Preparation method of fermented milk for inhibiting Helicobacter pylori

Abstract

The present invention discloses a preparation method of a fermented milk for inhibiting Helicobacter pylori, belonging to the technical field of microbial application. The compound fermented milk of Lactobacillus plantarum and a traditional starter has a strong effect of inhibiting H. pylori SS1; the fermented milk can tolerate gastrointestinal fluid digestion, inhibit H. pylori and its urease activity, and the antibacterial substances in the fermented supernatant are mainly protein substances sensitive to proteinase K; the fermented milk still has good quality and the effect of inhibiting H. pylori SS1 during refrigeration.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermented milk processing, and particularly relates to a preparation method of fermented milk for inhibiting Helicobacter pylori. Background Art

[0002] Helicobacter pylori (H. pylori) is the main pathogenic factor of diseases such as chronic gastritis, peptic ulcer and gastric cancer, and has been listed as a Class I carcinogen by the World Health Organization (Mommersteeg M C, Yu J, Peppelenbosch M P, et al. Genetic host factors in Helicobacter pylori-induced carcinogenesis: Emerging new paradigms[J]. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, 2018, 1869(1): 42-52). At the same time, it is also the only recognized bacterial carcinogen so far. In addition, recent studies have also found that H. pylori is related to extra-digestive tract diseases such as arteriosclerosis, hypertension and coronary heart disease.

[0003] With the increase in the antibiotic resistance of H. pylori (Ou Biyun, Liu Daihua. Current situation of antibiotic resistance of Helicobacter pylori in China[J]. Guide of China Medicine, 2013, 11(07): 68-69), the radical cure rate of the standard triple therapy has been decreasing year by year. The fourth national consensus report on the management of Helicobacter pylori infection in 2012 and the Maastricht IV consensus opinion both recommend the bismuth-containing quadruple therapy as the first-line treatment regimen for radical cure of H. pylori. However, long-term unreasonable use of antibiotics can cause disorders of the intestinal microbiota, and at the same time cause adverse reactions such as nausea, abdominal pain, diarrhea and loss of appetite, which in turn lead to a decrease in the tolerance and compliance of patients to eradication treatment (He Chenxi. Efficacy study of probiotics combined with triple therapy for eradicating H. pylori[D]. Hebei Medical University, 2014).

[0004] With the in vitro effect of probiotics against H. pylori and the confirmation of its clinical applications such as alleviating gastrointestinal symptoms by regulating the gut microbiota and host immune response, the treatment strategy for H. pylori has evolved from the combination of one or more antibiotics to the combination of antibiotics with proton pump inhibitors and gastric mucosal protectants, and then to the combination of probiotics with traditional therapies (Yoon J H, Baik G H, Sohn K M, et al. Trends in the eradication rates of Helicobacter pylori infection for eleven years[J]. World Journal of Gastroenterology: WJG, 2012, 18(45):6628).

[0005] Therefore, based on the current situation of H. pylori infection and treatment, it is meaningful to urgently develop fermented milk with good inhibitory effects. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing fermented milk that inhibits Helicobacter pylori (H. pylori). This fermented milk has the characteristics of inhibiting H. pylori, strong antibacterial ability, resistance to gastrointestinal juice digestion, stable quality during refrigeration, good taste, and delicate texture, laying a foundation for the future development of fermented milk or health products that inhibit Helicobacter pylori.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing fermented milk with the function of inhibiting Helicobacter pylori (H. pylori) includes the following steps:

[0009] (1) Selection of bacterial sources

[0010] Select a compound bacterium as the starter culture, and the compound bacterium is Lactobacillus plantarum (L. plantarum), Lactobacillus bulgaricus (L. bulgaricus), and Streptococcus thermophilus (S. thermophilus);

[0011] (2) Preparation of fermented milk

[0012] Inoculate the compound bacterium in step (1) into skim milk or reconstituted skim milk, and ferment at 37 - 45 °C for 8 - 18 h until coagulation occurs.

[0013] Based on the above technical solution, further, the viable cell number ratio of Lactobacillus plantarum (L. plantarum), Lactobacillus bulgaricus (L. bulgaricus), and Streptococcus thermophilus (S. thermophilus) in the compound bacterium is 1 - 2:1 - 2:1 - 2.

