Low-temperature fermented milk rich in vitamin K2 as well as preparation method and application of low-temperature fermented milk

Abstract

The invention discloses low-temperature fermented milk rich in vitamin K2 and a preparation method and application thereof.Raw materials of the low-temperature fermented milk rich in vitamin K2 comprise cow milk, sugar and a fermentation inoculant, the fermentation inoculant comprises lactococcus lactis subsp. Lactis MO-3 and lactococcus lactis HB-3, and the lactococcus lactis subsp. Lactis MO-3 and the lactococcus lactis HB-3 are compounded, so that the low-temperature fermented milk rich in vitamin K2 is obtained. The product texture can be effectively and obviously improved, the problem of granular caking is avoided, and meanwhile, the label cleaning attribute of the product is kept. The streptococcus salivarius subsp. Thermophilus and / or lactobacillus delbrueckii subsp. Bulgaricus are / is further compounded, so that the viable count content of the product can be increased, the requirements of the national standard GB 19302 can be effectively met, the fermentation time can be shortened, the production efficiency can be improved, and the flavor characteristics of the product can be enhanced. The invention also provides a new application of effectively improving the bone mineral density by increasing the content of vitamin K2 in the dairy product.

Description

Technical Field

[0001] The invention relates to the field of fermented milk, and in particular to low-temperature fermented milk rich in vitamin K2, and a preparation method and application thereof. Background Art

[0002] Vitamin K2 is an important fat-soluble coagulation vitamin that has a certain impact on bone health and cardiovascular disease. It not only promotes calcium absorption and plays an important role in maintaining bone health, but also keeps arteries clean and unobstructed. It is called a "revolutionary vitamin" and is also known as a "platinum vitamin" because of its extremely low content in food. Although the existing technology discloses that vitamin K2 plays an important role in maintaining bone health, it can be seen from the existing technology that it only promotes calcium absorption in bones to maintain bone health. And vitamin K2 has not yet been applied to dairy products, so there is no research to prove what effect vitamin K2 in dairy products will have.

[0003] At the same time, the vitamin K2 currently disclosed mainly comes from chemical synthesis and microbial fermentation. Chemical synthesis is easily affected by factors such as limited sources of precursor raw materials, the production of various by-products and low yields. Therefore, microbial fermentation is becoming more and more popular and has broad application prospects.

[0004] At present, the microorganisms that can synthesize vitamin K2 include lactic acid bacteria, Bacillus natto, and Flavobacterium. Currently, only lactic acid bacteria can be used in dairy products. Studies have shown that Lactococcus lactis can produce vitamin K2 during the fermentation process, but the product may appear granular after the fermentation is completed.

[0005] GB 19302 "National Food Safety Standard Fermented Milk" requires that the lactic acid bacteria in fermented milk products should be greater than 10 6 CFU / g(mL), and the test method is GB 4789.35 "Food Microbiology Test - Test for Lactic Acid Bacteria"; however, GB 4789.35 only stipulates the test methods for Bifidobacterium, Lactobacillus and Streptococcus thermophilus, and cannot test the number of live lactococci. As a result, when fermenting with Lactococcus lactis, the lactic acid bacteria count requirement in GB 19302 cannot be met. Summary of the invention

[0006] Therefore, the first technical problem to be solved by the present invention is that although the fermented milk prepared by Lactococcus lactis on the market can produce vitamin K2, its product texture may be granular, especially when fermented by Lactococcus lactis subspecies MO-3, the product texture appears obviously granular, so as to provide a low-temperature fermented milk rich in vitamin K2 and a preparation method thereof to solve the above problems.

[0007] To this end, the present invention provides the following technical solutions:

[0008] The invention discloses low-temperature fermented milk rich in vitamin K2. The raw materials include cow's milk, sugar and fermentation bacteria. The fermentation bacteria include Lactococcus lactis subspecies MO-3 and Lactococcus cremoris HB-3.

[0009] Lactococcus lactis subsp. lactis MO-3 was purchased from Chr. Hansen Trading Co., Ltd., and Lactococcus cremoris HB-3 was purchased from Chr. Hansen Trading Co., Ltd.

[0010] The addition amount of Lactococcus lactis subspecies MO-3 is 50-150 U / T, and the addition amount of Lactococcus cremoris HB-3 is 40-120 U / T;

[0011] And / or, the mass ratio of milk to sugar in the raw material is (92-95):(5-8).

