Lactobacillus helveticus and its use in goat's milk products: blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria

Abstract

Providing the use of Lactobacillus helveticus and blood glucose-lowering / uric acid-lowering peptide lactic acid bacteria in goat milk products. [Solution] Using raw or reconstituted goat's milk as a raw material, the goat's milk is fermented using Lactobacillus helveticus KD12, which produces α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides. By decomposing the proteins in the goat's milk into α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides, blood glucose-lowering and uric acid-lowering peptide-fermented goat's milk containing α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides is obtained. The α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides are separated and purified, and their amino acid sequences are identified to determine the positions of the α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides within the goat's milk proteins.

Description

【Technical Field】 【0001】 The present invention belongs to the technical field of fermentation engineering, and specifically relates to Lactobacillus helveticus and its use in lactobacillus goat dairy products with blood sugar-lowering and uric acid-lowering peptides. 【Background Art】 【0002】 According to the data of the "IDF Diabetes Atlas" (11th Edition) published by the International Diabetes Federation (IDF) in April 2025, the number of diabetic patients in the world has already reached 643 million, and one in nine adults (20 - 79 years old) suffers from diabetes, with the total number reaching 589 million. Currently, the treatment of diabetes mainly relies on drugs, especially α-glucosidase inhibitors (such as acarbose and miglitol). This type of drug competitively inhibits α-glucosidase in the intestinal epithelial mucosa and delays the rate at which carbohydrates are decomposed into monosaccharides in the intestine, thereby effectively regulating the postprandial blood glucose level of patients. 【0003】 Although drug treatment shows significant effects in blood glucose control, its side effects cannot be ignored. Common side effects include significant fluctuations in body weight, osteoporosis, gastrointestinal dysfunction, and an increased risk of cardiovascular diseases. These side effects not only affect the quality of life of patients but may also cause new health problems. Therefore, the search for a safer and more effective diabetes intervention strategy has become a hot spot in the research field. In view of the limitations of drug treatment, food-derived α-glucosidase inhibitory peptides have attracted wide attention. This type of peptide fragment can reduce the activity of α-glucosidase, thereby delaying the decomposition rate of carbohydrates and achieving blood sugar reduction. Although there are differences in the amino acid sequences and lengths of different peptide fragments, they all show good blood sugar-lowering effects and are collectively referred to as blood sugar-lowering peptides. Compared with chemically synthesized drugs, food-derived blood sugar-lowering peptides have higher safety and are suitable for long-term intake. 【0004】 Hyperuricemia is a metabolic disorder characterized by persistently elevated serum uric acid levels that exceed solubility limits, leading to the formation of sodium urate crystals. These crystals deposit in joints, kidneys, and other organs, triggering inflammatory responses and ultimately resulting in gout. Currently, treatment for hyperuricemia primarily relies on xanthine oxidase inhibitors (such as allopurinol and febuxostat). These drugs reduce uric acid production by inhibiting the activity of xanthine oxidase. However, these drugs also have a range of toxic side effects, including rashes, gastrointestinal reactions, bone marrow suppression, and liver dysfunction. 【0005】 Studies have shown a significant correlation and co-occurrence between diabetes and hyperuricemia. This correlation is primarily due to the effect of insulin on uric acid transport in the renal proximal tubules. High insulin concentrations promote sodium reabsorption in the tubules, thereby reducing uric acid excretion from the kidneys and leading to elevated serum uric acid levels. Therefore, scholars hypothesize a bidirectional causal relationship between hyperuricemia and insulin resistance. 【0006】 Given the limitations of drug therapy, dietary adjustments and moderate exercise have become important means of preventing and managing diabetes and hyperuricemia. Naturally derived key enzyme inhibitors for diabetes and hyperuricemia are highly valued due to their lack of side effects. In recent years, researchers have been actively studying non-pharmacological methods for lowering blood glucose and uric acid levels, particularly peptides that inhibit α-glucosidase and xanthine oxidase. These peptides are expected to effectively regulate patients' blood glucose and uric acid levels in a mild, non-toxic, and side-effect-free manner, potentially offering new solutions for the routine management of diabetes and hyperuricemia. [Overview of the Initiative] [Problems that the invention aims to solve] 【0007】 To solve the problems of the prior art, the present invention provides the use of Lactobacillus helveticus and lactic acid bacteria that produce blood glucose-lowering and uric acid-lowering peptides in goat