Microbial cell powder for mammals to produce milk rich in primary EPA (eicosapentaenoic acid) and preparation method
Microbial cell powder prepared by genetically engineered Yersinia lipase strains solves the problem of low EPA content in mammalian milk, achieving efficient synthesis and stable conversion of EPA, suitable for large-scale production and safe application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU HEVI BIOTECH CO LTD
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-10
AI Technical Summary
Existing technologies make it difficult to reliably supply EPA obtained from deep-sea fish. Chemical synthesis methods pose environmental pollution problems. EPA derived from microorganisms is easily decomposed and transformed in the digestive tract of mammals and is easily oxidized and decomposed during processing, resulting in low EPA content in milk and making it difficult to industrialize.
A genetically engineered strain of Yersinia lipolytica was used to prepare microbial cell powder through fermentation and inactivation. This powder was then added to mammalian feed. EPA in the form of triglycerides was efficiently synthesized in mammals and converted into milk. Emulsification and homogenization processes were employed to improve stability and dispersibility.
It significantly increases the EPA content in breast milk by 10-20 times, has high product stability, simple preparation process, controllable cost, and is suitable for large-scale production. EPA is easily absorbed and utilized by mammals after ingestion, and the EPA in breast milk is in its native form, which is easily absorbed by the human body.
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Figure CN121817328A_ABST
Abstract
Description
Technical Field
[0001] This invention specifically relates to a microbial cell powder that enables mammals to produce milk rich in native EPA and its preparation method. Background Technology
[0002] EPA, or eicosapentaenoic acid, is an important member of the Omega-3 unsaturated fatty acid family, possessing significant physiological functions and nutritional value. EPA effectively regulates lipid metabolism, lowers triglyceride and cholesterol levels in the blood, and reduces the risk of cardiovascular diseases such as atherosclerosis. Simultaneously, EPA plays a crucial role in anti-inflammation, regulating immune system function, and protecting nervous system health, making it significant for infant brain development, maintaining physical function in the elderly, and promoting health and well-being in the general population.
[0003] Currently, EPA is mainly obtained from deep-sea fish and their products. However, deep-sea fishery resources are limited, and overfishing can disrupt the marine ecological balance. Furthermore, the EPA content in fish products is greatly affected by factors such as species and growth environment, making it difficult to achieve a stable supply. In addition, the preparation of EPA through chemical synthesis methods has problems such as harsh reaction conditions, difficulty in controlling product purity, and environmental pollution, which limit its large-scale application.
[0004] While existing technologies have attempted to add microbially derived unsaturated fatty acids to dairy products, most methods involve direct addition after oil extraction or microencapsulation. These methods have several drawbacks: firstly, the extracted EPA oil is unstable and easily oxidized and decomposed during processing due to factors such as temperature, pressure, and oxygen, leading to the loss of active ingredients and off-flavors; secondly, directly added EPA is easily decomposed and converted in the digestive tract of mammals (especially ruminants), making it difficult to effectively deposit into milk, and the processing costs during addition are high, hindering industrial-scale promotion. Summary of the Invention
[0005] The purpose of this invention is to provide a microbial cell powder and its preparation method for enabling mammals to produce milk rich in native EPA. This microbial cell powder enables mammals to efficiently absorb and utilize EPA, significantly increasing the native EPA content in milk. It is also stable, has a simple preparation process, and controllable production costs, meeting the needs of large-scale production and market application.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a microbial cell powder that enables mammals to produce milk rich in native EPA, formulated from the following raw materials in the following mass percentages: 60%-70% EPA cells, 0.2%-0.3% emulsifier, 0.03%-0.07% antioxidant, 2%-4% filler, 5%-8% packaging material, 0.08%-0.12% dispersant, and 20%-25% water;
[0007] The EPA cells are genetically engineered Yersinia lipophila, with an intracellular triglyceride EPA accumulation of ≥25% (dry weight), and are obtained by inactivation at 121 degrees Celsius for 20–30 minutes.
[0008] The powder is used as a feed additive to enable mammals to produce milk rich in bioconvertible EPA.
