CN104894038B

CN104894038B - A kind of Lactococcus lactis subsp. lactis and its application in cheesemaking

Info

Publication number: CN104894038B
Application number: CN201510379257.7A
Authority: CN
Inventors: 黄宜; 焦晶凯; 刘振民; 莫蓓红; 韩瑨
Original assignee: Bright Dairy and Food Co Ltd
Current assignee: Bright Dairy and Food Co Ltd
Priority date: 2015-07-01
Filing date: 2015-07-01
Publication date: 2018-05-04
Anticipated expiration: 2035-07-01
Also published as: CN104894038A

Abstract

The invention discloses a kind of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis), preparation method and its application 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.10751.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.Available for the leavening for preparing cheese, and applied in cheese or other fermented food processing technologys.

Description

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

Technical field

The present invention relates to microorganism field, and in particular to a kind of Lactococcus lactis subsp. lactis and its in cheesemaking Using.

Background technology

Cheese, also known as cheese, there is miscellaneous taste, mouthfeel and form.Cheese is using milk as raw material, containing abundant Protein and lipid, have very high nutritive value.It is more next to the demand of cheese with our people's improvement of living standard It is more.In cheesemaking and maturation microorganism species play an important role, and promote the texture and flavor of product Formed.Formation of the main fermentation agent of cheese to cheese texture and characteristic flavor on basis has served extremely important.Main fermentation agent is dry Acid is produced in junket production process, it is possible to increase the activity of renin, helps to exclude whey, suppresses harmful bacterial growth, and produces Various enzymes participate in the formation of cheese characteristic flavor on basis and texture.

The production of most of cheese needs to 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 its speed being acidified and degree influence curdled milk simultaneously influence renin Activity.LAB can also produce 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 are select in production practices by long-term extensive high-quality cheese in itself, 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 thermophilic leavening suitable for Yoghourt.

At present, it is domestic that mesophilic new leavening is studied seldom, mainly it is keen to ferment agent for sour milk (mainly thermophilic breast Sour bacterium) screening and preparation research, and the screening study to the mesophilic property lactic acid bacteria available 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.

The content of the invention

The technical problems to be solved by the invention are, for it is current lack it is for cheesemaking, mesophilic, from China A kind of deficiency of the Lactococcus lactis subsp. lactis obtained in domestic microorganism resource, there is provided 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 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, and preserving number is： CGMCC No.10751。

It is isolated from the milk pimple sample in China Tibet, obtains the Lactococcus lactis subsp. lactis CGMCC No.10751.Lactococcus lactis subsp. lactis CGMCC No.10751 well-growns in breast, acid production speed is moderate, uses Good in production cheesy flavor, yield is high.The bacterial strain is identified, is as a result Lactococcus lactis subsp. lactis Lactococcus lactis subsp.lactis, are named as BD164.The bacterial strain is preserved in China on April 27th, 2015 Microbiological Culture Collection administration committee common micro-organisms center, and receive collection and register on the books numbering CGMCC No.10751, it is with following Microbiological Characteristics：

1st, morphologic characteristic

After when M17agar medium cultures 24 are small, bacterium colony is creamy white, and bacterium colony is smaller, and circular protrusions are glossy, bacterium Body is in oval.Mushroom out afterwards, bacterium colony is big and close after 2 days, and be creamy white yellowish.

2nd, 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；At 40 DEG C, 2 it is small when cheese cutting heating process under the conditions of viability it is good.

3rd, 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 separated out Clearly, curdled milk structural state is good, gives off a strong fragrance；Proteolytic activity is stronger, can by breaks down proteins for polypeptide, amino acid or its Its inorganic, small molecule organic compound；And there is phage resistance.

The two of technical scheme are：It is a kind of to prepare the Lactococcus lactis subsp. lactis CGMCC No.10751 Method, it comprises the following steps, cultivates the Lactococcus lactis subsp. lactis CGMCC No.10751 in the medium .

