A method for preparing high-quality fatty acids in cow milk and application thereof

Abstract

The application discloses a preparation method of high-quality fatty acid in cow milk and application thereof, and comprises the following steps: inoculating lactococcus lactis in sterilized cow milk to obtain cow milk with high-quality fatty acid after fermentation. The method has the advantages that the content of medium-chain fatty acid in dairy products is improved by endogenous enzyme of the strain, and the content of octanoic acid and decanoic acid is improved without changing the existing process.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a method for preparing high-quality fatty acids in milk and its application. Background Technology

[0002] Fatty acids are a class of compounds composed of carbon, hydrogen, and oxygen, and are the main components of neutral fats, phospholipids, and glycolipids.

[0003] Based on carbon chain length and degree of saturation, fatty acids can be classified into short-chain, medium-chain, and long-chain fatty acids, as well as saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, for example: Short-chain fatty acids: These have fewer than 6 carbon atoms in their carbon chain and are also called volatile fatty acids, such as butyric acid and hexanoic acid.

[0004] Medium-chain fatty acids: These have 6-12 carbon atoms in their carbon chain and are mainly composed of caprylic acid and capric acid.

[0005] Long-chain fatty acids: those with more than 12 carbon atoms in their carbon chain, such as palmitic acid, stearic acid, and oleic acid.

[0006] Saturated fatty acids: have no unsaturated bonds on their carbon and hydrogen atoms, such as palmitic acid and stearic acid.

[0007] Monounsaturated fatty acids: Their hydrocarbon chain has one unsaturated bond, such as oleic acid.

[0008] Polyunsaturated fatty acids: Their hydrocarbon chains have two or more unsaturated bonds, such as linoleic acid and linolenic acid.

[0009] In recent years, medium-chain fatty acids have attracted much attention due to their unique physiological functions. Compared with common long-chain fatty acids, medium-chain fatty acids have a more direct metabolic pathway in the human body, mainly manifested in the following physiological functions: (1) Rapid energy absorption and supply: Absorption pathway: It does not rely on carnitine transport; after absorption, it enters the liver directly via the portal vein, rather than through the lymphatic system.

[0010] Oxidation efficiency: It can be rapidly oxidized and decomposed in the liver to provide energy. Its metabolic rate is significantly faster than that of long-chain fatty acids, and it is not easy to accumulate in large quantities in adipose tissue.

[0011] (2) Health regulation effect: Immunity and inflammation: As an endogenous ligand of the GPR84 receptor, medium-chain fatty acids participate in regulating immune cell function and inflammatory responses, and are potential therapeutic targets for various inflammatory diseases.

[0012] (3) Cognition and aging: A study published in Nature in 2026 pointed out that medium-chain fatty acid metabolites produced by gut bacteria may be related to memory function in the aging process by activating gut immune cells and affecting vagal nerve signals.

[0013] (4) Gut health: Adequate intake helps maintain the intestinal mucosal barrier function and reduces the probability of inflammatory response.

[0014] Probiotic fermentation can effectively produce organic acids, but most are short-chain fatty acids. Only a few reports indicate that it can ferment to produce medium-chain fatty acids. Patent CN201810864199.0, "A High-Octaic Acid-Producing Lactobacillus helveticus and Its Applications," discloses a high-octanoic acid-producing Lactobacillus helveticus strain and its applications. This strain produces acid rapidly, with an octanoic acid yield of 66.47 μg / kg in 10 hours, showing potential as a primary fermenting agent. Furthermore, this strain can produce high levels of characteristic flavor compounds during the fermentation of skim milk, accounting for 7.01% of all volatile flavor compounds, giving dairy products an appealing, light aroma. However, this patent does not address the content of decanoic acid, which differs from the innovation of this patent. Patent CN116769635A, "A Strain of Enterococcus thamnoides and Its Applications," specifically relates to a high-octanoic acid-producing Enterococcus thamnoides strain and its applications. The strain was selected from high-quality cellar mud and is adapted to the environment of baijiu brewing. Experiments showed that when the strain fermented at 37℃ for 72 hours, the octanoic acid yield reached 5.45 g / L, which is higher than the octanoic acid yield of existing strains. Therefore, it can be used as an enhancing agent in baijiu brewing. The strain involved in this patent is not included in the "List of Edible Fungi" published by the National Health Commission, and it is mainly used in baijiu, which is inconsistent with the content of this patent.

