculated in the inoculum size of 2% (v/v) does not add sodium-chlor, do not regulate in the triangular flask of 10% degreasing milk medium of pH, cultivate 16 hours, obtain nutrient solution c for 30 DEG C;

D) well-grown bacterial strain of step (2) gained is inoculated in the inoculum size of 2% (v/v) does not add sodium-chlor, do not regulate in the triangular flask of 10% degreasing milk medium of pH, cultivate after 14 hours for 30 DEG C, in 40 DEG C of water-baths 2 hours, obtain nutrient solution d.

Nutrient solution b, nutrient solution c and nutrient solution d are carried out plate count according to the total number of bacterial colony measuring method described in GB4789.2-2010.

Selection meet b) in total viable count reach>=10 ⁵cfu/mL, c) in total viable count reach>=10 ⁷cfu/mL and 10 ⁶cfu/mL>=d) in total viable count reach>=10 ⁴the bacterial strain of cfu/mL condition, called after BD2263.

By Lactococcus lactis subsp.lactis BD2263 on April 27th, 2015 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, acquisition deposit number is: CGMCCNo.10749, culture title is BD2263, and Classification And Nomenclature is Lactococcus lactis subsp.lactis Lactococcus lactis subsp.lactis.

Feature is learned in morphology and the cultivation of embodiment 2 Lactococcus lactis subsp.lactis CGMCC No.10749

Cultural characteristic: adopt M17 agar substratum 30 DEG C to cultivate Lactococcus lactis subsp.lactis after CGMCCNo.107492 days, observe cultural characteristic.

The cultural characteristic of table 1 Lactococcus lactis subsp.lactis CGMCC No.10749 on substratum

Substratum	Bacterium colony quality	Bacterium colony surface color	Bacterium colony back side color
				M17 agar	Circular protrusions, glossy	Oyster white	Oyster white is with yellow slightly

The result of table 1 illustrates, Lactococcus lactis subsp.lactis CGMCC No.10749 is well-grown on M17 agar substratum, and cultivate after 24 hours, bacterium colony is creamy white, and bacterium colony is less, and circular protrusions is glossy, thalline oval.Ramp afterwards, after 2 days, greatly and closely, be creamy white bacterium colony yellowish.

The physiological and biochemical property of embodiment 3 Lactococcus lactis subsp.lactis CGMCC No.10749

A), product acid activity

Lactococcus lactis subsp.lactis CGMCC No.10749 is inoculated in the triangular flask of the degreasing milk medium that sterilizing is housed by the inoculum size of 1% (v/v), be placed in 30 DEG C of constant incubators to cultivate, within every 3 hours, take out a triangular flask, with the degreasing milk medium of non-inoculating lactic acid galactococcus lactic acid subspecies CGMCC No.10749 in contrast, survey pH value, result is as shown in table 2.

The rate of producing acid of table 2 Lactococcus lactis subsp.lactis CGMCC No.10749

	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 speed is very fast, after 24 hours, pH is only 4.30, produce sour power stronger, may be used for the pre-acidified speed of raw material milk, and being formed with of acid is beneficial to the strengthening function of rennin and the dissolving of Ruzhong calcium, is convenient to the formation of curd pieces.

B) the curdled milk time, in skimming milk and curdled milk viscosity measurement

Lactococcus lactis subsp.lactis CGMCC No.10749 is inoculated in the triangular flask of the degreasing milk medium that sterilizing is housed by the inoculum size of 1% (v/v), is placed in 30 DEG C of constant incubators and cultivates, until its curdled milk, keep 24 hours under 4 DEG C of environment.Measure the viscosity (mPa/s) of each fermentation yogurt respectively with rotational viscosimeter (purchased from German proRheo company) after curdled milk.Select No. 2 rotors during mensuration, speed is 64r/s.During mensuration, once, minute is 3min to every 15s value, and result is as shown in table 3.As shown in Table 3, Lactococcus lactis subsp.lactis CGMCC No.10749 curdled milk speed is fast, and curdled milk viscosity is higher.

The curdled milk time of table 3 Lactococcus lactis subsp.lactis CGMCC No.10749 and curdled milk viscosity

Bacterial strain	Curdled milk time/h	Viscosity mPa/s
			Lactococcus lactis subsp.lactis CGMCC No.10749	10	213

C), fermentation character test

Lactococcus lactis subsp.lactis CGMCC No.10749 is inoculated in the triangular flask of the degreasing milk medium that sterilizing is housed by the inoculum size of 1% (v/v), be placed in 30 DEG C of constant incubators to cultivate, until its curdled milk, investigate the quality of its curd taste, whey precipitation situation and grumeleuse, result is as shown in table 4.

