Grease feed additive with anti-inflammatory and lipid-lowering functions and application thereof

Abstract

The invention provides a grease feed additive with anti-inflammatory and lipid-lowering functions, and belongs to the field of animal feed additives. According to the feed additive taking the safflower seed oil and the linseed oil as core components, the fatty acid molar ratio (linoleic acid omega6 / linolenic acid omega3 = 3: 1) is optimized through accurate proportion control, and the metabolic health of high-fat diet animals is remarkably improved. According to animal experiment data, when the grease feed additive is added into animal feed for feeding, the weight gain can be reduced, the blood lipid spectrum can be improved, the expression of proinflammatory factors TNF-alpha and IL-6 can be inhibited, the composition of intestinal microbiota can be improved, lipid metabolism can be facilitated, and fat accumulation can be reduced in a high-fat diet mouse model. Meanwhile, the preparation method of the grease feed additive provided by the invention is simple and rapid, the components are safe and simple, and the grease feed additive has good market prospects and industrialization potential.

Description

Technical Field

[0001] The present invention relates to the field of animal feed additives, and particularly to an oil-based feed additive with anti-inflammatory and lipid-lowering functions prepared by optimizing the ratio of safflower oil to linseed oil. Background Art

[0002] Safflower (Carthamus tinctorius) oil, as a functional edible resource, is widely utilized globally for its health benefits, including enhancing cardiovascular health, reducing blood sugar levels, and alleviating inflammation. These benefits are attributed to its unique fatty acid composition. Safflower oil contains a high proportion of unsaturated fatty acids and a lower proportion of saturated fatty acids. Previous studies have shown that safflower oil is rich in 90% total unsaturated fatty acids, among which 76% are polyunsaturated fatty acids, mainly ω6. Excessive intake of ω6 fatty acids with insufficient ω3 fatty acids is associated with inflammation, metabolic disorders, and an increased risk of chronic diseases such as cardiovascular diseases, cancer, and neurodegenerative diseases. Previous studies have shown that consuming pure safflower oil has adverse effects, especially the need to address the imbalance in its fatty acid ratio (ω6 / ω3). Therefore, if linseed oil rich in ω3 is mixed with safflower oil, it can improve the fatty acid ratio in safflower oil, so as to achieve the effects of reducing weight gain, improving fat quality, enhancing glucose tolerance, improving blood lipid profiles, and reducing inflammation by lowering pro-inflammatory markers.

[0003] In the prior art, there are few feed additives based on safflower oil, and less focus on the combined functions of anti-inflammatory and lipid-lowering. For example, in patent CN114886041A, a feed additive is prepared with 90 - 95% pure safflower oil, 2 - 5% Portulaca oleracea, and 2 - 5% rice bran as raw materials, and its main function is to kill viruses and bacteria; although patent CN119214250A also uses safflower oil as a raw material for feed, its main function is to increase the milk production of sows, increase the number of immunoglobulins in milk, and improve the immunity of piglets. Among other feed additives with anti-inflammatory and lipid-lowering functions, most have complex compositions. There is no relevant report on animal feed with safflower-based blended oil additives in this field. Summary of the Invention

[0004] The present invention provides an oil-based feed additive with anti-inflammatory and lipid-lowering functions, which solves the deficiencies in the prior art. The preparation method of this feed additive is simple, rapid, and its ingredients are safe and simple; when added to animal feed as an additive, it has the effects of facilitating animal lipid metabolism and reducing the incidence of animal inflammation and metabolic syndrome.

[0005] The technical solutions adopted to achieve the above objectives of the present invention are as follows:

[0006] An oil-based feed additive with anti-inflammatory and lipid-lowering functions is prepared by the following method:

[0007] (1) Take safflower oil and linseed oil respectively, mix them with a sufficient amount of sulfuric acid methanol solution, then add heptadecanoic acid to obtain a mixed solution, and then dissolve the mixed solution in chloroform to prepare a safflower oil-chloroform mixture and a linseed oil-chloroform mixture;

[0008] (2) Incubate the safflower oil-chloroform mixture and the linseed oil-chloroform mixture at 75-95 °C;

[0009] (3) Add ultrapure water and n-hexane to the incubated safflower oil-chloroform mixture and linseed oil-chloroform mixture, mix well, and then perform centrifugation to obtain a safflower oil supernatant and a linseed oil supernatant;

[0010] (4) After filtering the safflower oil supernatant and the linseed oil supernatant, perform gas chromatography-mass spectrometry analysis to determine the contents of linoleic acid ω6 and linolenic acid ω3 in the safflower oil supernatant and the linseed oil supernatant;

[0011] (5) According to the contents of linoleic acid ω6 and linolenic acid ω3 determined in step (4), mix safflower oil and linseed oil so that the molar ratio of linoleic acid ω6 / linolenic acid ω3 in the mixed oil is 2.5-3.5:1; the prepared mixed oil is the oil-based feed additive with anti-inflammatory and lipid-lowering functions.