[0015] While some reports suggest that lactic acid bacteria enhance the synthesis of high-quality fatty acids, this has proven difficult to implement effectively in actual dairy production. Adding genetically modified microorganisms, though effective, faces regulatory hurdles, raising consumer concerns about product quality and further increasing costs. Therefore, utilizing fermented probiotics to produce high-quality fatty acids without altering existing processes can effectively enhance the nutritional value of milk while also improving its flavor and texture.

[0016] Currently, there is limited research on the effective production of high-quality fatty acids, especially medium-chain fatty acids, by lactic acid bacteria. The main reason is that there are still shortcomings in practical applications: (1) There is a lack of high-quality fatty acid production in actual fermentation of dairy products; (2) The preparation of high-quality fatty acid production processes for dairy fermentation is complex, and there is currently a lack of a safe and rapid alternative method. Summary of the Invention

[0017] To address the above problems, this invention provides a method for preparing high-quality fatty acids in milk and its application.

[0018] The invention aims to achieve the following: a method for preparing high-quality fatty acids in milk, comprising the following steps: inoculating sterilized milk with Lactococcus lactis and fermenting it to obtain milk with enhanced high-quality fatty acids.

[0019] A type of lactococcus, the strain being *Lactococcus lactis* ( Lactococcus lactis The strain name is INM3210, the strain accession number is GDMCCNo.68612, the accession date is July 14, 2026, the depositary institution is Guangdong Provincial Microbial Culture Collection Center, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0020] Furthermore, the colony characteristics are as follows: After 48 hours of culture on solid M17 agar plates, the colony diameter reaches 2-3 mm, the colonies are milky white, raised, round, smooth, and have neat edges.

[0021] An application of Lactococcus lactis in increasing lactic acid content in fermented dairy products.

[0022] An application of Lactococcus lactis in increasing the caprylic acid content in fermented dairy products.

[0023] An application of Lactococcus lactis in increasing the decanoic acid content in fermented dairy products.

[0024] An application of Lactococcus lactis in the production of a starter culture.

[0025] The advantages are: without changing the existing process, the content of medium-chain fatty acids in dairy products is improved by using endogenous enzymes from the bacterial strain, increasing the content of caprylic and capric acids. This not only enhances the functionality of fermented dairy products but also improves their taste. Attached Figure Description

[0026] Figure 1 This is a flowchart of the screening process for Lactococcus lactis.

[0027] Figure 2 This is a colony diagram of Lactococcus lactis.

[0028] Figure 3 This is a Gram staining observation of Lactococcus lactis.

[0029] Figure 4 This is the liquid phase diagram for Example 3. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments: Unless otherwise specified, the experimental methods used in the following implementation examples are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified.

[0031] Example 1, Appendix Figure 1-3 A method for preparing high-quality fatty acids in milk includes the following steps: inoculating sterilized milk with Lactococcus lactis and fermenting it to obtain milk with enhanced high-quality fatty acids.

[0032] A type of lactococcus, the strain being *Lactococcus lactis* ( Lactococcus lactis The strain name is INM3210, the strain accession number is GDMCCNo.68612, the accession date is July 14, 2026, the depositary institution is Guangdong Provincial Microbial Culture Collection Center, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0033] A type of lactococcus lactis has the following colony characteristics: after culturing on solid M17 agar plates for 48 hours, the colony diameter reaches 2-3 mm, the colony is milky white, raised, round, smooth, and has neat edges.

[0034] An application of Lactococcus lactis in increasing lactic acid content in fermented dairy products.

[0035] An application of Lactococcus lactis in increasing the caprylic acid content in fermented dairy products.

