A kind of preparation method of Lactobacillus casei and preparation thereof

Abstract

The present invention discloses a method for preparing Lactobacillus casei and a preparation thereof. The Lactobacillus casei was deposited in the China General Microbiological Culture Collection Center on July 6, 2022, with a deposit number of CGMCC No. 25194. The preparation method of the double-layer emulsion probiotic preparation comprises the following steps: (1) vortexing Lactobacillus casei and a probiotic protective agent solution to uniformly disperse them to form an inner water phase W1; (2) dissolving ursolic acid in oil to form an oil phase O, and mixing the oil phase O with the inner water phase W1 by high-speed shearing to obtain an oil-in-water type colostrum W1 / O; (3) using a suspension of colloidal particles as an external water phase W2, adding the external water phase W2 to the colostrum W1 / O, and mixing by medium-speed shearing to obtain a double-layer emulsion probiotic preparation with a W1 / O / W2 type double emulsion structure.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic preparation, and relates to a preparation method of a Lactobacillus casei and a preparation thereof, and more specifically, to the screening of a Lactobacillus casei with probiotic function and a probiotic preparation thereof based on a water-in-oil-in-water double-layer emulsion structure and a preparation method thereof. Background Art

[0002] Probiotics are defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host". Probiotics have many health benefits: stimulating the growth of intestinal microbiota, producing antimicrobial substances, eliminating potentially harmful bacteria, and strengthening the body's natural defense system. Probiotics have been proposed for the treatment of several intestinal diseases, including inflammatory bowel disease (IBD), irritable bowel syndrome, and infectious diarrhea.

[0003] At the same time, allergic diseases have become a global problem, and the prevalence of eczema, food allergies and asthma has increased dramatically during this period. The impact of allergic diseases on the healthcare system and society is often significant, and it is one of the most common causes of chronic and hospitalized diseases. Currently, most of the means of treating such diseases are with the help of medication, but medication has certain side effects, so effective and safe treatment methods have yet to be discovered. In addition, more and more studies have found that probiotic lactic acid bacteria have preventive and / or therapeutic effects on human allergic diseases. A large number of trials have used the method of taking probiotics to treat allergic diseases. This method is simple and can be used in combination with a variety of probiotics, which has great development advantages.

[0004] In recent years, a lot of research has been done to encapsulate probiotics. Water-in-oil-in-water (W / O / W) emulsion is a common emulsion system. Its dispersed phase itself is a water-in-oil (W / O) emulsion. The inner water phase W1 and the outer water phase W2 are easily miscible because they have the same polarity. The two-step emulsification method is the most common method for preparing multiple emulsions. Compared with the common oil-in-water (O / W) emulsion, W / O / W emulsion has the advantages of delivering and controlling the release of bioactive ingredients, masking bad odors, and reducing fat content. It is widely used in food, medicine, cosmetics and other fields. Encapsulating probiotics with W / O / W emulsions can improve the viability of probiotic cells during gastrointestinal digestion; increase the storage stability of probiotics and protect them from external factors (such as pH, oxygen, temperature, and light during storage); ensure the controlled and directional release of encapsulated substances in the gastrointestinal tract; increase the ability to add ideal concentrations of probiotics (from low to high); and adjust the properties of the emulsion (including size, charge, dispersibility, and chemical modification).

[0005] Ursolic Acid, also known as ursolic acid, is a triterpenoid compound found in natural plants. Studies in recent years have found that ursolic acid has a variety of biological activities, such as lowering blood sugar, anti-virus, anti-ulcer, anti-tumor and cardiovascular protection. Ursolic acid also has obvious antioxidant function, low toxicity, few adverse reactions, and has broad development prospects. However, its water solubility is poor and its oral bioavailability is low, which limits its use. Summary of the invention

[0006] The invention aims to overcome the deficiencies of the prior art and provides a method for preparing a lactobacillus casei preparation and a preparation thereof.

[0007] In order to achieve the above object, the technical solution provided by the present invention is:

[0008] The Lactobacillus casei was deposited in the China General Microbiological Culture Collection Center on July 6, 2022, with a deposit number of CGMCC No. 25194, and the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. The Lactobacillus casei can produce high amounts of exopolysaccharides. The produced exopolysaccharides have the ability to scavenge DPPH and -OH free radicals; they can stimulate the proliferation, NO release and phagocytosis rate of RAW264.7 macrophages; the exopolysaccharides of CGMCC No. 25194 and its two purified components EPS-1 and EPS-2 can inhibit the release of β-HEX through the RBL-2H3 cell degranulation model, regulate the release of HIS, IL-4 and TNF-α, have potential anti-allergic activity, and can be used to prepare foods with desensitizing effects and / or antioxidant activity.

[0009] The preparation method of the double-layer emulsion probiotic preparation based on the above-mentioned Lactobacillus casei comprises the following steps:

[0010] (1) vortexing the Lactobacillus casei and probiotic protective agent solutions to uniformly disperse them to form an inner aqueous phase W1; in the inner aqueous phase W1, the mass percentage content of the probiotic protective agent is 8-12%, and the colony count of Lactobacillus casei is not less than 1×10 7-8 CFU / mL; preferably, the probiotic protective agent is completely dissolved and hydrated overnight at 4°C, and the pH is adjusted to 7.0;

[0011] (2) dissolving ursolic acid in oil to form an oil phase O, and mixing the oil phase O with the internal water phase W1 by high-speed shearing to obtain a water-in-oil colostrum W1 / O; the mass percentage content of ursolic acid in the oil phase O is 1% to 5%; the volume ratio of the oil phase O to the internal water phase W1 is 1:1, 1:2, 1:3, 2:3 or 3:5; preferably, after adding ursolic acid to the oil phase O, stirring with a magnetic stirrer for 20 minutes to make it evenly dispersed;

[0012] (3) A suspension of colloidal particles is used as the external aqueous phase W2, and the external aqueous phase W2 is added to the colostrum W1 / O, and mixed by medium-speed shearing to obtain a double-layer emulsion probiotic preparation with a W1 / O / W2 double emulsion structure; the mass percentage content of the colloidal particles in the suspension is 0.1-0.5%. When preparing the suspension, the colloidal particles are added to water, treated in a water bath at 40-60°C for 10-20 minutes, dissolved, and then hydrated at 4°C overnight to obtain the preparation; the volume ratio of the external aqueous phase W2 to the colostrum W1 / O is 2:3.

[0013] Preferably, the probiotic protective agent described in step (1) is at least one of skim milk powder, whey protein isolate, trehalose, sucrose, stachyose, xylo-oligosaccharides, lactose and glycerol.

[0014] Preferably, the preparation method of the inner aqueous phase W1 in step (1) is: continuously activating the probiotics for three generations, expanding the culture, centrifuging and washing twice to obtain bacterial sludge, and suspending the bacterial sludge in a probiotic protective agent to prepare a probiotic suspension.

