Fermented milk whey capable of improving rheumatoid arthritis and application of fermented milk whey

Abstract

The invention belongs to the technical field of biology, and discloses fermented milk whey capable of improving rheumatoid arthritis and application of the fermented milk whey. The fermented milk whey disclosed by the invention is prepared by fermenting the lactobacillus helveticus (Lactobacillus helveticus) CCFM1501 or a fermentation solution of the lactobacillus helveticus (Lactobacillus helveticus) CCFM1501, and the preservation number of the lactobacillus helveticus (Lactobacillus helveticus) CCFM1501 is GDMCC (Lactobacillus helveticus) No: 66449. Lactobacillus helveticus (Lactobacillus helveticus) CCFM1501 fermented milk whey has the effect of relieving rheumatoid arthritis, so that the thickness of a posterior claw of a rat with rheumatoid arthritis is remarkably reduced, and knee joint injury is improved; the content of pro-inflammatory cytokines IL-1beta, IL-6 and TNF-alpha in the serum of the rat with rheumatoid arthritis is obviously reduced; the content of the arthritis specific antibody in the serum of the rheumatoid arthritis rat is obviously reduced, and potential safety hazards cannot be brought to the rheumatoid arthritis patient.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fermented whey that improves rheumatoid arthritis and its applications. Background Technology

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic, symmetrical polyarticular synovitis, accompanied by joint pain, swelling, and loss of function. In severe cases, it can lead to joint deformities and disability. Globally, the prevalence of RA is estimated at 0.5%–1%, with a significantly higher proportion of female patients than male patients. With increasing social pressure, changing living environments, and a rapidly aging population, the incidence of RA is on the rise, becoming a significant health issue affecting public quality of life. Current treatments primarily rely on nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, and biologics, but long-term use often results in adverse reactions. Therefore, the search for safer and more natural functional foods or dietary interventions that can regulate immunity and reduce joint inflammation and damage is receiving increasing attention.

[0003] Fermented milk whey is a byproduct of fermented milk production, containing various nutrients including proteins, amino acids, vitamins, and minerals. Currently, its main treatment methods are direct use as animal feed or direct wastewater treatment, leading to additional environmental pollution and costs, and constituting resource loss. Bioactive peptides are degradation products of proteins and have been reported to possess various biological activities, including antibacterial, antioxidant, antihypertensive, and immunomodulatory effects. These peptides can be obtained through microbial fermentation, digestive enzymatic hydrolysis, and other pathways. Among these, fermentation with specific *Lactobacillus helveticus* strains can release bioactive peptides with anti-inflammatory and immunomodulatory potential. However, no *Lactobacillus helveticus* strains capable of fermenting and producing fermented milk whey that improves rheumatoid arthritis have been found in current technology. Therefore, developing strains and products capable of producing such functional fermented milk whey is of great significance for meeting consumer health needs and also has broad market prospects. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a fermented whey that improves rheumatoid arthritis and its application.

[0005] To achieve the above and other related objectives, the first aspect of the present invention provides a fermented milk whey, which is obtained by fermenting raw milk with Lactobacillus helveticus CCFM1501 or its passaged strains, wherein the preservation number of Lactobacillus helveticus CCFM1501 is GDMCC No: 66449.

[0006] In a second aspect, the invention utilizes the aforementioned fermented whey in the preparation of products that aid in the improvement of rheumatoid arthritis.

[0007] The third aspect of the present invention provides a method for preparing the above-mentioned fermented milk whey, comprising: using an inoculum containing Lactobacillus helveticus CCFM1501 or a passaged strain as a starter culture, fermenting raw milk, and removing protein to obtain the fermented milk whey.

[0008] A fourth aspect of the present invention provides a product for assisting in the improvement of rheumatoid arthritis, the product comprising the above-mentioned fermented whey.

[0009] Compared with the prior art, the beneficial effects of the present invention include:

[0010] Lactobacillus helveticus is a type of probiotic and has been included in the "List of Strains that can be Used in Food". Therefore, the Lactobacillus helveticus CCFM1501 and its fermented milk whey screened by this invention will not pose any potential safety risks to patients with rheumatoid arthritis (RA).

[0011] The fermented whey of Lactobacillus helveticus CCFM1501 used in this invention has the effect of relieving rheumatoid arthritis, specifically manifested in: (1) significantly reducing the thickness of the hind paw of rats with rheumatoid arthritis and improving knee joint damage; (2) significantly reducing the content of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the serum of rats with rheumatoid arthritis; and (3) significantly reducing the content of arthritis-specific antibodies in the serum of rats with rheumatoid arthritis.

