Lactobacillus brevis as well as high-density culture method and application thereof

Abstract

The invention discloses a strain of Lactobacillus brevis as well as a high-density culture method and application thereof, the strain is Lactobacillus brevis X66, is obtained by separating and screening from a traditional dairy product dried milk cake in Inner Mongolia, is preserved in Guangdong Microbial Culture Collection Center on August 4, 2022, and has a preservation number of GDMCC No: 62641. The strain provided by the invention is safe, can tolerate gastrointestinal tract environment, has excellent self-coacervation capability and oxidation resistance, and can inhibit escherichia coli, staphylococcus aureus and pseudomonas aeruginosa. The invention provides a high-density culture method of the X66 strain, the viable count of X66 in optimized fermentation liquor exceeds 2 billion CFU / mL and is 2-3 times that before optimization, the viable count of freeze-dried powder prepared from the X66 strain reaches up to 10 billion CFU / g, and preconditions are provided for application of the strain in the fields of fermented dairy products, functional foods, nutritional dietary supplements and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus breadii X66 derived from Inner Mongolia milk tofu and a high-density culture method thereof. Background Art

[0002] Lactic acid bacteria are Gram-positive bacteria that use carbohydrates as the sole or main carbon source. The final fermentation product is mainly lactic acid, and they have strong tolerance to acidic environments. Lactic acid bacteria are widely distributed and have species diversity, including Lactobacillus, Pediococcus, and Bifidobacterium. Lactic acid bacteria can be used as probiotics or prebiotics. They can regulate the intestinal microecology of animals, inhibit the growth of harmful bacteria in the intestine, improve food digestion and absorption, lower blood pressure, resist oxidation, regulate the immune system, etc., and are widely used in the food industry. Lactobacillus crustorum also belongs to one of them. It was first isolated from long-term fermented yeast. It is a rod-shaped, usually regular, spore-free, facultative anaerobic bacterium. Lactobacillus crustorum has the potential to be used as a fermentation agent or as an ideal probiotic in functional foods and feeds. The results of Wang et al. showed that the addition of bread bacteria MN047 had the effect of reducing inflammatory responses and regulating intestinal flora in mice fed with high fat and high sugar. Qian et al. screened a strain of Lactobacillus bread that produces β-glucuronidase from raw cow milk, and used Lactobacillus bread and Bacillus subtilis that can produce cellulase to form a fermentation system, which improved the fermentation performance of tea seed meal and reduced the hemolytic activity of tea seed saponins.

[0003] There are many traditional fermented products in my country. Most of the research on lactic acid bacteria is focused on Lactobacillus plantarum and Lactobacillus casei, while there is less research on Lactobacillus brevis. At present, the main reasons limiting the widespread application of Lactobacillus brevis are: (1) The safety of the strain used is unknown, and it has not yet been included in the list of bacteria that can be used in food by the National Health Commission. (2) The strain yield is small and the production cost is high. Probiotics can only play a probiotic role when a certain number of probiotics is reached in the body. Functional foods with probiotics as active ingredients can achieve high-density cultivation of strains during the production process, which can reduce production costs and improve production efficiency. For example, the Chinese invention application with application number 201710157633.7 discloses "Lactobacillus brevis, antimicrobial peptides and their applications". A strain of Lactobacillus brevis was screened from sour mare's milk, and the antimicrobial peptides were isolated and purified and applied. However, the strain was not evaluated for safety, and the strain safety is unknown; secondly, the strain yield is unknown, and the use of high-density cultivation of the strain and preparation of fermented milk is not involved, so it cannot be used in fermented products.

[0004] Yogurt is very prone to post-acidification during storage, which seriously affects the taste and quality of the product. Post-acidification control of fermented milk is one of the difficulties and key points in fermented milk production. Post-acidification of fermented milk is mainly caused by lactobacillus continuing to ferment lactose to produce acid under acidic conditions, causing the pH value of fermented milk to drop to 3.5 or below. Therefore, the usual method is to improve the ratio of cocci to bacilli in the starter and add different types of sugars and their contents to alleviate or inhibit the growth and acid production of lactobacillus, but these methods are still very limited in their effect on alleviating severe post-acidification.

[0005] Therefore, the technical problem to be solved by the present invention is to provide a Lactobacillus strain that is safe for consumption and has high yield and quality and to apply it to dairy product fermentation, wherein high-density culture of probiotics is a key technology for realizing the industrial application of probiotics. Summary of the invention

[0006] The invention aims to provide a strain of Lactobacillus breadii X66 originating from Inner Mongolia milk tofu and a method for high-density culture thereof, so as to provide a theoretical basis for the research and practical application of Lactobacillus breadii originating from local areas.

[0007] In the first aspect of the present invention, a strain of Lactobacillus crustorum is provided, which is Lactobacillus crustorum X66, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 4, 2022, with a collection number of GDMCCNo: 62641.

[0008] In the second aspect of the present invention, a safety evaluation of the Lactobacillus breadii X66 is provided, which includes an antibiotic sensitivity test, a hemolytic test, an indole test, a nitroreductase test, and an aminodecarboxylase test of the strain, which proves that Lactobacillus breadii X66 is sensitive to ampicillin, erythromycin, tetracycline, and chloramphenicol, is non-hemolytic, has a negative indole test, a negative nitroreductase test, a negative aminodecarboxylase test, has no oral toxicity to mice, is at a safe and non-toxic level, and the strain is safe.

[0009] In the third aspect of the present invention, the tolerance of the Lactobacillus brevis X66 strain in an artificial simulated gastrointestinal fluid environment is provided. The survival rate of Lactobacillus brevis X66 after exposure to artificial simulated gastric fluid at different pH values ​​(pH=2, 2.5, 3, 4) for 3 hours is greater than 75%, and the survival rate after exposure to artificial intestinal fluid for 2 hours is greater than 100%.

[0010] In a fourth aspect of the present invention, the self-aggregation and antioxidant capacity of the Lactobacillus brevis X66 strain is provided. After culturing for 24 hours, the self-aggregation capacity of X66 reaches 93.39%. When the number of viable cells reaches 10 9 CFU / mL, X66 supernatant to DPPH, ·OH, O2 －The clearance rates of · were 94.82%, 80.18% and 49.13% respectively.

[0011] In a fifth aspect of the present invention, the inhibitory effect of the Lactobacillus brevis X66 strain on common pathogenic bacteria is provided. The inhibition zone size (cm) of the supernatant of Lactobacillus brevis X66 on Escherichia coli is 2.97-3.11, the inhibition zone size (cm) on Staphylococcus aureus is 2.88-2.92, and the inhibition zone size (cm) on Pseudomonas aeruginosa is 2.78-2.90.

[0012] In the sixth aspect of the present invention, the components in the basic MRS medium are optimized to provide a medium with a viable count of 2.63×10 9 CFU / mL high-density culture method of Lactobacillus bread X66.

[0013] In the seventh aspect of the present invention, a method for preparing the powder of Lactobacillus bread X66 is provided, which can obtain a viable bacterial count of 1.1×10 11 CFU / g of bacterial powder and is used in fermented milk.

[0014] Therefore, the bread lactobacillus of the present invention can be widely used in dairy processing and other functional products.

[0015] In order to achieve the above object, the present invention adopts the following technical solutions:

[0016] A strain of Lactobacillus crustorum X66 was isolated from a traditional Chinese dairy product, Inner Mongolian milk tofu. The biological characteristics of this strain of Lactobacillus crustorum X66 are: streaking on an MRS medium plate, culturing at 37°C for 48 hours, the colonies are round, convex, with smooth and neat edges, milky white, and a moist and smooth surface. Gram staining microscopy showed that it was a Gram-positive bacterium with a rod-shaped body and no spores (such as Figure 1 The results of physiological and biochemical tests showed that the strain could produce acid using maltose, fructose and glucose, and the KOH test was negative, the starch hydrolysis test was negative, the strain was facultatively anaerobic, and did not liquefy gelatin. X66 was identified as Lactobacillus breadii based on 16S rDNA and physiological and biochemical characteristics. Its 16S rDNA sequence is shown in SEQ NO: 1.