The fermentation character of table 4 Lactococcus lactis subsp.lactis CGMCC No.10749

Bacterial strain	Curdled milk situation	Judge
			Lactococcus lactis subsp.lactis CGMCC No.10749	Whey is separated out a lot, and structural state is partially hard	Fragrance is very dense

D), proteolytic activity test

Forint (Folin)-phenol reagent process measures the proteolytic activity of bacterial strain: get 1mL sample solution, add 2.5mL 0.28M NaOH solution, mixing, places 10min in about 25 DEG C.Add 0.75mL forint phenol reagent (purchased from traditional Chinese medicines reagent) again, shake up immediately, in about 35 DEG C reaction 15min, under 660nm, use ultraviolet spectrophotometer (purchased from Shanghai Techcomp Instrument Ltd.) to measure its light absorption value.Described sample solution is: be inoculated in the triangular flask of the degreasing milk medium that sterilizing is housed by the inoculum size of 1% (v/v) by Lactococcus lactis subsp.lactis CGMCC No.10749, is placed in 30 DEG C of constant incubators and cultivates the suspension after 24 hours.Found that, the OD of Lactococcus lactis subsp.lactis CGMCC No.10749 ₆₆₀be 0.93.Illustrate that Lactococcus lactis subsp.lactis CGMCC No.10749 proteolytic activity is stronger, and in Cheese during Ripening, breaks down proteins can be polypeptide, amino acid or other inorganic, small molecule organic compound by starter bacterial strain, plays an important role to the formation of cheesy flavour.

E), phage resistance

To ferment abnormal (cheese do not produce acid with bacterium or produce acid very slowly and curdled milk abnormal to be fermentation abnormal.) cheese raw dairy (obtaining from bright milk industry research institute pilot plant) as sample, being inoculated in by the inoculum size of 2% (v/v) by Lactococcus lactis subsp.lactis CGMCC No.10749 is equipped with in the triangular flask of sample, is placed in 30 DEG C of constant incubator fermentation culture 12 hours.Measure the pH value of the fermented liquid of fermentation culture, find Δ pH >=0.5, illustrate that Lactococcus lactis subsp.lactis CGMCC No.10749 has phage resistance.

By the data declaration of embodiment 2 ~ 3, described Lactococcus lactis subsp.lactis CGMCC No.10749 has following Microbiological Characteristics:

1, morphologic characteristic

In M17 agar culture medium culturing after 24 hours, bacterium colony is creamy white, and bacterium colony is less, and circular protrusions is glossy, thalline oval.Ramp afterwards, after 2 days, greatly and closely, be creamy white bacterium colony yellowish.

2, the characteristic learned is cultivated

3, physiological property

Acid producing ability is strong, and acid production speed is very fast; Curdled milk speed, curdled milk viscosity is high; Fermenting characteristic aspect, can separate out whey, and curdled milk structural state is good, give off a strong fragrance; Proteolytic activity is comparatively strong, can be polypeptide, amino acid or other inorganic, small molecule organic compound by breaks down proteins; And there is phage resistance.

The 16S rDNA sequential analysis of embodiment 4 Lactococcus lactis subsp.lactis CGMCC No.10749

16S rDNA sequential analysis: utilize 27F, the 16S rDNA fragment of 1492R primer amplification Lactococcus lactis subsp.lactis CGMCC No.10749, purifying, reclaims, and then uses sequenator ABI3730-XL to carry out DNA sequencing (upper Shanghai's style Sen Nuo bio tech ltd).Found that, the homology of the 16S rDNA sequence of Lactococcus lactis subsp.lactis CGMCC No.10749 and Lactococcus lactis subsp.lactis type strain Lactococcus lactis subsp.lactis Il1403 strain IL1403 is 99%.Due to when 16S rDNA sequence homology higher than 97% time, can think belong in of the same race, therefore yogurt coccus CGMCC No.10749 belongs to Lactococcus lactis subsp.lactis (Lactococcuslactis subsp.lactis).

