A strain of high temperature invertase-producing lactobacillus casei and its application

Abstract

This invention discloses a strain of *Lactobacillus casei* that produces thermostable invertase and its applications. This strain was deposited on August 14, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan, Hubei Province, China, with accession number CCTCCNO: M20231470. The *Lactobacillus casei* provided by this invention is easy to culture and can produce high levels of thermostable invertase. Its probiotic powder can rapidly degrade sucrose and prevent sucrose intolerance. The metabiotic prepared from this strain can also effectively degrade sucrose and prevent sucrose intolerance.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Lactobacillus casei* that produces thermo-invertase and its application in sucrose intolerance. Background Technology

[0002] Invertase, also known as β-fructofuranosidase, is a digestive enzyme that catalyzes the hydrolysis of sucrose into its fructose and glucose subunits, 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. Absence of brush border secretion due to genetics or temporary disease can lead to a deficiency or low secretion of intestinal invertase, resulting in the patient's inability to digest sucrose and thus sucrose intolerance.

[0003] Invertase (EC 3.2.1.26), also known as sucrase or β-D-fructofuranoside fructose hydrolase (EC 3.2.1.26), is widely distributed in the biological world and is an important class of hydrolytic enzymes in organisms. Currently, invertases mainly come from two sources: one is from microorganisms, including fungi, yeasts, and bacteria; the other is from plants, such as beets, artichokes, onions, asparagus, agave, and chicory.

[0004] In industrial production, Lactobacillus casei is mainly used for food fermentation and lactic acid production. In addition, as a probiotic, Lactobacillus casei can inhibit the growth of harmful bacteria, stimulate the immune system, and improve immune function. It plays a very important role in maintaining the stability of the intestinal microecological environment and in preventing or treating immune-related and metabolic disorders.

[0005] Invertases derived from Lactobacillus casei have been extensively studied, but invertases that can tolerate extremely high temperatures are relatively rare. Summary of the Invention

[0006] This invention provides a strain of *Lactobacillus casei* LC23 that produces extremely heat-resistant invertase. This strain of *Lactobacillus casei* was deposited on August 14, 2023, at the China Center for Type Culture Collection (CCTCC), located in Wuhan, Hubei Province, with accession number CCTCCNO:M20231470.

[0007] Preferably, the culture medium used for the Lactobacillus casei is MRS, cultured at 37°C under aerobic conditions.

[0008] Application of a thermo-invertase-producing *Lactobacillus casei*, wherein the *Lactobacillus casei* is capable of high-yield invertase production, with a reaction temperature of 0℃-100℃ and a pH of 5-7. The *Lactobacillus casei* exhibits good thermal stability at a reaction temperature of 0℃-100℃ and good stability at a pH of 5-7.

[0009] Preferably, the reaction temperature is 50°C and the pH is 5. Therefore, the optimal reaction temperature for the Lactobacillus casei-derived invertase is 50°C and the pH is 5.

[0010] Application of *Lactobacillus casei* producing a thermo-invertase, and application of probiotic powder prepared from *Lactobacillus casei* in sucrose intolerance. *Lactobacillus casei* LC23 and its produced thermo-invertase can effectively degrade sucrose.

[0011] Application of a thermo-invertase-producing Lactobacillus casei, and application of the metabiotic prepared from said Lactobacillus casei in sucrose intolerance.

[0012] The Lactobacillus casei LC23 and its produced high-temperature invertase provided by this invention can effectively degrade sucrose, and the metabiotic prepared by fermentation of Lactobacillus casei LC23 can also effectively degrade sucrose.

[0013] The live probiotic powder prepared from Lactobacillus casei LC23 invented in this invention and the sterilized metabiotic can effectively degrade sucrose and prevent sucrose intolerance.

[0014] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention isolates and identifies a strain of *Lactobacillus casei* LC23 that produces extremely heat-resistant invertase. This strain, the probiotic powder, and its metabiotics can all efficiently degrade sucrose and prevent sucrose intolerance. This is of great significance for alleviating sucrose intolerance symptoms because they can reduce the amount of undigested sucrose entering the large intestine, thereby reducing the risk of intestinal discomfort and diarrhea. Attached Figure Description

[0015] Figure 1 The colony morphology of the cheese bacillus strain in Example 1 is shown; the left side is the front view, and the right side is the back view.