[0017] A bread lactobacillus, the strain is bread lactobacillus (Lactobacillus crustorum) X66, which was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short) on August 4, 2022, with a preservation number of GDMCC No: 62641.

[0018] A high-density culture method of Lactobacillus bread comprises the following steps:

[0019] (1) inoculating the Lactobacillus bread X66 described in claim 1 into an MRS broth medium for activation to obtain a seed fermentation liquid;

[0020] (2) adding the seed fermentation liquid to the high-density culture medium, and culturing at a constant temperature of 36 to 38° C. for 16 to 18 hours to obtain a high-density fermentation liquid;

[0021] The high-density culture medium formula is: by mass, 100 parts of MRS broth culture medium, 3-5 parts of glucose, 2.5-3.5 parts of bacteriological peptone, 2.5-3.5 parts of beef extract powder, 2.5-3.5 parts of yeast extract powder, 0.2-0.4 parts of dipotassium hydrogen phosphate, 0.8-1.0 parts of sodium acetate, 0.08-0.12 parts of magnesium sulfate monohydrate, and 0.01-0.03 parts of manganese sulfate tetrahydrate.

[0022] Preferably, the high-density culture medium is prepared by adding distilled water to make up the volume of the above raw materials to 100 parts, adjusting the pH to 5.7-6.0, stirring to dissolve evenly, sterilizing and cooling.

[0023] Preferably, the sterilization condition is 0.08-0.10 MPa for 15-20 min.

[0024] Preferably, the activation method of the seed fermentation liquid is: inoculating the frozen Lactobacillus bread X66 in an MRS broth culture medium at a volume ratio of 2 to 6:100 for a first activation, inoculating the obtained once-activated seed fermentation liquid in an MRS broth culture medium at a volume ratio of 3 to 5:100 for a second activation, and obtaining a second-activated seed fermentation liquid.

[0025] Preferably, the bacterial content of the seed fermentation liquid is 0.8-1.0×10 9 CFU / mL.

[0026] Preferably, the bacterial content of the high-density fermentation broth is 2.0×10 9 CFU / mL and above.

[0027] A bread lactobacillus powder, wherein the high-density fermentation liquid prepared by the above method is freeze-dried to obtain a powder with a viable count of 1.1×10 11 CFU / g or above bacterial powder.

[0028] Preferably, the freeze-drying method is: centrifuge the high-density fermentation bacterial liquid to obtain a bacterial sludge precipitate, mix the protective agent with the bacterial sludge, put it into a -20°C refrigerator for pre-freezing (48 hours), and put the pre-frozen bacterial sludge precipitate into a vacuum freeze dryer for freeze drying (temperature -50 to -70°C, time 24 hours); preparation of the protective agent: in parts by mass, add 7 to 9 parts of trehalose, 4.70-4.74 parts of sodium glutamate, and 9.74 to 9.78 parts of skimmed milk powder, dilute to 100 parts with water, and sterilize at 110°C for 15 minutes to obtain the protective agent.

[0029] The application of the bread lactobacillus, high-density fermentation bacterial liquid or bread lactobacillus powder in the preparation of fermented milk.

[0030] Preferably, the bread lactobacillus, high-density fermentation liquid or bread lactobacillus powder is added with thermophilic streptococcus DMST-H2 and Lactobacillus delbrueckii DMLD-H1 for joint fermentation. The optimal compounding ratio of the bread lactobacillus X66 to thermophilic streptococcus DMST-H2 and Lactobacillus delbrueckii DMLD-H1 is 1:0.5:0.5.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] (1) The present invention discloses a strain of Lactobacillus bread X66, which is selected from a traditional fermented dairy product in China, Inner Mongolia milk tofu. The strain is proven to be safe through a series of safety experiments, and acute toxicology proves that it is actually non-toxic. Compared with imported strains from abroad, it is more suitable for the intestines of Chinese people.

[0033] (2) The present invention discloses a strain of Lactobacillus bread X66, which has good tolerance to the gastrointestinal environment, excellent self-aggregation ability, antioxidant ability, and can inhibit the growth of common pathogenic bacteria.

[0034] (3) The present invention discloses a high-density culture method for Lactobacillus bread X66, which optimizes the components in the basic MRS culture medium and can reach a viable count of 2.63×10 9 CFU / mL is 2.41 times that before the optimization of culture conditions. The subsequent rapid and large-scale proliferation of X66 can reduce the amount of starter and reduce the fermentation cost, which is beneficial to the production and application of this strain in the fields of functional foods and fermented foods.

[0035] (4) The present invention discloses a method for preparing Lactobacillus breadii X66 powder, wherein the high-density fermentation liquid is freeze-dried to obtain a powder with a viable count of 1.10×10 11CFU / g of bacterial powder, and determine the optimal ratio of composite bacteria to control the post-acidification of yogurt and improve product quality; the bread lactobacillus X66 is interacted with conventional fermentation bacteria to coordinate the fermentation to produce a composite ratio of fermented milk with good taste, which can also improve the sensory quality of fermented milk, increase the viscosity and water holding capacity of fermented milk, and effectively solve the problem of post-acidification of fermented milk.

[0036] The Lactobacillus crustorum X66 was deposited in Guangdong Microbiological Culture Collection Center on August 4, 2022, with a deposit number of GDMCC No: 62641. Deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0037] The Lactobacillus gasseri LGZ1029 was deposited in Guangdong Microbiological Culture Collection Center on April 16, 2019, with a deposit number of GDMCC No: 60641. Deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0038] The Lactobacillus delbrueckii DMLD-H1 was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 16, 2019, with a deposit number of GDMCC No. 60645. Deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0039] The Streptococcus thermophilus DMST-H2 was deposited in Guangdong Microbiological Culture Collection Center on April 16, 2019, and the deposit number is GDMCC No. 60642. Deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a morphological diagram of Lactobacillus bread X66 in Example 1.

[0041] Figure 2 It is the phylogenetic tree diagram of the Lactobacillus bread X66 strain in Example 1.

[0042] Figure 3 The figures are the growth conditions of the Lactobacillus breadii X66 strain on the blood agar plate of Example 3: (a) is the control hemolysis test result of Escherichia coli ATCC 25922; (b) is the hemolysis test result of Lactobacillus breadii X66.

[0043] Figure 4The indole experiment results of the Lactobacillus X66 strain in Example 3 are as follows: (a) is the experimental group with the addition of Escherichia coli ATCC25922 as a positive control; (b) is the experimental group with the addition of Lactobacillus X66; and (c) is the blank control group.

[0044] Figure 5 The nitroreductase test results of Lactobacillus bread X66 strain in Example 3 are shown in FIG. 1 : (a) is the experimental group with the addition of Escherichia coli ATCC 25922 as a positive control; (b) is the experimental group with the addition of Lactobacillus bread X66; and (c) is the blank control group.

[0045] Figure 6 1 is a graph showing the results of the aminodecarboxylase experiment of Lactobacillus bread X66 strain in Example 3: from top to bottom, they are the test results of the culture medium to which (a) arginine, (b) lysine, (c) tyrosine, and (d) histidine are added, wherein the left column is the experimental group to which Lactobacillus bread X66 is added, the middle column is the experimental group to which Escherichia coli ATCC 25922 is added as a positive control, and the right column is the blank control.

[0046] Figure 7 This is a graph showing the gastric juice tolerance results of Lactobacillus bread X66 in Example 4.

[0047] Figure 8 This is a graph showing the results of the self-aggregation ability test of three strains of lactic acid bacteria in Example 4.

[0048] 9 is a graph showing the DPPH clearance rate determination results of three lactic acid bacteria in Example 4, wherein (a) is Lactobacillus bread X66, (b) is Lactobacillus delbrueckii DMLD-H1, and (c) is Lactobacillus gasseri LGZ1029.

