Method for multi-bacterial synergistic fermentation of astragalus and application thereof

Abstract

The application discloses a method for multi-bacterial synergic fermentation of radix astragali and application thereof. Bacillus subtilis MY-6, lactobacillus plantarum MY-18 and enterococcus faecium MY-45 are used for step-by-step synergic fermentation, so that the content of active components such as flavones and polyphenols in radix astragali is efficiently improved, and the antioxidant activity is significantly enhanced. The obtained product can be used for improving the quality of livestock and aquatic meat, enhancing immunity, relieving heat stress, regulating the intestinal tract of pets and beautifying hair, and the process is mild and efficient, and realizes high-value utilization of radix astragali resources.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for multi-strain synergistic fermentation of Astragalus membranaceus, and the application of the fermentation products prepared by this method in improving meat quality, enhancing immunity, relieving heat stress, and improving intestinal health and hair luster in pets and fur-bearing animals. Background Technology

[0002] Astragalus membranaceus is a traditional Chinese medicinal herb used in both food and medicine. It is rich in flavonoids, polyphenols, polysaccharides, and other bioactive components, possessing physiological functions such as antioxidation, immune regulation, intestinal health promotion, and stress relief. In recent years, astragalus and its extracts have received widespread attention in the fields of functional foods, cosmetics, and feed additives, particularly showing promising applications in improving meat quality in farmed animals, enhancing immunity, alleviating heat stress, and improving the shine of pet and fur-bearing animals' coats.

[0003] Currently, the extraction of active ingredients from Astragalus membranaceus mainly relies on traditional methods such as water extraction and alcohol extraction, which have significant shortcomings. On the one hand, the dense cell wall structure of Astragalus membranaceus, with its large molecules such as cellulose, hemicellulose, and pectin, hinders the full release of effective components, resulting in a low extraction rate. On the other hand, high temperatures and organic solvents can easily damage the natural structure of active ingredients, reducing their biological activity. Furthermore, traditional extraction products tend to have relatively simple compositions, making it difficult to achieve synergistic effects from multiple active substances in Astragalus membranaceus. These problems severely restrict the high-value utilization of Astragalus membranaceus in functional foods, cosmetics, and animal husbandry.

[0004] To address the aforementioned issues, microbial fermentation technology has been gradually introduced into the development of Astragalus membranaceus resources. This technology offers advantages such as mild reaction conditions, abundant enzyme systems, and high conversion efficiency. Existing research indicates that fermenting Astragalus membranaceus with a single strain (such as Bacillus subtilis or Lactobacillus) can, to some extent, improve the release rate of active substances. However, the types of extracellular enzymes secreted by a single strain are limited, making it difficult to achieve complete degradation of the Astragalus membranaceus cell wall and fully convert macromolecular bound active ingredients, resulting in an unsatisfactory overall dissolution rate and bioconversion efficiency of active ingredients. Furthermore, existing fermentation processes often employ simple mixed fermentation of single or multiple strains, lacking a stepwise synergistic design between aerobic and anaerobic bacteria, thus failing to fully leverage the complementary and synergistic effects of multiple microbial enzyme systems.

[0005] More importantly, current technologies typically use Astragalus fermentation products directly as crude extracts, lacking graded separation and targeted application process design for different application scenarios. Due to the complex composition of active ingredients in fermentation products, different application scenarios have significantly different requirements for active components. Existing processes cannot achieve precise grading and function-oriented utilization of the products, resulting in the underutilization of the multifunctional value of Astragalus fermentation products. For example, in pig farming, existing meat quality improvers struggle to simultaneously address growth performance and multiple meat quality indicators; in aquaculture, their effects on improving fish crispness and collagen content are limited. The root cause of these problems lies in the lack of a technical means to effectively separate and target fermentation products according to application objectives.

[0006] Therefore, there is an urgent need to develop a fermentation technology that can efficiently release the active ingredients of Astragalus membranaceus, achieve synergistic effects among multiple microbial strains, and enable graded utilization of the product for different application goals. Based on the above needs, this invention provides a method for synergistic fermentation of Astragalus membranaceus by multiple microorganisms and its applications in improving meat quality, enhancing immunity, alleviating heat stress, and improving intestinal health and coat luster in pets and fur-bearing animals, thereby realizing the high-value and multifunctional utilization of Astragalus membranaceus resources. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method and application for multi-strain synergistic fermentation of Astragalus membranaceus. This method employs Bacillus subtilis, Lactobacillus plantarum, and Enterococcus faecalis for stepwise synergistic fermentation, fully leveraging the complementary enzyme systems and metabolic synergistic effects of different strains. This effectively promotes the release and transformation of active components such as total polyphenols and total flavonoids in Astragalus membranaceus, and significantly enhances the antioxidant activity of the fermentation products. The resulting fermentation products can be widely applied in animal husbandry, pet nutrition, and functional products, showing promising prospects in improving meat quality, enhancing immunity, alleviating heat stress, regulating intestinal health, and improving hair quality.

[0008] The technical solution adopted in this invention is as follows:

[0009] A method for multi-strain synergistic fermentation of Astragalus membranaceus includes the following steps:

[0010] (1) Remove impurities from Astragalus membranaceus, wash, dry, and pulverize it through a 60-mesh sieve. Add Astragalus membranaceus powder to pure water and mix at a material-to-liquid ratio of 1:10 (g / mL). First, heat the mixture to 70-75℃ and soften it in a water bath for 30-40 min. Then, cool it to 55-60℃ and sonicate it at 40-60 kHz for 1-1.5 h. After that, centrifuge it at 12000 r / min for 15 min, take the supernatant, sterilize it with high-pressure steam at 121℃ for 20 min, and cool it for later use.

[0011] (2) Inoculate with Bacillus subtilis seed liquid at an inoculation rate of 5% (v / v) of the fermentation substrate (supernatant) and let it ferment at 37℃ for 48 h;

[0012] (3) After the fermentation in step (2) is completed, adjust the pH of the fermentation liquid to 6.0-6.5, and then inoculate the seed liquid of *Lactobacillus plantarum* and *Enterococcus faecalis* at an inoculation rate of 5% (v / v) of the fermentation liquid volume, mix well, and let it stand for fermentation at 37℃ for 24 h under anaerobic conditions.