[0015] Preferably, the oil described in step (2) is at least one of soybean oil, peanut oil, rapeseed oil, corn oil, and sunflower oil; and the external aqueous phase W2 described in step (3) is at least one of pectin, locust bean gum, gellan gum, xanthan gum, and carrageenan.

[0016] Preferably, the high-speed shearing rate in step (2) is 6000-10000 rpm, and the shearing time is 1-5 min; the medium-speed shearing rate in step (3) is 5000-7000 rpm, and the shearing time is 1-3 min.

[0017] The probiotic preparation based on the ursolic acid W1 / O / W2 type Pickering emulsion structure is prepared according to the above method. The probiotic is at least one of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactococcus lactis or Streptococcus thermophilus.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are:

[0019] 1. The Pickering emulsion prepared by the present invention is a new W1 / O / W2 double-layer emulsion system stabilized by ursolic acid instead of traditional emulsifiers, with high content of probiotic cells in the inner water phase W1 and ursolic acid dispersed in the oil phase O. The emulsion is safe and non-toxic, has a high probiotic content, and greatly improved emulsion stability.

[0020] 2. The present invention adopts a double-layer emulsification system. First, the high internal phase processing technology can ensure the high content of probiotics in the inner water phase. Secondly, the antioxidant activity of ursolic acid can prevent the probiotic preparation from being oxidized and destroyed after storage and administration. In addition, the colloidal solution of the outer water phase has a very stable protective effect on the double-layer emulsion.

[0021] 3. The preparation process of the present invention is simple, which can not only improve the survival rate of probiotics during the shelf life of probiotics products, ensure the active functions of probiotics products, but also improve the bioavailability of ursolic acid. The present invention can be used for the development of new probiotic foods, medicines and health products.

[0022] In addition, ursolic acid can also be used as an emulsifier for medicines and foods. The present invention first screens a Lactobacillus casei with potential anti-allergic activity and explores the feasibility of using ursolic acid as an emulsifier to prepare a probiotic W1 / O / W2 Pickering emulsion. The emulsion prepared by this method can not only improve the survival rate and stress resistance of probiotics, but also improve the bioavailability of ursolic acid.

[0023] The first aspect of the present invention provides a strain of Lactobacillus casei. The second aspect of the present invention provides a method for preparing a Pickering emulsion based on W1 / O / W2 type for embedding CGMCC No.25194, wherein ursolic acid is used as an emulsifier. Compared with the traditional chemical synthetic emulsifier, ursolic acid is a natural compound in plants, and the amount used during emulsification is small, the operation is simple, and the anti-aggregation ability is strong. At the same time, ursolic acid has multiple biological activities, but its water solubility is poor and the oral absorption rate is low. The emulsion prepared by the method can improve the bioavailability of ursolic acid when used. Due to the introduction of ursolic acid, the problem that the traditional use of small molecule emulsifiers to prepare water-in-oil-in-water emulsions cannot be prepared or cannot be stably prepared is solved.

[0024] In short, the Lactobacillus casei provided by the present invention has good acid and bile salt tolerance and is a safe strain. The extracellular polysaccharide produced by the bacterium has good antioxidant capacity and can stimulate the proliferation, NO release and phagocytosis rate of RAW264.7 macrophages. At the same time, it was found through the RBL-2H3 cell degranulation model that the extracellular polysaccharide of Lactobacillus casei CGMCC No.25194 and its two components can inhibit the release of β-HEX, regulate the release of HIS, IL-4 and TNF-α, indicating that the polysaccharide produced by the bacterium may have potential anti-allergic activity. Lactobacillus casei CGMCC No.25194 is embedded in a double-layer emulsion. First, ursolic acid is added as an emulsifier to rapeseed oil and stirred to obtain an oil phase O, which is mixed with an inner water phase W1 containing Lactobacillus casei CGMCC No.25194 bacteria, and high-speed homogenization is performed to obtain colostrum W1 / O; after the outer water phase W2 is mixed with the colostrum W1 / O, high-speed homogenization is performed to obtain a W1 / O / W2 type double emulsion, i.e., a probiotic preparation. The invention adopts a double-layer emulsification system to embed Lactobacillus casei CGMCC No.25194. The obtained double-layer emulsion has strong stability and stable structure, and can greatly improve the survival rate of Lactobacillus casei CGMCC No.25194 during the storage of the bacterial agent. Secondly, the added natural active ingredient ursolic acid has biological activities such as anti-oxidation, which can prevent the emulsion structure from being oxidized and destroyed after storage and administration, improve the survival rate of Lactobacillus casei CGMCC No.25194, and enable Lactobacillus casei CGMCC No.25194 to smoothly reach the intestine to play a probiotic role. At the same time, the problem of low oral bioavailability of ursolic acid due to its poor water solubility can be solved, and the present invention can be used for the development of new probiotic foods, medicines and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Comparison of exopolysaccharide contents of different lactic acid bacteria;

[0026] Figure 2 Comparison of acid resistance of different lactic acid bacteria;

[0027] Figure 3 Comparison of bile salt tolerance of different lactic acid bacteria;

[0028] Figure 4 The survival ability of different lactic acid bacteria in simulated gastrointestinal fluid;

[0029] Figure 5 Colony morphology of Lactobacillus casei LZ9183;

[0030] Figure 6 Microscopic examination of Lactobacillus casei LZ9183;

[0031] Figure 7Comparison of the antioxidant capacity of exopolysaccharides of Lactobacillus casei LZ9183 and other lactic acid bacteria; DPPH(A) / hydroxyl(B) free radical scavenging ability of exopolysaccharides;

[0032] Figure 8 The regulatory activity of Lactobacillus casei LZ9183 and other lactic acid bacteria exopolysaccharides on RAW264.7 macrophages; the effects on proliferation activity (A) NO release (B) and phagocytic activity (C);

[0033] Fig. 9 Elution curve of Lactobacillus casei LZ9183 exopolysaccharide through DEAE-Cellulose-52 column;

[0034] Fig.10 The elution curves of the two components EPS-1 and EPS-2 of Lactobacillus casei LZ9183 through Sephadex G-100 column;

[0035] Fig.11 The inhibition rate of Lactobacillus casei LZ9183 exopolysaccharide and its two components on β-HEX release in RBL-2H3 cells;

[0036] Fig.12 Double-layer emulsion samples encapsulating probiotics;

[0037] Fig.13 Double-layer emulsion microstructure for encapsulating probiotics;

[0038] Fig.14 Particle size and Zeta potential results of the probiotic-encapsulated double-layer emulsion;

[0039] Fig.15 Storage stability results of the probiotic-encapsulated bilayer emulsion;

[0040] Fig.16 Storage stability results of the probiotic-encapsulated bilayer emulsion;

[0041] Fig.17 In vitro simulated digestion results of double-layer emulsions encapsulating probiotics. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0043] Example 1

[0044] 1. Preliminary screening of exopolysaccharide-producing lactic acid bacteria

[0045] 28 strains of lactic acid bacteria (milk and dairy products from the main mountainous pastures in Hunan) isolated and preserved in the laboratory were randomly selected for the study of exopolysaccharide production. The lactic acid bacteria were cultured on solid culture medium at 37°C for 48 hours, and the inoculation needle was touched to a single colony and slowly pulled out to compare whether the colonies of different strains had obvious stringing phenomenon, and the lactic acid bacteria with continuous stringing phenomenon were selected for further study.

