Preparation method and application of fermented milk rich in osteogenic growth peptide FPPQSVL

Abstract

The present invention discloses a preparation method and application of fermented milk rich in osteogenic growth peptide FPPQSVL, and belongs to the technical field of dairy product processing. The present invention prepares Lactobacillus helveticus fermented milk rich in osteogenic growth peptide FPPQSVL by inoculating Lactobacillus helveticus CCFM1263 into skimmed milk for fermentation. The fermented milk has the functions of significantly enhancing bone density, significantly increasing the number and thickness of trabeculae, significantly increasing bone volume fraction, significantly enhancing the bone biomechanics of rats, and significantly reducing the content of bone turnover biomarkers TRAP, CTX-1, and DPD in serum, while significantly increasing the content of BALP. The Lactobacillus helveticus fermented milk rich in osteogenic growth peptide FPPQSVL provided by the present invention can effectively prevent or treat osteoporosis, promote bone health, and is of great significance to the development of health products and medicines that promote bone health.

Description

Technical Field

[0001] The invention relates to a preparation method and application of fermented milk rich in osteogenic growth peptide FPPQSVL, belonging to the technical field of dairy product processing. Background Art

[0002] Osteoporosis is a systemic metabolic bone disease characterized by a decrease in bone mass and destruction of the microstructure of bone tissue. In healthy individuals, bone metabolism is maintained in equilibrium through a balance between bone resorption and bone formation. However, endocrine disorders, calcium malabsorption, nutrition, hormone regulation, aging, and many pathological processes disrupt this balance, ultimately leading to osteoporosis. Currently, the clinical treatment of osteoporosis is still mainly based on chemical drugs, but they often carry certain risks, and long-term use may cause diseases such as breast cancer and cardiovascular disease. Therefore, the search for safer and healthier functional foods that can promote bone formation and reverse bone structure damage is receiving increasing attention.

[0003] As living conditions improve, consumer demand for food is constantly evolving, with nutrition and health being a key concern for many. Fermented milk, made from bovine milk fermented by lactic acid bacteria, is an effective way to supplement nutrients such as protein and minerals. However, the current homogeneity of commercially available fermented milk products is a serious issue, gradually becoming a challenge for dairy companies. The rise of functional dairy products has changed this dynamic. As they align with consumers' pursuit of a healthier lifestyle, they have garnered significant attention and become a hot topic for competition among dairy companies. Peptides are protein degradation products, and a variety of bioactive peptides have been reported to exhibit benefits such as immune regulation, cardiovascular disease prevention, and bone health promotion. These peptides can be obtained through microbial fermentation, digestive enzymatic hydrolysis, genetic engineering synthesis, and chemical synthesis. However, digestive enzymatic hydrolysis is associated with numerous side effects, and many amino acid derivatives are unstable in water. Genetic engineering technology is still in its infancy and presents technical difficulties. Chemical synthesis methods are expensive, requiring high equipment and reagent costs, and their byproducts are harmful to the human body. Producing bioactive peptides through microbial fermentation overcomes these challenges, offering advantages such as low cost, safe ingredients, simple acquisition, and wide applicability, and therefore attracting increasing attention. Most bioactive peptides are composed of 2-20 amino acid residues and are easily digested and absorbed by the human body. Therefore, protein-rich dairy products are a good source of bioactive peptides. However, research on milk-derived osteogenic growth peptides is relatively limited, particularly those obtained through microbial fermentation. Furthermore, there are currently no naturally fermented dairy products with bioactive peptides that can prevent osteoporosis. Therefore, the development of a functional fermented milk for osteoporosis prevention is of great significance and holds broad market potential. Summary of the Invention

[0004] The present invention provides a method for preparing fermented milk rich in functional peptides and its application. The Lactobacillus helveticus was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, with a deposit number of GDMCC No. 62417.

[0005] Specifically, the present invention provides a method for preparing fermented milk rich in functional peptides. The method uses Lactobacillus helveticus CCFM1263 as a starter and ferments skim milk at 30-42° C. for 12-72 hours.

[0006] In one embodiment, the skim milk is prepared by dissolving 5-20% (w / w) skim milk powder in water.

[0007] In one embodiment, the method is to culture the purified Lactobacillus helveticus CCFM1263 in MRS medium and skim milk system adaptively until the bacterial concentration reaches 10 8 ~10 9 CFU / mL.

