Lactococcus lactis and fermented ovine colostrum preparations, methods of making and uses for improving age-related muscle loss

Abstract

This invention discloses a *Lactococcus faecium* strain, YS-IM01. This strain can efficiently hydrolyze sheep colostrum proteins and specifically release a large amount of leucine. This invention also provides a polypeptide composition comprising multiple active peptides with potential for muscle synthesis, immunomodulation, and antioxidant activity. This invention further provides a fermented sheep colostrum product obtained by fermenting sheep colostrum with the aforementioned *Lactococcus faecium*. This invention also provides a method for preparing the fermented sheep colostrum product, comprising steps including fermentation, low-temperature vacuum concentration, and vacuum freeze-drying. Simultaneously, this invention also provides a method for preparing a drug using *Lactococcus faecium* and the polypeptide composition to prevent and / or improve age-related muscle loss.

Description

Technical Field

[0001] This invention belongs to the field of probiotic fermentation, specifically relating to a strain of Lactococcus lactis, products made from fermented sheep colostrum, preparation methods, and its application in slowing down muscle loss. Background Technology

[0002] With the accelerating aging of the global population, sarcopenia has become an increasingly serious public health problem. This disease, characterized by a progressive, systemic decline in skeletal muscle mass, strength, and function, severely impacts the quality of life of older adults and significantly increases the risk of falls and fractures. Its underlying pathological mechanisms are complex, typically involving multiple factors such as weakened protein synthesis, enhanced catabolism, chronic low-grade inflammation, and decreased sensitivity of anabolism signaling pathways.

[0003] Among the many strategies for preventing and treating sarcopenia, nutritional support is widely recognized as a core approach. Numerous studies have shown that supplementing with high-quality protein and its hydrolysates, especially branched-chain amino acids (such as leucine) that specifically activate muscle synthesis pathways, is an effective intervention. Leucine is unique in that it not only builds muscle protein but also activates the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway to synthesize muscle protein. Sheep colostrum, as a nutrient-rich natural product, is abundant in protein, immunoglobulins, and various growth factors, making it an excellent source of high-quality amino acids. However, its large protein molecular structure may affect absorption efficiency in older adults with weakened digestive function.

[0004] Utilizing microbial fermentation technology to enhance the functional properties of food is a widely proven and effective approach. Selecting suitable probiotics for fermentation can not only break down large protein molecules into more easily absorbed small peptides and free amino acids, but may also generate new bioactive substances and even impart additional probiotic benefits to the product.

[0005] However, conventional fermentation strains (such as yogurt starter cultures) primarily function to produce acid and flavor, and their protease systems may not be capable of efficiently releasing specific functional amino acids (such as leucine) or generating specific bioactive peptides. Currently, there is a lack of probiotic strains and corresponding fermentation processes capable of efficiently hydrolyzing sheep colostrum proteins and selectively enriching bioactive components related to muscle synthesis and metabolism.

[0006] Therefore, targeted screening of probiotics with specific functional potential and optimization of their fermentation processes to develop novel functional fermented products that can effectively improve or prevent age-related muscle loss has significant scientific importance and application prospects.

[0007] Currently, there are numerous research reports on functional fermented dairy products. For example, patent application CN116941669A discloses a fermented dairy product with muscle-promoting functions and its applications. It uses *Streptococcus thermophilus* GDMCC No. 60810, screened from *Triplophysa yaks* in Qinghai, my country, as the core strain. This strain is inoculated into cow's milk, goat's milk, etc., and after homogenization and sterilization, fermented at 42℃ to obtain fermented milk. Animal experiments have confirmed that this fermented milk can significantly improve the morphology of muscle fibers in aging mice, increasing the cross-sectional area of ​​muscle fibers by 1.6 times, increasing the number of muscle nuclei by 1.9 times, and simultaneously increasing the grip strength of mice by 1.56 times.

[0008] However, this existing technology has certain limitations. First, although the thermophilic streptococci used in this technology are derived from traditional fermented foods, the screening process mainly relies on conventional fermentation performance and does not target specific functional targets related to muscle health. Second, while this existing technology has verified the effect of fermented milk on improving muscle fiber morphology and grip strength through animal experiments, it has not further explored its intrinsic mechanism of action, nor analyzed the fermentation products, thus failing to clarify the material basis of its functional activity. In contrast, this invention uses "leucine release" as the core indicator for targeted screening, ensuring the specific functional potential of the strains from the source, and combines it with peptidomics technology to clarify the material basis of its functional activity, providing a scientific basis for the efficacy of the fermentation products. Summary of the Invention

[0009] To address the problem of sarcopenia in the elderly, one aspect of this invention provides *Lactococcus lactis* YS-IM01, which can efficiently hydrolyze sheep colostrum proteins and specifically release leucine. Another aspect provides a polypeptide composition that can significantly downregulate the expression of key muscle atrophy genes MuRF1 and Atrogin-1, while simultaneously reducing serum levels of inflammatory factors TNF-α and IL-6. Furthermore, this invention provides a fermented sheep colostrum product rich in various bioactive peptides obtained by fermenting sheep colostrum using the aforementioned strain, and a method for preparing the fermented sheep colostrum product. Finally, this invention provides the application of the fermented sheep colostrum product in the preparation of a medicament for preventing or improving muscle loss.

[0010] This invention is achieved through the following technical solution: A strain of *Lactococcus cremoris*, YS-IM01, with accession number CGMCC NO. 37356, was deposited on January 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China (Institute of Microbiology, Chinese Academy of Sciences). The strain was viable at the time of deposit.

