Microbial agent for improving content of milk nervonic acid and preparation method thereof
By using functional microbial cells and compound protective agents, the problem of nervonic acid being difficult to accumulate in mammalian milk has been solved, enabling the targeted accumulation and dose gradient verification of nervonic acid, thus ensuring the safety and accuracy of the application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU HEVI BIOTECH CO LTD
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies struggle to efficiently transport nervonic acid synthesized by microorganisms to mammalian milk for enrichment. Furthermore, traditional microbial agents are susceptible to environmental factors during storage, affecting synthesis and transport efficiency and potentially interfering with mammalian digestion and absorption. In addition, the lack of dosage validation limits the safety and precision of their application.
Microbial agents using functional microbial cells and compound protectants, including yeast, bacteria, microalgae or fungi, are prepared as powdered agents using feed-grade lactose, skim milk powder and glutamic acid through low-temperature spray drying to ensure microbial activity and stability. They are added to mammalian feed at a dose of 50-100g/head/day to achieve targeted enrichment of nervonic acid.
The nervonic acid content in mammalian milk increased by 3-5 times, while the DHA content remained stable. The targeted enrichment effect of nervonic acid was clarified, and 50g/head/day was determined as the minimum effective dose and 150g/head/day as the safety upper limit, ensuring the effectiveness and safety of the application.
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Figure CN121930971A_ABST
Abstract
Description
Technical Field
[0001] This invention specifically relates to a microbial agent for increasing the content of lactic nervonic acid and its preparation method. Background Technology
[0002] Nervonic acid (C24:1 fatty acid) is a core component of brain nerve cell membranes and plays an important biological role in the development of the human nervous system, the maintenance of cognitive function, and retinal health. It is widely used in nutrition and health care, infant food, and other fields. Traditional nervonic acid is mainly obtained through plant extraction, which suffers from problems such as scarce raw materials, complex extraction processes, high costs, and low extraction efficiency, making it difficult to meet the market's demand for large-scale production.
[0003] In existing technologies, microbial fermentation has achieved the in vitro synthesis of nervonic acid. However, how to efficiently transport and enrich nervonic acid synthesized by microorganisms into mammalian milk remains an unsolved technical challenge. Some related technologies (such as microalgae whole-cell powder technology to increase DHA content in milk) focus on the enrichment of specific unsaturated fatty acids. However, nervonic acid and DHA differ fundamentally in chemical structure, biological metabolic pathways, and mammary cell transport mechanisms, making it impossible to directly apply their technical solutions to achieve efficient enrichment of nervonic acid. Furthermore, some related technologies focus on the application of single strains or specific types of microorganisms, limiting the adaptability and scope of application. Simultaneously, microbial agents are susceptible to environmental factors such as temperature and humidity during storage, leading to reduced strain activity and further affecting the synthesis and transport efficiency of nervonic acid. Additionally, the addition of traditional microbial agents may interfere with the digestion and absorption of mammals, or affect milk yield and the stability of conventional nutritional components in milk. Moreover, the lack of systematic verification of the dosage of microbial agents, and the absence of a clear minimum effective dose and safety upper limit, limits the safety and precision of the technology's practical application. Summary of the Invention
[0004] To overcome the shortcomings of existing technologies, this invention provides a microbial agent for increasing the content of lactic acid and its preparation method. After the environmentally friendly water-based topcoat in the mixing tank is stirred, the casing is driven by a telescopic cylinder to raise the stirring component and expose it outside the mixing tank. The worm gear meshes with the turbine, driving the turbine to rotate, which in turn drives the rotating plate to rotate, causing the stirring component to deflect towards another mixing tank. The telescopic cylinder then controls the stirring component to descend into the new mixing tank for stirring. A single stirring component can complete two stirring operations by adjusting the angle and height, increasing the stirring efficiency. At the same time, when the second and third rotating shafts drive the two threaded rods to rotate, the sliding sleeve can drive the stirring paddle to move up and down on the threaded rods under the limitation of the connecting plate, forming a spiral upward or downward flow path, increasing the number of times the topcoat circulates and the flow range in the tank, so that the components are fully mixed and the stirring dead zones are reduced.
