Lactobacillus casei capable of producing high-temperature converting enzyme and application of lactobacillus casei

Abstract

The invention discloses a lactobacillus casei strain for producing high temperature invertase and application thereof, the strain is preserved in China Center for Type Culture Collection (CCTCC) on August 14, 2023, the preservation address is Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M20231470. The lactobacillus casei provided by the invention is easy to culture and can produce high-temperature-resistant invertase at high yield, the probiotic powder of the lactobacillus casei can quickly degrade cane sugar and prevent cane sugar intolerance, and a metagen prepared from the lactobacillus casei can also effectively degrade cane sugar and prevent cane sugar intolerance.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus casei producing high-temperature invertase and application thereof in sucrose intolerance. Background Art

[0002] Invertase, also known as β-fructofuranosidase, is a digestive enzyme that catalyzes the hydrolysis of sucrose into its subunits fructose and glucose, as well as the hydrolysis of β-D-fructofuranosidic bonds in non-reducing sugars. It is secreted in the brush border of the small intestine. The lack of secretion of the brush border of the small intestine due to genetic or temporary diseases will lead to the lack of intestinal invertase or low secretion, resulting in the inability of patients to digest sucrose, thus causing sucrose intolerance.

[0003] Invertase Invertase (EC3.2.1.26), also known as sucrase, β-D-furanosyl fructohydrolase (EC3.2.1.26), is widely present in the biological world and is an important class of hydrolases in organisms. At present, there are two main sources of invertase, one is from microorganisms, including fungi, yeasts and bacteria. The other is from plants, such as beets, artichokes, onions, asparagus, agave and chicory. The main role of Lactobacillus casei in industrial production is to be used for food fermentation and production of lactic acid. In addition, as a probiotic, Lactobacillus casei can also inhibit the growth of harmful bacteria, stimulate the immune system, and improve immune function. It can maintain the stability of the intestinal microecological environment and prevent or treat immune-related and metabolic disorders. The disease plays a very important role.

[0004] The invertase from Lactobacillus casei has been extensively studied, but invertases that can withstand extremely high temperatures are rare. Summary of the invention

[0005] The invention provides a strain of Lactobacillus casei LC23 producing an extremely thermostable invertase. The Lactobacillus casei strain was deposited in China Center for Type Culture Collection (CCTCC) on August 14, 2023, with a deposit address of Wuhan City, Hubei Province, a deposit number of CCTCCNO: M20231470, and a classification name of Lacticaseibacillus casei LC23.

[0006] Preferably, the culture medium used for the Lactobacillus casei is MRS, and the culture is carried out at 37° C. under aerobic conditions.

[0007] The invention discloses an application of Lactobacillus casei that produces high-temperature invertase, wherein the Lactobacillus casei can produce high-quality invertase, the reaction temperature is 0-100°C, and the pH is 5-7. The Lactobacillus casei has good thermal stability when the reaction temperature is 0-100°C, and good stability when the pH is 5-7.

[0008] Preferably, the reaction temperature is 50°C and the pH is 5. Therefore, the optimal reaction temperature of the Lactobacillus casei-derived invertase is 50°C and the pH is 5.

[0009] The invention discloses an application of Lactobacillus casei producing a high-temperature invertase, and an application of probiotic powder prepared from the Lactobacillus casei in sucrose intolerance. The Lactobacillus casei LC23 and the high-temperature invertase produced by the Lactobacillus casei can effectively degrade sucrose.

[0010] Application of Lactobacillus casei producing high-temperature invertase, and application of postbiotics prepared by the Lactobacillus casei in sucrose intolerance.

[0011] The Lactobacillus casei LC23 provided by the present invention and the high-temperature invertase produced thereby can effectively degrade sucrose, and the postbiotics prepared by fermenting the Lactobacillus casei LC23 can also effectively degrade sucrose.