[0009] As a preferred embodiment of the present invention, the mass percentages of each raw material are as follows: 65.6% inactivated EPA engineered bacteria, 0.25% emulsifier, 0.05% antioxidant, 3% filler, 7% packaging material, 0.1% dispersant, and 24% water; the resulting powder contains ≥8% EPA by mass.
[0010] In a preferred embodiment of the present invention, the emulsifier is polyglycerol fatty acid ester; the antioxidant is vitamin C palmitate; the filler is maltodextrin with a DE value of 16–20; the packaging material is modified starch; and the dispersant is silica or talc.
[0011] As a preferred embodiment of the present invention, the finished powder has a particle size that passes through an 80–100 mesh sieve, and the external coating material is a 5% acrylic resin ethanol solution.
[0012] A method for preparing a microbial cell powder that enables mammals to produce milk rich in native EPA includes the following steps:
[0013] S1. The engineered yeast strain of Yersinia lipophila was inoculated into a fermentation medium containing glucose. After fermentation, the culture was centrifuged to obtain a bacterial sludge. The sludge was then inactivated by moist heat at 121 degrees Celsius for 20-30 minutes to obtain inactivated EPA engineered bacteria cells.
[0014] S2. Dissolve the packaging material and emulsifier in water, heat to 65 degrees Celsius ± 5 degrees Celsius, emulsify and shear for 5–10 minutes, add filler and continue shearing for 5 minutes to obtain the aqueous phase;
[0015] S3. Add antioxidants to inactivated bacterial cells and preheat to 50-60 degrees Celsius to obtain mud phase;
[0016] S4. Slowly add the mud phase to the water phase at a constant temperature of 50-60 degrees Celsius and stir to emulsify for 10 minutes.
[0017] S5. First homogenize at 20-30 MPa, then homogenize at 10 MPa.
[0018] S6, spray drying with inlet air temperature of 180–190 degrees Celsius and outlet air temperature of 80 degrees Celsius;
[0019] S7. Pass the dried material through an 80–100 mesh sieve, then add the dispersant and mix.
[0020] S8. Granulate and coat the product using a 5% acrylic resin ethanol solution, sieve, and then seal and package to obtain the finished product.
[0021] The use of microbial cell powder in mammalian feed involves mixing the powder into dairy cow or dairy sheep feed at a ratio of 0.5%–3% of the daily dry matter weight, feeding twice a day, with the feeding order being concentrate first, then powder, and finally roughage; this use increases the EPA content in milk by 10–20 times, without significantly affecting milk fat, protein, lactose, and dry matter.
[0022] As a preferred embodiment of the present invention, the addition amount is 1% or 2% of the dry matter weight of the diet, corresponding to EPA content in the milk of ≥ 8 mg / 100 mL and ≥ 18 mg / 100 mL, respectively.
[0023] Compared with existing technologies, the beneficial effects of this invention are as follows: Using genetically engineered strains as the core raw material, and by introducing exogenous EPA synthesis gene clusters and optimizing metabolic pathways, the invention achieves efficient synthesis and accumulation of EPA. Furthermore, EPA exists in the form of triglycerides within microbial cells, exhibiting structural stability and retaining cell integrity even after inactivation treatment. This avoids oxidative decomposition of EPA during processing and storage, improving product stability. The preparation process employs encapsulation, emulsification, and homogenization techniques, resulting in microbial cell powder with excellent dispersibility and intestinal release properties. After ingestion by mammals, EPA is efficiently released and absorbed in the intestines, and integrated into the milk fat globule membrane through in vivo metabolism, achieving native enrichment of EPA in milk. The resulting milk exhibits significantly increased EPA content in its native form, making it easier for the human body to absorb and utilize, thus solving the problem of EPA accumulation in traditional additive methods. To address the issues of easy loss and low conversion rate, the microbial strains in the raw materials are selected from safe, fast-growing chassis microorganisms with high oil content. After genetic engineering modification, they can be fermented using glucose or inexpensive carbon sources, resulting in low raw material costs and wide availability. The preparation process is mature, enabling large-scale production. Furthermore, adding them to feed does not affect the feed intake and milk production of mammals, and has no significant impact on conventional nutrients such as fat, protein, and dry matter in milk, ensuring high application safety. Attached Figure Description
[0024] Figure 1 This is a flowchart illustrating the construction of EPA-engineered bacteria and the preparation of microbial cell powder according to the present invention.