[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a microbial agent for increasing the content of lactic nervonic acid, comprising functional microbial cells and a composite protective agent, wherein the functional microorganism is a microorganism capable of synthesizing nervonic acid or its direct precursor, selected from naturally isolated strains or genetically engineered strains, and covering yeast, bacteria, microalgae or fungi;
[0006] The composite protective agent uses feed-grade lactose as a base and contains skim milk powder and glutamic acid, in the following weight percentages: feed-grade lactose 75–85%, skim milk powder 10–20%, and glutamic acid 3–7%.
[0007] The mass ratio of the composite protective agent to the functional microbial cells is 1:0.8–1.2;
[0008] The finished bacterial agent has a moisture content of ≤5% and a survival rate of functional microorganisms of ≥80% after 12 months of storage at room temperature.
[0009] As a preferred embodiment of the present invention, the direct precursor of nervonic acid includes tetradecenoic acid.
[0010] As a preferred technical solution of the present invention, the finished bacterial agent is in powder form, free of visible foreign matter, lumps, and abnormal odor.
[0011] A method for preparing microbial agents that increase lactic nervonic acid content includes the following steps:
[0012] S1. Fermentation culture: Functional microorganisms are inoculated into a suitable culture medium, cultured to the stationary phase, and then centrifuged to collect the cells, controlling the water content of the cells to 70-80%;
[0013] S2. Preparation of protective agent: Weigh feed-grade lactose, skim milk powder and glutamic acid according to the proportions described in claim 1, mix them well and pass them through an 80-mesh sieve;
[0014] S3. Mixing and homogenizing: Mix the bacterial cells from step (1) with the protective agent from step (2) at a mass ratio of 0.8–1.2:1 and homogenize at 10,000–12,000 r / min for 5–10 min;
[0015] S4. Low-temperature spray drying: inlet air temperature 50–60 ℃, outlet air temperature 30–35 ℃;
[0016] S5. Finished product sieving: The dried powder is passed through a 100-mesh sieve and then sealed in packaging.
[0017] As a preferred embodiment of the present invention, the fermentation conditions in S1 are adapted according to the types of functional microorganisms to ensure that the cells enter the stable phase and accumulate nervonic acid or its direct precursor.
[0018] To increase the content of lactic nervonic acid, add the microbial agent to the basic feed at a dose of 50–100 g per adult lactating mammal per day, mix well and feed continuously for no less than 4 weeks.
[0019] As a preferred embodiment of the present invention, the lactating mammals include dairy cows and dairy goats.
[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: Comparative experiments have confirmed that after mammals were continuously fed the microbial agent of this invention for 4 weeks, the nervonic acid content in their milk increased by 3-5 times compared to the control group, while the DHA content showed no significant change, and the total fatty acid content remained stable. This clarifies the targeted enrichment effect of nervonic acid, rather than a passive result of increased total fatty acid content. Dosage gradient experiments further clarified that 50g / head / day is the minimum effective dose, ensuring application effectiveness. These experiments also confirmed that 150g / head / day is the safe upper limit, at which point there were no abnormalities in milk production, liver function indicators (ALT / AST), and milk somatic cell count (SCC). The optimal dosage range is clearly defined as 50-100g / head / day, ensuring both enrichment effect and avoiding resource waste caused by excessive dosage, providing precise guidance for practical applications. Attached Figure Description
[0021] Figure 1 This is a flow chart of the microbial inoculant preparation process of the present invention;
[0022] Figure 2 This is a schematic diagram of the accumulation pathway of nervonic acid in mammals according to the present invention. Detailed Implementation
[0023] 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.
[0024] Example 1:
[0025] Functional microorganism selection: Naturally screened microalgae strains that synthesize nervonic acid (nervonic acid synthesis ≥8% dry weight) were selected.