[0008] In one embodiment, the method comprises: 6 ~10 8 The concentration of CFU / mL was inoculated into skim milk for fermentation.

[0009] In one embodiment, the skim milk is sterilized at 100-120° C. for 30 seconds to 30 minutes.

[0010] In one embodiment, the content of osteogenic growth peptide FPPQSVL in the fermented milk is 1 to 500 mg / L.

[0011] The present invention also provides the use of the fermented milk in preparing a health product for improving bone density.

[0012] In one embodiment, the content of osteogenic growth peptide FPPQSVL in the health product is ≥1 mg / L.

[0013] In one embodiment, the health care product includes granules, tablets, capsules, pills, liquid preparations or powders, the liquid preparations include milk drinks or oral liquids, and the tablets include cheese, milk tablets or candies.

[0014] In one embodiment, the health care product further comprises commonly used excipients.

[0015] The present invention also provides the use of the fermented milk in preparing medicines for preventing or treating osteoporosis.

[0016] In one embodiment, the fermented milk is dried and used as a medicinal powder.

[0017] In one embodiment, the preventive or therapeutic effects on osteoporosis induced by ovariectomized rats include but are not limited to the following aspects:

[0018] (1) Significantly increase bone density;

[0019] (2) Significantly increase the number and thickness of trabeculae;

[0020] (3) Significantly increase bone volume fraction;

[0021] (4) Significantly improve bone mechanical strength;

[0022] (5) Significantly reduced the level of tartrate phosphatase (TRAP) in rat serum;

[0023] (6) Significantly reduced the level of type I procollagen C-terminal peptide (CTX1) in rat serum;

[0024] (7) Significantly reduced the level of urinary deoxypyridinoline (DPD) in rat serum;

[0025] (8) Significantly increase the level of bone-specific alkaline phosphatase in college serum.

[0026] Beneficial effects:

[0027] 1. The Lactobacillus helveticus CCFM1263 used in the present invention is a food-safe and healthy strain that can be used to prepare various foods, medicines, and microbial preparations.

[0028] 2. The fermented milk of Lactobacillus helveticus CCFM1263 of the present invention is rich in osteogenic growth peptide FPPQSVL. Compared with the model group, the fermented milk can significantly enhance bone density by 15%, significantly increase the number and thickness of trabeculae by 30%, significantly increase the bone volume fraction by 30%, significantly enhance the bone biomechanical strength of rats by 28%, and significantly reduce the levels of TRAP, CTX-1, and DPD in serum, while significantly increasing the BALP content by 50%.

[0029] Biomaterial Deposit

[0030] The Lactobacillus helveticus CCFM1263 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, with the deposit number GDMCC No. 62417, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The content of osteogenic growth peptide FPPQSVL in different samples;

[0032] Figure 2 Reconstruction of trabecular bone in ovariectomized rats;

[0033] Figure 3 Effects on bone density in ovariectomized rats;

[0034] Figure 4 Effects on bone volume fraction in ovariectomized rats;

[0035] Figure 5 Trabecular bone thickness in ovariectomized rats;

[0036] Figure 6 Effects on bone biomechanics in ovariectomized rats;

[0037] Figure 7 Effects of transaminase on serum biochemical markers in ovariectomized rats; (a): TRAP; (b): CTX-1; (c): DPD; (d): BALP. DETAILED DESCRIPTION

[0038] The following is a detailed description of an embodiment of the present invention. It should be noted that this embodiment is descriptive rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0039] Unless otherwise specified, the raw materials used in the present invention are conventional commercial products; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0040] 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 used in the following examples was purchased from Bright Dairy Co., Ltd.; and the SPF-grade 7-week-old female SD rats used in the following examples were purchased from Weitong Lihua Laboratory Animal Co., Ltd. The standard sample used to detect the content of osteogenic growth peptide FPPQSVL was obtained by chemical synthesis and has a purity exceeding 98%.

[0041] Example 1 Preparation method of Lactobacillus helveticus CCFM1263 fermented milk

[0042] Screening and identification of S1 strain

[0043] 1. Prepare appropriate sample dilution gradient and culture

[0044] Take out Qula from Xining, Qinghai, which is stored in a -80℃ refrigerator and slowly thaw it on ice. After shaking and mixing, take 0.5mL of sample and add it to 4.5mL of sterile saline to complete a 10-fold dilution. After shaking and mixing, take out 0.5mL of dilution from the dilution and add it to 4.5mL of sterile saline to complete a second 10-fold dilution. Repeat this process until the fifth 10-fold dilution is completed (diluted to 10).-5 ), 100 μL was taken from each gradient dilution, evenly spread on MRS solid culture medium plates, and inverted to culture at 37°C for 48 h.