[0012] The cultivation process of Lactobacillus helveticus only requires culture medium and some control of culture conditions, and the cost is relatively low. Compared with expensive biological agents, it will not impose too much economic burden on patients with rheumatoid arthritis (RA). Attached Figure Description

[0013] Figure 1 The results show the peptide composition analysis of fermented whey from different strains of Lactobacillus helveticus, CCFM1263 and CCFM1501.

[0014] Figure 2 The data shows the concentrations of bone-related peptides in fermented whey from different strains of Lactobacillus helveticus, CCFM1263 and CCFM1501.

[0015] Figure 3 The figure shows the effect of Lactobacillus helveticus CCFM1501 fermented milk whey on the paw thickness of rats with rheumatoid arthritis; different letters in the figure indicate significant differences (p < 0.05).

[0016] Figure 4 The image shows the effect of fermented whey from Lactobacillus helveticus CCFM1501 on pathological sections of the knee joint of rats with rheumatoid arthritis.

[0017] Figure 5 The figure shows the effect of fermented whey from Lactobacillus helveticus CCFM1501 on serum IL-1β levels in rats with rheumatoid arthritis; in the figure, different letters above the bars indicate significant differences (p < 0.05).

[0018] Figure 6 The figure shows the effect of Lactobacillus helveticus CCFM1501 fermented milk whey on the serum IL-6 level in rats with rheumatoid arthritis; in the figure, different letters above the bars indicate significant differences (p<0.05).

[0019] Figure 7 The figure shows the effect of Lactobacillus helveticus CCFM1501 fermented milk whey on the serum TNF-α content in rats with rheumatoid arthritis; in the figure, different letters above the bars indicate significant differences (p < 0.05).

[0020] Figure 8The figure shows the effect of fermented whey from Lactobacillus helveticus CCFM1501 on serum CII-total IgG in rats with rheumatoid arthritis; in the figure, different letters above the bars indicate significant differences (p < 0.05).

[0021] Figure 9 The figure shows the effect of fermented whey from Lactobacillus helveticus CCFM1501 on CII-IgG1 levels in the serum of rats with rheumatoid arthritis; in the figure, different letters above the bars indicate significant differences (p < 0.05).

[0022] Figure 10 The figure shows the effect of fermented whey from Lactobacillus helveticus CCFM1501 on CII-IgG2a in the serum of rats with rheumatoid arthritis; in the figure, different letters above the bars indicate significant differences (p < 0.05).

[0023] Figure 11 The figure shows the effect of fermented whey from Lactobacillus helveticus CCFM1501 on CII-IgG2b in the serum of rats with rheumatoid arthritis; in the figure, different letters above the bars indicate significant differences (p < 0.05). Detailed Implementation

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0025] The present invention first provides a fermented milk whey, which is obtained by fermenting raw milk with Lactobacillus helveticus CCFM1501 or its passaged strain, wherein the preservation number of Lactobacillus helveticus CCFM1501 is GDMCC No: 66449.

[0026] The Lactobacillus of this invention is named Lactobacillus helveticus CCFM1501, with accession number GDMCC No: 66449, and was deposited on May 30, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0027] The Lactobacillus helveticus CCFM1501 of this invention was screened from dairy products in Xining, Qinghai, my country. It was identified as Lactobacillus helveticus by morphological observation and 16S rRNA gene sequencing.

[0028] The Lactobacillus helveticus CCFM1501 of this invention is a Gram-positive, rod-shaped bacterium.

[0029] In a preferred embodiment, the passaged strain has genetic similarity and functional equivalence to Lactobacillus helveticus CCFM1501.

[0030] In some embodiments of the present invention, the raw milk is fresh milk and / or skim milk, preferably skim milk.

[0031] In some embodiments of the present invention, the *Lactobacillus helveticus* CCFM1501 exists as live cells, dry cells, or immobilized cells. Hereinafter, "live cells" refers to *Lactobacillus helveticus* possessing metabolic, reproductive, or replication capabilities; "immobilized cells" refers to *Lactobacillus helveticus* fixed on a carrier, capable of carrying out life activities (growth, development, reproduction, heredity, and metabolism, etc.) within a certain spatial range. The dry cells are obtained by freeze-drying the *Lactobacillus helveticus* CCFM1501.

[0032] In some embodiments, the fermentation is carried out in a culture medium, preferably including but not limited to MRS medium, LB medium, and skim milk medium.