milk products. Using raw or reconstituted goat milk as a raw material, the goat milk is fermented with Lactobacillus helveticus KD12, which produces α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides, thereby decomposing the proteins in the goat milk into α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides, and obtaining fermented goat milk containing α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides. By inactivating the lactic acid bacteria in the obtained fermented goat milk, sterile blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk is obtained. Alternatively, a nutritional fortifier is added to the fermented goat milk and concentrated, and then spray-dried to obtain powder of blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk. Furthermore, by isolating and purifying α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides and identifying their amino acid sequences, the positions of these peptides within goat milk protein are determined. This allows the peptides to be provided for blood glucose-lowering and uric acid-lowering peptide foods, providing reference material for the development of blood glucose-lowering and uric acid-lowering drugs. [Means for solving the problem] 【0008】 To achieve the above objective, the present invention provides the following technical scheme: the use of Lactobacillus helveticus KD12 in the production of a blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk product. A blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk product is obtained by fermenting goat milk with Lactobacillus helveticus KD12. The Lactobacillus helveticus KD12 was deposited with the China Center for the Preservation of Typical Cultures on September 14, 2024, with deposit number CCTCC NO: M20241994. 【0009】 Furthermore, the aforementioned blood glucose-lowering and uric acid-lowering peptides can inhibit α-glucosidase and xanthine oxidase. 【0010】 Furthermore, the amino acid sequence of the blood glucose-lowering / uric acid-lowering peptide is as follows: PTVN, LPYP, VEPF, VAYGL, SLTLT, VLPVPQ, VMFPPQ, LAFNPTQ, FMAIPPK, FNPTQLE, KYIPIQY, FYQKFPQ, LTDDYKKYL, LKDYGGVSLPE, and AVATWLKPDPSQK. The blood glucose-lowering / uric acid-lowering peptide is used in the manufacture of blood glucose-lowering / uric acid-lowering foods or pharmaceuticals. 【0011】 The present invention also provides fermented goat's milk containing glucose-lowering and uric acid-lowering peptides. Fermented goat's milk containing glucose-lowering and uric acid-lowering peptides is obtained by fermenting goat's milk using Lactobacillus helveticus KD12. The Lactobacillus helveticus KD12 was deposited with the China Center for the Preservation of Typical Cultures on September 14, 2024, with deposit number CCTCC NO:M20241994. 【0012】 Furthermore, the aforementioned blood glucose-lowering and uric acid-lowering peptides can inhibit α-glucosidase and xanthine oxidase, with inhibition rates of 76.80% to 99.87% for α-glucosidase and 90.22% to 99.33% for xanthine oxidase. 【0013】 Furthermore, the amino acid sequences of the aforementioned blood glucose-lowering and uric acid-lowering peptides are as follows: PTVN, LPYP, VEPF, VAYGL, SLTLT, VLPVPQ, VMFPPQ, LAFNPTQ, FMAIPPK, FNPTQLE, KYIPIQY, FYQKFPQ, LTDDYKKYL, LKDYGGVSLPE, and AVATWLKPDPSQK. 【0014】 This invention also provides a method for producing fermented goat's milk with the above-mentioned blood glucose-lowering and uric acid-lowering peptides. The specific steps are as follows: Sterilized and cooled goat's milk is inoculated with either 0.01% to 0.05% Lactobacillus helveticus KD12 direct-addition fermenting agent, or 2% to 5% activated Lactobacillus helveticus KD12. Fermentation is carried out at a constant temperature of 37°C for 12 to 18 hours to obtain fermented goat's milk with glucose-lowering and uric acid-lowering peptides and lactic acid bacteria. 【0015】 The present invention also provides goat milk powder containing glucose-lowering and uric acid-lowering peptide lactic acid bacteria. The glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder is obtained by drying the above-mentioned glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat milk. The number of viable Lactobacillus helveticus KD12 cells in the milk powder is not less than 10 million CFU / g. 【0016】 This invention also provides a method for producing the above-mentioned blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder. The specific steps are as follows: By employing high-temperature spray drying or freeze-drying, goat milk fermented with lactic acid bacteria that produce blood glucose-lowering and uric acid-lowering peptides is directly dried to obtain goat milk powder that produces blood glucose-lowering and uric acid-lowering peptides. or To obtain blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat milk, whole fat goat milk powder, nutritional fortifiers, and one or more novel food ingredients are added to the blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat milk, and after the solid content is increased to 40% to 50%, high-temperature spray drying or low-temperature vacuum spray drying is performed to obtain blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder. 