Optimized feed for increasing polyunsaturated fatty acid in mutton and lamb feeding method

Abstract

The invention discloses a lamb feeding method for increasing polyunsaturated fatty acid in meat. According to the invention, lactobacillus reuteri is supplemented into the basic feed for sheep, so that polyunsaturated fatty acids in mutton can be increased, the tenderness of the mutton can be improved, and the proportion and area ratio of I-type muscle fibers in muscles can be increased. Meanwhile, the technical scheme also can regulate the rumen microbial community structure of the sheep, promote the degradation of rumen carbohydrates and the generation of muscle aromatic compounds, and regulate the metabolism of sheep muscle proteins. The proper addition amount of lactobacillus reuteri in the basic feed for sheep is determined, the obtained optimized feed can improve polyunsaturated fatty acid and tenderness of mutton, the slaughter performance of sheep is enhanced, and the economic income of pasturing households and the economic benefit level of enterprises can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of breeding, and in particular to a lamb feeding method for increasing polyunsaturated fatty acids in meat. Background Art

[0002] In recent years, as people's demand for a better life continues to grow, consumers' demand and expectations for mutton quality have also gradually increased. However, the current breeding methods for mutton sheep have shifted from traditional free-range grazing to intensive confinement or semi-confined confinement. This shift in breeding methods has led to varying degrees of decline in mutton flavor and quality.

[0003] To address this issue, it's crucial to find a way to improve the quality of lamb and meet consumer demand. Research has shown that grazing, in addition to adequate exercise, improves the antioxidant capacity and overall meat quality of sheep, as well as their intake of fresh forage (Huang, Yafeng, et al. "Feeding regimens affecting carcass and quality attributes of sheep and goat meat: A comprehensive review." Animal bioscience (2023). A unique microbial system in the rumen of ruminants synthesizes micronutrients, such as fatty acids and other essential nutrients, from crude fiber and dry matter. These synthesized compounds are transported through the bloodstream and deposited in animal tissues, where they significantly influence the taste of meat (Kyawt, Yin Yin, et al. "Dynamic changes of rumen microbiota and serum metabolome revealed increases in meat quality and growth performances of sheep fed biofermented ricestraw." Journal of Animal Science and Biotechnology 15.3(2024):1207-1226).

[0004] With the exception of fish oil, the primary fatty acids in all animal oils are saturated. However, unsaturated fatty acids play a significant role in the formation of body fat and are essential for the human body. Unsaturated fatty acids are classified into two types, monounsaturated and polyunsaturated, depending on the number of double bonds. These essential fatty acids help maintain normal body function and have positive effects on cardiovascular health, brain development, and the immune system (Zhou Yinghuan et al., "Research Progress on the Biological Functions of Polyunsaturated Fatty Acids and Their Application in Livestock and Poultry Production." China Animal Husbandry Journal). To improve the nutritional value of lamb (i.e., increase the polyunsaturated fatty acid content in lamb), overall regulation of polyunsaturated fatty acids by altering the dietary composition can be considered.

[0005] Microorganisms are widely used in animal husbandry. Adding microorganisms to feed can produce a variety of beneficial effects, such as improving animal digestibility, boosting immunity, and promoting weight gain. Based on this, this application aims to provide a method for regulating mutton quality to meet consumer demand and provide a theoretical basis for dietary adjustments to improve the quality of sheep meat under semi-intensive farming conditions. Summary of the Invention

[0006] The purpose of the present invention is to provide a lamb feeding method for increasing the polyunsaturated fatty acids in the meat, which improves the polyunsaturated fatty acid content and tenderness of the mutton by changing the diet and level, and has application value in the field of sheep breeding.

[0007] In order to achieve the above object, the present invention provides an optimized feed for increasing polyunsaturated fatty acids in mutton. The optimized feed is prepared by adding Lactobacillus reuteri to a basic diet, wherein the sheep is sheep, preferably Sunite sheep.

[0008] Preferably, the above-mentioned basic diet comprises, by weight, 55 parts of corn, 17 parts of wheat bran, 8 parts of soybean meal, 8 parts of rapeseed meal, 8 parts of cottonseed meal, and 4 parts of premix; The premix comprises, by weight, 0.2 parts of vitamin A, 0.1 parts of vitamin D3, 0.25 parts of vitamin E, 0.5 parts of cellulase, 3 parts of methionine, 2 parts of lysine, 0.07-0.10 parts of copper sulfate, 0.75 parts of ferrous sulfate, 0.32-0.5 parts of manganese sulfate, 0.34-0.40 parts of zinc sulfate, 0.5 parts of sodium nitrite, 0.35-0.5 parts of cobalt chloride, 0.4 parts of potassium iodide powder, 0.5-1 parts of antioxidant, 40 parts of calcium hydrogen phosphate, 3 parts of calcium carbonate, 7 parts of sodium chloride, 0.8 parts of pine needle powder, and 30-40 parts of medical stone. The premix is ​​implemented in accordance with GB / T18823-2010, calculated by weight, and stirred and mixed.

[0009] The optimized feed provided by the present invention has an addition amount of Lactobacillus reuteri of 2×10 10 CFU / bird / day.

[0010] The present invention also provides a lamb feeding method for increasing polyunsaturated fatty acids in meat, specifically referring to the "Meat Sheep Feeding Standard" (NY / Y816-2004), and conventionally feeding the lambs with the above-mentioned optimized feed for 3 to 4 months.