[0049] 10 is a graph showing the results of the hydroxyl radical scavenging rate determination of three lactic acid bacteria in Example 4, wherein (a) is Lactobacillus bread X66, (b) is Lactobacillus delbrueckii DMLD-H1, and (c) is Lactobacillus gasseri LGZ1029.

[0050] 11 is a graph showing the results of superoxide anion scavenging rate determination of three lactic acid bacteria in Example 4, wherein (a) is Lactobacillus bread X66, (b) is Lactobacillus delbrueckii DMLD-H1, and (c) is Lactobacillus gasseri LGZ1029.

[0051] Fig.12 The antibacterial circle diagrams of Lactobacillus X66 against different indicator bacteria in Example 4 are: (a) Escherichia coli ATCC25922, (b) Staphylococcus aureus ATCC 12598, and (c) Pseudomonas aeruginosa PAO1.

[0052] Fig.13 This is a graph showing the effect of the carbon source (glucose) on the growth of Lactobacillus bread X66 in Example 5.

[0053] 14 is a graph showing the effect of different nitrogen sources on the growth of Lactobacillus bread X66 in Example 5; a) bacteriological peptone, b) beef extract powder, c) yeast extract powder.

[0054] Fig.15 This is a graph showing the effects of different growth factors on the growth of Lactobacillus bread X66 in Example 5; a) manganese sulfate tetrahydrate, b) magnesium sulfate monohydrate, c) sodium acetate, d) K2HPO4.

[0055] Figure 16 is the 3D response surface and contour map of the interaction of each factor in the Box-Behnken experimental design in Example 5; wherein, (a) is the response surface and contour map of peptone and yeast extract powder; (b) is the response surface and contour map of peptone and magnesium sulfate; (c) is the response surface contour map of yeast extract powder and magnesium sulfate.

[0056] Fig.17 It is a sensory evaluation graph of fermented milk prepared by different compounding ratios of Lactobacillus thermophilus X66 (abbreviated as LC) and traditional starter (Streptococcus thermophilus + Lactobacillus delbrueckii DMLD-H1, i.e., Streptococcus thermophilus DMST-H2 + Lactobacillus delbrueckii DMLD-H1, abbreviated as SL) in Example 6.

[0057] Figure 18 is a graph showing the effect of different combinations of leavening agents on the rheological properties of fermented milk in Example 6; wherein, (a) is a graph showing the change of yield stress with shear rate; (b) is a graph showing the change of storage modulus with step time; and (c) is a graph showing the change of loss modulus with step time.

[0058] Fig.19 This is a graph showing the results of determining the monosaccharide and disaccharide contents in fermented milk obtained using different combinations of starter cultures in Example 6.

[0059] Fig. 20 This is a graph showing the results of determining the content of organic acids in fermented milk obtained using different combinations of starter cultures in Example 6.

[0060] Fig.21 This is a graph showing the water holding capacity and viable bacterial count of the fermented milk of the SL+LC group during the storage period in Example 6.

[0061] Fig. 22 This is a graph showing the pH and titratable acidity results of the fermented milk of the SL+LC group during the storage period in Example 6. DETAILED DESCRIPTION

[0062] The present invention provides a strain of Lactobacillus breadii X66 with good beneficial activity. Specifically, the present invention relates to a strain of Lactobacillus breadii X66 derived from traditional dairy milk tofu in Inner Mongolia, which is safe, tolerant to gastrointestinal environment, has self-aggregation ability, antioxidant ability and resistance to common pathogenic bacteria, and a high-density culture method of the strain. In order to better understand the present invention, the present invention is described and explained in detail in conjunction with specific embodiments below, but the protection scope of the present invention is not limited to the scope shown in the following specific embodiments.

[0063] Example 1: A strain of Lactobacillus breadii X66 derived from traditional fermented dairy products in China

[0064] The present embodiment is a strain of Lactobacillus bread X66 from milk tofu in Inner Mongolia, which is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No: 62641. The biological characteristics of the strain Lactobacillus bread X66 are: the colonies are round, convex, smooth and neat edges, milky white, and moist and smooth on the surface after streaking on an MRS medium plate and culturing at 37°C for 48h. Gram-staining microscopy shows that it is a Gram-positive bacterium with a rod-shaped body and no spores (such as Figure 1 The results of physiological and biochemical tests showed that the strain could produce acid using maltose, fructose and glucose, and was negative in KOH test and starch hydrolysis test. It was facultatively anaerobic and did not liquefy gelatin. X66 was identified as Lactobacillus breade based on 16S rDNA and physiological and biochemical characteristics. Its 16S rDNA sequence is shown in SEQ NO: 1, and the phylogenetic tree of Lactobacillus breade X66 is shown in Figure 2 .

[0065] Example 2: Isolation and purification of Lactobacillus bread X66

[0066] This embodiment is a method for separating and purifying a strain of Lactobacillus breadii X66 derived from traditional dairy product milk tofu in Inner Mongolia. The method is carried out in the following steps:

[0067] (1) Take about 10 g of Inner Mongolia milk tofu sample, add an appropriate amount of 0.9% NaCl solution into a conical flask, and oscillate at a constant temperature (170 r / min, 15 min) to fully dissolve and dilute it. -3 ~10 -7 5 dilution gradients, take 100-200uL of dilution solution, add it to the sterilized MRS-CaCO3 agar medium for coating, and place it in a 37℃ constant temperature incubator for inversion culture for 2-3 days;

[0068] (2) Observe the growth of the colonies and select characteristic single colonies after characteristic colonies grow on the plate;

[0069] (3) A single colony was streaked onto MRS solid medium and cultured at 37°C for 24 h. A single colony was streaked again. This process was repeated 6 times.

[0070] (4) Inoculate the purified single colony into liquid MRS medium and culture at 37°C until the bacterial solution becomes turbid.

[0071] (5) Under sterile conditions, take 0.5 mL of bacterial solution and 0.5 mL of 50% sterile glycerol into a 1.5 mL centrifuge tube, mix them in a 1:1 ratio of equal volumes, and store in a -20°C refrigerator.

[0072] The MRS liquid culture medium comprises, by mass ratio, 1 part of casein digest, 1 part of beef extract powder, 0.4 part of yeast extract powder, 0.2 part of triammonium citrate, 0.5 part of sodium acetate, 0.02 part of magnesium sulfate heptahydrate, 0.005 part of manganese sulfate tetrahydrate, 0.2 part of dipotassium hydrogen phosphate, 2 parts of glucose, 0.1 part of Tween-80, and distilled water to 100 parts, the pH before sterilization is 5.7±0.2, and sterilization is performed at 121° C. for 15 min.

[0073] Example 3: Safety evaluation of Lactobacillus bread X66

[0074] The strain frozen in a -20°C refrigerator was inoculated into a seed culture medium and activated 2 to 3 times to obtain a seed culture solution with a bacterial content of 0.8 to 1.0×10 9 CFU / mL. The seed culture medium is MRS liquid culture medium. Sterilize at 121°C for 15 min.

[0075] 3.1 Antibiotic sensitivity test of strains

[0076] The minimum inhibitory concentration of Lactobacillus bread X66 to ampicillin, erythromycin, tetracycline and chloramphenicol was determined by the microbroth dilution method. The detection concentration of ampicillin is 0.125-128μg / mL, the detection concentration of erythromycin is 0.0625-64μg / mL, and the detection concentrations of tetracycline and chloramphenicol are 0.5-512μg / mL and 0.125-128μg / mL, respectively. First, dilute the antibiotics twice to 10 times of all detection concentrations, take a sterile 96-well plate, add 10μL of antibiotic solution to wells 1-11, and then add 90μL of MH medium containing X66 bacterial solution to each well, and mix with a sterile pipette tip. The positive control is 90μL of the same concentration of the test bacterial solution and 10μL of distilled water, and the negative control is 90μL of MH medium and 10μL of distilled water. After culturing at 37℃ for 24h, the OD was measured with an enzyme reader. 600nm, compared with the corresponding critical values ​​in the guidelines of the European Food Safety Authority, the results of the X66 drug resistance test were obtained. The results are shown in Table 1. The bread lactobacillus X66 in this example is sensitive to ampicillin, erythromycin, tetracycline and chloramphenicol, and is safe.

