Lactobacillus helveticus for producing DPP-IV (dipeptidyl peptidase-IV) inhibitory peptide as well as hypoglycemic goat milk and preparation method thereof

Abstract

The invention discloses lactobacillus helveticus for producing DPP-IV (dipeptidyl peptidase-IV) inhibitory peptide and hypoglycemic goat milk and a preparation method thereof, goat milk is used as a raw material, and lactobacillus helveticus KD12 for producing the DPP-IV inhibitory peptide is used for fermenting the goat milk to obtain hypoglycemic lactobacillus fermented goat milk containing the DPP-IV inhibitory peptide. And drying the fermented goat milk to obtain the hypoglycemic lactic acid bacteria fermented goat milk powder containing the DPP-IV inhibitory peptide. Lactic acid bacteria of the hypoglycemic lactic acid bacteria fermented goat milk are inactivated to obtain hypoglycemic post-prebiotics goat milk, and the hypoglycemic post-prebiotics goat milk powder is obtained after the hypoglycemic post-prebiotics goat milk is concentrated and dried. The hypoglycemic goat dairy product provided by the invention has a relatively high DPP-IV inhibition rate, and can be used for inhibiting DPP-IV activity after consumers eat the hypoglycemic goat dairy product so as to reduce blood sugar and reduce the dosage of hypoglycemic drugs, so that side effects caused by the hypoglycemic drugs are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to Lactobacillus helveticus producing a DPP-IV inhibitory peptide, its hypoglycemic goat milk and a preparation method thereof. Background Art

[0002] Insulin is a hormone that regulates blood sugar. When the pancreas doesn't produce enough insulin or the body can't effectively use the insulin it produces, blood sugar levels rise above normal, eventually developing into diabetes. Diabetes can severely damage many of the body's systems, particularly the nerves and blood vessels.

[0003] Type 2 diabetes patients account for about 90% of the total number of diabetes patients, and the main manifestation is insulin resistance. Oral hypoglycemic drugs 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 insulin polypeptide (GIP). DPP-IV inhibitors can enhance the activity of GLP-1 and GIP, stimulate insulin secretion, and regulate blood sugar levels. Inhibiting DPP-IV is a key strategy for the effective treatment of type 2 diabetes;

[0004] While medications are effective in controlling blood sugar, they also come with numerous side effects, including significant weight loss, osteoporosis, gastrointestinal disturbances, and an increased risk of cardiovascular disease. Consequently, food-derived DPP-IV inhibitory peptides have garnered significant attention. These peptides can reduce DPP-IV activity. While their amino acid sequences and lengths vary, they all lower blood sugar, hence the name "hypoglycemic peptide." Compared to chemically synthesized drugs, food-derived hypoglycemic peptides are safer.

[0005] Currently, the main goat milk products 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 use lactic acid bacteria that produce DPP-IV inhibitory peptides to develop hypoglycemic goat milk products with DPP-IV inhibitory activity. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a DPP-IV inhibitory peptide-producing Lactobacillus helveticus, a hypoglycemic goat milk thereof, and a preparation method thereof. Fresh goat milk or reconstituted goat milk is used as raw material, and the goat milk is fermented with Lactobacillus helveticus KD12 producing a DPP-IV inhibitory peptide. Protein in the goat milk is decomposed into DPP-IV inhibitory peptides to obtain hypoglycemic lactic acid bacteria-fermented goat milk containing the DPP-IV inhibitory peptide. The lactic acid bacteria are inactivated to obtain hypoglycemic postbiotic goat milk. A nutritional enhancer is added to the milk, the milk is concentrated, and the milk is spray-dried to obtain hypoglycemic postbiotic goat milk powder. The DPP-IV inhibitory peptide is isolated and purified, and its amino acid sequence is identified to determine the location of the DPP-IV inhibitory peptide in the goat milk protein. The fermented goat milk is then dried to prepare hypoglycemic lactic acid bacteria-fermented goat milk powder containing the DPP-IV inhibitory peptide.