[0011] The optimized feed provided by the present invention can be applied in the field of sheep breeding, and can be particularly used to improve the quality of mutton, including the content of polyunsaturated fatty acids in mutton, the tenderness of mutton, and the generation of aromatic compounds in mutton.

[0012] The present invention has the following advantages: The technical solution provided by the present invention can effectively improve the rumen of relevant organisms, making their functions better, thereby promoting the healthy development of the overall breeding industry.

[0013] The application of the optimized feed and feeding method provided by the present invention to sheep breeding is conducive to reducing grazing behavior, which has positive significance for protecting natural grasslands and maintaining the balance and stability of the ecosystem, and makes an important contribution to creating a more harmonious and beautiful ecological environment.

[0014] The present invention discloses a lamb feeding method for increasing the polyunsaturated fatty acids in meat. By supplementing Lactobacillus reuteri, the proportion and area ratio of type I muscle fibers in muscles can be increased, the transformation of muscle fiber types from glycolysis to oxidation can be promoted, and the tenderness of lamb meat can be improved.

[0015] The present invention can promote the production of muscle aromatic compounds by supplementing Lactobacillus reuteri, significantly increase the glycoside hydrolase and glycosyltransferase genes in the rumen, enhance the degradation of sheep fat, protein and carbohydrates, improve the production of aromatic compounds in mutton, and enhance the slaughter performance of sheep, thereby promoting the improvement of the economic income of herders and the economic efficiency of enterprises.

[0016] This study revealed that supplementation with Lactobacillus reuteri had a positive effect on polyunsaturated fatty acids, volatile compounds, and gene expression related to these compounds in sheep. 9 CFU / g, and a constant addition of 4g / head / day of Lactobacillus reuteri can change the rumen microecological environment of sheep, adjust muscle fiber type, regulate protein metabolism, and improve the flavor and tenderness of mutton, bringing many beneficial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Results of the effect of adding Lactobacillus mucosae on the volatile flavor of sheep muscle in the basal diet of the present invention. Among them, A in the figure is the E-nose radar chart; B is the E-nose PCA chart; C is the statistical difference of the results of 10 electronic nose sensors; W2S is calculated using the Wilcoxon rank sum test, and other data are calculated using Student's t-test; P>0.05 is represented by "ns", and 0.01<P<0.05 is represented by "*".

[0018] Figure 2 Results of the effect of adding Lactobacillus mucosae on the muscle fiber type of sheep in the basal diet of the present invention. Among them, A and B in the figure are the ATPase staining results of the longissimus dorsi muscle of the control group and the treatment group, respectively; C is the cross-sectional area of muscle fibers; D is the diameter of muscle fibers; E is the area ratio of muscle fibers; F is the ratio of muscle fibers; G is the muscle fiber density; * indicates significance at P<0.05.

[0019] Figure 3 Results of the differential effect of adding or not adding Lactobacillus mucosae to the basal diet of the present invention on enzyme activity. Among them, A-E in the figure are the measurement results of TP (total protein), T-AOC (total antioxidant capacity), SDH (succinate dehydrogenase), MDH (malate dehydrogenase), and LDH (lactate dehydrogenase) in different groups, respectively, where * indicates significance at P<0.05, ** indicates significance at P<0.01, and *** indicates significance at P<0.001.

[0020] Figure 4 Results of the effect of adding Lactobacillus mucosae to the basal diet of the present invention on the rumen microbiota of Sunite lambs. Among them, A in the figure is the PCoA chart at the phylum level of CG and LG; B is the stacked bar chart of the top ten phyla with the highest relative abundances; C is the PCoA chart at the genus level of CG and LG; D is the stacked bar chart of the top 20 genera with the highest relative abundances; F is the statistical box plot of the differences in seven biomarkers at the genus level; the relative abundances of the seven genus-level biomarkers obtained using LEfSe were statistically analyzed for differences, where g_Alistipes, g_Paraprevotella, g_Schwartzia and g_ Selenomonas The Wilcoxon rank sum test was used for testing, and others were tested using Student's t-test; P>0.05 is represented by "ns", 0.01<P<0.05 is represented by "*", 0.001<P<0.05 is represented by "*", 0.001<0.005 is represented by "*". P>0.05 is represented by "ns", 0.01<P<0.05 is represented by "*", 0.001<P<0.01 is represented by "**", and P<0.001 is represented by "***".

[0021] Figure 5LEfSe dendrogram results of the effect of Lactobacillus reuteri supplementation on rumen microbial taxa in Sunite lambs.

[0022] Figure 6 The results of LDA linear discriminant analysis were presented to show the effect of Lactobacillus reuteri supplementation in feed on the composition of rumen microbiota in Sunite lambs. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0024] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0025] Experimental Example 1 Optimization of feed preparation The basal diets selected for this application are as follows: Calculated based on the growing and fattening period: by weight, it contains 55 parts of corn, 17 parts of wheat bran, 8 parts of soybean meal, 8 parts of rapeseed meal, 8 parts of cottonseed meal, and 4 parts of premix; The premix is ​​implemented in accordance with GB / T18823-2010, is calculated by weight and stirred and mixed, and includes: 0.2 parts of vitamin A, 0.1 parts of vitamin D3, 0.25 parts of vitamin E, 0.5 parts of cellulase, 3 parts of methionine, 2 parts of lysine, 0.07-0.10 parts of copper sulfate, 0.75 parts of ferrous sulfate, 0.32-0.5 parts of manganese sulfate, 0.34-0.40 parts of zinc sulfate, 0.5 parts of sodium nitrite, 0.35-0.5 parts of cobalt chloride, 0.4 parts of potassium iodide powder, 0.5-1 parts of antioxidant, 40 parts of calcium hydrogen phosphate, 3 parts of calcium carbonate, 7 parts of sodium chloride, 0.8 parts of pine needle powder, and 30-40 parts of medical stone.