[0016] Figure 2 This is a microscopic image of the Lactobacillus casei strain in Example 1.

[0017] Figure 3 This is a phylogenetic tree constructed based on sequence results for the *Chemophora cheesiensis* strain in Example 2;

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

[0019] Figure 5 This is a graph showing the effect of different pH values ​​on the activity of invertase in Example 4;

[0020] Figure 6 This is a graph showing the effect of Lactobacillus casei LC23 probiotics and its postbiotics on weight in an in vitro mouse experiment, as studied in Example 6.

[0021] Figure 7 This is a graph showing the effect of Lactobacillus casei LC23 probiotics and its postbiotics on bowel movements in an in vitro mouse experiment, as studied in Example 6. Specific implementation methods

[0022] The specific implementation method of the present invention will be described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

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

[0024] 1. Preparation of culture medium

[0025] MRS medium (g / L): Commercial MRS liquid medium. The solid medium was prepared by adding 20 g / L agar powder and autoclaving at 121°C for 20 min. The prepared solid plates were stored at 4°C.

[0026] Modified MRS medium (g / L): 10 g / L peptone, 5 g / L beef meal, 4 g / L yeast extract, 20 g / L sucrose (Note: sucrose must be prepared separately using a sterilized solution), 1 mL Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1000 mL distilled water, pH 6.2, autoclaved at 121°C for 20 min. For solid medium, add 20 g / L agar powder and 0.025 g / L bromocresol purple, autoclave at 121°C for 20 min, and pour into plates. Store the cooled and solidified plates at 4°C.

[0027] 2. Screening and acquisition of strains

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

[0029] Strain screening and acquisition: After serial dilution with sterile water, samples were spread onto MRS plates using a plate-spreading method. The plates were sealed with sealing film, and the date and name were written on them. After incubation at 37°C upside down for 2-3 days, colony morphology was observed, colony diameter was measured, and colony shape and color were recorded. Colonies were white with a yellowish-white reverse side. Colony morphology is shown in [reference needed]. Figure 1 Under a microscope, *Lactobacillus casei* is a distinct bacillus, and its microscopic morphology is shown in [the image / description]. Figure 2.

[0030] After single colonies grew, they were picked up with a toothpick and transferred to a modified MRS plate, one by one. The plates were sealed with sealing film and incubated upside down at 37°C for 2-3 days. Colonies that gradually changed color from purple to yellow were observed as potential invertase-producing strains. The chromogenic strain was inoculated into 50 mL / 250 mL shake flasks of modified MRS liquid medium and incubated on a shaker for 48 h at 37°C and 245 rpm / min. Invertase activity was measured every 24 h using the method described in the Codex Alimentarius Commission for Food (FCC VII). After initial screening with chromogenic plates and activity assays, a microbial strain with a significant color change, LC23 (CY5103-2), was obtained. Its 24-h and 48-h invertase activities reached 2.5 and 5 SU / mL, respectively, and its fructooligosaccharide content was measured at 0.12 mg / mL using high-performance liquid chromatography (GB / T 23528.2).

[0031] Example 2 Molecular biological identification and preservation of *Chemolyticus cheesinii* strains

[0032] 2.1 Extraction of bacterial DNA genome

[0033] MRS streak plate activation of Lactobacillus casei glycerol bacteria, incubated at 37°C for 3 days.

[0034] The bacterial culture was placed in a 1.5 mL EP tube and centrifuged at 12,000 rpm for 5 minutes, discarding the supernatant. After washing with 1.5 mL TE solution by centrifugation, the cells were resuspended in 567 μL TE solution and mixed well. 30 μL of 10% SDS and 3 μL of 20 mg / mL proteinase K (100 μg / mL) were added, mixed well, and incubated at 37°C for 1 hour. 100 μL of 15 mol / L NaCl was added, mixed thoroughly, followed by 8 μL of CTAB / NaCl, and incubated at 65°C for 10 minutes. An equal volume of chloroform / isoamyl alcohol (24:1) was added, mixed well, and centrifuged at 12,000 rpm for 5 minutes, retaining the supernatant. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the supernatant, mixed well, and centrifuged at 12,000 rpm for 5 minutes, retaining the supernatant. Add 0.6 times the volume of isopropanol and gently mix until DNA precipitates. Centrifuge at 12,000 rpm for 15 minutes to collect the DNA precipitate. Wash the DNA precipitate with 75% ethanol and centrifuge at 12,000 rpm for 5 minutes. Vacuum dry for 0.5 hours. Dissolve the genomic DNA precipitate in 50 μL 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] Using the extracted Lactobacillus casei genome as a template, PCR amplification was performed using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The reaction system was: a total volume of 50 μL, with 16 μL of sterile water added, and 2... TAQ Master Mix 25 μL, 27F 2 μL, 1492R 2 μL, Lactobacillus casei DNA template 5 μL. PCR reaction conditions: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 1 min, 50.3℃ annealing for 1 min, 72℃ extension for 1 min. 30 cycles. 72℃ extension for 7 min.

