A kind of antihypertensive peptide probiotic goat milk powder and preparation method thereof

Abstract

The invention provides a probiotic goat milk powder containing antihypertensive peptides and a preparation method thereof. Goat milk is used as a raw material, the goat milk is sterilized and then cooled, and a probiotic bacteria obtained by screening and fermenting goat milk to produce antihypertensive peptides, namely Lactobacillus rhamnosus KD5 (the strain has been preserved in the China Center for Type Culture Collection on September 7, 2023, and its preservation number is CCTCC NO: M20231641) is added, and the mixture is stirred evenly and fermented at a constant temperature. After the fermentation, probiotic fermented goat milk containing antihypertensive peptides is obtained. After the fermentation is completed, a nutritional enhancer for the middle-aged and elderly is added or not, and stirred evenly, and then the mixture is vacuum-low-temperature spray-dried to obtain the probiotic goat milk powder containing antihypertensive peptides. The goat milk powder has a high ACE inhibition rate and can be used by consumers to assist in lowering blood pressure.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation engineering, and specifically relates to antihypertensive peptide probiotic goat milk powder obtained by fermenting goat milk with Lactobacillus rhamnosus KD5 and then spray-drying at low temperature, and a preparation method thereof. Background Art

[0002] Currently, there are six major categories of drugs for treating hypertension: diuretics, calcium channel blockers, angiotensin II receptor antagonists (ARBs), angiotensin converting enzyme inhibitors (ACEIs), α-adrenergic antagonists, and β-blockers.

[0003] Among them, angiotensin converting enzyme inhibitors have good target organ protection and cardiovascular endpoint event prevention effects on hypertensive patients, and have clear antihypertensive effects and no adverse effects on glucose and lipid metabolism. Therefore, they are the fastest growing antihypertensive drugs today. Commonly used ACEI drugs include Captopril, Enalapril, Benazepril, Ramipril, Perindopril, Alacepril and Lisinopril, but these synthetic ACEIs can cause many adverse reactions, the most common of which is persistent dry cough. In addition, they can also cause hypotension, rash and taste disorders.

[0004] Since synthetic ACEI has many side effects and long-term use will produce multiple side effects, ACE inhibitory peptides derived from food have attracted people's attention. They are polypeptides that can reduce the activity of ACE. Although there are differences in their amino acid sequences and peptide lengths, they can all lower blood pressure, so they are also called antihypertensive peptides. Compared with chemically synthesized antihypertensive drugs, food-derived ACE inhibitory peptides have extremely high safety. Studies have shown that ACE inhibitory peptides have a better antihypertensive effect on patients with hypertension, but have no effect on people with normal blood pressure and no side effects. This selectivity of antihypertensive effect is not available in ordinary chemically synthesized antihypertensive drugs. Therefore, the industry has carried out various preparation studies on food-derived ACE inhibitory peptides.

[0005] Goat milk products mainly include infant formula goat milk powder, bulk milk powder and dry-mixed formulated goat milk powder. There are few product varieties, serious homogeneity and fierce competition. Therefore, it is necessary to develop functional goat milk powder. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a antihypertensive peptide probiotic goat milk powder and a preparation method thereof. Fresh goat milk or reconstituted goat milk is used as a raw material, and the goat milk is fermented by Lactobacillus rhamnosus KD5 to decompose the protein in the goat milk into antihypertensive peptides, and the antihypertensive peptides are separated and purified and the amino acid sequence is identified to determine the position of the antihypertensive peptides in the goat milk protein. At the same time, the fermented goat milk is vacuum-low-temperature spray-dried to obtain the antihypertensive peptide probiotic goat milk powder.

[0007] To achieve the above object, the present invention provides the following technical solution: a Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )KD5, deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M20231641.

[0008] Furthermore, the Lactobacillus rhamnosus KD5 is non-hemolytic, sensitive to ciprofloxacin, chloramphenicol, clindamycin, ceftriaxone, gentamicin, penicillin, and tetracycline, has a hydrophobicity of 52.28% in xylene, a self-aggregation rate of 58.86%, and a co-aggregation rate of more than 40% for Escherichia coli and Staphylococcus aureus.

[0009] The present invention also provides a blood pressure reducing dairy product, which is obtained by fermenting goat milk with the above-mentioned Lactobacillus rhamnosus KD5 to obtain the dairy product containing blood pressure reducing peptides.