[0026] The optimized feed is based on the basic diet, and Lactobacillus reuteri is accurately weighed and stirred into the feed. The viable count of the bacteria is 5×10 9 CFU / g, the constant dosage is 4g / animal / day, that is, the dosage is 2×10 10 CFU / bird / day.

[0027] Experimental Example 2 Animal feeding and mutton quality determination Eighteen three-month-old Sunite male sheep were randomly divided into two groups of nine each. Each group was fed a basal diet (control group, CG) or an optimized diet (treatment group, LG). Each group underwent a 7-day pilot study, followed by a 90-day experiment. The basal diet was formulated according to the "Standards for the Feeding of Mutton Sheep" (NY / Y816-2004). The sheep had free access to antibiotic-free water during the feeding period, and were monitored daily to ensure that all feed in the trough was consumed. After three to four months of feeding, samples were collected and the quality of the mutton was determined. The sheep were fasted for 12 hours before slaughter.

[0028] 1. Sample Collection Muscle samples: Within 45 minutes after slaughter, longissimus dorsi / biceps femoris samples (100 g) were collected from each sheep. A portion of the muscle tissue (60 g) was stored at 4°C for 24 hours for meat quality analysis. Another portion of the muscle tissue sample (15 g) was cut into six blocks of approximately 0.5 × 0.5 × 1 cm³ along the fiber direction. These blocks were then dehydrated and dried in cooled isopentane, then quickly immersed in liquid nitrogen for approximately 10 seconds for muscle fiber characterization analysis. The final portion of the muscle sample (25 g) was placed in a 2 mL cryovial and stored in liquid nitrogen for gene and enzyme activity analysis.

[0029] Gastrointestinal samples: Rumen and colon epithelial gene samples (5 g) were collected and placed in liquid nitrogen; rumen fluid (15 mL) and fecal samples (3 g) were collected in 15 mL enzyme-free sterile centrifuge tubes.

[0030] 2. Experimental Methods and Results 1. Determination of mutton quality indicators ① Color difference: The colorimeter was calibrated with a standardized white tile, 2° observer angle, 50 mm aperture, and light source D65. The brightness value of the longissimus dorsi muscle was measured ( L* )、Redness value( a* ) and yellowness value ( b* ).

[0031] ② Tenderness: Place a muscle block of approximately 2.5 cm × 3 cm × 5 cm in a sealed bag, cook in a water bath (75°C) for 45 min, take it out, cool to room temperature, and then cut it into 3 cm × 1 cm × 1 cm strips. Use a C-LM3B digital muscle tenderizer to measure its tenderness.

[0032] ③ pH: Use a carcass pH meter. The initial pH value is recorded as pH45min (measured 45 minutes after slaughter). After the meat sample is placed at 4°C for 24 hours to release acid, the pH value is measured again, recorded as pH24h. Calibrate the pH meter with standard calibration solution before measurement. Measure each sample three times, record, and calculate the average value.

[0033] ④ Cooking loss: Take a meat sample of about 50 g, weigh it accurately (recorded as m1), seal it, and cook it in a 75°C water bath for 45 min. When the core temperature of the meat sample reaches 75°C, keep it for 20 min, then quickly cool it to room temperature on ice, dry the surface moisture with absorbent paper, and weigh it again (recorded as m2). Use Formula 1 to calculate the cooking loss rate.

[0034] Formula 1 2. Determination of muscle long-chain fatty acids Following the method of Wang Baihui with some modifications (Wang Baihui. Effects of Rearing Methods on Gastrointestinal Microbiota, Fatty Acid Metabolism, and Meat Quality of Sunite Sheep and Their Mechanisms [D]. Inner Mongolia Agricultural University, 2019), approximately 0.4 g of the longissimus dorsi muscle was sampled and 1 mL of the ground meat was placed in a 15 mL centrifuge tube. 0.7 mL of 10 mol / L KOH and 5.3 mL of methanol were added to the centrifuge tube. The tube was incubated in a 55°C water bath for 1.5 hours, shaking vigorously for 5 seconds every 10 minutes. After cooling to room temperature (cold water may be used), 0.58 mL of 12 mol / L H₂SO₄ was added to each centrifuge tube. The tube was incubated in a 55°C water bath for 1.5 hours, shaking vigorously for 5 seconds every 15 minutes. After cooling to room temperature, 3 mL of n-hexane (chromatographic grade) was added to each centrifuge tube, vortexed for 5 minutes, and centrifuged at 1,500 r / min for 5 minutes. 1 mL of n-hexane was filtered through a 0.22 μm organic filter membrane into a sample vial for gas chromatography analysis. Gas chromatography-mass spectrometry conditions: Column: trans-type column (100 m x 0.25 mm x 0.20 μm), carrier gas: helium, carrier gas flow rate: 1 mL / min, inlet temperature: 240°C, injection volume: 1 μL, split ratio: 100:1. A temperature program was used: initial temperature: 60°C, hold: 1 min, then increase to 120°C at 20°C / min, hold: 1 min, then increase to 240°C at 5°C / min, hold: 15 min. MS conditions: ion source temperature: 300°C, transfer line temperature: 240°C, mass scan range: 50–500 (m / z), solvent delay time: 4 min.

