Lactobacillus paracasei FMBL l23249 FJX and use thereof

Abstract

Provided are a Lactobacillus paracasei FMBL L23249 FJX and the use thereof. The Lactobacillus paracasei FMBL L23249 FJX is deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097. The Lactobacillus paracasei FMBL L23249 FJX can be rapidly proliferated by means of efficiently utilizing a Cicer arietinum L. extract, and can be used for preparing Cicer arietinum L. yogurt. The Lactobacillus paracasei FMBL L23249 FJX can also adsorb heavy metals. Further provided is the use of the Lactobacillus paracasei FMBL L23249 FJX in the preparation of a drug, food, health care product or cosmetic for preventing and treating heavy metal poisoning. The Lactobacillus paracasei FMBL L23249 FJX has high adsorbability for the heavy metals lead and cadmium, with the adsorption rate for lead reaching 99.87%, and the adsorption rate for cadmium reaching 82.74%. The Lactobacillus paracasei FMBL L23249 FJX has good acid resistance, cholate resistance, drug resistance, self-aggregation and hydrophobicity, can inhibit pathogenic bacteria, has an antioxidant capacity, and has wide application prospects.

Description

Lactobacillus paracasei FMBL L23249 FJX and its application Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Lactobacillus paracasei FMBL L23249 FJX and an application thereof. Background Art

[0002] Chickpeas (Cicer arietinum L.) are a highly nutritious legume and a specialty agricultural product of Muli County, Xinjiang. They contain a variety of bioactive substances. For example, chickpeas contain approximately 5.89-10.1% galacto-oligosaccharides, a good source of natural prebiotics that can stimulate the growth and reproduction of probiotics and activate metabolic functions. Chickpea protein has high bioavailability and low allergenicity. Some chickpea peptides, formed after hydrolysis by microbial proteases, have excellent antibacterial properties. Chickpeas also contain a variety of vitamins and minerals that can meet the body's daily nutritional needs. Fermenting chickpea extract with probiotics not only enhances probiotic activity but also increases the postbiotics and active substances produced by probiotic metabolism, building on the nutrients and active ingredients already present in chickpea milk. This also imparts a unique flavor, improving the undesirable flavor profile of chickpeas. For example, invention patent CN102613381A discloses that probiotic peptides can be fermented using probiotics and chickpeas; invention patent CN115191559A discloses a method for preparing a chickpea milk fermented beverage. The prepared chickpea milk fermented beverage has significantly improved antioxidant capacity, DPPH free radical scavenging capacity, and ABTS+ free radical scavenging capacity, while functional indicators have both increased and decreased, with protein and fat indicators decreased, making it suitable for more people, such as those with obesity, hypertension, hyperlipidemia, and hyperglycemia. However, the ability of probiotics to ferment chickpea extract is strain-specific, and different strains vary in their ability to utilize chickpea extract.

[0003] Heavy metals are dense, high-atomic-mass elements that are non-biodegradable. If absorbed by humans or other plants and animals, they can seriously harm their health and growth. Lead is the second most abundant heavy metal found worldwide. It can enter the human body through food (65%), water (20%), and air (15%), causing neurological and renal impairment. Cadmium is primarily produced through human activities, metal smelting, chemical fuel combustion, and sewage sludge. Cadmium is highly toxic, and cadmium poisoning primarily disrupts the oxidative-antioxidant balance, causing various harmful effects on cellular molecules, leading to hypertension, renal tubular dysfunction, fractures, and cancer. Excessive cadmium levels can also cause serious damage to the nervous system, hematopoietic system, liver, and reproductive function of livestock and poultry. Currently, methods for removing heavy metals from contaminated systems include physical remediation, chemical precipitation, dialysis, ion exchange, reverse osmosis, and solvent extraction. However, most of these methods are costly and environmentally harmful. Biological methods, with their high efficiency, non-toxicity, environmental friendliness, and cost-effectiveness, have become a research hotspot for removing heavy metal pollution. Biological methods mainly use bacteria, fungi, yeast, etc. as adsorbents to adsorb heavy metals. For example, the invention patent CN110628678B discloses a heavy metal-resistant copper-greedy bacteria that can tolerate heavy metal copper and improve the absorption of Cu. 2+ The germination rate of contaminated seeds and the promotion of Cu 2+ The growth of polluted seedlings can effectively remove copper ions in the environment and can be used as Cu 2+ It can be used as a remediation agent for contaminated soil, but it cannot be used for heavy metal poisoning in humans or other animals.