[0010] Furthermore, the amino acid sequence of the antihypertensive peptide is:

[0011] FLDDD, SLPEW, IMGVPK, FAWPQ, LHLPLP, MPIQAF, EPINIF, LPQNILP, LPYPYY, TPVVVPPF, LAFNPTQL, KNRLNFL, FVVAPFPE, LLTTDVEK, KYIPIQY, and VPPFLQPE.

[0012] The present invention also provides a hypotensive peptide probiotic goat milk, which is prepared by fermenting goat milk with the above-mentioned Lactobacillus rhamnosus KD5, the inoculation amount of Lactobacillus rhamnosus KD5 is 0.01%-0.05%, the ACE inhibition rate is 65.90%-83.25%, and the pH is 3.56-4.59.

[0013] The present invention also provides a method for preparing antihypertensive peptide probiotic goat milk, the specific steps of which are as follows:

[0014] S1: adding 0.01% to 0.05% of freeze-dried powder of Lactobacillus rhamnosus KD5 into sterilized and cooled goat milk;

[0015] S2 was fermented at a constant temperature of 34°C to 40°C for 22h to 26h to obtain antihypertensive peptide probiotic fermented goat milk.

[0016] Furthermore, a nutritional enhancer for the middle-aged and elderly is added to the antihypertensive peptide probiotic fermented goat milk, and the nutritional enhancer for the middle-aged and elderly is one or more of phosphatidylserine, acetylneuraminic acid, and prebiotics.

[0017] The present invention also provides a hypotensive peptide probiotic goat milk powder, which is prepared by fermenting goat milk with the above-mentioned Lactobacillus rhamnosus KD5 and then spray-drying, wherein the inoculation amount of Lactobacillus rhamnosus KD5 is 0.01% to 0.05%, and the IC value of the hypotensive peptide probiotic goat milk powder on ACE inhibition is 0.01% to 0.05%. 50 The value was 0.0545 g / mL~0.0840 g / mL, the survival rate of probiotics was 53.19%~83.58%, and the number of viable probiotics in antihypertensive peptide probiotic goat milk powder was 4.41×10 8 CFU / g~6.94×10 8 CFU / g.

[0018] The present invention also provides a method for preparing antihypertensive peptide probiotic goat milk powder, the specific steps of which are as follows:

[0019] S1: adding 0.01% to 0.05% of freeze-dried powder of Lactobacillus rhamnosus KD5 into sterilized and cooled goat milk;

[0020] S2 is fermented at a constant temperature of 34°C to 40°C for 22h to 26h to obtain antihypertensive peptide probiotic fermented goat milk;

[0021] S3 prepares antihypertensive peptide probiotic goat milk powder by vacuum low-temperature spray drying of the antihypertensive peptide probiotic fermented goat milk.

[0022] Furthermore, the conditions for the vacuum low-temperature spray drying are: vacuum negative pressure of 0.03Mpa~0.036Mpa, and air inlet temperature of 65°C~75°C.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] The present invention provides a Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) KD5, and on September 7, 2023, it was preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M20231641. The rhamnosus Lactobacillus KD5 has good hydrophobic properties, and it is speculated that it has good intestinal adhesion ability; it is sensitive to 7 antibiotics such as chloramphenicol, clindamycin, ceftriaxone, gentamicin, and tetracycline, so it is not easy to develop drug resistance, has high safety, and has strong self-copolymerization ability, showing higher acid and bile resistance than strains Lactobacillus plantarum 7830 and Lactobacillus rhamnosus L20. In addition, rhamnosus Lactobacillus KD5 is derived from milk kefir grains, which has the advantages of being natural, green, and safe.

[0025] The invention provides a blood pressure reducing peptide probiotic goat milk. Lactobacillus rhamnosus KD5 is used to process and ferment goat milk, thereby enriching the types of goat milk products. The fermented goat milk has a higher ACE inhibition rate and can be used by consumers to assist in lowering blood pressure.