[0012] The live probiotic powder prepared from the Lactobacillus casei LC23 and the sterilized postbiotics invented by the present invention can effectively degrade sucrose and prevent sucrose intolerance.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the present invention isolates and identifies a strain of Lactobacillus casei LC23 that produces an extremely thermostable invertase, and the strain, probiotic powder and postbiotics thereof can all efficiently degrade sucrose and prevent sucrose intolerance. This is of great significance for alleviating the symptoms of sucrose intolerance, because they can reduce the amount of undigested sucrose entering the large intestine, thereby reducing the risk of intestinal discomfort and diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The colony morphology of the casei Bacillus casei strain in Example 1; the left is the front side, and the right is the back side;

[0015] Figure 2 The morphological diagram of the Lactobacillus casei strain under a microscope in Example 1;

[0016] Figure 3 The phylogenetic tree of the Bacillus casei strain constructed based on the sequence results in Example 2;

[0017] Figure 4 This is a graph showing the effect of different temperatures on the invertase activity in Example 4;

[0018] Figure 5 This is a graph showing the effect of different pH values ​​on the invertase activity in Example 4;

[0019] Figure 6 This is a graph showing the effect of Lactobacillus casei LC23 probiotics and its postbiotic samples on relieving sucrose intolerance in an in vitro mouse experiment in Example 6;

[0020] Figure 7 This is a graph showing the effect of Lactobacillus casei LC23 probiotics and its postbiotic samples on alleviating sucrose intolerance on defecation in an in vitro mouse experiment in Example 6. Specific implementation methods

[0022] The specific implementation method of the present invention is described in detail below in conjunction with examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0023] Example 1: Screening of Lactobacillus casei invertase strain

[0024] 1. Preparation of culture medium

[0025] MRS medium (g / L): Commercial MRS liquid medium. Its solid medium is based on this medium and 20g / L agar powder is added. It is sterilized by high pressure at 121℃ for 20min. The prepared solid plates are stored in a refrigerator at 4℃.

[0026] Improved MRS medium (g / L): 10g / L protein, 5g / L beef powder, 4g / L yeast powder, 20g / L sucrose (Note: sucrose needs to be sterilized separately), 1mL Tween 80, 2g / L potassium dihydrogen phosphate, 5g / L sodium acetate, 2g / L ammonium citrate, 0.2g / L magnesium sulfate, 0.05g / L manganese sulfate, 1000ml distilled water, pH 6.2, 121℃ autoclave for 20min. On this basis, the solid medium is added with 20g / L agar powder and bromocresol purple (0.025g / L), autoclaved at 121℃ for 20min, and then poured into the plate. The solid plate after cooling and solidification is stored in a 4℃ refrigerator.

[0027] 2. Screening and Acquisition of Strains

[0028] Sample collection: Soil samples were collected from a peach forest in Ningbo City, Zhejiang Province (longitude: 121.519 latitude: 29.986) on October 2, 2022 and stored in sterile bottles.

[0029] Screening and acquisition of strains: After the sample was diluted with sterile water, it was spread on the MRS plate by plate coating, sealed with sealing film, and the date and name were written. After inverting and culturing at 37°C for 2-3 days, the colony morphology was observed, the colony diameter was measured, and the colony shape and color were recorded; the colony was white, the back was yellow-white, and the colony morphology was Figure 1 . Under the microscope, Lactobacillus casei is a distinct bacillus, and its microscopic morphology is Figure 2 .

[0030] After a single colony grows, pick a single colony with a toothpick and place it on a modified MRS plate. One by one, seal it with a sealing film, and place the plate upside down in a 37°C incubator for 2 to 3 days. By observing the color change of the single colony, the colony whose color gradually changes from purple to yellow is a potential strain producing invertase. The color-developing strain was inoculated into a 50 ml / 250 ml shake bottle of modified MRS liquid culture medium and cultured in a shaker for 48 hours. The shaking conditions were: 37°C, 245 rpm / min. Samples were taken every 24 hours to determine the invertase activity. The detection method was based on the seventh edition of the Food Chemical Codex (FCC VII). After the initial screening of the color-developing plate and the activity determination, a microbial strain LC23 with significant color change was obtained, numbered CY5103-2. After testing, its 24h and 48h invertase activities reached 2.5 and 5SU / mL, respectively, and the oligofructose was measured by high-performance liquid chromatography (GB / T 23528.2) to obtain 0.12 mg / ml.