[0011] A polypeptide composition comprising polypeptides with the sequences LSPEVLNENLLR, LNENLLRF, and QAMEDAKQMK.

[0012] A fermented sheep colostrum product, obtained by fermenting sheep colostrum with the aforementioned Lactococcus fatensis YS-IM01.

[0013] The preparation method of the fermented sheep colostrum product includes the following steps: S1. The seed culture of Lactococcus fatiensis YS-IM01 was inoculated into sheep colostrum fermentation medium for fermentation to obtain fermentation broth; After S2 fermentation is completed, the fermentation liquid is concentrated under low temperature vacuum to obtain fermented sheep colostrum concentrate. S3. Add a freeze-drying protectant to the fermented sheep colostrum concentrate, and then freeze-dry, pulverize, and sieve to obtain the fermented sheep colostrum frozen product.

[0014] The viable count of the *Lactococcus fatii* seed culture YS-IM01 was 1~9×10⁻⁶. 9 CFU / mL; The seed liquid inoculation amount is 2% (v / v); The concentration of sheep colostrum in the sheep colostrum fermentation medium is 12-16 wt%. The sterilization conditions for the sheep colostrum fermentation medium are sterilization at 75-85℃ for 25-35 minutes; The fermentation conditions were: static fermentation at 33°C for 24-28 hours; The low-temperature vacuum concentration conditions are a temperature not exceeding 37°C and a vacuum degree of 0.08~0.09MPa. The concentration includes the step of concentrating the fermentation broth to 1 / 5 of its original volume. The freeze-drying protectant, by weight percentage, comprises 8% trehalose, 0.3% ascorbic acid, 1.2% monosodium glutamate, and the balance being water; The vacuum freeze-drying conditions are as follows: control the material layer thickness to be 8mm~10mm, pre-freeze at -80℃ for 5h, and then dry at a vacuum degree of no more than 5Pa and a cold trap temperature of -80℃ for 26h.

[0015] The use of Lactococcus lactis YS-IM01 in the preparation of drugs for the prevention and / or improvement of age-related muscle loss.

[0016] The use of the polypeptide composition in the preparation of a medicament for preventing and / or improving age-related muscle loss.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a strain of *Lactococcus faecium* YS-IM01 obtained through targeted screening based on leucine release levels. This bacterium can efficiently hydrolyze sheep colostrum proteins and specifically release leucine.

[0018] This invention provides a polypeptide composition. The composition comprises at least three polypeptides: LSPEVLNENLLR, LNENLLRF, and QAMEDAKQMK. These three polypeptides can significantly downregulate the expression of key muscle atrophy genes MuRF1 and Atrogin-1, while simultaneously reducing serum levels of inflammatory factors TNF-α and IL-6, thereby improving senile sarcopenia through both inhibiting muscle breakdown and alleviating chronic inflammation.

[0019] This invention provides a sheep colostrum enriched with a series of characteristic peptides such as LSPEVLNENLLR, LNENLLRF, and QAMEDAKQMK, which have functions of promoting muscle synthesis, immune regulation, and antioxidation. In key indicators such as maintaining body weight, increasing muscle mass and index, improving grip strength, and increasing muscle fiber cross-sectional area, its effects are significantly superior to unfermented sheep colostrum, probiotics alone, and even the leucine-positive control group, fully demonstrating the advantages of the synergistic effect of multiple active peptides.

[0020] This invention provides a method for preparing sheep colostrum (15% sheep colostrum, fermented at 33°C for 26 hours). This method yields a final product with a peptide yield of 9.56 mg / mL, of which 71.83% are small molecule peptides less than 3 kDa. Furthermore, by employing gentle low-temperature vacuum concentration and an optimized composite lyophilization protectant (trehalose, ascorbic acid, monosodium glutamate), the resulting lyophilized powder retains peptides well, exhibits good solubility, and is easy to store and use. Attached Figure Description

[0021] Figure 1 The graph shows the relationship between different sheep colostrum concentrations and peptide yield and the proportion of peptides less than 3 kDa in Example 2. Figure 2 The graph shows the relationship between different fermentation temperatures and peptide yield and the proportion of peptides less than 3 kDa in Example 2. Figure 3 The graph shows the relationship between different fermentation times and peptide yield and the proportion of peptides smaller than 3 kDa in Example 2. Figure 4 The mass spectrum of the peptide LSPEVLNENLLR is shown. Figure 5 The mass spectrum of the peptide LNENLLRF is shown; Figure 6 The mass spectrum of the polypeptide QAMEDAKQMK is shown. Figure 7The diagram shows HE staining of mouse muscles in different treatment groups. Figure 8 The diagram shows the relative expression levels of MuRF1 and Atrogin-1, genes associated with muscle atrophy, in mice under different treatment groups. Detailed Implementation

[0022] Example 1 This embodiment involved the targeted screening and identification of strains that produce muscle-synthesizing bioactive peptides.

[0023] The core problem of sarcopenia in the elderly lies in the imbalance between skeletal muscle protein synthesis and breakdown. Leucine, as a key activating signal of the mTORC1 pathway, is a crucial molecule for initiating muscle protein synthesis. Therefore, this embodiment aims to selectively screen for a lactic acid bacterium that can efficiently hydrolyze sheep colostrum proteins and specifically release leucine.

[0024] The targeted screening process for strains producing muscle-synthesizing bioactive peptides is as follows: (1) Isolation and purification of strains.