[0012] The sugar is white granulated sugar.

[0013] The second technical problem to be solved by the present invention is that the existing fermented milk fermented with Lactococcus lactis cannot meet the lactic acid bacteria count requirements of GB 19302 standard, so as to provide a low-temperature fermented milk rich in vitamin K2 and a preparation method thereof to solve the above problem.

[0014] To this end, the present invention provides the following technical solutions:

[0015] A low-temperature fermented milk rich in vitamin K2, the raw materials of which include cow's milk, sugar and fermentation bacteria, the fermentation bacteria at least include Lactococcus lactis subspecies MO-3, and the fermentation bacteria also include Streptococcus salivarius subspecies thermophilus and / or Lactobacillus delbrueckii subspecies bulgaricus.

[0016] The above-mentioned Streptococcus salivarius subsp. thermophilus can be Streptococcus salivarius subsp. thermophilus Y-3, or it can be a composite strain Premium1.0 containing Streptococcus salivarius subsp. thermophilus; the above-mentioned Lactobacillus delbrueckii subsp. bulgaricus can be Lactobacillus delbrueckii subsp. bulgaricus P233, or it can be a composite strain Premium1.0 containing Lactobacillus delbrueckii subsp. bulgaricus; the Premium1.0 is a composite strain of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0017] Streptococcus salivarius thermophilic subspecies Y-3 was purchased from Mengniu Hi-Tech Dairy (Beijing) Co., Ltd., Lactobacillus delbrueckii subspecies bulgaricus P233 was purchased from Mengniu Hi-Tech Dairy (Beijing) Co., Ltd., and the composite strain Premium 1.0 was purchased from Chr. Hansen Trading Co., Ltd.

[0018] The fermentation bacteria include Lactococcus lactis subspecies MO-3, Lactococcus cremoris HB-3 and Premium 1.0.

[0019] The addition amount of the Lactococcus lactis subspecies MO-3 is 50-150 U / T, the addition amount of the Lactococcus cremoris HB-3 is 40-120 U / T, and the addition amount of Premium 1.0 is less than 15 U / T.

[0020] The addition amount of the Premium 1.0 is preferably 5 to 15 U / T.

[0021] The sugar is sucrose;

[0022] And / or, the mass ratio of milk to white sugar in the raw materials is (92-95):(5-8).

[0023] A method for preparing low-temperature fermented milk rich in vitamin K2, comprising:

[0024] Mixing: Mix milk and sugar evenly to obtain a premix;

[0025] Sterilization: sterilize the premix;

[0026] Fermentation: Add fermentation bacteria to the sterilized premix and obtain low-temperature fermented milk after fermentation.

[0027] In the fermentation step, the fermentation temperature is 28-30°C, and / or the pH value at the fermentation end point is 4.50-4.70, preferably 4.60±0.05;

[0028] And / or, the sterilization method is pasteurization.

[0029] The third technical problem to be solved by the present invention is to develop a new application of vitamin K2 in dairy products. Specifically, the present invention provides an application of a dairy product rich in vitamin K2 in the preparation of a product for improving bone density.

[0030] Furthermore, the dairy product is yogurt or milk, preferably low-temperature fermented milk; more preferably, the content of vitamin K2 in the dairy product is 8-10 μg / 100g.

[0031] The technical solution of the present invention has the following advantages:

[0032] 1. The present invention provides a low-temperature fermented milk rich in vitamin K2, which is different from the conventional vitamin K2 added as a nutritional supplement through chemical synthesis. The vitamin K2 in the low-temperature fermented milk of the present invention is directly produced by fermenting cow milk with Lactococcus lactis subspecies MO-3 to promote calcium absorption. Therefore, the vitamin K2 in the low-temperature fermented dairy product does not have the defects of chemically synthesized vitamin K2;

[0033] At the same time, when Lactococcus lactis subspecies MO-3 is used to ferment cow's milk to directly produce vitamin K2, the product texture is prone to become granular. In the process of dairy product development, in order to solve the problem of granular agglomeration in the product texture, the conventional method is to add stabilizers or add strains containing more extracellular polysaccharides to improve the product texture. However, the inventors found in the research and development process that even if Streptococcus salivarius thermophilic subspecies Y-3 that can produce more extracellular polysaccharides is added, it cannot achieve better improvement. However, through the cooperation of specific strains Lactococcus cremoris HB-3 and Lactococcus lactis subspecies MO-3, the product texture can be effectively improved, the problem of granular agglomeration can be avoided, and the clean label attribute of the product can be maintained.