[0031] Example 2 Molecular Biological Identification and Preservation of Bacillus cheese Strains

[0032] 2.1 Extraction of bacterial DNA genome

[0033] The glycerol culture of Lactobacillus casei was activated by streaking on MRS plates and cultured in a 37°C incubator for 3 days.

[0034] Place the bacterial solution in a 1.5 ml EP tube, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant. Add 1.5 ml TE solution and centrifuge to wash, then dissolve and resuspend the bacteria in 567 μl TE solution and mix well. Add 30 μl 10% SDS and 3 μl 20 mg / ml proteinase K (100 μg / ml), mix well, and incubate at 37°C for 1 hour. Add 100 μl 15 mol / L NaCl, mix well, then add 8 μl CTAB / NaCl, mix well, and incubate at 65°C for 10 minutes. Add an equal volume of chloroform / isoamyl alcohol (24:1), mix well, centrifuge at 12,000 rpm for 5 minutes, and retain the supernatant. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) to the supernatant, mix well, centrifuge at 12,000 rpm for 5 minutes, and retain the supernatant. Add 0.6 times the volume of isopropanol, gently mix until the DNA precipitates, centrifuge at 12,000 rpm for 15 minutes to collect the DNA precipitate, wash the DNA precipitate with 75% ethanol, centrifuge at 12,000 rpm for 5 minutes, and vacuum dry for 0.5 hours. Dissolve the genomic DNA precipitate with 50 microliters of double distilled water to a final concentration of 20 μg / ml and store at -20°C.

[0035] 2.2 PCR amplification of 16S DNA:

[0036] The extracted Lactobacillus casei genome was used as a template, and the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for PCR amplification reaction. The reaction system was: 50 μL of total system, 16 μL of sterile water, 25 μL of 2*TAQ Master Mix, 2 μL of 27F, 2 μL of 1492R, and 5 μL of Lactobacillus casei DNA template. The PCR reaction conditions were: 94°C pre-denaturation for 3 minutes, 94°C denaturation for 1 minute, 50.3°C annealing for 1 minute, and 72°C extension for 1 minute. 30 cycles. 72°C extension for 7 minutes.

[0037] 2.3 16S rDNA sequencing and analysis

[0038] The target PCR product was recovered and purified using a DNA gel rapid purification kit (Beijing Quanshijin Biotechnology Co., Ltd.), and the purified PCR product was sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for sequencing. The gene sequence is shown in SEQ ID NO: 1.

[0039] The measured sequences were aligned in NCBI by BLAST, the sequences were analyzed by Mega 4.0 software, and the phylogenetic tree was constructed by the Neighbor-Joining method, such as Figure 3 The phylogenetic tree showed that the strain had a high affinity with Lactobacillus bacteria. Combined with the morphological characteristics, the strain was identified as Lactbacillus casei LC23 of the Lactobacillus bacteria. The strain was sent to the China Center for Type Culture Collection for preservation. Its preservation number is CCTCCNO: M20231470, the preservation date is August 14, 2023, and the address of the preservation unit is Wuhan, Wuhan University.

[0040] Example 3: Determination of Lactobacillus casei invertase activity

[0041] The detection method refers to the seventh edition of the Food Chemical Codex FCC VII, and the main steps are:

[0042] Pipette 5 ml of sucrose substrate solution into a test tube. Each sample requires 4 test tubes (enzyme solution reaction, enzyme blank reaction, glucose reaction and water reaction). All test tubes are placed in a 20°C water bath for 10 minutes. The enzyme solution samples are equilibrated at the same time under the same conditions.