[0004] Lactobacillus paracasei is a microorganism with beneficial activities for the human body. It can colonize the human intestinal tract, promoting a balanced intestinal flora and releasing active substances that influence physiological metabolism and benefit host health. Growing evidence indicates that metabolites produced by fermentation of plant-derived foods by Lactobacillus paracasei, such as short-chain fatty acids, bacteriocins, vitamins, and trimethylamine, can trigger probiotic effects in the body.

[0005] During research, the inventors isolated a strain of Lactobacillus paracasei (FMBL L23249 FJX) from the human intestinal tract. The strain can efficiently utilize chickpea extract to achieve rapid proliferation, making it more suitable for chickpea extract systems. The strain's viable cell count reached 9.17±0.011g (CFU / mL) after 12 hours of fermentation. After 21 days of storage at 4°C, the strain's viable cell count in chickpea extract remained above 8.32±0.031g (CFU / mL), making it suitable for use in yogurt preparation. The inventors also unexpectedly discovered that the strain has a high adsorption capacity for the heavy metals lead and cadmium, exhibiting excellent tolerance to lead ion concentrations of 0-300mg / L and remaining viable at cadmium ion concentrations of 300mg / L. The prepared Lactobacillus paracasei FMBL L23249 FJX is acid-resistant, bile-resistant, antioxidant, and has antibacterial properties. It can be used to prepare adsorbents with high adsorption capacity for heavy metals such as lead and cadmium, drugs that alleviate lead and / or cadmium toxicity and inhibit pathogenic bacteria, fermented foods, health products, food additives, feed, or microbial preparations, and has broad application prospects.

[0006] Summary of the Invention

[0007] The primary purpose of the present invention is to provide a Lacticaseibacillus paracasei FMBL L23249 FJX, wherein the Lacticaseibacillus paracasei FMBL L23249 FJX is deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

[0008] The second object of the present invention is to provide a bacterial agent containing the Lactobacillus paracasei FMBL L23249 FJX or its fermentation liquid.

[0009] The third object of the present invention is to provide a fermented product comprising the Lactobacillus paracasei FMBL L23249 FJX.

[0010] The fourth object of the present invention is to provide the use of the Lactobacillus paracasei FMBL L23249 FJX, the bacterial agent or the fermentation product in the preparation of a starter culture, a fermented food, a health product, a food additive or a dietary supplement.

[0011] Preferably, the fermented food is a fermented dairy product or a plant-based fermented beverage.

[0012] Preferably, the fermented milk product is fermented milk, a fermented milk beverage containing active probiotics, or an inactive fermented milk beverage.

[0013] Preferably, the fermented milk is yogurt or cheese.

[0014] The fifth object of the present invention is to provide the use of the Lactobacillus paracasei FMBL L23249 FJX, the bacterial agent or the fermentation product in removing heavy metals.

[0015] The sixth object of the present invention is to provide the use of the Lactobacillus paracasei FMBL L23249 FJX, the bacterial agent or the fermentation product in the preparation of medicines, health products and cosmetics for preventing and treating heavy metal poisoning.

[0016] Preferably, the heavy metal poisoning is chronic heavy metal poisoning.

[0017] Preferably, the heavy metal is cadmium and / or lead.

[0018] The seventh object of the present invention is to provide the use of the Lactobacillus paracasei FMBL L23249 FJX, the bacterial agent or the fermentation product in the preparation of antioxidant products.