[0012] Further, the added weight of the oil-based feed additive with anti-inflammatory and lipid-lowering functions added to animal feed is 1-3%.

[0013] Further, the added weight of the oil-based feed additive with anti-inflammatory and lipid-lowering functions added to animal feed is 2%.

[0014] Further, the molar ratio of linoleic acid ω6 / linolenic acid ω3 in the oil-based feed additive with anti-inflammatory and lipid-lowering functions is 3:1.

[0015] Further, in step (1), the volume ratios of safflower oil and linseed oil to the sulfuric acid methanol solution are both 1-3:1, and the added heptadecanoic acid

[0016] Further, in step (2), the incubation temperature is 85 °C and the incubation time is 2 hours.

[0017] Further, in step (3), the centrifugation time is 8 minutes and the rotation speed is 1000 rpm.

[0018] Further, in step (4), filtration is carried out using a 0.45 μm filter membrane.

[0019] Further, the conditions for gas chromatography - mass spectrometry analysis in step (4) are as follows: helium is used as the carrier gas with a flow rate of 1 mL / min and a split flow rate of 20 mL / min; the initial temperature is set at 170 °C for 1 minute, then gradually increased to 230 °C at a rate of 3 °C / min, and maintained at this temperature for 3 minutes.

[0020] Compared with the prior art, the oil - based feed additive provided by the present invention has the following advantages: 1. For the oil - based feed additive in the present invention, its raw materials are only safflower oil and linseed oil mixed in precise proportions. Its preparation method is very simple and fast, and the raw materials are safe and the components are simple. 2. After adding the oil - based feed additive provided by the present invention to animal feed and verified by animal experiments, it has many effects such as improving animal lipid metabolism, inhibiting inflammatory reactions, and regulating the intestinal flora. Description of the Drawings

[0021] Figure 1 It is a graph showing the change in body mass index of the diet supplement group in the embodiment of the present invention;

[0022] Figure 2 It is a graph showing the change in body weight of the HFD group in the embodiment of the present invention;

[0023] Figure 3 It is a graph of the glucose tolerance test in the embodiment of the present invention;

[0024] Figure 4 It is a graph comparing the relative expression levels of TNF - α and IL - 6 in mice in the embodiment of the present invention. Detailed Embodiments

[0025] Hereinafter, the applicant will make a detailed and specific description of the present invention in combination with the accompanying drawings and specific embodiments. It should be clear that the embodiments described here only represent a part of the numerous possible embodiments of the present invention, not all of them. All other embodiments that can be conceived by those of ordinary skill in the art without creative labor based on these embodiments of the present invention should be regarded as falling within the scope covered by the present invention.

[0026] Preparation of Oil - based Feed Additive

[0027] Analyze the fatty acid composition and content of the purchased safflower oil and linseed oil. The specific steps are as follows: First, derivatize the fatty acids to form fatty acid methyl esters (FAME): Take 10 mg of oil samples respectively. Mix 10 mg of oil samples with 4.5 mL of a solution containing sulfonic acid and methanol (this solution is prepared by mixing 95 mL of methanol and 5 mL of concentrated sulfuric acid), and add 0.1095 g of heptadecanoic acid (C17:0) dissolved in chloroform; obtain a safflower oil - chloroform mixture and a linseed oil - chloroform mixture.

[0028] Incubation mixture: Incubate the mixture at 85 °C for 2 hours.

[0029] Extraction and preparation of samples for analysis: Thoroughly mix the incubated mixture with 3 mL of ultrapure water and 3 mL of n-hexane. Then centrifuge at 1000 rpm for 8 minutes to obtain the supernatant, and filter the supernatant through a 0.45 μm filter membrane.

[0030] Perform gas chromatography - mass spectrometry analysis: Use helium as the carrier gas with a flow rate of 1 mL / min and a split flow rate of 20 mL / min. Set the initial temperature to 170 °C for 1 minute, then gradually increase the temperature to 230 °C at a rate of 3 °C / min and hold at this temperature for 3 minutes.

[0031] Data processing: Determine the elution protocol for different fatty acid contents according to Chinese national standard (GB 5009.168 - 2016). Process the original GC - MS data using Xcalibur software.

[0032] Calibration analysis: Inject and analyze a mixed standard solution containing 37 components of fatty acid methyl esters (product number DRE - A50000091HP, Laboratory of the Government Chemist, UK) to determine the elution time of fatty acid methyl esters.