[0026] The invention provides a blood pressure reducing peptide probiotic goat milk powder. The production process adopts vacuum low-temperature spray drying. The viable bacteria count of the prepared blood pressure reducing peptide probiotic goat milk powder is significantly higher than that of high-temperature spray drying, thereby avoiding the problems of low probiotic survival rate and viable bacteria count caused by conventional high-temperature spray drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The bacterial (a) and colony (b) morphology of Lactobacillus rhamnosus KD5;

[0028] Figure 2 MALDI-TOF-MS identification image of Lactobacillus rhamnosus KD5;

[0029] Figure 3 Hemolytic activity of Lactobacillus rhamnosus KD5 and Escherichia coli;

[0030] Figure 4 Antibiotic susceptibility of Lactobacillus rhamnosus KD5;

[0031] Figure 5 Tolerance of Lactobacillus rhamnosus KD5 to simulated gastric fluid (a) and intestinal fluid (b);

[0032] Figure 6 The hydrophobicity of Lactobacillus rhamnosus KD5;

[0033] Figure 7 The self-aggregation rate and co-aggregation of Lactobacillus rhamnosus KD5;

[0034] Figure 8 Coaggregation rate of Lactobacillus rhamnosus KD5 with Escherichia coli (a) and Staphylococcus aureus (b);

[0035] Fig. 9 ACE inhibition rate of goat milk fermented by Lactobacillus rhamnosus KD5;

[0036] Fig.10 Effects of fermentation conditions on the production of antihypertensive peptides by Lactobacillus rhamnosus KD5 in goat milk (a: temperature, b: inoculation amount, c: fermentation time);

[0037] Figures 11 to 26 Secondary mass spectrum of 16 peptides in antihypertensive peptide powder. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0039] The present invention provides a hypotensive peptide probiotic goat milk powder, wherein the probiotic is Lactobacillus rhamnosus KD5, which is classified and named Lacticaseibacillus rhamnosus , the deposit number is CCTCC NO: M20231641, the deposit time is September 7, 2023, the depository is China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0040] The invention provides a method for preparing antihypertensive peptide probiotic goat milk powder. Fresh goat milk or reconstituted goat milk is used as a raw material, Lactobacillus rhamnosus KD-5 is used to ferment the goat milk, proteins in the goat milk are decomposed into antihypertensive peptides, the antihypertensive peptides are separated and purified, and the amino acid sequence is identified to determine the position of the antihypertensive peptides in the goat milk protein, and the fermented goat milk is spray-dried at low temperature to prepare the antihypertensive peptide probiotic goat milk powder.

[0041] Example 1 Isolation and identification of Lactobacillus rhamnosus KD5

[0042] 1. Isolation of Lactobacillus rhamnosus KD5

[0043] Weigh a certain amount of whole goat milk powder, add distilled water in a ratio of 1:7 (w / v) to make reconstituted goat milk, sterilize at 115°C for 10 min, add milk kefir grains (purchased from the Taobao store Tibet Linzhi Natural Snow Lotus Mushroom, the website is: https: / / item.taobao.com / item.htm?_u=u3mt71o1c4a&id=16772532386&spm=a1z09.2.0.0.7f4d2e8dwTV9kw), ferment at 25°C for 22 h to obtain goat milk kefir, filter out the kefir grains after fermentation, rinse with sterilized saline to activate once, repeat the operation and continue to activate 4 times, crush the goat milk kefir obtained after the fourth activation, stir evenly, take 5 mL and inject it into 45 mL of sterile saline to mix well to make a diluent. Take 1 mL of the diluent and inject it into 9 mL of sterile saline and shake vigorously to make the bacteria evenly distributed. Repeat the above steps until the dilution factor reaches 10. -6 ~10 -8. Pipette 0.1mL of the diluent and spread it on an MRS agar plate (the ingredients are as follows: 10g peptone, 5g beef powder, 4g yeast powder, 2g potassium dihydrogen phosphate, 2g ammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar, 1000mL distilled water), and culture at 37ºC for 48h until obvious single colonies are formed. Select a plate with 30-80 single colonies from the culture medium, pick out typical colonies, and purify them by streaking on the MRS solid plate culture medium several times until the colony morphology on the entire plate is consistent. Select a single colony and inoculate it into MRS broth (10g peptone, 5g beef powder, 4g yeast powder, 2g potassium dihydrogen phosphate, 2g ammonium citrate, 5g sodium acetate, 20g glucose, Tween 80 1mL, magnesium sulfate 0.2g, manganese sulfate 0.05g, distilled water 1000mL) and then culture at 37℃ for 24h. Then, aseptically take 0.1mL of the shaken culture solution into a lyophilization tube in an ultra-clean workbench, add 0.1mL of sterile skim milk, mix well, freeze-dry, and store in a refrigerator.

[0044] 2. Identification of Lactobacillus rhamnosus KD5

[0045] 2.1 Colony and bacterial morphology

[0046] After Lactobacillus rhamnosus KD5 was cultured in MRS agar medium for 48 hours, 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 (b) The bacterial morphology of Lactobacillus rhamnosus KD5 after toluidine blue staining is long rod-shaped, and some of the bacteria are bent. Figure 1 (a).