[0013] (4) After fermentation, centrifuge at 4℃ and 12000 r / min for 15 min, and collect the supernatant and precipitate respectively to obtain the multi-strain synergistic fermentation product of Astragalus membranaceus.

[0014] Furthermore, the variable-temperature heating and ultrasonic coupling extraction process used in step (1) has a significant synergistic effect: on the one hand, the use of 150-200 mesh ultrafine grinding significantly increases the solid-liquid contact area; on the other hand, the high temperature of 70-75℃ is first used to soften and expand the cellulose and pectin in the Astragalus cell wall, while reducing the viscosity of the pure water solvent and accelerating molecular thermal motion; then, ultrasonic cell wall breaking is performed at 55-60℃ (the optimal temperature range for ultrasonic cavitation effect), and the cavitation microjets are used to completely break the cell wall, promoting the efficient release of Astragalus macromolecular polysaccharides and bound flavonoids, polyphenols and other components into the aqueous phase. This physical coupling pretreatment provides a substrate with extremely high concentration and rich nutrients for subsequent multi-strain synergistic fermentation, completely solving the technical bottleneck of low active ingredient content in the supernatant of traditional Astragalus water extraction.

[0015] Furthermore, the method employs a stepwise synergistic fermentation process. First, Bacillus subtilis secretes proteases, cellulases, and various extracellular enzymes under aerobic conditions to initially degrade the cell walls and macromolecules of Astragalus membranaceus. Then, Lactobacillus plantarum and Enterococcus faecalis are used to continue fermentation under anaerobic conditions to promote the further release and biotransformation of active ingredients such as phenols and flavonoids, thereby enhancing the functional activity of Astragalus membranaceus fermentation products.

[0016] The present invention also provides a multi-strain synergistic fermentation product of Astragalus membranaceus prepared by the above method.

[0017] This invention also provides the application of the multi-strain co-fermented Astragalus product in improving the content of active ingredients and antioxidant capacity. Experimental results showed that, compared with unfermented Astragalus, the total polyphenol content significantly increased from 1397.5 μg / g to 4650.0 μg / g after multi-strain co-fermentation, and the total flavonoid content increased from 1865.3 μg / g to 3920.0 μg / g, representing increases of approximately 3.0 times and 2.0 times, respectively. The DPPH, ABTS, and hydroxyl radical scavenging rates increased from 68.5%, 62.0%, and 65.0% to 94.0%, 92.5%, and 93.0%, respectively, indicating that the fermentation product has significantly enhanced antioxidant capacity.

[0018] This invention also provides the application of the multi-strain synergistic fermentation product of Astragalus membranaceus in the fields of animal husbandry and pet nutrition, specifically including at least one of the following:

[0019] (1) Application in improving the meat quality of livestock, poultry and aquatic animals;

[0020] (2) Application in enhancing animal immunity;

[0021] (3) Application in alleviating heat stress in animals;

[0022] (4) Application in improving the gut health of pets and fur-bearing animals;

[0023] (5) Application in improving the luster and quality of the coat of pets and fur-bearing animals.

[0024] Furthermore, the improvement in meat quality includes increasing at least one of muscle firmness, elasticity, gelatinousness, chewiness, and collagen content; the relief of heat stress includes improving rumen pH and increasing total volatile fatty acid content.

[0025] The present invention also provides a feed additive, pet nutritional preparation or functional product comprising the above-mentioned multi-strain synergistic fermentation product of Astragalus membranaceus.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. Multi-strain synergistic fermentation for higher fermentation efficiency. This invention employs Bacillus subtilis, Lactobacillus plantarum, and Enterococcus faecalis for multi-strain synergistic fermentation, taking into account the metabolic characteristics of both aerobic and anaerobic bacteria. This fully leverages the complementary effects of extracellular enzyme systems and metabolites from different bacterial species, which is more conducive to the disruption of Astragalus membranaceus cell wall structure and the release of bound active ingredients compared to single-strain fermentation or simple mixed-strain fermentation.

[0028] 2. Significantly increases the content of active ingredients in Astragalus membranaceus. This invention significantly increases the content of major active ingredients such as total polyphenols and total flavonoids in Astragalus membranaceus through synergistic fermentation, providing a good material basis for its subsequent functional development and high-value utilization.

[0029] 3. Significantly enhanced antioxidant activity. The fermentation product exhibits strong scavenging ability against DPPH, ABTS, and hydroxyl radicals, indicating that it possesses good antioxidant properties, which can support animal health regulation and the development of functional products.

[0030] 4. Wide range of applications and high functional value. The fermented Astragalus product obtained by this invention can not only be used in livestock and aquaculture to improve meat quality, enhance immunity and relieve heat stress, but also in the fields of regulating intestinal health and improving hair quality in pets and fur-bearing animals, and has high industrial application value.

[0031] 5. The process conditions are mild and suitable for widespread application. The process of this invention is simple, the fermentation conditions are mild, and the raw materials are widely available, making it easy to scale up production. It is suitable for widespread application in feed additives, pet nutrition products, and related functional products. Attached Figure Description

[0032] Figure 1 Effects of multi-strain co-fermentation of Astragalus membranaceus on the content of total polyphenols and total flavonoids

[0033] Figure 2 Effects of multi-strain co-fermentation of Astragalus membranaceus on DPPH, ABTS, and hydroxyl radical scavenging rates

[0034] Figure 3 The effects of multi-strain synergistic fermentation of Astragalus membranaceus on the muscle hardness, elasticity, gelatinous texture, and chewiness of tilapia.

[0035] Figure 4 Effects of multi-strain co-fermentation of Astragalus membranaceus on collagen content in tilapia muscle

[0036] Figure 5 Effects of multi-strain synergistic fermentation of Astragalus membranaceus on rumen pH and total volatile fatty acid content in heat-stressed beef cattle

[0037] Figure 6 The effects of multi-strain synergistic fermentation of Astragalus membranaceus on kitten growth, gut microbiota, and coat quality. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Activation of the strain

[0040] 1.1 Preparation of Culture Medium

[0041] Preparation of LB agar medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, add ultrapure water to 1 L, adjust pH to 7.0-7.2. Autoclave at 121℃ for 20 min.

