Preparation method of a calcium absorption promoting probiotic and application thereof

Abstract

This invention discloses a method for preparing a calcium absorption-enhancing postbiotic and its application, belonging to the field of food processing technology. The invention utilizes *Lactobacillus helveticus* CCFM1263 inoculated into a casein solution at a specific ratio for fermentation to prepare a postbiotic containing the calcium absorption-enhancing peptide SFSDIPNPIGSENSEK. This postbiotic significantly increases serum calcium and bone calcium levels. The postbiotic provided by this invention can effectively promote calcium absorption and bone health, and is of great significance for developing products that improve calcium absorption efficiency.

Description

Technical Field

[0001] This invention relates to a method for preparing a calcium-absorbing prebiotic and its application, belonging to the field of food processing technology. Background Technology

[0002] Calcium is the most abundant mineral element in the human body, playing a variety of biological functions. Calcium intake is crucial for bone growth and metabolism. Children and adolescents have even greater calcium requirements during their rapid growth period, and calcium accumulation can reach peak bone mass in early life. Although serum calcium can be maintained within the normal range through bone resorption, dietary calcium intake is the only source of calcium for human bones. Low calcium intake and bioavailability can lead to calcium deficiency, and long-term calcium deficiency can cause cramps, osteoporosis, and rickets.

[0003] While dietary intake or calcium supplementation can provide more calcium, limited calcium-rich foods and dietary habits mean that people don't always consume sufficient amounts. For patients with insufficient dietary calcium intake, products that promote calcium absorption can help improve this situation. While post-biotics have been developed to regulate gut microbiota balance and enhance immunity, their impact on calcium absorption remains unknown, and the mechanism by which the active ingredients in post-biotics affect calcium absorption is also unclear. Therefore, developing a post-biotic product that promotes calcium absorption is of great significance and has broad market potential. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a method for preparing a calcium-absorbing metagener and its application. The purpose is to develop a calcium-absorbing metagener by using a calcium-absorbing active peptide contained in the metagener.

[0005] The first technical solution provided by the present invention is a method for preparing a calcium absorption-promoting postbiotic. The method involves inoculating Lactobacillus helveticus CCFM1263 into an αs1-casein solution and fermenting it to obtain a fermentation broth. The fermentation broth is then used to prepare a postbiotic containing the calcium absorption-promoting peptide SFSDIPNPIGSENSEK.

[0006] In one embodiment, the *Lactobacillus helveticus* CCFM1263 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 17, 2022, with accession number GDMCC No. 62417. The *Lactobacillus helveticus* CCFM1263 has been disclosed in patent CN117481196A.

[0007] In one embodiment, the method involves successively culturing purified Lactobacillus helveticus CCFM1263 in MRS medium and a milk system until the bacterial concentration reaches 10. 6~10 9 CFU / mL.

[0008] In one embodiment, the method uses activated Lactobacillus helveticus CCFM1263 at 10 6 ~10 9 The solution was inoculated with CFU / mL of αs1-casein for fermentation.

[0009] In one embodiment, the concentration of the αs1-casein solution is 5–20% (w / w).

[0010] In one embodiment, the casein includes, but is not limited to, bovine casein and sheep casein.

[0011] In one embodiment, the fermentation temperature is 30–42°C, and the fermentation time is 12–72 h.

[0012] In one embodiment, the fermentation broth is centrifuged, the supernatant is collected, and the supernatant is sterilized to obtain the postgenetic agent.

[0013] In one embodiment, the sterilization conditions are 100–120°C for 30 seconds to 30 minutes.

[0014] In one embodiment, the content of the calcium absorption-promoting peptide SFSDIPNPIGSENSEK in the metagenerium is 0.2–200 mg / L.

[0015] The second technical solution provided by the present invention is an epigenetic agent prepared using the first technical solution, wherein the epigenetic agent contains the calcium absorption-promoting peptide SFSDIPNPIGSENSEK.

[0016] In one embodiment, the post-genetic agent contains a large number of milk-derived polypeptides (450-550).