[0036] An application of Lactococcus lactis in increasing the decanoic acid content in fermented dairy products.

[0037] The Lactococcus lactis strain described in this invention can effectively increase the content of caprylic acid and capric acid in cow's milk.

[0038] The Lactococcus lactis strain described in this invention enhances the flavor and texture of fermented milk products.

[0039] The Lactococcus lactis strain described in this invention improves the content of medium-chain fatty acids in dairy products and increases the content of caprylic acid and capric acid through endogenous enzymes in the strain.

[0040] The screening method for Lactococcus lactis of the present invention is universal and can be widely used in the screening of strains to increase the content of medium-chain fatty acids in milk.

[0041] The lactococcus lactis strain provided by this invention can enhance the flavor of fermented milk, and the fermented dairy products will not have any off-flavors. It can be mixed with other products, thus preserving the nutrition and flavor of the dairy products while enhancing their functionality.

[0042] According to the production process of fermented milk products, the inoculation amount in dairy products is 10. 4 -10 8 After fermentation with cfu / mL Lactococcus lactis for 4-6 hours at 37-42℃, the levels of caprylic acid and capric acid in the fermented milk were measured, and the fermented milk product underwent sensory evaluation.

[0043] Liquid M17 medium: a commercial product.

[0044] Solid M17 medium: Add 2% agar to liquid M17 medium.

[0045] The screening medium was sterilized fresh milk (sterilization conditions: 95℃, 15 min), cooled to room temperature.

[0046] 1. Preliminary screening of bacterial strains The thermophilic subspecies of Streptococcus salivarius INM25-ST has been accessed under the preservation number CGMCCNO.15446 and has been published under the patent: 201910707050.6; Lactobacillus delbrueckii subsp. bulgaricus INM25-LB has been accessed under CGMCC No. 15445 and is published under patent number 201910694197.6. Eight strains of *Lactococcus lactis* preserved in the laboratory were inoculated into liquid M17 medium at a 1% inoculum concentration and cultured at 37°C for 24 hours. Then, at a 1% inoculum concentration, *Lactobacillus delbrueckii* subsp. bulgaricus INM25-LB and *Streptococcus salivarius* subsp. thermophilicus INM25-ST were inoculated into sterilized fresh milk and cultured statically at 37°C for 4–6 hours until the final bacterial count reached 10⁻⁶. 8 CFU / mL. After post-fermentation at 4℃ for 6 hours, 1.0 mL of fermented milk was added to an equal volume of sterile water, mixed well, and centrifuged at 10000 rpm for 10 minutes. The supernatant was obtained as the sample. After filtering through a 0.22 μm aqueous filter membrane, physicochemical indicators were tested. The screening procedure is as follows: Figure 1 The initial screening results are shown in Table 1.

[0047] High performance liquid chromatography (HPLC) detection: flow rate 1 mL / min; analytical column, CAPCELLPAKADME-HRS5, 5 μm, 4.6 × 250 mm; temperature, 35 ℃; gradient conditions: 10%-100% acetonitrile-water (0.2% phosphoric acid), 30 min; absorption detection wavelength: 215 nm.

[0048] Table 1. Content of caprylic and capric acid in fermented fresh milk

[0049] Strains NM0101, NM0107, and NM0108, which simultaneously produce high levels of both caprylic and capric acid, were selected for secondary screening.

[0050] 3. Secondary screening of strains The strains NM0101, NM0107, and INM0108 selected in the initial screening were reactivated and cultured. Following the initial screening method and referring to the national standard GB19302-2025, flavored yogurt was produced and subjected to sensory evaluation. The results are shown in Table 2. Table 2 Sensory Analysis

[0051] Note: Each indicator has a maximum score of 5 points, and the higher the score, the better the result.

[0052] As shown in Table 2, NM0101 had the best sensory evaluation results. As a target strain for secondary screening, its morphological observation on solid M17 agar plates revealed colonies with a diameter of 1-2 mm, a milky-white color, raised and round shape, a smooth surface, and neat edges. The results are as follows: Figure 2 Gram staining followed by microscopic observation yielded the following results: Figure 3 As shown.