[0035] 3. Electronic nose measurement Lamb was thawed at 4°C, the fascia removed, and minced. The sample temperature was maintained at room temperature. A 5 g sample was weighed and placed in a 15 mL injection vial, sealed with double plastic wrap, and equilibrated at room temperature for 2 h before electronic nose testing. The sensors used in the PEN3 electronic nose and their performance are shown in Table 1 below. Preliminary experiments revealed that the electronic tongue data from the sample stabilized after 90 s. Therefore, the electronic nose testing method was set to a sampling frequency of 1 Hz, an air intake frequency of 200 mL / min, a rinsing time of 90 s, and an injection time of 120 s. The sample was named and stored.

[0036] Table 1 Sensors used in the electronic nose (PEN3) and their performance

[0037] The effects of optimized feed on lamb flavor were determined as follows: It is known that small changes in volatile compounds can lead to changes in the response of the electronic nose sensor, allowing it to more accurately represent the overall volatile flavor compounds in meat (Han Yuanxi, et al. Analysis of volatile flavor characteristics of different types of air-dried meat by electronic nose combined with HS-GC-IMS technology [J / OL]. Food Industry Science and Technology). Although electronic noses have advantages in identifying specific odors, their ability to identify complex odor mixtures is limited. The current comprehensive analysis helps to correlate the comprehensive taste determined by the electronic nose with the volatile compounds identified by gas chromatography-mass spectrometry (GC-MS). By extracting the response intensity (response value) of 10 sensors in the Sunite lamb electronic nose, the aroma fingerprints of similar foods from different parts and feeding methods were established, and the results are shown in Figure 2. Figure 1 As shown in Figures A and C, the CG group is the control group and the LG group is the treatment group. The results show that the W1C, W1S, and W3C sensors have different aroma intensities for different groups of meat samples, while the response intensities of the other seven sensors in the LG group are roughly the same. Principal component analysis (PCA) analysis showed no significant differences between the two groups of samples (see the results). Figure 1 The responses of electronic sensors W5S and W1W were helpful in distinguishing lamb breeds, while sensors W1C, W1S, and W3C were mainly sensitive to methane and aromatic compounds (see Table 1), indicating that the addition of Lactobacillus reuteri to the basal diet promoted the formation of aromatic compounds in lamb muscle.

[0038] At the same time, the effects of optimized feed on sheep slaughter performance and meat quality were further determined. Meat quality includes sensory quality and nutritional components. The pH value reflects the level of muscle glycogen degradation after lamb slaughter, and the pH value of lambs reflects the level of muscle glycogen degradation after animal slaughter (Li Ping, et al. Effects of different feeding methods on the quality characteristics of Guizhou black goat meat [J]. Chinese Herbivore Science). The results of the effects on the slaughter performance and meat quality of Sunite sheep obtained after measurement are shown in Table 2. It can be seen that the pH45min and pH24h of the CG group and the LG group are within the normal range. The pH24h of the LG group was significantly higher than that of the CG group (P<0.05), and the yellowness value between the two groups ( b* )、Redness value( a* ) and brightness values ​​( L* ) showed no significant difference (P>0.05). Shear force was negatively correlated with meat tenderness, with sheep in the LG group showing significantly higher tenderness than those in the CG group (P<0.05). Slaughter performance, reflecting animal development, includes metrics such as carcass weight, dressing percentage, net meat weight, and backfat thickness. The LG group showed higher dressing percentage and carcass weight compared to the CG group (P<0.05). These results suggest that dietary supplementation with Lactobacillus reuteri can improve meat quality by increasing the dressing percentage and tenderness of sheep.

[0039] Table 2 Effects of optimized feed on slaughter performance and meat quality of Sunite sheep

[0040] Note: CG = control group; LG = Lactobacillus reuteri group (n = 9).

[0041] For further testing of the effects of optimized diets on sheep muscle fibers, see Figure 2 The results of the effect of adding Lactobacillus reuteri to the basal diet on the muscle fiber type of sheep. The ultrastructure of muscle fibers was observed by ATPase histochemical staining. Under a 100x microscope, the sheep muscle fibers were structurally intact and tightly arranged, and there was no interstitial breakage or expansion. According to the histochemical staining of ATPase, muscle fibers can be divided into three types: dark type I muscle fibers, bright type IIA muscle fibers and intermediate type IIB muscle fibers. These classifications reflect the differences in the pH stability range of ATPase in different fiber types (Tan Dandan, et al. Study on the composition and characteristics of muscle fiber types in different parts of sheep [J / OL]. China Animal Husbandry Journal). Whether it is the control group ( Figure 2 A) or the treatment group ( Figure 2 In Figure B), the muscle fiber composition of the longissimus dorsi muscle of sheep follows the pattern of IIB>IIA>I. Software analysis was used to calculate Figure 2The muscle fiber biometric index shown in the figure. In sheep longissimus dorsi muscle, type IIB muscle fibers account for approximately 50%, with the remainder being oxidized myoglobin (I+IIA). Addition of Lactobacillus reuteri had no significant effect on the cross-sectional area and diameter of each type of myoglobin ( Figure 2 C and D) (P>0.05). Figure 2 E and Figure 2 As shown in Figure F, the addition of Lactobacillus reuteri significantly increased the area and proportion of type I muscle fibers, while reducing the area and proportion of type IIB muscle fibers (P<0.05) ( Figure 2 G in ).