[0034] 2. The present invention provides a low-temperature fermented milk rich in vitamin K2. Since the low-temperature fermented milk rich in vitamin K2 is prepared by adding Lactococcus lactis subspecies MO-3 and Lactococcus creamer HB-3, wherein the fermented milk obtained by fermenting Lactococcus lactis subspecies MO-3 and Lactococcus creamer HB-3 cannot meet the number of lactic acid bacteria required by GB 19302 standard, the present invention further improves the number of lactic acid bacteria in the product by compounding Streptococcus salivarius thermophilus subspecies and / or Lactobacillus delbrueckii subspecies bulgaricus, thereby effectively meeting the requirements of national standard GB 19302; at the same time, the present invention can not only improve the number of live bacteria in the product by adding Streptococcus salivarius thermophilus subspecies and / or Lactobacillus delbrueckii subspecies bulgaricus, but also shorten the fermentation time, increase the production efficiency, and enhance the flavor characteristics of the product.

[0035] 3. The low-temperature fermented milk rich in vitamin K2 provided by the present invention, when meeting the requirements of national standard GB 19302, needs to additionally add thermophilic Streptococcus salivarius subsp. thermophilus and / or Lactobacillus delbrueckii subsp. bulgaricus. The additionally added thermophilic Streptococcus salivarius subsp. thermophilus and / or Lactobacillus delbrueckii subsp. bulgaricus will significantly affect the content of vitamin K2 produced by fermentation of Lactococcus lactis subsp. lactis MO-3, resulting in a significant decrease in the content of vitamin K2 in the fermented milk; therefore, in order to further solve the problem that the content of vitamin K2 in the fermented milk is significantly reduced after adding thermophilic Streptococcus salivarius subsp. thermophilus and / or Lactobacillus delbrueckii subsp. bulgaricus, the present invention preferably uses a composite strain Premium 1.0 having both thermophilic Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and optimizes the addition amount of Premium 1.0, reducing its addition amount to less than 15 U / T, which can meet the requirements of national standard GB 19302 while not significantly affecting the content of vitamin K2 in the fermented milk, and has a significant effect.

[0036] 4. The present invention provides an application of a vitamin K2-rich dairy product in the preparation of a product for improving bone density, so that the dairy product is enriched with vitamin K2, which can effectively achieve the effect of improving bone density; specifically, the effect of the technical solution is verified using a zebrafish model, and a normal control group and a model control group are set up. Compared with the model control group, when the dairy product is enriched with 8-10 μg / 100g of vitamin K2, the fluorescence intensity of the skull can be significantly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 This is a comparison diagram of tissue states of Group 1 and Group 3 in Example 1 of the present invention;

[0039] Figure 2 The fluorescence intensity microphotographs of the skulls of individual zebrafish in each group in Example 4 of the present invention are shown;

[0040] Figure 3 This is a comparison diagram of the fluorescence intensity of the skulls of zebrafish in each group whose dairy product is milk in Example 4 of the present invention;

[0041] Figure 4 This is a comparison chart of the fluorescence intensities of the skulls of zebrafish in each group whose dairy product is low-temperature fermented milk in Example 4 of the present invention. DETAILED DESCRIPTION

[0042] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0043] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0044] The raw materials used in the present invention are all conventional commercially available raw materials, and the percentages of the raw materials are all mass percentages.

[0045] Embodiment 1:

[0046] A low-temperature fermented milk rich in vitamin K2 is fermented with the formula in Table 1 as a fermentation base material, and is fermented at 30° C. to a fermentation end point pH of about 4.6, with a pH deviation of no more than ±0.05.

[0047] Table 1

[0048]

[0049] The tissue status and extracellular polysaccharide content of each group in Table 1 above were observed and tested, and the results are shown in Tables 2 and Figure 1 shown.

[0050] Table 2

[0051]

[0052]

[0053] In the above Table 2, “-” means not detected.