[0047] 2.2. Identification of bacterial species

[0048] 2.2.1. Identification by fully automatic microbial mass spectrometer (VITEK MS, BioMérieux, France)

[0049] Lactic acid bacteria were inoculated on Columbia blood agar plates and cultured at 37°C for 24 hours. Pinpoint-sized colonies were evenly spread on the target plate, and 1 μL of matrix solution was added and mixed. After drying, the samples were loaded onto the fully automatic microbial mass spectrometer for sample analysis. The strain name was determined based on the characteristic peaks after comparison with the VITEK-MS mass spectrometer research library. The MALDI-TOF-MS identification diagram is shown in Figure 2 .

[0050] 22.2 Automatic microbial identification instrument (VITEK 2, BioMerieux, France)

[0051] Take the lactic acid bacteria colony, add 3 ml of 0.45% saline, adjust to a bacterial suspension with a McFarland turbidity of 0.5, insert the bacterial suspension into the Gram-positive bacillus identification card and place it on the microbial identification instrument. After the bacterial suspension is put on the machine, the biochemical reaction results are obtained. According to the biochemical results, the ASE8.01 database is compared to identify and determine the name of the bacterial species. The biochemical reaction identification results are shown in Table 1.

[0052] Table 1 Biochemical reaction identification results of Lactobacillus rhamnosus KD5

[0053]

[0054] Note: +. Biochemical reaction positive; –. Biochemical reaction negative

[0055] The mass spectrometry characteristic peaks of KD10 and KD5 were analyzed using the Launch pad software of the VITEK-MS mass spectrometer research library (RUO). Figure 2 The mass spectrum results of Lactobacillus rhamnosus KD5, the mass spectrum peaks m / z 2068.83, 2208.07, 4450.12, 4491.45, 5018.64, 5393.42, 7528.71, 8041.42, 8192.85, 15058.32, 16083.52 are the characteristic peaks of Lactobacillus rhamnosus, the identification results are in line with the credibility of more than 99%, Table 1 is the biochemical reaction results of Lactobacillus rhamnosus KD5, compared with the AES8.01 database, KD5 is Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), the identification results were all in line with the credibility of more than 99%. In summary, the two methods identified strain KD5 as Lactobacillus rhamnosus, and it was deposited in the China Center for Type Culture Collection on September 7, 2023, with the deposit numbers of CCTCC NO: M20231641.

[0056] Example 2 Test of probiotic properties of Lactobacillus rhamnosus KD5

[0057] 1. Hemolytic assay

[0058] Activated Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, Lactobacillus rhamnosus L20 and hemolytic Escherichia coli were spread on the surface of Columbia blood agar supplemented with 5% sterile defibrinated sheep blood and cultured at 37°C for 48 hours. The hemolytic activity of the probiotics was evaluated based on different halos. The results are shown in Figure 3 .

[0059] Depend on Figure 3It can be seen that an obvious transparent halo was observed on the Columbia blood agar plates inoculated with hemolytic Escherichia coli, which is β-hemolysis, while no hemolysis was observed on the Columbia blood agar plates inoculated with Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, and Lactobacillus rhamnosus L20, indicating that these three strains are non-hemolytic, that is, they are all safe strains. The lack of hemolysis ensures the safety of the strain and is used as one of the screening criteria for probiotic strains.

[0060] 2. Antibiotic Sensitivity Determination

[0061] A total of 9 antibiotics, including chloramphenicol (C, 30 μg), clindamycin (CC, 2 μg), ciprofloxacin (CIP, 5 μg), ceftriaxone (CTR, 30 μg), gentamicin (GM, 10 μg), penicillin (PEN, 10U), streptomycin (S, 10 μg), tetracycline (TET, 30 μg), and vancomycin (VAN, 30 μg), were used to evaluate the antibiotic resistance of Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, and Lactobacillus rhamnosus L20. The results are shown in the table. Figure 4 .

[0062] Depend on Figure 4 It can be seen that these three probiotics are generally sensitive to penicillin (PEN), with an inhibition zone ranging from 34.26 to 44.38 mm; Lactobacillus plantarum 7830 and Lactobacillus rhamnosus L20 are resistant to ciprofloxacin (CIP), streptomycin (S) and vancomycin (VAN), while Lactobacillus rhamnosus KD5 is sensitive to ciprofloxacin (CIP) with an inhibition zone of 10.71 mm; the inhibition zones of Lactobacillus rhamnosus KD5 to tetracycline (TET) and clindamycin (CC) are 28.71 mm and 26.31 mm, respectively. In summary, Lactobacillus rhamnosus KD5 is sensitive to seven antibiotics, including chloramphenicol (C), clindamycin (CC), ceftriaxone (CTR), gentamicin (GM), tetracycline (TET), etc., is not easy to develop drug resistance, and has high safety.