Wherein, the culture medium is the culture medium of this area routine, and it is sub- can to grow the Lactococcus lactis Kind CGMCC No.10751, preferably MRS culture mediums, M17agar culture mediums or degreasing milk medium.The MRS Culture medium is the MRS culture mediums of this area routine, it is preferred that it includes 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, it includes 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 area routine, preferably 10% degreasing milk medium.By skimmed milk powder It is dissolved in the water to mix and accounts for the skimmed milk powder and institute up to the degreasing milk medium, the percentage for the skimmed milk powder State the mass percent of water gross mass.The M17agar culture mediums are the M17agar culture mediums of this area routine, it is preferred that It includes following constituent：4.5~5.5g/L of phytone, 4~5g/L of yeast extract, 4~5g/L of polyprotein peptone, β -18~20g/L of phosphoglycerol disodium, 0.4~0.5g/L of ascorbic acid, 2.2~2.8g/L of beef extract, bitter salt 1 2.5~3g/L of~1.5g/L and agar powder.More preferably, it includes following constituent：Phytone 5g/L, yeast extraction Thing 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 and agar powder 2.5g/L.

The temperature of the culture is the temperature of this area routine, can grow the Lactococcus lactis subsp. lactis CGMCC No.10751, preferably 25~35 DEG C, are more preferably 28~30 DEG C.The time of the culture is normal for this area The time of rule, preferably 24~72 it is small when, more preferably for 24~48 it is small when.The pH of the culture is the pH of this area routine, The Lactococcus lactis subsp. lactis CGMCC No.10751, preferably 4~6 can be grown, is more preferably 4~5.

It is preferred that the step of carrying out seed culture using seed culture medium is further included before the culture.The seed Cultivate as the seed culture of this area routine.The seed culture medium is the seed culture medium of this area 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 area routine, preferably 16~32 it is small when, more preferably for 16~24 it is small when.The temperature of the seed culture is the temperature of this area routine, preferably 28 ~32 DEG C.The inoculum concentration of the seed culture is the inoculum concentration of this area routine, preferably 1~2%, the percentage is body Product percentage.The algebraically of the activation of the seed culture is the algebraically of this area routine, preferably 2~4 generations, is more preferably 2 ~3 generations.It is preferred that the Lactococcus lactis subsp. lactis CGMCC No.10751 are to freeze state, the seed culture bag The step of including heating.The temperature of the heating is the temperature of this area 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 area routine, is preferably Shaking culture or fermentation tank culture.

The three of technical solution provided by the invention are：The Lactococcus lactis subsp. lactis CGMCC No.10751 are dry Application in junket production.

The cheese is the cheese of this area 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 10~24 months no wheys can be stored under non-refrigerated conditions and separates out or becomes sour.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can be combined, each preferably real up to the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is：The present invention provides a kind of Lactococcus lactis subsp. lactis CGMCC No.10751, its growth performance in breast is good, and phage resistance is good, and acid production speed is moderate, good for producing cheesy flavor, Yield is high.Available for the leavening for preparing 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 Compile：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 Compile：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 Compile：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 LM79, and Classification And Nomenclature is Leuconostoc mesenteroides Leuconostoc mesenteroides。

Embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality Apply among a scope.The experimental method of actual conditions is not specified in the following example, according to conventional methods and conditions, or according to business Product specification selects.

The acquisition of 1 Lactococcus lactis subsp. lactis CGMCC No.10751 of embodiment

(1), activate：The milk pimple sample for being derived from Tibet is taken out from cryopreservation tube, is inoculated with the inoculum concentration of 2% (v/v) In have passed through 121 DEG C, (M17 broth bouillons are the M17Agar that does not contain agar in the M17 broth bouillons of sterilizing in 15 minutes Culture medium, purchased from OXOID companies of Britain) 30 DEG C of cultures 36 it is small when, 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 Co., Ltd, skimmed milk powder is dissolved in the water to mix accounts for up to degreasing milk medium, the percentage for the skimmed milk powder The mass percent of the skimmed milk powder and the water gross mass) in, 30 DEG C of cultures 16 obtain nutrient solution when small.By nutrient solution according to Total plate count assay method described in GB4789.2-2010 carries out plate count.Measure total viable count and reach >=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, do not adjusted in 10% degreasing milk medium of pH, when 30 DEG C of cultures 16 are small, obtains nutrient solution.By nutrient solution according to After step method processing described in GB4789.2-2010, when 37 DEG C of cultures 48 are small.

48 it is small when after, measured by colony counting method, total viable count reaches >=10⁷cfu/mL。

(3), selected by temperature：

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, when 15 DEG C of cultures 16 are small, nutrient 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, when 30 DEG C of cultures 16 are small, nutrient 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, 30 DEG C culture 14 it is small when after, when 40 DEG C of water-baths 2 are small, must cultivate Liquid d.