【0017】 The present invention also provides goat's milk containing inactivated blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria. The inactivated blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat's milk is obtained by heat-treating the above-mentioned blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat's milk at 90°C for 15 minutes, or at 116°C for 10 minutes. [Effects of the Invention] 【0018】 Compared to the prior art, the present invention has at least the following beneficial effects. 【0019】 The present invention provides Lactobacillus helveticus KD12 that produces an α-glucosidase inhibitory peptide and a xanthine oxidase inhibitory peptide. It was deposited with the China Center for Type Culture Collection on September 14, 2024, and the deposit number was CCTCC NO: M20241994. Lactobacillus helveticus KD12 is derived from kefir grains and has the advantages of being natural, green, and safe. 【0020】 The present invention provides a lactic acid bacteria-fermented goat milk with blood sugar-lowering and uric acid-lowering peptides. Goat milk is fermented using Lactobacillus helveticus KD12 that produces an α-glucosidase inhibitory peptide and a xanthine oxidase inhibitory peptide. The fermented goat milk has a high α-glucosidase inhibition rate and xanthine oxidase inhibition rate and can be used by consumers for blood sugar lowering and uric acid lowering. 【0021】 The present invention also provides blood sugar-lowering and uric acid-lowering inhibitory peptides. These are derived from goat milk proteins and belong to food-derived bioactive peptides. By using these as food supplements for blood sugar lowering and uric acid lowering, the dosage of blood sugar-lowering and uric acid-lowering drugs can be reduced and side effects can be alleviated. They can also be used in the development of blood sugar-lowering and uric acid-lowering drugs. 【0022】 The present invention provides a lactic acid bacteria goat milk powder with blood sugar-lowering and uric acid-lowering peptides. The manufacturing process employs conventional spray drying or vacuum freeze drying. The viable count of the produced lactic acid bacteria goat milk powder with blood sugar-lowering and uric acid-lowering peptides all meets the requirement that the viable count in the national standard is 10 6 CFU / g or more. Since conventional spray drying can utilize the existing production equipment of enterprises, it is suitable for industrial production. When vacuum freeze drying is adopted, the viable count of lactic acid bacteria is high and the rehydration characteristics are also good. 【0023】 The present invention provides an inactivated hypoglycemic and hypouricemic peptide lactic acid bacterium goat milk, which has a high α-glucosidase inhibition rate and xanthine oxidase inhibition rate. Since there are no viable lactic acid bacteria, it does not require a refrigeration chain to maintain low temperature during transportation and sales, and has a long shelf life, thus reducing transportation and sales costs. Furthermore, it can be used by consumers for hypoglycemic and hypouricemic purposes. 【Brief Description of the Drawings】 【0024】 [Figure 1] It is a diagram showing the α-glucosidase inhibition rate and xanthine oxidase inhibition rate of fermented goat milk by 4 strains of lactic acid bacteria. [Figure 2] It is a diagram showing the effect of simulated gastrointestinal fluid digestion on the hypoglycemic and hypouricemic activities of Lactobacillus helveticus KD12 fermented goat milk. [Figure 3] It is a diagram showing the effect of subculture type and direct-inoculation type fermenting agents on the hypoglycemic and hypouricemic activities of Lactobacillus helveticus KD12 fermented goat milk. [Figure 4] It is a chromatogram of the component separation of Lactobacillus helveticus KD12 fermented goat milk. [Figure 5] It is a diagram showing the α-glucosidase inhibition rate and xanthine oxidase inhibition rate of each component of Lactobacillus helveticus KD12 fermented goat milk. [Figure 6] It is a secondary mass spectrometry diagram of 15 kinds of peptides in the FG2 component. [Figure 7] It is a secondary mass spectrometry diagram of 15 kinds of peptides in the FG2 component. [Figure 8] It is a secondary mass spectrometry diagram of 15 kinds of peptides in the FG2 component. [Figure 9] It is a secondary mass spectrometry diagram of 15 kinds of peptides in the FG2 component. [Figure 10] It is a secondary mass spectrometry diagram of 15 kinds of peptides in the FG2 component. [Figure 11] It is a secondary mass spectrometry diagram of 15 kinds of peptides in the FG2 component. [Figure 12] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 13] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 14] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 15] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 16] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 17] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 18] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 19] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 20] This is a secondary mass spectrometry diagram of 15 different peptides in the FG2 component. [Figure 21a] These are HPLC purity analysis and primary mass spectrometry diagrams for FYQKFPQ. [Figure 21b] These are HPLC purity analysis and primary mass spectrometry diagrams for FYQKFPQ. [Figure 22a] These are HPLC purity analysis and primary mass spectrometry diagrams for VEPF. [Figure 22b] These are HPLC purity analysis and primary mass spectrometry diagrams for VEPF. [Figure 23a] These are HPLC purity analysis and primary mass spectrometry diagrams for FNPTQLE. [Figure 23b] These are HPLC purity analysis and primary mass spectrometry diagrams for FNPTQLE. [Figure 24] This figure shows the α-glucosidase inhibition rate and xanthine oxidase inhibition rate of sterilized blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria in goat's milk. [Modes for carrying out the invention] 【0025】 The present invention will be further described below with reference to the drawings and specific embodiments. 