[0063] 3. Simulated gastrointestinal fluid tolerance

[0064] Prepare simulated gastric fluid: accurately weigh 4 g of pepsin, adjust the pH of the sterilized PBS solution to 2.0 with 1 mol / L HCl solution, and then make up to 100 mL with 0.1 mol / L HCl solution. After filtering and sterilizing, store at 4°C for later use.

[0065] Prepare simulated intestinal fluid: accurately weigh 0.2g trypsin and 1.8g ox bile salt, adjust the pH of the sterilized PBS solution to 8.0 with 1mol / L NaOH solution, and then make up to 100mL with 0.1mol / L NaOH solution. After filtering and sterilizing, store at 4℃ for later use.

[0066] 3.1 Simulated gastric fluid test

[0067] Inoculate 5% of the inoculum into the prepared artificial gastric juice with Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, and Lactobacillus rhamnosus L20, and place the mixture in a shaker at 37°C and 210 r / min for 3 hours. Collect the treated fluid at 0h, 1h, 2h, and 3h, and determine the number of viable bacteria. The results are shown in Table 1. Figure 5 (a).

[0068] 3.2 Simulated intestinal fluid test

[0069] The bacterial liquid treated in simulated gastric fluid for 3 hours was centrifuged and washed twice, and the bacterial sludge was collected and inoculated into the prepared artificial intestinal fluid. The treatment method was the same as that of gastric fluid. The results are shown in Figure 5 (b).

[0070] Depend on Figure 5 It is known that the survival rate of each strain in the simulated gastrointestinal fluid decreased with the extension of treatment time, and the survival rate was between 13.88% and 38.36%; the survival rate of strain 7830 was the highest, and the final survival rate was 38.36%; the survival rate of L20 was the lowest, and the final survival rate was only 13.88%; in the 3 hours of gastric juice treatment, the decrease in survival rate was significantly higher than that in intestinal fluid; in the first 3 hours of gastric juice treatment, the survival rate ranking of the three probiotics was: 7830>KD5>L20; in the last 2 hours of intestinal juice treatment, the survival rate of KD5 was better than that of strains 7830 and L20. It can be seen that the survival rate of probiotics in intestinal fluid is greatly affected by the early gastric juice treatment, and the decrease in survival rate in the later intestinal juice treatment gradually slows down.

[0071] 4. Hydrophobicity Determination

[0072] The hydrophobicity of Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, and Lactobacillus rhamnosus L20 was determined by the following formula. The results are shown in Figure 6 .

[0073]

[0074] Wherein: A is the absorbance before mixing with hydrophobic solvent; A0 is the absorbance after mixing with hydrophobic solvent.

[0075] Depend on Figure 6 It can be seen that the hydrophobicity of the three strains in xylene ranges from 21% to 52.28%, among which KD5 and 7830 have higher hydrophobicity. （P＜0.05）, 52.28% and 36.86% respectively. The surface hydrophobicity of bacteria mainly depends on the non-polar groups on the surface of bacteria, which may be related to structures such as surface proteins, pili, lipoteichoic acid and capsule. The hydrophobicity of probiotics directly reflects its adhesion ability to intestinal epithelial cells. In the present invention, KD5 shows good hydrophobicity, and it is speculated that it has good intestinal adhesion ability.

[0076] 5. Autoagglutination and coagglutination assay

[0077] 5.1 The formula for calculating the autoagglutination rate of Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, and Lactobacillus rhamnosus L20 is as follows:

[0078]

[0079] Where: A0 is the OD of bacterial suspension at 0h 600nm Value; A t OD measured at different time points after standing 600nm value.

[0080] 5.2 The coaggregation calculation formula of Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, Lactobacillus rhamnosus L20 and pathogenic bacteria is as follows:

[0081] The standard pathogenic strains used were Escherichia coli DC and Staphylococcus aureus JP stored frozen in the laboratory.

[0082]

[0083] Where: Apro+Apat is the OD of the mixed bacterial suspension at 0h 600nm Value; Amix is ​​OD at different time points 600nm value.