[0019] The eighth object of the present invention is to provide the use of the Lactobacillus paracasei FMBL L23249 FJX, the bacterial agent or the fermentation product in the preparation of an antibacterial product.

[0020] The ninth object of the present invention is to provide a dairy product prepared using the Lactobacillus paracasei FMBL L23249 FJX, the bacterial agent or the fermentation product.

[0021] Preferably, the dairy product is soy yogurt, pea yogurt, chickpea yogurt, kidney bean yogurt or black bean yogurt.

[0022] Preferably, the dairy product is chickpea yogurt.

[0023] The beneficial effects of the present invention are as follows: the present invention provides a Lactobacillus paracasei FMBL L23249 FJX suitable for chickpea fermentation, wherein the Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097. After 8 hours of fermentation, the viable count can reach 8.79±0.01lg (CFU / mL), and after 12 hours of fermentation, it can be balanced at 9.17±0.01lg (CFU / mL); it can efficiently utilize chickpea extract to achieve rapid proliferation. After 16 hours of fermentation, the pH value is 3.76 and the titratable acidity is 56T°; it is more suitable for the system of chickpea extract. The viable bacterial count of the Lactobacillus paracasei FMBL L23249 FJX can reach 9.17±0.011g (CFU / mL) after fermentation for 12 hours, and the viable bacterial count of the Lactobacillus paracasei FMBL L23249 FJX in chickpea extract can be maintained at above 8.32±0.031g (CFU / mL) after storage at 4°C for 21 days, and the Lactobacillus paracasei FMBL L23249 FJX can be used to prepare yogurt; the Lactobacillus paracasei FMBL L23249 FJX is used in the adsorption of heavy metals or in the preparation of medicines, foods, health products or cosmetics for preventing and treating heavy metal poisoning; the Lactobacillus plantarum has strong lead and cadmium adsorption capacity, with a lead adsorption rate of 99.87% and a cadmium adsorption rate of 82.74% ...%. FJX has good tolerance to lead ion concentrations of 0-300 mg / L and can still survive at a cadmium ion concentration of 300 mg / L; it has an inhibitory effect on diarrheagenic Escherichia coli, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli, and Listeria monocytogenes; it is also resistant to acid and bile salts; it has the ability to utilize carbohydrates and good antioxidant capacity, and can be used to prepare adsorbents with high adsorption capacity for heavy metals lead and cadmium, drugs that alleviate the toxicity of lead and / or cadmium and inhibit pathogenic bacteria, fermented foods, health products, food additives, feed or microbial preparations, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Colony image of strain FMBL L23249 FJX

[0025] Note: a. Cell morphology; b. Cell morphology after Gram staining

[0026] Fig. 2 Phylogenetic tree of Lactobacillus paracasei FMBL L23249 FJX

[0027] Figure 3 Adsorption rate of heavy metals lead and cadmium by different strains

[0028] Figure 4 Infrared spectrum scanning results of Lactobacillus paracasei FMBL L23249 FJX before and after lead and cadmium adsorption DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are described in detail below through several examples to further illustrate the present invention, but no limitation is imposed on the claims of the present invention.

[0030] The chickpeas used in the following examples were commercially available.

[0031] Lacticaseibacillus paracasei CGMCC1.2345 was purchased from the China General Microbiological Culture Collection Center; Lacticaseibacillus paracasei FMBL L23249 FJX and other control strains were isolated from the feces of healthy people by researchers from the Food Microbiology and Biotechnology Research Center, School of Food Science and Technology, Shihezi University, and preserved in the laboratory.

[0032] In the following examples, the LAMVAB agar medium has the following formula: peptone 10.0 g, beef extract 8.0 g, yeast extract 4.0 g, glucose 20.0 g, Tween 80 1.0 mL, K2HPO4 2.0 g, sodium acetate 5.0 g, diammonium hydrogen citrate 2.0 g, MgSO4·7H2O 0.2 g, MnSO4·4H2O 0.05 g, bromocresol green 0.05 g, agar 18.0 g, L-cysteine ​​hydrochloride 0.5 g, vancomycin hydrochloride 20.0 mg, pH 5.0.