[0042] Preparation of LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, add ultrapure water to 1 L, and adjust pH to 7.0-7.2. Autoclave at 121℃ for 20 min.

[0043] Preparation of MRS agar medium: 10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L anhydrous sodium acetate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 20 g / L agar powder, add ultrapure water to 1 L, adjust pH to 6.0-6.4. Autoclave at 121℃ for 20 min.

[0044] Preparation of MRS liquid culture medium: 10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L anhydrous sodium acetate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, add ultrapure water to 1 L, adjust pH to 6.0-6.4. Autoclave at 121℃ for 20 min.

[0045] 1.2 Strain Culture

[0046] Bacillus subtilis MY-6: In a clean bench, transfer 100 μL of the frozen target bacterial culture from an EP tube to the culture medium. Then, streak the tube onto LB agar using a sterilized inoculation loop. Seal the tube with sealing film and incubate aerobically at 37°C for 24 h. Pick typical colonies from the agar medium and inoculate them into test tubes containing 5 mL of LB liquid medium. Incubate at 37°C with shaking at 180 r / min for 24 h.

[0047] Lactobacillus plantarum MY-18: In a clean bench, take 100 μL of EP tube containing the frozen target bacterial culture and streak it onto MRS agar medium. Seal the tube with sealing film and place it in an anaerobic jar. Place a gas-generating anaerobic bag in the anaerobic jar to ensure an anaerobic environment. Incubate at 37°C for 24 h. Pick typical colonies from the above agar medium and inoculate them into anaerobic tubes containing 5 mL of MRS liquid medium. Incubate at 37°C for 24 h.

[0048] Enterococcus faecalis MY-45: In a clean bench, take 100 μL of the frozen target bacterial culture from an EP tube, streak it onto MRS agar medium, seal the tube with sealing film, and place it in an anaerobic jar. Place a gas-generating anaerobic bag inside the anaerobic jar to ensure an anaerobic environment. Incubate at 37°C for 24 h. Pick typical colonies from the agar medium and inoculate them into anaerobic tubes containing 5 mL of MRS liquid medium. Incubate statically at 37°C for 24 h.

[0049] Example 2: Fermentation of Astragalus membranaceus

[0050] 2.1 Bacillus subtilis fermentation

[0051] Astragalus membranaceus was cleaned, dried, and pulverized through a 60-mesh sieve. 20 g of the pulverized astragalus was added to a fermentation flask along with 200 mL of purified water. The flask was then placed in a 70°C water bath for 30 min to soften the tissue. After the tissue had fully absorbed water and softened, it was transferred to an ultrasonic cleaner. The water temperature was controlled at 60°C, and the ultrasonic frequency was adjusted to 40 kHz for continuous ultrasonic treatment for 1.5 h. The mixture was centrifuged at 12000 rpm for 15 min, and the supernatant was collected and sterilized by steam at 121°C for 20 min. The mixture was then cooled and used for inoculation with Bacillus subtilis at a rate of 5% (v / v), and fermented at 37°C for 48 h.

[0052] 2.2 Fermentation by *Lactobacillus plantarum* and *Enterococcus faecalis*

[0053] After Bacillus subtilis fermentation, the pH of the fermentation broth was adjusted to 6.0-6.5, and *Lactobacillus plantarum* and *Enterococcus faecalis* seed culture were inoculated at 5% (v / v) respectively. After mixing, the mixture was placed under anaerobic conditions at 37℃ for 24 h of static fermentation. After fermentation, the fermentation broth was centrifuged at 4℃ and 12000 r / min for 15 min, and the supernatant and precipitate were collected separately for later use.

[0054] Example 3: Determination of component content

[0055] 3.1 Determination of total polyphenol content by Folin-Ciocalten phenol colorimetric method

[0056] Accurately weigh 50 mg of gallic acid standard into a 500 mL volumetric flask. Prepare a 0.1 mg / mL standard solution with distilled water. Pipette 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard sample solution into stoppered tubes, respectively. Add deionized water to a final volume of 1 mL, then add 4 mL of deionized water and 1 mL of Folin-Ciocalten. Mix thoroughly, and finally add 4 mL of 10% NaCO3. Vortex and react at 30°C for 15 min. Measure the absorbance at 760 nm. Use a blank tube as a control group. Sample determination: Determine the absorbance at 760 nm according to the standard curve method and calculate using the standard equation.

[0057] 3.2 Determination of total flavonoid content by aluminum nitrate colorimetric method

[0058] Weigh 20 mg of rutin reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with 70% ethanol. Accurately weigh 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the reference standard solution, add 1 mL of 5% sodium nitrite solution, mix well, and let stand for 5 min. Then add 1 mL of 10% aluminum nitrate solution, mix well, and let stand for 5 min. Add 10 mL of 1 M NaOH solution and dilute to the mark with 70% ethanol. After standing for 20 min, measure the absorbance at 510 nm. Sample determination: Determine the absorbance at 510 nm according to the standard curve method and calculate using the standard equation.

[0059] like Figure 1 As shown, compared with the unfermented control group, the total polyphenol content in the multi-strain synergistic fermentation group significantly increased from 98.5 μg / g to 1850.0 μg / g, and the total flavonoid content significantly increased from 65.3 μg / g to 1120.0 μg / g, representing increases of approximately 18.8 times and 17.2 times, respectively. This indicates that multi-strain synergistic fermentation can significantly promote the release and accumulation of phenolic and flavonoid active ingredients in Astragalus membranaceus.

[0060] Example 4 Antioxidant Activity Test

[0061] 4.1 DPPH free radical scavenging rate experiment

[0062] Take 1 mL of sample and add 1 mL of DPPH ethanol solution (0.2 mM). React at 25°C in the dark for 30 min and measure the absorbance of the sample at a wavelength of 517 nm. The DPPH free radical scavenging rate is determined according to the following formula, where A1 is the absorbance of the sample; A2 is the absorbance of the sample without DPPH solution; and A0 is the absorbance of the blank tube.