[0033] Mix the safflower oil and linseed oil after determining the fatty acid components. For example, safflower oil containing 74.40% linoleic acid ω6 (relative molecular mass 280) is mixed with linseed oil containing 47.84% linolenic acid ω3 (relative molecular mass 278) (the densities of the finished safflower oil and the finished linseed oil are almost the same). Mix 13.33 mL of safflower oil with 6.67 mL of linseed oil, and after mixing, a mixed oil with a molar ratio of linoleic acid ω6 / linolenic acid ω3 of 3.1:1 can be obtained. This mixed oil is the lipid feed additive. Add this lipid feed additive to 1 kg of basal feed without other oils (total lipid content accounts for 2%); the components of the basal feed include: 400 g of corn flour, 200 g of soybean meal, 70 g of fish meal, 230 g of flour, 30 g of yeast, 20 g of dicalcium phosphate, 15 g of calcium carbonate, and 5 g of salt per kg of feed.

[0034] Mix the basal feed components and the lipid feed additive evenly and granulate, then store at 20 - 22 °C for use in subsequent biological experiments.

[0035] Biological experiments

[0036] Experimental materials: Six - week - old male Kunming mice (weighing 40 - 48 g) were purchased from the Experimental Animal Center of Huazhong Agricultural University. All animal experimental protocols were approved by the Hubei Experimental Animal Monitoring Station (license number: SCXK2020 - 0019).

[0037] Main instruments: Thermo Fisher Scientific ISQ7000 instrument: Thermo Fisher Scientific; Bio-Rad CT003142 real-time PCR system: Bio-Rad; NanoPhotometer-N80 spectrometer: Implen GmbH, Germany.

[0038] Experimental protocol: Three to four mice were housed per cage, and two to three cages were assigned to each group for different dietary interventions. They had free access to the designated feed and pure water and were maintained under a 12-hour light / 12-hour dark cycle with a relative humidity of 45%. One week before the experiment started, after the mice had acclimated to the animal house environment, they were randomly divided into five dietary groups (10 mice per group): (1) control group (basal diet + 2% soybean oil); (2) ω6 group (soybean oil was replaced with safflower oil, hereinafter referred to as the ω6 group); (3) ω3 group (soybean oil was replaced with linseed oil, hereinafter referred to as the ω6 group); (4) 5:1 group (soybean oil was replaced with a mixture of safflower oil and linseed oil to achieve an ω6 / ω3 ratio of 5:1, hereinafter referred to as the 5:1 group); (5) 3:1 group (soybean oil was replaced with a mixture of safflower oil and linseed oil to achieve an ω6 / ω3 ratio of 3:1, hereinafter referred to as the 3:1 group).

[0039] The mice were provided with normal feed and supplemented with the corresponding oils for seven weeks, followed by a high-fat diet and oral gavage (400 μl / 100 g per day) for an additional eight weeks. General health indicators such as mouse body weight, obesity tendency, food intake, fasting blood glucose level, and glucose tolerance were analyzed. In addition, lipids, inflammatory factors, and gut microbiota in different tissues were also detected to explore the potential mechanisms of the action of optimized oil additives.

[0040] Experimental results: Physiological indices of the mice were detected in various aspects. The detailed results showed that the intake of the ω6 group without ω3 supplementation could lead to weight gain, reduced glucose tolerance, and poor blood lipid levels in mice. However, compared with the ω6 group supplemented with pure safflower oil, the mice in the 3:1 group showed a significant reduction in weight gain, a decrease in fat mass, and improved glucose tolerance. The specific experimental results are as follows:

[0041] The changes in body mass index of the five dietary supplement groups were as Figure 1 shown, where (a) is the body mass index graph, and it can be seen that after 42 days of control, it was significantly different from the ω6 group, and the ω6 group was significantly different from the 3:1 group; (b) is the weight gain graph, and (c) is the fasting blood glucose level graph.

[0042] The weight change graph of the five HFD groups (high-fat diet and oral gavage) was as Figure 2As shown, where (a) is the body mass index graph. It can be seen that for C-ω-6, ω-3P = 0.01, 0.001, ω-6 / ω-3, 5:1, 3:1P = 0.001, 0.003, 0.001, and P < 0.05 indicates a significant difference. Thus, it can be known that compared with the control group, the body mass index of the ω6 group increased significantly, but ω6 had no effect on the fasting blood glucose level. Compared with the pure ω6 group, the pure ω3 group and the ω6 / ω3 optimized groups could control the degree of weight gain to a certain extent; (b) is the weight gain graph, and (c) is the fasting blood glucose level graph.

