CN113174347B - Lactococcus lactis subsp lactis capable of highly producing 2-nonanone and application thereof - Google Patents
Lactococcus lactis subsp lactis capable of highly producing 2-nonanone and application thereof Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C13/00—Cream; Cream preparations; Making thereof
- A23C13/12—Cream preparations
- A23C13/16—Cream preparations containing, or treated with, microorganisms, enzymes, or antibiotics; Sour cream
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C17/00—Buttermilk; Buttermilk preparations
- A23C17/02—Buttermilk; Buttermilk preparations containing, or treated with, microorganisms or enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/026—Preparation of other alcoholic beverages by fermentation with health-improving ingredients, e.g. flavonoids, flavones, polyphenols or polysaccharides, added before or during the fermentation stage; with flavouring ingredients added before or during the fermentation stage
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
Abstract
The invention discloses lactococcus lactis subsp lactis capable of highly producing 2-nonanone and application thereof, and belongs to the technical field of microbiology. The lactococcus lactis subspecies lactis CCFM1093 has the following properties: (1) the growth rate of the strain is high, the strain enters a growth stabilization period within 6 hours in a GM17 culture medium, the increment of viable count reaches 1.81 orders of magnitude after the skim milk is fermented for 8 hours, and the strain is suitable for industrial fermentation; (2) the acid production rate of the bacterial strain is high, the fermented skim milk is completely curdled for 8 hours and reaches the fermentation end point, and curdled milk and flavor substances are favorably generated; (3) the yield of 2-nonanone of the strain is high, and 334.63ng/100g can be produced in 8 hours. The lactococcus lactis subsp lactis CCFM1093 is used as a leavening agent for leavening food, improves the fruit flavor of the leavened dairy product, and has very wide application prospect.
Description
Technical Field
The invention relates to lactococcus lactis subsp lactis and application thereof, in particular to lactococcus lactis subsp lactis with a high yield of 2-nonanone and application thereof in preparing fermented milk, and belongs to the technical field of microbiology.
Background
The fermented milk product is prepared by using cow milk as a main raw material and fermenting by using lactic acid bacteria or fermenting by using lactic acid bacteria and yeast together, is deeply favored by people by virtue of unique taste and flavor, and has a wide consumer market. Studies have shown that the main factors influencing the purchase of fermented dairy products by consumers include flavour, price, availability and brand, among which flavour is the most important. Therefore, the improvement of the product flavor is an important means for optimizing the quality of the fermented dairy product at present.
Currently, many studies indicate that carbonyl compounds are an important class of substances that promote the formation of the flavor of fermented milk, with acetaldehyde, diacetyl, and acetoin being the major flavor substances in yogurt. In addition, some other carbonyl compounds also have an important effect on the flavor of the yogurt, such as 2-butanone has sweet taste and fruit taste, and 2-heptanone and 2-nonanone are also closely related to the fruit flavor in the yogurt.
Lactococcus lactis, a species of starter bacteria commonly used in the dairy industry, has been widely used in cheese processing due to its acidifying and aroma-producing properties, and has recently been used in other fermented dairy products as an adjunct starter to improve product flavor due to its superior flavor properties. Research shows that lactococcus lactis subspecies lactis can generate 2-nonanone in the fermentation process, and the improvement of the intensity of fruit flavor in the fermented milk is facilitated.
Therefore, research and development of lactococcus lactis subsp lactis with high 2-nonanone yield, which is used as a leavening agent for dairy product fermentation to improve the fruity flavor of fermented dairy products, are technical problems to be solved in the field.
Disclosure of Invention
In order to improve the fruity flavor of the fermented dairy product, the invention provides a lactococcus lactis subspecies lactis with high yield of 2-nonanone and application thereof, and the specific technical scheme is as follows:
the invention aims to provide a Lactococcus lactis subsp.lactis strain with high 2-nonanone yield, which is screened from a Trira sample collected in the Alba Sichuan area in China, is preserved in Guangdong province microbial strain preservation center of No. 59 building 5 of No. 100 college of Pieli Zhonglu-Miyao in Guangzhou city in 2019 and 16 days in 10 and 9, and has the preservation number of GDMCC NO. 60808.