[0014] Based on the above technical solution, further, the Lactobacillus plantarum is Lactobacillus plantarum IF-5014 or Lactobacillus plantarum IF-3083, the Streptococcus thermophilus is Streptococcus thermophilus 6038, and the Lactobacillus bulgaricus is Lactobacillus bulgaricus 6047.

[0015] Based on the above technical solution, further, the concentration of each bacterium in the compound bacteria (Lactobacillus plantarum, Lactobacillus bulgaricus, and Streptococcus thermophilus) is 10 7 -10 9 CFU / mL.

[0016] Based on the above technical solution, further, in step (2), the concentration of the skim milk or reconstituted skim milk is 11%-12% (w / v).

[0017] Based on the above technical solution, further, in step (2), 6%-8% (w / v) of granulated sugar is not added or added to the skim milk or reconstituted skim milk.

[0018] Based on the above technical solution, further, in step (2), the fermentation temperature is 37°C.

[0019] Based on the above technical solution, further, in step (2), the total inoculation amount of the compound bacteria is 2%-4% (v / v).

[0020] The present invention also relates to a fermented milk with the function of inhibiting Helicobacter pylori (H. pylori) obtained by the above preparation method.

[0021] The present invention ferments reconstituted skim milk with compound bacteria, and screens out the strain L. plantarum IF-3083 with the strongest comprehensive ability to inhibit H. pylori. L. plantarum IF-3083 is compounded and fermented with traditional starters (S. thermophilus 6038 and L. bulgaricus 6047), the activities of inhibiting H. pylori SS1 during simulated gastrointestinal fluid digestion and refrigeration of the compounded fermented milk and the product quality are measured, and the types of antibacterial substances in the fermentation supernatant are preliminarily determined by the Oxford cup method and the microscale resazurin colorimetric method.

[0022] Based on the above technical solutions, further, the sugared fermented milk prepared by compounding L. plantarum IF-3083 and traditional starters (S. thermophilus 6038 and L. bulgaricus 6047) has the best antibacterial property.

[0023] Based on the above technical solutions, further, the fermented milk prepared by compounding L. plantarum IF-3083 still has antibacterial property after gastrointestinal digestion.

[0024] Based on the above technical solutions, further, the antibacterial substances in the supernatant of the fermented milk prepared by compounding L. plantarum IF-3083 are preliminarily determined to be protein substances sensitive to proteinase K, as well as a very small amount of acid and hydrogen peroxide.

[0025] Based on the above technical solutions, further, the supernatant of the fermented milk prepared by compounding L. plantarum IF-3083 has a strong inhibition rate on the urease of H. pylori SS1.

[0026] Based on the above technical solutions, further, the fermented milk prepared by compounding L. plantarum IF-3083 has good product quality and increasingly enhanced antibacterial property during refrigeration.

[0027] Beneficial effects:

[0028] (1) The fermented milk prepared by compounding L. Plantarum IF-3083 and traditional starters has a strong inhibitory effect on H. pylori SS1 and storage stability.

[0029] (2) The fermented milk can withstand gastrointestinal juice digestion, its fermentation supernatant can reduce the urease activity of H. pylori SS1, and the antibacterial substances in the fermentation supernatant are mainly protein substances most sensitive to proteinase K.

[0030] (3) The fermented milk still has good quality and an inhibitory effect on H. pylori SS1 during refrigeration. Description of the drawings

[0031] Figure 1 For the quality comparative analysis of the compounded fermented milk, where (a) viable cell count, (b) pH, (c) titratable acidity, (d) antibacterial zone diameter. Note: The viable cell count refers to the total number of colonies of lactic acid bacteria in the fermented milk; different lowercase (uppercase) letters in the figure indicate the significant difference comparison of the supernatant of the fermented milk with (without) added granulated sugar; * indicates a significant difference in the antibacterial property between the supernatant of the skim milk with and without added granulated sugar (p < 0.05).

[0032] Figure 2To simulate the effect of gastrointestinal fluid digestion on the antibacterial property of the supernatant of the compound fermented milk of L. plantarum IF-3083, including (a) antibacterial zone and (b) diameter of antibacterial zone. Note: In the figure, from left to right are the fermented supernatants treated with simulated gastric juice, intestinal juice, and gastrointestinal fluid in sequence.

