Lactobacillus helveticus producing dpp-iv inhibitory peptide, hypoglycemic goat milk and preparation method thereof

Abstract

The application discloses a DPP-IV inhibiting peptide-producing lactobacillus helveticus, a goat milk with blood sugar reduction and a preparation method thereof, and relates to taking goat milk as raw material, fermenting the goat milk by using the DPP-IV inhibiting peptide-producing lactobacillus helveticus KD12 to obtain the goat milk fermented by the lactic acid bacteria containing DPP-IV inhibiting peptide with blood sugar reduction, and preparing the goat milk powder fermented by the lactic acid bacteria containing DPP-IV inhibiting peptide with blood sugar reduction through drying after fermentation. The lactic acid bacteria of the goat milk fermented by the lactic acid bacteria with blood sugar reduction is inactivated to obtain the goat milk with postbiotics with blood sugar reduction, and after concentration, the goat milk powder with postbiotics with blood sugar reduction is obtained through drying. The goat milk with blood sugar reduction provided by the application has a high DPP-IV inhibition rate, can be used for inhibiting DPP-IV activity after being eaten by consumers, thereby reducing blood sugar, reducing the dosage of blood sugar reduction drugs, and thereby reducing the side effects caused by the blood sugar reduction drugs.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering technology, specifically to a type of Lactobacillus helveticus that produces DPP-IV inhibitory peptides, its hypoglycemic goat milk, and its preparation method. Background Technology

[0002] Insulin is a hormone that regulates blood sugar. When the pancreas cannot produce enough insulin or the body cannot effectively use the insulin it produces, blood sugar levels will exceed normal values, eventually leading to diabetes. Diabetes causes serious damage to many systems in the body, especially the nerves and blood vessels.

[0003] Type 2 diabetes accounts for approximately 90% of all diabetes cases, primarily characterized by insulin resistance. Oral hypoglycemic agents for type 2 diabetes include dipeptidyl peptidase-IV (DPP-IV) inhibitors, such as sitagliptin, saxagliptin, and alogliptin. DPP-IV is a serine protease widely distributed in human tissues. It is responsible for the degradation and inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). DPP-IV inhibitors can enhance GLP-1 and GIP activity, stimulate insulin secretion, and regulate blood glucose levels. Inhibiting DPP-IV is a key strategy for the effective treatment of type 2 diabetes.

[0004] While drug therapy is effective in controlling blood sugar, it also carries many side effects such as significant weight changes, osteoporosis, gastrointestinal disorders, and increased risk of cardiovascular disease. Therefore, food-derived DPP-IV inhibitory peptides have attracted attention. These are peptides that reduce DPP-IV activity. Although their amino acid sequences and peptide lengths differ, they all lower blood sugar, hence they are also known as hypoglycemic peptides. Compared to chemically synthesized drugs, food-derived hypoglycemic peptides are generally safer.

[0005] Currently, goat milk products mainly include infant formula goat milk powder, modified milk powder, pure goat milk, yogurt goat milk, and a small amount of milk beverages. The products are highly homogenized and the competition is fierce. Therefore, it is necessary to develop goat milk products that inhibit DPP-IV activity by using lactic acid bacteria that produce DPP-IV inhibitory peptides. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a *Lactobacillus helveticus* producing DPP-IV inhibitory peptides, its hypoglycemic goat milk, and a preparation method thereof. Using fresh or reconstituted goat milk as raw material, *Lactobacillus helveticus* KD12, which produces DPP-IV inhibitory peptides, is used to ferment the goat milk. This process breaks down the proteins in the goat milk into peptides that inhibit DPP-IV, resulting in hypoglycemic lactic acid bacteria-fermented goat milk containing DPP-IV inhibitory peptides. After inactivating the lactic acid bacteria, hypoglycemic post-biotic goat milk is obtained. This milk is then fortified with nutritional agents and concentrated, followed by spray drying to obtain hypoglycemic post-biotic goat milk powder. The DPP-IV inhibitory peptides are isolated, purified, and their amino acid sequence is identified, determining their position within the goat milk protein. Simultaneously, the fermented goat milk is dried to prepare hypoglycemic lactic acid bacteria-fermented goat milk powder containing DPP-IV inhibitory peptides.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a *Lactobacillus helveticus* KD12 that produces DPP-IV inhibitory peptide, classified and named... Lactobacillus helveticus This strain was deposited at the China Center for Type Culture Collection on September 14, 2024, with accession number CCTCC NO: M20241994.