[0077] The MH medium comprises, by mass ratio, 30 parts of beef (from which extract powder is extracted), 0.15 parts of soluble starch, 1.75 parts of casein hydrolysate, and distilled water to make up to 100 parts. The pH value before sterilization is 7.3±0.2, and the medium is sterilized at 121° C. for 15 minutes.

[0078] Table 1 Minimum inhibitory concentration of antibiotics against Lactobacillus bisporus X66

[0079]

[0080] Note: EFSA is the abbreviation for European Food Safety Authority.

[0081] 3.2 Hemolysis experiment

[0082] The activated Lactobacillus bread X66 and the quality control strain Escherichia coli ATCC 25922 were streaked on Columbia blood agar using a sterilized inoculation needle, and a blank control was made at the same time. The colonies were cultured at 37°C for 48 hours, and the presence of obvious hemolysis zones around the colonies was observed and photographed for record. Figure 3 Shown in the figure, the right side is the growth of Lactobacillus brevis X66 on blood agar plate, and the left side is the growth of quality control strain Escherichia coli on blood agar plate. The quality control strain selected in the present embodiment has hemolyticity, and hemolysis circle appears around. Compared with the quality control strain, there is no obvious hemolysis circle formation around Lactobacillus brevis X66, that is, Lactobacillus brevis will not cause hemolysis damage to human body, and is safe.

[0083] 3.3 Indole experiment

[0084] The activated Lactobacillus bread X66 seed fermentation liquid and the positive control strain Escherichia coli liquid were inoculated into the peptone water culture medium at a rate of 3% (v / v), and a blank control was set up at the same time. The culture was cultured at 37°C for 72 hours, and 8 to 10 drops of indole reagent were added to observe the experimental results. The appearance of a red ring at the interface of the two liquid layers was positive, and the absence of color change was negative. The experimental results are as follows Figure 4 As shown in the figure, after adding the indole reagent, a clear red ring appeared at the junction of the two liquid layers in the peptone water medium inoculated with the positive control Escherichia coli, and the indole test result was positive. The peptone water medium inoculated with X66 and the blank control had no obvious color change, and the indole test result was negative.

[0085] The peptone water culture medium is, by mass ratio, 1 part of bacteriological peptone and 0.5 part of sodium chloride, distilled water is added to 100 parts, stirred evenly, the pH value of the culture medium is adjusted to 7.8, dispensed into conical bottles, and sterilized at 121° C. for 15 minutes.

[0086] 3.4 Nitroreductase assay

[0087] The activated Lactobacillus bread X66 and the positive control strain Escherichia coli were inoculated into the nitroreductase detection medium at a rate of 3% (v / v), and a blank control group was set up at the same time. The culture was cultured at 37°C for 72 hours. 8 to 10 drops of α-naphthylamine solution and p-aminobenzenesulfonic acid solution were added to the culture medium in sequence. After gently shaking, the color change of the culture medium was observed and the results were recorded by taking photos. If the culture medium turns red, it means that the nitroreductase test result is positive, otherwise it is negative. The results are as follows Figure 5 As shown, the result of the nitroreductase test of Lactobacillus bread X66 was negative. There was no active nitroreductase in the Lactobacillus bread X66 strain and it would not reduce nitrate to nitrite.

[0088] The nitroreductase detection medium is, by mass ratio, 1 part of bacteriological peptone, 0.1 part of potassium nitrate, adding distilled water to 100 parts, stirring evenly, adjusting the pH value of the medium to 7.4, dispensing into conical bottles, and sterilizing by high pressure at 121° C. for 15 minutes.

[0089] 3.5 Aminodecarboxylase assay

[0090] The activated Lactobacillus bread X66 and the positive control strain Escherichia coli were inoculated in a decarboxylase medium at a rate of 3% (v / v) and cultured at 37°C for 5 times to induce the production of decarboxylase. 200 μL was inoculated in 2 mL of a decarboxylase medium containing histidine, arginine, tyrosine, and lysine at a final concentration of 0.5%. The medium was cultured at 37°C for 3 days to detect the production of histamine, putrescine, spermidine, and tyramine. If the culture medium changes from yellow to purple, it is positive; otherwise, it is negative. The experimental results are as follows Figure 6 As shown, the culture media with the addition of four kinds of amino acids of Escherichia coli were all purple in color, indicating a positive result; while the culture media with Lactobacillus bread X66 were all yellow, indicating a negative result, which was consistent with the blank control result, indicating that X66 did not produce amino decarboxylase during its proliferation and metabolism, thus avoiding the formation of biogenic amines.

[0091] The aminodecarboxylase culture medium comprises, by mass ratio, 0.5 parts of tryptone, 0.8 parts of beef extract, 0.4 parts of yeast extract, 0.05 parts of Tween-80, 0.02 parts of magnesium sulfate, 0.005 parts of manganese sulfate, 0.004 parts of iron sulfate, 0.01 parts of calcium carbonate and 0.006 parts of bromocresol purple, distilled water is added to 100 parts, the mixture is stirred evenly, the pH value of the culture medium is adjusted to 5.3, and the mixture is sterilized by high pressure at 121° C. for 15 minutes.

[0092] 3.6 Oral acute toxicity study in mice

[0093] Lactobacillus bread X66 was prepared into 1×10 8 CFU / mL of bacterial liquid was commissioned to Guangzhou Customs Technical Center to conduct an oral acute toxicity experiment on mice of X66 in accordance with the "National Food Safety Standard Acute Oral Toxicity Experiment" (Experimental Animal Use License No. SYXK (Guangdong) 2018-0086). 20 half-male and half-female SPF-grade Kunming mice (weight 20.0-22.0g, experimental animal production license No. SCXK (Guangdong) 2018-0002, quality certificate No. 44007200105037) were purchased from the Guangdong Medical Experimental Animal Center. The room temperature was 22±1℃ and the humidity was 62±5%. 50.00g of bacterial liquid was weighed and mixed with ultrapure water to 100mL for experimental liquid. The experimental liquid was administered orally once with a gavage needle at a rate of 0.2mL / 10g body weight. The mice were fasted for 4h before gavage and had free access to water. Water. A normal diet was given 1 hour after gavage; the experimental observation period was 14 days, and the poisoning symptoms, number of deaths and time of death of the mice were recorded. During this period, the mice were weighed once a week and recorded. The results are shown in Table 2. After 14 days of oral administration of X66 bacterial solution, no clinical abnormalities, eye abnormalities or signs of death were found in the mice. After the experiment, the mice were dissected and no abnormalities were found with the naked eye. Although the weight of the mice gradually increased during the study, the difference was not significant. The acute oral toxicity LD50 of the oral solution containing X66 to male and female KM mice was>10.0g / kg body weight, which is a practically non-toxic level.

[0094] Table 2 Results of the acute oral toxicity test of X66 bacterial solution on mice

[0095]

[0096] Example 4: Study on the beneficial activity of Lactobacillus bread X66

[0097] 4.1 Artificial gastrointestinal environment tolerance experiment

[0098] The activated Lactobacillus bread X66 seed fermentation liquid was inoculated into artificial simulated gastric fluid at pH = 2.0, 2.5, 3.0, and 4.0 at an inoculation rate of 10% (v / v), and an equal volume of distilled water was used as a blank group. The culture was carried out at 37°C, and samples were taken at 0h, 1h, 2h, and 3h to count the number of viable bacteria. Figure 7 As shown, Lactobacillus bread X66 survived well in artificial gastric juice of different pH values. At the same time, the survival rate of X66 in artificial gastric juice of different pH values ​​(the lowest was 76.29%) was higher than that in an equal volume of distilled water. The survival rate of X66 in a pH 2.5 environment was higher than that in other pH environments. Within 1 to 2 hours, the survival rate of X66 in pH 2.5, 3.0, and 4.0 environments increased, and the survival rate in gastric juice of pH 2.0 and the blank experiment was basically the same, which indicates that the X66 strain has a strong acid resistance and can adapt to an acidic environment of 2.5 to 4.0 and proliferate normally.