[0007] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a DPP-IV inhibitory peptide-producing Lactobacillus helveticus KD12, classified and named Lactobacillus helveticus, which was deposited in the China Center for Type Culture Collection on September 14, 2024, and its deposit number is 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 glucose-lowering lactic acid bacteria fermented goat milk. The above-mentioned Lactobacillus helveticus KD12 is used to ferment goat milk to obtain goat milk containing DPP-IV inhibitory peptides, i.e., glucose-lowering lactic acid bacteria fermented goat milk, whose 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 with hypoglycemic lactic acid bacteria are as follows: sequences 2 to 13: FPKYP, AGPFTPT, AIPY, KYIPIQY, PIQY, VLPVPQ, VLGP, LKALP, GKNKSQS, IRISHEL, FSDIPNP and PVPQ.

[0011] Furthermore, the inoculation amount of Lactobacillus helveticus KD12 is 0.01%-0.05%, the fermentation time is 18h-24h, and the fermentation temperature is 34°C-40°C.

[0012] The present invention also provides a hypoglycemic lactic acid bacteria fermented goat milk powder, which is prepared by drying the hypoglycemic lactic acid bacteria fermented goat milk. The viable number of Lactobacillus helveticus KD1 in the hypoglycemic lactic acid bacteria goat milk powder is 1.68×10 7 CFU / g~1.47×10 9CFU / g, and the DPP-IV inhibition rate was 27.87%-85.75%.

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

[0014] or

[0015] Prebiotics and full-fat goat milk powder are added to the above-mentioned hypoglycemic lactic acid bacteria goat milk, the mixture is evenly mixed, and the mixture is then subjected to high-temperature spray drying or vacuum low-temperature spray drying to obtain hypoglycemic lactic acid bacteria goat milk powder.

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

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

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

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

[0020] The present invention provides a Lactobacillus helveticus KD12 that produces a DPP-IV inhibitory peptide, and on September 14, 2024, it was deposited in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M20241994. Lactobacillus helveticus KD12 is derived from milk kefir grains and has the advantages of being natural, green, and safe.

[0021] The present invention provides a blood sugar-lowering lactic acid bacteria fermented goat milk. The goat milk is fermented by using Lactobacillus helveticus KD12 which produces a DPP-IV inhibitory peptide. The fermented goat milk has a high DPP-IV inhibition rate and can be used for consumers to lower blood sugar.

[0022] The present invention provides a hypoglycemic lactic acid bacteria fermented goat milk powder. The production process adopts conventional high-temperature spray drying or 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 not less than 10 viable bacteria count. 6 In order to meet the requirements of CFU / g, conventional high-temperature spray drying can utilize the company's existing production equipment to facilitate industrial production, and vacuum low-temperature spray drying can further increase the number of live lactic acid bacteria in goat milk powder products.

[0023] The present invention provides a hypoglycemic postbiotic goat milk, which is an inactivated hypoglycemic lactic acid bacteria fermented goat milk with a high DPP-IV inhibition rate. Since it does not contain live lactic acid bacteria, it does not require a cold chain to maintain a low temperature during transportation and sales, and has a long shelf life, thereby reducing transportation and sales costs. It can also be used to lower blood sugar for consumers.

[0024] The present invention provides a blood sugar lowering postbiotic goat milk powder, which is a goat milk powder added with a nutritional enhancer, has no live lactic acid bacteria but has a high DPP-IV inhibition rate. Since it has no live lactic acid bacteria and is a solid product, it is easy to transport and carry, and is used for consumers to lower blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 is the phylogenetic tree of Lactobacillus helveticus KD12;

[0027] Figure 3 is the DPP-IV inhibition rate of goat milk fermented by four lactic acid bacteria strains;

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

[0029] Figure 5 is the DPP-IV inhibition rate of each component of goat milk fermented by Lactobacillus helveticus KD12;