[0033] MRS liquid medium: peptone 10 g; beef extract 10 g; yeast extract 5 g; glucose 20 g; Tween 80 1 mL; K2HPO4 2 g; sodium acetate 5 g; diammonium hydrogen citrate 2 g; MgSO4·7H2O 0.58 g; MnSO4·4H2O 0.25 g, deionized water 1000 mL;

[0034] M17 broth medium; M17 solid medium (M17 broth medium plus 2% agar);

[0035] PYG medium: peptone 10.0 g, yeast extract 5.0 g, glucose 1.0 g, agar 20.0 g, deionized water 1000 mL;

[0036] TSA medium: 15.0 g tryptone, 5.0 g soy peptone, 5.0 g sodium chloride, 15 g agar, 1000 mL deionized water;

[0037] Nutrient broth agar medium: peptone 5.0 g, beef extract 3.0 g, sodium chloride 5.0 g, agar 20.0 g, deionized water 1000 mL.

[0038] Example 1: Isolation and identification of strains

[0039] Fresh stool samples were collected from healthy Uyghur individuals in Aksu Prefecture, Xinjiang, placed in sterile stool collection tubes, stored in a vehicle refrigerator at -18 to -15°C, and transported to the laboratory within 12 hours. Strains were isolated and purified using the gradient dilution plate method.

[0040] Weigh 1.0 g of feces into 9.0 mL of normal saline and perform a gradient dilution. Pipette 0.1 mL of the fecal sample dilution solution and evenly spread it onto LAMVAB agar medium. Incubate anaerobically at 37°C for 24-36 hours. Based on the characteristics of the colony, randomly select suspected Lactobacillus colonies surrounded by yellow or light yellow transparent circles. After three purifications, combine Gram staining and hydrogen peroxide catalase assay to preliminarily screen for Gram-positive, catalase-negative strains with rod-shaped cell morphology. Enrich the strains and prepare a bacterial suspension.

[0041] The bacterial genomic DNA was extracted using a rapid bacterial genomic DNA extraction kit and the groEL gene primers were used:

[0042] GroEL-Lac-F:5'-GCY GGT GCW AAC CCN GTT GG-3'

[0043] GroEL-Lac-R:5'-AAN GTN CCV CGV ATC TTG TT-3'

[0044] The amplified product was amplified and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing after quality inspection. The obtained sequence was submitted to the GenBank database for BLAST online comparison.

[0045] The colony characteristics and microscopic characteristics of the strain FMBL L23249 FJX are shown in Figure 1, and the phylogenetic tree is shown in Figure 2. After isolation and identification, the strain FMBL L23249 FJX was obtained, which belongs to Lactobacillus paracasei, Latin name: Lacticaseibacillus paracasei, and was named Lacticaseibacillus paracasei FMBL L23249 FJX. It was deposited in the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M 20231097, and the deposit address is: Wuhan University, China, Tel: 027-68754052, Fax: 027-68754833.

[0046] In the following examples, Lacticaseibacillus paracasei FMBL L23249 FJX is abbreviated as Lacticaseibacillus paracasei FMBL L23249 FJX.

[0047] Example 2: Preparation of chickpea extract

[0048] The preparation method of the chickpea extract is as follows:

[0049] (1) Weigh 100g of chickpeas, ensure that they are free of pests and diseases and mechanical damage, rinse them repeatedly with running water to clean the mud and impurities on the surface, drain and set aside.

[0050] (2) Add the washed chickpeas to 300 ml of drinking water and soak at room temperature for 12 hours to allow the chickpea particles to swell naturally and be in a semi-germinated state. Then filter and discard the soaking water, and drain the semi-germinated chickpeas for later use.