[0045] 2. Line separation and purification

[0046] Remove the plate with colonies, select the gradient plate with obvious single colonies, pick colonies with different colony morphologies, and perform secondary streaking until all single colonies are purified.

[0047] 3. Strain preservation

[0048] After purification, a single colony of each strain was picked into 5 mL of MRS liquid medium and cultured at 37°C for 24 h. After mixing, 1 mL of the bacterial solution was transferred to a bacterial preservation tube and centrifuged at 6000 rpm for 3 min. The supernatant was discarded, and 1 mL of 30% (v / v) sterile glycerol solution was added, resuspended, and stored at -80°C.

[0049] 4. 16S rDNA sequence amplification

[0050] After mixing, aspirate 1 mL of bacterial suspension and centrifuge at 6000 rpm for 3 minutes. The supernatant was decanted, washed twice with sterile water, and centrifuged to discard the supernatant. This bacterial slurry was used as a template for PCR amplification. The amplification system consisted of 20 μL of template, 0.5 μL of each bidirectional primer, 10 μL of Taq MasterMix, and 8 μL of ddH2O. Primers used were 27F: AGA GTT TGA TCC TGG CCTCA (SEQ ID No. 1) and 1492R: GGT TAC CTT GTT ACG ACT T (SEQ ID No. 2). Amplification conditions were as follows: initial denaturation at 94°C for 10 minutes; denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds; extension at 72°C for 2 minutes; 30 cycles; a final extension at 72°C for 10 minutes; and a hold at 12°C for 10 minutes.

[0051] 5. Sequencing and identification

[0052] The PCR sample was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing, and the amplified nucleotide sequence sequencing results of the strain number DQHXN-Q32M42 ​​were obtained. The sequencing results were compared with the National Center for Biotechnology Information (NCBI) sequence database (http: / / www.ncbi.nlm.nih.gov / blast) by BLAST, and it was determined that the strain provided by the present invention was Lactobacillus helveticus.

[0053] Preparation of S2 inoculum

[0054] Lactobacillus helveticus CCFM1263 was inoculated into MRS medium and cultured at 37°C for 18-24 hours. The cells were subcultured 2-3 times and then washed twice with sterile saline and resuspended until the bacterial concentration reached 10 8 ~10 9 CFU / mL.

[0055] S3 inoculation culture

[0056] 11% (w / w) skim milk was sterilized at 100-120℃ for 30s-30min, and then the bacterial suspension was inoculated into the sterilized skim milk to adjust the bacterial concentration to 10 6 ~10 8 CFU / mL, ferment at 30-42°C for 12-72h to obtain Lactobacillus helveticus fermented milk.

[0057] Example 2 Determination of osteogenic growth peptide FPPQSVL content

[0058] 1. Preparation of Peptide Samples

[0059] The Lactobacillus helveticus fermented milk sample in Example 1 was added with 50% (v / v) lactic acid or 1M sodium hydroxide solution to adjust the sample pH to 4.6, and centrifuged at 12000g and 4°C for 10 min. The supernatant was taken and filtered through a 0.45 μm organic filter membrane. Subsequently, the supernatant was centrifuged at 4000g and 4°C for 15 min using a 10 kDa ultrafiltration centrifuge tube. The filtrate was the fermented milk polypeptide sample.

[0060] 2. Determination of osteogenic growth peptide FPPQSVL content

[0061] The content of osteogenic growth peptide (FPPQSVL) in fermented milk was determined using a triple quadrupole linear ion trap liquid chromatography-mass spectrometry (LC-MS / MS) with an established targeted MRM monitoring method. A standard stock solution of FPPQSVL was prepared using purified aqueous solution. A series of standard solutions were prepared to generate a calibration curve for FPPQSVL, and the content of the samples was calculated using the external standard method.

[0062] Liquid chromatography parameters: chromatographic column BEH T3 column (1.7 μm, 2.1×100 mm), mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile solution, column temperature 40° C., injection volume 2 μL, flow rate 0.3 mL / min.