[0099] Preparation of the PBS buffer solution: by mass ratio, 0.8 parts of NaCl, 0.02 parts of KH2PO4, 0.115 parts of Na2HPO4, add distilled water to 100 parts, and stir evenly. Preparation of the artificial gastric juice: 0.3 parts of pepsin, add PBS buffer solution to 100 parts, and adjust the pH to 2.0, 2.5, 3.0, 4.0 with 1N HCl. After fully dissolved, filter and sterilize with a microporous membrane with a pore size of 0.22 μm.

[0100] The activated Lactobacillus bread X66 seed fermentation liquid was inoculated into artificial intestinal fluid at an inoculum volume of 10% (v / v), and an equal volume of distilled water was used as a blank group, cultured at 37°C, and samples were taken at 0h, 2h, 4h, 6h, and 8h to count the number of viable bacteria. As shown in Table 3, Lactobacillus bread X66 survived well after being treated in simulated intestinal fluid, and the survival rate exceeded 100% after exposure to artificial intestinal fluid for 2 to 8h.

[0101] The preparation of the artificial intestinal fluid: by weight ratio, add 0.1 part of trypsin to 100 parts of PBS buffer solution, adjust the pH to 8.0 with 1N NaOH, and after fully dissolving, filter and sterilize with a microporous membrane with a pore size of 0.22 μm.

[0102] Table 3 Changes in the number of live bacteria of Lactobacillus bisporus X66 in artificial intestinal fluid

[0103]

[0104] 4.2 Determination of self-cohesion ability

[0105] Take 10 mL of each test bacterial solution into a centrifuge tube, centrifuge at 10000 rpm for 5 min at 4 °C, discard the supernatant, collect the bacterial precipitate, wash twice with sterile PBS, and adjust the bacterial concentration to 108 CFU / mL, use a vortex shaker to mix the X66 suspension, measure the absorbance at 600nm, incubate at 37℃, take the upper bacterial suspension at 4, 8 and 24h, and measure the absorbance at 600nm.

[0106]

[0107] Among them, A t and A0 are the OD of the test sample and control sample, respectively. 600nm value.

[0108] The activated Lactobacillus gasseri LGZ1029 and Lactobacillus delbrueckii DMLD-H1 were subjected to comparative experiments with Lactobacillus bread X66 in the same manner as above. Figure 8 As shown, the self-aggregation ability of the three strains increased over time. At 24 hours, the self-aggregation ability of X66 exceeded 90%, which was higher than that of Lactobacillus delbrueckii DMLD-H1 and Lactobacillus gasseri LGZ1029.

[0109] 4.3 Antioxidant capacity determination

[0110] 4.3.1DPPH free radical scavenging experiment

[0111] After activation, the concentration of Lactobacillus bisporus X66 was adjusted to 10 9 CFU / ml, respectively test the DPPH free radical scavenging ability of bacterial suspension, supernatant and intracellular extract diluted 10, 100 and 1000 times. Mix 1mL of the sample to be tested with 1mL of 0.2mmol / LDPPH ethanol solution, react in the dark at room temperature for 30min, and measure the absorbance at 517nm. DPPH free radical scavenging rate

[0112]

[0113] Among them, A r A is the absorbance measured after the sample to be tested is mixed with DPPH ethanol solution. s To measure the absorbance by replacing the DPPH ethanol solution with an equal volume of anhydrous ethanol, A tThe absorbance was measured by using PBS buffer instead of the sample. The above-mentioned supernatant is the supernatant obtained after centrifugation of the bacterial solution at this concentration, and the above-mentioned intracellular extract is the intracellular extract obtained after the bacterial solution at this concentration was broken by a cell disruptor for 30 minutes and then centrifuged. The DPPH free radical scavenging rate of Lactobacillus gasseri LGZ1029 and Lactobacillus delbrueckii DMLD-H1 was determined by the same method as above. As shown in Figure 9, Lactobacillus bread X66 has a strong antioxidant capacity, and as the bacterial concentration increases, the antioxidant activity gradually increases. When the number of viable cells reaches 10 9 CFU / mL, the scavenging rate of X66 supernatant to DPPH was over 90%, which was slightly lower than that of Lactobacillus gasseri LGZ1029 and slightly higher than that of Lactobacillus delbrueckii DMLD-H1.

[0114] 4.3.2 Hydroxyl radical scavenging experiment

[0115] After activation, the concentration of Lactobacillus bisporus X66 was adjusted to 10 9 CFU / ml, respectively detect the hydroxyl radical scavenging ability of bacterial suspension, supernatant and intracellular extract diluted 10, 100 and 1000 times. Add 1.0mL of 0.02mol / L PBS buffer, 0.5mL of 2.5mmol / L o-phenanthroline ethanol solution, 0.5mL of 2.5mmol / L ferrous sulfate solution, 0.5mL of 20mmol / L hydrogen peroxide solution, and then add 0.5mL of the sample to be tested. After 60min of constant temperature reaction at 37℃, centrifuge at 3500r / min for 10min, and measure the absorbance A at 536nm. o .

[0116] Among them, A o A is the absorbance measured after adding hydrogen peroxide solution and reacting with the sample to be tested. p The absorbance is measured by replacing the sample with PBS buffer, A q The absorbance was measured by replacing the sample and hydrogen peroxide solution with PBS buffer. The hydroxyl radical scavenging rate of Lactobacillus gasseri LGZ1029 and Lactobacillus delbrueckii DMLD-H1 was determined by the same method as above. As shown in Figure 10, when the number of viable cells reached 10 9 CFU / mL, the scavenging rate of X66 supernatant on hydroxyl free radicals exceeded 80%, which was significantly higher than that of Lactobacillus gasseri LGZ1029 and Lactobacillus delbrueckii DMLD-H1.

[0117] 4.3.3 Superoxide anion scavenging experiment

[0118] After activation, the concentration of Lactobacillus bisporus X66 was adjusted to 10 9CFU / ml, detect the superoxide anion scavenging ability of bacterial suspension, supernatant and intracellular extract diluted 10, 100 and 1000 times respectively. Add 2mL of 150mmol / L pH 8.2 Tris-HCl solution and 0.5mL of the sample to be tested, preheat at 25℃ for 10min, then add 0.5mL of 1.2mmol / L pyrogallol aqueous solution preheated at 25℃, bath at the same temperature for 20min, centrifuge at 3500r / min for 10min, and measure the absorbance at 325nm.

[0119]

[0120] Among them, A j The absorbance is measured after adding pyrogallol aqueous solution and the sample to be tested in warm water bath, A k The absorbance was measured by replacing the pyrogallol aqueous solution with PBS buffer. m The absorbance is measured by replacing the sample with PBS buffer, A n The absorbance of the sample to be tested was measured by replacing the pyrogallol aqueous solution with PBS buffer. The superoxide anion scavenging rate of Lactobacillus gasseri LGZ1029 and Lactobacillus delbrueckii DMLD-H1 was determined by the same method as above. As shown in Figure 11, the superoxide anion scavenging rate of the supernatant of the three strains increased with the increase of concentration. When the number of viable cells reached 10 9 CFU / mL, the supernatant of X66 had a maximum scavenging rate of superoxide anions of 49.13%, which was lower than that of Lactobacillus gasseri LGZ1029 and higher than that of Lactobacillus delbrueckii DMLD-H1.