[0084] Autoagglutination refers to the aggregation of cells themselves, which protects the central cell from harmful substances from the outside world. Figure 7 It is known that with the extension of time, the self-agglutination rate of the strains showed an upward trend. After 24 hours, the ranking of the self-agglutination rate was: KD5>L20>7830, ranging from 31% to 58.86%.

[0085] In addition to the ability to self-aggregate, probiotics also have the ability to co-aggregate with pathogens. This ability allows probiotics to encapsulate pathogens and prevent them from colonizing in the gastrointestinal tract, which helps the health of the human digestive system. The co-aggregation rate results of Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830, and Lactobacillus rhamnosus L20 with pathogenic bacteria are shown in the figure below. Figure 8 shown.

[0086] Depend on Figure 8 It is known that the coaggregation rates measured for different pathogens are not much different. Figure 8 a), after 24 hours, the ranking of coagulation rate was: KD5>L20>7830, ranging from 46.41% to 48.01%; for Escherichia coli ( Figure 8 b) The overall trend of the coaggregation rate ranking after 24 hours is similar to that of Staphylococcus aureus, ranging from 41.65% to 43.47%, and the coaggregation rate of KD5 is 42.12%. Studies have found that probiotics with higher self-aggregation ability also have better coaggregation effects on pathogenic bacteria. This shows that the KD5 in the present invention has strong self-aggregation and copolymerization abilities.

[0087] Example 3 Study on the production of antihypertensive peptides by fermenting goat milk with Lactobacillus rhamnosus KD5

[0088] 1. Antihypertensive peptide activity of goat milk fermented with Lactobacillus rhamnosus KD5

[0089] The lyophilized powders of Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830 and Lactobacillus rhamnosus L20 were inoculated at an inoculum rate of 0.01% into goat milk sterilized at 95°C for 10 min and cooled to 37°C, fermented at 37°C for 22 h, and centrifuged to obtain the supernatant. The ACE inhibition rate was determined using an angiotensin converting enzyme inhibitor activity detection kit (BC5575-100T / 96S, Beijing Solarbio Technology Co., Ltd., the determination steps are shown in the instruction manual https: / / solarbio.com / goodsInfo?id=215539), and the antihypertensive drug captopril (0.25 μmol / L) was used as a control. The results are as follows: Fig. 9 shown.

[0090] Depend on Fig. 9 It can be seen that the fermented milk of the three strains of bacteria all contain antihypertensive peptides. The ACE inhibition rates of the supernatants of milk fermented by Lactobacillus rhamnosus KD5, Lactobacillus plantarum 7830 and Lactobacillus rhamnosus L20 were 68.56%, 21.04% and 31.63%, respectively. The ACE inhibition rate of 0.25 μmol / L antihypertensive drug captopril was 36.75%. It can be seen that the antihypertensive peptide activity in milk fermented by Lactobacillus rhamnosus KD5 was significantly higher than that of the other two strains and 0.25 μmol / L antihypertensive drug captopril.

[0091] 2. Effect of fermentation conditions on the production of antihypertensive peptides by Lactobacillus rhamnosus KD5 fermentation of goat milk

[0092] The freeze-dried powder of Lactobacillus rhamnosus KD5 was inoculated into goat milk as a starter to study the effects of inoculation amount (0.01%, 0.02%, 0.03%, 0.04%, 0.05%, w / v), fermentation temperature (31℃, 34℃, 37℃, 40℃, 43℃) and fermentation time (20h, 22h, 24h, 26h, 28h) on the production of antihypertensive peptides by Lactobacillus rhamnosus KD5 fermented goat milk, and the ACE inhibition rate and pH of fermented goat milk were determined. The basic experimental conditions were fermentation time of 22h, fermentation temperature of 37℃, and inoculation amount of 0.01%. The results are shown in Fig.10 shown.

[0093] Depend on Fig.10 It can be seen that with the increase of fermentation temperature, the ACE inhibition in goat milk fermented by Lactobacillus rhamnosus KD5 first increased and then decreased, and the pH first decreased and then increased, showing opposite trends. The ACE inhibition rate ranged from 40.82% to 79.59%. This is because the increase in temperature is beneficial to Lactobacillus rhamnosus KD5, which metabolizes to produce more antihypertensive peptides, but if the temperature is too high, the bacteria will age prematurely, resulting in less antihypertensive peptides.