[0033] In some embodiments, the culture medium may be MRS medium, the formulation of which, exemplary, includes: peptone, beef extract, glucose, sodium acetate, yeast extract, diammonium citrate, K₂PO₄, MgSO₄·7H₂O, MnSO₄·H₂O, and Tween 80. The culture temperature is 30-40°C, for example, 30°C, 32°C, 35°C, 36°C, 37°C, 38°C, etc.; the culture time is 18-24 hours, for example, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, etc. In one specific embodiment, *Lactobacillus helveticus* CCFM1501 is inoculated into MRS medium and cultured at 37°C for 24 hours.

[0034] In some embodiments, the fermented milk whey contains active peptides FPPQSVL, NE19 (NIPPLTQTPVVVPPFLQPE), IPP, VPP, AF10 (ARHPHPHLSF), SFSDIPNPIGSENSEK, TVMFPPQ, VLGPVRGPFP, TEIPTIN, QGPIVLNPWDQVKR, IVLNPWDQVKRN, and KEDVPSER.

[0035] Preferably, based on fermented milk whey, the concentration of the active peptide FPPQSVL is ≥0.5 mg / L, the concentration of the active peptide NE19 is ≥17.1 mg / L, the concentration of the active peptide IPP is ≥1.9 mg / L, and the concentration of the active peptide VPP is ≥7.6 mg / L.

[0036] The present invention also provides the application of the above-mentioned fermented milk whey in the preparation of products that help improve rheumatoid arthritis.

[0037] In some embodiments, the product can alleviate pathological damage to the knee joint. For example, it can reduce joint swelling and decrease joint thickness.

[0038] In some embodiments, the product can alleviate the progression of arthritis. In some embodiments, the product can reduce the levels of type II collagen-specific antibodies IgG, IgG1, IgG2a, and IgG2b.

[0039] In some embodiments, the product can reduce inflammatory factor levels and decrease the levels of IL-1β, IL-6, and TNF-α.

[0040] In some embodiments, the product may include one or more of food, medicine, or health products.

[0041] In one embodiment, the product further includes a food, health product, or pharmaceutically acceptable carrier or excipient.

[0042] "Acceptable in food, health products or medicines" means that when the drug is properly administered to animals or humans, it will not produce adverse, allergic or other adverse reactions.

[0043] "A carrier or excipient acceptable for use in food, health products, or pharmaceuticals" should be compatible with the active ingredient, meaning it can be mixed with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of substances that can serve as carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffer solutions, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0044] The dosage form of the product is not specifically limited, such as oral liquid, lyophilized powder, capsule, tablet, granule or gummy.

[0045] The present invention also provides a method for preparing the above-mentioned fermented milk whey, comprising: using an inoculum containing Lactobacillus helveticus CCFM1501 or its passaged strain as a starter culture, fermenting raw milk, and removing protein to obtain the fermented milk whey.

[0046] In some embodiments of the present invention, the raw milk is fresh milk and / or skim milk, preferably skim milk.

[0047] In one embodiment, the skim milk is prepared by dissolving 5-20% (w / w) of skim milk powder in water.

[0048] In one embodiment, the method uses a culture medium obtained by adapting purified Lactobacillus shelveticus CCFM1501 or its passaged strains to MRS medium and / or a skim milk system as a starter culture, wherein the bacterial concentration in the fermentation broth reaches 1×10⁻⁶. 8 ~1×10 9 CFU / mL.

[0049] In one embodiment, the concentration of *Lactobacillus helveticus* CCFM1501 or its passaged strain in the bacterial agent is 1 × 10⁻⁶. 8 ~5×10 8 CFU / mL, which can be 1×10 8 ~2×10 8 CFU / mL, 2×10 8 ~3×10 8 CFU / mL, 3×10 8 ~4×10 8 CFU / mL, 4×10 8 ~5×10 8 CFU / mL; preferably, the inoculum amount of Lactobacillus helveticus CCFM1501 or its passaged strain is 2% to 4% (v / v).

[0050] In one embodiment, and in some embodiments, the fermentation is carried out in a culture medium, preferably including but not limited to MRS medium, LB medium, and skim milk medium.

[0051] In some embodiments, the culture medium may be MRS medium, the formulation of which includes, for example, peptone, beef extract, glucose, sodium acetate, yeast extract, diammonium hydrogen citrate, K2PO4, MgSO4·7H2O, MnSO4·H2O, and Tween 80.

[0052] In some embodiments, the fermentation time is 12-72 hours, specifically 12-24 hours, 24-36 hours, 36-48 hours, or 48-72 hours; the fermentation temperature is 30-42°C, specifically 30-32°C, 32-34°C, 34-37°C, 37-38°C, 38-40°C, or 40-42°C, preferably 37°C. The fermentation is anaerobic; *Lactobacillus helveticus* is an anaerobic bacterium that can rapidly grow and reproduce under static culture conditions.