[0046] 2. Rescreening of exopolysaccharide-producing lactic acid bacteria

[0047] The strains after initial screening were inoculated into 50 mL of liquid culture medium, cultured at 37°C for 24 h, centrifuged for 10 min (4°C, 10,000 r / min) to remove the bacteria, and the fermentation supernatant was boiled in water for 10 min. After cooling to room temperature, 80% (w / w) trichloroacetic acid solution was added to a final concentration of 6% (w / v), and allowed to stand at 4°C for 12 h. The protein precipitate was removed by centrifugation for 10 min (4°C, 10,000 r / min). Three volumes of anhydrous ethanol were added to the supernatant, allowed to stand at 4°C for 12 h, and centrifuged for 20 min (4°C, 10,000 r / min) to collect the crude polysaccharide precipitate. The precipitate was dissolved by adding an appropriate amount of distilled water, and dialyzed at 4°C for 72 h using a dialysis bag with a molecular weight cutoff of 8000-14000 D. The water was changed every 8 h. After the dialysis, the polysaccharide solution was fixed to 50 ml with distilled water, and the polysaccharide content was determined by the phenol-sulfuric acid method. The results of the comparison of the extracellular polysaccharide yields of each strain are shown in Figure 1 .

[0048] from Figure 1 It can be seen that the polysaccharide content of strains numbered 12, 77, 79, 83, 89, 91, 150, 154, LZ9183, 194, 260, and 285 was higher than that of the control strain LGG (577.33±8.64mg / L) (P≤0.05), and there was no significant difference between 302, Y10, and Y28 and the control strain (P>0.05). Based on the laboratory's preliminary research foundation and comprehensive consideration of yield, the final selected strains for the polysaccharide yield rescreening were 77, 89, 91, 154, LZ9183, 260, 285, Y06, Y07, and Y10.

[0049] 3. Acid and bile salt resistance test

[0050] 3% lactic acid bacteria suspension was inoculated into liquid culture medium at pH=2, pH=3 and containing ox bile salt (3.0 g / L) respectively, and OD was measured. 600nm Value A0, culture at 37℃ for 12h, measure OD 600nm Value A1, in ΔOD 600nm =A1-A0 indicates the strain's acid and bile resistance. The test results for acid and bile resistance are shown in Figure 2 and Figure 3 .

[0051] Depend on Figure 2 It can be seen that when the pH of the strain culture environment is 2, the acid tolerance of strains 89, LZ9183, 260, and Y10 is higher than that of the control strain LGG (ΔOD value = 0.0251 ± 0.0029); when pH = 3, the acid tolerance of 89, LZ9183, and 260 is also higher than that of the control strain LGG (ΔOD value = 0.0343 ± 0.0024), and the acid resistance of Y10 reaches a level similar to that of LGG, which indicates that the four lactic acid bacteria 89, LZ9183, 260, and Y10 have strong tolerance to acid, among which the acid resistance of LZ9183 and 260 is more prominent, which is significantly different from the control strain (P≤0.05). When the pH of the culture medium was 2, the ΔOD value of LZ9183 was 0.0297±0.0031, and the ΔOD value of 260 was 0.0340±0.0026; when the pH of the culture medium was 3, the ΔOD value of LZ9183 was 0.0360±0.0030, and the ΔOD value of 260 was 0.0437±0.0040; under the same acid environment, there was no significant difference in the acid resistance of the two strains (P>0.05).

[0052] Depend on Figure 3 It can be seen that in the environment without bile salt addition, the ΔOD values ​​of 89 and 285 were higher than the control strain LGG (ΔOD value = 1.009 ± 0.0021) (P ≤ 0.05), and there was no significant difference in the growth of the four strains 154, LZ9183, 260, and Y10 compared with the control bacteria (P> 0.05), indicating that most strains can grow well under bile salt-free conditions. In the environment with bile salt addition, the growth of the control bacteria LGG was significantly inhibited, and its ΔOD value was 0.2250 ± 0.0287. Among the other ten lactic acid bacteria, the ΔOD values ​​of 89, LZ9183, 285, and Y06 were all higher than that of LGG, and the ΔOD values ​​of strains 89 and LZ9183 were significantly different from the ΔOD value of LGG (P ≤ 0.05). This shows that these four strains can also grow relatively stably in an environment with bile salts and have a strong ability to tolerate bile salts.

[0053] 4. Simulated gastrointestinal fluid test

[0054] Take 1 mL of lactic acid bacteria suspension and use the plate count method to determine the number of live bacteria at 0 h. Take another 1 mL of lactic acid bacteria suspension and add it to 9 mL of simulated gastric juice and intestinal juice, vortex and shake for 30 seconds to mix evenly, and culture it in a 37°C incubator for 3 hours. After taking it out, use the plate count method to determine the number of live bacteria at 3 hours. The survival rate of LAB in gastrointestinal juice is calculated according to the following formula:

[0055]

[0056] The results of the simulated gastrointestinal fluid test are shown in Figure 4 .Depend on Figure 4 It can be seen that in the environment of simulated artificial gastrointestinal fluid, the 10 strains of lactic acid bacteria screened all have a high survival rate. The survival rate of gastric juice is above 78%, and the survival rate of intestinal fluid is as low as 79%. There are differences in the survival rates of strains in gastric juice and intestinal fluid, such as the survival rate of Y06 in gastric juice is as high as 128.48±3.65%, and the survival rate in intestinal fluid is as high as 163.96±5.04%. In the environment of simulated gastric fluid, only the survival rates of LZ9183, Y06, and Y07 were higher than the control strain LGG (100.29±1.40%), and the survival rates of 77, 285, and Y10 were not significantly different from those of the control strain (P>0.05), which may be due to the specificity of the strain or species. In the simulated intestinal fluid environment, the survival rates of strains 89, LZ9183, and Y06 were higher than those of the control strain LGG (97.36±2.09%), and the survival rates of strains 77 and Y10 were similar to those of the control strains and had no significant difference (P>0.05). Based on the above results, it was found that strains 89, LZ9183, 285, Y06, Y07, and Y10 all had strong tolerance in simulated gastrointestinal fluid, and therefore had the potential to pass smoothly through human gastrointestinal fluid, and strains Y06 and LZ9183 performed particularly well in this test.