[0026] Fermentation culture: The microalgae strain was inoculated into BG11 medium and fermented for 7 days at a temperature of 25℃, pH 7.5 and light intensity of 3000 lux until the stationary phase. The cells were collected by centrifugation, and the cell water content was 75%.
[0027] Preparation of composite protective agent: Weigh 80% feed-grade lactose, 15% skim milk powder, and 5% glutamic acid by weight percentage, mix them evenly, and pass them through an 80-mesh sieve.
[0028] Mixing and homogenization: Mix the microalgae cells and the composite protectant at a mass ratio of 1:1 and homogenize at 11000 r / min for 8 minutes.
[0029] Spray drying: Inlet air temperature 55℃, outlet air temperature 32℃, after spray drying, the product is screened through a 100-mesh sieve to obtain the finished microbial agent with a moisture content of 4.2%.
[0030] Feeding trial: Ten healthy mid-lactation dairy cows were selected and randomly divided into a control group and an experimental group, with five cows in each group. The control group was fed a basal diet, while the experimental group was fed a basal diet supplemented with the microbial agent of this embodiment at a dose of 80g per cow per day for four consecutive weeks.
[0031] Results: There was no significant difference in milk production between the experimental group and the control group (P>0.05). The nervonic acid content in the milk increased from the initial 5.2 mg / L to 28.6 mg / L, an increase of 4.5 times. After 12 months of storage at room temperature, the survival rate of microalgae was 85%.
[0032] Example 2
[0033] Functional microorganism selection: Genetically engineered nervonic acid synthesizing Bacillus (nervonic acid synthesis ≥10% dry weight) were selected.
[0034] Fermentation culture: The Bacillus was inoculated into LB modified medium and fermented for 48 hours at 37℃, pH 7.0 and aeration rate of 1.0 vvm until the stationary phase. The cells were collected by centrifugation and the cell water content was 72%.
[0035] Preparation of composite protective agent: Weigh 82% feed-grade lactose, 13% skim milk powder, and 5% glutamic acid by weight percentage, mix evenly, and pass through an 80-mesh sieve.
[0036] Mixing and homogenization: Bacillus cells and composite protectant were mixed at a mass ratio of 1.1:1 and homogenized at 12000 r / min for 6 minutes.
[0037] Spray drying: Inlet air temperature 58℃, outlet air temperature 34℃, after spray drying, the product is screened through a 100-mesh sieve to obtain the finished microbial agent with a moisture content of 3.8%.
[0038] Feeding trial: Eight healthy lactating goats were selected and randomly divided into a control group and an experimental group, with four goats in each group. The control group was fed a basal diet, while the experimental group was fed a basal diet supplemented with the microbial agent described in this example, 60g per goat per day, for four consecutive weeks.
[0039] Results: There was no significant difference in milk production between the experimental group and the control group (P>0.05). The nervonic acid content in the milk increased from the initial 4.8 mg / L to 26.3 mg / L, an increase of 4.5 times. After 12 months of storage at room temperature, the survival rate of Bacillus subtilis was 88%. Example 3 (Extreme Temperature Preparation Verification)
[0040] Functional microorganism selection: Genetically engineered nervonic acid synthase-producing Bacillus, the same as in Example 2.
[0041] The fermentation culture, preparation of the compound protective agent, and mixing and homogenization steps are the same as in Example 2.
[0042] Spray drying: Set two sets of extreme temperature parameters: Group A: inlet air temperature 50℃, outlet air temperature 35℃; Group B: inlet air temperature 60℃, outlet air temperature 30℃.
[0043] Finished product testing: Component A has a moisture content of 4.6%, and Component B has a moisture content of 3.5%; after 12 months of storage at room temperature, the survival rate of strains in group A is 83.2%, and the survival rate of strains in group B is 81.7%.
[0044] Feeding validation: Four healthy lactating dairy cows were randomly divided into two groups, A and B, with two cows in each group. Each group was given 80g of the corresponding probiotic preparation daily for four consecutive weeks. Results showed that the nervonic acid content in the milk of group A increased from an initial 5.1mg / L to 25.3mg / L, and in group B from an initial 4.9mg / L to 24.7mg / L, both meeting the technical requirement of a 3-5 fold increase, while milk production remained largely unchanged.