[0063] DPPH free radical scavenging rate (%) =

[0064] 4.2 Determination of ABTS free radical scavenging rate

[0065] Accurately weigh a certain amount of ABTS and dissolve it in acetate buffer (pH 4.5, 20 mM) to prepare a 7 mM ABTS acetate buffer solution. Accurately weigh potassium persulfate and dissolve it in deionized water to prepare a 2.45 mM potassium persulfate solution. Mix ABTS and potassium persulfate solution (1:1) thoroughly and let stand at 25°C in the dark for 12–16 h to obtain the ABTS+ stock solution. Dilute the ABTS+ stock solution with acetate buffer to make its absorbance at 734 nm 0.7 ± 0.02. Finally, accurately pipette 2 mL of sample solution and mix it thoroughly with 2 mL of ABTS+ stock solution (25°C, 20 min), and measure the absorbance at 734 nm after the reaction. Where A1 is the sample absorbance; A2 is the absorbance of the sample with acetate buffer instead of ABTS+ solution; A0 is the absorbance of the blank tube.

[0066] ABTS free radical scavenging rate (%) =

[0067] 4.3 Determination of hydroxyl radical scavenging rate

[0068] Prepare a 9 mM 7H₂O·FeSO₄ solution, an 8.8 M H₂O₂ solution, and a 9 mM salicylic acid solution. Accurately pipette 1 mL of the sample solution, add 1 mL of 7H₂O·FeSO₄ solution and 1 mL of salicylic acid solution sequentially, mix thoroughly, and finally add 1 mL of H₂O₂. React at 37℃ for 30 min, and measure the absorbance of the sample after the reaction at 510 nm. Where A1 is the sample absorbance; A2 is the absorbance of the sample with deionized water instead of H₂O₂ solution; and A0 is the absorbance of the blank tube.

[0069] Hydroxyl radical scavenging rate (%) =

[0070] like Figure 2 As shown, compared with the unfermented control group, the DPPH, ABTS and hydroxyl radical scavenging rates of the multi-strain synergistic fermentation group increased from 28.5%, 32.0% and 25.0% to 94.0%, 92.5% and 93.0%, respectively, which are approximately 3.3 times, 2.9 times and 3.7 times higher, respectively. This indicates that the antioxidant activity of Astragalus membranaceus was significantly enhanced after fermentation, and it has a good free radical scavenging ability.

[0071] Example 5: Application in improving pork quality

[0072] 5.1 Experimental Design and Grouping

[0073] (1) Experimental subjects: 120 three-way crossbred commercial pigs with good health, 60 days old and initial weight of 25±2kg were selected and randomly divided into 3 groups, with 4 replicates in each group and 10 pigs in each replicate (half male and half female) to eliminate the interference of individual differences on the experimental results.

[0074] (2) Experimental period: The experiment covered the entire fattening stage for a total of 90 days, which is consistent with the conventional breeding cycle of commercial pigs.

[0075] (3) Feeding environment: All three groups of experimental pigs were raised in the same pig house, with the same ambient temperature, humidity, light and ventilation conditions; they had free access to feed and water, ensuring that the feeding and management conditions of each group were completely consistent.

[0076] (4) Experimental grouping

[0077] Blank control group: fed a basal diet without any functional additives.

[0078] Standard additive group: A combination of commercially available standard meat quality improvers added to the basal diet.

[0079] Experimental group: The multi-strain synergistic fermented Astragalus preparation of this invention was added to the basal diet at a level of 1%.

[0080] 5.2 Growth performance indicators

[0081] Initial and final weight: On day 1 and day 90 of the experiment, each head was weighed after fasting for 12 hours. The average initial weight, average final weight and average daily weight gain for each group were calculated (ADG = (final weight - initial weight) / number of days of the experiment).

[0082] Average Daily Feed Intake (ADFI): The total feed intake of each group is recorded daily and summarized weekly to calculate the average daily feed intake over the entire period.

[0083] Feed conversion ratio (FCR): The feed conversion ratio is calculated based on ADFI and ADG (FCR=ADFI / ADG). The lower the feed conversion ratio, the higher the feed conversion efficiency.

[0084] Table 1 Comparison of growth performance indicators of finishing pigs in different treatment groups

[0085] Average initial body weight (kg) 25.6±1.6 24.9±1.6 25.9±1.5 Average final body weight (kg) 117.5±4.0 122.8±3.6 129.5±3.2 Average daily weight gain (ADG, g) 1030.2±45.2 1092.5±41.8 1160.8±37.5 Average daily feed intake (kg / d) 2.88±0.11 2.84±0.09 2.81±0.08 Material weight ratio (FCR) 2.80±0.07 2.60±0.05 2.42±0.04

[0086] There were no significant differences in initial body weight among the groups (P > 0.05), indicating consistent baseline characteristics and comparable experimental results. Compared with the blank control group, the average final body weight and average daily weight gain of the additive group of this invention were significantly increased, and superior to those of the conventional additive group. Simultaneously, the feed conversion ratio was significantly reduced, while daily feed intake showed no significant difference. This demonstrates that the multi-strain synergistic fermentation of Astragalus membranaceus preparation of this invention can significantly promote the growth of fattening pigs and improve feed conversion efficiency without affecting feed intake, thus possessing good application value.

[0087] 5.3 Quality Indicators for Pork

[0088] Meat color: 45 minutes and 24 hours after slaughter, the L value (brightness), a value (redness), and b value (yellowness) of the central area of ​​the eye muscle were measured using a colorimeter. The meat color was then blind-scored by three professional evaluators, and the average result was taken.

[0089] pH: 45 min post-slaughter (pH1), 24 h post-slaughter (pH2) 24 A pH meter was inserted into the center of the eye muscle, and each sample was measured twice, and the average value was taken.

[0090] Drip loss: Within 2 hours post-slaughter, standard samples of eye muscle were taken, accurately weighed, and then suspended and refrigerated at 4°C for 48 hours. After being removed, dried, and weighed again, the drip loss rate was calculated. Each sample was tested in 4 parallel trials, and the average value was taken.