【0026】 This invention provides for the use of Lactobacillus helveticus KD12 and its blood glucose-lowering / uric acid-lowering peptide lactic acid bacteria in goat milk products. The lactic acid bacteria is Lactobacillus helveticus KD12, with the taxonomic name Lactobacillus helveticus, the deposit number CCTCC NO:M20241994, the deposit date September 14, 2024, the depositary institution China Center for the Preservation of Typical Cultures, located at Wuhan University, 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, postal code: 430072. 【0027】 This invention provides the use of Lactobacillus helveticus KD12 and its properties in goat milk products containing glucose-lowering and uric acid-lowering peptide lactic acid bacteria. Using raw or reconstituted goat milk as a raw material, the goat milk is fermented with Lactobacillus helveticus KD12 to decompose the proteins in the goat milk into α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides, thereby obtaining glucose-lowering and uric acid-lowering peptide lactic acid bacteria-fermented goat milk containing α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides. By inactivating the lactic acid bacteria in the obtained fermented goat milk, sterile glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk is obtained. Furthermore, after concentrating the glucose-lowering and uric acid-lowering peptide lactic acid bacteria-fermented goat milk with or without the addition of nutritional fortifiers, glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder is obtained by spray drying. Furthermore, by isolating and purifying α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides and identifying their amino acid sequences, we determined their positions within goat milk protein. This allows us to provide blood glucose-lowering and uric acid-lowering peptides for blood glucose-lowering and uric acid-lowering peptide foods, providing a reference for the development of blood glucose-lowering and uric acid-lowering drugs. 【0028】 Example 1: Isolation and identification of Lactobacillus helveticus KD12 【0029】 1. Isolation of Lactobacillus helveticus KD12, which produces glucose-lowering and uric acid-lowering peptides. 【0030】 Milk kefir granules were inoculated at 3% into sterile, cooled goat's milk and fermented at room temperature for 24 hours to obtain the sample. After a series of dilutions, lactic acid bacteria were isolated by plate smearing, and repeated primary and secondary screenings were performed to obtain one strain of lactic acid bacteria with high viability for producing blood glucose-lowering and uric acid-lowering peptides. 【0031】 2. Identification of Lactobacillus helveticus KD12, which produces glucose-lowering and uric acid-lowering peptides. 【0032】 2.1 Colony and Cellular Morphology 【0033】 Lactobacillus helveticus KD12, which produces blood glucose-lowering and uric acid-lowering peptides, was cultured on MRS agar for 48 hours, and distinct colonies formed on the MRS medium. The colonies were circular with well-defined edges, milky white, moist and smooth on the surface, and did not produce any pigment. After toluidine blue staining, the bacterial morphology of Lactobacillus helveticus KD12 producing α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides was long and rod-shaped, with some bacteria exhibiting curvature. 【0034】 2.2 Identification of bacterial species 【0035】 The bacterial strain was identified using 16S rDNA. After amplifying and purifying the target fragment, the strain was characterized based on homology analysis of the 16S rDNA partial gene fragment. By comparing the obtained sequence with the nucleotide sequence in the NCBI database, the strain was confirmed to be Lactobacillus helveticus. Its nucleotide sequence is shown in Sequence ID No. 1: 【0036】 The strain in question was named Lactobacillus helveticus KD12. Lactobacillus helveticus KD12 was deposited on September 14, 2024, at the China Center for the Preservation of Typical Cultures, Wuhan University, 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with deposit number CCTCC NO:M20241994. 【0037】 Example 2: Preparation of goat's milk fermented with glucose-lowering and uric acid-lowering peptides. 【0038】 1. Blood glucose-lowering and uric acid-lowering peptide activity of fermented goat's milk by four strains of lactic acid bacteria. 【0039】 Freeze-dried fungal powders of Lactobacillus helveticus KD12, Lactobacillus rhamnosus KD5, Lactobacillus plantarum ATCC 8014, and Bifidobacterium bifidum BB01 were inoculated into goat's milk that had been sterilized at 95°C for 10 minutes and then cooled to activate the fungi. The mixtures were then fermented at a constant temperature of 37°C for 24 hours, and this activation process was repeated three times. The four activated fungal strains were each inoculated at a 3% dose into sterilized and cooled goat's milk and cultured at 37°C for 18 hours. The supernatant was obtained by centrifugation, and its α-glucosidase inhibition rate, xanthine oxidase inhibition rate, pH value, and acidity were measured. The hypoglycemic agent acarbose (0.124 mmol / L) and the uric acid-lowering agent allopurinol (0.294 mmol / L) were used as controls, and the results are shown in Figure 1. 