[0030] Figures 6-17 This is the MS / MS spectrum of 16 peptides in the FG4 fraction;

[0031] Figure 18 HPLC purity analysis chart of FPKYP;

[0032] Figure 19 is the primary chromatogram of FPKYP;

[0033] Figure 20 is the inhibition rate of DPP-IV at different concentrations of FPKYP;

[0034] Figure 21 is the DPP-IV inhibition rate in hypoglycemic postbiotic goat milk;

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

[0036] Figure 23 is the inhibition rate of DPP-IV at different concentrations of sitagliptin. DETAILED DESCRIPTION

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

[0038] The present invention provides a Lactobacillus helveticus that produces a DPP-IV inhibitory peptide. The Lactobacillus helveticus is named Lactobacillus helveticus KD12, classified as Lactobacillus helveticus, with a preservation number of CCTCC NO: M20241994, a preservation date of September 14, 2024, and a preservation unit of the China Center for Type Culture Collection, with an address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a zip code of 430072.

[0039] The invention provides an application of Lactobacillus helveticus producing a DPP-IV inhibitory peptide in a hypoglycemic goat milk product. The invention comprises the following steps: using fresh goat milk or reconstituted goat milk as a raw material, fermenting the goat milk with Lactobacillus helveticus KD12 producing a DPP-IV inhibitory peptide, decomposing proteins in the goat milk into peptides inhibiting DPP-IV, and obtaining hypoglycemic lactic acid bacteria fermented goat milk containing the DPP-IV inhibitory peptide; 1) inactivating lactic acid bacteria in the goat milk fermented with the DPP-IV inhibitory peptide to obtain hypoglycemic postbiotic goat milk; adding a nutritional enhancer and concentrating the milk; and spray-drying the milk to obtain hypoglycemic postbiotic goat milk powder; separating and purifying the DPP-IV inhibitory peptide and identifying its amino acid sequence to determine the position of the DPP-IV inhibitory peptide in goat milk protein; and 2) drying the hypoglycemic lactic acid bacteria fermented goat milk containing the DPP-IV inhibitory peptide to obtain hypoglycemic lactic acid bacteria fermented goat milk powder containing the DPP-IV inhibitory peptide.

[0040] Example 1 Isolation and identification of Lactobacillus helveticus KD12

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

[0042] 3% milk kefir grains were inoculated into sterilized and cooled goat milk, fermented at room temperature for 24 hours, and used as a sample. After serial dilution, 30 strains of lactic acid bacteria were isolated using the plate spreading method. After repeated primary and secondary screening, a 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 bacterial morphology

[0045] After culturing DPP-IV inhibitory peptide-producing Lactobacillus helveticus KD12 in MRS agar medium for 48 h, it formed obvious colonies on the MRS medium. The colonies were round, with neat edges, milky white, moist and smooth surface, and no pigment was produced. Figure 1(Left), the bacterial morphology of Lactobacillus helveticus KD12 producing DPP-IV inhibitory peptide after toluidine blue staining is long rod-shaped, and some bacteria are bent. Figure 1 (right).

[0046] 2.2. Identification of bacterial species

[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 a partial 16S rDNA gene fragment. The obtained sequence was compared with base sequences in the NCBI database and analyzed, confirming that the strain was Lactobacillus helveticus. Its base pair sequence (SEQ ID NO: 1) is as follows:

[0048]

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

[0050] Example 2 Preparation of hypoglycemic lactic acid bacteria fermented goat milk

[0051] 1.3 Antidiabetic Peptide Activity of Goat Milk Fermented by Lactic Acid Bacteria

[0052] The freeze-dried bacterial powders of Lactobacillus helveticus KD12, Lactobacillus plantarum ATCC 8014, Lactobacillus fermentans KD-1, and Lactobacillus helveticus KD-3 were inoculated into goat milk sterilized at 95°C for 10 minutes and cooled to 37°C for activation, and fermented at 37°C for 22 hours, which was repeated three times. The four activated strains were inoculated into the sterilized and cooled goat milk at an inoculum size of 3%, cultured at 37°C for 18 hours, and the supernatants were obtained by centrifugation. The inhibition rate of DPP-IV was determined, and the hypoglycemic drug sitagliptin (4.91 μmol / L) was used as a control. The results are shown in FIG. Figure 3 shown.