[0054] The growth of lactic acid bacteria during the fermentation process is generally accompanied by the production of exopolysaccharides (EPS). Exopolysaccharides are a good natural biological thickener that can increase the viscosity and water retention of dairy products, thereby improving the texture of the product. However, through the above-mentioned results of the tissue state and exopolysaccharide content of low-temperature fermented milk, it can be seen that the exopolysaccharide content of the thermophilic Streptococcus salivarius subspecies Y-3 is the highest, but its effect on improving the tissue state is not obvious. Therefore, it is not the case that adding strains that produce more exopolysaccharides can better improve the tissue state of low-temperature fermented milk. The present invention can only effectively improve the tissue state so that it does not show a granular feel by combining specific strains of Lactococcus creamerii HB-3 with Lactococcus lactis subspecies MO-3. Figure 1As shown, the Figure 1 (a) is the organizational status diagram of group 1, and (b) is the organizational status diagram of group 3.

[0055] Embodiment 2:

[0056] A low-temperature fermented milk rich in vitamin K2 is fermented with the formula in Table 3 as a base material and fermented at 30° C. to a fermentation end point of pH 4.6.

[0057] Table 3

[0058]

[0059] The number of lactic acid bacteria (CFU / g) in each group in Table 3 above was tested, and the results are shown in Table 4 below.

[0060] Table 4

[0061] Fermentation time Number of lactic acid bacteria (CFU / g) Group 3 9.0h / Group 4 7.9h / Group 9 8.5h <![CDATA[3.2×10 8 ]]> Group 10 7.5h <![CDATA[1.9×10 8 ]]> Group 11 8.5h <![CDATA[1.1×10 8 ]]> Group 12 7.4h <![CDATA[1.7×10 8 ]]> Group 13 8.2h <![CDATA[3.1×10 8 ]]> Group 14 7.5h <![CDATA[4.4×10 8 ]]>

[0062] In the above Table 4, “ / ” means not detected.

[0063] The test results of the lactic acid bacteria count (CFU / g) of the low-temperature fermented milk show that the lactic acid bacteria count of the product can be effectively increased by compounding Streptococcus salivarius thermophilus subspecies and / or Lactobacillus delbrueckii subspecies bulgaricus, thereby meeting the requirements of national standard GB19302. At the same time, the present invention can not only increase the content of viable bacteria count of the product by adding Streptococcus salivarius thermophilus subspecies and / or Lactobacillus delbrueckii subspecies bulgaricus, but also shorten the fermentation time, increase production efficiency, and enhance the flavor characteristics of the product.

[0064] Embodiment 3:

[0065] A low-temperature fermented milk rich in vitamin K2 is fermented with the formula in Table 5 as a fermentation base material and fermented at 30° C. to a fermentation end point of pH 4.6.

[0066] Table 5

[0067]

[0068] The number of lactic acid bacteria (CFU / g) and the content of vitamin K2 (μg / 100g) in each group in Table 5 above were tested, and the results are shown in Table 6 below.

[0069] Table 6

[0070] <![CDATA[Vitamin K2 content (μg / 100g)]]> Number of lactic acid bacteria (CFU / g) Group 3 11 / Group 4 9 / Group 13 7 <![CDATA[3.1×10 8 ]]> Group 14 6 <![CDATA[4.4×10 8 ]]> Group 15 10 <![CDATA[1.3×10 8 ]]> Group 16 8 <![CDATA[2.4×10 8 ]]>

[0071] In the above Table 6, “ / ” means not detected.

[0072] The test results of the lactic acid bacteria count (CFU / g) and the vitamin K2 content (μg / 100g) of the low-temperature fermented milk show that when the addition amount of thermophilic Streptococcus salivarius and Lactobacillus delbrueckii subspecies bulgaricus is too high, the growth of lactococci will be inhibited, thereby affecting the product flavor and vitamin K2 content; the present invention reduces the addition amount of the composite strain Premium1.0 composed of thermophilic Streptococcus salivarius and Lactobacillus delbrueckii subspecies bulgaricus to less than 15U / T, which does not significantly affect the vitamin K2 content in the fermented milk. At the same time, based on the requirements of the number of lactic acid bacteria in the national standard GB 19302, when the addition amount of Premium1.0 is guaranteed to be above 5U / T, the requirement of the number of lactic acid bacteria in the fermented milk being greater than 10 can be effectively met. 6 The requirements of the national standard GB 19302 for CFU / g(mL).