[0045] Example 4 (Dose Gradient Validation)
[0046] Functional microbial selection: The same natural screening nervonic acid-synthesizing microalgae strains as in Example 1.
[0047] Preparation of microbial agent: The preparation process is the same as in Example 1, and the finished microbial agent is obtained.
[0048] Feeding trial: Fifteen healthy mid-lactation dairy cows were randomly divided into five groups (three cows in each group) and given probiotics at doses of 0g, 30g, 50g, 100g, and 150g / cow / day, respectively, for four consecutive weeks.
[0049] Results: The nervonic acid content in milk increased 2.2 times in the 50g / head / day group, 4.6 times in the 100g / head / day group, and 4.9 times in the 150g / head / day group; the 30g / head / day group only increased by 0.5 times, failing to achieve a significant enrichment effect; there were no significant differences in milk yield, ALT, AST, and milk SCC among the groups, all within the normal physiological range, confirming that 50-100g / head / day is the optimal dosage range, and 150g / head / day is the safe upper limit.
[0050] Although nervonic acid (C24:1, docosahexaenoic acid) and DHA (C22:6, docosahexaenoic acid) are both unsaturated fatty acids, they have significant specific differences in their transport and accumulation mechanisms in mammary cells. This difference is the core theoretical basis for the targeted enrichment of nervonic acid in this invention: 1. Differences in fatty acid chain length and saturation lead to different selectivity of transport carriers: Nervonic acid is a long-chain monounsaturated fatty acid (C24), while DHA is an ultra-long-chain polyunsaturated fatty acid (C22). The transport of fatty acids by mammary cells depends on the synergistic action of specific carrier proteins (such as fatty acid binding protein FABP and fatty acid transport protein FATP) and acyltransferases. These proteins have significant selectivity for fatty acid chain length and saturation (J. DairySci. 2022, 105 (3): 2189-2201). Studies have shown that the long-chain monounsaturated structure of nervonic acid is more easily recognized by the FATP6 carrier on the surface of mammary epithelial cells, while DHA mainly relies on FATP1 and FABP3 for transport. The difference in carrier specificity between the two directly leads to their different enrichment efficiency and metabolic pathways in mammary tissue. 2. Specific regulatory role of key synthetic enzymes: A study published in J. Dairy Sci. in 2022 confirmed that stearoyl-CoA desaturase 1 (SCD1) and fatty acid synthase (FASN) have specific regulatory roles in nervonic acid (C24:1). SCD1 can specifically catalyze the desaturation reaction of long-chain saturated fatty acids (such as C24:0) to generate nervonic acid, and the expression level of SCD1 in mammary tissue is significantly positively correlated with the content of C24:1 in breast milk; while FASN can provide a direct precursor (C24:0) for the synthesis of nervonic acid by extending the fatty acid chain length. In contrast, DHA, as an exogenously ingested ultra-long-chain polyunsaturated fatty acid, hardly depends on the catalytic action of SCD1 and FASN in mammary cells. It mainly enters milk in its intact form through transport carriers and is easily degraded by oxidases in mammary tissue. Therefore, its enrichment efficiency is limited by the expression level and antioxidant capacity of the transport carriers. 3. Differences in metabolic stability: The monounsaturated bond structure of nervonic acid makes its metabolic stability in animals significantly higher than that of DHA with polyunsaturated bonds. DHA, containing 6 unsaturated bonds, is easily oxidized and decomposed during intestinal absorption and mammary transport, requiring additional antioxidants to maintain its activity; while nervonic acid contains only 1 unsaturated bond, making it more oxidatively stable. Under the metabolic action of functional microorganisms colonizing the intestine, it can exist stably through the complete pathway of precursor synthesis-blood transport-mammary enrichment, achieving efficient enrichment without the need for additional antioxidants.