[0008] Furthermore, the base pair sequence of the Lactobacillus helveticus KD12 is as shown in Sequence 1.

[0009] The present invention also provides a hypoglycemic lactic acid bacteria fermented goat milk, wherein the above-mentioned Lactobacillus helveticus KD12 ferments goat milk to obtain goat milk containing DPP-IV inhibitory peptide, namely hypoglycemic lactic acid bacteria fermented goat milk, and its DPP-IV inhibition rate is 68.23%-92.72%.

[0010] Furthermore, the amino acid sequences of the DPP-IV inhibitory peptides in goat milk fermented by hypoglycemic lactic acid bacteria are as follows: sequences 2-13: FPKYP, AGPFTPT, AIPY, KYIPIQY, VLPVPQ, VLGP, LKALP, GKNKSQS, IRISHEL, FSDIPNP and PVPQ.

[0011] Furthermore, the inoculum size of Lactobacillus helveticus KD12 was 0.01%-0.05%, the fermentation time was 18h-24h, and the fermentation temperature was 34℃-40℃.

[0012] This invention also provides a hypoglycemic lactic acid bacteria fermented goat milk powder, which is obtained by drying the aforementioned hypoglycemic lactic acid bacteria fermented goat milk. The hypoglycemic lactic acid bacteria goat milk powder contains 1.68 × 10⁻⁶ viable Lactobacillus helveticus KD12 bacteria. 7 CFU / g ~1.47×10 9 CFU / g, with a DPP-IV inhibition rate of 27.87%-85.75%.

[0013] Furthermore, the above-mentioned hypoglycemic lactic acid bacteria goat milk is directly spray-dried at high temperature or freeze-dried under vacuum to obtain hypoglycemic lactic acid bacteria goat milk powder;

[0014] or

[0015] Prebiotics and whole goat milk powder are added to the above-mentioned hypoglycemic lactic acid bacteria goat milk, and after being mixed evenly, hypoglycemic lactic acid bacteria goat milk powder is obtained by high-temperature spray drying or vacuum low-temperature spray drying.

[0016] The present invention also provides a hypoglycemic prebiotic goat milk, which is obtained by sterilizing and inactivating the above-mentioned hypoglycemic lactic acid bacteria fermented goat milk, and the DPP-IV inhibition rate is 81.50%-85.83%.

[0017] Furthermore, the heat sterilization is performed at 90°C for 15 minutes or at 116°C for 10 minutes.

[0018] The present invention also provides a hypoglycemic prebiotic goat milk powder, which is prepared by concentrating the above-mentioned hypoglycemic prebiotic goat milk to a solid content of 45%-52% and then spray drying it.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] This invention provides a *Lactobacillus helveticus* that produces DPP-IV inhibitory peptides (…). Lactobacillus helveticus Lactobacillus helveticus KD12 was deposited at the China Center for Type Culture Collection on September 14, 2024, with accession number CCTCC NO: M20241994. Lactobacillus helveticus KD12 is derived from milk kefir grains and has the advantages of being natural, green, and safe.

[0021] This invention provides a hypoglycemic lactic acid bacteria fermented goat milk, which uses Lactobacillus helveticus KD12, which produces DPP-IV inhibitory peptides, to ferment goat milk. The fermented goat milk has a high DPP-IV inhibition rate and can be used by consumers to lower blood sugar.

[0022] This invention provides a hypoglycemic lactic acid bacteria fermented goat milk powder. The production process employs conventional high-temperature spray drying, vacuum low-temperature spray drying, or vacuum freeze drying. The viable bacteria count of the prepared hypoglycemic lactic acid bacteria goat milk powder is higher than the national standard of no less than 10. 6 For the CFU / g requirement, conventional high-temperature spray drying can utilize the company's existing production equipment, facilitating industrial production. Vacuum low-temperature spray drying can further increase the number of live lactic acid bacteria in goat milk powder products.

[0023] This invention provides a hypoglycemic prebiotic goat milk, which is a hypoglycemic lactic acid bacteria fermented goat milk with a high DPP-IV inhibition rate. Because it does not contain live lactic acid bacteria, it does not require cold chain maintenance during transportation and sales, and has a long shelf life, reducing transportation and sales costs. It can also be used by consumers to lower blood sugar.