[0094] With the increase of inoculum size, the ACE inhibition in goat milk fermented by Lactobacillus rhamnosus KD5 first increased and then decreased, and the pH gradually decreased. This may be because the increase in inoculum size is conducive to the metabolism and production of more antihypertensive peptides. However, the excessive inoculum size causes the nutrients in the goat milk to be consumed too quickly, resulting in the decomposition of antihypertensive peptides. The ACE inhibition rate ranges from 68.06% to 82.25%.

[0095] As the fermentation time increased, the ACE inhibition rate and pH of goat milk fermented by Lactobacillus rhamnosus KD5 gradually decreased, and the ACE inhibition rate ranged from 51.02% to 72.11%;

[0096] Taking the ACE inhibition rate higher than 65% as the goal, it can be seen that the suitable conditions for fermenting goat milk with Lactobacillus rhamnosus KD5 to produce antihypertensive peptides are: inoculation amount 0.01%~0.05%, temperature 31℃~40℃, and fermentation time 22h~26h.

[0097] Example 4 Preparation of antihypertensive peptides and peptide spectrum identification

[0098] The whey obtained by centrifugation of probiotic fermented goat milk with antihypertensive peptides was separated by ultrafiltration to obtain three components (A:>3kDa, B:1~3k Da and C:<1k Da). As shown in Table 4, the ACE inhibitory activity of the peptides is closely related to their relative molecular mass, and the ACE inhibition rate increases with the decrease of molecular weight. The antihypertensive peptide in component C has the best activity, with an ACE inhibition rate of 85.9%. The above results show that the activity of the 1k Da peptide component is improved after ultrafiltration, and it has a strong ACE inhibitory activity, indicating that the antihypertensive peptides are mainly concentrated in component C.

[0099] Table 2 Antihypertensive peptide activity of each ultrafiltration fraction

[0100]

[0101] Component C was further purified by Sephadex G-15, and the ACE inhibition rates of different components were compared. The component with the highest ACE inhibition rate was identified by HPLC-MS / MS, and 16 peptides were obtained (Table 3).

[0102] Table 3 Amino acid sequences of antihypertensive peptides

[0103]

[0104] As shown in Table 3, the number of amino acids in the 16 peptides in the antihypertensive peptide powder ranges from 5 to 8, and the molecular weight ranges from 623.24Da to 903.53Da. They come from β-casein, αs1 and αs2-casein, κ-casein, α-lactalbumin, β-lactoglobulin and lactoferrin from goat milk.

[0105] The secondary mass spectra of each goat milk antihypertensive peptide are shown in Figure 11 to Figure 26 .

[0106] Example 5 Preparation of antihypertensive peptide probiotic goat milk powder

[0107] The antihypertensive peptide probiotics fermented goat milk was dried by high temperature spray drying (small spray dryer, YM-6000Y, Shanghai Yuming Instrument Co., Ltd.) and vacuum low temperature spray drying (vacuum low temperature spray dryer, BILON-VSD1500, Biron Company). The high temperature spray drying conditions were 170℃ inlet air temperature and 85℃ outlet air temperature. The vacuum low temperature spray drying conditions were 65℃, 70℃ and 75℃, respectively. The negative pressure of drying was 0.03-0.04Mpa. The viable bacteria counts before and after spray drying were measured, the survival rate and viable bacteria count of probiotics were calculated, and the ACE inhibitory activity (IC 50 The results are shown in Table 4.

[0108] Table 4 Effects of high temperature and vacuum low temperature spray drying on the number of viable probiotics and the activity of antihypertensive peptides

[0109]

[0110] As shown in Table 4, the viable count of the antihypertensive peptide probiotic goat milk powder prepared by vacuum low-temperature spray drying is 4.41×10 8 CFU / g~6.94×10 8 CFU / g, probiotic survival rate was 53.19%~83.58%, ACE inhibitory activity IC 50The range was 0.055 g / mL to 0.084 g / mL, and the number of viable bacteria and the survival rate were significantly higher than those of the antihypertensive peptide probiotic goat milk powder obtained by high-temperature spray drying. The ACE inhibitory activity IC 50 Significantly lower than the antihypertensive peptide probiotic goat milk powder prepared by high temperature spray drying, ACE inhibitory activity IC 50 The smaller the value, the stronger the antihypertensive peptide activity is, and a lower concentration can inhibit ACE enzyme activity by 50%. It can be seen that vacuum low-temperature spray drying can improve the survival rate, viable bacteria count and ACE inhibitory activity of probiotics.