[0025] Figure 2 This is a flowchart illustrating the application of the microbial cell powder of this invention and the production process of milk rich in native EPA. Detailed Implementation
[0026] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0027] Example 1:
[0028] Construction of EPA engineered bacteria: 1. Selection of chassis microorganisms: Yersinia lipolytica, which has a high safety level, fast growth rate, and high lipid accumulation, was selected as the chassis microorganism; 2. Genetic engineering modification: 1) Introduction of exogenous EPA synthesis gene clusters: Key gene clusters for EPA synthesis (including acetyl-CoA carboxylase gene, fatty acid synthase gene, desaturase gene, and elongation enzyme gene) were cloned from Pseudomonas marineis and the recombinant expression vector pPICZαA-EPA was constructed; 2) Optimization of endogenous metabolic pathways: The endogenous acetyl-CoA synthesis pathway of Yersinia lipolytica was optimized using CRISPR-Cas9 technology to enhance the flow of carbon sources to fatty acid synthesis; 3) Knockout of competitive metabolic pathway genes: Lipase genes involved in lipid degradation and competing genes involved in the synthesis of other fatty acids were knocked out in Yersinia lipolytica to reduce the ineffective consumption of carbon sources. 3. Screening of engineered strains: The recombinant expression vector was transformed into competent cells of Yersinia lipolyticis. EPA production was detected by Zeocin resistance screening and high-performance liquid chromatography to obtain an engineered strain that can use glucose as a substrate and accumulate EPA to more than 25% of the cell dry weight.
[0029] Example 2:
[0030] Preparation of Microbial Cell Powder: 1. Raw material formula (by mass percentage): EPA cells 65.6%, polyglycerol fatty acid ester 0.25%, vitamin C palmitate 0.05%, maltodextrin with DE value 18 3%, modified starch 7%, silica 0.1%, water 24%; 2. Preparation steps: 1) Preparation of inactivated EPA engineered bacteria cells: The engineered strain constructed in Example 1 was inoculated into a glucose-containing fermentation medium and fermented at 30°C and 200 rpm for 72 hours. The fermentation broth was concentrated by centrifugation at 8000 rpm for 15 minutes to obtain bacterial sludge, and then inactivated by moist heat sterilization at 121°C for 25 minutes to obtain EPA cells; 2) Preparation of aqueous phase: 7 kg of modified starch and 0.25 kg of polyglycerol fatty acid ester were added to 24 kg of water and stirred for 5 minutes until completely dissolved. The mixture was then transferred to an emulsification shearing tank, heated to 65°C, and emulsified and sheared for 8 minutes to completely gelatinize the modified starch. 3 kg of maltodextrin was added, and shearing was continued for 5 minutes. 3) Preparation of mud phase: Add 0.05 kg of vitamin C palmitate to 65.6 kg of EPA cells, stir evenly and preheat to 55 degrees Celsius to obtain mud phase; 4) Mixing and emulsification: Under continuous stirring, slowly add mud phase to aqueous phase, maintain system temperature at 55 degrees Celsius, emulsify for 10 minutes to obtain homogeneous mixed emulsion; 5) Homogenization: Homogenize the mixed emulsion once at 25 MPa pressure, and then once at 10 MPa pressure; 6) Spray drying: Send the homogenized material into a spray dryer, control the inlet air temperature at 185 degrees Celsius and the outlet air temperature at 80 degrees Celsius for spray drying; 7) Premixing: Pass the dried powder through a 100-mesh sieve, add 0.1 kg of silica, and mix evenly; 8) Granulation and coating: Add the premixed powder to a granulation and coating machine, use 5% acrylic resin solution for granulation and coating, and after coating, pass through an 80-mesh sieve. The microbial cell powder is obtained by sieving through a fine mesh, sealing and packaging.