[0053] In one embodiment, the protein extraction includes the following steps: adding a protein precipitation reagent to the fermentation broth obtained from fermentation to adjust the pH, separating and taking the supernatant, and filtering to obtain fermented milk whey.

[0054] In one embodiment, the protein precipitation reagent is selected from trichloroacetic acid, sulfosalicylic acid, trifluoroacetic acid, or sodium hydroxide, preferably 10% trichloroacetic acid or 1 mol / L sodium hydroxide.

[0055] In one embodiment, the pH is selected from 4-5, and can be 4.1-4.2, 4.2-4.4, 4.4-4.6, 4.6-4.8, 4.8-5.0, preferably 4.6.

[0056] In some embodiments of the present invention, the separation is performed by centrifugation. Preferably, the centrifugation speed is 10000~14000×g, which can be 10000×g, 11000×g, 12000×g, 13000×g, or 14000×g, and is more preferably 12000×g. Preferably, the centrifugation time is 5~15 min, which can be 5~10 min or 10~15 min, and is more preferably 10 min.

[0057] In one embodiment, the filtration is performed using a microporous membrane, the pore size of which is selected from 0.2 to 0.3 μm, and can be 0.22 μm.

[0058] The present invention also provides a product for assisting in the improvement of rheumatoid arthritis, the product comprising the aforementioned Lactobacillus helveticus.

[0059] In some embodiments, the product may include one or more of food, medicine, or health products.

[0060] In one embodiment, the product further includes a carrier or excipients.

[0061] In this document, the term "carrier" includes any solvent, stabilizer, or combination thereof, which are known to those skilled in the art. Its use in products for adjuvant improvement of rheumatoid arthritis is covered, except in cases where any conventional carrier is incompatible with the active ingredient. In this document, the term "excipient" may include any solvent, etc., suitable for a specific target dosage form. Their use is also within the scope of this disclosure, except for any conventional excipients that are incompatible with the *Lactobacillus helveticus* of this disclosure, such as resulting in any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition.

[0062] Preferably, the excipients include any one or a combination of at least two of the following: fillers, pH adjusters, antioxidants, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, antibacterial agents, or buffers.

[0063] In this article, food generally refers to substances that are edible or drinkable by humans, including processed foods, semi-finished products, food ingredients, and items that are traditionally both food and medicinal materials. Food does not include items intended for therapeutic purposes.

[0064] In this document, when the product is a drug, the carrier or excipient is a pharmaceutically acceptable carrier or excipient.

[0065] "Pharmaceutical acceptable" means that when a drug is properly administered to animals or humans, it will not produce adverse, allergic, or other adverse reactions.

[0066] "Pharmaceutical-acceptable carriers or excipients" should be compatible with the active ingredient, meaning they can be mixed with it without significantly reducing the drug's efficacy under normal circumstances.

[0067] The preservation information of the strain applied for is as follows:

[0068] Strain name: Lactobacillus helveticus CCFM1501;

[0069] Accession number: GDMCC No: 66449;

[0070] Classification and naming: Lactobacillus helveticus;

[0071] Deposit date: May 30, 2025;

[0072] Preservation institution: Guangdong Provincial Center for Microbial Culture Collection;

[0073] Abbreviation of depositary institution: GDMCC;

[0074] Address of the storage unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0075] The female Wistar rats used in the following examples were purchased from Zhejiang Vital River Co., Ltd.; the bovine type II collagen solution and Freund's incomplete adjuvant used in the following examples were purchased from Chondrex; the ELISA kits for detecting IL-1β, IL-6, and TNF-α used in the following examples were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.; and the ELISA kits for total type II collagen-specific antibody IgG, type II collagen-specific antibody IgG1, type II collagen-specific antibody IgG2a, and type II collagen-specific antibody IgG2b were purchased from Nanjing Senbeiga Co., Ltd.

[0076] The reagents used to prepare MRS liquid / solid culture media in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.; the skim milk powder involved in the following examples was purchased from Bright Dairy & Food Co., Ltd.; and the standards used to detect bone-related peptides were purchased from Shanghai Ketai Biotechnology Co., Ltd.

[0077] In this invention, unless otherwise specified, "%" or percentage used to describe concentration or proportion refers to weight percentage.

[0078] The culture media involved in the following examples are as follows:

[0079] MRS liquid culture medium: 10 g tryptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 2 g diammonium citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, 1 mL Tween 80, add water to 1000 mL.