[0042] The tenderness of meat is directly related to the diameter and cross-sectional area of ​​muscle fibers. As livestock and poultry grow, the increase in muscle fiber diameter and cross-sectional area will lead to a decrease in fiber density, an increase in shear force, and a decrease in meat tenderness (see Katekhaye, MA, Kumar, KN, Reddy, KJ, et al. (2018). Comparison of Chicken Varieties: Muscle Fiber Diameter, pH, Color, Tenderness in Pectoralis major Muscle. J. Meat Sci. 13(1)). The results of this experiment showed that compared with the control group, the diameter and cross-sectional area of ​​type IIA and IIB muscle fibers in the treated group showed a slight downward trend, although it was not statistically significant, which may have an impact on the tenderness of meat ( Figure 2 C and D in the figure). Previous studies have shown that glycolytic fiber causes a faster drop in pH and a higher water loss rate. This may be due to the rapid glycolytic metabolism and reliance on glycogenolysis for energy, resulting in a high lactic acid content and a rapid drop in pH (see Picard, B., & Gagaoua, M. (2020). Muscle Fiber Properties in Cattle and Their Relationships with Meat Qualities: An Overview. J Agric Food Chem. 68 (22)). The shear force of the Lactobacillus reuteri group was significantly lower than that of the control group (P<0.05), which is due to the higher proportion of type I muscle fibers and the lower proportion of type IIB muscle fibers.

[0043] To further determine the effect of optimized feed on sheep muscle enzyme activity, explore muscle fiber type and conversion mechanism, the energy metabolism and antioxidant enzyme activities in the organism were measured. The difference in enzyme activity between the two groups was obtained as follows Figure 3As shown, the C group was the control group fed with the basic diet, and the L group was the treatment group fed with the optimized diet. The results showed that the activities of TP, T-AOC, and MDH in the L group were slightly increased compared with the C group, but there was no statistical significance. Lactobacillus reuteri increased SDH (P<0.01) and decreased LDH (P<0.001) activity in sheep muscle, indicating a regulatory effect on metabolic enzyme activity. In this study, LDH activity in the muscle of the L group was decreased (P<0.001), consistent with the observed decrease in the area and proportion of type IIB muscle fibers in this group (P<0.05). MDH contributes to fatty acid synthesis, while SDH participates in the tricarboxylic acid cycle, supplying electrons for the respiratory chain of eukaryotic cells, both of which indicate aerobic metabolic levels (see Naseri, NN, Bonica, J., Xu, H., et al. (2016). Novel Metabolic Abnormalities in the Tricarboxylic Acid Cycle in Peripheral CellsFrom Huntington's Disease Patients. PloS one. 11 (9)). The number and area ratio of type I muscle fibers increased in group L (P<0.05), and SDH activity was enhanced in this group (P<0.01).

[0044] Finally, using the long-chain fatty acid analysis method and parameters described above, fatty acids were determined in the muscles of sheep after different treatments. The results are shown in Table 3. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1n9c), linoleic acid (C18:2n6c), and cis-11-eicosenoic acid (C20:1n9) were the most abundant fatty acids found in longissimus thoracis muscle of sheep. Overall, individual fatty acid profiles differed between treatments. Decanoic acid (C10:0), linoleic acid (C18:2n6c), γ-linolenic acid (C18:3n6), arachidonic acid (C20:4n6), and docosahexaenoic acid (C22:6) were higher in LG than in CG, while erucic acid (C22:1n9) and tricosanoic acid (C23:0) were higher in CG than in LG (P < 0.05). Compared with CG, LG showed significantly increased levels of PUFAs (C18:2n6c and C18:3n6). Linoleic acid, an essential fatty acid, can lower blood cholesterol levels and promote lipid metabolism. In this study, supplementation with Lactobacillus reuteri significantly increased DHA (P < 0.05). Erucic acid content was significantly reduced in LG compared with CG (P < 0.05). This finding suggests that Lactobacillus reuteri controls the production of long fatty acids through differentially expressed proteins, leading to increased concentrations of specific long fatty acids, such as linoleic acid (C18:2n6c), γ-linolenic acid (C18:3n6), and DHA (C22:6), in meat. This regulation mitigates the negative effects of erucic acid on steatosis.

[0045] Table 3 Determination of fatty acid content in sheep muscle (%) 1

[0046] Note: CG = control group; LG = Lactobacillus reuteri group (n = 9). SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; 1 indicates the reported fatty acid content is less than 0.1% of the total lipids.

[0047] 4. The effects of adding Lactobacillus reuteri on the activities of enzymes related to fat metabolism in sheep were tested as follows: Accurately weigh approximately 0.5 g of muscle tissue sample and place it in a centrifuge tube. Add 9 times the volume of physiological saline and homogenize at high speed (8,000 r / min, 30 s) in an ice bath. Centrifuge the prepared 10% tissue homogenate at 4°C (4,000 r / min, 10 min), and collect the supernatant in a new centrifuge tube. After sample pretreatment, measure relevant indicators according to the steps in the Nanjing Jiancheng kit operating instructions.