[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 TransGen Biotech Co., Ltd.). The purified PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The gene sequence is shown in SEQ ID NO: 1.

[0039] The obtained sequences were aligned to NCBI using BLAST, analyzed using Mega 4.0 software, and a phylogenetic tree was constructed using the Neighbor-Joining method. Figure 3 As shown.

[0040] Phylogenetic analysis revealed a high degree of affinity between this strain and bacteria of the genus *Lactobacillus*. Based on morphological characteristics, the strain was identified as *Lactobacillus casei* LC23. This strain was deposited at the China Center for Type Culture Collection (CCTCCNO: M20231470) on August 14, 2023, at Wuhan University, Wuhan.

[0041] Example 3: Determination of invertase activity in Lactobacillus casei

[0042] The testing method refers to FCC VII, 7th edition of the Codex Alimentarius Commission, and the main steps are as follows:

[0043] Pipette 5 ml of sucrose substrate solution into a test tube. Each sample requires 4 test tubes (enzyme reaction, enzyme blank reaction, glucose reaction, and water reaction). Place all test tubes in a 20°C water bath for equilibration for 10 minutes. Equilibrate the enzyme solution sample simultaneously under the same conditions.

[0044] Enzyme solution blank treatment: boiling water bath for 10 minutes, ice bath for 5 minutes;

[0045] Start the reaction: Add 1 ml of enzyme solution, enzyme blank, glucose and water to 5 ml of sucrose substrate solution at certain time intervals;

[0046] The reaction was allowed to proceed precisely for 30 minutes. At regular intervals, 1.5 ml of the reaction mixture was then added to 3.5 ml of DNS working solution to terminate the reaction and mix thoroughly.

[0047] Boil in water for 10 minutes, then in an ice bath for 5 minutes. Add 20 ml of deionized water, mix well, and let stand for at least 10 minutes before measuring the absorbance at a wavelength of 515 nm.

[0048] One SU unit: under the above reaction conditions, 1 mg of sucrose is converted into glucose and fructose in 5 minutes.

[0049] Example 4. Purification and property determination of crude invertase solution

[0050] Obtaining crude invertase from the original strain of *Lactobacillus casei*: A single colony of *Lactobacillus casei* LC23 on an MRS plate was inoculated into 50 mL (250 mL shake flask) of MRS liquid medium and incubated on a shaker for 36 hours at 37°C and 200 rpm. The supernatant was collected by centrifugation at 4000 rpm, which is the crude invertase solution.

[0051] The crude enzyme solution was concentrated and purified by passing it through a PALL Microsep ultrafiltration centrifuge tube 10K (MCP010C41) and then its properties were determined.

[0052] 4.1 Temperature properties of invertase

[0053] 1) Optimal reaction temperature of invertase

[0054] The crude enzyme solution was appropriately diluted and enzymatic hydrolysis was carried out at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃, respectively. The activity of *Lactobacillus casei* invertase was determined according to the experimental method in Example 3. The enzyme activity measured at the optimal reaction temperature was taken as 100%, and the relative enzyme activity at different temperatures was calculated. Figure 4 The experimental results showed that the optimal reaction temperature for Lactobacillus casei invertase was 50℃, at which temperature the enzyme activity was highest and the catalytic efficiency was optimal.

[0055] 2) Thermostability of Invertase

[0056] The crude enzyme solution was appropriately diluted and pretreated for 30 minutes at ice bath, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃, respectively. After cooling the samples to room temperature, the activity of *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 4 The results showed that Lactobacillus casei invertase had good thermal stability at low and medium temperatures, but its stability decreased at high temperatures, especially above 70°C, where enzyme activity decreased significantly.