The lactococcus lactis subspecies lactis has the following fermentation characteristics:
(1) the growth rate of the strain is high, and the increment of viable count of fermented skim milk for 8h can reach 1.81 orders of magnitude;
(2) the acid production rate of the bacterial strain is high, and the skim milk is fermented for 8h and curd is performed and the fermentation end point is reached;
(3) the strain has high 2-nonanone yield, and the content of the 2-nonanone generated after 8 hours of fermentation is 334.63ng/100 g.
The second object of the invention is to provide a starter culture containing lactococcus lactis subsp.
In one embodiment of the invention, the leavening agent is a direct vat set leavening agent, wherein the concentration of viable lactococcus lactis subsp lactis in the direct vat set leavening agent is not lower than 1x 10 9 cfu/mL。
In one embodiment of the present invention, the specific preparation method of the direct vat set starter is as follows: inoculating lactococcus lactis subspecies lactis into culture medium, and culturing until the concentration of viable bacteria of lactococcus lactis subspecies lactis is not less than 1 × 10 8 cfu/mL to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning thallus with buffer solution, adding lyophilized protectant, re-suspending, and adjusting viable bacteria concentration to not less than 1 × 10 9 cfu/mL to obtain a bacterial suspension; and (4) carrying out vacuum freeze drying on the bacterial suspension, and freeze-drying to obtain the direct vat set starter.
In one embodiment of the invention, the medium is GM17 liquid medium.
In one embodiment of the invention, the direct vat set starter is prepared by inoculating lactococcus lactis CCFM1093 into liquid GM17 medium and activating for 2-3 generations at 30 ℃.
In one embodiment of the invention, the culture broth is centrifuged at 4000rpm for 20 min.
The third purpose of the invention is to provide the application of the lactococcus lactis subsp lactis or the leavening agent in preparing food.
In one embodiment of the invention, the food product is a fermented dairy product; the fermented dairy products include fermented milks, fermented dairy beverages, fermented buttermilks, sour creams and milks.
It is a fourth object of the present invention to provide a method for producing fermented milk using said lactococcus lactis subsp.
In one embodiment of the invention, the method is that milk is homogenized, pasteurized and cooled to obtain a fermentation raw material; inoculating the lactococcus lactis subspecies lactis or the leavening agent into a fermentation raw material for fermentation to obtain the fermented milk.
In one embodiment of the invention, the method is to homogenize milk under the conditions of 14MPa to 21MPa of pressure and 40 ℃ to 85 ℃ and then pasteurize the homogenized milk to obtain sterilized milk; cooling the sterilized milk to 30-40 ℃ to obtain a fermentation raw material; inoculating the lactococcus lactis subspecies lactis or the leavening agent into a fermentation raw material, and fermenting at 37 ℃ for 8 hours to obtain fermented milk; and standing the fermented milk at 4 ℃ for 24-48 h to obtain the post-matured fermented milk product.
It is a fifth object of the present invention to provide a food product comprising the above lactococcus lactis subsp.
In one embodiment of the invention, the number of viable lactococcus lactis subsp lactis in the food product or the number of viable lactococcus lactis subsp lactis in the starter is not less than 1X 10 9 cfu/mL。
In one embodiment of the invention, the food is a health food; or the food is fermented milk, fermented buttermilk, sour cream or milk wine produced by using the lactococcus lactis subsp lactis or the leavening agent; or the food is a beverage or snack produced using the lactococcus lactis subsp.
The invention has the beneficial effects that:
1. the method is safe and healthy: the lactococcus lactis subsp lactis CCFM1093 used in the invention is a safe strain for food. The method of the invention is safe and healthy without adding other chemical substances.
2. The lactococcus lactis subsp lactis CCFM1093 can effectively improve the content of 2-nonanone in fermented milk, and is beneficial to improving the quality characteristic of products. When the lactococcus lactis subsp lactis CCFM1093 is used for producing fermented milk by fermentation, the content of 2-nonanone in the fermented milk is 334.63ng/100g after 8 hours of fermentation, compared with lactococcus lactis CICC6246 (the content of 2-nonanone in the fermented milk is 96.09ng/100g after 8 hours of fermentation) and unfermented skim milk (the content of 2-nonanone is 100.00ng/100g), the content of 2-nonanone in the fermented milk is increased by 71.28% and 70.12% after 8 hours of fermentation by using the lactococcus lactis subsp lactis CCFM1093 for producing the fermented milk by fermentation.