[0121] 4.4 Pathogen inhibition experiment

[0122] Staphylococcus aureus ATCC 12598, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa PAO1 were used as indicator strains. Sterilized LB medium was used to dilute the three strains to an OD of 600nm is 0.5±0.01. Prepare LB medium with an agar content of 0.75%, sterilize and cool to about 40°C for use. Add 160μL of the diluted bacterial solution to 100mL of the above LB medium, mix well and pour into the plate, and put it in a 4°C refrigerator for use. The activated Lactobacillus bread X66 seed fermentation liquid is inoculated into MRS medium at a 4% inoculation amount, cultured at 37°C for 16-18h, centrifuged at 4°C and 8000r / min for 15min, and the supernatant is collected and filtered through a 0.22μm filter membrane to obtain the fermentation supernatant. Use a hole puncher with a diameter of 8mm to punch holes on the plates of the above three bacteria, add 100μL of fermentation supernatant to each hole, and after sufficient diffusion, place it in a 37°C constant temperature incubator for 8-12h, observe the size of the inhibition zone and measure and record the diameter.

[0123] The above LB agar medium, calculated by mass, contains 1 part of sodium chloride, 0.5 part of yeast extract powder, 1 part of bacteriological peptone, 0.75 part of agar, and distilled water to 100 parts, with a pH of 7.0±0.2. Fig.12 The results showed that the supernatant of X66 had a certain inhibitory effect on common pathogenic bacteria Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, among which the inhibitory effect of X66 supernatant on Escherichia coli was slightly stronger than that on Staphylococcus aureus and Pseudomonas aeruginosa.

[0124] Table 4 The size of inhibition zone of Lactobacillus X66 against different indicator bacteria

[0125]

[0126]

[0127] Example 5 Study on high density culture method of Lactobacillus bread X66

[0128] The strain frozen in a -20°C refrigerator was inoculated into a seed culture medium and activated 2 to 3 times to obtain a seed culture solution. The bacterial content of the seed starter was 0.8 to 1.0×10 9 CFU / mL. In a ratio of 4% (v / v), the activated seed fermentation liquid is inoculated into a high-density culture medium, and fermented and cultured at 37°C. The number of viable bacteria is counted after 16-18 hours. The high-density culture medium is: based on the MRS broth culture medium, by mass ratio, 3-5 parts of glucose, 2.5-3.5 parts of bacteriological peptone, 2.5-3.5 parts of beef extract powder, 2.5-3.5 parts of yeast extract powder, 0.2-0.4 parts of dipotassium hydrogen phosphate, 0.8-1.0 parts of sodium acetate, 0.08-0.12 parts of magnesium sulfate monohydrate, 0.01-0.03 parts of manganese sulfate tetrahydrate, add distilled water to 100 parts, stir evenly, and adjust the pH of the culture medium to 6.8. After stirring evenly, sterilize at 0.10MPa for 15 minutes, cool to 30°C for use.

[0129] The high-density culture was obtained by the following single factor optimization, Plackett-Burmen experimental design and Box-Behnken optimization experiment, as follows:

[0130] 5.1 Single factor optimization for high-density culture

[0131] 5.1.1 Carbon source optimization

[0132] Based on MRS medium, 2, 4, 6, and 8 portions of glucose were added respectively to observe the changes in the number of viable bacteria of X66. Fig.13As shown in the figure, with the increase of glucose addition, the number of live bacteria increased first and then decreased. Too high glucose concentration will inhibit the proliferation of Lactobacillus bread. When the amount of glucose added was 4 parts, the number of live bacteria of X66 reached the highest value of 1.34×10 9 CFU / mL.

[0133] 5.1.2 Nitrogen source optimization

[0134] (1) Bacteriological peptone optimization

[0135] Based on MRS medium, 1, 1.5, 2, 2.5, 3, and 3.5 portions of bacteriological peptone were added respectively to observe the changes in the number of viable bacteria of X66. As shown in Figure 14(a), when the amount of bacteriological peptone added was 3 portions, the number of viable bacteria of X66 reached the maximum value of 1.38×10 9 CFU / mL, when the added amount continued to increase, the number of viable bacteria decreased, indicating that the bacteria could not utilize excessive bacteriological peptone.

[0136] (2) Optimization of beef extract powder

[0137] Based on MRS medium, 1, 1.5, 2, 2.5, 3, and 3.5 portions of beef extract powder were added, respectively, and the changes in the number of viable bacteria of X66 were observed. As shown in Figure 14(b), when the amount of beef extract powder added was 3 portions, the number of viable bacteria of X66 reached the maximum value of 1.61×10 9 CFU / mL, too much beef extract powder is not conducive to obtaining a high-concentration bacterial suspension.

[0138] (3) Yeast extract powder optimization

[0139] Based on MRS medium, 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 portions of yeast extract powder were added respectively to observe the changes in the number of viable bacteria of X66. As shown in Figure 14(c), when the amount of yeast extract powder added was 3 portions, the number of viable bacteria of X66 reached the maximum value of 1.62×10 9 CFU / mL, after adding 3 copies, the number of live bacteria decreased, indicating that the utilization rate of yeast extract powder by the bacteria has reached its limit, and too much nitrogen source will have a certain inhibitory effect.

[0140] 5.1.3 Growth Factor Optimization

[0141] (1) Optimization of manganese sulfate tetrahydrate

[0142] Based on MRS medium, 0.005, 0.01, 0.015, 0.02, 0.025, and 0.03 parts of manganese sulfate tetrahydrate were added respectively to observe the changes in the number of viable bacteria of X66. Fig.15 As shown in (a), when the addition amount of manganese sulfate tetrahydrate was 0.02 parts, the number of viable bacteria of X66 reached the maximum value of 1.38×10 9CFU / mL, and the number of viable bacteria decreased with the continued addition of manganese sulfate tetrahydrate.

[0143] (2) Optimization of magnesium sulfate monohydrate

[0144] Based on MRS medium, 0.02, 0.04, 0.06, 0.08, 0.1, and 0.12 parts of magnesium sulfate monohydrate were added respectively to observe the changes in the number of viable bacteria of X66. Fig.15 As shown in (b), when the addition amount of magnesium sulfate monohydrate is 0.1 part, the number of viable bacteria of X66 is the maximum value of 1.30×10 9 CFU / mL.

[0145] (3) Sodium acetate optimization

[0146] Based on MRS medium, 0.3, 0.5, 0.7, 0.9, and 1.1 parts of sodium acetate were added respectively to observe the changes in the number of viable bacteria of X66. Fig.15 As shown in (c), when the amount of sodium acetate added was 0.9 parts, the number of viable bacteria of X66 reached a maximum of 1.87×10 9 CFU / mL, the amount of sodium acetate added continued to increase, and the number of viable bacteria tended to be stable.

[0147] (4) Optimization of potassium dihydrogen phosphate

[0148] Based on MRS medium, 0.1, 0.3, 0.5, 0.7, and 0.9 portions of potassium dihydrogen phosphate were added respectively to observe the changes in the number of viable bacteria of X66. Fig.15 As shown in (d), when the amount of potassium hydrogen phosphate added was 0.3 parts, the number of viable bacteria of X66 reached a maximum of 1.51×10 9 CFU / mL, as the amount of potassium dihydrogen phosphate added gradually increased, the number of viable bacteria decreased, indicating that excessive addition of potassium dihydrogen phosphate is not conducive to the proliferation of the bacteria.

[0149] 5.2 Culture medium formulation optimization experiment

[0150] Design Expert11 software was used to design the culture medium optimization experiment. In the above single factor analysis, eight single factors were selected for analysis, including glucose, bacteriological peptone, beef extract powder, yeast extract powder, manganese sulfate tetrahydrate, magnesium sulfate monohydrate, sodium acetate, and dipotassium hydrogen phosphate. The experimental results showed that when the culture medium contained (MRS culture medium as the base medium) 4 parts of glucose, 3 parts of bacteriological peptone, 3 parts of beef extract powder, 3 parts of yeast extract powder, 0.02 parts of manganese sulfate tetrahydrate, 0.1 parts of magnesium sulfate monohydrate, 0.9 parts of sodium acetate, and 0.3 parts of dipotassium hydrogen phosphate, the obtained bacterial suspension had the largest number of viable bacteria in each single factor optimization experimental group. Therefore, these concentrations were selected as the benchmark for the Plackett-Burmen experiment and Box-Behnken optimization experiment.