[0031] 3. Finished Product Testing: The prepared microbial cell powder was tested for relevant indicators, and the results are as follows:
[0032] Inspection items Standard requirements Test results in conclusion Appearance It is a yellowish powder with a characteristic odor, free from burnt, rancid, ammonia-like, or bitter smells; it contains no visible impurities, clumps, or insects. This product is in powder form, with a yellowish-brown color; it has a characteristic odor, without any burnt, rancid, or ammonia-like bitter smells or other off-odors; it is free of visible impurities, clumps, and insect infestation. Compliant Crude protein (on a dry basis) ≥ 8.0 % 11.8 % Compliant Total lipid content (on a dry basis) ≥ 25.0 % 30.5 % Compliant EPA (on a dry basis) ≥ 8.0 % 8.3 % Compliant Moisture ≤ 7.0 % 2.2 % Compliant salmonella Not detectable Not detected Compliant
[0033] Example 3:
[0034] Application of microbial cell powder in dairy cow feeding
[0035] 1. Selection and grouping of experimental animals: Nine healthy Holstein lactating cows of similar age, parity, lactation time and physiological state were selected and randomly divided into three groups of three cows each: control group, experimental group one and experimental group two. 2. Experimental diet: (1) Basal diet: 5 kg / head / day of concentrate feed, and roughage (straw + sugarcane tail) was fed in the conventional proportion to keep the basal diet of each group consistent. (2) Feed of experimental groups: Experimental group one added the microbial cell powder of the present invention to the basal diet (added at 1% of the dry matter weight of the diet, about 100g / head / day); Experimental group two added the microbial cell powder of the present invention to the basal diet (added at 2% of the dry matter weight of the diet, about 200g / head / day); the control group did not add microbial cell powder. 3. Feeding management: The experimental cows were tethered and fed twice a day, with water three times a day and milking twice a day. The feeding order was: concentrate feed first, then microbial cell powder, and finally roughage. The experiment lasted for 30 days, with the first 7 days being the pre-trial period and the last 23 days being the formal trial period. The management measures were consistent during the experiment. The results of the experiment were as follows: (1) Feed intake and milk production: There was no significant difference in daily feed intake between the experimental group and the control group (P>0.05), indicating that the addition of microbial cell powder did not affect the feed intake of dairy cows. The statistical results of milk production in each group are as follows. There was no significant difference between the groups (P>0.05), indicating that the powder had no adverse effect on the milk production of dairy cows.
[0036] Group Milk production on day 1 Milk production on day 15 Milk production on day 30 Control group (no powder added) 20.5 ± 1.6 21.0 ± 0.7 20.8 ± 0.9 Experimental group 1 (added 100 g / head / day) 20.9 ± 1.3 21.5 ± 0.5 21.2 ± 0.6 Experimental group 2 (added 200 g / head / day) 20.3 ± 0.9 21.3 ± 1.1 20.7 ± 0.8
[0037] Note: The difference between groups was not significant (P>0.05), indicating that the addition of microbial cell powder did not affect the milk yield of dairy cows;
[0038] Breast milk composition analysis: After the trial period, the composition of breast milk in each group was analyzed, and the results are as follows:
[0039] Group Fat(%) protein(%) lactose(%) Dry matter (%) EPA (mg / 100ml) control group 3.12±0.31 2.35±0.22 4.33±0.12 11.68±0.43 0.8±0.2 Experimental Group 1 3.15±0.06 2.29±0.18 4.02±0.25 11.26±0.55 8.5±1.3 Experimental Group 2 3.02±0.14 2.30±0.27 4.41±0.15 11.50±0.38 18.6±2.1
[0040] Note: 1. There were no significant differences among the groups in terms of fat, protein, lactose, and dry matter (P>0.05);
[0041] 2. EPA content: The difference between the experimental group and the control group was extremely significant (P<0.01), and the experimental group 2 was significantly higher than that of the experimental group 1 (P<0.01).
