CN104894037B

CN104894037B - A kind of Lactococcus lactis subsp. lactis and its purposes in cheesemaking

Info

Publication number: CN104894037B
Application number: CN201510378621.8A
Authority: CN
Inventors: 黄宜; 刘振民; 莫蓓红; 朱培; 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: 2018-02-13
Anticipated expiration: 2035-07-01
Also published as: CN104894037A

Abstract

The invention discloses a kind of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis), preparation method and its purposes 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.10752.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 purposes 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 Purposes.

Background technology

Cheese, also known as cheese, there are miscellaneous taste, mouthfeel and form.Cheese is using milk as raw material, containing abundant Protein and lipid, there is very high nutritive value.It is more next to the demand of cheese with our people's growth in the 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, help to exclude whey, suppress harmful bacterial growth, and produce 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..The most of leavening used now 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 applied to completely newly using the abundant microorganism resource exploitation of China The fermentation strain of cheese starter is significant.

The content of the invention

The technical problems to be solved by the invention are, for lack at present for it is 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 purposes in cheesemaking.Described 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.10752。

It is isolated from the raw milk in China Shanghai, obtains described Lactococcus lactis subsp. lactis CGMCC No.10752.Described Lactococcus lactis subsp. lactis CGMCC No.10752 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), is named as BD401.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 numbering CGMCC No.10752, it has following Microbiological Characteristics：

1st, morphologic characteristic

After M17agar medium cultures 24 hours, 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；Viability is good under the conditions of 40 DEG C, the cheese cutting heating process of 2 hours.

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 be 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 described Lactococcus lactis subsp. lactis CGMCC No.10752 Method, it comprises the following steps, cultivates described Lactococcus lactis subsp. lactis CGMCC No.10752 in the medium .

Wherein, described culture medium is the conventional culture medium in this area, and it is sub- can to grow described Lactococcus lactis Kind CGMCC No.10752, preferably MRS culture mediums, M17agar culture mediums or degreasing milk medium.Described MRS Culture medium is the conventional MRS culture mediums in this area, 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 Degreasing milk medium is stated as the conventional degreasing milk medium in this area, preferably 10% degreasing milk medium.By skimmed milk powder It is dissolved in the water to mix and produces 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.Described M17agar culture mediums are the conventional M17agar culture mediums in this area, 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 conventional temperature in this area, can grow described Lactococcus lactis subsp. lactis CGMCC No.10752, preferably 25~35 DEG C, it is more preferably 28~30 DEG C.The time of the culture is that this area is normal The time of rule, preferably 24~72 hours, it is more preferably 24~48 hours.The pH of the culture is the conventional pH in this area, It can grow described Lactococcus lactis subsp. lactis CGMCC No.10752, preferably 4~6, it is more preferably 4~5.

It is preferred that the step of also carrying out seed culture including the use of seed culture medium before described culture.Described seed Cultivate as the conventional seed culture in this area.Described seed culture medium is the conventional seed culture medium in this area, preferably Degreasing milk medium, it is more preferably 10~12% degreasing milk mediums, described percentage is mass percent.Described degreasing Breast culture bacterium sterilizes by 115 DEG C, 15~20min.The time of the seed culture is this area conventional time, preferably 16~32 hours, be more preferably 16~24 hours.The temperature of the seed culture is the conventional temperature in this area, preferably 28 ~32 DEG C.The inoculum concentration of the seed culture is the conventional inoculum concentration in this area, preferably 1~2%, the percentage is body Product percentage.The algebraically of the activation of the seed culture is the conventional algebraically in this area, in preferably 2~4 generations, is more preferably 2 ~3 generations.It is preferred that described Lactococcus lactis subsp. lactis CGMCC No.10752 are to freeze state, the seed culture bag The step of including heating.The temperature of the heating is the conventional temperature in this area, and preferably 10~34 DEG C, be more preferably 20~28 ℃。

The method of the culture is the method for the conventional culture in this area, preferably Shaking culture or fermentation tank culture.

The three of technical scheme provided by the invention are：Described Lactococcus lactis subsp. lactis CGMCC No.10752 are dry Purposes in junket production.

Described cheese is the conventional cheese in this area, 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 deposited under non-refrigerated conditions and separates out or becomes sour.

It on the basis of common sense in the field is met, above-mentioned each optimum condition, can be combined, it is each preferably real to produce the present invention Example.

Agents useful for same and raw material of 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.10752, 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 described reality Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business Product specification selects.

The yogurt coccus lactic acid subspecies CGMCC No.10752 of embodiment 1 acquisition

(1), activate：The raw milk for being derived from Shanghai is taken out from cryopreservation tube, with 2% (v/v) inoculum concentration be inoculated in through Crossed 121 DEG C, 15 minutes sterilizing M17 broth bouillons in (M17 broth bouillons be do not contain agar M17 Agar culture Base, 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 2% (v/v) inoculum concentration It is inoculated in sodium chloride content 4%, (skimmed milk powder is purchased from New Zealand Westland cooperation dairy industries to pH5 10% degreasing milk medium Co., Ltd, skimmed milk powder, which is dissolved in the water, to mix produces degreasing milk medium, 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 nutrient solution in 16 hours.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 2% (v/v) inoculum concentration and is not added with Sodium chloride, do not adjust in pH 10% degreasing milk medium, 30 DEG C are cultivated 16 hours, obtain nutrient solution.By nutrient solution according to After step method processing described in GB4789.2-2010,37 DEG C are cultivated 48 hours.