[0024] This invention provides a blood sugar-lowering prebiotic goat milk powder, which is a goat milk powder with added nutritional fortifiers, no live lactic acid bacteria but with a high DPP-IV inhibition rate. Because it does not contain live lactic acid bacteria and is a solid product, it is easy to transport and carry, and is used by consumers to lower blood sugar. Attached Figure Description

[0025] Figure 1 The colony (left) and cell (right) morphology of Lactobacillus helveticus KD12;

[0026] Figure 2 Phylogenetic tree of Lactobacillus helveticus KD12;

[0027] Figure 3 The DPP-IV inhibition rate of four strains of lactic acid bacteria fermenting goat milk;

[0028] Figure 4 Preparative chromatogram for the separation of goat milk components fermented by Lactobacillus helveticus KD12;

[0029] Figure 5 The DPP-IV inhibition rate of various components of goat milk fermented with Lactobacillus helveticus KD12;

[0030] Figure 6-17 The images show the secondary mass spectra of each DPP-IV repressor peptide in the FG4 fraction.

[0031] Figure 18 This is the HPLC purity analysis chromatogram for FPKYP.

[0032] Figure 19 This is the primary chromatography for FPKYP;

[0033] Figure 20 The inhibition rate of DPP-IV by different concentrations of FPKYP;

[0034] Figure 21 The DPP-IV inhibition rate in the blood of goat milk after glucose reduction;

[0035] Figure 22 The morphology and appearance of goat milk powder fermented with hypoglycemic lactic acid bacteria;

[0036] Figure 23 The inhibition rate of DPP-IV by different concentrations of sitagliptin is shown. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0038] This invention provides a *Lactobacillus helveticus* species that produces DPP-IV inhibitory peptides, named *Lactobacillus helveticus* KD12, and classified as... Lactobacillus helveticus The accession number is CCTCC NO: M20241994, the accession date is September 14, 2024, the depositary institution is China Center for Type Culture Collection, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0039] This invention provides the application of *Lactobacillus helveticus* producing DPP-IV inhibitory peptides in hypoglycemic goat milk products. Using fresh or reconstituted goat milk as raw material, *Lactobacillus helveticus* KD12 producing DPP-IV inhibitory peptides is used to ferment the goat milk, breaking down the goat milk proteins into DPP-IV inhibitory peptides, resulting in hypoglycemic lactic acid bacteria fermented goat milk containing DPP-IV inhibitory peptides. 1) After inactivating the lactic acid bacteria in the hypoglycemic lactic acid bacteria fermented goat milk containing DPP-IV inhibitory peptides, hypoglycemic post-biotic goat milk is obtained. This is then fortified with a nutrient agent and concentrated, followed by spray drying to obtain hypoglycemic post-biotic goat milk powder. The DPP-IV inhibitory peptides are isolated, purified, and their amino acid sequence is identified, determining their position in goat milk proteins. 2) Hypoglycemic lactic acid bacteria fermented goat milk containing DPP-IV inhibitory peptides is dried to prepare hypoglycemic lactic acid bacteria fermented goat milk powder containing DPP-IV inhibitory peptides.

[0040] Example 1: Isolation and Identification of Lactobacillus helveticus KD12

[0041] 1. Isolation of DPP-IV inhibitory peptide from Lactobacillus helveticus KD12

[0042] Milk kefir grains were inoculated at 3% into sterilized and cooled goat milk. After fermentation at room temperature for 24 hours, the samples were used as samples. After serial dilution, 30 strains of lactic acid bacteria were isolated by plate coating. After repeated primary and secondary screening, a strain of lactic acid bacteria with a high DPP-IV inhibition rate was obtained.

[0043] 2. Identification of Lactobacillus helveticus KD12 producing DPP-IV inhibitory peptide

[0044] 2.1 Colony and cell morphology

[0045] After culturing *Lactobacillus helveticus* KD12, which produces DPP-IV inhibitory peptide, in MRS agar medium for 48 hours, it formed distinct, round colonies with regular edges, a milky white color, and a moist, smooth surface. It did not produce pigment. Figure 1(Left) The bacterial morphology of *Lactobacillus helveticus* KD12, which produces DPP-IV inhibitory peptide, after toluidine blue staining is that of long rods, with some bacteria showing curvature. Figure 1 (right).