[0057] 3) The optimal reaction pH of the invertase

[0058] Enzymatic hydrolysis was performed at different temperatures and at 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 invertase activity was determined according to the method described in Example 2. The relative enzyme activity at different pH values ​​was calculated with the highest enzyme activity defined as 100%. Figure 5 The experimental results showed that the relative enzyme activity of the invertase reached 100% at pH 5.0, which was the highest among all tested pH values. Therefore, the optimal reaction pH for the invertase was determined to be 5.0.

[0059] 4) pH stability of invertase

[0060] The crude enzyme solution was appropriately diluted and pretreated for 30 minutes at pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively. The invertase activity was then measured according to the method described in Example 2. The relative enzyme activity at different pH values ​​was calculated with the highest enzyme activity defined as 100%. Figure 5 The experimental results show that the invertase exhibits the highest stability at pH 7.0, meaning that it can maintain good activity and stability under near-neutral conditions. This is of positive significance for the long-term storage and application of the invertase.

[0061] Example 5: Preparation of Lactobacillus casei LC23 probiotic powder, preparation of postbiotics, and determination of hydrolysis activity.

[0062] Lactobacillus casei LC23, cultured on MRS medium for 48 hours, was harvested by low-temperature centrifugation, and its wet weight was determined. Skim milk powder (10% concentration) was added at a 1:1 ratio, followed by freeze-drying. After 48-72 hours, probiotic Lactobacillus casei LC23 powder was obtained. The viable count was determined to be 2 x 10⁻⁶ according to the National Food Safety Standard for Microbiological Examination of Food (GB 4789.35). 11 CFU / g. Following the method in Example 3, the invertase activity of Lactobacillus casei LC23 probiotic powder was measured to be 60 SU / g.

[0063] After 48 hours of culture in MRS medium, the fermentation broth of *Lactobacillus casei* LC23 was mixed with 1-5% (m / m) skim milk powder, pasteurized (85°C, 30 min), and then spray-dried (inlet air temperature 125-175°C, outlet air temperature 75-85°C) to obtain post-biotics of *Lactobacillus casei* LC23. Following the method in Example 3, the post-biotic activity of *Lactobacillus casei* LC23 was measured to be 50 SU / g.

[0064] Example 6: Determination of the effect of Lactobacillus casei LC23 probiotic powder on alleviating sucrose intolerance.

[0065] 1. Dynamic in vitro digestion study of the response of Lactobacillus casei LC23 probiotic and its postbiotics to sucrose.

[0066] 1.1 Reagent preparation and instruments

[0067] Gastric juice simulation solution (SGF): SGF electrolyte stock solution + CaCl2(H2O)2 + 6M HCl + deionized water + pepsin (adjust pH to 1.57±0.02, then add pepsin, the final concentration of pepsin in the gastric juice simulation solution is 4000 U / mL).

[0068] Simulated intestinal fluid (SIF): SIF electrolyte reserve + CaCl2(H2O)2 + 6M HCl + deionized water + pancreatic enzyme + bile salts (Pancreatic enzyme and bile salts are added after adjusting the pH to 6.8. The final concentration of pancreatic enzyme in the simulated intestinal fluid is 200 U / mL, and the concentration of bile salts is 20 mM).

[0069] Test samples (250ml): 2g Lactobacillus casei LC23 probiotic powder; 2g Lactobacillus casei LC23 probiotic powder + 10g sucrose; 2g Lactobacillus casei LC23 postbiotic + 10g sucrose

[0070] Instrument: In vitro biomimetic human gastrointestinal digestive system (Xiaodong Yijian Suzhou Instrument Equipment Co., Ltd.)

[0071] 1.2 Dynamic in vitro gastrointestinal digestion

[0072] Continuous gastrointestinal digestion experiment (one sample per test): simulated gastrointestinal digestion for 240 minutes, sampling location: terminal small intestine.

[0073] Properly install the sterilized gastrointestinal model and disposable sterile syringe, and draw the prepared digestive fluid simulation solution (electrolyte solution filtered and sterilized) into the syringe and connect it correctly to the model.

[0074] Operating parameters of the dynamic in vitro gastrointestinal digestion device: system temperature is set to 37℃, the injection volume is not more than 250ml, the injection is done in one go, the injection time is 0.5 min, the gastric peristalsis frequency is 3 times / min, the pyloric opening size is 8-13 mm, the pyloric opening frequency is 1 time the pylorus opens once for every gastric peristalsis, and the fasting gastric fluid is 23 mL.