[0042] The test results showed that the EPA content in the milk of the experimental group was significantly different from that of the control group (P<0.01), and the EPA content of experimental group two was significantly higher than that of experimental group one (P<0.01). However, there were no significant differences in the conventional components such as fat, protein, lactose, and dry matter in the milk of each group (P>0.05). This indicates that the microbial cell powder of the present invention can significantly increase the native EPA content in dairy cow milk without affecting the conventional nutritional quality of milk. The EPA cells are engineered inactivated bacterial cells, which are genetically engineered Yersinia lipolytica, with an intracellular triglyceride EPA accumulation of ≥25% (dry weight), and are obtained by inactivation by moist heat at 121 degrees Celsius for 20–30 minutes. The core functional microorganisms used in this invention for preparing microbial cell powders are not limited by species, acquisition method, or taxonomic status. Any microorganism capable of autonomously synthesizing EPA (eicosapentaenoic acid) and whose intracellular EPA accumulates in the form of triglycerides with an accumulation amount (dry weight) that meets the product preparation requirements is applicable to this invention. This includes, but is not limited to, engineered strains obtained through genetic engineering and natural strains isolated from nature. The microbial categories cover various microbial species with EPA synthesis capabilities, such as yeast, bacteria, and microalgae.
[0043] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.
[0044] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.
Claims
1. A microbial cell powder that enables mammals to produce milk rich in native EPA, characterized in that, It is formulated from the following raw materials in the following weight percentages: EPA cells 60%-70%, emulsifier 0.2%-0.3%, antioxidant 0.03%-0.07%, filler 2%-4%, packaging material 5%-8%, dispersant 0.08%-0.12%, and water 20%-25%.
2. The microbial cell powder for producing EPA-rich milk from mammals according to claim 1, characterized in that: The powder is used as a feed additive to enable mammals to produce milk rich in bioconvertible EPA.
3. The microbial cell powder for producing EPA-rich milk from mammals according to claim 1, characterized in that: The percentage of each raw material by mass is as follows: EPA cells 65.6%, emulsifier 0.25%, antioxidant 0.05%, filler 3%, packaging material 7%, dispersant 0.1%, and water 24%; the resulting powder contains ≥8% EPA by mass.
4. The microbial cell powder for producing EPA-rich milk from mammals according to any one of claims 1-3, characterized in that: The emulsifier is polyglycerol fatty acid ester; the antioxidant is vitamin C palmitate; the filler is maltodextrin with a DE value of 16–20; the packaging material is modified starch; and the dispersant is silica or talc.
5. The microbial cell powder for producing EPA-rich milk from mammals according to any one of claims 1-4, characterized in that: The finished powder has a particle size that passes through an 80–100 mesh sieve, and the external coating material is a 5% acrylic resin ethanol solution.
6. A method for preparing the microbial cell powder according to any one of claims 1-5, which enables mammals to produce milk rich in native EPA, characterized in that, Includes the following steps: S1. Inoculate the engineered Yersinia lipophila into a fermentation medium containing glucose. After fermentation, centrifuge to obtain bacterial sludge. Inactivate the bacteria by moist heat at 121 degrees Celsius for 20-30 minutes to obtain EPA cells. S2. Dissolve the packaging material and emulsifier in water, heat to 65 degrees Celsius ± 5 degrees Celsius, emulsify and shear for 5–10 minutes, add filler and continue shearing for 5 minutes to obtain the aqueous phase; S3. Add antioxidants to inactivated bacterial cells and preheat to 50-60 degrees Celsius to obtain mud phase; S4. Slowly add the mud phase to the water phase at a constant temperature of 50-60 degrees Celsius and stir to emulsify for 10 minutes. S5. First homogenize at 20-30 MPa, then homogenize at 10 MPa. S6, spray drying with inlet air temperature of 180–190 degrees Celsius and outlet air temperature of 80 degrees Celsius; S7. Pass the dried material through an 80–100 mesh sieve, then add the dispersant and mix. S8. Granulate and coat the product using a 5% acrylic resin ethanol solution, sieve, and then seal and package to obtain the finished product.
7. The use of the microbial cell powder according to any one of claims 1–5 in mammalian feed, characterized in that: The powder is mixed into dairy cow or dairy sheep feed at a ratio of 0.5%–3% of the dry matter weight of the diet, and fed twice a day. The feeding order is concentrate first, then powder, and finally roughage. The above use increases the EPA content in milk by 10–20 times, and has no significant effect on milk fat, protein, lactose and dry matter.
8. The use of the microbial cell powder according to claim 7 in mammalian feed, characterized in that: The addition amount is 1% or 2% of the dry matter weight of the diet, corresponding to EPA content in milk of ≥ 8 mg / 100 mL and ≥ 18 mg / 100 mL, respectively.