[0025] Take 10 mL of fresh goat milk sample and perform 10-fold serial dilutions using sterile physiological saline. Spread 100 μL of each serial dilution onto MRS solid medium and incubate at 37°C for 48 h. After colonies have grown, select typical lactic acid bacteria colony morphology (round, milky white, smooth and moist surface, neat edges, and a raised center) and repeatedly streak them on MRS solid medium for purification until a pure culture is obtained. Examine the pure culture under Gram staining microscopy, retaining Gram-positive, cocci or short rod-shaped strains arranged in pairs or short chains.

[0026] Through isolation, purification, and microscopic examination, a total of 18 strains were obtained in this experiment, which were frozen at -80℃ with glycerol for later use.

[0027] (2) Preliminary screening of strains.

[0028] To target specific strains, a dual screening system with leucine release as the core indicator was designed.

[0029] Preparation of primary screening culture medium: Group A (Protein hydrolysis capacity assessment): Prepare a 10% (w / v) sheep colostrum solution and sterilize at 80℃ for 30 min.

[0030] Group B (Metabolic Preference Assessment): A 10% (w / v) sheep colostrum solution was prepared, and 0.05% (w / v) neutral protease was added. The solution was hydrolyzed at 50°C for 40 min, followed by sterilization at 80°C for 30 min to inactivate the enzyme. This pretreatment exposed more restriction enzyme sites to assess the strain's potential for utilizing short peptides and releasing leucine.

[0031] Strain activation and inoculation: The 18 preserved bacterial strains were activated and prepared into seed solutions. In 96-well plates, each strain was placed in 4 parallel wells (2 wells for group A and 2 wells for group B). 200 μL of the corresponding culture medium was added to each well, and the inoculum was 2% and incubated at 37°C for 24 h.

[0032] Testing indicators: Degree of protein hydrolysis: The amino nitrogen release from the fermentation broth of group A was determined using the o-phthalaldehyde (OPA) method; Relative quantification of leucine: The chromatographic peak area of ​​leucine in the fermentation broths of groups A and B was determined by HPLC.

[0033] Initial screening results: The initial screening results of 18 strains are shown in Table 1.

[0034] Table 1 Initial screening results of strains Screening criteria were set for the above indicators: ① The degree of protein hydrolysis in group A was greater than 15%; ② The relative peak area of ​​leucine in group A was greater than 130.00×103AU; ③ The relative peak area of ​​leucine in group B was greater than that in group A, indicating that the strains preferred to utilize pre-hydrolyzed products and release leucine.

[0035] As can be seen from the initial screening results in Table 1, only N41S1 and N42S9 among the 18 strains met all three criteria.

[0036] (3) Re-screening of strains.

[0037] N41S1 and N42S9 were inoculated at a rate of 2% into 20 mL of natural sheep colostrum medium (10%, w / v) and fermented at 37°C for 24 h. The absolute contents of leucine and total BCAAs in the fermentation broth were determined. The results are shown in Table 2. Table 2 Comparison of fermentation performance of dominant strains after 24 hours Note: Δ represents the difference between the 24h value and the 0h value.

[0038] As shown in Table 2, strain N41S1 exhibits significantly higher levels of leucine and total BCAAs release compared to N42S9, indicating its ability to release key muscle-building factors from sheep colostrum proteins more efficiently. Therefore, N41S1 was selected as the core strain of this invention.

[0039] (4) Molecular biological identification of strain YS-IM01 (N41S1).

[0040] Single colonies of strain N41S1 were picked and placed in PCR lysis buffer, and lysed in an 80°C water bath for 30 min to obtain bacterial DNA template.

[0041] The universal primers for bacterial 16S rRNA gene amplification are as follows: 27F: 5´-CAGAGTTTGATCCTGGCTCAG-3´; 1492R: 5´-GGTTACCTTGTTACGACTT-3´.

[0042] The reaction mixture (25 μL) consisted of: 1 μL each of primers 27F and 1492R, 0.5 μL of bacterial DNA template, 12.5 μL of 2×RapidTaqMaster Mix, and 10 μL of ddH2O. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 30 cycles (94℃ denaturation for 25 s, 55℃ annealing for 25 s, 72℃ extension for 25 s), followed by holding at 72℃ for 5 min to ensure complete extension; storage at 4℃. After PCR, the reaction mixture was electrophoresed on a 1.5% agarose gel using 1×TAE (50 mmol / L Tris-acetic acid, 1 mmol / L EDTA, pH 8.0).

[0043] After sequencing, the sequencing results were compared with GenBank using BLAST. The results showed 99.9% homology with *Lactococcus cremoris*. Based on morphological and physiological-biochemical characteristics, this strain was identified as *Lactococcus cremoris* and named YS-IM01. It was deposited on January 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.37356. Its 16S rRNA gene sequence is shown in SEQ ID NO. 1.

[0044] SEQ ID NO:1:

[0045] Example 2 This embodiment optimized the process of fermenting sheep colostrum using strain YS-IM01.

[0046] To further enrich the active ingredients (small molecule peptides and leucine) that promote muscle synthesis, key fermentation parameters were optimized in this embodiment. The evaluation indicators were defined as peptide yield, which reflects the total amount of active substances, and the proportion of peptides less than 3 kDa, which reflects absorption potential.

[0047] (1) Single-factor experimental design Single-factor screening of sheep colostrum concentration Weigh out sheep colostrum powder separately and prepare 5%, 10%, and 15% (w / v) sheep colostrum solutions with distilled water. Adjust the pH to 6.8, mix thoroughly, and sterilize at 80°C for 30 min. After cooling to room temperature, inoculate with the *Lactococcus lactis* seed culture prepared in Example 1 (5 × 10⁻⁶ viable cells) at a 2% (v / v) inoculation rate. 9 (CFU / mL), and fermented at 37℃ for 24 hours.