[0057] 5. Safety evaluation

[0058] (1) Hemolysis test: 5 μL of lactic acid bacteria culture solution was spotted on a blood agar plate and cultured at 37°C for 72 h. Staphylococcus aureus was used as the positive control group. The hemolytic activity of the strain was determined by observing whether there was a transparent hemolysis zone around the colony on the blood agar plate.

[0059] (2) Decarboxylase test: A lysine decarboxylase test kit was used, with Staphylococcus aureus as the positive control group. The color change of the liquid in the vial was used to determine whether the strain produced bioamines.

[0060] (3) Antibiotic sensitivity test: 20 antibiotic-sensitive paper discs were lightly pressed onto the surface of a solid culture medium containing lactic acid bacteria, and the antibiotic sensitivity of LAB was determined by measuring the diameter of the inhibition zone.

[0061] (4) The results of hemolytic activity and lysine decarboxylase activity of different strains are shown in Table 1.

[0062] Table 1 Hemolytic and decarboxylase activities of lactic acid bacteria

[0063]

[0064] Note: a “-” indicates non-hemolytic, “+” indicates hemolytic; b“-” means decarboxylase negative, and “γ” means decarboxylase positive.

[0065] As shown in Table 1, with Staphylococcus aureus as the positive control strain, none of the 11 tested strains produced hemolysis, and the lysine decarboxylase activity was negative. The above results show that all the tested bacteria were initially judged to be safe strains in terms of bioamine production and hemolysis.

[0066] The results of the sensitivity test of the strains to different antibiotics are shown in Table 2.

[0067] Table 2 Sensitivity of strains to 20 antibiotics

[0068]

[0069]

[0070] Note: d is the diameter of the inhibition zone of antibiotic sensitive tablets against lactic acid bacteria, respectively: "-" d<5mm; "+": 5mm≤d<15mm;

[0071] “++”: 15mm≤d<25mm; "+++": d≥25mm indicates the sensitivity of antibiotics to lactic acid bacteria.

[0072] As can be seen from Table 2, there are certain differences in the sensitivity of the 11 lactic acid bacteria to 20 antibiotics. They are more sensitive to ampicillin, cephalexin, cefazolin, etc., and less sensitive to carbenicillin, neomycin, cefoperazone, etc. When lactic acid bacteria preparations are used together with antibiotics with inhibition zones greater than 15 mm, such as strain 77 and minocycline (inhibition zone diameter ≥ 25 mm), the survival rate of lactic acid bacteria will be affected.

[0073] 6. Identification of strains

[0074] After activating strain LZ9183, single colonies were obtained by streaking method and their characteristics were observed. Figure 5 It can be seen that the colony is milky white, moist, and has neat edges. Figure 6 It can be seen that the cell microscopic morphology (X100) is rod-shaped, without spores, and Gram-positive.

[0075] The PCR amplification product of 16S rDNA was sequenced, and the sequencing results were logged on to the NCBI website for Blast sequence comparison. The results showed that the 16S rDNA homology of strain LZ9183 and Lactobacillus casei was more than 99%, and the bacteria were confirmed to be Lactobacillus casei. The Lactobacillus casei was deposited in the China General Microbiological Culture Collection Management Center on July 6, 2022, with a deposit number of CGMCC No. 25194, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0076] Example 2

[0077] 1. Extraction of Extracellular Polysaccharides

[0078] Four strains of lactic acid bacteria including LZ9183 were activated three times to prepare bacterial suspension, which was inoculated into 1L liquid culture medium at a 3% inoculation rate. Extracellular polysaccharides were extracted by water extraction and alcohol precipitation. After dialysis, the polysaccharide aqueous solution was freeze-dried to obtain extracellular polysaccharide powder, which was dissolved in double distilled water.

[0079] 2. Antioxidant activity of extracellular polysaccharides

[0080] (1) DPPH free radical scavenging ability: Ascorbic acid solution with the same concentration gradient as polysaccharide was prepared as a control. 1 mL of LAB EPS solution or The control solution and an equal amount of 100 μmol / LDPPH solution (dissolved in anhydrous ethanol) were added to a brown test tube, mixed and placed in a dark place for 30 min, taken out and centrifuged for 10 min (8000 r / min), and the supernatant was taken to test the OD of each group. 517nm The DPPH clearance rate of LAB extracellular polysaccharide was calculated as follows:

[0081]

[0082] Where:

[0083] A1—indicates the absorbance value of the test group;

[0084] A2—represents the absorbance value of the control group (absolute ethanol instead of DPPH solution);

[0085] A0—represents the blank absorbance value of the test group (distilled water replaces the sample).

[0086] (2) -OH radical scavenging ability: An ascorbic acid solution with the same concentration gradient as that of the polysaccharide was prepared, and the -OH radical scavenging ability of the polysaccharide solution was tested using an -OH radical scavenging ability test kit.

[0087] The results of the antioxidant activity of extracellular polysaccharides are shown in Figure 7 .from Figure 7 It can be seen that the hydroxyl radical scavenging ability of a single extracellular polysaccharide is concentration-dependent, and the hydroxyl radical scavenging rates of each concentration group of the same polysaccharide are significantly different (P≤0.05). When the concentration of the polysaccharide solution is 1000μg / mL, the scavenging activity is in the following order: LZ9183>Y10>89>285, and the scavenging rates are 45.86%, 44.10%, 39.93% and 38.27%, respectively.

[0088] 3. Immunomodulatory activity of extracellular polysaccharides

[0089] ① Effect on NO secretion by RAW264.7 macrophages: Cell culture and grouping were the same as above, LPS (1 μg / mL) was used as the positive control group, and a blank control was set up at the same time. After 24 h of culture, 50 μL of supernatant was aspirated from each well of the 96-well plate, and the Griess reagent kit was used to react with it in the dark, and the OD490nm value of each group was measured.

[0090] ② Effect on phagocytosis of neutral red by RAW264.7 macrophages: Remove the cell culture medium from each well, wash with PBS, then add 100 μL of 0.072% neutral red solution to each well, incubate at 37°C for 30 min, then absorb the neutral red solution, wash with PBS until there is no obvious purple-red color in the well, then add 100 μL of RIPA lysis solution to each well, let stand at 4°C for 12 h to fully lyse the cells, and measure the OD of each group 570nm The neutral red phagocytic activity was calculated as follows:

[0091] The immunomodulatory effect of extracellular polysaccharides on RAW264.7 macrophages is shown in Figure 8 . After adding polysaccharides to the cell culture medium, the NO concentration increased in a concentration-dependent manner, and there were significant differences among the concentration groups (P≤0.05). The experimental results showed that the four crude extracellular polysaccharides can effectively increase the NO secretion of RAW264.7 macrophages. The NO concentration of LZ9183 extracellular polysaccharide was 64.28% higher than that of the positive control LPS group at a concentration of 1000μg / mL (P≤0.05), indicating that it may have the potential to be an immunostimulant. The phagocytic neutral red experiment was used to observe the effects of strain 89, LZ9183, 285, and Y10 extracellular polysaccharides on the phagocytic activity of macrophages. The four polysaccharides can enhance the phagocytic activity of RAW264.7 macrophages, among which Y10 and LZ9183 extracellular polysaccharides have a more obvious effect on enhancing the ability of RAW264.7 macrophages to phagocytose neutral red.