Nutrient solution b, nutrient solution c and nutrient 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 BD164.

By Lactococcus lactis subsp. lactis BD164 on April 27th, 2015 in Chinese microorganism strain preservation conservator Meeting common micro-organisms center (CGMCC) preservation, obtaining deposit number is：CGMCC No.10751, culture title are BD164, 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.10751 of embodiment and culture

Cultural characteristic：Using 30 DEG C of culture Lactococcus lactis subsp. lactis CGMCC No.107512 days of M17agar culture mediums Afterwards, cultural characteristic is observed.

Cultural characteristics of the 1 Lactococcus lactis subsp. lactis CGMCC No.10751 of table on culture medium

Culture medium	Bacterium colony quality	Bacterium colony surface color	Bacterium colony back side color
				M17agar	Circular protrusions, it is glossy	Milky	Milky band yellow slightly

The result explanation of table 1, Lactococcus lactis subsp. lactis CGMCC No.10751 grow good on M17agar culture mediums Well, after when culture 24 is small, bacterium colony is creamy white, and bacterium colony is smaller, circular protrusions, glossy, thalline oval.It is rapid afterwards 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.10751 of embodiment

A), product acid activity

Lactococcus lactis subsp. lactis CGMCC No.10751 are inoculated in equipped with sterilizing by the inoculum concentration of 1% (v/v) In the triangular flask of degreasing milk medium, be placed in 30 DEG C of constant incubator cultures, it is every 3 it is small when take out a triangular flask, not to be inoculated with The degreasing milk medium of Lactococcus lactis subsp. lactis CGMCC No.10751 surveys pH value, the results are shown in Table 2 as control.

The rate of producing acid of 2 Lactococcus lactis subsp. lactis CGMCC No.10751 of table

	0 it is small when	3 it is small when	6 it is small when	9 it is small when	12 it is small when	15 it is small when	18 it is small when	21 it is small when	24 it is small when
										pH	6.35	6.20	5.86	5.54	5.2	5.05	4.92	4.79	4.68

As shown in Table 2, Lactococcus lactis subsp. lactis CGMCC No.10751 acid production speeds are slower, 24 it is small when after, pH is only For 4.68, it is stronger to produce sour power, can be used for the pre-acidified speed of raw material milk, and is formationed of acid has the function beneficial to reinforcing renin The dissolving of calcium in effect and breast, easy to the formation of ziega.

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

Lactococcus lactis subsp. lactis CGMCC No.10751 are inoculated in equipped with sterilizing by the inoculum concentration of 1% (v/v) In the triangular flask of degreasing milk medium, 30 DEG C of constant incubator cultures are placed in, when holding 24 is small 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).Measure No. 2 rotors of Shi Xuanyong, speed 64r/s.During measure per 15s values once, minute 3min, the results are shown in Table 3.By Table 3 understands that Lactococcus lactis subsp. lactis CGMCC No.10751 curdled milk speed is fast, and curdled milk viscosity is higher.

The curdled milk time of 3 Lactococcus lactis subsp. lactis CGMCC No.10751 of table and curdled milk viscosity

Bacterial strain	The curdled milk time/h	Viscosity mPa/s
			Lactococcus lactis subsp. lactis CGMCC No.10751	15	121

C), fermentation character is tested

Lactococcus lactis subsp. lactis CGMCC No.10751 are inoculated in equipped with sterilizing by the inoculum concentration of 1% (v/v) In the triangular flask of 10% degreasing milk medium, be placed in 30 DEG C of constant incubator cultures, until its curdled milk, investigate its curd taste, Whey separates out situation and the quality of grumeleuse, and the results are shown in Table 4.

The fermentation character of 4 Lactococcus lactis subsp. lactis CGMCC No.10751 of table

Bacterial strain	Curdled milk situation	Judge
			Lactococcus lactis subsp. lactis CGMCC No.10751	Whey separates out：It is more, structural state:Preferably	It is general fragrant