[0151] Table 5 Plackett-Burmen test results

[0152]

[0153]

[0154] The results of the Plackett-Burmen experiment are shown in Table 5. The data in Table 5 were analyzed using Design Expert 11 software, and the results are as follows:

[0155] Table 6 Plackett-Burmen experiment analysis table

[0156]

[0157] As can be seen from Table 6, magnesium sulfate, bacteriological peptone and yeast extract powder have the highest contribution to the growth of Lactobacillus bread X66, which are 18.13%, 23.45% and 20.27% respectively. The results show that among the eight promoting factors, the addition of magnesium sulfate, bacteriological peptone and yeast extract powder has a more obvious growth promoting effect on Lactobacillus bread X66 than the others, and the contribution values ​​of these three factors are much higher than other factors. Therefore, magnesium sulfate, bacteriological peptone and yeast extract powder were selected as research objects for the next Box-Behnken optimization experiment.

[0158] 5.3 Analysis of Box-Behnken optimization experimental results

[0159] Table 7 Box-Behnken test results

[0160]

[0161] Table 8 Box-Behnken experiment analysis table

[0162]

[0163]

[0164] The results of the Box-Behnken experiment are shown in Table 7. As can be seen from Table 8, the p of the fitted model is less than 0.0001, and the determination coefficient R 2 =0.9954, indicating that the model fits the actual situation well. According to the above analysis, the multiple regression equation of the number of viable bacteria Y on the independent variables bacteriological peptone (A), yeast extract powder (B), and magnesium sulfate (C) is:

[0165] Y=26.65333-0.71250A+0.35000B+0.46250C+0.100000AB+0.17500AC+0.050000BC-6.68917A 2 -4.16417B 2 -6.43917C 2

[0166] The equation shows that the linear relationship between the number of live bacteria in the fermentation broth of Lactobacillus bread X66 and the three influencing factors is significant, that is, this modeling method is reliable. According to the regression equation, the interaction coefficient of AC in the interaction term coefficient is large, indicating that the interaction effect between magnesium sulfate and bacteriological peptone is large, while the interaction coefficient of BC is small, indicating that the interaction effect between yeast extract powder and magnesium sulfate is small.

[0167] In order to test the validity of the equation, the variance analysis was performed on the model of the number of live bacteria in the fermentation broth of Lactobacillus bread X66, and the partial regression coefficients of the three factors were tested. The regression coefficient of A in the first-order term was more significant than that of other factors, indicating that the concentration of bacteriological peptone had a more significant promoting effect on the growth of X66. 2 =0.9954, indicating that the equation fits the actual growth situation of Lactobacillus X66 well.

[0168] The first-order partial derivative of the second-order equation is obtained. When the response value Y (viable count) is at its maximum, the factor levels are 2.48 parts of bacteriological peptone (A), 3.02 parts of yeast extract powder (B), and 0.10 parts of magnesium sulfate (C). At this time, the theoretical prediction of the concentration of Lactobacillus bread X66 is 2.67×10 9 CFU / mL, the culture medium obtained by this formula was verified in the experiment, and the actual bacterial concentration was 2.63×10 9 CFU / mL, close to the theoretical value.

[0169] Therefore, the formula of the optimal high-density culture medium of the present invention is: by mass, 100 parts of MRS broth culture medium, 2.48 parts of bacteriological peptone, 3.02 parts of yeast extract powder, and 0.10 parts of magnesium sulfate.

[0170] Example 6: Application of Lactobacillus bread X66 in fermented milk

[0171] Preparation of cryoprotectant: by weight, 8 parts of trehalose, 4.72 parts of sodium glutamate, 9.76 parts of skim milk powder, dilute to 100 parts with distilled water, sterilize at 110° C. for 15 min to obtain a cryoprotectant solution.

[0172] Preparation of starter: Take an appropriate amount of X66 bacterial solution (4℃, 8000r / min, 15min) that has been reactivated with optimized culture medium and centrifuged to obtain bacterial sludge precipitate. Mix the protective agent with the bacterial sludge according to the ratio of protective agent: bacterial sludge = 1:1 (weight ratio), put it in a -20℃ refrigerator for pre-freezing for 48h, and put the pre-frozen bacterial sludge precipitate into a vacuum freeze dryer for freeze drying for 24h. Count the bacterial powder obtained by freeze drying according to the national standard. The results showed that the number of live bacteria of Lactobacillus bread X66 powder was 1.1×10 11 CFU / g, stored in a -20℃ refrigerator for future use. The traditional starter culture is freeze-dried powder of Lactobacillus delbrueckii DMLD-H1 and Streptococcus thermophilus DMST-H2, obtained by freeze-drying, with a viable count of 10 10 CFU / g. The control group, Coulson fermentation agent, was purchased from Guangzhou Yukaishen Biotechnology Co., Ltd., with a viable count of 10 10 CFU / g, stored in a -20℃ refrigerator for later use.

[0173] Preparation of fermented milk: Weigh 12 parts of whole milk powder and 8 parts of white sugar by mass and put them in a fermented milk bottle, add 100 parts of distilled water, heat in a water bath at 85°C for 30 minutes, cool to room temperature, inoculate the starter, put it in a 43°C incubator and ferment until the milk is completely coagulated, then put the fermented milk in a 4°C refrigerator for 12 hours for post-fermentation.

[0174] 6.1 Determination of the optimal compound ratio of fermentation agents

[0175] Lactobacillus baker's X66 (LC) and traditional starter (Streptococcus thermophilus DMST-H2 + Lactobacillus delbrueckii DMLD-H1, SL) were added into the starter at a mass ratio of 0:1, 1:9, 1:5, 1:3, 1:1, 3:1, 5:1, 9:1, and 1:0, and the inoculum amount was 100 parts of reconstituted milk plus 1 part of bacterial powder. The cells were fermented at 43°C for 4h and post-ripened at 4°C for 12h. The quality of the fermented milk was analyzed and the relevant basic physicochemical properties and sensory measurements were carried out to determine the optimal compound ratio.

[0176] Table 9 Effects of different compounding ratios on basic physicochemical properties and sensory properties of fermented milk

[0177]

[0178] Note: The compounding ratio in the table is the ratio of Lactobacillus bisporus X66: (Streptococcus thermophilus DMST-H2: Lactobacillus delbrueckii DMLD-H1 = 1:1)

[0179] As shown in Table 9, when the proportion of Lactobacillus bisporus is too high, the fermented milk has insufficient acidity and flavor, and the coagulation state is poor at the same fermentation time; when the proportion of Lactobacillus bisporus is too low, the fermented milk acidity and flavor meet the standard requirements, but the water holding capacity is low, which is not conducive to the storage stability of the fermented milk. When the compound ratio is 1:1, the fermented milk has a good coagulation state, moderate acidity, and a water holding capacity level of 51.91±0.01%. Sensory evaluation of fermented milk with different compound ratios is shown in Figure 9. Fig.17 Combined with sensory evaluation, it was found that the color, texture, smell and taste of the fermented milk were all at a relatively good level and relatively balanced. In summary, this study selected a 1:1 bacterial strain compound ratio for subsequent research on fermented milk.

[0180] 6.2 Determination of rheological properties of fermented milk

[0181] The rheological properties of the samples were measured at 25 ± 1 °C using a HAAKE RheoStress 600HP (Karlsruhe, Germany) viscometer equipped with a Z34DIN, Ti sensor (gap 1 mm). The stirred fermented milk sample was pipetted and 2 mL of the sample was placed on the rheometer plate. The shear rate was increased from 0.01 to 200 rad / s within 180 s and kept constant at 200 rad / s until the system was completely destroyed. The flow curve was obtained by decreasing the shear rate from 200 rad / s to 0 within 180 s. The frequency sweep was performed with a fixed amplitude of 1% in the range of 0.1 to 100 Hz to determine the viscoelasticity of the fermented milk.