[0048] Single-factor screening of fermentation temperature Prepare a 15% (w / v) sheep colostrum solution and sterilize at 80℃ for 30 min. After cooling, inoculate with YS-IM01 seed culture at a 2% inoculation rate (5 × 10⁻⁶ viable bacteria). 9 (CFU / mL), and fermented at constant temperature for 24 hours at 33℃, 37℃, and 42℃ respectively.

[0049] Fermentation time single-factor screening Prepare a 15% (w / v) sheep colostrum solution and sterilize at 80℃ for 30 min. After cooling, inoculate with YS-IM01 seed culture at a 2% inoculation rate (5 × 10⁻⁶ viable bacteria). 9 (CFU / mL), and were fermented at 33℃ for 18h, 24h, and 30h respectively.

[0050] Peptide yield determination method Take 2 mL of fermentation broth, add an equal volume of 20% (w / v) trichloroacetic acid solution, mix well, let stand for 10 min, and centrifuge at 4℃ and 8000 rpm for 15 min. Take the supernatant and determine the peptide content using the Lowry method, plotting a standard curve with casein as the standard. Peptide yield is expressed as the number of milligrams of peptide per milliliter of fermentation broth (mg / mL).

[0051] Method for determining the proportion of peptides smaller than 3kDa Take 2 mL of fermentation broth and centrifuge at 10000 rpm for 10 min at 4℃, then collect the supernatant. Centrifuge in an ultrafiltration centrifuge tube (molecular weight cutoff 3 kDa) at 5000 rpm for 30 min, and collect the filtrate. Measure the total peptide content before ultrafiltration and the peptide content of the filtrate after ultrafiltration. The percentage of peptides less than 3 kDa (%) = (peptide content of filtrate / total peptide content before ultrafiltration) × 100%.

[0052] Results analysis: The effect of sheep colostrum concentration: The fermentation temperature was fixed at 37℃, and the fermentation time was 24 hours. The peptide yield and the proportion of peptides less than 3 kDa in the fermented sheep colostrum were determined, and the results are as follows: Figure 1 As shown, both peptide yield and the proportion of peptides less than 3 kDa reached their highest values ​​at a 15% sheep colostrum concentration.

[0053] The effect of fermentation temperature: The concentration of sheep colostrum was fixed at 15%, and the fermentation time was 24 hours. The peptide yield and the proportion of peptides less than 3 kDa in the fermented sheep colostrum were determined, and the results are as follows: Figure 2 As shown, strain YS-IM01 exhibited the highest peptide yield and proportion of small peptides at 33℃, indicating that low temperature (33℃) is more conducive to the hydrolysis of its protease system.

[0054] The effect of fermentation time: The concentration of sheep colostrum was fixed at 15%, and the fermentation temperature was 33℃. The peptide yield and the proportion of peptides less than 3 kDa in the fermented sheep colostrum were determined, and the results are as follows: Figure 3 As shown, the peptide yield was close to its peak at 24 h of fermentation, and the proportion of peptides less than 3 kDa reached its highest level; at 30 h, the proportion of small molecule peptides decreased, which may have been due to excessive hydrolysis.

[0055] (2) Orthogonal optimization experimental design To obtain the optimal combination, based on the results of single-factor experiments, L9(3) was adopted. 4 Orthogonal optimization.

[0056] Factor A (colostrum concentration): 12%, 15%, 18%; Factor B (fermentation temperature): 31℃, 33℃, 35℃; Factor C (fermentation time): 22h, 24h, 26h.

[0057] Results analysis: The results of the orthogonal experiment are shown in Table 3.

[0058] Table 3. Orthogonal experimental design and results Table 3 shows that group 5 (A2B2C3) is the optimal combination, namely: 15% sheep colostrum concentration, fermentation temperature 33℃, and fermentation time 26h. Under these conditions, the peptide yield reached 9.56 mg / mL, and the proportion of small molecule peptides (less than 3 kDa) reached 71.83%, both significantly better than other combinations. Compared with the 24h initially selected in the single-factor experiment, extending the fermentation time by 2h increased the peptide yield by 12.3% and the proportion of small molecule peptides by 8.5%, indicating that 26h is the key point for this strain to hydrolyze proteins and accumulate small molecule bioactive peptides. Therefore, 15% sheep colostrum and fermentation at 33℃ for 26h were determined as the subsequent process.

[0059] Example 3 This embodiment describes the preparation of fermented sheep colostrum products.

[0060] (1) Preparation of highly active seed liquid.

[0061] Lactococcus fatii YS-IM01, frozen in glycerol at -80℃, was streaked onto MRS solid medium for activation and cultured at 37℃ for 48 h. Single colonies were picked and inoculated into MRS liquid medium, incubated statically at 37℃ for 24 h, and passaged twice to restore good viability. The activated bacterial solution was centrifuged at 4℃ and 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline and resuspended. The bacterial concentration was adjusted to 1~9×10⁻⁹. 9 The CFU / mL concentration yielded a highly active seed solution, which was then stored at 4°C for later use.

[0062] (2) Preparation of fermented sheep colostrum.

[0063] Weigh out sheep colostrum powder and prepare a 15% (w / v) sheep colostrum solution with warm water. After thorough mixing, sterilize at 80℃ for 30 minutes. After cooling to room temperature, inoculate the above-mentioned highly active seed liquid at a 2% (v / v) inoculation rate, stir at low speed until homogeneous, and let it ferment at 33℃ for 26 hours. After fermentation, immediately transfer the fermentation broth to 4℃ to stop fermentation. To prevent stratification of the fermented milk, homogenize the cooled fermentation broth twice under 30 MPa pressure to obtain a homogeneous, non-stratified fermented sheep colostrum.