[0046] 2.2. Strain Identification

[0047] The strain was identified using 16S rDNA. After amplification and purification of the target fragment, the strain was characterized based on homology analysis of partial 16S rDNA gene fragments. The obtained sequence was compared and analyzed with the base sequences in the NCBI database, confirming that the strain was *Lactobacillus helveticus*. Lactobacillus helveticus Its base pair sequence (Sequence 1) is as follows:

[0048]

[0049] The strain was named Lactobacillus helveticus ( Lactobacillus helveticus KD12, Lactobacillus helveticus ( Lactobacillus helveticus The KD12 strain was deposited on September 14, 2024 at the China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M20241994.

[0050] Example 2: Preparation of Blood Sugar-Lowering Lactic Acid Bacteria Fermented Goat Milk

[0051] 1.4 Hypoglycemic peptide activity of goat milk fermented by lactic acid bacteria strains

[0052] Lyophilized bacterial powders of *Lactobacillus helveticus* KD12, *Lactobacillus plantarum* ATCC 8014, *Lactobacillus fermentum* KD-1, and *Lactobacillus helveticus* KD-3 were inoculated into goat milk sterilized at 95℃ for 10 min and cooled to 37℃ for activation. Fermentation was carried out at 37℃ for 22 h, repeated three times. The four activated bacterial strains were then inoculated into sterilized and cooled goat milk at a 3% inoculum size and cultured at 37℃ for 18 h. The supernatant was obtained by centrifugation, and its inhibition rate against DPP-IV was determined. Sitagliptin (4.91 μmol / L) was used as a control. The results are as follows: Figure 3 As shown.

[0053] Depend on Figure 3 It was found that all four strains of bacteria exhibited inhibitory activity against DPP-IV in fermented goat milk. The DPP-IV inhibition rates of the supernatant of fermented goat milk by *Lactobacillus helveticus* KD12, *Lactobacillus plantarum* ATCC 8014, *Lactobacillus fermentum* KD-1, and *Lactobacillus helveticus* KD-3 were 80.17%, 27.56%, 33.52%, and 26.70%, respectively. This indicates that the DPP-IV inhibition rate of fermented goat milk by *Lactobacillus helveticus* KD12 was significantly higher than that of the other three strains and the hypoglycemic drug sitagliptin at 4.91 μmol / L.

[0054] 2. Effects of fermentation conditions on the hypoglycemic activity of hypoglycemic lactic acid bacteria fermenting goat milk

[0055] The freeze-dried Lactobacillus helveticus KD12 was used as a starter culture in goat milk. The effects of fermentation time (18h, 21h, 24h), fermentation temperature (34℃, 37℃, 40℃), and inoculum size (0.01%, 0.03%, 0.05%, w / v) on the production of hypoglycemic peptides in fermented goat milk by Lactobacillus helveticus KD12 were studied. The hypoglycemic peptide activity and pH value of the fermented goat milk were measured. The results are shown in Table 1.

[0056] Table 1. Effects of fermentation conditions on the activity of hypoglycemic peptides in goat milk fermented with Lactobacillus helveticus KD12

[0057]

[0058] As shown in Table 1, when the fermentation time was 18-24 h, the fermentation temperature was 34℃-40℃, and the inoculum size was 0.01%-0.05% (w / v), the DPP-IV inhibition rate of Lactobacillus helveticus KD12 fermented goat milk was 68.23%-93.77%, which is equivalent to the DPP-IV inhibition effect of sitagliptin at concentrations of 5.67 μmol / L-9.16 μmol / L.

[0059] Using the DPP-IV inhibition rate ≥65% as an indicator, the suitable fermentation conditions for Lactobacillus helveticus KD12 to produce hypoglycemic peptides from goat milk are: fermentation time of 18h-24h, fermentation temperature of 34℃-40℃, and inoculum size of 0.01%-0.05% (w / v).

[0060] Example 3: Preparation, identification, and hypoglycemic activity study of DPP-IV inhibitory peptide

[0061] Goat milk fermented with hypoglycemic lactic acid bacteria containing DPP-IV inhibitory peptide was centrifuged to obtain whey, which was then ultrafiltered through a membrane with a molecular weight cutoff of less than 10 kDa. The permeate was freeze-dried to obtain lyophilized powder, which was then dissolved in ultrapure water. The components were collected by preparative high-performance liquid chromatography. Figure 4 The absorption wavelength was 220 nm, and the flow rate was 10.0 mL / min. A Gemini-NX 10μC18 100A column was used for preparation; mobile phase A consisted of 0.1% trifluoroacetic acid in acetonitrile (TFA-ACN); mobile phase B consisted of 0.1% trifluoroacetic acid in ultrapure water (TFA-H2O). After freeze-drying the collected fractions, their DPP-IV inhibition rate was determined, and the results are as follows: Figure 5 As shown, by Figure 5 It can be seen that the DPP-IV inhibition rate of component FG4 is significantly higher than that of other components, indicating that the hypoglycemic peptides are mainly concentrated in component FG4.