[0053] Depend on Figure 3 It can be seen that the fermented goat milk of the four strains of bacteria all have inhibitory activity against DPP-IV. The ACE inhibition rates of the supernatants of goat milk fermented by Lactobacillus helveticus KD12, Lactobacillus plantarum ATCC 8014, Lactobacillus mucosa KD-1 and Lactobacillus helveticus KD-3 were 80.17%, 27.56%, 33.52%2 and 6.70%, respectively. It can be seen that the DPP-IV inhibition rate in goat milk fermented by Lactobacillus helveticus KD12 was significantly higher than that of the other three strains and 4.91 μmol / L of the hypoglycemic drug sitagliptin.

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

[0055] The freeze-dried powder of Lactobacillus helveticus KD12 was inoculated into goat milk as a starter. The effects of fermentation time (18 h, 21 h, 24 h), fermentation temperature (34 ° C, 37 ° C, 40 ° C) and inoculation amount (0.01%, 0.03%, 0.05%, w / v) on the production of hypoglycemic peptides by Lactobacillus helveticus KD12 fermented goat milk 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 goat milk hypoglycemic peptides fermented by Lactobacillus helveticus KD12

[0057]

[0058] As can be seen from Table 1, when the fermentation time is 18h-24h, the fermentation temperature is 34℃-40℃, and the inoculation size is 0.01%-0.05% (w / v), the DPP-IV inhibition rate of goat milk fermented by Lactobacillus helveticus KD12 is 68.23%-93.77%, which is equivalent to the inhibitory effect of sitagliptin on DPP-IV at a concentration of 5.67μmol / L-9.16μmol / L.

[0059] Taking the DPP-IV inhibition rate ≥ 65% as an indicator, it can be seen that the suitable fermentation conditions for Lactobacillus helveticus KD12 to ferment goat milk to produce hypoglycemic peptides are: fermentation time 18h-24h, fermentation temperature 34℃-40℃, and inoculation amount 0.01%-0.05% (w / v).

[0060] Example 3 Preparation, Identification and Hypoglycemic Activity Study of DPP-IV Inhibitory Peptides

[0061] The whey was obtained by centrifugation of goat milk fermented with glucose-lowering lactic acid bacteria containing DPP-IV inhibitory peptides, and then ultrafiltered through a filter membrane with a molecular weight cutoff of less than 10 kDa. The permeate was freeze-dried to obtain a lyophilized powder, and the lyophilized sample was dissolved in ultrapure water and the components were collected by preparative high performance liquid chromatography ( Figure 4 ), absorption wavelength 220 nm, flow rate 10.0 mL / min. Preparative column Gemini-NX 10μC18 100A; mobile phase A: 0.1% trifluoroacetic acid in acetonitrile (TFA-ACN); mobile phase B: 0.1% trifluoroacetic acid in ultrapure water (TFA-H2O). The collected fractions were freeze-dried and their DPP-IV inhibition rates were determined. The results are shown in Figure 2. 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 glucose-lowering peptides are mainly concentrated in component FG4.