[0043] The glucose tolerance test was conducted in the last week when the mice were sacrificed. The fasting glucose level was measured before glucose administration (0 minute), and then the blood glucose levels were measured at 15 minutes, 30 minutes, 60 minutes, and 120 minutes after administration. The results of the glucose tolerance test are as Figure 3 shown. Figure a is the graph of the glucose tolerance test results of mice. Figure b is the blood glucose AUC curve of mice. The larger the AUC value, the greater the blood glucose fluctuation and the worse the blood glucose control. It can be seen from the figure that compared with the control group, ω6 in safflower oil may cause impaired glucose metabolism. However, the group with optimized ω6 / ω3 ratio could significantly improve glucose tolerance and control the range of blood glucose fluctuation to a certain extent compared with the pure ω6 group. (* in the figure indicates significant difference from group C, and # indicates significant difference from the ω6 group).

[0044] Figure 4 It is the comparison graph of the relative expression levels of TNF-α and IL-6 in mice. It can be seen from the figure that the 5:1 group and the 3:1 group showed beneficial changes in the serum lipid profile, including reduced triglyceride, total cholesterol, and LDL-C levels. At the same time, the expression of pro-inflammatory markers such as TNF-α and IL-6 in the liver tissue decreased significantly. The expression of key genes involved in lipid metabolism studied in the liver, muscle, and adipose tissues revealed an increase in fatty acid transport and oxidation in the 5:1 group and the 3:1 group, and an increase in fatty acid synthesis and fat accumulation in the pure ω6 group.

[0045] Microbiota analysis revealed that the 3:1 group with optimized ω6 / ω3 ratio in the present invention showed increased microbial diversity and enrichment of beneficial bacteria such as Helicobacteraceae, as well as a more favorable Firmicutes / Bacteroidetes ratio, which is related to the reduction of obesity risk. In addition, the optimized 3:1 group supplementation reduced pro-inflammatory microbiota such as Deferribacteraceae, which is related to the reduction of fat accumulation and the improvement of lipid metabolism.

Claims

1. An oil-based feed additive with anti-inflammatory and lipid-lowering functions, characterized in that Prepared by the following method: (1) Respectively take safflower oil and linseed oil, mix them with a sufficient amount of sulfuric acid methanol solution, then add heptadecanoic acid to obtain a mixed solution, and then dissolve the mixed solution in chloroform to prepare a safflower oil-chloroform mixture and a linseed oil-chloroform mixture; (2) Incubate the safflower oil-chloroform mixture and the linseed oil-chloroform mixture at 75-95 °C; (3) Add ultrapure water and n-hexane to the incubated safflower oil-chloroform mixture and linseed oil-chloroform mixture, mix well, and then perform centrifugation to obtain a safflower oil supernatant and a linseed oil supernatant; (4) After filtering the safflower oil supernatant and the linseed oil supernatant, perform gas chromatography-mass spectrometry analysis to determine the contents of linoleic acid ω6 and linolenic acid ω3 in the safflower oil supernatant and the linseed oil supernatant; (5) According to the contents of linoleic acid ω6 and linolenic acid ω3 determined in step (4), mix safflower oil and linseed oil so that the molar ratio of linoleic acid ω6 / linolenic acid ω3 in the mixed oil after mixing is 2.5-3.5:1; the prepared mixed oil is an oil-based feed additive with anti-inflammatory and lipid-lowering functions.

2. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 1, wherein: The added weight of the oil-based feed additive with anti-inflammatory and lipid-lowering functions added to animal feed is 1-3%.

3. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 2, wherein: The added weight of the oil-based feed additive with anti-inflammatory and lipid-lowering functions added to animal feed is 2%.

4. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 1, wherein: The molar ratio of linoleic acid ω6 / linolenic acid ω3 in the oil-based feed additive with anti-inflammatory and lipid-lowering functions is 3:

1.

5. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 1, wherein: In step (1), the volume ratios of safflower oil and linseed oil to the sulfuric acid methanol solution are both 1-3:

1.

6. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 1, wherein: In step (2), the incubation temperature is 85 °C and the incubation time is 2 hours.

7. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 1, characterized in that: In step (3), the centrifugation time is 8 minutes and the rotation speed is 1000 rpm.

8. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 1, characterized in that: In step (4), filter with a 0.45 μm filter membrane.

9. The lipid feed additive with anti-inflammatory and lipid-lowering functions according to claim 1, wherein: The conditions for gas chromatography-mass spectrometry analysis in step (4) are: use helium as the carrier gas, the flow rate is 1 mL / min, and the split flow rate is 20 mL / min; set the initial temperature to 170 °C for 1 minute, then gradually increase to 230 °C at a rate of 3 °C / min, and maintain at this temperature for 3 minutes.

10. Use of the oil-based feed additive described in claim 1 in the preparation of animal feed for improving animal lipid metabolism, inhibiting inflammatory reactions, and regulating intestinal flora.

Images