[0051] (3) Add 600 ml of 60°C drinking water and 0.45% α-amylase (1000 u / g) to the semi-germinated chickpeas, grind for 8 min, and enzymolyze at 60°C for 24 min.

[0052] (4) The chickpea slurry after enzymatic hydrolysis was boiled for 5 minutes, cooled, filtered through a 100-mesh filter cloth, and packaged to obtain chickpea extract.

[0053] (5) The chickpea extract obtained in (4) was sterilized at 105°C for 10 min, cooled to room temperature, and then refrigerated at 4°C for later use.

[0054] Example 3: Viable Cell Count of Lactobacillus paracasei FMBL L23249 FJX

[0055] Lactobacillus paracasei FMBL L23249 FJX and other control strains were activated and cultured for 2-3 generations. The seed solution was centrifuged and the fermentation supernatant was removed. The bacterial sludge was washed with sterile saline and resuspended in sterile saline. The final concentration was 1.0×10 7 Chickpea extract was inoculated with an inoculum of 100 CFU / mL. The inoculated chickpea extract was then placed in a 37°C incubator for fermentation. 1 mL of the chickpea extract was sampled at 8, 10, 12, and 16 hours of fermentation for viable bacterial counts. Specific data are shown in Tables 1 and 2.

[0056] Table 1 Composition of strains in the experimental and control groups

[0057] Table 2 Changes in viable bacterial count during fermentation of chickpea extract by Lactobacillus paracasei FMBL L23249 FJX (lg (CFU / mL))

[0058] Note: Different letters in the same row and column indicate significant differences

[0059] As shown in Table 2, the viable count of Lactobacillus paracasei FMBL L23249 FJX reached 8.79±0.01lg (CFU / mL) after 8h of fermentation, and reached a balance of 9.17±0.01lg (CFU / mL) after 12h of fermentation. In contrast, the viable count of Lactobacillus paracasei in control group 8, which had the highest viable count, could only reach 8.81±0.01lg (CFU / mL) after 16h of fermentation. Therefore, Lactobacillus paracasei FMBL L23249 FJX can efficiently utilize chickpea extract to achieve rapid proliferation.

[0060] Example 4: pH value and titratable acidity of Lactobacillus paracasei FMBL L23249 FJX

[0061] Lactobacillus paracasei FMBL L23249 FJX and the control strain were activated and cultured for 2-3 generations. The seed liquid was centrifuged to remove the supernatant fermentation liquid. The bacterial sludge was washed with sterile saline and resuspended in sterile saline. The final concentration was 1.0×10 7 Chickpea extract was inoculated with an inoculum of 100 CFU / mL. The inoculated chickpea extract was then placed in a 37°C incubator for fermentation. 10 mL samples were taken from the chickpea extract at 8, 10, 12, and 16 hours of fermentation for pH and titratable acidity determination. The results are shown in Tables 3 and 4.

[0062] Table 3 Changes in pH and titratable acidity during fermentation of chickpea extract by Lactobacillus paracasei FMBL L23249 FJX

[0063] Note: Different letters in the same row have significant differences

[0064] Table 4 Changes in pH and titratable acidity during fermentation of chickpea extract by Lactobacillus paracasei CGMCC1.2345

[0065] Note: Different letters in the same row have significant differences

[0066] Comparison of Tables 3 and 4 shows that after 16 hours of fermentation, the pH of Lactobacillus paracasei FMBL L23249 FJX was 3.76, with a titratable acidity of 56°; whereas, the pH of Lactobacillus paracasei CGMCC 1.2345 was 3.93, with a titratable acidity of 42°. Both meet the acidity requirements for fermented beverages. Furthermore, within the same titratable acidity range, the viable cell count of Lactobacillus paracasei FMBL L23249 FJX was much higher than that of Lactobacillus paracasei CGMCC 1.2345. Furthermore, Example 2 shows that Lactobacillus paracasei FMBL L23249 FJX is more suitable for the chickpea extract system.