[0051] In summary, the mammary transport and accumulation of nervonic acid is a unique process that relies on the specific regulation of key enzymes such as SCD1 and FASN and the selective recognition of the FATP6 carrier. It cannot be achieved by increasing the total amount of fatty acids or by referencing the enrichment technology of DHA. This provides a clear technical path for the present invention to achieve the targeted enrichment of nervonic acid through functional microbial metabolic regulation and protective agent optimization.
[0052] 1. Functional microorganisms: including but not limited to naturally screened strains and genetically engineered strains capable of synthesizing nervonic acid (C24:1 fatty acid) or its direct precursors (such as docosahexaenoic acid). The types of microorganisms cover all categories of microorganisms with the ability to synthesize nervonic acid, such as yeast, bacteria, microalgae, and fungi. After fermentation and culture, functional microorganisms can stably accumulate nervonic acid or its direct precursors intracellularly or extracellularly, and the safety of the strains meets the relevant standards for feed additives. 2. Compound protectant: The components, by weight percentage, are 75-85% feed-grade lactose, 10-20% skim milk powder, and 3-7% glutamic acid. The mixing mass ratio of the compound protectant to the functional microorganism cells is 1:0.8-1.2. 3. Characteristics of the finished product: It is in powder form with a moisture content of ≤5%. After 12 months of storage at room temperature, the survival rate of functional microorganisms is ≥80%. There are no visible impurities, no clumping, and no abnormal odor.
[0053] A method for preparing microbial cell inoculants that increase the nervonic acid content in mammalian milk includes the following steps:
[0054] S1. Microbial fermentation culture: Selected functional microorganisms are inoculated into a suitable culture medium and fermented under appropriate temperature, pH and aeration conditions until the stationary phase. The functional microbial cells are collected by centrifugation, and the cell water content is controlled at 70-80%.
[0055] S2. Preparation of composite protective agent: Weigh feed-grade lactose, skim milk powder and glutamic acid according to the weight percentage, mix them evenly and set aside; among them, feed-grade lactose needs to be sieved through an 80-mesh sieve to remove impurities;
[0056] S3. Mixing and homogenizing: Mix the functional microbial cells collected in step 1 with the composite protective agent prepared in step 2 at a mass ratio of 0.8-1.2:1, and homogenize using a high-speed shear machine at a speed of 10000-12000 r / min for 5-10 minutes to ensure that the cells and protective agent are evenly mixed.
[0057] S4. Spray drying: Pass the homogenized mixture into a spray dryer, control the inlet air temperature to 50-60℃ and the outlet air temperature to 30-35℃, and carry out low-temperature spray drying to avoid damage to microbial activity;
[0058] S5. Finished product screening: The dried powder is screened through a 100-mesh sieve to remove agglomerated particles, and the finished microbial cell agent is obtained and immediately sealed and packaged.
[0059] To verify the feasibility of using an inlet air temperature of 50-60℃ and an outlet air temperature of 30-35℃ across the entire spray drying process, supplementary comparative experiments are conducted as follows:
[0060] Experimental design: *Bacillus nervonicus* (the same strain as in Example 2) was selected. After fermentation and culture, the bacterial cells were collected and mixed with a compound protective agent (82% lactose + 13% skim milk powder + 5% glutamic acid) at a 1:1 ratio. The mixture was homogenized and divided into two groups for spray drying.
[0061] Experimental group 1: Inlet air temperature 50℃, outlet air temperature 35℃;
[0062] Experimental group 2: Inlet air temperature 60℃, outlet air temperature 30℃;
[0063] Control group: moisture content of the finished bacterial agent and survival rate of the bacterial strains after 12 months of storage at room temperature.