[0080] MRS solid medium is obtained by adding 1.5% agar based on the total weight of the liquid medium to the above.

[0081] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0082] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0083] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0084] Example 1 – Screening and identification of Lactobacillus helveticus CCFM1501

[0085] 1. Screening

[0086] The samples were derived from dairy products in Xining, Qinghai Province, and stored in 30% (v / v) glycerol at -80°C. After thawing at low temperature, the samples were aseptically mixed and 0.5 mL was added to a 10 mL centrifuge tube containing 4.5 mL of physiological saline to obtain 10... -1 Diluent, repeat the above dilution steps to obtain 10 -2 10 -310 -4 10 -5 10 -6 Diluents: 100 μL of each gradient dilution was spread onto MRS solid medium and incubated at 37°C for 72 h to obtain dilution plates. Typical colonies from the dilution plates were streaked onto MRS solid medium and incubated at 37°C for 48 h to obtain purified colonies. The purified colonies were inoculated into MRS liquid medium and incubated at 37°C for 48 h to obtain strain DQHXNS10M5, named CCFM1501.

[0087] 2. Identification

[0088] The genome of CCFM1501 was extracted and 16S amplified. The 16S rDNA amplification conditions were: 95℃ for 5 min; 35 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min); 72℃ for 10 min. Amplification primers: 27F: (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R: (5'-TACGGCTACCTTGTTACGACTT-3'). The purification and sequence alignment of the amplification products were performed according to the method described in the literature (Turroni F et al. Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract[J]. Appl EnvironMicrob. 2009; 75(6):1534–45). The 16S rDNA of CCFM1501 was amplified and sequenced (by Suzhou Genewiz Biotechnology Co., Ltd.). The 16S rDNA sequence of CCFM1501 obtained from the sequencing analysis was compared with NCBI. The results showed that this strain is Lactobacillus helveticus and named Lactobacillus helveticus CCFM1501.

[0089] Example 2: Preparation of fermented whey from Lactobacillus helveticus CCFM1501

[0090] The *Lactobacillus helveticus* CCFM1501 obtained in Example 1 was inoculated into MRS liquid medium and cultured at 37°C for 18–24 h. The culture was then subcultured 2–3 times until the bacterial concentration reached 10⁻⁶. 8 ~10 9 CFU / mL.

[0091] 11% (w / w) skim milk was sterilized at 100–120°C for 30–30 seconds. Then, the bacterial culture activated in MRS was taken out, centrifuged at 6000 × g for 5 minutes, and resuspended in 11% (w / w) skim milk to the same volume. The culture was then inoculated into 11% (w / w) skim milk at an inoculation rate of 2% (v / v) and fermented at 37°C for 12–72 hours to obtain Lactobacillus helveticus CCFM1501 fermented milk.

[0092] After adjusting the pH of the fermented milk of Lactobacillus helveticus CCFM1501 to 4.6 by adding 10% trichloroacetic acid or 1M sodium hydroxide solution, centrifuge at 12000×g and 4℃ for 10min, take the supernatant, and then filter it through a 0.22 μm organic filter membrane to obtain the whey of fermented milk of Lactobacillus helveticus CCFM1501.

[0093] Example 3: Peptidomics analysis of whey fermented by Lactobacillus helveticus CCFM1501

[0094] The whey from the Lactobacillus helveticus CCFM1501 fermented milk in Example 2 was centrifuged at 4000×g, 4℃ for 15 min using a 10kDa ultrafiltration centrifuge tube. The filtrate was collected and lyophilized. The lyophilized sample was desalted using a Pierce™ desalting column and then subjected to peptidomics analysis.

[0095] The Orbitrap Exploris 240 high-resolution mass spectrometry system (Thermo Fisher Scientific, USA) was used for the analysis, equipped with a UHPLC ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA) and Xcalibur data acquisition software (AB Thermo Fisher Scientific, USA). The acquired data were searched using Compound Discoverer software and the Mzcloud mass spectrometry database. The chromatographic conditions were as follows: Thermo Hypersil Gold column (1.9 μm, 2.1 × 100 mm column); column temperature 50℃; injection volume 2 μL; mobile phase A: acetonitrile; mobile phase B: 0.1% formic acid / water; flow rate 0.3 mL / min; gradient elution program (45 min): 95% B phase as the initial concentration; 0 min–2 min, B phase maintained at 95%; 2–37 min, B phase changed from 95% to 1%; 37–42 min, B phase maintained at 1%; 42.1–45 min, B phase maintained at 95%.