[0073] Embodiment 4:

[0074] A use of a vitamin K2-rich dairy product in the preparation of a product for improving bone density, comprising the following steps: testing bone density using a normal control group, an experimental group 1-3, a comparison group 1-2, chondroitin sulfate, and a model control group. The specific settings are as follows:

[0075] 4.1. Sample information

[0076] Chondroitin sulfate: chondroitin sulfate A sodium salt (hereinafter referred to as chondroitin sulfate), the solvent is ultrapure water;

[0077] Model control group: ultrapure water;

[0078] Comparative group 1: pure milk;

[0079] Comparative group 2: low-temperature fermented milk; the low-temperature fermented milk is: the same formula as group 15 is used, and only 5-10 U / T of Premium 1.0 is added as a starter and fermented under the same conditions to obtain the low-temperature fermented milk;

[0080] Implementation group 1: pure milk containing vitamin K2; the pure milk containing vitamin K2 is: vitamin K2 is added to the pure milk of control group 1 so that the content reaches 8-10 μg / 100g;

[0081] Implementation group 2: low-temperature fermented milk containing vitamin K2; the low-temperature fermented milk containing vitamin K2 is: vitamin K2 is added to the low-temperature fermented milk of control group 2 so that the content reaches 8-10 μg / 100g;

[0082] Implementation Group 3: Low-temperature fermented milk containing vitamin K2; The low-temperature fermented milk containing vitamin K2 is: low-temperature fermented milk corresponding to Group 15 in Example 3, with a vitamin K2 content of about 8-10 μg / 100g.

[0083] 4.2 Experimental Animals

[0084] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458), IACUC ethics review number: IACUC-2023-7319-01.

[0085] 4.3 Instruments, consumables and reagents

[0086] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); electric focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); precision electronic balance (CP214, OHAUS, America); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China).

[0087] Sodium percarbonate (Shanghai Aladdin Biochemical Technology Co., Ltd., China); dexamethasone (Shanghai Aladdin Biochemical Technology Co., Ltd., China); chondroitin sulfate A sodium salt (Shanghai Aladdin Biochemical Technology Co., Ltd., China); Alizarin red (Shanghai MacLean Biochemical Technology Co., Ltd., China).

[0088] 4.4 Evaluation of bone density enhancement efficacy

[0089] 3dpf wild-type AB strain zebrafish were randomly selected in beakers, 30 zebrafish were treated in each beaker, and sodium percarbonate with a concentration of 100μg / mL was added as an oxygenator, and the capacity of each beaker was 20mL. Except for the normal control group, the remaining groups were given dexamethasone to establish a zebrafish osteoporosis model; dexamethasone was given at the same time as the corresponding samples of each group, wherein the amount of dexamethasone given was 2μΜ, the amount of chondroitin sulfate given was 500μg / mL, and the sample amount in the implementation group 1-3 and the comparison group 1-2 was 33.3mg / mL.

[0090] After 4 days of treatment at 28°C, samples were collected and stained with alizarin red. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The photographic results are shown in the figure. Figure 2As shown, NIS-Elements D3.20 advanced image processing software was used to analyze and collect data, analyze the fluorescence intensity of zebrafish skull, and use the statistical analysis results of this indicator to evaluate the efficacy of samples in enhancing bone density. The statistical analysis results are expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis between the two groups. The analysis results are shown in Figure 3-Figure 4 As shown, p < 0.05 indicates that the difference is statistically significant. Figure 3 and Figure 4 * indicates significant difference compared with the model control group. Figure 3 A in the middle indicates significant difference compared with comparison group 1. Figure 4 B in the middle indicates a significant difference compared with control group 2. Figure 4 C in the middle indicates a significant difference compared with implementation group 2.

[0091] 4.5 Results Analysis

[0092] The higher the absolute value of skull fluorescence intensity, the better the effect of enhancing bone density.

[0093] pass Figure 3 It can be seen that the fluorescence intensity of the skull of the zebrafish model control group is lower than that of the normal control group. After taking chondroitin sulfate, the fluorescence intensity of the skull of the zebrafish is improved, which proves that the zebrafish osteoporosis model of the present invention is successfully established. Figure 3 Comparison of the data between implementation group 1 and control group 1 shows that the fluorescence intensity of the zebrafish skull was significantly improved in the pure milk with added vitamin K2 in implementation group 1 compared with the pure milk in control group 1, proving that adding vitamin K2 to milk can significantly increase bone density.