[0075] Sample introduction: Dissolve the sample in 200g of sterile water and introduce it into the simulated esophagus in one go to start the simulated dynamic gastrointestinal digestion.

[0076] Sampling: After all samples collected from the end of the small intestine are mixed evenly, 1 mL of liquid nitrogen is taken, quick-frozen, and then stored at -40°C.

[0077] Samples were taken for determination of the bacterial colon delivery rate and sucrose hydrolysis rate. The experimental results are shown in the table below:

[0078]

[0079] The colonic delivery rate of the strains showed that the survival rate of *Lactobacillus casei* LC23 increased from 15% to 18% after the addition of sucrose. This suggests that sucrose may have a protective effect on *Lactobacillus casei* LC23, or that *Lactobacillus casei* LC23 can utilize sucrose as a carbon source, thereby improving its delivery rate. The sucrose hydrolysis rate results showed that the mixture of *Lactobacillus casei* LC23 probiotic and sucrose had an extremely high sucrose hydrolysis rate of 99.1%, while the mixture of *Lactobacillus casei* LC23 probiotic powder and sucrose also had a very high sucrose hydrolysis rate of 98.3%. This indicates that *Lactobacillus casei* LC23 probiotic powder and its probiotic can effectively degrade sucrose. This is significant for alleviating sucrose intolerance symptoms, as it can reduce the amount of undigested sucrose entering the large intestine, thereby reducing the risk of intestinal discomfort and diarrhea.

[0080] 2. In vitro mouse experiment to study the effect of Lactobacillus casei LC23 postbiotic sample on alleviating sucrose intolerance.

[0081] Thirty SPF-grade laboratory mice, weighing 80 ± 5 g, were acclimatized to the experimental environment and diet for one week. They were then randomly divided into a control group, a *Lactobacillus casei* LC23 probiotic group, and a *Lactobacillus casei* LC23 postbiotic group, with five males and five females in each group. A sucrose intolerance model was induced by feeding a high-sucrose diet, with symptoms including recurrent diarrhea. Dietary intervention was used: the control group received a sucrose-containing diet; the probiotic powder group received a sucrose-containing diet and *Lactobacillus casei* LC23 probiotic powder; and the postbiotic group received a sucrose-containing diet and *Lactobacillus casei* LC23 postbiotic powder. The effects of each group on alleviating sucrose intolerance were observed. All rats were acclimatized to the experimental environment and diet for one week. During the experiment, each group of mice was fed the corresponding diet for one week. Body weight and defecation frequency data were collected before and after the experiment to assess changes in sucrose intolerance symptoms. Figure 6 , Figure 7 Experimental data showed that all groups of mice gained weight during the experiment, but the probiotic powder group showed the most significant increase. Regarding defecation frequency, the control group experienced a slight increase in defecation frequency with no improvement in diarrhea, while both the probiotic powder and post-biotic groups showed a decrease in defecation frequency. The probiotic powder group showed the most significant reduction, with a substantial improvement in diarrhea and a change in stool from loose to soft. This indicates that *Lactobacillus casei* LC23 probiotic powder and its post-biotic have a certain effect on alleviating sucrose intolerance, especially in reducing defecation frequency. This provides experimental evidence for further research on the application of *Lactobacillus casei* LC23 in alleviating sucrose intolerance.

Claims

1. A strain of *Lactobacillus casei* LC23 that produces thermo-convertase, characterized by: The *Lactobacillus casei* was isolated from soil and deposited on August 14, 2023, at the China Center for Type Culture Collection (CCTCC), located in Wuhan, Hubei Province, with accession number CCTCC NO: M20231470.

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

3. The application of the *Lactobacillus casei* producing thermo-invertase as described in claim 1, characterized in that: The Lactobacillus casei is capable of producing a thermo-invertase, the reaction temperature of which is 0℃-100℃ and the pH is 5-7.

4. The application of the *Lactobacillus casei* producing thermo-invertase according to claim 3, characterized in that: The reaction temperature was 50℃ and the pH was 5.

5. The application of the *Lactobacillus casei* producing thermo-invertase as described in claim 1, characterized in that: The application of Lactobacillus casei in the preparation of probiotic powder.

6. The application of the *Lactobacillus casei* producing thermo-invertase as described in claim 1, characterized in that: The application of Lactobacillus casei in the preparation of postbiotics.

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