[0043] Enzyme solution blank treatment: boiling water bath for 10 minutes, ice bath for 5 minutes;

[0044] Start the reaction: add 1 ml of enzyme solution, enzyme solution blank, glucose and water to 5 ml of sucrose substrate solution at regular time intervals;

[0045] Allow to react for 30 minutes, pipette 1.5 ml of the reaction mixture into 3.5 ml of DNS working solution at regular intervals to terminate the reaction and mix well;

[0046] Boil in water for 10 minutes, ice bath for 5 minutes, then add 20 ml of deionized water, mix well, leave for more than 10 minutes and measure the absorbance at a wavelength of 515 nm.

[0047] One SU unit: under the above reaction conditions, 1 mg of sucrose is converted into glucose and fructose in 5 minutes.

[0048] Example 4. Purification and property determination of crude invertase solution

[0049] Obtaining crude invertase solution of original strain of Lactobacillus casei: Inoculate a single colony of Lactobacillus casei LC23 on an MRS plate into 50 ml (250 ml shake flask) of MRS liquid culture medium and culture on a shaker for 36 hours at 37°C and 200 rpm. Centrifuge at 4000 rpm to collect the supernatant, which is the crude invertase solution.

[0050] The crude enzyme solution was concentrated and purified by PALL Microsep ultrafiltration centrifuge tube 10K (MCP010C41) and then its properties were determined.

[0051] 4.1 Temperature properties of invertase

[0052] 1) Optimal reaction temperature of invertase

[0053] The crude enzyme solution was diluted appropriately, and enzymolysis was performed at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C, respectively, and the activity of the Lactobacillus casei invertase was determined according to the experimental method in Example 3. The enzyme activity measured at the optimal reaction temperature was 100%, and the relative enzyme activity at different temperatures was calculated ( Figure 4 The experimental results show that the optimal reaction temperature of Lactobacillus casei invertase is 50°C, at which the enzyme activity is the highest and the catalytic efficiency is the best.

[0054] 2) Thermal stability of invertase

[0055] The crude enzyme solution was diluted appropriately and pretreated for 30 minutes at ice bath, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C, respectively. After the sample was cooled to room temperature, the activity of the Lactobacillus casei invertase was determined according to the experimental method in Example 3. The relative enzyme activity at different temperatures was calculated with the highest residual enzyme activity as 100%. Figure 4The results showed that the invertase of Lactobacillus casei had good thermal stability at low and medium temperatures, but its stability decreased at high temperatures, especially above 70°C, where the enzyme activity decreased significantly.

[0056] 3) Optimal reaction pH of invertase

[0057] The enzymatic hydrolysis reaction was carried out at different temperatures and pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0, and the activity of the invertase was determined according to the method in Example 2. The relative enzyme activity at different pH values ​​was calculated with the highest enzyme activity as 100%. Figure 5 ). The experimental results show that at pH 5.0, the relative enzyme activity of the invertase reaches 100%, which is the highest among all the tested pH values. Therefore, the optimal reaction pH of the invertase is determined to be 5.0.

[0058] 4) pH stability of invertase

[0059] The crude enzyme solution was diluted appropriately and pretreated for 30 minutes at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0, respectively, and the activity of the invertase was determined according to the method in Example 2. The relative enzyme activity at different pH values ​​was calculated with the highest enzyme activity as 100%. Figure 5 ). The experimental results show that invertase shows the highest stability at pH 7.0, which means that in near-neutral conditions, invertase can maintain good activity and stability. This has positive significance for the long-term storage and application of invertase.