Wherein, primer pair sequence as above is: 27F:agagtttgat cctggctcag; 1492R:ctacggctac cttgttacga (its nucleotide sequence is respectively as shown in sequence table SEQ ID NO.2 and SEQ IDNO.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 embodiment 5 Lactococcus lactis subsp.lactis CGMCC No.10749

Frozen described Lactococcus lactis subsp.lactis CGMCC No.10749 is slowly warming up to 10 DEG C.

Seed culture medium: (skimmed milk powder is purchased from New Zealand Westland cooperation dairy industry company limited for 10% degreasing milk medium, skimmed milk powder is dissolved in the water and mixes and obtain degreasing milk medium, described per-cent is the mass percent that described skimmed milk powder accounts for described skimmed milk powder and described water total mass), described per-cent is mass percent.This skimming milk cultivates bacterium through 115 DEG C, 15min sterilizing.

The culture temperature 28 DEG C of seed culture medium in seed bottle, culture cycle 32h, inoculum size is 1%, and described per-cent is the volume percent accounting for fermention medium.Activated for 2 generations in seed culture medium.

Fermention medium: peptone 8g/L, extractum carnis 9g/L, glucose 15g/L, yeast extract paste 4g/L, ammonium citrate 1.8g/L, dipotassium hydrogen phosphate 1.8g/L, sodium acetate 4.8g/L, magnesium sulfate 0.5g/L, manganous sulfate 0.5g/L and tween 80 g/L.

Culture temperature is 25 DEG C, culture cycle 72h, and cultivating pH is 4.

The fermentation of embodiment 6 Lactococcus lactis subsp.lactis CGMCC No.10749

Frozen described Lactococcus lactis subsp.lactis CGMCC No.10749 is slowly warming up to 20 DEG C.

Seed culture medium: 12% degreasing milk medium, described per-cent is mass percent.This skimming milk cultivates bacterium through 115 DEG C, 20min sterilizing.

The culture temperature 32 DEG C of seed culture medium in seed bottle, culture cycle 16h, inoculum size is 2%, and described per-cent is the volume percent accounting for fermention medium.Activated for 4 generations in seed culture medium.

Fermention medium: peptone 12g/L, extractum carnis 12g/L, glucose 20g/L, yeast extract paste 6g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 2.0g/L, sodium acetate 5.2g/L, magnesium sulfate 0.6g/L, manganous sulfate 0.55g/L and polysorbate85 g/L.

Culture temperature is 35 DEG C, culture cycle 24h, and cultivating pH is 6.

The fermentation of embodiment 7 Lactococcus lactis subsp.lactis CGMCC No.10749

Frozen described Lactococcus lactis subsp.lactis CGMCC No.10749 is slowly warming up to 28 DEG C.

The culture temperature 32 DEG C of seed culture medium in seed bottle, culture cycle 16h, inoculum size is 2%, and described per-cent is the volume percent accounting for fermention medium.Activated for 3 generations in seed culture medium.

Fermention medium: peptone 10g/L, extractum carnis 10g/L, glucose 20g/L, yeast extract paste 5g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.5g/L, manganous sulfate 0.5g/L and tween 80 g/L.

Culture temperature is 28 DEG C, culture cycle 48h, and cultivating pH is 5.

The fermentation of embodiment 8 Lactococcus lactis subsp.lactis CGMCC No.10749

Frozen described Lactococcus lactis subsp.lactis CGMCC No.10749 is slowly warming up to 34 DEG C.

Seed culture medium: 10% degreasing milk medium, described per-cent is mass percent.This skimming milk cultivates bacterium through 115 DEG C, 15min sterilizing.

Culture temperature is 30 DEG C, culture cycle 48h, and cultivating pH is 5.

The fermentation of embodiment 9 Lactococcus lactis subsp.lactis CGMCC No.10749

The culture temperature 28 DEG C of seed culture medium in seed bottle, culture cycle 32h, inoculum size is 1%, and described per-cent is the volume percent accounting for fermention medium.Activated for 4 generations in seed culture medium.

Fermention medium: phytone 4.5g/L, yeast extract 4g/L, polyprotein peptone 4g/L, β-Phosphoric acid glycerol esters disodium 18g/L, xitix 0.4g/L, beef extract 2.2g/L and bitter salt 1g/L and agar powder 2.5g/L.

Culture temperature is 35 DEG C, culture cycle 24h, and cultivating pH is 4.