【0040】 Figure 1(a) shows that fermented goat milk from all four strains of bacteria exhibits inhibitory activity against α-glucosidase. The α-glucosidase inhibition rates of the fermented goat milk supernatants of Lactobacillus helveticus KD12, Lactobacillus rhamnosus KD5, Lactobacillus plantarum ATCC 8014, and Bifidobacterium bifidum BB01 were 95.32%, 6.83%, 1.33%, and 8.76%, respectively. The α-glucosidase inhibition rate of the positive control, the hypoglycemic agent acarbose (0.124 mmol / L), was 83.30%. Therefore, the hypoglycemic activity of Lactobacillus helveticus KD12 fermented goat milk was significantly higher than that of the other three strains and the hypoglycemic agent acarbose at 0.124 mmol / L. 【0041】 Figure 1(b) shows that fermented goat milk from all four strains of bacteria exhibits inhibitory activity against xanthine oxidase. The xanthine oxidase inhibition rates of the fermented goat milk supernatants of Lactobacillus helveticus KD12, Lactobacillus rhamnosus KD5, Lactobacillus plantarum ATCC 8014, and Bifidobacterium bifidum BB01 were 97.57%, 65.20%, 3.47%, and 24.24%, respectively. The xanthine oxidase inhibition rate of the positive control, the uric acid-lowering drug allopurinol (0.294 mmol / L), was 77.50%. Therefore, the uric acid-lowering activity of Lactobacillus helveticus KD12 fermented goat milk was significantly higher than that of the other three strains and the uric acid-lowering drug allopurinol at 0.294 mmol / L. 【0042】 2. Effects of simulated gastrointestinal fluid digestion on the blood glucose-lowering and uric acid-lowering activity of Lactobacillus helveticus KD12 fermented goat's milk. 【0043】 Pepsin and trypsin in the human digestive system hydrolyze high-molecular-weight proteins and polypeptides in milk into lower-molecular-weight polypeptides, but this process may inhibit or enhance the functional properties of bioactive peptides. Therefore, it is necessary to investigate the functional stability of α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides in Lactobacillus helveticus KD12 fermented goat milk after digestion with simulated gastrointestinal fluid. In vitro simulated gastrointestinal digestion can be used to study the intestinal degradation of α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides and to explore their bioavailability and bioactivity in the body. 【0044】 A study of simulated gastrointestinal fluid digestion was conducted using fermented goat's milk containing Lactobacillus helveticus KD12. pH values ​​were adjusted using 1 mol / L HCl solution and 1 mol / L NaOH solution. 【0045】 Simulated gastric juice digestion test: After adjusting the pH of Lactobacillus helveticus KD12 fermented goat milk to 1.5, pepsin was added at a concentration of 2% (w / v), mixed uniformly, and reacted at 37°C for 2 hours. After inactivating the enzymes at 98°C for 10 minutes, the whey sample was collected by centrifugation, and the inhibition rates against α-glucosidase and xanthine oxidase were measured. 【0046】 Simulated intestinal fluid digestion test: After adjusting the pH of Lactobacillus helveticus KD12 fermented goat milk to 1.5, pepsin was added at a concentration of 2% (w / v), mixed uniformly, and reacted at 37°C for 2 hours. After adjusting the pH again to 7.5, trypsin was added at a concentration of 2% (w / v), mixed uniformly, and reacted at 37°C for 4 hours. After inactivating the enzymes at 98°C for 10 minutes, the whey sample was collected by centrifugation, and the inhibition rates for three types of enzymes were measured. 【0047】 Figure 2 shows the results of the effects of simulated gastrointestinal fluid digestion on the blood glucose-lowering and uric acid-lowering activity of Lactobacillus helveticus KD12 fermented goat's milk. 【0048】 Figure 2 shows that Lactobacillus helveticus KD12 fermented goat milk did not show any significant change in its α-glucosidase inhibition rate or xanthine oxidase inhibition rate after treatment with simulated gastric juice (p>0.05). However, both rates significantly decreased after treatment with simulated intestinal juice (p<0.05). The α-glucosidase inhibition rate decreased from 98.53% to 81.83%, and the xanthine oxidase inhibition rate decreased from 99.81% to 75.60%. 【0049】 3. Effects of subculturing and direct-addition fermentation on the blood glucose-lowering and uric acid-lowering activity of Lactobacillus helveticus KD12 fermented goat's milk. 【0050】 After activating the Lactobacillus helveticus KD12 strain through culture, the bacterial sediment was collected by centrifugation and washed twice with sterile physiological saline. After collecting the bacterial sediment by centrifugation, a freeze-drying protective agent was added at a fixed rate, mixed uniformly, and freeze-dried to obtain a bacterial powder, which was used as a direct-addition fermentation agent. This was compared with subculturing fermentation. The inoculation amount for the direct-addition fermentation agent was 0.01% (w / v), and the inoculation amount for subculturing was 3% (v / v). The cultures were incubated at 37°C, and samples were taken every 3 hours to measure pH, acidity, and the inhibitory activity of α-glucosidase and xanthine oxidase. The results are shown in Figure 3. 