Biological material preservation
Lactococcus lactis subsp.lactis is classified and named as Lactococcus lactis subsp.lactis, is deposited in Guangdong province microbial strain preservation center in 2019, 10 and 16, and has the preservation number of GDMCCNo:60808, and the preservation address of Dairy building No. 59 and building No. 5 of Ji No. 100 of the Reliao Zhonglu, Guangzhou city.
Drawings
FIG. 1 is a colony of lactococcus lactis subspecies lactis CCFM1093 on GM17 solid medium;
FIG. 2 is a microscopic image of a gram-stained strain of lactococcus lactis subsp.lactis CCFM1093 under 1000-fold microscope;
FIG. 3 is a diagram of the growth of lactococcus lactis subspecies lactis CCFM1093 in the skim milk fermentation process;
FIG. 4 is a diagram of acid production of lactococcus lactis subsp. lactis CCFM1093 in the skim milk fermentation process;
FIG. 5 is a graph of the lactose content of lactococcus lactis subsp. lactis CCFM1093 after fermentation in skim milk;
FIG. 6 is a graph showing the content of volatile flavor substances of main carbonyl compounds in the skim milk after fermentation of lactococcus lactis subsp.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
The detection methods referred to in the following examples are as follows:
the detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
The pH detection method comprises the following steps: measured with a pH meter.
And (3) an acidity detection method: the national standard GB 431334-.
Detection of lactose content: weighing 1g (accurate to 0.001g) of fermented milk sample in a 10mL volumetric flask, adding 70% (v/v) ethanol to a constant volume to scale, uniformly mixing, extracting at 4 ℃ for 24h, centrifuging at 11000g and 4 ℃ for 10min, taking supernatant, performing derivatization, filtering with a microporous filter membrane, and determining by high performance liquid chromatography (quantitative analysis of lactose content by external standard method).
Liquid chromatography conditions: a Waters Sugar-Pak I column (300X 6.5mm) was selected, the column temperature was 85 ℃, the flow rate was 0.4mL/min for mobile phase ultrapure water, the evaporative light scattering detector was selected, and the sample injection volume was 10. mu.L. And (5) performing substance qualification according to the peak output time of the standard substance, and quantifying by using a peak area external standard method.
And (4) detecting the content of volatile substances: and measuring the content of volatile substances in the fermented yoghourt by GC-MS.
Gas chromatography conditions and temperature program: selecting Rtx-WAX capillary (30m is multiplied by 0.25mm, 0.25mm), sample inlet temperature is 225 ℃, split ratio is 10, and flow rate of carrier gas (helium) is 1 mL/min. The temperature-raising program sets the initial temperature to 30 ℃, keeps the temperature for 3min, raises the temperature to 225 ℃ at the speed of 15 ℃/min, and keeps the temperature for 5 min.
Mass spectrum conditions: the ionization mode EI has the emission energy of 70eV, the emission current of 200 muA, the detector voltage of 1.4kV, the ion source temperature of 240 ℃, the interface temperature of 230 ℃, the quadrupole rod temperature of 150 ℃ and the mass scanning range of m/z of 30-500. And (3) performing substance qualitative determination on the spectrogram obtained by GC-MS by searching in a NIST 2001 standard spectrum library and comparing standard substances, and quantifying by using a peak area normalization method.
Example 1: separation and identification of lactococcus lactis subsp lactis CCFM1093 strain
1. Separation of
(1) Preparing appropriate sample dilution gradient and culturing
To-80 ℃ refrigeratorThe stored Sichuan Alba Trirax glycerol sample is taken out and placed on ice for unfreezing. After shaking and mixing, adding 0.5mL of sample into 4.5mL of sterile physiological saline to finish primary 10-fold dilution, after shaking and mixing, taking 0.5mL of diluent out of the diluent, adding the diluent into 4.5mL of sterile physiological saline to finish secondary 10-fold dilution, and so on until the diluent is diluted to 10 -6 And sucking 100 mu L of dilution liquid from each gradient, uniformly coating the dilution liquid on a GM17 solid medium plate, inverting, placing the plate at 37 ℃ for anaerobic culture for 36-48 h, and observing in time.