[0064] (3) Low temperature vacuum concentration.

[0065] To reduce the pressure of subsequent drying processes and enrich active ingredients, a vacuum centrifugal concentrator was used to concentrate the fermentation broth. The water bath temperature was controlled to not exceed 37℃, the vacuum degree was maintained at 0.08~0.09MPa, and the rotation speed was 1200r / min. The process ended when the fermentation broth volume was concentrated to 1 / 5 of its original volume, at which point the solid content of the concentrated broth was approximately 32~35%. The temperature remained below 37℃ throughout the entire concentration process to maximize the protection of heat-sensitive peptides and probiotic activity.

[0066] (4) Vacuum freeze drying.

[0067] Preliminary experiments revealed that direct freeze-drying of the concentrate resulted in significant loss of bacterial cells (survival rate <50%) and peptides. Therefore, a freeze-drying protection system suitable for the YS-IM01 strain and capable of improving peptide retention was screened.

[0068] The final composition of the preservative was determined as follows: 8% (w / w) trehalose, 0.3% ascorbic acid, and 1.2% monosodium glutamate were added by weight of the concentrate. After thorough stirring and dissolution, the mixture was allowed to stand for equilibration for 15 minutes. The concentrate with the preservative was dispensed into freeze-drying trays, with the material layer thickness controlled at 8-10 mm. The samples were pre-frozen at -80℃ for 5 hours to ensure the center temperature was below -45℃. After pre-freezing, the samples were quickly transferred to a vacuum freeze dryer with the following drying parameters set: cold trap temperature -80℃, vacuum degree ≤5Pa, main drying for 20 hours, desorption drying for 6 hours, and a total drying time of 26 hours. After drying, high-purity nitrogen was immediately introduced into the drying chamber to break the vacuum, and the freeze-dried blocks were quickly removed under conditions of relative humidity <20%. The blocks were then pulverized using a high-speed grinder, passed through a 120-mesh sieve, and the sieve-underfill material was collected as the finished product: fermented sheep colostrum freeze-dried powder. The finished product was stored at temperatures below -18℃.

[0069] (5) Quality analysis of the finished product of fermented sheep colostrum freeze-dried powder.

[0070] The basic indicators of the prepared fermented sheep colostrum freeze-dried powder were tested, and the results are shown in Table 4.

[0071] Table 4. Results of Basic Physicochemical and Microbiological Indicators of Fermented Goat Colostrum Powder As shown in the table above, the product has a moisture content of 3.24% and a viable bacteria count of 5.37 × 10⁻⁶. 9 CFU / g, solubility 98.13%, pH value 6.18, excellent quality.

[0072] (6) Protein degradation related indicators.

[0073] To compare the changes in protein and peptide content in the samples before and after fermentation, the soluble protein content was determined using the Folin-phenol reagent method; the free amino acid content was determined using the ninhydrin colorimetric method; and the peptide content was determined using trichloroacetic acid precipitation combined with the Lowry method. The degree of protein hydrolysis was calculated as (amino nitrogen content after hydrolysis / total nitrogen content) × 100%. The results are shown in Table 5.

[0074] Table 5 Comparison of protein and peptide content in sheep colostrum before and after fermentation As shown in the table above, the soluble protein content in the freeze-dried powder decreased significantly after fermentation, while the content of free amino acids and peptides increased significantly, with a protein hydrolysis degree as high as 86.47%. The peptide content (92.86 mg / g) is very close to the theoretical value (approximately 95.6 mg / g) of the fermentation broth in Example 2 after conversion to dry basis, indicating that the freeze-drying process has a good retention effect on peptides.

[0075] (7) Determination of leucine content.

[0076] The leucine content in the samples was determined using an automated amino acid analyzer, and the results are shown in Table 6.

[0077] Table 6 Comparison of leucine content before and after sheep colostrum fermentation As shown in the table above, the leucine content in the freeze-dried sheep colostrum powder after fermentation reached 12.68 mg / g, which is approximately 2.98 times higher than the 4.25 mg / g before fermentation. This indicates that the YS-IM01 strain can effectively degrade sheep colostrum proteins and specifically release leucine during fermentation.

[0078] Example 4 In this embodiment, peptidomics analysis and bioactive peptide identification were performed on fermented sheep colostrum powder.

[0079] To elucidate the material basis of YS-IM01 fermentation products, this invention uses LC-MS / MS technology to compare and analyze the changes in peptide spectra of sheep colostrum before and after fermentation, and identifies specific peptides enriched therein.

[0080] (1) Sample preparation: Control group: 10% (w / v) sterile sheep colostrum solution that has not been fermented.

[0081] Experimental group: Fermented sheep colostrum solution prepared according to the optimal process in Example 2.

[0082] Add an appropriate amount of 8M UA solution to the sample and incubate in a boiling water bath for 5 min. Transfer the solution to a 10KD ultrafiltration centrifuge tube and centrifuge at 12000 rpm. After centrifugation, add 0.25% acetic acid solution and centrifuge at 12000 rpm for 20 min. Repeat twice. Collect the lower peptide filtrate, dry it, add an appropriate amount of 0.1% TFA solution, and desalt it using a C18 cartridge for LC-MS analysis.