[0062] The peptide and amino acid sequences of component FG4 were identified by HPLC-MS / MS. First, separation was performed using high-performance liquid chromatography (HPLC). Solution A was a 0.1% formic acid aqueous solution, and solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile 84%). The HPLC column (0.15mm*150mm, RP-C18, Column Technology Inc.) was equilibrated with 95% solution A. The sample was loaded into a Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) via an autosampler and then separated by the HPLC column. The relevant HPLC gradient settings were as follows:

[0063] From 0 to 50 minutes, the linear gradient of solution B increased from 4% to 50%; from 50 to 54 minutes, the linear gradient of solution B increased from 50% to 100%; from 54 to 60 minutes, solution B was maintained at 100%.

[0064] The hypoglycemic peptides were separated by capillary high-performance liquid chromatography (HPLC) and then analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (ThermoFisher). Positive ion detection was used, with an analysis time of 60 min. Ten fragment spectra were acquired after each full scan (MS2 scan). The raw mass spectrometry files were retrieved from the goat milk protein database using MaxQuant 1.5.5.1 software, and the analytical results of the hypoglycemic peptides were obtained (Table 2).

[0065] Table 2. Amino acid sequences and sources of DPP-IV inhibitory peptides.

[0066]

[0067] As shown in Table 2, the number of amino acids in the 11 peptides in the hypoglycemic peptide powder ranges from 4 to 7, and the molecular weight ranges from 384.24 Da to 956.48 Da. They are derived from β-casein, αs1 and αs2-casein, κ-casein, β-lactoglobulin, lactoferrin and osteopontin in goat milk.

[0068] Secondary mass spectra of each DPP-IV inhibitory peptide are shown below. Figures 6-17 .

[0069] FPKYP was synthesized in a solid phase. The purity analysis and primary chromatographic results of the synthesized FPKYP are as follows: Figure 18 and Figure 19 As shown in the figure, the purity of the synthesized FPKYP was 97.99%. Different concentrations (0-8 μmol / L) were prepared to obtain the inhibition rates against DPP-IV as shown in the figure. Figure 20 As shown, by Figure 20 The IC50 for DPP-IV inhibition can be calculated. 50 The value was 2.14 μmol / mL.

[0070] Example 4 Preparation of hypoglycemic prebiotic goat milk and hypoglycemic prebiotic goat milk powder

[0071] Glucose-lowering lactic acid bacteria were fermented in goat milk and then treated at 90℃ for 15 min or sterilized at 116℃ for 10 min to obtain the hypoglycemic precursor. The supernatant was then centrifuged, and its DPP-IV inhibition rate was determined. The results are as follows: Figure 21 As shown. By Figure 21It can be seen that no live lactic acid bacteria were detected after heat treatment, but the DPP-IV inhibition rate of goat milk fermented with hypoglycemic lactic acid bacteria increased slightly to 81.50%-85.83%, but there was no significant difference from the untreated group. This indicates that the lactic acid bacteria were inactivated after heat treatment, but their hypoglycemic peptide activity did not change significantly.

[0072] After concentrating the hypoglycemic prebiotic goat milk to a solid content of 45%-52%, the hypoglycemic prebiotic goat milk powder was obtained by conventional spray drying. After reconstitution at 12.5% ​​(w / v), its DPP-IV inhibition rate was 76.25%-82.78%.

[0073] Example 5: Preparation of goat milk powder fermented with hypoglycemic lactic acid bacteria

[0074] 5% inulin and 22.5% whole goat milk powder were added to goat milk fermented with hypoglycemic lactic acid bacteria to achieve a solids content of approximately 40%. Whole goat milk with a 40% solids content was used as a control. High-temperature spray drying (small spray dryer, YM-6000Y, Shanghai Yuming Instrument Co., Ltd.) and low-temperature vacuum spray drying (vacuum low-temperature spray dryer, BILON-VSD1500, Bilang Company) were performed. The high-temperature spray drying conditions were: inlet air temperature 130℃, outlet temperature 70℃, and peristaltic pump speed 8%. The low-temperature vacuum spray drying conditions were: inlet air temperature 70℃, outlet temperature 60℃, and peristaltic pump speed 40%. Four types of goat milk powder were obtained, including two hypoglycemic lactic acid bacteria goat milk powders and two control goat milk powders.