[0062] The peptide and amino acid sequence of component FG4 were identified by HPLC-MS / MS. High-performance liquid chromatography (HPLC) separation was performed using solution A, which consisted of 0.1% formic acid in water and solution B, which consisted of 0.1% formic acid in acetonitrile (84% acetonitrile). A liquid chromatography column (0.15 mm x 150 mm, RP-C18, Column Technology Inc.) was equilibrated with 95% solution A. The sample was loaded via an autosampler onto Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) and then separated on the liquid chromatography column using the following liquid phase gradient settings:

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

[0064] The glucose-lowering peptides were separated by capillary high-performance liquid chromatography and analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (ThermoFisher). Positive ion detection was used, and the analysis lasted 60 minutes. Ten MS2 fragmentation spectra were acquired after each full scan. The mass spectrometry raw files were searched against a goat milk protein database using MaxQuant 1.5.5.1 software. The glucose-lowering peptide analysis results 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 12 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 of goat milk.

[0068] The secondary mass spectra of each DPP-IV inhibitory peptide are shown in Figures 6-17 .

[0069] FPKYP was synthesized by solid phase, and the purity of the synthesized FPKYP was analyzed by primary chromatography. Figure 18 and Figure 19 The results show that the purity of the synthesized FPKYP is 97.99%. It was prepared into different concentrations (0-8 μmol / L) and the inhibition rate of DPP-IV was obtained as shown in the following table. Figure 20 As shown by Figure 20 The IC of DPP-IV inhibition can be calculated 50 The value was 2.14 μmol / mL.

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

[0071] The hypoglycemic postbiotics were obtained by fermenting goat milk with hypoglycemic lactic acid bacteria at 90°C for 15 min or sterilizing at 116°C for 10 min. The supernatant was centrifuged and the DPP-IV inhibition rate was determined. Figure 21 As shown. Figure 21It can be seen that after heat treatment, no live lactic acid bacteria were detected, 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 compared with the untreated group, indicating that after heat treatment, the lactic acid bacteria were inactivated, but the hypoglycemic peptide activity did not change significantly.

[0072] The hypoglycemic postbiotic goat milk is concentrated to a solid content of 45%-52%, and then conventionally spray-dried to obtain the hypoglycemic postbiotic goat milk powder. After reconstitution to 12.5% ​​(w / v), the DPP-IV inhibition rate is 76.25%-82.78%.

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

[0074] 5% inulin and 22.5% whole goat milk powder were added to goat milk fermented with glucose-lowering lactic acid bacteria to achieve a solid content of approximately 40%. The whole goat milk with a solid content of 40% was used as a control. The milk was then subjected to high-temperature spray drying (a small spray dryer, YM-6000Y, Shanghai Yuming Instrument Co., Ltd.) and low-temperature vacuum spray drying (a vacuum low-temperature spray dryer, BILON-VSD1500, Biron Corporation). The high-temperature spray drying conditions were: an inlet air temperature of 130°C, an outlet air temperature of 70°C, and a peristaltic pump speed of 8%; the low-temperature vacuum spray drying conditions were: an inlet air temperature of 70°C, an outlet air temperature of 60°C, and a peristaltic pump speed of 40%. A total of four types of goat milk powder were obtained, including two types of glucose-lowering lactic acid bacteria goat milk powder and two types of control goat milk powder.

[0075] The samples were named as 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). The four goat milk powder samples were observed with the naked eye and subjected to colorimetric analysis. The number of viable bacteria was determined and the survival rate was calculated. After rehydration, the DPP-IV inhibition rate was determined. The results are as follows: Figure 22 As shown in Table 3.

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

[0077] Table 3 Chroma comparison of glucose-lowering lactic acid bacteria fermented goat milk powder and control goat milk powder

[0078]

[0079] As shown in Table 3, the L* value of the sugar-reducing lactic acid bacteria goat milk powder prepared by high temperature or low temperature spray drying is lower than that of the control group, and the a* value and b* value are both improved. The color difference DE of the sugar-reducing lactic acid bacteria goat milk powder prepared by high temperature or low temperature spray drying and the control group are 1.54 and 2.2.42 NBS, respectively, which are perceptible visual perception values. The increase in the b* value of the sugar-reducing lactic acid bacteria goat milk powder prepared by high temperature or low temperature spray drying will cause the color to be yellowish, which is consistent with the Figure 22 The results were consistent with those observed with the naked eye.