The fermentation of embodiment 10 Lactococcus lactis subsp.lactis CGMCC No.10749

Seed culture medium: 12% degreasing milk medium, described per-cent is mass percent.This skimming milk cultivates bacterium through 115 DEG C, 15min sterilizing.

Fermention medium: phytone 5.5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-Phosphoric acid glycerol esters disodium 20g/L, xitix 0.5g/L, beef extract 2.8g/L and bitter salt 1.5g/L and agar powder 3g/L.

Culture temperature is 30 DEG C, culture cycle 48h, and cultivating pH is 4.

The fermentation of embodiment 11 Lactococcus lactis subsp.lactis CGMCC No.10749

Seed culture medium: 10% degreasing milk medium, described per-cent is mass percent.This skimming milk cultivates bacterium through 115 DEG C, 20min sterilizing.

The culture temperature 28 DEG C of seed culture medium in seed bottle, culture cycle 32h, inoculum size is 1%, and described per-cent is the volume percent accounting for fermention medium.Activated for 3 generations in seed culture medium.

Fermention medium phytone 5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-Phosphoric acid glycerol esters disodium 19g/L, xitix 0.5g/L, beef extract 2.5g/L, bitter salt 1.2g/L and agar powder 2.5g/L.

Culture temperature is 28 DEG C, culture cycle 24h, and cultivating pH is 5.

Effect example 1

(1) Lactococcus lactis subsp.lactis BD164, Lactococcus lactis subsp.lactis BD2263, Lactococcus lactis subsp.lactis BD401 and Leuconostoc mesenteroides LM79 are inoculated in the degreasing milk medium (purchased from New Zealand Westland cooperation dairy industry company limited) of 10% (w/v) sterilizing by the inoculum size of 2% (v/v), be placed in 30 DEG C of constant incubators and cultivate 24 hours, activated for 2 generations, obtain the bacterial strain of activation.The bacterial strain of activation is inoculated in the degreasing milk medium of 10% (w/v) sterilizing by the inoculum size of 2% (v/v), is placed in 30 DEG C of constant incubators and cultivates and carry out enlarged culturing in 24 hours, repeat 3 times, acquisition culture.

(2) culture of step (1) gained is obtained Lactococcus lactis subsp.lactis BD164, Lactococcus lactis subsp.lactis BD2263, Lactococcus lactis subsp.lactis BD401 and Leuconostoc mesenteroides LM79, then be the ratio mixing of 1:1:1:1 according to the viable count of described Lactococcus lactis subsp.lactis BD401, described Lactococcus lactis subsp.lactis BD2263, described Lactococcus lactis subsp.lactis BD164 and described Leuconostoc mesenteroides LM79, obtain cheese starter.

Effect example 2 cheese starter produces Gouda cheese

1) 100kg fresh cow milk (purchased from pasture, Kingsoft) after filtration, adds 0.01% calcium chloride, stirs, and at 72 DEG C, is cooled to 30 DEG C after 15s pasteurize, and described per-cent is mass percent.Cheese starter obtained for effect example 1 is poured in the fresh cow milk after pasteurize, makes the viable count of the cheese starter in fresh cow milk reach 10 ⁷cfu/mL, slowly stirs 15min.Leave standstill 32 DEG C of fermentations and add 20mg/100L rennin (purchased from Danisco A/S BJ Rep Office) (wherein after pH reduces by 0.2, rennin dissolves with the sterilized water of 10 times of quality before use, stirring 2min makes it be uniformly dissolved), obtain curdled milk after 30min.

2) by step 1) obtained curd cutting becomes the cubical grumeleuse of length of side 5mm, discharges the whey of cumulative volume 35% after slowly stirring after 15min; Point to add 50 DEG C for 2 times, volume is the water of fresh cow milk cumulative volume 25%, stir 20min and reach 35 DEG C to curd temperature.The whey pH continuing to be stirred to discharge is 6.1, whole whey of draining.Afterwards grumeleuse is entered mould, squeezing is shaped, the demoulding is taken out.Working concentration is salt solution 12 DEG C of salt marsh 20h of 20%, and described per-cent is mass percent.Vacuum packaging afterwards, 10 DEG C, humidity 90%RH, ripe 5 weeks, obtain young Gouda cheese.