[0033] Figure 3 For the preliminary determination of antibacterial substances in the supernatant of the compound fermented milk of L. plantarum IF-3083, where (a) and (b) are respectively the antibacterial zone and the diameter of the antibacterial zone by the Oxford cup double-layer plate agar diffusion method, and (c) is the microscale resazurin colorimetric method. Note: In figures a - c, from left to right are the supernatants without treatment, treated with pepsin, trypsin, proteinase K, catalase, and neutralized with sodium hydroxide; in figure c, the abbreviations of groups, with 6 parallels in each group (×6), where the negative control (negative) refers to BB broth without H. pylori.

[0034] Figure 4 For the effect of the supernatant of the compound fermented milk of L. plantarum IF-3083 on the urease activity of H. pylori SS1. Note: In figures a - c, from left to right are the fermented supernatants without treatment, treated with pepsin, trypsin, proteinase K, catalase, and neutralized with sodium hydroxide.

[0035] Figure 5 For the quality and antibacterial property of the compound fermented milk of L. plantarum IF-3083 during refrigeration, including (a) pH and titratable acidity, (b) viable cell count, (c) diameter of antibacterial zone of the fermented milk and its supernatant, (d) water holding capacity, (e) texture, and (f) sensory evaluation. Note: The viable cell count refers to the total number of colonies of lactic acid bacteria in the fermented milk; D1, D4, D7, D11, and D14 respectively represent that the fermented milk is stored at 4°C for 1, 4, 7, 11, and 14 days. Specific implementation manners

[0036] The following non-limiting examples can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0037] Example 1: Source and culture of strains

[0038] L.plantarum IF-5014 and L.plantarum IF-3083 are probiotics with strong inhibitory activity against H. pylori SS1 screened from the feces of healthy infants in the early stage of the laboratory (Gao F, Sui L, Mu G, Zhu X, Qian F. Screening of potential probiotics with anti-Helicobacter pylori activity from infant feces through principal component analysis[J]. Food Bioscience, 2021, 42, 101045.). Lactobacillus rhamnosus ATCC 53103 (LGG), S. thermophilus 6038 and L. bulgaricus 6047 were purchased from the China General Microbiological Culture Collection Center (CGMCC), and H. pylori SS1 was preserved by the Key Laboratory of Probiotic Functional Characteristics Research in Dalian, Dalian Polytechnic University.

[0039] L.plantarum IF-5014, L.plantarum IF-3083, S.thermophilus 6038, L.bulgaricus 6047 and LGG were inoculated into MRS liquid medium at an inoculum size of 2% (v / v) respectively, cultured at 37 °C for 18 h, activated for 2 generations, centrifuged at 4 °C and 5000 r / min for 10 min, resuspended in PBS buffer (pH 7.4) and adjusted to a concentration of 10 9 CFU / mL for use, namely L.plantarum IF-5014 bacterial suspension, L.plantarum IF-3083 bacterial suspension, S.thermophilus 6038 bacterial suspension, L.bulgaricus 6047 and LGG bacterial suspension respectively.

[0040] H. pylori SS1 was subcultured on Columbia blood agar medium containing H. pylori selective additive (Qingdao Haibo Biotech, 1% selective additive), and cultured at 37 °C under microaerophilic conditions (85% N2, 10% CO2, 5% O2) for 72 h; after being activated for 2 generations, the cells were washed with PBS buffer (pH 7.4), centrifuged at 4 °C and 5000 r / min for 10 min, resuspended in PBS buffer and adjusted to a concentration of 10 7 CFU / mL for use, which is the H. pylori SS1 bacterial suspension.

[0041] Example 2: Selection of antibacterial strains

[0042] Experiment 1: L. plantarum IF-3083 was compounded with traditional starters S. thermophilus 6038 and L. bulgaricus 6047 (1:1:1): The bacterial suspensions of the three strains were inoculated into 12% (w / v) reconstituted skim milk with or without 6% (w / v) granulated sugar at a total inoculation amount of 2% (v / v), and fermented at 37°C for 18 h. The fermented milk was named: Group 3083.