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

[0081]

[0082] As shown in Table 4, low-temperature vacuum spray drying can significantly increase the survival rate of lactic acid bacteria, and its viable bacterial count is 4.72 times that of high-temperature spray drying. Spray drying conditions and the type of heat-resistant protective agent have a significant impact on the viable bacterial count and survival rate of bacteria in milk powder. However, the viable bacterial count in goat milk powder fermented with hypoglycemic lactic acid bacteria is significantly higher than the national standard, that is, not less than 1.0×10 6 CFU / g requirements.

[0083] Compared with the non-spray-dried goat milk fermented with glucose-lowering lactic acid bacteria, the DPP-IV inhibition rate of the glucose-lowering lactic acid bacteria fermented goat milk powder after rehydration was significantly decreased (p < 0.05). This is because the addition of heat-resistant protective agents before spray drying increased the solid content. Rehydration at the same concentration after drying is equivalent to diluting the glucose-lowering and uric acid-lowering peptides, resulting in a decrease in the inhibition rate. The solid content can be increased by low-temperature concentration, thereby reducing the amount of heat-resistant protective agent whole-fat goat milk powder added to improve 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 goat milk fermented with hypoglycemic lactic acid bacteria was directly spray-dried and freeze-dried by vacuum. The obtained goat milk powder was expressed as SDP and FDP, respectively. The number of viable bacteria 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 Live cell count and DPP-IV inhibition rate of glucose-lowering lactic acid bacteria fermented goat milk powder prepared by direct drying

[0086]

[0087] As shown in Table 5, direct high-temperature spray drying and vacuum freeze drying of goat milk fermented with glucose-lowering lactic acid bacteria had a significant effect on the number of viable lactic acid bacteria, but had little effect on the DPP-IV inhibition rate, indicating that the activity of DPP-IV inhibitory peptides is not sensitive to temperature changes, thus maintaining their activity.

[0088] In summary, the present invention provides a DPP-IV inhibitory peptide-producing Lactobacillus helveticus, its hypoglycemic goat milk, and a preparation method thereof. Goat milk is fermented with Lactobacillus helveticus KD12, which produces a DPP-IV inhibitory peptide, to obtain hypoglycemic lactic acid bacteria-fermented goat milk containing the DPP-IV inhibitory peptide. The fermented goat milk is then added with prebiotics and a heat protectant and dried to produce hypoglycemic lactic acid bacteria-fermented goat milk powder containing the DPP-IV inhibitory peptide. The lactic acid bacteria in the hypoglycemic lactic acid bacteria-fermented goat milk are inactivated to obtain hypoglycemic postbiotic goat milk, which is then concentrated and dried to obtain hypoglycemic postbiotic goat milk powder. The hypoglycemic goat milk products provided by the present invention all have a high DPP-IV inhibition rate and can be used by consumers to inhibit DPP-IV activity and thereby lower blood sugar, reducing the dosage of hypoglycemic medications and thus minimizing the side effects of hypoglycemic medications. Furthermore, the present invention decomposes proteins in goat milk into DPP-IV inhibitory peptides, isolates and purifies the DPP-IV inhibitory peptides, and identifies their amino acid sequences, thereby determining the location of the DPP-IV inhibitory peptides within goat milk proteins.

[0089] Method for calculating relevant parameters of the aforementioned Lactobacillus helveticus producing a DPP-IV inhibitory peptide and its application in hypoglycemic goat dairy products:

[0090] 1. Dipeptidyl peptidase-IV (DPP-IV) inhibitory activity assay

[0091] Take a 96-well microtiter plate and add the reaction reagents as shown in Table 2-5. Use a microplate reader to detect the absorbance of the reaction solution at 405nm. Sitagliptin is used as a positive control. The inhibitory rate of sitagliptin concentration on DPP-IV is shown in Figure 23 .