[0092] Example 3

[0093] 1. Extraction of Extracellular Polysaccharides

[0094] Four strains of lactic acid bacteria including LZ9183 were activated three times to prepare bacterial suspension, which was inoculated into 1L liquid culture medium at a 3% inoculation rate. Extracellular polysaccharides were extracted by water extraction and alcohol precipitation. After dialysis, the polysaccharide aqueous solution was freeze-dried to obtain extracellular polysaccharide powder, which was dissolved in double distilled water.

[0095] 2. Purification of Crude Exopolysaccharides

[0096] (1) Ion exchange column separation and purification: DEAE-Cellulose-52 dry powder was first soaked in distilled water to remove impurities, then alternately soaked in 0.5M HCl and NaOH and washed until neutral, wet packed, and added to a chromatography column (40×2.6cm). After the chromatography column was balanced, 10mg / mL polysaccharide sample was added to the chromatography column and eluted with deionized water, 0.1M, 0.3M and 0.5M NaCl solutions in sequence, with an elution rate of 1.2mL / min. The eluted solution was collected using an automatic collection device, and the collection volume of each tube was 5mL. The polysaccharide content in the collection tube was tracked and monitored using the phenol-sulfuric acid method, and an elution curve was drawn. The solutions in the same elution peak were collected and combined, and different components of extracellular polysaccharides were obtained by reduced pressure concentration, dialysis and freeze-drying.

[0097] (2) Gel column separation and purification: After Sephadex G-100 dry powder is soaked and impurities are removed, it is also wet packed and slowly poured into a chromatography column (60×1.6 cm). After the chromatography column is stable, 5 mg / mL of the extracellular polysaccharide component solution of strain LZ9183 purified by ion exchange is loaded and eluted with deionized water. The elution rate is 0.5 mL / min, and the elution solution is collected by an automatic collection device. The collection volume of each tube is 5 mL. The polysaccharide content in the collection tube is tracked and monitored by the phenol-sulfuric acid method, and an elution curve is drawn. The solutions in the same elution peak are collected and combined, and the extracellular polysaccharide component with a relatively uniform molecular weight is obtained by reduced pressure concentration, dialysis and freeze-drying.

[0098] from Fig. 9 It can be seen that the extracellular polysaccharide of strain LZ9183 was divided into three components after separation by anion exchange column. According to the order of peaks, the first component was named EPS-1, which was eluted with deionized water, indicating that component EPS-1 was uncharged and was a neutral polysaccharide. After collecting 35 tubes, the elution solution was changed to 0.1 mol / L NaCl solution, and component EPS-2 was eluted at this time, indicating that EPS-2 was an acidic polysaccharide or a complex polysaccharide that may have acidic groups and was negatively charged. The elution curves of the two components after Sephadex G-100 gel column are shown in Figure 2. Fig.10As shown, both polysaccharide components obtained a single peak after elution, indicating that both components may be polysaccharides with relatively uniform molecular weight. The liquid in the peak tube was collected, combined, dialyzed, and freeze-dried to obtain purified polysaccharide samples, which were named EPS-1 and EPS-2, respectively.

[0099] (3) Determination of antiallergic activity of Lactobacillus casei LZ9183 extracellular polysaccharide

[0100] RBL-2H3 cells were cultured in DMEM medium containing FBS and 1% double antibody.

[0101] ② Preparation of cell culture medium containing polysaccharides: The freeze-dried Lactobacillus casei LZ9183 extracellular polysaccharides and their purified components were added to the culture medium, filtered through a 0.22 μm microporous filter membrane, and the polysaccharide concentrations in the culture medium were adjusted to 100 μg / mL, 250 μg / mL and 500 μg / mL, respectively, and used as the culture medium for the experimental group (Group A) for subsequent experiments.

[0102] ③ Establishment and grouping of RBL-2H3 degranulation model: RBL-2H3 cells in the logarithmic growth phase were digested and cell suspension (2.0×10 5 cells / mL), 1 mL of cell suspension was inoculated into a 6-well plate, 3 mL of complete medium was added, and the cells were divided into the experimental group (group A), model group (group B), positive control group (group Y) and blank group (group K). After culturing in an incubator for 24 h (37°C, 5% CO2), the RBL-2H3 degranulation model was established as follows.

[0103] The original culture medium was removed from the BL-2H3 cells cultured for 24 hours, and the cells were washed twice with PBS buffer. 1 mL of culture medium containing 0.5 μL / mL anti-DNP-IgE was added to group A, group B and group Y, and 1 mL of culture medium was added to group K, and the cells were cultured for 12 hours; the culture medium was removed, the cells were washed twice, 1 mL of culture medium containing polysaccharide was added to group A, 1 mL of culture medium containing 500 μg / mL difen fumarate was added to group Y, and 1 mL of culture medium was added to groups B and K, and the cells were incubated for 2 hours; the culture medium was removed, the cells were washed twice, 1 mL of culture medium containing 0.25 μg / mL DNP-BSA was added to groups A, group B and group Y, and 1 mL of culture medium was added to group K, and the cells were placed in an incubator.

[0104] ④ RBL-2H3 cell β-hexosaminidase (β-HEX) release inhibition test

[0105] Configuration of related reagents:

[0106] CS buffer: weigh 1.5500 g of sodium citrate and 1.2175 g of citric acid, add distilled water to make up to 100 mL.

[0107] Color development solution: Weigh 34.20 mg PNP-Nag, dissolve it in CS buffer, make up to 100 mL, filter it with a 0.22 μm microporous filter membrane, and store it in a -20 °C refrigerator for later use.

[0108] Stop solution: prepare Na2CO3 / NaHCO3 buffer solution, adjust the pH to 11.0, and filter through a 0.22μm microporous filter membrane for later use.

[0109] Determination method: After adding DNP-BSA to each test group and culturing for 0.5h, take the supernatant, centrifuge for 5min (1000r / min, 4℃), add 50μL supernatant and colorimetric solution to the 96-well plate in turn, incubate at 37℃ for 1h, add 200μL stop solution, and measure the OD405nm value. The calculation formula of the inhibition rate of polysaccharide on β-HEX release of RBL-2H3 cells is as follows:

[0110] Depend on Fig.11 It can be seen that Lactobacillus casei LZ9183 exopolysaccharide and the two purified components have an inhibitory effect on the release of β-HEX from RBL-2H3 cells, and the inhibition rate is between 23.64±0.705 and 52.293±0.153%. In the concentration range of 100μg / mL-500μg / mL, the inhibition rate of crude polysaccharide and purified components is on the rise, and the inhibition rate of β-HEX release between each concentration group of exopolysaccharide has significant differences (P≤0.05).