[0043] The fermented milk was centrifuged (4°C, 8000 r / min, 15 min), and after filtration through a 0.45 μm filter membrane, it was the fermented supernatant. The Oxford cup double-layer plate agar diffusion method was used to compare its antibacterial property. The specific steps were as follows: A double-layer blood plate was prepared using Columbia agar medium containing 7% (v / v) sterile defibrinated sheep blood. 500 μL of the H. pylori SS1 bacterial suspension prepared in Example 1 was added to the plate. After the bacterial suspension was fully adhered, 100 μL of the fermented supernatant was pipetted into the well. It was cultured at 37°C under microaerophilic conditions (85% N2, 10% CO2, 5% O2) for 72 h, and the diameter of the antibacterial zone was measured. The pH of the fermented milk was measured with a pH meter; the titratable acidity of the fermented milk was measured with an automatic potentiometric titrator; the fermented milk was appropriately diluted with 0.85% sterile saline and poured onto a sterile MRS solid medium to count the lactic acid bacteria in the fermented milk.

[0044] Experiment 2: L. plantarum IF-5014 was compounded with S. thermophilus 6038 and L. bulgaricus 6047 (1:1:1): The bacterial suspensions of the three strains were inoculated into 12% (w / v) skim milk with or without 6% (w / v) granulated sugar at a total inoculation amount of 2% (v / v), and fermented at 37°C for 18 h. The fermented milk was named: Group 5014. The antibacterial property, viable count, pH, and titratable acidity of its fermented supernatant were measured as described in Experiment 1.

[0045] Experiment 3: LGG was compounded with S. thermophilus 6038 and L. bulgaricus 6047 (1:1:1): The bacterial suspensions of the three strains were inoculated into 12% (w / v) reconstituted skim milk with or without 6% (w / v) granulated sugar at a total inoculation amount of 2% (v / v), and fermented at 37°C for 18 h. The fermented milk was named: Group LGG (as a control). The antibacterial property, viable count, pH, and titratable acidity of its fermented supernatant were measured as described in Experiment 1.

[0046] Experiment 4: Compound fermentation of L. plantarum IF-3083, L. plantarum IF-5014, S. thermophilus 6038 and L. bulgaricus 6047 (1:1:1:1): The bacterial suspensions of the four strains were inoculated into 12% (w / v) reconstituted skim milk with or without (w / v) granulated sugar at a total inoculum of 2% (v / v), and fermented at 37 °C for 18 h. The fermented milk was named: Group 3083 + 5014, and the antibacterial activity, viable cell count, pH and titratable acidity of its fermentation supernatant were measured as described in Experiment 1.

[0047] As Figure 1 It can be seen from the experimental results that compared with the sugar-free group, the viable cell count, titratable acidity and antibacterial activity in the compound fermented milk with 6% (w / v) sugar added were significantly increased (p < 0.05), and the pH value was significantly decreased (p < 0.05). It indicates that the strains can better utilize granulated sugar to produce other antibacterial substances and improve their antibacterial activity against H. pylori SS1.

[0048] In the compound fermented milk with sugar, the experimental results were as follows:

[0049] Antibacterial activity: Group IF-3083 ≈ Group LGG > Group IF-5014 > Group 3083 + 5014 (p < 0.05);

[0050] Viable cell count: Group IF-3083 ≈ Group IF-5014 > Group LGG > Group 3083 + 5014 (p < 0.05);

[0051] pH value: Group IF-3083 ≈ Group IF-5014 < Group LGG < Group 3083 + 5014 (p < 0.05);

[0052] Titratable acidity: Group LGG > Group IF-3083 ≈ Group IF-5014 > Group 3083 + 5014 (p < 0.05).

[0053] It can be seen that the compound fermented milk containing sugar of L. plantarum IF-3083 not only has a high viable cell count and acidity, but also has the strongest antibacterial effect on H. pylori SS1. Therefore, L. plantarum IF-3083 was selected to be compounded with traditional starters and inoculated into 6% (w / v) granulated sugar reconstituted skim milk to prepare fermented milk.

[0054] Example 3: Comparison of antibacterial activity of fermented milk supernatant before and after simulated gastrointestinal fluid digestion

[0055] (1) Preparation of simulated gastrointestinal fluid

[0056] Pepsin was added to the autoclaved NaCl solution (0.15 M, pH 2.0) at 0.66% (w / v). After filtration through a 0.22 μm filter membrane, artificial simulated gastric juice was obtained.