[0048] Lipid metabolism is a complex series of biochemical processes in the body, encompassing lipid synthesis, degradation, transport, and storage. Enzymes play a crucial role in this process. AC plays a crucial role in the cAMP signaling pathway, catalyzing the conversion of ATP (adenosine triphosphate) to cAMP, which in turn activates PKA (protein kinase A) and influences the synthesis and degradation of fatty acids (Li Mingyang. Study on the Expression Patterns and Correlations of Genes Related to Intramuscular Fat Deposition and Fat Metabolism in Duolang Sheep [D]. Tarim University). AC activity was significantly increased in LG, while ATP activity was significantly decreased (P < 0.001). Therefore, the addition of Lactobacillus reuteri may regulate lipid metabolism by activating the cAMP signaling pathway. HSL, the rate-limiting enzyme in lipolysis, was found to have enhanced HSL activity and increased total PUFA content in LG. MDA activity in LG was lower than that in CG, and the relative content of flavor compounds associated with lipid oxidation was lower, indicating that Lactobacillus reuteri can delay lipid oxidation.

[0049] cAMP plays a role in lipid metabolism by activating PKA. PKA can inhibit the activity of acetyl-CoA carboxylase 1 (ACC1) and acetyl-CoA carboxylase 2 (ACC2) by phosphorylating them, thereby reducing fatty acid synthesis. It can also promote the activation of HSL, thereby increasing fatty acid release. Furthermore, the cAMP signaling pathway promotes the entry of fatty acids into mitochondria for β-oxidation by activating carnitine palmitoyltransferase I (CPT1), thus regulating fatty acid oxidation (Ke Bilun, Tan Siwei, Yi Jiazhi, et al. Sodium butyrate increases mitochondrial biogenesis and fat oxidation in a fatty liver cell model by activating the cAMP pathway [J]. New Medicine).

[0050] Table 4 shows the effects of the optimized diet supplemented with Lactobacillus reuteri on the activities of lipid metabolism-related enzymes in sheep, as determined using the aforementioned method. As can be seen, the relative expression levels of all genes in the LG group were higher than in the CG group, and fatty acid synthesis and release were also higher in the LG group than in the CG group. In summary, the addition of Lactobacillus reuteri influences the meat quality, flavor, and fatty acid composition of lambs by regulating lipid metabolism.

[0051] Table 4 Effects of Lactobacillus reuteri on activities of enzymes related to lipid metabolism in sheep

[0052] Note: CG = control group; LG = Lactobacillus reuteri group (n = 9). CPT1, carnitine palmitoyltransferase 1; PKA, protein kinase A; HSL, hormone-sensitive triglyceride lipase; ACC1, acetyl-CoA carboxylase 1; ACC2, acetyl-CoA carboxylase 2.

[0053] 5. Effects of feed optimization on rumen metabolites in sheep Metabolites in the sheep rumen were measured. Specific metabolites are shown in Table 5. The effects of the optimized diet supplemented with Lactobacillus reuteri on rumen metabolites are shown in Table 5. Analysis of rumen short-chain fatty acids revealed that propionate content was significantly higher in the Lactobacillus reuteri group than in the control group (P < 0.05), while other SCFAs did not differ significantly between the two groups (P > 0.05). Rumen pH was significantly lower in the Lactobacillus reuteri group than in the control group (P < 0.05), likely due to the production of large amounts of volatile fatty acids from polysaccharides by dominant rumen bacteria such as Bacteroides and Prevotella (Chloe, Matthews, et al. The rumen microbiome: a crucial consideration when optimizing milk and meat production and nitrogen utilization efficiency. [J]. Gut Microbes, 2018, 10: 0.). Other research results have shown that as the gastrointestinal metabolite propionate increases, tight junction protein gene expression also increases, thereby improving intestinal barrier function (1, Dan, Liang, et al. Potatoresistant starch inhibits diet-induced obesity by modifying the compositionof intestinal microbiota and their metabolites in obese mice.[J].Int J BiolMacromol, 2021, 180: 0. 2, Xue, M., et al. Propolis modulates the gutmicrobiota and improves the intestinal mucosal barrier function in diabeticrats. Biomedicine&Pharmacotherapy, 118, Article 109393). The results of this study showed that acetate, propionate, and butyrate are the two major groups of rumen metabolites.Zhang Wenjie et al. found that significantly increased acetic acid and propionic acid content in the rumen can improve the daily weight gain of Kazakh sheep (Zhang, W., et al Effects of dietary addition of ellagic acid on rumen metabolism, nutrient apparent digestibility, and growth performance in Kazakh sheep. Frontiers in Veterinary Science, 11, Article 1334026). At the same time, short-chain fatty acids (propionic acid) produced by microorganisms have also been found to bind to GRP43 and enhance histone acetylation, thereby increasing the expression levels of occludin and ZO-1 tight junction proteins and increasing gastrointestinal mucus levels (He, KY, et al. Akkermansia muciniphila protects the intestine from irradiation-induced injury by secretion of propionic acid. Gut Microbes, 15(2), Article 2293312.) and the integrity of the small intestinal epithelial barrier.