[0053] The NM0101 strain obtained through the above screening was identified by 16S sequencing. Comparative analysis showed that the 16S rRNA sequence of the strain obtained in this invention was similar to that of *Lactococcus lactis*. Lactococcus lactis With a similarity of 100.00%, this strain can be identified as *Lactococcus lactis*, suitable for use in the food fermentation industry. It is named *Lactococcus lactis*. Lactococcus lactis The strain, named INM3210, has the accession number GDMCC No. 68612, was deposited on July 14, 2026, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Therefore, in this application, INM3210 and NM0101 refer to the same strain. The NM0101 strain of this invention has been genetically identified, and its gene sequence is as follows: Example 2: Detection of the genetic stability of Lactococcus lactis INM3210 Lactococcus lactis INM3210 from Example 1 was passaged 10 times on screening medium. The samples from the 1st, 5th, and 10th generations were centrifuged at 10,000 rpm for 10 min, and the supernatant was used as the sample. Physicochemical indicators were analyzed, and the methods for determining octanoic acid and decanoic acid content were the same as in Example 1. Sensory evaluation was also performed to assess its passage stability; specific data are shown in Table 3.

[0054] Table 3. Results of the passaging stability test

[0055] Table 3 shows that the fermentation performance of Lactococcus lactis strains INM3210 from different generations was normal, the medium-chain fatty acid content varied within 10%, the genetic stability of the strains was good, and they met the requirements for production and use.

[0056] Example 3: Preparation of flavored fermented milk using Lactococcus lactis INM3210 Sterilized fresh milk was inoculated with Lactococcus lactis INM3210, and flavored fermented milk was prepared according to the national standard GB19302-2025. The concentration of the inoculated fermentation bacteria was 10... 4 -10 8 At CFU / mL, using NM0102 as the control strain, the concentrations of *Lactobacillus delbrueckii* subsp. bulgaricus INM25-LB and *Streptococcus salivarius* subsp. *thermophilus* INM25-ST were 1×10⁻⁶. 6 The method for detecting caprylic and capric acid (CFU / mL) in flavored fermented milk was described in Example 1, and the results are shown in Table 4. Table 4: Content of Caprylic and Capric Acid in Flavored Fermented Milk

[0057] Table 5 Sensory evaluation results of flavored fermented milk

[0058] Note: Each assessment item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all scores. The results in Tables 4 and 5 show that inoculating flavored fermented milk with *Lactococcus lactis* INM3210 significantly increases the content of caprylic and capric acids, and the higher the inoculation amount, the higher the content of caprylic and capric acids. However, excessively high or low inoculation concentrations of *Lactococcus lactis* INM3210 have a significant impact on the content of caprylic and capric acids, and also affect the sourness, sweetness, and taste of the flavored fermented milk. The optimal inoculation concentration is 1.0 × 10⁻⁶. 7 CFU / mL, this concentration is sufficient to produce a sufficient variety and quantity of high-quality fatty acids, as shown in the results. Figure 4It can also improve the flavor and texture of flavored fermented milk.

[0059] Example 4: Preparation of fermented milk-containing beverages using Lactococcus lactis INM3210 The concentration of Lactococcus lactis INM3210 was 10. 7 The inoculation concentration of fermented milk beverages was determined using a dose of CFU / mL. Another group was inoculated with NM0102 as a control group. The standards for preparation were: national standard GB19302-2025 "Dairy Beverages" and GB19302-2025 "Fermented Milk". The proportion of fermented milk was greater than 80%. The content of caprylic acid and capric acid in the fermented milk beverages was tested according to the method in Example 1. A professional evaluator was asked to conduct a sensory evaluation of the flavor of the flavored milk beverages. The results are recorded in Table 6.