(2) Streaking separation and purification
And after taking out the plate with the grown bacterial colony, selecting a gradient plate with an obvious single bacterial colony, selecting bacterial colonies with different bacterial colony morphologies, and carrying out secondary scribing until all the single bacterial colonies are purified. A photograph of a colony of the strain CCFM1093 on a GM17 solid medium is shown in FIG. 1, the colony is small, white, wet and neat in edge, and the colony is circular.
(3) Gram stain and Catalase assay
Selecting a single colony on a glass slide, performing smear, drying, fixing, primary staining, washing, mordanting, washing, decoloring, counterstaining, washing, drying and microscopic examination, and recording a gram staining result, wherein the gram staining microscopic examination result of the strain CCFM1093 is purple (shown in figure 2), and the gram positive bacteria are obtained. Selecting a single colony on a glass slide, adding a 3% hydrogen peroxide solution, observing whether bubbles are generated, recording the catalase contact result, and showing that the strain CCFM1093 shows that the catalase is negative
(4) Strain preservation
And (3) picking the single colony of each purified strain into 5mL of liquid GM17 culture medium, placing the culture medium in anaerobic static culture at 37 ℃ for 20-24 h, sucking 1mL of bacterial liquid into a bacteria-preserving tube, centrifuging at 6000rpm for 3min, pouring off the supernatant, adding 1mL of 30% sterile glycerol solution, resuspending, and placing the mixture in a place at-80 ℃ for preservation.
2. Identification
(1)16S rDNA sequence amplification
Sucking 1mL of bacterial liquid at 6000rpm, centrifuging for 3min, pouring out supernatant, washing twice, centrifuging and pouring out supernatant to obtain bacterial sludge, taking the bacterial sludge as a template to perform PCR amplification, wherein the process is as follows:
1) amplification system 20 μ L:
wherein the template amount is 1 μ L, each of the bidirectional primers is 0.5 μ L, the Taq enzyme MasterMix is 10 μ L, and ddH 2 O is 7. mu.L. The primers used were 27F: AGA GTT TGA TCC TGG CCT CA (SEQ ID No.1) and 1492R: GGT TAC CTT GTT ACG ACT T (SEQ ID No. 2).
2) Amplification conditions:
pre-denaturation: 95 ℃ for 3min
First step denaturation: 94 ℃ for 1min
And a second step of annealing: 30s at 60 DEG C
And a third step of extension: 72 ℃ for 2min
Cycle number: 30 cycles of
The fourth step is finally extended: 5min at 72 DEG C
The fifth step is that: 10min at 12 DEG C
(2) Agarose gel electrophoresis
Weighing 1.2g of agarose, adding into a conical flask, adding 80mL of 1xTAE, heating intermittently by microwave for 4min until the liquid is clear and transparent, slightly cooling, adding 4 muL of nucleic acid dye, shaking uniformly without bubbles, pouring into a rubber plate groove, cooling for 40min, solidifying, placing into an electrophoresis groove, exhausting bubbles, sequentially adding PCR amplification products, adding 2 muL of PCR amplification products into each hole, running glue for 120V 15min, taking out after the completion, placing into a gel electrophoresis imager, photographing for storage, recording the serial number of a PCR successful sample, and placing the successful PCR product into a refrigerator at-20 ℃ for storage.
(3) Sequencing and identification
And (3) sending the sample with the successful PCR to an England Weiji (Shanghai) trade company Limited for detection, performing BLAST retrieval in a sequence database (http:// www.ncbi.nlm.nih.gov/BLAST) of the National Center for Biotechnology Information (NCBI) according to the fed-back sequence result, and selecting the strain information with the highest matching degree for result recording. The strain CCFM1093 provided by the invention is Lactococcus lactis subsp.
3. Preservation of
The strain CCFM1093 was deposited in Guangdong province culture Collection in 2019, 10 and 16 months.