[0063] The results of the determination of the content of fermented milk osteogenic growth peptide FPPQSVL are as follows Figure 1 As shown. Figure 1The FPPQSVL content in milk whey fermented with Lactobacillus helveticus CCFM1263 was 19.87 mg / L, while the FPPQSVL content in milk whey fermented with Lactobacillus helveticus ATCC15009 and unfermented bovine milk whey was 5.13 mg / L and 1.63 mg / L, respectively. Therefore, milk fermented with Lactobacillus helveticus CCFM1263 has a high content of the osteogenic growth peptide FPPQSVL.

[0064] Example 3 Effect of Lactobacillus helveticus CCFM1263 fermented milk on osteoporosis induced by ovariectomized rats

[0065] 1. Animal Experimental Protocol

[0066] Thirty 7-week-old SPF female SD rats were housed in a constant temperature and humidity enclosure with equal day and night intervals. After one week of adaptive feeding, they were randomly divided into five groups of six rats each: a sham operation group, a model group, an estradiol-positive group given by oral gavage, a group given by oral gavage with Lactobacillus helveticus ATCC15009 fermented milk, and a group given by oral gavage with Lactobacillus helveticus CCCFM1263 fermented milk. ATCC15009 is the model strain of Lactobacillus helveticus. The experiment lasted 15 weeks: the first week was an adaptation period for the rats, during which they were housed at a temperature of 22±2°C, a humidity of 40-70%, and a 12-hour day and night cycle. They were given free access to a rat breeding formula purchased from Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd. Experiments were conducted after one week of adaptation to a normal diet. After the first week, rats in the model control group, positive control group, Lactobacillus helveticus ATCC15009 fermented milk group, and Lactobacillus helveticus CCCFM1263 fermented milk group were anesthetized with isoflurane inhalation and underwent bilateral dorsal ovarian removal surgery, with the incisions sutured in two layers (OVX). The sham-operated group underwent a similar procedure, with surrounding adipose tissue removed without ovarian removal, and the incisions sutured in two layers. Rats in each group were injected with penicillin at a dose of 10,000 U / 100 g for three consecutive days postoperatively to combat infection. After two weeks of recovery, rats were gavaged daily starting in the fourth week until the end of the experiment, with daily gavages performed according to Table 1.

[0067] Table 1 Animal experimental groups and treatment methods

[0068]

[0069] 2. Analysis of rat bone morphomechanical indicators

[0070] After gavage, rats were anesthetized with 1% sodium pentobarbital (40 mg / kg) via intraperitoneal injection. The femurs and tibias were removed and wrapped in saline-soaked gauze. Bones requiring long-term storage were stored at -80°C, while the remaining bones were stored at 4°C.

[0071] Rat bone morphomechanics were measured using Micro-CT. Specific imaging conditions were: scanning voltage 90 kV, current 88 μA, field of view 86 μm, high-resolution scanning mode, scanning time 4 minutes, and a 0.1 mm Cu filter. Image and data analysis were performed using the instrument's own software. Volumetric parameters were identical for all analyzed bone sites: the distal femur.

[0072] Depend on Figure 2 It can be seen that the number of trabeculae in ovariectomized rats was significantly reduced. Compared with the model group, estradiol and CCFM1263 fermented milk could significantly increase the number of trabeculae, and the effect of CCFM1263 fermented milk was significantly higher than that of ATCC15009 fermented milk, tending to the blank group and Yangshen group.

[0073] Depend on Figure 3 Compared with the sham operation group, the bone density of rats after ovariectomy was significantly reduced. After intervention with ATCC15009 fermented milk and CCFM1263 fermented milk, the bone density was restored to 94% and 95% of normal rats, respectively. Compared with the model group, the bone density increased by 14% and 15%, respectively, after intervention with ATCC15009 fermented milk and CCFM1263 fermented milk.

[0074] Depend on Figure 4 Compared with the sham operation group, the bone volume fraction of ovariectomized rats was significantly reduced. However, after CCFM1263 fermented milk treatment, the bone volume fraction returned to 73% of the normal rats. Compared with the model group, ATCC15009 fermented milk treatment did not significantly change the bone volume fraction, but CCFM1263 fermented milk treatment significantly increased the bone volume fraction by 30%.

[0075] Depend on Figure 5 Compared to the sham-operated group, trabecular bone thickness was significantly reduced in ovariectomized rats. However, CCFM1263 fermented milk treatment restored trabecular bone thickness to 88% of normal rats. Compared to the model group, ATCC15009 fermented milk treatment did not significantly change trabecular bone thickness, but CCFM1263 fermented milk treatment significantly increased trabecular bone thickness by 30%.