[0060] Table 6. Detection and sensory evaluation results of fermented dairy beverages

[0061] Note: Each sensory evaluation item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all items. As shown in Table 6, inoculating fermented dairy beverages with 10... 7 Using CFU / mL Lactococcus lactis INM3210 to prepare fermented dairy beverages can significantly increase the content of caprylic and capric acids in fermented dairy beverages. In addition, it can improve the sensory evaluation of fermented dairy beverages, increase their aroma, enhance their flavor and taste, and make them fragrant, sweet and sour.

[0062] Example 5: Preparation of soft cheese using Lactococcus lactis INM3210 The concentration of Lactococcus lactis INM3210 was 10. 7 The inoculum concentration for soft cheese was determined using a dose of CFU / mL, and a control group was inoculated with NM0102. The production standard was based on the national standard GB5420-2021 "Cheese". The caprylic and capric acid content in the soft cheese was tested according to the method described in Example 1. Simultaneously, a professional evaluator conducted a sensory evaluation of the flavor of the soft cheese, and the results are recorded in Table 7.

[0063] Table 7. Detection and sensory evaluation results of soft cheeses

[0064] Note: Each sensory evaluation item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all items. As shown in Table 7, inoculating the raw milk with 10 7Using CFU / mL Lactococcus lactis INM3210 to make soft cheese can significantly increase the content of caprylic and capric acids in soft cheese. In addition, it can improve the sensory evaluation of soft cheese, increase its aroma, enhance its flavor and texture, and make the cheese fragrant and sweet and sour.

[0065] Example 6: Fermented butter made with Lactococcus lactis INM3210 The concentration of Lactococcus lactis INM3210 was 10. 7 The inoculum concentration for fermented butter was determined using a dose of CFU / mL, and a control group was inoculated with NM0102. The fermented butter production standard was based on the national standard GB19646-2025 "Whipped Cream, Butter and Anhydrous Butter." The caprylic and capric acid content in the fermented butter was tested according to the method described in Example 1. Simultaneously, a professional evaluator conducted a sensory evaluation of the fermented butter's flavor, and the results are recorded in Table 8.

[0066] Table 8. Detection and sensory evaluation results of caprylic and capric acids in fermented butter.

[0067] Note: Each sensory evaluation item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all items. As shown in Table 8, inoculating the raw milk with 10 7 Using CFU / mL Lactococcus lactis INM3210 to make fermented butter can significantly increase the content of caprylic and capric acid in fermented butter. In addition, it can improve the sensory evaluation of fermented butter, increase its aroma, enhance its flavor and taste, and make fermented butter fragrant and sweet and sour.

[0068] Based on the results of the above embodiments, the Lactococcus lactis INM3210 provided by the present invention can be applied to a variety of dairy products. Without changing the existing process, it can significantly increase the content of caprylic and capric acid in the relevant products, while also improving the aroma and taste of dairy products, thus significantly improving the quality of dairy products.

[0069] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, which fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention patent should be determined by the appended claims. The above descriptions are only preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in this invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of this invention.

Claims

1. A method for preparing high-quality fatty acids in milk, characterized in that: Includes the following steps: Milk with enhanced fatty acids is obtained by inoculating sterilized milk with Lactococcus lactis and fermenting it.

2. A lactococcus lactis strain as described in claim 1, characterized in that: The strain is Lactococcus lactis ( Lactococcus lactis The strain name is INM3210, the strain accession number is GDMCCNo.68612, the accession date is July 14, 2026, the depositary institution is Guangdong Provincial Microbial Culture Collection Center, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

3. The lactococcus lactis according to claim 3, characterized in that: The colony characteristics are as follows: After 48 hours of culture on solid M17 plate medium, the colony diameter reaches 2-3 mm. The colonies are milky white, raised, round, smooth, and have neat edges.

4. An application of the Lactococcus lactis according to claim 2 in increasing the lactic acid content in fermented dairy products.

5. An application of the Lactococcus lactis according to claim 2 in increasing the caprylic acid content in fermented dairy products.

6. An application of the Lactococcus lactis according to claim 2 in increasing the decanoic acid content in fermented dairy products.

7. An application of the Lactococcus lactis according to claim 2 in the preparation of a starter culture.

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