[0017] In one embodiment, the metagenerogen contains a calcium absorption-promoting peptide SFSDIPNPIGSENSEK ≥ 0.2 mg / L.

[0018] The third technical solution provided by the present invention is the application of the method described in the first technical solution or the post-biotic described in the second technical solution in the preparation of products that promote calcium absorption and / or prevent calcium deficiency.

[0019] In one embodiment, the product is a pharmaceutical, food, or health product.

[0020] In one embodiment, the product includes granules, tablets, capsules, pills, liquid formulations, or powders, wherein the liquid formulation includes milk beverages or oral liquids, and the tablets include effervescent tablets, milk tablets, or candies.

[0021] In one embodiment, the product also includes commonly used auxiliary materials.

[0022] In one embodiment, the effects on rats fed a low-calcium diet include, but are not limited to, the following aspects:

[0023] (1) Significantly increased serum calcium levels in rats;

[0024] (2) Significantly increased the calcium content in rat bones.

[0025] The fourth technical solution provided by the present invention is the application of the method described in the first technical solution or the postbiotic described in the second technical solution in the preparation of food containing the calcium absorption-promoting peptide SFSDIPNPIGSENSEK.

[0026] Beneficial effects

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

[0028] 2. The metabiotic prepared from Lactobacillus helveticus CCFM1263 of the present invention can significantly increase serum calcium content by 15% and bone calcium content by 23% compared with the model group. Attached Figure Description

[0029] Figure 1 This is the effect on serum calcium levels in rats;

[0030] Figure 2 It affects the bone calcium content of rats. Detailed Implementation

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0032] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0033] The reagents used to prepare MRS liquid / solid culture media in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.; the casein powder involved in the following examples was purchased from Ala Foods Ingredients Group; and the SPF-grade 3-week-old male SD rats involved in the following examples were purchased from Vital River Laboratory Animal Co., Ltd.

[0034] Example 1: A method for preparing a calcium absorption-enhancing post-biotic.

[0035] Preparation of S1 inoculum

[0036] Under aseptic conditions, Lactobacillus helveticus CCFM1263 stored at 80°C was streaked onto agar plates to screen for single colonies. Lactobacillus helveticus CCFM1263 was then inoculated into MRS medium and cultured at 30–42°C for 18–24 h. The medium was passaged three times, then washed twice with sterile physiological saline and resuspended.

[0037] Preparation of S2 post-biotic

[0038] Prepare an 8% (w / w) αs1-casein solution and sterilize it at 100–120°C for 30–30 seconds. Then, inoculate the bacterial suspension into the sterilized solution and adjust the bacterial concentration to 10. 6 ~10 9 CFU / mL, fermented at 30–42℃ for 12–72 h, followed by centrifugation. The centrifuged liquid was then sterilized by heating at 105℃ for 10 min to obtain post-biotics.

[0039] Experimental Example 2: Determination of post-biotic peptide profiles and content of calcium absorption-promoting peptides

[0040] 1. Preparation of peptide samples

[0041] The post-genetic peptide sample from Example 1 was adjusted to pH 4.6 by adding 50% (v / v) lactic acid or 1M sodium hydroxide solution. It was then centrifuged at 12000g and 4°C for 10 min. The supernatant was collected, filtered through a 0.45μm organic filter membrane, and then centrifuged at 4000g and 4°C for 15 min using a 10kDa ultrafiltration centrifuge tube. The filtrate was the post-genetic peptide sample.

[0042] 2. Peptide profiling

[0043] Mobile phase A consisted of 0.1% (v / v) formic acid and 2% (v / v) acetonitrile, while mobile phase B consisted of 0.1% (v / v) formic acid and 90% (v / v) acetonitrile. The sample was redissolved in mobile phase A to a concentration of 0.5 μg / μL. The sample was then analyzed using an Acclaim PepMap™ RSLC (50 μm × 15 cm, 2 μm) system on an EASY nLC 1200 system. The sample was separated using an elution gradient of 6%–20% B for 0–40 min; 20%–32% B for 40–52 min; and 32%–80% B for 52–56 min, with a flow rate of 0.2 μL / min.