The sensory evaluation of effect example 3 cheese

This subjective appreciation personnel comprise the personnel that 12 are engaged in food research, are all familiar with subjective appreciation precaution and standards of grading.Total score is 50 points, and mark evaluation is in accordance with table 5.Each assessment officer independently evaluates, and use clear water to gargle during each replacing sample, result is as shown in table 6.Wherein, control group for use direct-throwing cheese starter CHOOZIT RM 32 (purchased from red Knicks (China) company limited), according to effect example 2 step obtained by cheese.

Table 5 cheese subjective appreciation method

Table 6 cheese results of sensory evaluation

The result of table 6 illustrates, the difference of Gouda cheese (control group) in quality and between the cheese of effect example 2 gained that commercial fermentation agent makes is not remarkable; In the scoring of taste smell, the cheese of effect example 2 gained grows scoring in smell and bitter taste and total score all higher than control group in the distinctive cheese feature of cheese.Therefore, the cheese of effect example 2 gained has good quality and better grows smell.

Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. a Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis), it is characterized in that, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is: CGMCC No.10749.

2. prepare a method of Lactococcus lactis subsp.lactis CGMCC No.10749, it is characterized in that, it comprises the following steps, cultivates Lactococcus lactis subsp.lactis CGMCCNo.10749 in the medium.

3. method as claimed in claim 2, it is characterized in that, described substratum is MRS substratum, M17agar substratum or degreasing milk medium; Described MRS substratum comprises following moiety: peptone 8 ~ 12g/L, extractum carnis 9 ~ 12g/L, glucose 15 ~ 20g/L, yeast extract paste 4 ~ 6g/L, ammonium citrate 1.8 ~ 2g/L, dipotassium hydrogen phosphate 1.8 ~ 2.0g/L, sodium acetate 4.8 ~ 5.2g/L, magnesium sulfate 0.5 ~ 0.6g/L, manganous sulfate 0.5 ~ 0.55g/L and tween 80 ~ 85g/L; Described degreasing milk medium is 10% degreasing milk medium, and described per-cent is mass percent; And/or described M17agar substratum comprises following moiety: phytone 4.5 ~ 5.5g/L, yeast extract 4 ~ 5g/L, polyprotein peptone 4 ~ 5g/L, β-Phosphoric acid glycerol esters disodium 18 ~ 20g/L, xitix 0.4 ~ 0.5g/L, beef extract 2.2 ~ 2.8g/L, bitter salt 1 ~ 1.5g/L and agar powder 2.5 ~ 3g/L.

4. method as claimed in claim 3, it is characterized in that, described MRS substratum comprises following moiety: peptone 10g/L, extractum carnis 10g/L, glucose 20g/L, yeast extract paste 5g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 2g/L, sodium acetate 5g/L, magnesium sulfate 0.5g/L, manganous sulfate 0.5g/L and tween 80 g/L; And/or described M17agar substratum comprises following moiety: phytone 5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-Phosphoric acid glycerol esters disodium 19g/L, xitix 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, it is characterized in that, the temperature of described cultivation is 25 ~ 35 DEG C; The time of described cultivation is 24 ~ 72 hours; And/or the pH of described cultivation is 4 ~ 6.

6. method as claimed in claim 5, it is characterized in that, the temperature of described cultivation is 28 ~ 30 DEG C; The time of described cultivation is 24 ~ 48 hours; And/or the pH of described cultivation is 4 ~ 5.

7. method as claimed in claim 2, is characterized in that, also comprise the step using seed culture medium to carry out seed culture before described cultivation, described seed culture medium is degreasing milk medium; Described skimming milk cultivates bacterium through 115 DEG C, 15 ~ 20min sterilizing; The time of described seed culture is 16 ~ 32 hours; The temperature of described seed culture is 28 ~ 32 DEG C; The inoculum size of described seed culture is 1 ~ 2%, and described per-cent is volume percent; And/or the algebraically of the activation of described seed culture was 2 ~ 4 generations.

8. method as claimed in claim 7, it is characterized in that, described seed culture medium is 10 ~ 12% degreasing milk mediums, and described per-cent is mass percent; The time stating seed culture is 16 ~ 24 hours; And/or the algebraically of the activation of described seed culture was 2 ~ 3 generations.

9. method as claimed in claim 7, it is characterized in that, described seed culture comprises the step of intensification; And/or the temperature of described intensification is 10 ~ 34 DEG C, is preferably 20 ~ 28 DEG C.

10. the application of Lactococcus lactis subsp.lactis CGMCC No.10749 in cheesemaking as claimed in claim 1.