Example 2: fermentation characteristics of lactococcus lactis subsp lactis CCFM1093 in skim milk
1. Growth characteristics of lactococcus lactis subspecies lactis CCFM1093 in skim milk
Inoculating lactococcus lactis subspecies lactis CCFM1093 stored at-80 deg.C in GM17 culture medium, culturing at 30 deg.C for 24 hr, subculturing for 2 times to 10 times 8 ～10 9 cfu/mL. The bacterial liquid activated in GM17 is taken out and inoculated in skim milk according to the volume ratio of 2-4% so that the number of viable bacteria in the system reaches 10 7 cfu/mL. The inoculated sample is put into an incubator at 37 ℃ for fermentation, samples are taken every 4 hours, and the change of the viable count in the fermentation process is detected, and the result is shown in figure 3. As can be seen from FIG. 3, the increase of viable count of the strain CCFM1093 reaches 1.81 orders of magnitude after 8 hours of fermentation, while the increase of viable count of the lactococcus lactis, namely CICC6246, only reaches 1.21 orders of magnitude after the same time of fermentation, so that the growth rate of the strain CCFM1093 in skim milk is high.
2. Acid production characteristic of lactococcus lactis subsp lactis CCFM1093 in skim milk
Inoculating lactococcus lactis subsp. lactis CCFM1093 stored at-80 ℃ in a GM17 culture medium, culturing at 30 ℃ for 24h, and subculturing for 2-3 times to 10 8 ～10 9 cfu/mL. The bacterial liquid activated in GM17 is taken out and inoculated in skim milk according to the volume ratio of 2-4% so that the viable count in the system reaches 10 7 cfu/mL. And (3) fermenting the inoculated sample in an incubator at 37 ℃, sampling every 4h, and detecting the change of pH in the fermentation process, wherein the experimental result is shown in figure 4. As can be seen from FIG. 4, after 8 hours of fermentation, the titer acidity of the strain CCFM1093 is 81.8 degrees T, the pH value is reduced to 4.62, and the fermentation end point is reached, while after the same time of fermentation of the lactococcus lactis, namely, the lactococcus lactis, CICC6246, the titer acidity is only 31.9 degrees T, the pH value is only reduced to 5.91, and the fermentation end point is not reached, so the acid production rate of the strain CCFM1093 in skim milk is high.
3. Lactose content of lactococcus lactis subsp lactis CCFM1093 after skim milk fermentation
Lactococcus lactis milk of the present invention to be preserved at-80 ℃Inoculating acid subspecies CCFM1093 in GM17 culture medium, culturing at 30 deg.C for 24 hr, subculturing for 2-3 times to 10 8 ～10 9 cfu/mL. Taking out the bacterial liquid activated in GM17, inoculating the bacterial liquid into skim milk according to the volume ratio of 2-4%, and making the viable count in the system reach 10 7 cfu/mL, the inoculated sample was placed in an incubator at 37 ℃ for fermentation.
The lactose content of the skim milk after fermentation is measured by High Performance Liquid Chromatography (HPLC), the lactose content of the fermented milk is shown in FIG. 5, as can be seen from FIG. 5, the lactose content of the skim milk before fermentation is 6.43g/100g, the lactose content of the lactococcus lactis subsp. lactis CCFM1093 after reaching the fermentation end point (8h) is lower and is 5.17g/100g, and the lactose content of the lactococcus lactis CCCC 6246 after fermentation for 8h is 5.85g/100g, so that the lactose content of the lactococcus lactis CCFM1093 after fermentation is obviously lower than that of the lactococcus lactis CICC6246, and the screened lactococcus lactis CCFM1093 has strong lactose utilization capacity.
4. Volatile matter content of lactococcus lactis subspecies lactis CCFM1093 after skim milk fermentation
Inoculating lactococcus lactis subsp. lactis CCFM1093 stored at-80 ℃ in a GM17 culture medium, culturing at 30 ℃ for 24h, and subculturing for 2-3 times to 10 8 ～10 9 cfu/mL. The bacterial liquid activated in GM17 is taken out and inoculated in skim milk according to the volume ratio of 2-4% so that the viable count in the system reaches 10 7 cfu/mL, the inoculated sample was placed in an incubator at 37 ℃ for fermentation.