[0111] ⑤ Effects of crude polysaccharide and its two components on the release of activated cytokines in RBL-2H3 cells

[0112] After adding DNP-BSA to each group and culturing for 1h and 4h, the supernatant was taken and centrifuged for 5min (1000r / min, 4℃), and the supernatant was retained for later use. The release of HIS (histamine) (1h) and IL-4 (interleukin 4) and TNF-α (tumor necrosis factor) (4h) in the culture medium of the RBL-2H3 cell degranulation model was determined using an enzyme-linked immunosorbent assay kit (ELISA kit).

[0113] The release of HIS, IL-4 and TNF-α from RBL-2H3 cells after treatment with crude polysaccharide, EPS-1 and EPS-2 was measured by ELISA kit. The results are shown in Table 3.

[0114] Table 3 Effects of crude polysaccharide of Lactobacillus casei LZ9183 and its two components on activation of HIS, IL-4 and TNF-α release in RBL-2H3 cells

[0115]

[0116] According to the HIS content of each group in Table 3, the secretion of HIS in the model group (group B) and the normal group (group K) was significantly different (P≤0.05), which was 27.713±1.21ng / mL and 15.88±0.653ng / mL, respectively, indicating that the degranulation model of RBL-2H3 cells was successfully established. The HIS release level of cells treated with different concentrations of crude polysaccharides and polysaccharide components was lower than that of the model group (group B) (27.713±1.214ng / mL), especially the RBL-2H3 cells treated with EPS-1 at a concentration of 500μg / mL, whose HIS release was 16.265±0.761ng / mL, which was very close to the histamine release of the normal group (15.88±0.653ng / mL), and there was no significant difference between the two (P>0.05). The order of HIS release of RBL-2H3 cells treated with crude polysaccharide, EPS-1 and EPS-2 at a concentration of 500 μg / mL was crude polysaccharide>EPS-2>EPS-1, and there was a significant difference (P≤0.05). The above results showed that EPS-1 could effectively reduce the release of histamine. After treatment with crude polysaccharide, EPS-1 and EPS-2, the release of TNF-α of RBL-2H3 cells was lower than that of the model group, and the release of each group decreased with the increase of polysaccharide concentration. The release of TNF-α in each concentration group of EPS-1 was significantly different from that in the model group (P≤0.05), indicating that EPS-1 had a certain inhibitory effect on the release of TNF-α of RBL-2H3 cells. After treatment with crude polysaccharides EPS, EPS-1 and EPS-2, the release of IL-4 from RBL-2H3 cells decreased, and the release amount of the high concentration group (500 μg / mL) was close to that of the positive control group (Y group) (15.753±0.882 pg / mL).

[0117] In summary, crude polysaccharide of Lactobacillus casei LZ9183 and its two polysaccharide components can downregulate the release of HIS, IL-4 and TNF-α in the RBL-2H3 cell degranulation model to a level close to that of the normal group, indicating that it can alleviate IgE-mediated allergic reactions. Among them, component EPS-1 performed relatively well and may have potential anti-allergic activity.

[0118] Example 4

[0119] 1. Preparation of probiotic double-layer emulsion

[0120] (1) Preparation of bacterial suspension: Probiotic Lactobacillus casei CGMCC No.25194 is inoculated in MRS medium, activated for 3 generations, cultured at 37°C for 24 hours, the activation inoculum amount is 2% (v / v), the activated strain is also inoculated in MRS medium at a ratio of 2% (v / v), cultured for 24 hours and used as the final bacterial liquid for standby use; the activated bacterial suspension is subjected to refrigerated centrifugation (9000 rpm, 10 min), the supernatant is discarded, and the suspension is washed with physiological saline, and the process is repeated twice to obtain bacterial bodies for use in preparing emulsion.

[0121] (2) Preparation of the internal aqueous phase W1: Prepare a 12% (w / v) skim milk solution as a probiotic protective agent, pasteurize at 90°C for 20 min, adjust the pH to 7.0 after cooling to room temperature, and hydrate at 4°C overnight. After mixing the bacteria and the protective agent, vortex them to evenly disperse them to form the internal aqueous phase W1.

[0122] (3) Preparation of oil phase O: 0.6 g of ursolic acid was weighed and added to 15 g of rapeseed oil. The mixture was stirred at 80° C. and 1000 rpm for 20 to 30 min. The oil phase O of the primary emulsion was obtained after stirring evenly.

[0123] (4) Preparation of W1 / O primary emulsion: The internal water phase W1 is dripped into the oil phase O while stirring, the mass ratio of the internal water phase W1 to the oil phase O is 2:3, and emulsification is carried out at 6000-10000 rpm for 1-5 min to obtain colostrum W1 / O.

[0124] (5) Preparation of external aqueous phase W2: 0.3 g of pectin was added to 97 g of deionized water, stirred for 30 min to fully dissolve it, and then placed in a 4°C refrigerator overnight to fully hydrate, to obtain a 0.3% (w / v) pectin solution, which is the external aqueous phase W2.

[0125] (6) Preparation of W1 / O / W2 type double-layer emulsion preparation: colostrum W1 / O and external aqueous phase W2 are mixed in a mass ratio of 2:3, and sheared at a speed of 5000-6000 rpm for 2-4 min to obtain a double-layer emulsion W1 / O / W2, which is stored at 4-25°C to obtain a probiotic liquid preparation. Fig.12 , 13 .

[0126] 2. Study on the storage stability of probiotic double-layer emulsion

[0127] The number of viable bacteria in free probiotics (blank group) and embedded probiotic emulsion (experimental group) was measured after storage at 4°C for 1, 7, 14, 30, and 45 days. 1.5 mL of sample was taken in a centrifuge tube and centrifuged at 15800 × g for 10 min to destroy the stability of the emulsion. Then, vortex mixing was performed, 1 mL of emulsion was taken with a pipette and diluted to an appropriate multiple with physiological saline (9 g / L NaCl), then spread on MRS solid culture medium, and then counted after static culture at 37°C for 48 hours. The average number was taken after three parallel experiments.

[0128] The results are as follows Fig.14 As shown in the data, the initial cell count of the experimental group was 9.35log CFU / mL, and the cell count after 45 days of storage was 8.53log CFU / mL, a decrease of 0.82log CFU / mL; the initial cell count of the blank group was 9.99log CFU / mL, and the cell count after 45 days of storage was 8.13log CFU / mL, a decrease of 1.77log CFU / mL. Compared with the blank group, the experimental group significantly improved the survival rate of probiotics, indicating that the W1 / O / W2 double-layer emulsion can reduce the inactivation of probiotics and enable them to better exert their physiological activity in the body.