[0054] In addition, supplementation of Lactobacillus reuteri in the diet may promote an increase in the body's energy demand and active mitochondrial metabolism, and cause changes in the gastrointestinal mucosal morphology and immune function of sheep (Yu, J., et al. DietaryClostridium butyricum and Bacillus subtilis promote goose growth by improving intestinal structure and function, antioxidative capacity and microbialcomposition. Animals, 11(11), 3174.). In summary, feeding Lactobacillus reuteri in this study may affect the expression of tight junction proteins by regulating propionic acid, thereby improving rumen epithelial immunity and absorption function.

[0055] Table 5 Effects of Lactobacillus reuteri on metabolites in the rumen of sheep (mmoL / L)

[0056] 6. Effect of optimized feed on rumen microbial diversity in sheep Metagenomic sequencing has been used to study microbial community diversity, species composition, gene function, and metabolic networks. In this experiment, the rumen microbiome was determined through sample DNA extraction → DNA detection → PCR amplification → Illumina HiSeq4000 platform sequencing → data information analysis (determined by Meiji Bioassay). The samples included the control group CG and the treatment group LG. The rumen microbiome diversity of the samples was determined and the results were referenced. Figure 4 , as follows:

[0057] PCoA1 explains 65.24% of the variation in the phylum-level dataset, and PCoA2 explains 21.10%. Figure 4 As shown in A. Figure 4 In Figure C, PCoA1 explained 65.92% of the genus-level variation in the dataset, and PCoA2 explained 12.78%. The rumen microbiota of Mongolian lambs in CG was mainly located in the second and third quadrants, while the rumen microbiota of Mongolian lambs in LG was mainly located in the right quadrant, indicating a regional clustering pattern.

[0058] ① Microbial composition at the phylum level The microbiome composition of rumen samples at different taxonomic levels was analyzed, and the results were as follows: Figure 4 As shown in B. At the phylum level, the top 10 genera with the highest relative abundance in the rumen microbiota of Sunite lambs are Bacteroidetes, Firmicutes, Proteobacteria, Chytridiomycota, Fibrobacteres, Ascomycota, Mucoromycota, Synergistetes, Euryarchaeota, and Actinobacteria Bacteroidetes and Firmicutes were the most abundant phyla at the phylum level in rumen samples, representing the dominant phyla (approximately 60.90%). Bacteroidetes are involved in the breakdown of both fiber and non-fiber diets and are the primary nitrogen-metabolizing microbial community. They play a crucial role in maintaining gastrointestinal health and balance, influencing the composition of short-chain fatty acids, nutrient digestion, and energy absorption within the gastrointestinal tract. Fibrobacteria are widely distributed in the rumen of ruminants, and cellulase, present in the pericytoplasm, breaks down cellulose for absorption (Losang Donzhu, Pingcuo Zhandui, Zhang Qiang, et al. Comparative Analysis of Rumen Fermentation Parameters and Microbial Diversity in Wild and Domestic Yaks [J]. Heilongjiang Animal Husbandry and Veterinary Medicine). Furthermore, Fibrobacteria promote the fermentation of substrates in fiber-rich foods into acetic and propionic acids. To further evaluate the effects of Lactobacillus reuteri intervention on the rumen microbiota composition of Sunite lambs, linear discriminant analysis (LDA) statistical analysis was used to observe changes at each taxonomic level.

[0059] The LEfSe method was used to explore the differential microbiota and biomarkers between the CG and LG groups, and the LEfSe dendrogram of the effects of Lactobacillus reuteri supplementation on the rumen microbial taxa of Sunite lambs was obtained. Figure 5As shown in the figure, compared with CG, the Bacteroidetes in the rumen flora of LG Sunite lambs increased significantly, while the Ascomycota decreased significantly.

[0060] The LDA scores of the two treatment groups were as follows Figure 6 As shown in the figure, the hierarchical distribution of important enriched species in all samples, where |LDA|>4 is considered a sign of difference between the two groups, and different colors represent different groups. LDA>0 means that its abundance in LG is greater than that in CG, and LDA<0 means that its abundance in CG is higher than that in LG. It can be seen that g_Prevotella、o_ Bacteroidales,s_Prevotella_sp_ne3005 and f_Prevotellaceae The abundance of Prevotella is increasing (LDA>5.2). Prevotella has been shown to be involved in vitamin synthesis in the body, including a wider range of fiber-related enzymes used to digest available fiber. In addition, increasing the ratio of Prevotella to Bacteroidales can enhance the body's ability to degrade carbohydrates and effectively inhibit excessive obesity. s_Selenomonas_ruminantium, g_Selenomonas, f_ Selenomonadaceae, and c_Neocallimastig Levels of β-lactamase (β-lactamase) were significantly increased (P < 0.05). Compared to CG, LG showed lower microbial abundance (capacity to utilize other microbial fermentation products). This may be due to the supplementation of Lactobacillus reuteri during fermentation, which produces glucans and fructans as carbon sources for bacterial growth (Han Jun, Wang Yi, Zhang Kaiping, et al. Biological Characterization of Lactobacillus reuteri JBR3 and Effects of Protectants on Its Viability [J / OL]. Food Industry Science and Technology). A key reason for the high abundance of Prevotella is its preferential metabolism of plant polysaccharides. Microbial fermentation of fiber substrates produces short-chain fatty acids, which activate gastrointestinal epithelial receptors (GPR family) by activating adenosine monophosphate-activated protein kinase (AMPK), serine / threonine kinase (Akt), and extracellular signal-regulated kinase (ERK) 1 / 2 signaling pathways. These pathways are involved in intramuscular fat accumulation and fatty acid metabolism, serving as sources of fat deposits, sex-specific flavor precursors, and other substances. This suggests that Lactobacillus reuteri intervention may enhance the digestion and breakdown of dietary carbohydrates and fiber in the feed by increasing the abundance of Bacteroidetes and Fibrobacteriaceae in the rumen.