[0096] Using the representative Lactobacillus helveticus fermentation strain CCFM1263 as a positive control, the fermented whey of this strain can produce osteogenic peptides FPPQSVL, AF10 (ARHPHPHLSF), AY17 (AAGGPGAPADPGRPTGY), and NE19 (NIPPLTQTPVVVPPFLQPE), as well as calcium absorption-promoting peptide SFSDIPNPIGSENSEK, which have a promoting effect on bone development and have been reported in the literature many times.

[0097] like Figure 1 As shown, 134 peptides were detected in the whey of CCFM1501 fermented milk and 123 peptides were detected in the whey of CCFM1263 fermented milk, which is 11 more peptides than CCFM1263; and there are 17 differential peptides between CCFM1501 and CCFM1263. The specific analysis is shown in Table 1.

[0098] Table 1. Composition of bioactive peptides in Lactobacillus helveticus fermented whey

[0099]

[0100] Note: - indicates that it does not contain, + indicates that it contains.

[0101] The concentration of bone-related peptides in fermented milk was determined using a triple quadrupole linear ion trap liquid chromatography-mass spectrometry (LC-MS) system and a suitable targeted MRM monitoring method was established. Standard curves of these bone-related peptides were plotted, and the concentration of bone-related peptides in the whey of fermented milk from two different bacterial strains was preliminarily and roughly determined.

[0102] like Figure 2 As shown, the concentrations of FPPQSVL, NE19 (NIPPLTQTPVVVPPFLQPE), IPP, and VPP in CCFM1263 fermented milk whey were 0.1 mg / L, 5.6 mg / L, 0.4 mg / L, and 1.9 mg / L, respectively, while the concentrations of the corresponding peptides in CCFM1501 fermented milk whey were 0.5 mg / L, 17.1 mg / L, 1.9 mg / L, and 7.6 mg / L, respectively, all higher than those in CCFM1263 fermented milk whey.

[0103] Example 4: Effect of Lactobacillus helveticus CCFM1501 fermented milk whey on hind paw thickness in rats with rheumatoid arthritis

[0104] 1. Laboratory animals

[0105] Six-week-old female SPF-grade Wistar rats were housed in IVC cages, with four rats per cage. The room temperature was 22-24℃ and the humidity was 40-60%. The rats were housed in a 12-hour / 12-hour day-night cycle with free access to food and water.

[0106] 2. Experimental Methods

[0107] (1) Establishment of a rat model of rheumatoid arthritis

[0108] The experiment lasted five weeks, starting one week before modeling and continuing until the end of the experiment. The second and third weeks were the modeling period. On the first day of modeling, equal volumes of bovine type II collagen solution (Chondrex, 20022) and Freund's incomplete adjuvant (Chondrex, 7002) were mixed and emulsified to form a complete emulsion. Rats were anesthetized with isoflurane, fixed, and their tail base was disinfected with 75% alcohol. The rats were then given their first immunization (on day 8). For the first immunization, 0.2 mL of the complete emulsion was accurately aspirated and injected subcutaneously at a distance of 1.5 cm from the tail base. One week later, a booster immunization was performed using the same method, i.e., 0.2 mL of the complete emulsion was accurately aspirated and injected subcutaneously at a distance of 2.0 cm from the tail base. Rats in the normal group were injected with the same volume of sterile saline using the same method.

[0109] (2) Experimental grouping and drug administration

[0110] Rats were randomly divided into three groups of eight each: a normal group, a model group, and a CCFM1501 fermented whey group (rats were administered CCFM1501 fermented whey via gavage). Rats in the normal and model groups were administered 1 mL of physiological saline via gavage daily, while rats in the CCFM1501 fermented whey group were administered 1 mL of physiological saline solution containing 1.25 mg / (kg BW) CCFM1501 fermented whey (the CCFM1501 fermented whey was lyophilized and then reconstituted in physiological saline for gavage) via gavage daily. The gavage period lasted from the first week to the fifth week, a total of 35 days.

[0111] After modeling, the joint thickness of each group of rats was measured using a micrometer, and hematoxylin-eosin pathological sections of the rat knee joints were prepared. The measurement results are shown below. Figure 3-4 .

[0112] Figure 3 The results showed that joint swelling began approximately 4 days after the first immunization, i.e., on day 12 of the experiment. By day 35, the joint thickness of rats in the model group was significantly higher than that in the normal group (p < 0.05), while the joint thickness of rats in the CCFM1501 fermented milk whey group was significantly lower than that in the model group (p < 0.05). Specifically, the joint thicknesses of rats in the control group, model group, and CCFM1501 fermented milk whey group were 5.10, 7.48, and 6.79 mm, respectively.