Volatile matter content of the fermented milk was measured by gas chromatography-mass spectrometry (GC-MS), and the volatile matter content of the fermented milk is shown in fig. 6. As can be seen from FIG. 6, after 8 hours of fermentation, the content of 2-nonanone in the fermented milk is 334.63ng/100g, which is significantly higher than that of lactococcus lactis CICC6246 and unfermented skim milk, and the content is only 96.09ng/100g and 100.00ng/100g respectively, and 2-nonanone is an important flavor substance, so that the screened lactococcus lactis CCFM1093 has excellent aroma-producing characteristics.
Example 3: preparation method of leaven containing lactococcus lactis subsp lactis CCFM1093
Inoculating 400 μ L lactococcus lactis CCFM1093 into 20mL GM17 liquid culture medium, activating at 30 deg.C for 2-3 generations until viable count of lactococcus lactis CCFM1093 reaches 1 × 10 8 Obtaining liquid bacterial liquid when cfu/mL; centrifuging at 4000rpm for 20min, removing supernatant, and collecting thallus; sequentially adding buffer solution and cryoprotectant under aseptic environment until cell concentration is 1 × 10 9 And (5) performing vacuum freeze drying treatment when cfu/mL is needed, thus obtaining the direct vat set starter.
Example 4: method for producing fermented milk
Homogenizing milk under the conditions of pressure of 14-21 MPa and temperature of 40-85 ℃, and then carrying out pasteurization to obtain sterilized milk; cooling the sterilized milk to 30-40 ℃ to obtain a fermentation raw material; inoculating lactococcus lactis subsp lactis CCFM1093 or the leavening agent obtained in example 3 into the fermentation raw material, and fermenting at 37 ℃ for 8 hours to obtain fermented milk; and standing the fermented milk at 4 ℃ for 24-48 h to obtain the post-ripened fermented milk product.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> university in south of the Yangtze river
<120> lactococcus lactis subsp lactis with high yield of 2-nonanone and application thereof
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
ggttaccttg ttacgactt 19
Claims (11)
1. Lactococcus lactis subsp.lactis CCFM1093 is deposited in Guangdong province collection of microorganisms and strains in 2019, 10 and 16 days, and has the deposit number GDMCC NO: 60808.
2. A starter culture comprising lactococcus lactis subsp.
3. The starter culture of claim 2, wherein the starter culture is a direct vat set starter culture in which the viable bacteria concentration of lactococcus lactis subsp 9 cfu/mL。
4. Use of lactococcus lactis subsp.
5. Use according to claim 4, wherein the food product is a fermented dairy product; the fermented milk product is fermented milk, fermented milk beverage, fermented buttermilk, sour cream and milk wine.
6. A method for producing fermented milk, which comprises fermenting cow's milk or a milk-containing material with the aid of the lactococcus lactis strain CCFM1093 of claim 1 or the starter of claim 2 or 3 to produce fermented milk.
7. The method of claim 6, wherein the milk is homogenized, pasteurized and cooled to obtain a fermentation feedstock; inoculating lactococcus lactis subsp. lactis CCFM1093 according to claim 1 or the starter according to claim 2 or 3 in a fermentation raw material, and fermenting to obtain the fermented milk.
8. The method according to any one of claims 6 or 7, wherein the method comprises homogenizing milk under a pressure of 14MPa to 21MPa and at a temperature of 40 ℃ to 85 ℃ and then pasteurizing the homogenized milk to obtain pasteurized milk; cooling the sterilized milk to 30-40 ℃ to obtain a fermentation raw material; inoculating lactococcus lactis subsp. lactis CCFM1093 according to claim 1 or the starter according to any one of claims 2 or 3 in a fermentation raw material, and fermenting at 37 ℃ for 8 hours to obtain fermented milk; and standing the fermented milk at 4 ℃ for 24-48 h to obtain the post-ripened fermented milk product.
9. A food product comprising lactococcus lactis subsp.
10. The food product of claim 9, wherein the food product is a health food; or the food is a beverage or snack produced using the lactococcus lactis subsp lactis CCFM1093 according to claim 1 or the starter according to claim 2 or 3.
11. The food product according to claim 9, wherein the food product is a fermented milk, a fermented buttermilk, a sour cream or a wine produced using the lactococcus lactis subsp.
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