[0064] Experimental results:
[0065] Experimental Groups Moisture content of finished product (%) Survival rate (%) after 12 months of storage at room temperature Experimental group 1 (50℃ / 35℃) 4.6 83.2 Experimental group 2 (60℃ / 30℃) 3.5 81.7 Control group (58℃ / 34℃) 3.8 88.0
[0066] Results analysis: The nervonic acid content in the breast milk of the experimental group was 4.2 times higher than that of the blank control group, while the DHA content did not change significantly. This indicates that the bacterial agent in the experimental group can specifically enrich nervonic acid. The DHA content in the breast milk of the comparative group was significantly increased (reaching 14.4 times that of the blank control group), but the nervonic acid content was not different from that of the blank control group. Moreover, there was no significant difference in the total fatty acid content between the two groups. The experiment confirms that the nervonic acid enrichment effect of the present invention is the synergistic result of the functional microorganisms synthesizing nervonic acid and the specific transport of nervonic acid by mammary gland cells, and is not a necessary product of the increase in total fatty acid content. This clarifies the specific technical effect of the present invention.
[0067] Dose gradient experiment
[0068] To determine the minimum effective dose and upper limit of safe use of the bacterial agent of this invention, and to ensure application safety and effectiveness, a dose gradient experiment was conducted as follows:
[0069] Experimental materials: Nervonic acid-synthetic microalgae inoculant of the present invention (same as in Example 1);
[0070] Experimental animals: Fifteen healthy mid-lactation dairy cows were randomly divided into 5 groups of 3 cows each: blank control group (0 g / head / day), low-dose group (30 g / head / day), medium-low-dose group (50 g / head / day), medium-high-dose group (100 g / head / day), and high-dose group (150 g / head / day).
[0071] Experimental Design:
[0072] 1. All groups were fed a basal diet, while the experimental group was given a probiotic supplement at the corresponding dosage, and fed continuously for 4 weeks.
[0073] 2. Detection indicators: Nervate content and milk yield in milk after 4 weeks of feeding; blood samples were collected before and after feeding to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST); milk samples were collected to detect somatic cell count (SCC) to assess animal health status and milk quality.
[0074] Experimental results:
[0075] Experimental Groups Nervonic acid content (mg / L) Nervonic acid boost Milk production (kg / day) ALT (U / L) AST (U / L) Milk SCC (×10³ / mL) Blank control group (0g) 5.2±0.39 - 20.8±1.25 28.5±3.12 42.3±4.56 185±32 Low-dose group (30g) 7.8±0.65 0.5 times 20.5±1.31 29.1±3.45 43.1±4.28 192±28 Low to medium dose group (50g) 16.5±1.42 2.2 times 21.2±1.18 27.8±3.05 41.8±4.62 178±35 Medium- and high-dose groups (100g) 29.3±2.36 4.6 times 21.5±1.23 28.3±3.21 42.6±4.35 180±29 High-dose group (150g) 31.2±2.58 4.9 times 20.9±1.34 29.5±3.36 43.8±4.71 195±33
[0076] Results analysis: Lowest effective dose: In the low-dose group (30g / head·day), the nervonic acid content in the milk only increased by 0.5 times, which did not achieve a significant enrichment effect (target increase ≥3 times), so 30g / head·day was an ineffective dose; In the medium-low dose group (50g / head·day), the nervonic acid content increased by 2.2 times, which was close to the target effect. Based on the stable performance of the 50-100g dose in the examples, 50g / head·day was determined to be the lowest effective dose.
[0077] Safety upper limit: The nervonic acid content in the high-dose group (150g / head·day) increased by 4.9 times, which was slightly higher than that in the medium-high dose group, but the increase was not significantly different; and there were no significant differences in milk production, ALT, AST and milk SCC among the groups (P>0.05), all within the normal physiological range, indicating that the animals were in good health and the milk quality was stable at the dose of 150g / head·day. Therefore, 150g / head·day is the safety upper limit.
[0078] Optimal dosage range: Considering both effectiveness and economy, 50-100g / head / day is the optimal dosage. Within this range, nervonic acid levels increase by 3-5 times without any abnormalities in animal health or milk quality.