[0057] Trypsin and bile salts were dissolved in phosphate buffer (0.1 M, pH 8.0) at 1.5% (w / v) and 0.15% (w / v) respectively. After filtration through a 0.22 μm filter membrane, artificial simulated intestinal juice was obtained.

[0058] (2) Simulated gastrointestinal fluid digestion

[0059] Simulated gastric juice digestion: The supernatant of the co-fermented milk of L. plantarum IF-3083 and traditional starter culture (IF-3083 group) in Example 2 was mixed with simulated gastric juice at a ratio of 1:3 (v / v), incubated in a shaker at 37 °C (100 rpm, 2 h), and then heated in a boiling water bath for 5 min to inactivate the enzyme activity.

[0060] Simulated intestinal juice digestion: The supernatant of the co-fermented milk of L. plantarum IF-3083 and traditional starter culture (IF-3083 group) in Example 2 was mixed with simulated intestinal juice at a ratio of 1:2 (v / v), incubated in a shaker at 37 °C (100 rpm, 3 h), and then heated in a boiling water bath for 5 min to inactivate the enzyme activity.

[0061] Simulated gastrointestinal continuous digestion: The sample after simulated gastric juice digestion was subjected to simulated intestinal juice digestion.

[0062] The samples before and after simulated digestion were compared for their antibacterial activity against H. pylori SS1 using the Oxford cup double-layer plate agar diffusion method as described in Example 1.

[0063] The experimental results are as Figure 2 shown. After the fermentation supernatant was treated successively with simulated gastric juice, simulated intestinal juice, and gastrointestinal juice, its antibacterial activity decreased significantly (p < 0.05). Among them, gastrointestinal juice digestion < intestinal juice digestion < gastric juice digestion (p < 0.05). The antibacterial diameter of the fermentation supernatant after simulated gastrointestinal juice digestion was about 11 mm, that is, it still retained a certain ability to inhibit H. pylori SS1.

[0064] Example 4: Analysis of antibacterial substances in the supernatant of fermented milk

[0065] Oxford cup double-layer plate agar diffusion method: As described in Example 2, the supernatant of the fermented milk obtained by the compound fermentation of L. plantarum IF-3083 and traditional starter (IF-3083 group) was harvested (recorded as the untreated group), and the pH of the supernatant was adjusted to 6.0 with 1 mol / L NaOH solution (recorded as the neutralization group); pepsin, trypsin, proteinase K and catalase with a final concentration of 1.0 mg / mL were added to the supernatant, and then the pH was adjusted to the optimal pH for enzymatic reaction with 1 mol / L NaOH or HCl solution. The optimal pH values of pepsin, trypsin, proteinase K and catalase were 2.0, 8.1, 7.3 and 7.0 respectively, and the mixture was incubated in a constant temperature water bath at 37 °C for 2 h. Then the pH of the treated supernatant was adjusted to be the same as that of the original fermented supernatant (recorded as the enzyme digestion treatment group). Using the Oxford cup method with the untreated group as the control, the antibacterial substances in the fermented milk supernatant were preliminarily inferred.

[0066] Microscale resazurin colorimetric method: As described above, the fermented supernatants obtained by neutralization and enzyme digestion were harvested. At the same time, the H. pylori SS1 bacterial solution was adjusted to an OD 600 ≈0.5 ± 0.05 with BB broth containing 10% (v / v) fetal bovine serum. 160 μL of the H. pylori SS1 bacterial suspension prepared in Example 1 and 20 μL of the supernatant were added to a 96-well plate and incubated at 37 °C under microaerophilic conditions (85% N2, 10% CO2, 5% O2) for 72 h. Then 20 μL of resazurin solution (200 μg / mL) was added to the well plate. After incubation for 0.5 h, the antibacterial substances in the supernatant were preliminarily determined by color change.

[0067] The experimental results are as Figure 3 (a - b) shown. Compared with the untreated group, the diameters of the antibacterial zones in the enzyme digestion treatment group and the neutralization group decreased significantly (p < 0.05). The order was: untreated group > pepsin > trypsin ≈ catalase ≈ neutralization group ≈ proteinase K. This indicates that the antibacterial substances in the fermented supernatant are mainly protein substances that are relatively sensitive to proteinase K, as well as a very small amount of acid and hydrogen peroxide.