[0060] Example 5 Preparation of Lactobacillus casei LC23 probiotic powder and preparation of postbiotics and determination of hydrolysis activity

[0061] After culturing Lactobacillus casei LC23 in MRS medium for 48 hours, the bacteria were collected by low-temperature centrifugation, the wet weight was measured, skim milk powder (concentration 10%) was added in a ratio of 1:1, and then freeze-dried. After 48-72 hours, the probiotic Lactobacillus casei LC23 bacterial powder was obtained. According to the National Food Safety Standard Food Microbiology Test for Lactic Acid Bacteria (GB 4789.35), the viable count was 2x10 11 According to the method in Example 3, the invertase activity of Lactobacillus casei LC23 probiotic powder was measured to be 60 SU / g.

[0062] 1-5% (m / m) skim milk powder was added to the fermentation liquid of Lactobacillus casei LC23 after 48h of culture in MRS medium, and after pasteurization (85°C, 30min), spray drying (inlet air temperature 125-175°C, outlet air temperature 75-85°C) was performed to obtain Lactobacillus casei LC23 postbiotics. According to the method in Example 3, the activity of Lactobacillus casei LC23 postbiotics was measured to be 50SU / g.

[0063] Example 6 Determination of the effect of Lactobacillus casei LC23 probiotic powder on alleviating sucrose intolerance

[0064] 1. Dynamic in vitro digestion to study the response of Lactobacillus casei LC23 probiotics and their postbiotics to sucrose

[0065] 1.1 Reagent preparation and instruments

[0066] Gastric fluid simulation solution (SGF): SGF electrolyte stock solution + CaCl2(H2O)2 + 6M HCl + deionized water + pepsin (adjust the pH to 1.57±0.02, then add pepsin, the final concentration of pepsin in the gastric fluid simulation solution is 4000U / mL).

[0067] Simulated intestinal fluid (SIF): SIF electrolyte stock solution + CaCl2(H2O)2 + 6M HCl + deionized water + pancreatic enzymes + bile salts (adjust the pH to 6.8 and add pancreatic enzymes and bile salts. The final concentration of pancreatic enzymes in the simulated intestinal fluid is 200U / mL, and the bile salts is 20mM.

[0068] Test sample (250ml): 2g Lactobacillus casei LC23 probiotic powder; 2g Lactobacillus casei LC23 probiotic powder + 10g sucrose; 2g Lactobacillus casei LC23 postbiotic + 10g sucrose Instrument: In vitro ecological human gastric (intestinal) digestive system (Xiaodong Yijian Suzhou Instrument Equipment Co., Ltd.) 1.2 Dynamic in vitro gastrointestinal digestion

[0069] Gastrointestinal continuous digestion experiment (one sample for one test): simulated gastrointestinal digestion is expected to last 240 minutes, sampling location: the end of the small intestine.

[0070] Correctly install the sterilized gastrointestinal model and disposable sterile syringe, and suck the digestive fluid simulation solution (electrolyte solution filtered and sterilized) prepared on the same day into the syringe and correctly connect it to the model.

[0071] The operating parameters of the dynamic in vitro gastrointestinal digestion equipment were as follows: the system temperature was set at 37°C, the injection volume did not exceed 250 ml, and the injection time was 0.5 min through a one-time injection method. The gastric motility frequency was 3 times / min, the pyloric opening size was 8-13 mm, and the pyloric opening frequency was 1 time the gastric motility occurred and the pyloric opening was 1 time. The fasting gastric juice was 23 mL.

[0072] Injection: The sample was dissolved in 200 g of sterile water and introduced into the simulated esophagus at once to start simulating dynamic gastrointestinal digestion.

[0073] Sampling: After all samples collected from the end of the small intestine are mixed evenly, 1 mL of liquid nitrogen is taken for quick freezing and then stored at -40°C.