[0111] In summary, the present invention provides a antihypertensive peptide probiotic goat milk powder and a preparation method thereof. Goat milk is used as a raw material, the goat milk is sterilized and then cooled, and the screened probiotic bacteria for fermenting goat milk to produce antihypertensive peptides, namely Lactobacillus rhamnosus KD5, is added and stirred evenly, and then fermented at a constant temperature. After the fermentation is completed, probiotic fermented goat milk containing antihypertensive peptides is obtained. After adding or not adding a nutritional enhancer for the middle-aged and elderly, stirring evenly, the powder is vacuum-low-temperature spray-dried to obtain the antihypertensive peptide probiotic goat milk powder. The goat milk powder has a high ACE inhibition rate and can be used by consumers to assist in lowering blood pressure.

Claims

1. A Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) KD5, deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M20231641.

2. A Lactobacillus rhamnosus KD5 according to claim 1, characterized in that The Lactobacillus rhamnosus KD5 is non-hemolytic, sensitive to ciprofloxacin, chloramphenicol, clindamycin, ceftriaxone, gentamicin, penicillin and tetracycline, has a hydrophobicity of 52.28% in xylene, a self-aggregation rate of 58.86%, and a co-aggregation rate of more than 40% for Escherichia coli and Staphylococcus aureus.

3. A blood pressure reducing dairy product, characterized in that: The milk product containing antihypertensive peptides is obtained by fermenting goat milk with the Lactobacillus rhamnosus KD5 described in claim 1.

4. The blood pressure reducing dairy product according to claim 3, characterized in that: The amino acid sequence of the antihypertensive peptide is: FLDDD, SLPEW, IMGVPK, FAWPQ, LHLPLP, MPIQAF, EPINIF, LPQNILP, LPYPYY, TPVVVPPF, LAFNPTQL, KNRLNFL, FVVAPFPE, LLTTDVEK, KYIPIQY, and VPPFLQPE.

5. A blood pressure lowering peptide probiotic goat milk, characterized in that: The method is prepared by fermenting goat milk with the rhamnosus lactobacillus KD5 described in claim 1, wherein the inoculation amount of rhamnosus lactobacillus KD5 is 0.01%-0.05%, the ACE inhibition rate is 65.90%-83.25%, and the pH is 3.56-4.

59.

6. A method for preparing antihypertensive peptide probiotic goat milk, characterized in that: The specific steps are as follows: S1: adding 0.01% to 0.05% of freeze-dried powder of Lactobacillus rhamnosus KD5 into sterilized and cooled goat milk; S2 was fermented at a constant temperature of 34°C to 40°C for 22h to 26h to obtain antihypertensive peptide probiotic fermented goat milk.

7. The method for preparing antihypertensive peptide probiotic goat milk according to claim 6, characterized in that: A nutritional enhancer for the middle-aged and elderly is also added to the antihypertensive peptide probiotic fermented goat milk. The nutritional enhancer for the middle-aged and elderly is one or more of phosphatidylserine, acetylneuraminic acid and prebiotics.

8. A blood pressure lowering peptide probiotic goat milk powder, characterized in that: The method is prepared by fermenting goat milk with the rhamnosus lactobacillus KD5 according to claim 1 and then spray drying the fermented goat milk, wherein the inoculation amount of rhamnosus lactobacillus KD5 is 0.01% to 0.05%, and the IC value of the antihypertensive peptide probiotic goat milk powder on ACE inhibition is 50 The value was 0.0545 g / mL~ 0.0840 g / mL, the survival rate of probiotics was 53.19%~83.58%, and the number of viable probiotics in antihypertensive peptide probiotic goat milk powder was 4.41×10 8 CFU / g~6.94×10 8 CFU / g.

9. A method for preparing antihypertensive peptide probiotic goat milk powder, characterized in that: The specific steps are as follows: S1: adding 0.01% to 0.05% of freeze-dried powder of Lactobacillus rhamnosus KD5 into sterilized and cooled goat milk; S2 is fermented at a constant temperature of 34°C to 40°C for 22h to 26h to obtain antihypertensive peptide probiotic fermented goat milk; S3 prepares antihypertensive peptide probiotic goat milk powder by vacuum low-temperature spray drying of the antihypertensive peptide probiotic fermented goat milk.

10. The method for preparing a blood pressure reducing peptide probiotic goat milk powder according to claim 9, characterized in that: The conditions for the vacuum low-temperature spray drying are: vacuum negative pressure of 0.03Mpa~0.036Mpa, and air inlet temperature of 65°C~75°C.

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