[0068] Similarly, as Figure 3 (c) shown, compared with the untreated control group, the colors of the treated groups became lighter after resazurin color development, especially the proteinase K treatment group showed the most obvious color change. This phenomenon is because after the supernatant was treated, its antibacterial property decreased, the activity of H. pylori SS1 increased, and its reducing property was strong, resulting in obvious resazurin color development. This result further proves that the supernatant of the compound fermented milk of L. plantarum IF-3083 contains antibacterial substances that are sensitive to proteinase K.

[0069] Therefore, the conclusion is drawn that the antibacterial substances in the fermented supernatant are mainly protein substances that are most sensitive to proteinase K, as well as a very small amount of acid and hydrogen peroxide.

[0070] Example 5: Effect of Fermented Milk Supernatant on Urease Activity of H. pylori SS1

[0071] Using the urea-phenol red method, preheat the H. pylori SS1 bacterial suspension prepared in Example 1 and the supernatants of the reconstituted fermented milk with Lactobacillus plantarum IF-3083 harvested in Example 4 and traditional starter cultures (untreated group, neutralized group, and enzyme-digested group) at 37 °C for 30 min. Add the H. pylori SS1 bacterial suspension (40 μl) and the supernatant (10 μl) into a 96-well plate, shake well, and incubate at 37 °C under microaerophilic conditions (85% N2, 10% CO2, 5% O2) for 48 h. Add the preheated (37 °C) urea-phenol red indicator (150 μl) into the well plate and react for 5 min. By measuring OD 550 , calculate the urease inhibition rate (%) through the following formula.

[0072]

[0073] Among them, OD1 is the OD of the untreated group 550 ; OD2 is the OD of each experimental group 550 .

[0074] The experimental results are as Figure 4 shown. Compared with the untreated group, the urease inhibition rates of the supernatants in the enzyme-digested groups with protease K, pepsin, trypsin, etc. all decreased significantly (p < 0.05); among them, after treatment with protease K, the urease inhibition rate decreased most significantly; after treatment with catalase and neutralization, the urease inhibition rate of its supernatant did not decrease significantly (p > 0.05). From this, a preliminary conclusion is drawn that there are protein substances in the supernatant that are most sensitive to protease K, relatively sensitive to pepsin and trypsin, and have urease inhibitory activity. At the same time, there are very small amounts of acid and hydrogen peroxide in the fermented supernatant.

[0075] Example 6: Quality Analysis of Reconstituted Fermented Milk Inhibiting H. pylori during Refrigeration

[0076] Mix Lactobacillus plantarum IF-3083 with traditional starter cultures (Streptococcus thermophilus 6038 and Lactobacillus bulgaricus 6047) in a ratio of 1:1:1, and add them to 12% (w / v) reconstituted skim milk with 6% (w / v) granulated sugar at an inoculation amount of 4% (v / v), and ferment at 37 °C for 8 h. After coagulation, refrigerate at 4 °C. Measure its pH, titratable acidity, viable cell count, water holding capacity, and texture on the 1st, 4th, 7th, 11th, and 14th days (D1 - D14) respectively.

[0077] (1) Determination of pH, titratable acidity, viable count, and antibacterial activity

[0078] The determination was carried out as described in Example 2.

[0079] The experimental results are as Figure 5 (a - c) shown. During refrigeration (D1 - D14), the pH of the fermented milk fermented by the compounding of IF - 3083 and the traditional starter decreased gradually, and the titratable acidity increased gradually. The viable count increased at D4 first, then decreased at D7, and tended to be stable during D7 - D14. The antibacterial activities of the fermented milk and its supernatant against H. pylori SS1 increased with the increase of refrigeration time, and the antibacterial diameters reached the maximum values of 47 mm and 43.69 mm at D14, respectively.

[0080] (2) Determination of water - holding capacity

[0081] Slowly add 10 g of fermented milk into a centrifuge tube, centrifuge at 4 °C (8000×g, 15 min), discard the supernatant, and aspirate the residual moisture. Weigh the centrifuge tube and the precipitate, and calculate the water - holding capacity value WHC through the following formula.