[0075] The samples were named high-temperature lactic acid bacteria goat milk powder (GSF), high-temperature goat control milk powder (GSD), low-temperature lactic acid bacteria goat milk powder (DSF), and low-temperature goat control milk powder (DSD), respectively. The four goat milk powder samples were visually observed and subjected to color analysis. The viable bacteria count was determined and the survival rate was calculated. After rehydration, the DPP-IV inhibition rate was measured. The results are as follows: Figure 22 As shown in Table 3.

[0076] Depend on Figure 22 It can be seen that all four types of goat milk powder are white or light yellow powders. Under careful observation with the naked eye, a slight color difference can be found between the hypoglycemic lactic acid bacteria fermented goat milk powder and the control group. The color of the control group sample is close to the standard white color, while the hypoglycemic lactic acid bacteria fermented goat milk powder sample is slightly milky yellow, which may be due to the Maillard reaction occurring under high temperature conditions during the drying process.

[0077] Table 3. Comparison of color between hypoglycemic lactic acid bacteria fermented goat milk powder and control goat milk powder

[0078]

[0079] Table 3 shows that the L* values ​​of the hypoglycemic lactic acid bacteria goat milk powder prepared by high-temperature or low-temperature spray drying were lower than those of the control group, while the a* and b* values ​​were increased. The color difference (DE) values ​​(1.54 and 2.242 NBS) between the hypoglycemic lactic acid bacteria goat milk powder prepared by high-temperature or low-temperature spray drying and the control group were respectively, which are perceptible visual values. The increase in the +b* value of the hypoglycemic lactic acid bacteria goat milk powder prepared by high-temperature or low-temperature spray drying leads to a more yellow color, which is related to... Figure 22 The results observed with the naked eye are consistent.

[0080] Table 4. Viable bacteria count, survival rate, and DPP-IV inhibition rate of goat milk powder fermented with hypoglycemic lactic acid bacteria.

[0081]

[0082] Table 4 shows that low-temperature vacuum spray drying significantly improves the survival rate of lactic acid bacteria, with the viable count being 4.72 times that of high-temperature spray drying. Spray drying conditions and the type of heat protectant significantly affect the viable count and survival rate of bacteria in milk powder. However, the viable count in the goat milk powder fermented with hypoglycemic lactic acid bacteria was significantly higher than the national standard requirement of not less than 1.0 × 10⁻⁶. 6 Requirements for CFU / g.

[0083] Compared with unspray-dried hypoglycemic lactic acid bacteria fermented goat milk, the DPP-IV inhibition rate of rehydrated hypoglycemic lactic acid bacteria fermented goat milk powder was significantly lower (p < 0.05). This is because the addition of a heat protectant before spray drying increased the solids content. Rehydration at the same concentration after drying is equivalent to diluting the hypoglycemic and uric acid-lowering peptides, resulting in a decrease in the inhibition rate. The solids content can be increased by low-temperature concentration, thereby reducing the amount of heat protectant added to whole goat milk powder and improving its DPP-IV inhibition rate. Low-temperature vacuum spray drying and high-temperature spray drying had no significant effect on the DPP-IV inhibition rate of goat milk (p > 0.05).

[0084] The blood sugar-lowering lactic acid bacteria fermented goat milk was directly spray-dried at high temperature and freeze-dried under vacuum. The resulting goat milk powder was represented by SDP and FDP, respectively. The viable bacteria count was determined and the survival rate was calculated. After rehydration, the DPP-IV inhibition rate was determined. The results are shown in Table 5.

[0085] Table 5. Viable bacterial count and DPP-IV inhibition rate of hypoglycemic lactic acid bacteria fermented goat milk powder prepared by direct drying.

[0086]

[0087] As shown in Table 5, directly subjecting goat milk fermented with hypoglycemic lactic acid bacteria to high-temperature spray drying and vacuum freeze drying has a significant impact on the viable count of lactic acid bacteria, but little impact on the DPP-IV inhibition rate. This indicates that the activity of the DPP-IV inhibitory peptide is not sensitive to temperature changes, thus maintaining its activity.