After 48 hours, determined 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 2% (v/v) inoculum concentration 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, obtain nutrient solution b；

C) well-grown bacterial strain obtained by step (2) is inoculated in 2% (v/v) inoculum concentration 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, obtain nutrient solution c；

D) well-grown bacterial strain obtained by step (2) is inoculated in 2% (v/v) inoculum concentration 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.

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 BD401.

By Lactococcus lactis subsp. lactis BD401 on April 27th, 2015 in Chinese microorganism strain preservation conservator Meeting common micro-organisms center (CGMCC) preservation, obtaining deposit number is：CGMCC No.10752, culture title are BD401, Classification And Nomenclature is Lactococcus lactis subsp. lactis Lactococcus lactis subsp.lactis.

Feature is learned in the Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 2 morphology and culture

Cultural characteristic：Using 30 DEG C of culture Lactococcus lactis subsp. lactis CGMCC No.107522 days of M17agar culture mediums Afterwards, cultural characteristic is observed.

Cultural characteristics of the Lactococcus lactis subsp. lactis CGMCC No.10752 of table 1 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.10752 grow good on M17agar culture mediums Good, after cultivating 24 hours, 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 Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 3 physiological and biochemical property

A), product acid activity

Lactococcus lactis subsp. lactis CGMCC No.10752 are inoculated in equipped with sterilizing by 1% (v/v) inoculum concentration 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 Lactococcus lactis subsp. lactis CGMCC No.10752 degreasing milk medium surveys pH value, as a result as shown in table 2 as control.

The Lactococcus lactis subsp. lactis CGMCC No.10752 of table 2 rate of producing acid

	0 hour	3 hours	6 hours	9 hours	12 hours	15 hours	18 hours	21 hours	24 hours
										pH	6.34	6.32	5.88	5.55	5.24	5.08	4.95	4.80	4.73

As shown in Table 2, Lactococcus lactis subsp. lactis CGMCC No.10752 acid production speeds are slower, and after 24 hours, pH is only For 4.73, produce that sour power is stronger, 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, is easy to 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.10752 are inoculated in equipped with sterilizing by 1% (v/v) inoculum concentration 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. Determine the viscosity (mPa/s) of each fermentation yogurt after curdled milk respectively with rotational viscometer (being purchased from German proRheo companies).Measure No. 2 rotors of Shi Xuanyong, speed 64r/s.During measure per 15s values once, minute 3min, as a result as shown in table 3.By Table 3 understands that Lactococcus lactis subsp. lactis CGMCC No.10752 curdled milk speed is slower, and curdled milk viscosity is higher.

The Lactococcus lactis subsp. lactis CGMCC No.10752 of table 3 curdled milk time and curdled milk viscosity

Bacterial strain	The curdled milk time/h	Viscosity mPa/s
			Lactococcus lactis subsp. lactis CGMCC No.10752	18	185

C), fermentation character is tested

Lactococcus lactis subsp. lactis CGMCC No.10752 are inoculated in equipped with sterilizing by 1% (v/v) inoculum concentration 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, as a result as shown in table 4.

The Lactococcus lactis subsp. lactis CGMCC No.10752 of table 4 fermentation character

Bacterial strain	Curdled milk situation	Judge
			Lactococcus lactis subsp. lactis CGMCC No.10752	Whey separates out：It is more, structural state:Very well	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, 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, (Shanghai day U.S. scientific instrument are purchased from using ultraviolet specrophotometer under 660nm Co., Ltd) determine its light absorption value.Described sample solution is：Lactococcus lactis subsp. lactis CGMCC No.10752 are pressed 1% (v/v) inoculum concentration 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 find, Lactococcus lactis subsp. lactis CGMCC No.10752 OD₆₆₀For 0.8.Illustrate breast Yogurt coccus lactic acid subspecies CGMCC No.10752 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.10752 is inoculated in the triangular flask equipped with sample by 2% (v/v) inoculum concentration, 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 determined, Δ pH >=0.5 is found, illustrates Lactococcus lactis subsp. lactis CGMCC No.10752 has phage resistance.

Illustrate that the Lactococcus lactis subsp. lactis CGMCC No.10752 have following by the data of embodiment 2~3 Microbiological Characteristics：

1st, morphologic characteristic

After M17agar medium cultures 24 hours, 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 slower, and curdled milk viscosity is high；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 be polypeptide, amino acid or its Its inorganic, small molecule organic compound；And there is phage resistance.

The Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 4 16S rDNA sequence analyses

16S rDNA sequence analyses：Utilize 27F, 1492R primers amplification Lactococcus lactis subsp. lactis CGMCC No.10752 16S rDNA fragments, purify, recovery, then carrying out DNA sequencing using sequenator ABI3730-XL, (the upper Shanghai's style is gloomy Promise bio tech ltd).As a result find, Lactococcus lactis subsp. lactis CGMCC No.10752 and Lactococcus lactis Subspecies type strain Lactococcus lactis subsp.lactis Il1403strain IL1403 16S rDNA sequences Homology is 100%.Due to when 16S rDNA sequence homologies are higher than 97%, it is believed that be of the same race in category, therefore yogurt Coccus CGMCC No.10752 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 Lactococcus lactis subsp. lactis CGMCC No.10752 16S rRNA gene sequencing is as shown in SEQ ID NO.1.

The Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 5 fermentation

The Lactococcus lactis subsp. lactis CGMCC No.10752 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, which is dissolved in the water, to mix produces degreasing milk medium, 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), described 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%, described 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 Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 6 fermentation

The Lactococcus lactis subsp. lactis CGMCC No.10752 frozen are to slowly warm up to 20 DEG C.

Seed culture medium：12% degreasing milk medium, described 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%, described 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 Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 7 fermentation

The Lactococcus lactis subsp. lactis CGMCC No.10752 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%, described 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 Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 8 fermentation

The Lactococcus lactis subsp. lactis CGMCC No.10752 frozen are to slowly warm up to 34 DEG C.

Seed culture medium：10% degreasing milk medium, described 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 Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 9 fermentation

28 DEG C, cultivation cycle 32h of the cultivation temperature of seed culture medium, inoculum concentration 1%, described 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 Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 10 fermentation

Seed culture medium：12% degreasing milk medium, described 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 Lactococcus lactis subsp. lactis CGMCC No.10752 of embodiment 11 fermentation

Seed culture medium：10% degreasing milk medium, described 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%, described 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 2% (v/v) inoculum concentration In base (being purchased from New Zealand Westland cooperation dairy industries Co., Ltd), 30 DEG C of constant incubator cultures 24 hours, activation 2 are placed in 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 2% (v/v) inoculum concentration In base, it is placed in 30 DEG C of constant incubator cultures and is enlarged culture in 24 hours, be repeated 3 times, obtains 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 Leukonid LM79 viable count is 1:1:1:1 ratio mixing, obtains cheese starter.

The cheese starter of effect example 2 produces Gouda cheese

1) 100kg fresh cow milks (being purchased from Kingsoft pasture) are filtered, add 0.01% calcium chloride, stir, at 72 DEG C, 30 DEG C are cooled to after 15s pasteurizes, 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, it 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 before use with the sterilized water of 10 times of quality, 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, whole wheys of draining.Grumeleuse is entered into mould, squeezing shaping afterwards, taken off Mould takes out.Concentration is 20% 12 DEG C of salt marsh 20h of salt solution, and the percentage is mass percent.It is vacuum-packed afterwards, 10 DEG C, humidity 90%RH, ripe 5 weeks, produce young Gouda cheese.

The sensory evaluation of the cheese of effect example 3

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 changing sample every time Gargle, as a result as 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), obtained cheese the step of according to effect example 2.

The cheese subjective appreciation method of table 5

The cheese results of sensory evaluation of table 6

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 grown in cheese feature Scoring and total score in terms of smell, bitter taste are all higher than control group.Therefore, the cheese of the gained of effect example 2 has well Quality and preferably grow smell.

It should be understood that after the above of the present invention has been read, those skilled in the art can make various to the present invention Change or modification, these equivalent form of values equally fall within the application appended claims limited range.

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.10752.
A kind of 2. method for preparing Lactococcus lactis subsp. lactis CGMCC No.10752, it is characterised in that it includes following Step, Lactococcus lactis subsp. lactis CGMCC No.10752 are cultivated in the medium.
3. method as claimed in claim 2, it is characterised in that described culture medium is that MRS culture mediums or M17 agar are cultivated Base or degreasing milk medium；Described MRS culture mediums include following constituent：8 ~ 12g/L of peptone, 9 ~ 12g/ of beef extract L, 15 ~ 20g/L of glucose, the g/L of yeast extract 4 ~ 6,1.8 ~ 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 For 10% degreasing milk medium, the percentage is mass percent；Described M17 agar culture mediums include following composition into Point：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, it is characterised in that described 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；Described M17 agar culture mediums include following composition Composition：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.
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 hours；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 hours；The pH of the culture is 4 ~ 5.
7. method as claimed in claim 2, it is characterised in that also planted before described culture including the use of seed culture medium The step of son culture, described seed culture medium is degreasing milk medium；Described skimmed milk culture bacterium 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；The algebraically of the activation of the seed culture was 2 ~ 4 generations.
8. method as claimed in claim 7, it is characterised in that described seed culture medium is 10 ~ 12% degreasing milk mediums, Described percentage is mass percent；The time of the seed culture is 16 ~ 24 hours；The generation of the activation of the seed culture Number was 2 ~ 3 generations.
9. method as claimed in 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. purposes of the Lactococcus lactis subsp. lactis CGMCC No.10752 as claimed in claim 1 in cheesemaking.