D), proteolytic activity is tested

The proteolytic activity of forint (Folin)-phenol reagent process measure bacterial strain：1mL sample solutions are taken, add 2.5mL 0.28M NaOH solutions, mix, and 10min is placed in 25 DEG C or so.0.75mL forint phenol reagent (being purchased from traditional Chinese medicines reagent) is added, Shake up immediately, react 15min in 35 DEG C or so, using ultraviolet specrophotometer (purchased from the U.S. scientific instrument in Shanghai day under 660nm Co., Ltd) measure its light absorption value.The sample solution is：Lactococcus lactis subsp. lactis CGMCC No.10751 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 24 it is small when after suspension.It turns out that the OD of Lactococcus lactis subsp. lactis CGMCC No.10751₆₆₀For 0.82.Illustrate breast Yogurt coccus lactic acid subspecies CGMCC No.10751 proteolytic activities are stronger, and in Cheese during Ripening, leavening bacterial strain Can be polypeptide, amino acid or other inorganic, small molecule organic compounds by breaks down proteins, the formation to cheesy flavor rises emphatically Act 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 By the use of raw milk (at bright milk industry research institute pilot plant obtain) as sample, by Lactococcus lactis subsp. lactis CGMCC No.10751 is inoculated in the triangular flask equipped with sample by the inoculum concentration of 2% (v/v), is placed in 30 DEG C of constant incubator fermented and cultureds 12 it is small when.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.10751 has phage resistance.

Illustrate that the Lactococcus lactis subsp. lactis CGMCC No.10751 have following by the data of embodiment 2~3 Microbiological Characteristics：

1st, morphologic characteristic

After when M17agar medium cultures 24 are small, bacterium colony is creamy white, and bacterium colony is smaller, and circular protrusions are glossy, bacterium Body oval.Mushroom out afterwards, bacterium colony is big and close after 2 days, and be creamy white yellowish.

2nd, the characteristic that culture is learned

3rd, physiological property

Acid producing ability is strong, and acid production speed is slower；Curdled milk speed is relatively fast, and curdled milk viscosity is higher；In terms of fermenting characteristic, energy Whey is separated out, curdled milk structural state is good, gives off a strong fragrance；Proteolytic activity is stronger, can be polypeptide by breaks down proteins, amino Sour or other inorganic, small molecule organic compounds；And there is phage resistance.

The 16S rDNA sequence analyses of 4 Lactococcus lactis subsp. lactis CGMCC No.10751 of embodiment

16S rDNA sequence analyses：Utilize 27F, 1492R primer amplification Lactococcus lactis subsp. lactis CGMCC The 16S rDNA fragments of No.10751, purify, recycling, and then carrying out DNA sequencing using sequenator ABI3730-XL, (the upper Shanghai's style is gloomy Promise bio tech ltd).It turns out that Lactococcus lactis subsp. lactis CGMCC No.10751 and Lactococcus lactis The 16S rDNA sequences of subspecies type strain Lactococcus lactis subsp.lactis Il1403strain IL1403 Homology is 100%.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.10751 belong to Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis).

Wherein, primer pair sequence as described above is：27F：agagtttgat cctggctcag；1492R： Ctacggctac cttgttacga (nucleotide sequence is respectively as shown in sequence table SEQ ID NO.2 and SEQ ID NO.3).Breast The result of the 16S rRNA gene sequencing of yogurt coccus lactic acid subspecies CGMCC No.10751 is as shown in SEQ ID NO.1.

The fermentation of 5 Lactococcus lactis subsp. lactis CGMCC No.10751 of embodiment

The Lactococcus lactis subsp. lactis CGMCC No.10751 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 Department, skimmed milk powder is dissolved in the water to mix accounts for described de- up to degreasing milk medium, the percentage for the skimmed milk powder 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.10751 of embodiment

The Lactococcus lactis subsp. lactis CGMCC No.10751 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, cultivation cycle 24h, and culture pH is 6.

The fermentation of 7 Lactococcus lactis subsp. lactis CGMCC No.10751 of embodiment

The Lactococcus lactis subsp. lactis CGMCC No.10751 frozen are to slowly warm up to 28 DEG C.

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.10751 of embodiment

The Lactococcus lactis subsp. lactis CGMCC No.10751 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.

Cultivation temperature is 30 DEG C, cultivation cycle 48h, and culture pH is 5.

The fermentation of 9 Lactococcus lactis subsp. lactis CGMCC No.10751 of embodiment

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, cultivation cycle 24h, and culture pH is 4.

The fermentation of 10 Lactococcus lactis subsp. lactis CGMCC No.10751 of embodiment

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.

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.10751 of embodiment

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, cultivation cycle 24h, and culture pH is 5.