[0129] 3. Investigation of particle size and Zeta potential of probiotic double-layer emulsion during storage

[0130] The particle size and Zeta potential of the embedded probiotic emulsion were measured after storage at 4°C for 1, 7, 14, 30 and 45 days. The particle size of the emulsion was determined by Master sizer 3000 laser particle size analyzer. The dispersion medium was water (refractive index 1.33), the refractive index of the emulsion was set to 1.59, and the volume average diameter d(4, 3) was used to characterize the size of the emulsion droplet particle size. The Zeta potential of the emulsion droplets was determined by Nano-ZS potential meter. Each sample was measured in 3 groups in parallel and the results were averaged.

[0131] The results are as follows Fig.15 As shown in the figure, the particle size of the emulsion after storage for 45 days remained between 31-40 μm, with no significant change. Zeta potential can reflect the charge properties between droplets and is an important parameter for studying the physical stability of emulsions. Figure 4 It can be seen that the potential value of the W1 / O / W2 emulsion did not change significantly during storage, indicating that the multiple emulsion formed with ursolic acid as an emulsifier and 0.3% (w / v) pectin as the external aqueous phase can effectively prevent aggregation between droplets and has a positive effect on the stability of the emulsion.

[0132] Example 5

[0133] 1. Preparation of probiotic double-layer emulsion

[0134] (1) Preparation of bacterial suspension: Probiotic Lactobacillus casei CGMCC No.25194 is inoculated into MRS medium, activated for 3 generations, cultured at 37°C for 24 hours, the activation inoculum amount is 2% (v / v), the activated strain is also inoculated into MRS medium at a ratio of 2% (v / v), cultured for 24 hours and used as the final bacterial liquid for standby; the activated bacterial suspension is subjected to refrigerated centrifugation (9000 rpm, 10 min), the supernatant is discarded, and washing is performed with physiological saline, and after repeating twice, bacterial bodies are obtained for use in embedding preparation.

[0135] (2) Preparation of the internal aqueous phase W1: Prepare a 12% (w / v) skim milk solution as a probiotic protective agent, pasteurize at 90°C for 20 min, adjust the pH to 7.0 after cooling to room temperature, and hydrate at 4°C overnight. After mixing the bacteria and the protective agent, vortex them to evenly disperse them to form the internal aqueous phase W1.

[0136] (3) Preparation of oil phase O: 0.6 g of ursolic acid was weighed and added to 15 g of rapeseed oil. The mixture was stirred at 80° C. and 1000 rpm for 20 to 30 min. The oil phase O of the primary emulsion was obtained after stirring evenly.

[0137] (4) Preparation of W1 / O primary emulsion: The inner water phase W1 is dripped into the oil phase O while stirring, the mass ratio of the inner water phase W1 to the oil phase O is 2:3, and the emulsification is carried out at 6000-10000 rpm and shearing for 1-5 min to obtain colostrum W1 / O.

[0138] (5) Preparation of external aqueous phase W2: 0.3 g of gellan gum was added to 97 g of deionized water, stirred for 30 min to fully dissolve it, and then placed in a 4°C refrigerator overnight to fully hydrate, to obtain a 0.3% (w / v) gellan gum solution, which is the external aqueous phase W2.

[0139] (6) Preparation of W1 / O / W2 double-layer emulsion preparation: colostrum W1 / O and external aqueous phase W2 were mixed in a mass ratio of 2:3, and sheared at a medium speed of 5000-6000 rpm for 2-4 min to obtain a double-layer emulsion W1 / O / W2, which was stored at 4-25°C to obtain a probiotic liquid preparation.

[0140] 2. Study on the storage stability of probiotic double-layer emulsion

[0141] The number of viable bacteria in free probiotics (blank group) and embedded probiotic emulsion (experimental group) was measured after storage at 4°C for 1, 7, 14, 30, and 45 days. 1.5 mL of sample was taken in a centrifuge tube and centrifuged at 15800 × g for 10 min to destroy the stability of the emulsion. Then, vortex mixing was performed, 1 mL of emulsion was taken with a pipette and diluted to an appropriate multiple with physiological saline (9 g / L NaCl), then spread on MRS solid culture medium, and then counted after static culture at 37°C for 48 hours. The average number was taken after three parallel experiments.

[0142] The results are as follows Fig.16 As shown, the initial cell count of the experimental group was 9.36log CFU / mL, and the cell count after 45 days of storage was 8.76log CFU / mL, which decreased by 0.60log CFU / mL. The initial cell count of the blank group was 9.99log CFU / mL, and the cell count after 45 days of storage was 8.13log CFU / mL, which decreased by 1.77log CFU / mL. Compared with the blank group, the experimental group significantly improved the survival rate of probiotics, indicating that the W1 / O / W2 double-layer emulsion can reduce the inactivation of probiotics and have a significant protective effect on the activity of probiotics during long-term storage.

[0143] Example 6

[0144] 1. Preparation of probiotic double-layer emulsion

[0145] (1) Preparation of bacterial suspension: Probiotic Lactobacillus casei CGMCC No.25194 is inoculated into MRS medium, activated for 3 generations, cultured at 37°C for 24 hours, the activation inoculum amount is 2% (v / v), the activated strain is also inoculated into MRS medium at a ratio of 2% (v / v), cultured for 24 hours and used as the final bacterial liquid for standby; the activated bacterial suspension is subjected to refrigerated centrifugation (9000 rpm, 10 min), the supernatant is discarded, and washing is performed with physiological saline, and after repeating twice, bacterial bodies are obtained for use in embedding preparation.

[0146] (2) Preparation of the internal aqueous phase W1: Prepare a 12% (m / v) skim milk solution as a probiotic protective agent, pasteurize at 90°C for 20 min, cool to room temperature, adjust the pH to 7.0, hydrate at 4°C overnight, mix the bacteria and the protective agent, and vortex to evenly disperse them to form the internal aqueous phase W1.

[0147] (3) Preparation of oil phase O: 0.6 g of ursolic acid was weighed and added to 15 g of rapeseed oil. The mixture was stirred at 80° C. and 1000 rpm for 20 to 30 min. The oil phase O of the primary emulsion was obtained after stirring evenly.

[0148] (4) Preparation of W1 / O primary emulsion: The inner water phase W1 is dripped into the oil phase O while stirring, the mass ratio of the inner water phase W1 to the oil phase O is 2:3, and the emulsification is carried out at 6000-10000 rpm and shearing for 1-5 min to obtain colostrum W1 / O.

[0149] (5) Preparation of external aqueous phase W2: 0.5 g of pectin was added to 95 g of deionized water, stirred for 30 min to fully dissolve it, and then placed in a 4°C refrigerator overnight to fully hydrate, to obtain a 0.55 (m / v) pectin solution, which is the external aqueous phase W2.