【0051】 Figure 3 shows that in subculture fermentation, the decrease in pH and increase in acidity progressed rapidly. Between 0 and 9 hours, the pH decreased from 6.12 to 4.32, and the acidity increased from 17.5°T to 80°T. On the other hand, after direct inoculation with fermenting agent, the decrease in pH was slow between 0 and 9 hours, with the pH decreasing from 6.67 to 5.58 and the acidity increasing from 10°T to 36°T. Between 9 and 12 hours, the pH decreased rapidly, and after 12 hours of fermentation, both were below pH 4.5. After 15 hours, the pH values ​​of goat milk fermented by direct inoculation and subculture fermentation showed a tendency to coincide, reaching approximately 3.65. The pH decrease curve and acidity increase curve for goat milk fermented with direct inoculation with fermenting agent both showed an "S" shape, suggesting that there is an adaptation period for using Lactobacillus helveticus KD12 goat milk after direct inoculation with fermenting agent. 【0052】 The α-glucosidase and xanthine oxidase inhibition rates of subcultured fermented goat's milk at 9 hours were 21.12% and 44.02%, respectively, and thereafter rapidly increased, reaching 90.37% and 90.22% after 12 hours, and 99.87% and 99.21% after 18 hours. No α-glucosidase inhibition or xanthine oxidase inhibition was observed in fermented goat's milk inoculated with direct-addition fermentation agents at 9 hours. However, these inhibition rates increased rapidly thereafter, reaching 76.80% and 92.46% respectively at 12 hours of fermentation, and rising to 99.60% and 99.33% respectively at 18 hours of fermentation. 【0053】 Therefore, it was shown that lactic acid bacteria-fermented goat milk with high blood glucose-lowering and uric acid-lowering peptide activity can be obtained regardless of whether a subculture or direct-addition fermentation agent is used to ferment the goat milk. 【0054】 Example 3: Preparation, identification, and study of blood glucose-lowering and uric acid-lowering peptides and their blood glucose-lowering and uric acid-lowering activities. 【0055】 Whey was obtained by centrifuging goat's milk fermented with lactic acid bacteria containing α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides that lower blood glucose and uric acid levels. Furthermore, ultrafiltration was performed using a filter membrane with a molecular weight of less than 3 kDa, and the permeate was freeze-dried to obtain freeze-dried powder. The freeze-dried sample was dissolved in ultrapure water, and components were collected using high-performance liquid chromatography. F1 to F4 are components collected at 2-3 minutes, 3-4 minutes, 8-10 minutes, and 10-14 minutes, respectively (Figure 4). After freeze-drying the collected components, their α-glucosidase inhibition rate and xanthine oxidase inhibition rate were measured. The results are shown in Figure 4. From Figure 5, the α-glucosidase inhibition rate and xanthine oxidase inhibition rate of component F2 were significantly higher than those of the other components, indicating that the blood glucose and uric acid-lowering peptides are mainly present in component F2. 【0056】 Component F2 was analyzed by HPLC-MS / MS to identify the amino acid sequences (SEQ ID NOs. 2-16) and origin of its blood glucose-lowering and uric acid-lowering peptides. The results are shown in Table 1. 【0057】 [Table 1] 【0058】 Table 1 shows that the 15 peptides in the freeze-dried powder containing blood glucose-lowering and uric acid-lowering peptides have 4 to 13 amino acids and a molecular weight range of 429.22 Da to 1439.78 Da, and that these are derived from goat's milk β-casein, αs1 and αs2-casein, κ-casein, α-lactalbumin, β-lactoglobulin, lactoferrin, and osteopontin. 【0059】 Secondary mass spectrometry data for each blood glucose-lowering and uric acid-lowering peptide are shown in Figures 6 to 20. 【0060】 To analyze the structure-activity relationship of blood glucose-lowering and uric acid-lowering peptides, FYQKFPQ, VEPF, and FNPTQLE were selected and synthesized using solid-phase synthesis. HPLC purity analysis and primary mass spectrometry of the three synthesized peptides are shown in Figures 21-23. The results showed that the purities of the synthesized FYQKFPQ, VEPF, and FNPTQLE were 98.17%, 97.68%, and 98.52%, respectively. These peptides were prepared at different concentrations, and their inhibition rates against α-glucosidase and xanthine oxidase were obtained. The IC50 of the inhibition rates against α-glucosidase and xanthine oxidase was then calculated. 50 The values ​​are shown in Table 2. 【0061】 [Table 2] 【0062】 Table 2 shows that FYQKFPQ has the strongest α-glucosidase inhibitory effect, and its IC 50 The value was only 84 μmol / L, but it did not have a xanthine oxidase inhibitory effect. On the other hand, both VEPF and FNPTQLE had α-glucosidase and xanthine oxidase inhibitory effects, and VEPF's IC50 50 The smaller value indicates that the inhibitory effect is superior. 【0063】 Example 4: Preparation of goat's milk powder containing glucose-lowering and uric acid-lowering peptide lactic acid bacteria. 