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 (being purchased from New Zealand Westland cooperation dairy industries Co., Ltd), be placed in 30 DEG C of constant incubator cultures 24 it is small when, activation 2 In generation, obtain 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) In base, be placed in 30 DEG C of constant incubator cultures 24 it is small when be enlarged culture, 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) are filtered, add 0.01% calcium chloride, stir evenly, 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 Pour into the fresh cow milk after pasteurize, the viable count of the cheese starter in fresh cow milk is reached 10⁷Cfu/mL, is slowly stirred 15min.Stand 32 DEG C of fermentations to pH reductions by 0.2 and add 20mg/100L renins (being purchased from Danisco A/S BJ Rep Office) (wherein, afterwards Renin is dissolved with the sterile water of 10 times of quality before use, and stirring 2min is uniformly dissolved it), after 30min curdled milk.

2) the cubical grumeleuse by curd cutting made from step 1) into length of side 5mm, is discharged after being slowly stirred rear 15min The whey of cumulative volume 35%；Divide 2 50 DEG C of additions, the water that volume is fresh cow milk cumulative volume 25%, stirring 20min to curd temperature Reach 35 DEG C.The whey pH for continuing stirring to discharge is 6.1, exhausts whole wheys.Grumeleuse is entered into mould, squeezing shaping afterwards, is taken off Mould takes out.Concentration is 20% 12 DEG C of salt marsh 20h of brine, and the percentage is mass percent.It is vacuum-packed afterwards, 10 DEG C, humidity 90%RH, ripe 5 weeks, up to youth 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 fraction is evaluated in accordance with table 5.Each assessment officer is independently evaluated, and clear water is used when replacing sample every time Gargle, 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

Table 6 result explanation, commercial fermentation agent make Gouda cheese (control group) in terms of quality with effect example 2 Difference between the cheese of gained is not notable；In smell scoring is grown, the cheese of the gained of effect example 2 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 the gained of effect example 2 Grow with good quality and preferably smell.

It is to be understood that after the above of the present invention has been read, 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

A kind of 1. Lactococcus lactis subsp. lactis（Lactococcus lactis subsp. lactis）, it is characterised in that its China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited in, preserving number is：CGMCC No.10751.
A kind of 2. method for preparing Lactococcus lactis subsp. lactis CGMCC No.10751, it is characterised in that it includes following Step, cultivates Lactococcus lactis subsp. lactis CGMCC No.10751 in the medium.
3. method as claimed in claim 2, it is characterised 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；And/or the M17 agar Culture medium includes following constituent：4.5 ~ 5.5g/L of phytone, 4 ~ 5g/L of yeast extract, 4 ~ 5g/ of polyprotein peptone L, 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 2.5 ~ 3g/L of ~ 1.5g/L and agar powder.
4. method as claimed in claim 3, it is characterised 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；The degreasing milk medium is 10% degreasing milk medium, The percentage is mass percent；And/or the M17 agar culture mediums include following constituent：Vegetable protein Peptone 5g/L, yeast extract 5g/L, polyprotein peptone 5g/L, β-phosphoglycerol disodium 19g/L, ascorbic acid 0.5g/L, beef leaching Cream 2.5g/L, bitter salt 1.2g/L and agar powder 2.5g/L.
5. method as claimed in claim 2, it is characterised in that the temperature of the culture is 25 ~ 35 DEG C；The time of the culture For 24 ~ 72 it is small when；And/or the pH of the culture is 4 ~ 6.
6. method as claimed in claim 5, it is characterised in that the temperature of the culture is 28 ~ 30 DEG C；The time of the culture For 24 ~ 48 it is small when；And/or the pH of the culture is 4 ~ 5.
7. method as claimed in claim 2, it is characterised in that the culture is further included carries 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；When the time of the seed culture is 16 ~ 32 small；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, it is characterised in that the seed culture medium is 10 ~ 12% degreasing milk mediums, The percentage is mass percent；When the time of the seed culture is 16 ~ 24 small；And/or the work of the seed culture The algebraically of change was 2 ~ 3 generations.
9. the method for claim 7, it is characterised in that the seed culture includes the step of heating, the heating Temperature is 10 ~ 34 DEG C.
10. method as claimed in claim 9, it is characterised in that the temperature of the heating is 20 ~ 28 DEG C.
11. applications of the Lactococcus lactis subsp. lactis CGMCC No.10751 in cheesemaking as claimed in claim 1.