[0091] Water-binding capacity: Within 2 hours after slaughter, standard samples of eye muscle were taken and held at 35 kg pressure for 5 minutes. The water-binding capacity was calculated based on the mass difference before and after pressurization. Each sample was tested in parallel, and the average value was taken.

[0092] Marbling: 24 hours post-slaughter, a cross-section of the eye muscle was taken, and the marbling of the meat sample was scored. The average score was calculated through blind scoring by three professional scorers. The scoring criteria are as follows:

[0093] 1 point: There is almost no visible fat deposit on the surface of the muscle, the overall color is dark red, and the texture is lean and dry.

[0094] 2 points: There are very few scattered fat spots. The fat particles are relatively large and isolated, unevenly distributed, mainly concentrated at the edge of the muscle or in individual areas, while the muscle as a whole remains predominantly red.

[0095] 3 points: The number of fat spots is moderate and they are relatively evenly distributed, with some fine fat striations beginning to appear. The muscles are a mix of red and white, and the proportions are harmonious.

[0096] 4 points: Abundant fat deposits, with fine fat striations covering the entire muscle cross-section, forming a clear network structure. White fat blends harmoniously with red muscle in a balanced proportion.

[0097] 5 points: Extremely rich fat deposits, forming a dense, fine, and uniform net-like or snowflake-like texture. The muscle cross-section presents a beautiful "marble" or "frost" appearance.

[0098] Intramuscular fat: 24 hours after slaughter, meat samples from the same cross section of the eye muscle were taken, the peripheral fascia and extramuscular fat were removed, the meat was ground evenly and samples were prepared. The intramuscular fat content was determined by Soxhlet extraction. Each sample was measured twice in parallel, and the average value was taken.

[0099] Moisture content: Take a meat sample with the same consistency as intramuscular fat and determine the moisture content using the direct drying method. Each sample was tested twice in parallel, and the average value was taken.

[0100] Tenderness: 24 hours after slaughter, take meat samples (2cm×2cm×5cm) from the middle of the eye muscle and measure the shear force using a texture analyzer (cross-cross method, shearing speed 2mm / s).

[0101] Table 2 Results of Pork Quality Indicators

[0102] flesh color <![CDATA[45 min L * Value (brightness) 54.0±1.7 50.8±1.4 47.5±1.2 <![CDATA[45 min a * Value (Redness) 12.3±0.7 14.6±0.6 16.4±0.8 <![CDATA[45 min b * Value (yellowness) 8.5±0.5 7.9±0.4 7.3±0.3 <![CDATA[24 h L * Value (brightness) 56.5±1.8 53.0±1.5 49.8±1.3 <![CDATA[24 h a * Value (Redness) 10.7±0.6 13.0±0.5 14.6±0.7 <![CDATA[24 h b * Value (yellowness) 9.1±0.6 8.3±0.4 7.6±0.4 pH value <![CDATA[pH1(45 min)]]> 6.20±0.13 6.33±0.10 6.50±0.09 <![CDATA[pH 24 (24 h)]]> 5.56±0.08 5.68±0.07 5.84±0.06 Water retention Drip loss (%) 3.9±0.2 3.2±0.1 2.3±0.1 Water system capacity (%) 65.2±2.1 69.5±1.8 73.8±1.5 Marble pattern Rating (1-5 points) 2.0±0.3 2.7±0.2 3.6±0.2 Intramuscular fat content(%) 1.8±0.2 2.4±0.2 3.1±0.2 Moisture content(%) 73.5±1.0 73.2±0.9 73.0±0.8 tenderness Shear force (N) 46.0±2.5 39.0±1.9 31.6±1.6

[0103] As shown in Table 2, compared with the blank control group, the pork quality of the experimental group of this invention was comprehensively improved, and the effect was better than that of the conventional additive group. The meat color was bright red and uniform, which can effectively reduce the risk of PSE meat; the muscle metabolism process was more stable; the muscle had a stronger water retention capacity, and the meat was more tender and juicy; the fat distribution was more uniform; the meat was more tender and palatable.

[0104] Example 6: Application in improving the crispness of fish meat

[0105] 6.1 Experimental Design and Grouping

[0106] (1) Experimental subjects: 180 healthy, disease-free Nile tilapia of uniform size with an initial weight of 210±18 g were randomly divided into 3 groups, with 3 replicates in each group and 20 fish in each replicate. There was no significant difference in the initial weight among the groups (P>0.05).

[0107] (2) Experimental period: 60 days. The experimental fish were raised in a recirculating aquaculture system.

[0108] (3) Rearing environment: water temperature 26~28 ℃, dissolved oxygen ≥5.0 mg / L, pH 7.2~7.5, ammonia nitrogen <0.2 mg / L, nitrite <0.1 mg / L, feed twice a day (8:30, 16:30), the amount of feed is 3%~4% of the fish body weight, and the aquaculture management conditions remain consistent.

[0109] (3) Experimental grouping:

[0110] Blank control group: fed a basal diet without any functional additives;

[0111] Standard group: Commercially available aquatic meat quality improver additives were added to the basal diet;

[0112] Experimental group of this invention: 0.4% of the multi-strain synergistic fermentation preparation of Astragalus membranaceus of this invention was added to the basal diet.

[0113] 6.2 Sample Collection and Index Measurement

[0114] Sample collection: After the experiment, the fish were fasted for 24 hours. Twelve fish were randomly selected from each group for dissection. The white muscle on the left side of the back was removed, and the fascia and fat were removed. The samples were divided into two parts. One part was refrigerated at 4 ℃ and its textural properties were determined within 24 hours. The other part was stored at -80 ℃ for collagen content detection.

[0115] Measurement Indicators and Methods

[0116] (1) Muscle texture characteristics

[0117] Back muscle was harvested and cut into 1 cm thick sections. Texture analysis was performed using a texture analyzer with a 6 mm diameter cylindrical probe, applying bidirectional compression at a speed of 0.50 mm / s to a target compression depth of 0.5 cm. Hardness, elasticity, gelatinous texture, and chewiness were recorded. Each sample was measured in triplicate, and the average value was taken.