[0113] Depend on Figure 4It can be seen that the knee joint of the model group rats was severely damaged, and a large number of chondrocytes and osteocytes were necrotic and dissolved in the joint tissue, replaced by proliferating connective tissue. Pannus was seen on the surface, the synovium was significantly thickened, accompanied by lymphocyte infiltration and epithelial hyperplasia; the CCFM1501 fermented milk whey group could significantly alleviate the pathological damage of the knee joint.

[0114] It is evident that Lactobacillus helveticus CCFM1501 fermented whey can be used as an adjunct treatment to alleviate symptoms of rheumatoid arthritis.

[0115] Example 6: Effect of Lactobacillus helveticus CCFM1501 on the levels of pro-inflammatory factors in the serum of rheumatoid arthritis rats

[0116] The animal experimental methods are described in Example 5.

[0117] In rheumatoid arthritis (RA), activated immune cells (such as T cells, B cells, and macrophages) and activated synovial fibroblasts secrete large amounts of various cytokines. Through a complex autocrine and paracrine network, these cytokines regulate the abnormal activation, infiltration, differentiation, and survival of immune cells, thereby driving the disease progression. Among these, pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β play a dominant role in synovial tissue. They not only exacerbate local inflammation but also directly stimulate synovial hyperplasia, the formation of invasive pannus, and activate osteoclasts, leading to joint bone erosion. When these cytokines are released from the diseased joints into the circulatory system, their serum concentration becomes a key indicator for measuring the level of systemic inflammation and immune response in the body.

[0118] After the experiment, blood was collected from the rats, and they were euthanized. Rat serum was collected, and the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the serum of each group of rats were measured using an ELISA kit. The results are shown below. Figure 5-7 .

[0119] like Figure 5 As shown, the concentration of IL-1β in the serum of rats in the model group was 104.95 pg / mL, which was significantly higher than that in the normal group (50.96 pg / mL) (p < 0.05). Compared with the model group, the serum IL-1β level in rats in the CCFM1501 fermented milk whey group was significantly lower (p < 0.05), at 42.34 pg / mL, a decrease of 59.66%.

[0120] like Figure 6As shown, the concentration of IL-6 in the serum of rats in the model group was 133.91 pg / mL, which was significantly higher than that in the normal group (53.42 pg / mL) (p < 0.05). Compared with the model group, the serum IL-6 level in rats in the CCFM1501 fermented milk whey group was significantly lower (p < 0.05), at 73.22 pg / mL, a decrease of 45.32%.

[0121] like Figure 7 As shown, the concentration of TNF-α in the serum of rats in the model group was 364.41 pg / mL, which was significantly higher than that in the normal group (146.5 pg / mL) (p < 0.05). Compared with the model group, the serum TNF-α level in rats in the CCFM1501 fermented milk whey group was significantly lower (p < 0.05), at 209.79 pg / mL, a decrease of 42.43%.

[0122] This indicates that fermented whey from Lactobacillus helveticus CCFM1501 can reduce the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the serum of rats with rheumatoid arthritis.

[0123] Example 7: Effect of Lactobacillus helveticus CCFM1501 on the levels of type II collagen-specific antibody IgG and its subtypes in the serum of rheumatoid arthritis rats.

[0124] The animal experimental methods are described in Example 5.

[0125] Anti-CII IgG antibodies are specific antibodies against type II collagen. In the CIA model, the production of anti-CII IgG is crucial for disease development, inducing or exacerbating arthritis, and can be used to assess the severity of rheumatoid arthritis. Reducing the production of autoantibodies can effectively block the progression of arthritis.

[0126] After the experiment, blood was collected from the rats, and they were euthanized. Rat serum was collected, and the levels of total type II collagen-specific antibody IgG, type II collagen-specific antibody IgG1, type II collagen-specific antibody IgG2a, and type II collagen-specific antibody IgG2b in each group of rat serum were measured using an ELISA kit. The results are shown below. Figure 8-11 .

[0127] like Figure 8 As shown, the concentration of total type II collagen-specific antibody IgG in the model group rats was 9.27 μg / L, which was significantly higher than that in the normal group (5.21 μg / L) (p < 0.05); compared with the CCFM1501 fermented milk whey group rats in the model group rats, the level of total type II collagen-specific antibody IgG in the serum was significantly lower (p < 0.05), at 5.24 μg / L, a decrease of 43.47%.