[0083] (2) LC-MS / MS analysis: A suitable amount of peptide was taken from each sample and chromatographically separated using a Vanquish Neo UHPLC system. Buffer solutions: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile content 80%). The column was equilibrated with 96% Solution A. After the sample was injected into the Trap Column (PepMap Neo 5 µm C18 300 µm x 5 mm), it underwent gradient separation through the analytical column (μPAC Neo High Throughput column). The HPLC gradient settings were as follows: 0 min–0.1 min, linear gradient of Solution B from 4%–8%; 0.1 min–1.2 min, linear gradient of Solution B from 8%–10%; 1.2 min–11.2 min, linear gradient of Solution B from 10%–28%; 11.2 min–13.5 min, linear gradient of Solution B from 28%–45%; 13.5 min–15 min, Solution B maintained at 99%. After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using an Orbitrap Astral mass spectrometer. The analysis time was 15 min, the electrospray voltage was 2.2 kV, the detection mode was positive ion, the precursor ion scan range was 380-980 m / z, the first-stage mass spectrometry resolution was 240,000, the AGC target was 500%, and the first-stage maximum IT was 3 ms. The second-stage mass spectrometry resolution was 80,000, the AGC target was 500%, the second-stage maximum IT was 3 ms, the RF-lens was 40%, the MS2 activation type was HCD, the isolation window was 2Th, the normalized collision energy was 25%, and the cycle time was 0.6.

[0084] Results analysis: The mass spectrometry results were analyzed using a database to screen for peptides whose abundance increased significantly (≥2-fold) after fermentation and whose molecular weight was <3kDa. The results are shown in Table 7.

[0085] Table 7 Peptide Identification Table The top 10 most abundant peptides newly generated after fermentation are shown in Table 8.

[0086] Table 8. Top 10 most abundant newly produced peptides in fermented sheep colostrum. In line with the core objectives of this invention (improving muscle loss and enhancing muscle function), in-depth analysis was conducted on the identified peptides, and three core peptides were selected: ① Muscle-building peptide: LSPEVLNENLLR (mass spectrum shown) Figure 4 ) The abundance of this peptide increased significantly by 7.09-fold after fermentation. Its sequence is rich in leucine (L), containing a total of four leucine residues. Leucine is a key signaling molecule that activates the mTORC1 pathway, a core switch in skeletal muscle protein synthesis. This peptide may be absorbed intact in the digestive tract or act as a sustained-release carrier of leucine, continuously providing signals and substrates for muscle synthesis and directly promoting myofibril hypertrophy.

[0087] ②Immune modulation and anti-inflammatory peptides: LNENLLRF (mass spectra see...) Figure 5 ) This peptide is newly generated after fermentation and is abundant. Sequence analysis showed that it is highly homologous to the core fragment of Isracidin (RPKHPIKHQGLPQEVLNENLLRF), a well-known immunomodulatory peptide derived from milk protein, indicating that it may have immunomodulatory activity. One of the core pathological mechanisms of sarcopenia in the elderly is chronic low-grade inflammation. This peptide may reduce inflammation-mediated muscle protein breakdown by regulating the balance of inflammatory factors and reducing serum TNF-α and IL-6 levels.

[0088] ③ Antioxidant protective peptide: QAMEDAKQMK (mass spectrum shown) Figure 6 ) The abundance of this peptide increased significantly by 6.68-fold after fermentation, and its sequence contains easily oxidized methionine (M) residues. Such peptides can act as "sacrificial" antioxidants, preferentially capturing and eliminating excess free radicals generated during aging or exercise, protecting muscle cell membranes and functional proteins from oxidative damage, and providing a healthy cellular environment for muscle protein synthesis.

[0089] In summary, the active peptides enriched in the fermented sheep colostrum product of the present invention do not act as a single component, but rather through a synergistic network of core peptide segments such as LSPEVLNENLLR (muscle synthesis promoter), LNENLLRF (anti-inflammatory), and QAMEDAKQMK (antioxidant), they work together to improve muscle loss and enhance muscle function from three dimensions: promoting protein synthesis, inhibiting inflammatory degradation, and protecting cells from oxidative damage.

[0090] Example 5 This embodiment validates the effect of fermented sheep colostrum on improving sarcopenia in a mouse model.

[0091] (1) Mouse feeding and grouping.

[0092] Fifty SPF-grade, 18-month-old male C57BL / 6J naturally aging mice, weighing 35±2g, were selected. The rearing environment was controlled at 22±2℃, 55±10% humidity, and a 12-hour light-dark cycle, with free access to water and food. After one week of acclimatization, the mice were randomly divided into 5 groups of 10 mice each, with each group receiving 10 mL / kg via gavage. The intervention lasted for 8 consecutive weeks. The grouping and intervention protocols are as follows: Natural aging model group: daily gavage with physiological saline; Positive control group: Leucine solution was administered by gavage daily (dose of 200 mg / kg, leucine purity ≥98%). sheep colostrum group: daily gavage administration of unfermented sheep colostrum solution (gavage dose based on protein content, maintaining the same protein level as the fermented sheep colostrum group); Probiotic group: Daily gavage administration of YS-IM01 probiotic solution (dose 2×10⁻⁶). 9 CFU / kg); Fermented sheep colostrum group: daily gavage administration of the fermented sheep colostrum lyophilized powder solution prepared in Example 3 (dosage: 1.5 g / kg).

[0093] (2) Detection indicators and methods.

[0094] Body weight and forelimb grip strength measurement: During the intervention, the mice were weighed at fixed times each week to record the trend of weight changes. Before the end of the intervention, the grip strength of the mice's forelimbs was measured using a grip strength meter to assess muscle function.