[0118] like Figure 3 As shown, compared with the blank control group, the muscle hardness, elasticity, gelatinous texture and chewiness of the conventional group of tilapia were improved to a certain extent; the above texture indicators of the experimental group of this invention were significantly higher than those of the blank control group and the conventional group (P<0.05), and the muscle firmness, crispness and chewiness were greatly improved.

[0119] (2) Muscle collagen content

[0120] The hydroxyproline colorimetric method was used to determine muscle collagen content. A hydroxyproline assay kit was used, and the manufacturer's operating procedures were strictly followed. Based on the principle that hydroxyproline oxidation products react with dimethylaminobenzaldehyde to form a purple-red chromophore, the absorbance was measured using a spectrophotometer, and the collagen concentration was calculated. Each sample was tested in duplicate, and the results were averaged.

[0121] Depend on Figure 4 It can be seen that the collagen content in the muscle of the experimental group of this invention was significantly higher than that of the blank control group and the conventional group (P<0.01), and the difference was significant. The increase in collagen content can directly enhance the toughness and density of muscle fibers, which is the core material basis for improving the crispness of fish meat.

[0122] Adding 0.4% of a multi-strain synergistic fermented Astragalus preparation to the basic diet of Nile tilapia can significantly increase muscle collagen content, optimize muscle texture, and effectively improve the crispness, firmness, and palatability of the fish meat. The effect is superior to commercially available conventional aquatic meat quality improvers, and it has good application prospects in aquaculture meat quality improvement.

[0123] Example 7: Application in enhancing the immunity and antioxidant capacity of tilapia

[0124] 7.1 Experimental Design and Grouping

[0125] (1) Experimental subjects: 450 healthy, disease-free, uniformly sized tilapia with an initial weight of 25.80±0.45 g were selected and randomly divided into 5 groups, with 3 replicates in each group and 30 fish in each replicate. There was no significant difference in the initial weight among the groups (P>0.05).

[0126] (2) Feeding and management: The experiment was conducted in an indoor recirculating aquaculture system with a culture water volume of 150 L / tank; water temperature of 28~31℃, dissolved oxygen ≥5.0 mg / L, pH 7.2~7.6, ammonia nitrogen <0.05 mg / L, and nitrite <0.1 mg / L; feeding was done twice a day (8:00 and 17:00), with a feeding amount of 2%~5% of the fish body weight, and the experimental period was 8 weeks.

[0127] (3) Experimental grouping

[0128] Blank control group: fed a basal diet without any functional additives;

[0129] Low-dose group: 0.5% of multi-strain co-fermented Astragalus preparation was added to the basal diet;

[0130] Medium-dose group: 1.0% of a multi-strain co-fermented Astragalus preparation was added to the basal diet;

[0131] High-dose group: 1.5% of multi-strain synergistic fermented Astragalus preparation was added to the basal diet;

[0132] Ultra-high dose group: 2.0% of multi-strain synergistic fermented Astragalus preparation was added to the basal diet.

[0133] 7.2 Sample Collection and Index Measurement

[0134] (1) Sample collection

[0135] After the experiment, the fish were fasted for 24 hours. Five fish were randomly selected from each replicate, and blood was collected from their tail veins. The blood was left to stand at 4°C for 1 hour, then centrifuged at 4°C and 6000 r / min for 10 minutes to separate the serum, which was then stored at −80°C for later use.

[0136] (2) Immunological marker measurement

[0137] Serum nitric oxide synthase (NOS), catalase (CAT), superoxide dismutase (SOD), and acid phosphatase (ACP) activities were measured using a kit.

[0138] Serum phenol oxidase (PO) activity was measured using L-DOPA as a substrate. 10 μL of serum was added to a 96-well microplate, followed by 200 μL of 0.1 mol / L phosphate buffer (pH 7.0) to each well. Finally, 10 μL of 0.01 mol / L L-DOPA was added to each sample well. After incubation at room temperature for 10 min, the plate was immediately placed in a microplate reader, and enzyme activity was measured at 490 nm. OD0.05 per milliliter of sample per minute was used as the experimental data. 490nm An increase of 0.001 is defined as one unit of enzyme activity.

[0139] Serum protein concentration determination: Protein concentration was determined using the Coomassie brilliant blue method (Bradford, 1976), with bovine serum albumin as the standard.

[0140] Table 3. Immune and Antioxidant Indicators

[0141] control group 13.12±1.71ⁿ 47.35±3.42ⁿ 106.42±3.11ⁿ 51.46±2.03ⁿ 14.85±0.52ⁿ 0.5% group 15.68±1.65ᵃᵇ 50.16±4.77ᵃᵇ 113.57±2.43ᵃᵇ 75.33±4.12ᵇ 15.11±0.61ᵃᵇ 1.0% group 17.24±0.55ᵃᵇ 54.82±5.11ᵃᵇ 118.23±2.07ᵃᵇ 118.42±7.65ᶜ 28.36±1.24ᵇ 1.5% group 19.85±1.08ᵃ 63.47±2.63ᵃ 129.65±1.88ᵃ 138.64±6.39ᵈ 33.67±1.18ᶜ 2.0% group 18.11±0.97ᵃᵇ 57.69±4.24ᵃᵇ 109.74±4.92ᵃᵇ 115.37±9.82ᶜ 29.82±1.07ᵇ

[0142] Adding a multi-strain synergistic fermented Astragalus preparation to the basal diet can significantly enhance the activity of serum immune-related enzymes (NOS, PO, ACP) and antioxidant enzymes (SOD, CAT) in Nile tilapia. The overall dose-response effect is characterized by increased activity at low doses, optimal activity at medium doses, and a slight decrease at high doses. The immune and antioxidant indicators of the 1.5% addition group were significantly higher than those of other groups, indicating that the preparation can effectively enhance the non-specific immunity and antioxidant capacity of tilapia. 1.5% is the optimal addition dose for enhancing immune and antioxidant functions in tilapia farming.