[0067] Example 5. Changes in viable cell count and pH value of Lactobacillus paracasei FMBL L23249 FJX during storage

[0068] Lactobacillus paracasei FMBL L23249 FJX and the control strain Lactobacillus paracasei CGMCC1.2345 were activated and cultured for 2-3 generations. The seed liquid was centrifuged to remove the supernatant fermentation liquid. The bacterial sludge was washed with sterile saline and resuspended in sterile saline. The final concentration was 1.0×10 7 Chickpea extract was inoculated with an inoculum of 100 CFU / mL and then fermented in a 37°C incubator. 10 mL of the chickpea extract was sampled at 12 h, 7 d, 14 d, and 21 d for viable bacterial count and pH determination.

[0069] Table 5 Changes in viable cell count and pH value of chickpea extract fermented by Lactobacillus paracasei FMBL L23249 FJX during storage

[0070] Note: Different letters in the same row have significant differences

[0071] Table 6 Changes in viable cell count and pH value of chickpea extract fermented by Lactobacillus paracasei CGMCC 1.2345 during storage

[0072] Note: Different letters in the same row have significant differences

[0073] Comparison of Table 5 and Table 6 shows that, according to the preparation method provided by the present invention, the viable count of Lactobacillus paracasei FMBL L23249 FJX fermented chickpea extract can reach 9.17±0.011g (CFU / mL) after fermentation for 12h, and the viable count of Lactobacillus paracasei FMBL L23249 FJX in the chickpea extract can be maintained at above 8.32±0.031g (CFU / mL) after storage at 4°C for 21d; while the viable count of Lactobacillus paracasei CGMCC 1.2345 prepared in the control group was only 8.63±0.021g (CFU / mL) after fermentation for 12h, and the viable count of Lactobacillus paracasei CGMCC1.2345 in the chickpea extract could only be maintained at 7.74±0.021g (CFU / mL) after storage at 4°C for 21d. After 12 h of fermentation and 21 days of storage at 4°C, the viable cell count of Lactobacillus paracasei CGMCC1.2345 was significantly lower than that of Lactobacillus paracasei FMBL L23249 FJX. Therefore, Lactobacillus paracasei FMBL L23249 FJX is more suitable for preparing chickpea yogurt.

[0074] Example 6: Adsorption capacity of Lactobacillus paracasei FMBL L23249 FJX for heavy metals lead and cadmium

[0075] 1. Test strains

[0076] Lactobacillus plantarum FMBL L23290 WP, Lactobacillus paracasei FMBL L23249 FJX, Lactobacillus paracasei FMBL L23284 WP, Lactobacillus rhamnosus FMBL L23136 YWT, and Lactobacillus mucilaginosus reuteri were all isolated from the feces of healthy individuals. Lactobacillus plantarum FMBL L23290 WP, Lactobacillus paracasei FMBL L23284 WP, Lactobacillus rhamnosus FMBL L23136 YWT, and Lactobacillus mucilaginosus reuteri were deposited at the Food Microbiology and Biotechnology Research Center, College of Food Science and Engineering, Shihezi University.

[0077] 2. Adsorption Experiment

[0078] (1) The strain to be tested was inoculated into liquid MRS medium and cultured at 37°C for 24 h for activation.

[0079] (2) The activated strain in step (1) was centrifuged at 8000 rpm and 4°C for 20 min to collect the bacterial precipitate, and the bacterial precipitate was washed three times with PBS buffer (pH = 7.4) to obtain the bacterial precipitate to be tested.

[0080] (3) Prepare the bacterial precipitate obtained in step (2) into a 50g / L bacterial suspension; prepare lead nitrate and cadmium nitrate into 50mg / L lead ion solution and cadmium ion solution; add the bacterial suspension into 50mg / L lead and cadmium ion solution respectively, so that the final bacterial concentration in the solution is 5g / L; place in a shaker at 150rpm and 37℃ for 1h, centrifuge and take the supernatant, and determine the content of lead and cadmium in the supernatant by atomic spectrophotometry. Calculate the adsorption rate by the following formula. C0 is the content of lead and cadmium ions in the adsorbed lead solution, C I is the content of lead and cadmium ions in the solution after adsorption.