[0182] As shown in Figure 18(a), the fermented milk of different groups was fitted according to the Herschel-Bulkley equation. The yield stress τ0 increased with the increase of shear rate, and the rate of increase gradually decreased. The addition of Lactobacillus X66 made the rheological curve closer to that of the Control group, which helped to improve the rheological properties of fermented milk.

[0183] As shown in Figures 18(b) and (c), the dynamic time-varying curves of the viscoelasticity of fermented milk with different combinations show that, with the increase of step time, the storage modulus (G') and loss modulus (G") first rapidly decrease to a certain level and then remain stable. G' and G" are closely related to the gel network structure of fermented milk. The higher the G' and G" at the same step time, the tighter the three-dimensional gel network structure of the fermented milk. The G' and G" of the fermented milk in the SL+LC group are higher than those in the SL group and lower than those in the Control group, indicating that the addition of X66 helps to improve the density of the gel structure of the fermented milk and make the micelles more tightly bound, thereby optimizing the viscoelasticity of the fermented milk.

[0184] 6.3 Determination of sugar and organic acid content in fermented milk

[0185] The content of sugars and organic acids was determined by high performance liquid chromatography. The results of monosaccharide and disaccharide content determination are as follows Fig.19 The glucose content of the fermented milk in the SL+LC group was 955.21 μg / mL, which was more than three times that of the SL group and the Control group. The total content of disaccharides fermented in the SL+LC group was the lowest, while the total content of monosaccharides was the highest, indicating that Lactobacillus X66 can promote the utilization of lactose and sucrose by the composite bacteria, decompose them into monosaccharides such as glucose and galactose, and thus improve sugar metabolism.

[0186] The organic acid contents of the three groups of fermented milk are as follows Fig. 20 As shown in the figure, the L-lactic acid content of the fermented milk in the SL+LC group was significantly higher than that in the SL group and the Control group, and the D-lactic acid content was basically the same. The results show that adding Lactobacillus brevis X66 to fermentation can produce more L-lactic acid, and traditional starter does not affect the acid production of X66 and even has a certain promoting effect. Lactobacillus brevis fermentation can produce a variety of organic acids such as formic acid, acetic acid, and propionic acid. After adding X66, the acetic acid content in the fermented milk increased by 951.12 μg / mL compared with the SL group, and the acetic acid content accounted for a large proportion, which may be related to the high content of acetic acid produced by Lactobacillus brevis fermentation. Combined with the results of sugar determination, the polysaccharide content produced by the SL+LC group fermentation increased, which can further promote sugar decomposition and metabolism to produce organic acids.

[0187] 6.4 Determination of stability of fermented milk during storage

[0188] Changes in viable bacterial count and water holding capacity of fermented milk in the SL+LC group during the 4-week storage period Fig.21 The results showed that the water holding capacity of fermented milk decreased from 57.48% to 45.63% during the storage period, fluctuated between 38% and 45% in the middle three weeks, and a small amount of whey precipitated in the late storage period. The number of viable bacteria in the fermented milk after 12 hours after reaching the fermentation end point was 9.59×10 8 CFU / mL, which meets the requirements of the national standard for the number of live bacteria in fermented milk. During the storage period, the number of live bacteria remained basically the same in the first week, and then began to decrease gradually. At 28 days, the change in the number of live bacteria became smaller, and the final number of live bacteria was 2.59×10 7 CFU / mL, that is, the number of live bacteria in fermented milk remains at a high level during the storage period, which meets the lactic acid bacteria count of GB19302-2010 "National Food Safety Standard Fermented Milk" ≥ 1×10 6 CFU / mL requirements.

[0189] Changes of pH and titratable acidity of fermented milk in SL+LC group during 4 weeks of storage Fig. 22The results show that the pH of fermented milk decreased first and then stabilized during the storage period, while the titratable acidity gradually increased from 73.08°T to 89.47°T within 0-21 days, and began to decrease after 21 days, reaching 83.19°T at 28 days. This shows that the pH and acidity of fermented milk can be stabilized at a good level during the storage period, which meets the requirement of acidity ≥ 70°T in GB19302-2010 "National Food Safety Standard Fermented Milk".

Claims

1. A strain of Lactobacillus baker's, characterized in that The strain is Lactobacillus crustorum X66, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 4, 2022, with the deposit number GDMCC No: 62641.

2. A high-density culture method for Lactobacillus bread, characterized in that: The steps include: (1) inoculating the Lactobacillus bread X66 described in claim 1 into MRS broth medium for activation to obtain a seed fermentation liquid; (2) adding the seed fermentation liquid to the high-density culture medium, and culturing at a constant temperature of 36 to 38° C. for 16 to 18 hours to obtain a high-density fermentation liquid; The high-density culture medium formula is: by mass, 100 parts of MRS broth culture medium, 3-5 parts of glucose, 2.5-3.5 parts of bacteriological peptone, 2.5-3.5 parts of beef extract powder, 2.5-3.5 parts of yeast extract powder, 0.2-0.4 parts of dipotassium hydrogen phosphate, 0.8-1.0 parts of sodium acetate, 0.08-0.12 parts of magnesium sulfate monohydrate, and 0.01-0.03 parts of manganese sulfate tetrahydrate.

3. The high-density culture method according to claim 2, characterized in that: Preparation of the high-density culture medium: dilute the above raw materials to 100 parts with distilled water, adjust the pH to 5.7-6.0, stir and dissolve evenly, sterilize and cool.

4. The high-density culture method according to claim 3, characterized in that: The sterilization condition is 0.08-0.10 MPa sterilization for 15-20 min.

5. The high-density culture method according to any one of claims 2 to 4, characterized in that: The method for activating the seed fermentation liquid comprises the following steps: inoculating frozen Lactobacillus breadii X66 in an MRS broth medium at a volume ratio of 2 to 6:100 for a first activation, inoculating the obtained once-activated seed fermentation liquid in an MRS broth medium at a volume ratio of 3 to 5:100 for a second activation, and obtaining a second-activated seed fermentation liquid.

6. The high-density culture method according to any one of claims 2 to 4, characterized in that: The bacterial content of the seed fermentation liquid is 0.8-1.0×10 9 CFU / mL; the bacterial content of the high-density fermentation broth is 2.0×10 9 CFU / mL and above.

7. A bread Lactobacillus powder, characterized in that: The high-density fermentation bacterial liquid prepared by the method according to any one of claims 2 to 6 is freeze-dried to obtain a viable bacterial count of 1.1×10 11 CFU / g or above bacterial powder.

8. The bread Lactobacillus powder according to claim 7, characterized in that The freeze-drying method comprises the following steps: centrifuging a high-density fermented bacterial liquid to obtain a bacterial sludge precipitate, uniformly mixing a protective agent with the bacterial sludge, pre-freezing the mixture in a refrigerator, and freeze-drying the pre-frozen bacterial sludge precipitate; and preparing the protective agent: by weight, adding 7 to 9 parts of trehalose, 4.70 to 4.74 parts of sodium glutamate, and 9.74 to 9.78 parts of skimmed milk powder, diluting the mixture to 100 parts with water, and sterilizing the mixture to obtain the protective agent.

9. Use of the Lactobacillus breadi according to claim 1, the high-density fermentation solution prepared by the method according to any one of claims 2 to 6, or the Lactobacillus breadi powder according to claim 7 or 8 in preparing fermented milk.

10. The use according to claim 9, characterized in that: Streptococcus thermophilus DMST-H2 and Lactobacillus delbrueckii DMLD-H1 are added to the bread lactobacillus, high-density fermentation bacterial liquid or bread lactobacillus powder for joint fermentation.

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