[0061] ② Microbial composition at the genus level The results are as follows Figure 4 As shown in D. At the genus level, the relative abundances of the first nine genera are Prevotella (25.92%), Bacteroides (2.83%), Succinimonas (1.96%), Selenomonas (1.76%), Ruminococcus (1.25%), Clostridium (1.14%), Alistipes (1.13%), Succiniclasticum (1.07%) and Fibrobacter (1.03%). The bacterial genera that showed significant differences between the two groups were Selenomonas (P=0.004), Prevotella (P=0.038), Bacteroides (P=0.038), Paraprevotella (P=0.041), Fibrobacter (P=0.008), and Schwartzia (P=0.024). It is worth noting that low-abundance groups are still important members of the microbial community. In the process of screening differential genera (see Figure 4 ), and found that although the LEfSe method will g_ Alistipes However, after Wilcoxon rank sum test, there was no significant difference between the two groups. g_Alistipes The relative abundance of was large and the two methods were different. Therefore, the common markers in the two methods were selected as the differential genera, i.e. g_Bacteroides, g_Fibrobacter, g_Prevotella, g_ Schwartzia, g_Selenomonas, and g_Paraprevotella .

[0062] LG Prevotella, Bacteroides, Paraprevotella, and Fibrobacte The relative abundance of Figure 4 F in ). Prevotella It can effectively degrade trypsin, protect gastrointestinal IgA from being degraded by trypsin, and enhance the body's immunity. Prevotella It has been found that arabinoxylan and oligofructose are used to ferment propionate. The main byproducts of anaerobic respiration in Bacteroides are acetate, isovalerate, and succinate (Zhao Wei. Study on the Effects and Regulatory Mechanisms of Rumen Microbiota Transplantation on Lamb Gastrointestinal Microbiota [D]. Northwest Agriculture and Forestry University). They are also frequently used as substrates for the synthesis of cholesterol and long-chain fatty acids. Short-chain fatty acids, as quorum sensing signaling molecules, can activate signaling pathways related to fatty acid deposition. Previous studies have shown that acetate can be directly converted into acetyl-CoA, directly providing an acetyl group for histone acetylation. The metabolite, acetyl-CoA, is further activated by acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). Cells can synthesize fatty acids (both unsaturated and saturated) based on acetyl-CoA. Acetyl-CoA is both a product of the fatty acid oxidation pathway and a substrate for the fatty acid synthesis pathway. Fatty acids are important precursors for the formation of mutton fat flavor (Zhang Xia. Research on the efficient synthesis of medium-chain fatty acids through the reverse fatty acid β-oxidation pathway [D]. Nanjing Agricultural University). It can be seen that dietary Lactobacillus reuteri has a significant effect on the rumen microbiome of lambs.

[0063] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An optimized feed for increasing polyunsaturated fatty acids in mutton, characterized in that: The optimized feed is prepared by adding Lactobacillus reuteri to the basic diet.

2. The optimized feed according to claim 1, characterized in that The basic diet comprises, by weight, 55 parts of corn, 17 parts of wheat bran, 8 parts of soybean meal, 8 parts of rapeseed meal, 8 parts of cottonseed meal, and 4 parts of premix; The premix comprises, by weight: 0.2 parts of vitamin A, 0.1 parts of vitamin D3, 0.25 parts of vitamin E, 0.5 parts of cellulase, 3 parts of methionine, 2 parts of lysine, 0.07-0.10 parts of copper sulfate, 0.75 parts of ferrous sulfate, 0.32-0.5 parts of manganese sulfate, 0.34-0.40 parts of zinc sulfate, 0.5 parts of sodium nitrite, 0.35-0.5 parts of cobalt chloride, 0.4 parts of potassium iodide powder, 0.5-1 parts of antioxidant, 40 parts of calcium hydrogen phosphate, 3 parts of calcium carbonate, 7 parts of sodium chloride, 0.8 parts of pine needle powder, and 30-40 parts of medical stone; the premix is ​​implemented in accordance with GB / T18823-2010, calculated according to the mass fraction, and stirred and mixed.

3. The optimized feed according to claim 2, characterized in that The amount of Lactobacillus reuteri added is 2×10 10 CFU / bird / day.

4. The optimized feed according to claim 1, characterized in that The sheep are sheep.

5. The optimized feed according to claim 4, characterized in that The sheep are Sunite sheep.

6. A lamb feeding method for increasing polyunsaturated fatty acids in meat, characterized in that: With reference to the "Meat Sheep Feeding Standards" (NY / Y816-2004), lambs are fed with the optimized feed described in claim 3 for 3 to 4 months.

7. Application of the optimized feed as described in any one of claims 1 to 5 in the field of sheep breeding.

8. The use according to claim 7, characterized in that: The application comprises improving the quality of mutton.

9. The use according to claim 8, characterized in that: The qualities include the content of polyunsaturated fatty acids in the mutton, the tenderness of the mutton, and the generation of aromatic compounds in the mutton.

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