[0128] like Figure 9 As shown, the concentration of type II collagen-specific antibody IgG1 in the model group rats was 6.87 μg / L, which was significantly higher than that in the normal group (3.69 μg / L) (p < 0.05). Compared with the model group rats, the level of type II collagen-specific antibody IgG1 in the serum of rats in the CCFM1501 fermented milk whey group was significantly lower (p < 0.05), at 4.40 μg / L, a decrease of 35.95%.

[0129] like Figure 10 As shown, the concentration of type II collagen-specific antibody IgG2a in the model group rats was 4.81 μg / L, which was significantly higher than that in the normal group (1.71 μg / L) (p < 0.05). Compared with the model group rats, the level of type II collagen-specific antibody IgG2a in the serum of rats in the CCFM1501 fermented milk whey group was significantly lower (p < 0.05), at 2.41 μg / L, a decrease of 49.90%.

[0130] like Figure 11 As shown, the concentration of type II collagen-specific antibody IgG2b in the model group rats was 1.80 μg / L, which was significantly higher than that in the normal group (0.77 μg / L) (p < 0.05). Compared with the model group rats, the level of type II collagen-specific antibody IgG2b in the serum of rats in the CCFM1501 fermented milk whey group was significantly lower (p < 0.05), at 1.14 μg / L, a decrease of 36.67%.

[0131] It is evident that fermented whey from Lactobacillus helveticus CCFM1501 can reduce the levels of total type II collagen-specific antibody IgG, type II collagen-specific antibody IgG1, type II collagen-specific antibody IgG2a, and type II collagen-specific antibody IgG2b in the serum of rats with rheumatoid arthritis, thereby alleviating the symptoms of arthritis in rats.

[0132] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0133] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0134] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A fermented milk whey, wherein the fermented milk whey is obtained by fermenting raw milk with Lactobacillus helveticus CCFM1501 or its passaged strains, wherein the preservation number of Lactobacillus helveticus CCFM1501 is GDMCC No: 66449.

2. The fermented whey as described in claim 1, characterized in that, The fermented whey contains the active peptides FPPQSVL, NE19, IPP, VPP, AF10, SFSDIPNPIGSENSEK, TVMFPPQ, VLGPVRGPFP, TEIPTIN, QGPIVLNPWDQVKR, IVLNPWDQVKRN, and KEDVPSER. And / or, the passaged strain has genetic similarity and functional equivalence to Lactobacillus helveticus CCFM1501.

3. The fermented whey as described in claim 2, characterized in that, Based on fermented milk whey, the concentration of the active peptide FPPQSVL is ≥0.5 mg / L, the concentration of the active peptide NE19 is ≥17.1 mg / L, the concentration of the active peptide IPP is ≥1.9 mg / L, and the concentration of the active peptide VPP is ≥7.6 mg / L.

4. The use of fermented whey as described in any one of claims 1 to 3 in the preparation of products that help improve rheumatoid arthritis.

5. The application as described in claim 4, characterized in that, The product has one or more of the following effects: 1) Relieves pathological damage to the knee joint; 2) Relieves the progression of arthritis; 3) Reduce the level of inflammatory factors.

6. The application as described in claim 4, characterized in that, The products include any one or more of the following: food, medicine, or health products.

7. The method for preparing fermented milk whey as described in any one of claims 1-3, comprising: Using a starter culture containing Lactobacillus helveticus CCFM1501 or its passaged strains as a starter culture, the raw milk is fermented, and the protein is removed to obtain the fermented milk whey.

8. The preparation method according to claim 7, characterized in that, The bacterial agent contains Lactobacillus helveticus CCFM1501 or its passaged strains at a concentration of 1×10⁻⁶. 8 ~5×10 8 CFU / mL, inoculation amount is 2% to 4% (v / v); And / or, the fermentation time is 12~72h; And / or, the fermentation temperature is 30~42℃; And / or, the raw milk is selected from fresh milk and / or skim milk; And / or, the protein removal includes the following steps: adding a protein precipitation reagent to the fermentation broth obtained from fermentation to adjust the pH, separating and taking the supernatant, and filtering to obtain fermented milk whey.

9. The preparation method according to claim 8, characterized in that, The separation method is centrifugation, preferably, the centrifugation speed is 10000~14000×g, and the centrifugation time is 5~15min; And / or, the protein precipitation reagent is selected from one or more of trichloroacetic acid, sulfosalicylic acid, trifluoroacetic acid, or sodium hydroxide; And / or, the filtration is performed using a microporous membrane, the pore size of which is selected from 0.2 to 0.3 μm.

10. A product for assisting in the improvement of rheumatoid arthritis, the product comprising the fermented whey as described in any one of claims 1-3.

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