[0044] The eluted peptides were directly ionized into a Q-Exactive mass spectrometer for continuous MS scanning, followed by three MS / MS scans. MS analysis was performed in positive ion and data-dependent analysis (DDA) mode, with an ion source voltage of 2.3 kV, an MS spectral acquisition range of m / z 150–2000, and a resolution of 60,000. MS / MS data acquisition range was m / z 180–2000, and the scan time was 0.1 s.

[0045] The number of milk-derived polypeptides in the postbiotic was measured to be 450–550.

[0046] 3. Determination of the content of calcium absorption-promoting peptides SFSDIPNPIGSENSEK

[0047] The content of calcium absorption-promoting peptide SFSDIPNPIGSENSEK in post-biotics was determined using a triple quadrupole linear ion trap liquid chromatography-mass spectrometry (LC-MS) system with a suitable targeted MRM monitoring method. A series of mixed standard solutions of SFSDIPNPIGSENSEK were prepared by using purified aqueous solution to prepare standard samples. A standard curve for SFSDIPNPIGSENSEK was plotted, and the content of the samples was calculated using the external standard method.

[0048] Liquid chromatography parameters: BEH T3 column (1.7 μm, 2.1 × 100 mm), mobile phase A is an aqueous solution containing 0.1% formic acid, mobile phase B is acetonitrile solution, column temperature is 40℃, injection volume is 2 μL, and flow rate is 0.3 mL / min.

[0049] The content of SFSDIPNPIGSENSEK in the post-biotic was determined to be 1.42 mg / L.

[0050] Experiment Example 3: Effects of Calcium Absorption Promoters on Low-Calcium Diet Rats

[0051] 1. Animal experimental protocol

[0052] Three-week-old female SPF-grade SD rats were housed in a barrier environment with equal day and night cycles and constant temperature and humidity. The rats were randomly divided into three groups of eight rats each: a control group, a model group, and a post-natal susceptibility group (LHW). The control group was fed a normal calcium diet (5g Ca / kg), while the model and LHW groups were fed a low-calcium diet (1g Ca / kg). The control and model groups were administered physiological saline by gavage, while the LHW group was administered 120mg LHW / kg BW by gavage. The experiment lasted for 7 weeks.

[0053] 2. Serum calcium and bone calcium content analysis

[0054] According to the national food safety standard GB5009.92-2016, the serum calcium and bone calcium content were determined by flame atomic absorption spectrometry.

[0055] Depend on Figure 1 and Figure 2 It can be seen that, compared with the model group, the serum calcium content and bone calcium content of rats after metagenic intervention increased by 15% and 23%, respectively.

[0056] In conclusion, the analysis of serum calcium and bone calcium in rats showed that the post-biotic obtained from the fermentation of Lactobacillus helveticus CCCFM1263 can improve calcium absorption efficiency.

[0057] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of post-natal vitamins in the preparation of pharmaceuticals that promote calcium absorption and / or prevent calcium deficiency, characterized in that, The method for preparing the postbiotic is as follows: *Lactobacillus helveticus* CCFM1263 is inoculated into an αs1-casein solution and fermented to obtain a fermentation broth. The fermentation broth is then used to prepare a postbiotic containing the calcium absorption-promoting peptide SFSDIPNPIGSENSEK. The *Lactobacillus helveticus* (… Lactobacillus helveticus CCFM1263 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 17, 2022, with accession number GDMCC No. 62417. The concentration of the αs1-casein solution is 5-20% (w / w), and the content of the calcium absorption-promoting peptide SFSDIPNPIGSENSEK in the metabiotic is not less than 1.42 mg / L.

2. The application according to claim 1, characterized in that, The Lactobacillus helveticus CCFM1263 was inoculated at a concentration of 10 6 ~10 9 CFU / mL.

3. The application according to claim 1, characterized in that, The fermentation temperature is 30~42℃, and the fermentation time is 12~72 h.

4. The application according to claim 1, characterized in that, The promotion of calcium absorption and / or prevention of calcium deficiency includes at least one of the following effects: (1) Increase the serum calcium content of individuals; (2) Increase the calcium content in individual bones.

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