[0150] (6) Preparation of W1 / O / W2 double-layer emulsion preparation: colostrum W1 / O and external aqueous phase W2 were mixed in a mass ratio of 2:3, and sheared at a medium speed of 5000-6000 rpm for 2-4 min to obtain a double-layer emulsion W1 / O / W2, which was stored at 4-25°C to obtain a probiotic liquid preparation.

[0151] 2. In vitro simulated digestion experiment of probiotic double-layer emulsion

[0152] (1) Preparation of simulated gastric fluid (SGF): 0.1 g of pepsin was added to 100 mL of ultrapure water to prepare simulated gastric fluid, and the pH value was adjusted to 2.0 with 1 mol / L hydrochloric acid. The solution was sterilized by filtration with a 0.22 μm microporous filter membrane and set aside for use.

[0153] (2) Preparation of simulated intestinal fluid (SIF): 0.1 g of pancreatic enzyme was added to 100 mL of 0.05 mol / L potassium dihydrogen phosphate buffer to prepare simulated intestinal fluid, and the pH value was adjusted to 6.8 with 0.5 mol / L sodium hydroxide. The solution was sterilized by filtration with a 0.22 μm microporous filter and set aside for use.

[0154] (3) Continuous simulated gastrointestinal fluid tolerance test

[0155] Take fresh emulsion and free Lactobacillus casei No.25194 respectively and place them in a centrifuge tube, add 9 times the volume of artificial gastric juice preheated at 37°C, then put it in a shaker, react for 3 hours at 37°C, 150r / min, take the solution after artificial gastric juice reaction for 3 hours, add 9 times the volume of artificial intestinal juice preheated at 37°C, then put it in a water bath shaker, react for 3 hours at 37°C, 125r / min, and take a sample every 1 hour. Take 1.5mL sample in a centrifuge tube, centrifuge at 15800×g for 10min to destroy the stability of the emulsion. Then, vortex mix, take 1mL of emulsion with a pipette, dilute it to a suitable multiple with physiological saline (9g / L NaCl), apply it on MRS solid culture medium, then count after 37°C static culture for 48h, and perform three parallel experiments to take the average.

[0156] The results are as follows Fig.17As shown, the number of live bacteria in the blank group (free No. 25194 without embedding) decreased by 1.11 log CFU / mL after 3 hours of artificial gastric juice digestion, indicating that the probiotics used in the present invention have a strong tolerance to gastric juice. However, after the experimental group was embedded in the W1 / O / W2 double-layer emulsion, the number of live bacteria in the system decreased by 0.89 log CFU / mL after 3 hours of gastric juice digestion, indicating that the W1 / O / W2 double-layer emulsion prepared by this method under the acidic conditions of simulated artificial gastric juice has a better protective effect on probiotics than the blank group.

[0157] After the samples after simulated gastric juice were transferred to artificial intestinal juice, the number of viable bacteria decreased only in the initial 1 hour, but during the 3 hours of artificial intestinal juice reaction, the number of viable bacteria remained in the range of (8.06-7.99) Log CFU / mL. Compared with the blank group, the number of viable bacteria of probiotics in the experimental group was still greatly improved, indicating that the use of the W1 / O / W2 double-layer emulsion prepared by this method in the intestinal environment has a good protective effect on probiotics.

Claims

1. A Lactobacillus casei, characterized in that The Lactobacillus casei was deposited in the China General Microbiological Culture Collection Center on July 6, 2022, with the deposit number CGMCC No.25194.

2. The application of Lactobacillus casei as claimed in claim 1 in preparing an exopolysaccharide and producing a probiotic preparation.

3. The use according to claim 2, characterized in that The extracellular polysaccharide can scavenge DPPH free radicals and -OH free radicals.

4. The use according to claim 2, characterized in that The extracellular polysaccharide can stimulate the proliferation, NO release and phagocytosis rate of RAW264.7 macrophages.

5. The use according to claim 2, characterized in that After purification, the extracellular polysaccharide has two components, EPS-1 and EPS-2. The extracellular polysaccharide and its two purified components, EPS-1 and EPS-2, can inhibit the release of β-HEX and regulate the release amounts of HIS, IL-4 and TNF-α through the RBL-2H3 cell degranulation model, and have potential anti-allergic activity.

6. A method for preparing a double-layer emulsion probiotic preparation based on Lactobacillus casei according to claim 1, characterized in that: The method comprises the following steps: (1) Vortexing the Lactobacillus casei and probiotic protective agent solutions to uniformly disperse them to form an inner aqueous phase W1; in the inner aqueous phase W1, the mass percentage content of the probiotic protective agent is 8-12%, and the colony count of Lactobacillus casei is not less than 1×10 7 -8 CFU / mL; (2) dissolving ursolic acid in oil to form an oil phase O, and mixing the oil phase O with the internal water phase W1 by high-speed shearing to obtain a water-in-oil colostrum W1 / O; the mass percentage content of ursolic acid in the oil phase O is 1% to 5%; the volume ratio of the oil phase O to the internal water phase W1 is 1:1, 1:2, 1:3, 2:3 or 3:5; (3) The colloidal particle suspension is used as the external aqueous phase W2, and the external aqueous phase W2 is added to the colostrum W1 / O, and mixed by medium-speed shearing to obtain a double-layer emulsion probiotic preparation with a W1 / O / W2 double emulsion structure; the mass percentage content of the colloidal particles in the suspension is 0.1-0.5%. When preparing the suspension, the colloidal particles are added to water, treated in a water bath at 40-60°C for 10-20 min, dissolved, and then hydrated at 4°C overnight to obtain the preparation; the volume ratio of the external aqueous phase W2 to the colostrum W1 / O is 2:

3.

7. The method according to claim 6, characterized in that The probiotic protective agent described in step (1) is at least one of skimmed milk powder, whey protein isolate, trehalose, sucrose, stachyose, xylo-oligosaccharides, lactose and glycerol.

8. The method according to claim 6, characterized in that The oil described in step (2) is at least one of soybean oil, peanut oil, rapeseed oil, corn oil, and sunflower oil; the external aqueous phase W2 described in step (3) is at least one of pectin, locust bean gum, gellan gum, xanthan gum, and carrageenan.

9. The method according to claim 6, characterized in that The high-speed shearing rate in step (2) is 6000-10000 rpm, and the shearing time is 1-5 min; the medium-speed shearing rate in step (3) is 5000-7000 rpm, and the shearing time is 1-3 min.

10. A probiotic preparation based on ursolic acid W1 / O / W2 Pickering emulsion structure, characterized in that: The probiotic preparation is prepared according to the method according to any one of claims 6 to 9, and the probiotic is at least one of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactococcus lactis or Streptococcus thermophilus.

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