[0143] Example 8: Application in alleviating heat stress and improving rumen fermentation in ruminants

[0144] 8.1 Experimental Design and Grouping

[0145] (1) Experimental subjects: Sixty healthy Simmental beef cattle around 14 months old and weighing (435±20) kg were randomly divided into 4 groups, with 3 replicates in each group and 5 cattle in each replicate.

[0146] (2) Experiment period: The experiment was conducted during the high-temperature season in summer and lasted for 45 days. The temperature and humidity index (THI) of the cattle shed was controlled within the range of 78 to 89 by artificially adjusting the ambient temperature and humidity to simulate a moderate heat stress environment.

[0147] (3) Feeding and management: The feeding and management conditions of each group are the same, and they have free access to total mixed ration (TMR) and free access to water.

[0148] (4) Experimental grouping:

[0149] Blank control group: fed with basal TMR diet.

[0150] Experimental Group 1 (low dose): 0.1% of the multi-strain synergistic fermentation Astragalus preparation of this invention was added to the basal diet.

[0151] Experimental group 2 (medium dose): 0.2% of the multi-strain synergistic fermentation Astragalus preparation of this invention was added to the basal diet.

[0152] Experimental Group 3 (High Dose): 0.3% of the Astragalus membranaceus preparation of this invention, which is co-fermented by multiple microorganisms, was added to the basal diet.

[0153] 8.2 Measurement Indicators and Methods

[0154] (1) Growth performance: Fasting weight was measured at the beginning and end of the experiment to calculate the average daily weight gain; daily feed intake was recorded to calculate dry matter intake and feed conversion ratio. (2) Serum immune indicators: Blood was collected from the jugular vein at the end of the experiment to separate serum and measure the levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and tumor necrosis factor-α (TNF-α). (3) Rumen fermentation parameters: Rumen fluid was collected orally before morning feeding the day after the end of the experiment, and the pH value was measured immediately. After centrifugation and filtration, the concentrations of total volatile fatty acids, acetic acid, propionic acid, and ammonia nitrogen were measured, and the ethyl acetate-propionic acid ratio was calculated.

[0155] Table 4 Comparison of growth performance and rumen fermentation parameters of beef cattle in different treatment groups

[0156] Average daily weight gain (g / d) 1100±45 1210±52 1265±48 1280±50 Dry matter intake (kg / d) 9.8±0.5 10.5±0.6 11.3±0.5 11.4±0.6 Serum IgG (g / L) 12.5±1.2 15.8±1.3 17.2±1.4 17.5±1.3 TNF-α (ng / L) 85.6±6.5 72.3±5.8 65.4±5.2 63.8±5.0 Rumen pH 6.12±0.08 6.28±0.09 6.35±0.07 6.37±0.08 Total volatile fatty acids (mmol / L) 85.6±4.2 92.5±4.8 98.7±5.1 99.2±5.0 Acetic acid / propionic acid ratio 3.45±0.12 3.62±0.14 3.78±0.13 3.80±0.15 Ammonia nitrogen concentration (mg / dL) 16.5±1.2 13.2±1.0 11.8±0.9 11.5±0.8

[0157] As shown in Table 4 and Figure 5 As shown, under heat stress conditions, the addition of the multi-strain synergistic fermented Astragalus preparation of this invention to the diet can significantly improve the average daily weight gain and dry matter intake of beef cattle, increase serum immunoglobulin levels, reduce pro-inflammatory factor content, and enhance the body's immune function. Simultaneously, it improves rumen fermentation parameters, manifested as stable pH, increased total volatile fatty acid concentration, optimized ethylene-propyl ratio, and decreased ammonia nitrogen concentration. Considering both growth performance and rumen fermentation indicators, 0.2% is the recommended addition amount under the conditions of this experiment. This invention can serve as a green functional feed additive to alleviate heat stress in ruminants, and has good application prospects in summer dairy cattle, beef cattle, and sheep farming.

[0158] Example 9: Application in improving gut health and coat shine in pet cats

[0159] 9.1 Experimental Design and Grouping

[0160] (1) Experimental subjects: 48 healthy weaned Chinese rural kittens of about 50 days old and similar weight were selected, with half males and half females.

[0161] (2) Experimental period: 30 days.

[0162] (3) Feeding and management: Individual cage feeding, feeding twice a day at fixed times and in fixed quantities, free access to water, and consistent routine immunization and deworming procedures.

[0163] (4) Experimental grouping:

[0164] Blank control group: fed a basic complete kitten diet.

[0165] Experimental group 1 (low dose): 0.2% of the multi-strain synergistic fermentation Astragalus preparation of this invention was added to the basal diet.

[0166] Experimental group 2 (medium dose): 0.4% of the multi-strain synergistic fermentation Astragalus preparation of this invention was added to the basal diet.

[0167] Experimental Group 3 (High Dose): 0.6% of the Astragalus membranaceus preparation of this invention, which is co-fermented by multiple microorganisms, was added to the basal diet.

[0168] 9.2 Measurement Indicators and Methods

[0169] (1) Growth performance: Weigh the animals on an empty stomach at the beginning and end of the experiment and calculate the average daily weight gain; record the feed intake and calculate the feed conversion ratio.

[0170] (2) Serum indicators: At the end of the experiment, fasting blood was collected and serum was separated to determine the levels of superoxide dismutase (SOD), malondialdehyde (MDA), total antioxidant capacity (T-AOC), and immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM).

[0171] (3) Intestinal flora: Fresh feces were collected at the end of the experiment, and the number of Bifidobacterium, Lactobacillus, Escherichia coli and Staphylococcus aureus was determined by real-time PCR.

[0172] (4) Coat quality: Three professionals will conduct a blind evaluation to comprehensively score the coat density, softness and luster (0-10 points); the thickness and flatness of the hair cuticles will be observed under a microscope.

[0173] Table 5. Comparison of growth performance, gut microbiota, and coat quality in kittens from different treatment groups.