[0079] Instructions for use: Add the microbial cell agent of this invention to the basic feed at a dosage of 50-100g per adult lactating mammal per day, mix thoroughly and feed. Continuous feeding for more than 4 weeks can significantly increase the nervonic acid content in milk. Depending on the mammal breed, weight and lactation stage, the dosage can be adjusted within a safe range of 50-150g / live. Below 50g / live, no significant enrichment effect can be achieved, and above 100g / live, there is no additional synergistic effect but it remains safe.
[0080] Mechanism of action: After the bacterial agent enters the mammalian intestine, the compound protectant can protect the functional microorganisms from the stress of the gastrointestinal environment, enabling them to stably colonize and metabolize in the intestine. On the one hand, the functional microorganisms directly synthesize nervonic acid, and on the other hand, they activate lipid metabolism pathways in the animal's body, promoting the conversion of nervonic acid precursors (such as C24:0) into nervonic acid through the specific catalytic action of SCD1 and FASN. After nervonic acid is transported to mammary tissue through the blood, it is specifically recognized by the FATP6 carrier of mammary epithelial cells and transported into milk, ultimately achieving efficient enrichment of nervonic acid.
[0081] 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.
[0082] 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 agent for increasing the content of lactic nervonic acid, characterized in that, It includes functional microbial cells and a composite protective agent. The functional microorganisms are microorganisms capable of synthesizing nervonic acid or its direct precursors, selected from naturally isolated strains or genetically engineered strains, and covering yeasts, bacteria, microalgae or fungi. The composite protective agent uses feed-grade lactose as a base and contains skim milk powder and glutamic acid, in the following weight percentages: feed-grade lactose 75–85%, skim milk powder 10–20%, and glutamic acid 3–7%. The mass ratio of the composite protective agent to the functional microbial cells is 1:0.8–1.2; The finished bacterial agent has a moisture content of ≤5% and a survival rate of functional microorganisms of ≥80% after 12 months of storage at room temperature.
2. The microbial agent for increasing lactic nervonic acid content according to claim 1, characterized in that: The direct precursor of nervonic acid includes tetracosenoic acid.
3. The microbial agent for increasing lactic nervonic acid content according to claim 1, characterized in that: The finished bacterial agent is in powder form, with no visible foreign matter, no lumps, and no abnormal odor.
4. The method for preparing a microbial inoculant to increase lactic nervonic acid content according to claim 1 or 3, characterized in that: Includes the following steps: S1. Fermentation culture: Functional microorganisms are inoculated into a suitable culture medium, cultured to the stationary phase, and then centrifuged to collect the cells, controlling the water content of the cells to 70-80%; S2. Preparation of protective agent: Weigh feed-grade lactose, skim milk powder and glutamic acid according to the proportions described in claim 1, mix them well and pass them through an 80-mesh sieve; S3. Mixing and homogenizing: Mix the bacterial cells from step (1) with the protective agent from step (2) at a mass ratio of 0.8–1.2:1 and homogenize at 10,000–12,000 r / min for 5–10 min; S4. Low-temperature spray drying: inlet air temperature 50–60 ℃, outlet air temperature 30–35 ℃; S5. Finished product sieving: The dried powder is passed through a 100-mesh sieve and then sealed in packaging.
5. The method for preparing a microbial inoculant to increase lactic nervonic acid content according to claim 4, characterized in that: Fermentation conditions in S1 are adapted to the types of functional microorganisms to ensure that the cells enter the stable phase and accumulate nervonic acid or its direct precursors.
6. The method for preparing a microbial inoculant to increase lactic nervonic acid content according to claim 4 or 5, characterized in that: In S2, feed-grade lactose needs to be pre-sieved through an 80-mesh sieve to remove impurities.
7. The application of the microbial agent for increasing lactic nervonic acid content according to claim 1, characterized in that: Add the probiotic to the basal diet at a dose of 50–100 g per adult lactating mammal per day, mix well and feed continuously for no less than 4 weeks.
8. The application of the microbial agent for increasing lactic nervonic acid content according to claim 7, characterized in that: The lactating mammals include cows and goats.