【0064】 Using the fermented goat milk with blood glucose-lowering and uric acid-lowering peptides prepared in Example 2 as a raw material, blood glucose-lowering and uric acid-lowering peptides goat milk powders A and B were obtained by direct high-temperature spray drying (small spray dryer, YM-6000Y, Shanghai Yuming Instruments Co., Ltd.) and freeze-drying. Alternatively, 5% inulin and 22.5% whole fat goat milk powder were added to the fermented goat milk with blood glucose-lowering and uric acid-lowering peptides to bring the solid content to 40%, and blood glucose-lowering and uric acid-lowering peptides goat milk powders C and D were obtained by high-temperature spray drying and low-temperature vacuum spray drying (vacuum low-temperature spray dryer, BILON-VSD1500, Bilon Co., Ltd.), respectively. The conditions for high-temperature spray drying were an intake air temperature of 130°C, an exhaust air temperature of 70°C, and a peristaltic pump speed of 8%. The conditions for low-temperature vacuum spray drying were an intake air temperature of 70°C, an exhaust air temperature of 60°C, and a peristaltic pump speed of 40%. Four types of blood glucose-lowering and uric acid-lowering peptides and lactic acid bacteria goat's milk powder were reconstituted at 12.5%, and their α-glucosidase inhibition rates and xanthine oxidase inhibition rates were measured. The results are shown in Table 3. 【0065】 [Table 3] 【0066】 Table 3 shows that the α-glucosidase inhibition rate and xanthine oxidase inhibition rate of goat milk powder containing glucose-lowering and uric acid-lowering peptides prepared by direct high-temperature spray drying and freeze-drying were significantly higher than those of goat milk powder containing glucose-lowering and uric acid-lowering peptides prepared by inulin and whole-fat goat milk powder. This is because the addition of protective agents increased the solid content in the fermented goat milk, and rehydrating to the same concentration after drying diluted the glucose-lowering and uric acid-lowering peptides, leading to a decrease in the inhibition rate. The number of viable cells was highest in goat milk powder containing glucose-lowering and uric acid-lowering peptides prepared by freeze-drying, and lowest in goat milk powder containing glucose-lowering and uric acid-lowering peptides prepared by direct spray-drying. On the other hand, the number of viable cells in goat milk powder containing glucose-lowering and uric acid-lowering peptides prepared by inulin and whole-fat goat milk powder was intermediate between the two. However, the number of viable bacteria in the four types of blood glucose-lowering / uric acid-lowering peptide lactic acid bacteria in goat's milk powder was 1.0 × 10⁶ in all cases. 6It significantly exceeded the national standard requirement of CFU / g. 【0067】 Example 5: Preparation of goat's milk containing inactivated blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria. 【0068】 Inactivated goat milk fermented with lactic acid bacteria containing blood glucose-lowering and uric acid-lowering peptides was obtained by heat sterilization at 90°C for 15 minutes or at 116°C for 10 minutes. The supernatant was collected by centrifugation, and its α-glucosidase inhibition rate and xanthine oxidase inhibition rate were measured. The results are shown in Figure 24. From Figure 24, although no live lactic acid bacteria were detected after heat treatment, there was no significant difference in the α-glucosidase inhibition rate and xanthine oxidase inhibition rate compared to the untreated group. This indicates that although the lactic acid bacteria are inactivated by heat treatment, there is no significant change in their blood glucose-lowering and uric acid-lowering peptide activity. 【0069】 In summary, the present invention provides the use of Lactobacillus helveticus KD12 and its properties in goat milk products containing lactic acid bacteria that produce blood glucose-lowering and uric acid-lowering peptides. Using raw or reconstituted goat milk as a raw material, the goat milk is fermented using Lactobacillus helveticus KD12, which produces α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides, thereby decomposing the proteins in the goat milk into α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides to obtain fermented goat milk containing α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides. By inactivating the lactic acid bacteria in the obtained fermented goat milk, sterile goat milk containing blood glucose-lowering and uric acid-lowering peptides is obtained. Furthermore, after concentrating the fermented goat milk containing blood glucose-lowering and uric acid-lowering peptides, with or without the addition of nutritional fortifiers, spray-drying is performed to obtain powder of goat milk containing blood glucose-lowering and uric acid-lowering peptides. Furthermore, by isolating and purifying α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides and identifying their amino acid sequences, we determined their positions within goat milk protein. This provides a reference for the development of foods containing blood glucose-lowering and uric acid-lowering peptides, as well as for the development of blood glucose-lowering and uric acid-lowering drugs. 【0070】 The calculation method for the parameters related to the use of Lactobacillus helveticus KD12 and glucose-lowering / uric acid-lowering peptide lactic acid bacteria in goat milk products is as follows. 【0071】 1. Measurement of α-glucosidase inhibitory activity 【0072】 Reaction reagents were added to a 96-well microtiter plate according to the design shown in Table 4. The absorbance of the reaction solution at 405 nm was measured using an enzyme labeling analyzer, and acarbose was used as a positive control. 【0073】 [Table 4] 【0074】 【number】 【0075】 2. Measurement of xanthine oxidase inhibitory activity 【0076】 A 96-well microtiter plate was used, and reaction reagents were added according to the design shown in Table 5. The absorbance of the reaction solution at 295 nm was measured using an enzyme labeling analyzer, and allopurinol was used as a positive control. 【0077】 [Table 5] 【0078】 【number】 【0079】 3. Measurement of the number of viable lactic acid bacteria 【0080】 Refer to the method described in Chinese National Standard GB 4789.35-2016. [Deposit Certificate] JPEG0007872646000009.jpg192168