[0174] Average daily weight gain (g / d) 12.5±1.8 15.2±2.0 17.8±2.2 19.5±2.1 Material weight ratio 3.85±0.32 3.42±0.28 3.15±0.25 2.98±0.24 Serum SOD (U / mL) 85.6±6.5 102.5±7.2 118.6±8.1 125.4±7.8 Serum IgG (g / L) 8.5±1.1 10.8±1.3 12.5±1.4 13.2±1.5 Bifidobacterium (1g CFU / g) 8.2±0.4 9.3±0.5 9.8±0.5 10.2±0.6 Escherichia coli (1g CFU / g) 7.5±0.5 6.8±0.4 6.2±0.3 5.9±0.3 Overall coat color score 6.2±0.7 7.8±0.6 8.6±0.5 9.0±0.6 Cuticle adhesion generally better good Excellent

[0175] As shown in Table 5 and Figure 6As shown, adding the multi-strain synergistic fermented Astragalus preparation of this invention to the diet can significantly increase the average daily weight gain of kittens, reduce the feed conversion ratio, and promote growth and development; increase serum antioxidant enzyme activity, reduce malondialdehyde content, and enhance the body's antioxidant capacity; increase immunoglobulin levels and enhance immune function; and simultaneously significantly promote the proliferation of Bifidobacteria and Lactobacillus, inhibit Escherichia coli and Staphylococcus aureus, and optimize the intestinal flora structure. Regarding coat quality, the experimental group of kittens showed significant improvements in cuticle thickness and conformation, as well as increased coat luster and smoothness. Based on all indicators, 0.6% is the recommended addition amount under the conditions of this experiment. This invention can be used as a functional additive for pet-specific intestinal conditioning and coat enhancement, and has important application value in the development of high-end pet foods and pet health products.

Claims

1. A method for multi-strain synergistic fermentation of Astragalus membranaceus, characterized in that, Includes the following steps: (1) Remove impurities from Astragalus membranaceus, wash, dry, crush and sieve, add pure water, pre-treat by constant temperature heating and ultrasonic coupling, centrifuge to take the supernatant, sterilize to obtain Astragalus membranaceus fermentation substrate; (2) Inoculate with Bacillus subtilis at an inoculation rate of 5% of the volume of Astragalus membranaceus fermentation substrate and ferment under aerobic conditions; (3) After the fermentation in step (2) is completed, adjust the pH of the fermentation liquid to 6.0-6.5, and then inoculate with *Lactobacillus plantarum* and *Enterococcus faecalis* at an inoculation rate of 5% of the fermentation liquid volume, and continue fermentation under anaerobic conditions. (4) After fermentation, centrifuge and collect the supernatant and precipitate respectively to obtain the multi-strain synergistic fermentation product of Astragalus membranaceus.

2. The method for multi-strain synergistic fermentation of Astragalus membranaceus according to claim 1, characterized in that, In step (1), the Astragalus membranaceus is pulverized and passed through a 60-mesh sieve.

3. The method for multi-strain synergistic fermentation of Astragalus membranaceus according to claim 1, characterized in that, In step (1), the ratio of Astragalus membranaceus to purified water is 1:10 (g / mL).

4. The method for multi-strain synergistic fermentation of Astragalus membranaceus according to claim 1, characterized in that, The conditions for the constant temperature heating and ultrasonic coupling pretreatment in step (1) are as follows: first, heat the mixture to 70~75℃ and soften it by constant temperature extraction for 30~40 min, then lower the system temperature to 55~60℃ and ultrasonically treat it at 40~60 kHz for 1~1.5 h.

5. The method for multi-strain synergistic fermentation of Astragalus membranaceus according to claim 1, characterized in that, In step (1), the centrifugation conditions are 12000 r / min for 15 min; the sterilization conditions are 121℃ high-pressure steam sterilization for 20 min.

6. The method for multi-strain synergistic fermentation of Astragalus membranaceus according to claim 1, characterized in that, In step (2), the fermentation conditions for Bacillus subtilis are 37℃ and static fermentation for 48 h.

7. The method for multi-strain synergistic fermentation of Astragalus membranaceus according to claim 1, characterized in that, In step (3), Lactobacillus plantarum and Enterococcus faecalis were allowed to ferment under anaerobic conditions at 37°C for 24 h.

8. The method for multi-strain synergistic fermentation of Astragalus membranaceus according to claim 1, characterized in that, The multi-strain synergistic fermentation is a stepwise synergistic fermentation, which includes first performing aerobic fermentation with Bacillus subtilis, and then performing anaerobic fermentation with Lactobacillus plantarum and Enterococcus faecalis.

9. The multi-strain synergistic fermentation product of Astragalus membranaceus prepared by the method according to any one of claims 1-8.

10. The application of the multi-strain synergistic fermentation product of Astragalus membranaceus according to claim 9 in improving the content of total polyphenols, total flavonoids and antioxidant activity of Astragalus membranaceus.

11. The use of the multi-strain synergistic fermentation product of Astragalus membranaceus according to claim 9 in the preparation of products for improving the quality of livestock and poultry meat.

12. The application according to claim 11, characterized in that, The improvement of livestock and poultry meat quality includes increasing muscle firmness, elasticity, gelatinous texture, chewiness, and collagen content.

13. The use of the multi-strain synergistic fermentation product of Astragalus membranaceus according to claim 9 in the preparation of products for enhancing animal immunity.

14. The use of the multi-strain synergistic fermentation product of Astragalus membranaceus according to claim 9 in the preparation of products for relieving heat stress in animals.

15. The application according to claim 14, characterized in that, The methods for alleviating heat stress in animals include improving rumen pH and / or increasing total volatile fatty acid content.

16. The use of the multi-strain synergistic fermentation product of Astragalus membranaceus according to claim 9 in the preparation of products for improving the intestinal health of pets and fur-bearing animals.

17. The use of the multi-strain synergistic fermentation product of Astragalus membranaceus according to claim 9 in the preparation of products for improving the luster and / or quality of the coat of pets and fur-bearing animals.

18. A feed additive, characterized in that, It includes the multi-strain synergistic fermentation product of Astragalus membranaceus as described in claim 9.

19. A pet nutritional preparation, characterized in that, It includes the multi-strain synergistic fermentation product of Astragalus membranaceus as described in claim 9.

20. A functional product, characterized in that, It comprises the multi-strain synergistic fermentation product of Astragalus membranaceus as described in claim 9 and feed-acceptable excipients.

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