[0074] The samples were taken for determination of strain colon delivery rate and sucrose hydrolysis rate. The experimental results are shown in the following table:

[0075]

[0076] From the results of the strain colon delivery rate, the survival rate of Lactobacillus casei LC23 increased from 15% to 18% after the addition of sucrose. This suggests that sucrose may have a certain protective effect on Lactobacillus casei LC23, or that Lactobacillus casei LC23 is able to use sucrose as a carbon source, thereby improving its delivery rate. The results of the sucrose hydrolysis rate showed that the mixture of Lactobacillus casei LC23 postbiotics and sucrose had an extremely high sucrose hydrolysis rate of 99.1%, while the sucrose hydrolysis rate of the mixture of Lactobacillus casei LC23 probiotic powder and sucrose was also very high, at 98.3%. This shows that Lactobacillus casei LC23 probiotic powder and its postbiotics can effectively degrade sucrose. This is of great significance for alleviating the symptoms of sucrose intolerance, because they can reduce the amount of undigested sucrose entering the large intestine, thereby reducing the risk of intestinal discomfort and diarrhea.

[0077] 2. In vitro mouse experiment to determine the effect of Lactobacillus casei LC23 postbiotic samples on alleviating sucrose intolerance

[0078] Thirty SPF experimental mice with a body weight of 80±5g were used. All rats were allowed to adapt to the experimental environment and feed for 1 week and randomly divided into a control group, a Lactobacillus casei LC23 probiotic group and a Lactobacillus casei LC23 postbiotic group, with 5 males and 5 females in each group. A sucrose intolerance model was induced by feeding a feed with a high sucrose content, and its symptoms were multiple diarrhea. Through feed intervention, the control group was given a feed containing sucrose, the probiotic powder group was given a feed containing sucrose and Lactobacillus casei LC23 probiotic powder, and the postbiotic group was given a feed containing sucrose and Lactobacillus casei LC23 postbiotic group to observe the effect of each group in alleviating sucrose intolerance. All rats were allowed to adapt to the experimental environment and feed for 1 week. During the experiment, each group of mice was given the corresponding feed for one week. The weight and defecation frequency data of mice were collected before and after the experiment to evaluate the changes in the symptoms of sucrose intolerance ( Figure 6 , Figure 7 ). Experimental data showed that mice in all groups gained weight during the experiment, but the weight gain in the probiotic powder group was the most significant. In terms of the number of bowel movements, the control group had a slight increase in bowel movements, and the diarrhea condition did not improve, while the number of bowel movements in the probiotic powder group and the postbiotic group decreased. Among them, the reduction in the probiotic powder group was the most significant, the diarrhea condition was greatly improved, and the feces changed from loose stools to soft stools. This shows that Lactobacillus casei LC23 probiotic powder and its postbiotics have a certain effect on alleviating sucrose intolerance, especially in reducing the number of bowel movements. This provides an experimental basis for further research on the application of Lactobacillus casei LC23 in alleviating sucrose intolerance.

Claims

1. A strain of Lactobacillus casei LC23 producing a high temperature invertase, characterized in that: The Lactobacillus casei was isolated from the soil. The Lactobacillus casei was deposited in the China Center for Type Culture Collection (CCTCC) on August 14, 2023, with the deposit address being Wuhan City, Hubei Province, with the deposit number: CCTCCNO:M20231470.

2. The Lactobacillus casei producing thermophilic invertase according to claim 1, characterized in that The culture medium used for the Lactobacillus casei is MRS, which is cultured at 37° C. under aerobic conditions.

3. An application of Lactobacillus casei producing a high temperature invertase, characterized in that: The Lactobacillus casei can produce high levels of invertase, the reaction temperature is 0°C-100°C, and the pH is 5-7.

4. The application of the Lactobacillus casei producing high temperature invertase according to claim 3, characterized in that: The reaction temperature was 50°C and the pH was 5.

5. An application of Lactobacillus casei producing a high temperature invertase, characterized in that: Application of the probiotic powder prepared by Lactobacillus casei in sucrose intolerance.

6. An application of Lactobacillus casei producing a high temperature invertase, characterized in that: Application of the postbiotics prepared by Lactobacillus casei in sucrose intolerance.

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