[0092] Table 6 Amount of each reagent added during DPP-IV inhibitory activity determination (unit: μL)

[0093]

[0094]

[0095] 2. Chromaticity analysis

[0096] A Minolta CM-5 spectrophotometer was used for analysis. Samples were zeroed and calibrated with a white plate before analysis. Each sample was measured three times and the average value was obtained. The color difference between the sample and the control was expressed as DE, which was calculated according to Equation 2.

[0097]

[0098] 3 Lactic acid bacteria viable count determination

[0099] Reference to the method in GB 4789.35-2016.

Claims

1. A Lactobacillus helveticus KD12 producing a DPP-IV inhibitory peptide, characterized in that: The strain was classified and named Lactobacillus shelveticus. It was deposited in the China Center for Type Culture Collection on September 14, 2024, and its deposit number is CCTCCNO: M20241994.

2. The Lactobacillus helveticus KD12 producing a DPP-IV inhibitory peptide according to claim 1, characterized in that: The base pair sequence of the Lactobacillus helveticus KD12 is as shown in Sequence 1.

3. A hypoglycemic lactic acid bacteria fermented goat milk, characterized in that: The goat milk is fermented with the Lactobacillus helveticus KD12 according to claim 1 or 2 to obtain goat milk containing DPP-IV inhibitory peptide, i.e., goat milk fermented with hypoglycemic lactic acid bacteria, with a DPP-IV inhibition rate of 68.23%-92.72%.

4. The glucose-lowering lactic acid bacteria fermented goat milk according to claim 3, characterized in that: The amino acid sequences of the DPP-IV inhibitory peptides in goat milk fermented with hypoglycemic lactic acid bacteria are as follows: sequences 2 to 13: FPKYP, AGPFTPT, AIPY, KYIPIQY, PIQY, VLPVPQ, VLGP, LKALP, GKNKSQS, IRISHEL, FSDIPNP and PVPQ.

5. The glucose-lowering lactic acid bacteria fermented goat milk according to claim 3, characterized in that: The inoculation amount of Lactobacillus helveticus KD12 is 0.01%-0.05%, the fermentation time is 18h-24h, and the fermentation temperature is 34°C-40°C.

6. A hypoglycemic lactic acid bacteria fermented goat milk powder, characterized in that: The hypoglycemic lactic acid bacteria fermented goat milk according to claim 3 is dried to obtain the lactic acid bacteria goat milk powder, wherein the viable number of Lactobacillus helveticus KD1 in the lactic acid bacteria goat milk powder is 1.68×10 7 CFU / g~1.47×10 9 CFU / g, and the DPP-IV inhibition rate was 27.87%-85.75%.

7. The method for preparing a hypoglycemic lactic acid bacteria fermented goat milk powder according to claim 6, characterized in that: Directly spray-drying or vacuum freeze-drying the hypoglycemic lactic acid bacteria goat milk according to claim 3 to obtain hypoglycemic lactic acid bacteria goat milk powder; or Prebiotics and full-fat goat milk powder are added to the hypoglycemic lactic acid bacteria goat milk according to claim 3, and the mixture is evenly mixed and then subjected to high-temperature spray drying or vacuum low-temperature spray drying to obtain hypoglycemic lactic acid bacteria goat milk powder.

8. A hypoglycemic postbiotic goat milk, characterized in that: The hypoglycemic lactic acid bacteria fermented goat milk according to claim 3 is sterilized and inactivated to obtain hypoglycemic postbiotic goat milk, and its DPP-IV inhibition rate is 81.50%-85.83%.

9. The hypoglycemic postbiotic goat milk according to claim 8, characterized in that: The heat sterilization is carried out at 90° C. for 15 minutes or at 116° C. for 10 minutes.

10. A hypoglycemic postbiotic goat milk powder, characterized in that: The hypoglycemic postbiotic goat milk according to claim 8 is concentrated to a solid content of 45%-52%, and then spray-dried to obtain hypoglycemic postbiotic goat milk powder.

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