[0094] Figure 4 Compared with the low temperature fermented milk in the control group 2 Figure 3 The milk in the comparison group 1 also significantly increased the fluorescence intensity of the zebrafish skull. The results showed that fermented milk has a higher effect on improving bone density than milk, which may be due to the lactic acid produced by milk during the fermentation process, which is more conducive to the absorption and utilization of calcium. Figure 4 The data comparison between implementation group 2 and control group 2 showed that the fluorescence intensity of zebrafish skulls was significantly improved compared with the low-temperature fermented milk in control group 1, which proved that adding vitamin K2 to low-temperature fermented milk can significantly improve bone density. Figure 4Comparison of the data between implementation group 3 and implementation group 2 shows that the fluorescence intensity of zebrafish skulls was significantly improved in low-temperature fermented milk with endogenous fermentation to produce vitamin K2 compared with low-temperature fermented milk with exogenous vitamin K2 added in implementation group 2, proving that low-temperature fermented milk with endogenous fermentation to produce vitamin K2 can significantly increase bone density. The reason why the bone density of implementation group 3 was significantly higher than that of implementation group 2 may be that the vitamin K2 produced by endogenous fermentation of the strain is more stable and is not affected by heat treatment during processing, and certain metabolites can be produced during the fermentation process to maintain the stability of vitamin K2.

[0095] Obviously, the above embodiments are only examples for clear explanation, and are not intended to limit the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A low-temperature fermented milk rich in vitamin K2, characterized in that: The raw materials include milk, sugar and fermentation bacteria, and the fermentation bacteria include Lactococcus lactis subspecies MO-3 and Lactococcus cremoris HB-3.

2. The low-temperature fermented milk according to claim 1, characterized in that The addition amount of Lactococcus lactis subspecies MO-3 is 50-150 U / T, and the addition amount of Lactococcus cremoris HB-3 is 40-120 U / T; And / or, the mass ratio of milk to sugar in the raw material is (92-95):(5-8); And / or, the sugar is sucrose.

3. A low-temperature fermented milk rich in vitamin K2, characterized in that: The raw materials include milk, sugar and fermentation bacteria, wherein the fermentation bacteria at least include Lactococcus lactis subspecies MO-3, and the fermentation bacteria further include Streptococcus salivarius subspecies thermophilus and / or Lactobacillus delbrueckii subspecies bulgaricus.

4. The low-temperature fermented milk according to claim 3, characterized in that The fermentation agent includes Lactococcus lactis subspecies MO-3, Lactococcus cremoris HB-3 and Premium 1.0; the Premium 1.0 is a composite strain of Streptococcus salivarius thermophilus subspecies and Lactobacillus delbrueckii subspecies bulgaricus; And / or, the sugar is sucrose; the mass ratio of milk to sucrose in the raw material is (92-95):(5-8).

5. The low-temperature fermented milk according to claim 4, characterized in that: The addition amount of the lactococcus lactis subspecies MO-3 is 50-150 U / T, the addition amount of the lactococcus cremoris HB-3 is 40-120 U / T, and the addition amount of Premium 1.0 is above 5 U / T.

6. The low-temperature fermented milk according to claim 5, characterized in that: The addition amount of the Premium 1.0 is 5 to 15 U / T.

7. The method for preparing low-temperature fermented milk rich in vitamin K2 according to any one of claims 1 to 6, characterized in that: include: Mixing: Mix milk and sugar evenly to obtain a premix; Sterilization: sterilize the premix; Fermentation: Add fermentation bacteria to the sterilized premix and obtain low-temperature fermented milk after fermentation.

8. The preparation method according to claim 7, characterized in that: In the fermentation step, the fermentation temperature is 28-30°C, and / or the pH value at the fermentation end point is 4.50-4.70; And / or, the sterilization method is pasteurization.

9. Use of a dairy product rich in vitamin K2 in preparing a product for improving bone density.

10. The use according to claim 9, characterized in that: The dairy product is the low-temperature fermented milk according to any one of claims 1 to 6, and preferably, the content of vitamin K2 in the dairy product is 8-10 μg / 100g.

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