Claims

[Claim 1] The use of Lactobacillus helveticus KD12 in the production of a goat milk product containing glucose-lowering and uric acid-lowering peptide lactic acid bacteria, wherein the goat milk product containing glucose-lowering and uric acid-lowering peptide lactic acid bacteria is obtained by fermenting goat milk using Lactobacillus helveticus KD12, and the Lactobacillus helveticus KD12 was deposited with the China Center for the Preservation of Typical Cultures on September 14, 2024, with deposit number CCTCC NO: M20241994. The use of Lactobacillus helveticus KD12 in the production of lactic acid bacteria goat milk products, characterized in that the blood glucose-lowering / uric acid-lowering peptide includes peptides represented by the amino acid sequences PTVN, LPYP, VEPF, VAYGL, SLTLT, VLPVPQ, VMFPPPQ, LAFNPTQ, FMAIPPK, FNPTQLE, KYIPIQY, FYQKFPQ, LDTDYKKYL, LKDYGGVSLPE, and AVATWLKPDPSQK. [Claim 2] The aforementioned blood glucose-lowering and uric acid-lowering peptides are α-glucosidase and xanthine oxidase. The use according to claim 1, characterized in that it can inhibit ze. [Claim 3] This is a fermented goat's milk product containing glucose-lowering and uric acid-lowering peptides, obtained by fermenting goat's milk using Lactobacillus helveticus KD12. The Lactobacillus helveticus KD12 was deposited with the China Center for the Preservation of Typical Cultures on September 14, 2024, with deposit number CCTCC NO: M20241994. The blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat's milk is characterized by containing peptides represented by the amino acid sequences PTVN, LPYP, VEPF, VAYGL, SLTLT, VLPVPQ, VMFPPPQ, LAFNPTQ, FMAIPPK, FNPTQLE, KYIPIQY, FYQKFPQ, LDTDYKKYL, LKDYGGVSLPE, and AVATWLKPDPSQK. [Claim 4] The blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat milk according to claim 3, characterized in that the aforementioned blood glucose-lowering and uric acid-lowering peptide can inhibit α-glucosidase and xanthine oxidase, with an inhibition rate of 76.80% to 99.87% for α-glucosidase and an inhibition rate of 90.22% to 99.33% for xanthine oxidase. [Claim 5] A method for producing fermented goat's milk with blood glucose-lowering and uric acid-lowering peptides according to claim 3 or 4, the specific steps being as follows: Sterilized and cooled goat's milk is inoculated with either 0.01% to 0.05% Lactobacillus helveticus KD12 direct-addition fermenting agent, or 2% to 5% activated Lactobacillus helveticus KD12. A method for producing fermented goat milk fermented with blood glucose-lowering and uric acid-lowering peptides according to claim 3 or 4, characterized by carrying out constant temperature fermentation at 37°C for 12 to 18 hours to obtain fermented goat milk fermented with blood glucose-lowering and uric acid-lowering peptides. [Claim 6] A goat milk powder containing glucose-lowering and uric acid-lowering peptide lactic acid bacteria, characterized in that the glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder is obtained by drying the glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat milk powder according to claim 3 or 4, and the number of viable Lactobacillus helveticus KD12 bacteria in the milk powder is not less than 10 million CFU / g. [Claim 7] A method for producing blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder according to claim 6, the specific steps being as follows: By employing high-temperature spray drying or freeze-drying, goat milk fermented with lactic acid bacteria that produce blood glucose-lowering and uric acid-lowering peptides is directly dried to obtain goat milk powder that produces blood glucose-lowering and uric acid-lowering peptides. or A method for producing blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder according to claim 6, characterized by adding whole fat goat milk powder, a nutritional fortifier, and one or more new resource food ingredients to blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat milk to adjust the solid content to 40% to 50%, and then performing high-temperature spray drying or low-temperature vacuum spray drying to obtain blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk powder. [Claim 8] Inactivated blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk, characterized in that the inactivated blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria goat milk is obtained by heat-treating the blood glucose-lowering and uric acid-lowering peptide lactic acid bacteria fermented goat milk described in claim 3 or 4 at 90°C for 15 minutes or at 116°C for 10 minutes.

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