[0095] Muscle tissue sample collection: After the final intervention, mice were fasted for 12 hours but allowed free access to water, and their fasting body weight was measured. After anesthesia with intraperitoneal injection of sodium pentobarbital (50 mg / kg), mice were euthanized by cervical dislocation. The bilateral gastrocnemius muscles were rapidly dissected and separated. Surface bloodstains were rinsed with pre-cooled physiological saline, and moisture was absorbed with filter paper. The wet weight of the gastrocnemius muscles was measured using an electronic balance, and the muscle index was calculated using the formula: Muscle Index (%) = Gastrocnemius muscle weight (g) / Fasting body weight of mouse (g) × 100%. The left gastrocnemius muscle was fixed in 4% paraformaldehyde solution for histological observation; the right gastrocnemius muscle was flash-frozen in liquid nitrogen and then transferred to a -80°C ultra-low temperature freezer for gene expression detection.

[0096] Morphological observation of muscle tissue: The fixed left gastrocnemius muscle was dehydrated using a gradient process (70%, 80%, 90%, 95%, and anhydrous ethanol for 1 hour each), cleared with xylene, and embedded in paraffin. 5 μm thick serial sections were prepared and baked at 60°C for 2 hours. After dewaxing to water, the sections were stained with hematoxylin and eosin (HE): hematoxylin staining for 5 minutes, differentiation with 1% hydrochloric acid ethanol for 30 seconds, blueing with tap water for 10 minutes, eosin staining for 30 seconds, gradient dehydration, clearing with xylene, and mounting with neutral resin. Muscle fiber morphology was observed under an optical microscope. Five non-overlapping fields of view were randomly selected from each section, and the cross-sectional area (CSA) of muscle fibers was measured using Image-Pro Plus software. At least 50 muscle fibers were measured in each field of view.

[0097] Serum biochemical markers determination: Blood was collected from the orbital venous plexus of mice before sacrifice. The blood was placed in 1.5 mL centrifuge tubes, incubated at 4°C for 30 min, and then centrifuged at 3000 rpm at 4°C for 15 min. The serum was separated, aliquoted, and stored at -80°C. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured using an ELISA kit.

[0098] q-PCR detection of gene expression related to muscle atrophy: Approximately 50 mg of a right gastrocnemius muscle sample stored at -80℃ was ground into powder using liquid nitrogen. Total RNA was extracted using 1 mL of Trizol reagent (Invitrogen). The purity (A260 / A280 ratio 1.8-2.0) and concentration of the RNA were determined using a nucleic acid protein analyzer. 1 μg of the total RNA was used to synthesize cDNA using a reverse transcription kit (TaKaRa). The reaction conditions were: incubation at 42℃ for 30 min, followed by heating at 85℃ for 5 min to terminate the reaction. The synthesized cDNA was stored at -20℃.

[0099] Using cDNA as a template, amplification was performed using a real-time quantitative PCR instrument to detect the relative mRNA expression levels of muscle atrophy-related genes MuRF1 (muscle ring finger protein 1) and Atrogin-1 (atrophy box F protein). The internal reference gene was GAPDH, and the primer sequences are shown in Table 9 below.

[0100] Table 9 Primers for MuRF1 and Atrogin-1 The PCR amplification system consisted of 20 μL (10 μL SYBR Green Mix, 0.8 μL each of forward and reverse primers, 2 μL cDNA template, and 6.4 μL sterile deionized water); reaction conditions were: 95℃ pre-denaturation for 30 s, followed by 40 cycles (95℃ denaturation for 5 s, 60℃ annealing for 15 s, and 72℃ extension for 10 s). After each cycle, melting curve analysis was performed to confirm the specificity of the amplified product. The relative mRNA expression level of the target gene was calculated using the 2^(-ΔΔCt) method.

[0101] (3) Experimental results.

[0102] Effects of fermented sheep colostrum on the body weight of aged mice.

[0103] During the intervention period, no mice in any group died, and their mental state, appetite, and defecation were normal, with no abnormal reactions. Body weight is a fundamental indicator reflecting the overall nutritional and metabolic status of the body; sarcopenia in older adults is often accompanied by weight loss. After 8 weeks of intervention, the model group mice showed a significant decrease in body weight. Compared with the model group, all intervention groups showed varying degrees of slowed weight loss. The fermented sheep colostrum group had the highest body weight, which was not significantly different from the positive control group but significantly higher than the sheep colostrum group, probiotic group, and model group. Specific results are shown in Table 10 below.

[0104] Table 10. Changes in mouse body weight in each group Note: Different lowercase letters after the data in the same column indicate significant differences between groups (P<0.05), the same applies below.

[0105] Effects of fermented sheep colostrum on muscle weight, muscle index and forelimb grip strength in aged mice.

[0106] As the main skeletal muscle of the hind limb, the gastrocnemius muscle's weight and muscle index directly reflect the level of muscle protein deposition, while forelimb grip strength is a commonly used indicator for assessing muscle function. As shown in Table 11, the fermented sheep colostrum group performed best in all three indicators: gastrocnemius muscle weight, muscle index, and forelimb grip strength. Its gastrocnemius muscle weight (0.147±0.01) and muscle index (0.413±0.03) were significantly higher than other groups; its forelimb grip strength (1.37±0.11) was comparable to the positive control group but significantly better than the sheep colostrum group and the probiotic group. This indicates that fermented sheep colostrum can effectively promote muscle protein deposition and improve muscle function.

[0107] Table 11 Muscle weight, muscle index, and gripping strength of mice in each group (4) Observation of muscle tissue morphology.