[0082]

[0083] Where, W1 is the mass of the centrifuge tube in g; W2 is the total mass of the centrifuge tube and the fermented milk in g; W3 is the total mass of the centrifuge tube and the sour milk after discarding the supernatant in g.

[0084] The experimental results are as Figure 5 (d) shown. The water - holding capacity of the fermented milk increased continuously from D1 - D7, reached the maximum value of 40.5% at D7, and then began to decline and tended to be stable at 35.2% - 36.2%, but the water - holding capacities at D11 and D14 were still higher than that at D1.

[0085] (3) Texture determination

[0086] Use a TA.XTPLUS type dairy product quality detector (StableMicroSystem, UK) to conduct a texture (TPA) test on the fermented milk sample. Test parameters: pre - test speed is 1.70 mm / s, test speed is 1.70 mm / s, post - test speed is 10.00 mm / s, test distance is 15.00 mm, trigger mode is automatic, and the minimum trigger force is 0.5 g.

[0087] The experimental results are as Figure 5 (e) shown. Compared with D1 of refrigeration, both the hardness and viscosity of the fermented milk increased with the increase of refrigeration time, which may be related to post - acidification and casein hydration.

[0088] (4) Sensory evaluation

[0089] Prepare a sensory evaluation scoring form 1, and conduct a sensory evaluation on the fermented milk prepared by compounding L. plantarum IF-3083 with traditional starters (S. thermophilus 6038 and L. bulgaricus 6047) through color, taste, flavor and texture.

[0090] The experimental results are as Figure 5 (f) shown. The sensory evaluation scores of the compounded fermented milk continued to increase during refrigeration from D1 to D11, reached the maximum value at D11, and then stabilized at 70-71 from D11 to D14, indicating that the product quality and sensory properties were stable.

[0091] Therefore, it is concluded that the compounded fermented milk of L. plantarum IF-3083 and traditional starters still has good quality and the effect of inhibiting H. pylori SS1 during refrigeration.

[0092] Table 1 Sensory evaluation scoring form

[0093]

[0094]

[0095] The above embodiments are only used for exemplifying and explaining the present invention, and are not intended to limit the present invention to the scope of the described embodiments. In addition, those skilled in the art can understand that the present invention is not limited to the above embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the scope claimed by the present invention.

Claims

1. A method for preparing fermented milk with the function of inhibiting Helicobacter pylori (H. pylori), characterized in that, It includes the following steps: (1) Selection of the bacterial source Select a compound bacterium as the starter culture. The compound bacterium is Lactobacillus plantarum (L. plantarum), Lactobacillus bulgaricus (L. bulgaricus), and Streptococcus thermophilus (S. thermophilus); The viable count ratio of Lactobacillus plantarum (L. plantarum), Lactobacillus bulgaricus (L. bulgaricus), and Streptococcus thermophilus (S. thermophilus) in the compound bacterium is 1 - 2:1 - 2:1 - 2; The Lactobacillus plantarum (L. plantarum) is Lactobacillus plantarum (L. plantarum) IF - 3083, the Streptococcus thermophilus (S. thermophilus) is Streptococcus thermophilus (S. thermophilus) 6038, and the Lactobacillus bulgaricus (L. bulgaricus) is Lactobacillus bulgaricus (L. bulgaricus) 6047; (2) Preparation of fermented milk Inoculate the compound bacterium in step (1) into skim milk or reconstituted skim milk, and ferment at 37 - 45 °C for 8 - 18 h until coagulation of the milk; Add 6% - 8% (w / v) of granulated sugar to the skim milk or reconstituted skim milk.

2. The preparation method according to claim 1, characterized in that, The concentration of each bacterium in the single bacterium or compound bacteria is 10 7 -10 9 CFU / mL.

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the skim milk or reconstituted skim milk is 11% - 12% (w / v).

4. The preparation method according to claim 1, wherein In step (2), the fermentation temperature is 37 °C.

5. The preparation method according to claim 1, wherein In step (2), the total inoculation amount of the compound bacterium is 2% - 4% (v / v).

6. Fermented milk with the function of inhibiting Helicobacter pylori (H. pylori) obtained by the preparation method according to claim 1.

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