[0088] In summary, this invention provides a DPP-IV inhibitory peptide-producing *Lactobacillus helveticus*, its hypoglycemic goat milk, and a preparation method thereof. Using goat milk as raw material, *Lactobacillus helveticus* KD12, which produces the DPP-IV inhibitory peptide, is used to ferment the goat milk, resulting in hypoglycemic lactic acid bacteria fermented goat milk containing the DPP-IV inhibitory peptide. The fermented goat milk is then dried with the addition of prebiotics and a heat protectant to obtain hypoglycemic lactic acid bacteria fermented goat milk powder containing the DPP-IV inhibitory peptide. After inactivating the lactic acid bacteria from the hypoglycemic lactic acid bacteria fermented goat milk, hypoglycemic post-biotic goat milk is obtained, which is then concentrated and dried to obtain hypoglycemic post-biotic goat milk powder. The hypoglycemic goat milk products provided by this invention all exhibit high DPP-IV inhibition rates and can be used by consumers to inhibit DPP-IV activity, thereby lowering blood sugar and reducing the dosage of hypoglycemic drugs, thus minimizing the side effects of hypoglycemic drugs. Furthermore, this invention also decomposes the proteins in goat milk into DPP-IV inhibitory peptides, and isolates, purifies, and identifies the amino acid sequence of the DPP-IV inhibitory peptides, determining their location within the goat milk protein.

[0089] The calculation method for relevant parameters of the above-mentioned Lactobacillus helveticus producing DPP-IV inhibitory peptide and its application in hypoglycemic goat milk products:

[0090] 1. Assay of dipeptidyl peptidase-IV (DPP-IV) inhibitory activity

[0091] Take a 96-well microtiter plate and add reaction reagents according to the design shown in Table 2-5. Detect the absorbance of the reaction solution at 405 nm using an ELISA reader. Use sitagliptin as a positive control. The inhibition rate of sitagliptin concentration on DPP-IV is shown in [Table 2-5]. Figure 23 .

[0092] Table 6. Amounts of each reagent added during the DPP-IV inhibitory activity assay (unit: μL)

[0093]

[0094] (1)

[0095] 2. Colorimetric analysis

[0096] Analysis was performed using a Minolta CM-5 spectrophotometer. Samples were zeroed and calibrated with a white plate before analysis. Each sample was measured three times, and the average value was taken. The color difference between the sample and the control group is represented by DE, which is calculated using Formula 2.

[0097] (2)

[0098] 3. Determination of viable lactic acid bacteria count

[0099] Refer to the method in national standard GB 4789.35-2016.

Claims

1. A type of Lactobacillus helveticus KD12, characterized in that, Category naming Lactobacillus helveticus This strain was deposited at the China Center for Type Culture Collection on September 14, 2024, with accession number CCTCC NO: M20241994.

2. The *Lactobacillus helveticus* KD12 according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of Lactobacillus helveticus KD12 is shown in SEQ ID NO.

1.

3. A hypoglycemic lactic acid bacteria fermented goat milk powder, characterized in that, Blood sugar-lowering lactic acid bacteria fermented goat milk is obtained by fermenting goat milk with *Lactobacillus helveticus* KD12 as described in claim 1 or 2. The blood sugar-lowering lactic acid bacteria fermented goat milk is then dried to obtain blood sugar-lowering lactic acid bacteria fermented goat milk powder. The viable count of *Lactobacillus helveticus* KD12 in the blood sugar-lowering lactic acid bacteria fermented goat milk powder is 1.68 × 10⁻⁶. 7 CFU / g ~1.47×10 9 CFU / g, with a DPP-IV inhibition rate of 27.87%-85.75%.

4. The method for preparing hypoglycemic lactic acid bacteria fermented goat milk powder according to claim 3, characterized in that, The blood sugar-lowering lactic acid bacteria fermented goat milk is directly spray-dried at high temperature or freeze-dried under vacuum to obtain blood sugar-lowering lactic acid bacteria fermented goat milk powder.

5. The method for preparing hypoglycemic lactic acid bacteria fermented goat milk powder according to claim 3, characterized in that, Prebiotics and whole goat milk powder are added to the hypoglycemic lactic acid bacteria fermented goat milk, and after being mixed evenly, the mixture is dried by high-temperature spray drying or vacuum low-temperature spray drying to obtain hypoglycemic lactic acid bacteria fermented goat milk powder.

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