[0076] 3. Analysis of rat bone mechanical indicators

[0077] After gavage, rats were anesthetized with 1% sodium pentobarbital (40 mg / kg) via intraperitoneal injection. The femurs and tibias were removed and wrapped in saline-soaked gauze. Bones requiring long-term storage were stored at -80°C, while the remaining bones were stored at 4°C.

[0078] The maximum load and deflection of the bone were analyzed using a texture analyzer with a shear probe. The maximum breaking force (i.e., the maximum load on the bone) was measured using a three-point bending test at a test speed of 1 mm / s. The bone was placed horizontally, anterior surface facing up, and the probe was compressed at a test speed of 1 mm / s until fracture occurred. The load was applied vertically to the center of the bone, with the brackets spaced 16 mm apart.

[0079] Depend on Figure 6 Compared with the sham operation group, the bone mechanics of ovariectomized rats was significantly reduced. However, intervention with ATCC15009 and CCFM1263 fermented milk restored the bone mechanics to 88% and 94% of normal rats, respectively. Compared with the model group, the maximum load increased by 19% and 28%, respectively, after intervention with ATCC15009 and CCFM1263 fermented milk.

[0080] 4. Analysis of Rat Bone Turnover Serum Biomarkers

[0081] After gavage, rats were anesthetized with 1% sodium pentobarbital (40 mg / kg) via intraperitoneal injection. Blood was collected from the abdominal aorta and centrifuged at 5000 g for 15 minutes at 4°C to obtain serum. Serum was stored at -80°C to avoid repeated freezing.

[0082] The levels of tartrate phosphatase (TRAP), type I procollagen C-terminal peptide (CTX-1), urinary deoxypyridinoline (DPD) and bone-specific alkaline phosphatase (BALP) in rat serum were determined by ELISA kits. Figure 7 shown.

[0083] Depend on Figure 7 It can be seen that the TRAP, CTX-1 and DPD levels of rats were significantly increased, while the BALP content was significantly decreased after ovariectomy; compared with the model group, there was no significant change after intervention with ATCC15009 fermented milk, while the BALP content was significantly reduced by 33%, 27% and 27% respectively after intervention with CCFM1263 fermented milk, and the BALP content was significantly increased by 50%, restoring all indicators to the level equivalent to that of the control group.

[0084] In summary, the bone morphology analysis, bone mechanics analysis and bone turnover serum biomarker analysis experiments in rats showed that CCFM1263 fermented milk can effectively prevent the occurrence and development of osteoporosis.

[0085] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing fermented milk rich in osteogenic growth peptide FPPQSVL, characterized in that: Lactobacillus helveticus ( Lactobacillus helveticus ) CCFM1263 was inoculated into cow's milk and fermented at 30-42°C for 12-72 h. The Lactobacillus helveticus CCFM1263 was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, with the deposit number GDMCC No. 62417.

2. The method according to claim 1, wherein The bacterial concentration of Lactobacillus helveticus CCFM1263 in the fermented milk is 10 8 ~10 9 CFU / mL.

3. The method according to claim 1, characterized in that The milk is skim milk.

4. The fermented milk prepared by the method according to any one of claims 1 to 3, characterized in that The fermented milk contains osteogenic growth peptide FPPQSVL.

5. Use of the fermented milk according to claim 4 in preparing a health product for improving bone density.

6. The use according to claim 5, characterized in that The content of osteogenic growth peptide FPPQSVL in the health product is ≥1 mg / L.

7. The use according to claim 5, characterized in that The health care products include granules, tablets, capsules, pills, liquid preparations or powders, the liquid preparations include milk drinks or oral liquids, and the tablets include cheese, milk tablets or candies.

8. The use according to claim 7, characterized in that The health care product also includes common auxiliary materials.

9. Use of the fermented milk according to claim 4 in preparing a medicament for preventing or treating osteoporosis, characterized in that: The prevention or treatment of osteoporosis includes: (1) Increase bone density; (2) Increase the number and thickness of trabeculae; (3) Increase bone volume fraction; (4) Improve bone mechanical strength; (5) Reduce the level of tartrate-resistant acid phosphatase in rat serum; (6) Reduce the content of urinary deoxypyridinoline in rat serum; (7) Increase the content of bone-specific alkaline phosphatase in rat serum.

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