[0108] Muscle fiber cross-sectional area is a direct indicator of muscle fiber hypertrophy, and its reduction is the core pathological change in sarcopenia. HE staining results of the gastrocnemius muscle in each group of mice are shown below. Figure 7 As shown, the muscle fibers in the model group were loosely arranged, uneven in size, and exhibited significant atrophy. Muscle fiber morphology improved in all intervention groups, with the fermented sheep colostrum group showing the fullest and most orderly muscle fibers. Quantitative analysis (Table 12) indicated that its muscle fiber cross-sectional area (1728.53±193.87) was the largest, showing no significant difference from the positive control group, but significantly higher than other intervention groups.

[0109] Table 12 Cross-sectional area of ​​gastrocnemius muscle fibers in each group of mice (5) Serum inflammatory factor levels.

[0110] TNF-α and IL-6 are core pro-inflammatory cytokines mediating chronic low-grade inflammation, often abnormally elevated in sarcopenia of the elderly, and promote muscle protein breakdown by activating pathways such as NF-κB. As shown in Table 13, the fermented sheep colostrum group most effectively reduced serum levels of pro-inflammatory factors TNF-α and IL-6 (62.47±6.18 and 36.93±4.41, respectively), significantly better than all other groups. This reveals their prominent role in alleviating age-related chronic low-grade inflammation.

[0111] Table 13 Serum marker levels in each group of mice (6) Expression of genes related to muscle atrophy.

[0112] MuRF1 and Atrogin-1 are skeletal muscle-specific E3 ubiquitin ligases, upregulated in sarcopenia, mediating the ubiquitination and degradation of muscle proteins. Figure 8 As shown, compared with the natural aging model group, all intervention groups significantly downregulated the mRNA expression of MuRF1 and Atrogin-1, two muscle-specific E3 ubiquitin ligases. The fermented sheep colostrum group exhibited the strongest inhibitory effect and the lowest expression levels. This indicates that fermented sheep colostrum can effectively inhibit the ubiquitination and degradation pathway of muscle proteins.

[0113] In summary, the fermented sheep colostrum prepared by this invention can significantly improve sarcopenia in the elderly. Its mechanism of action is mainly manifested in: ① promoting synthesis: significantly increasing muscle weight and muscle fiber cross-sectional area, improving grip strength; ② inhibiting degradation: significantly downregulating the expression of MuRF1 and Atrogin-1, reducing muscle protein degradation; ③ alleviating inflammation: significantly reducing serum TNF-α and IL-6 levels, improving chronic inflammatory states. Intergroup comparisons showed that its overall effect was not only superior to unfermented sheep colostrum and YS-IM01 probiotics alone, but even superior to the leucine positive control. This fully demonstrates that the fermentation process not only preserves the nutrients of the raw materials, but also achieves a significant enhancement of efficacy by generating a variety of synergistic active peptides.

Claims

1. A type of Lactococcus lactis YS-IM01, characterized in that: Its accession number is CGMCC NO.37356, and its Latin name is Lactococcus cremoris.

2. A polypeptide composition, characterized in that: The polypeptide composition is isolated from sheep colostrum fermented by Lactococcus lactis YS-IM01 as described in claim 1, and includes polypeptides with the sequences LSPEVLNENLLR, LNENLLRF, and QAMEDAKQMK.

3. A fermented sheep colostrum product, characterized in that: It is obtained by fermenting sheep colostrum with Lactococcus fatensis YS-IM01 as described in claim 1.

4. A method for preparing the fermented sheep colostrum product according to claim 3, characterized in that: Includes the following steps: S1. The seed culture of Lactococcus fatiensis YS-IM01 was inoculated into sheep colostrum fermentation medium for fermentation to obtain fermentation broth; S2 The fermentation broth is concentrated under low temperature and vacuum to obtain fermented sheep colostrum concentrate; S3 adds a freeze-drying protectant to the fermented sheep colostrum concentrate, and obtains the fermented sheep colostrum product by vacuum freeze-drying, pulverizing, and sieving.

5. The method for preparing fermented sheep colostrum product as described in claim 4, characterized in that: The viable count of the *Lactococcus fatii* seed culture YS-IM01 was 1~9×10⁻⁶. 9 CFU / mL; The seed liquid inoculation amount is 2% (v / v).

6. The method for preparing fermented sheep colostrum product as described in claim 4, characterized in that: The concentration of sheep colostrum in the sheep colostrum fermentation medium is 12-16 wt%. The sterilization conditions for the sheep colostrum fermentation medium are sterilization at 75-85℃ for 25-35 minutes.

7. The method for preparing fermented sheep colostrum product as described in claim 4, characterized in that: The fermentation conditions were: static fermentation at 33℃ for 24-28 hours; The low-temperature vacuum concentration conditions are: temperature not exceeding 37°C and vacuum degree 0.08~0.09MPa; The concentration includes the step of concentrating the fermentation broth to 1 / 5 of its original volume.

8. The method for preparing fermented sheep colostrum product as described in claim 4, characterized in that: The freeze-drying protectant, by weight percentage, comprises 8% trehalose, 0.3% ascorbic acid, 1.2% monosodium glutamate, and the balance being water; The vacuum freeze-drying conditions are as follows: material layer thickness 8mm~10mm, pre-freezing at -80℃ for 5h, and then drying at a vacuum degree not greater than 5Pa and a cold trap temperature of -80℃ for 26h.

9. The use of the Lactococcus lactis YS-IM01 of claim 1 in the preparation of a medicament for preventing and / or improving age-related muscle loss.

10. Use of the polypeptide composition of claim 2 in the preparation of a medicament for preventing and / or improving age-related muscle loss.

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