[0081] 3. Results

[0082] The results, as shown in Figure 3, show that the adsorption rates of lead by each strain ranged from 55% to 100%, and the adsorption rates for cadmium ranged from 70% to 90%. Different strains showed significant differences in their adsorption capacity for heavy metal ions. Lactobacillus paracasei FMBL L23249 FJX exhibited the best adsorption of heavy metals, with adsorption rates for lead ions of 99.87% and cadmium ions of 82.74%, significantly higher than those of other strains.

[0083] Example 7 Effects of different concentrations of lead and cadmium ions on the growth of Lactobacillus paracasei FMBL L23249 FJX

[0084] 1. Experimental Procedure

[0085] (1) Filter the lead nitrate and cadmium nitrate solutions through a filter membrane and add them to MRS liquid culture medium to prepare liquid culture medium with lead ion concentrations of (50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L) and cadmium ion concentrations of (50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L), respectively.

[0086] (2) Add the activated Lactobacillus paracasei FMBL L23249 FJX to the liquid culture medium prepared in step (1) at a 2% inoculum volume. Incubate at 37°C for 24 hours and measure the OD value using a spectrophotometer. 600 The liquid culture medium without heavy metal ions was used as the control.

[0087] Table 7 Activity of Lactobacillus paracasei FMBL L23249 FJX in cadmium-containing medium

[0088] Table 8 Activity of Lactobacillus paracasei FMBL L23249 FJX in lead-containing medium

[0089] 2. Results

[0090] Tables 7 and 8 show the effects of different lead and cadmium ion concentrations on the growth of Lactobacillus paracasei FMBL L23249 FJX. The growth of Lactobacillus paracasei FMBL L23249 FJX was largely unaffected in the lead ion concentration range of 0-300 mg / L, demonstrating good tolerance. The growth of Lactobacillus paracasei FMBL L23249 FJX was largely unaffected in the cadmium ion concentration range of 0-100 mg / L. However, when the cadmium ion concentration was ≥200 mg / L, the growth activity of Lactobacillus paracasei FMBL L23249 FJX gradually weakened with increasing cadmium ion concentration, but remained significantly higher than that of other strains. At a cadmium ion concentration of 300 mg / L, Lactobacillus paracasei FMBL L23249 FJX remained viable, demonstrating a certain degree of tolerance to cadmium ions.

[0091] Example 8: Infrared spectroscopy detection of related groups adsorbing lead and cadmium ions by Lactobacillus paracasei FMBL L23249 FJX

[0092] 1. Experimental Procedure

[0093] (1) Prepare 50 mg / L lead nitrate and cadmium nitrate into lead ion solution and cadmium ion solution respectively, sterilize and set aside.

[0094] (2) Activate Lactobacillus paracasei FMBL L23249 FJX and collect the cells by centrifugation. Wash the cell pellet three times with PBS buffer and then add it to the lead ion solution and cadmium ion solution prepared in step (1) to a final cell concentration of 5 g / L. Incubate the cells at 37° C. for 1 h on a shaker at 150 rpm, and collect the cell pellet by centrifugation.

[0095] (3) Freeze-dry the bacterial precipitate from step (2). Grind the dried bacterial powder and potassium bromide in a ratio of 1:50, mix well, and press into thin slices. Measure with a Fourier transform infrared spectrometer. -1 The sample is scanned within the range and its spectrum is recorded.

[0096] 2. Results

[0097] The results are shown in Figure 4. It can be seen that the spectral peak intensity increased before and after lead and cadmium stress, and the characteristic peak position changed, which indicates that functional groups such as carbonyl, phosphate, and hydroxyl groups on the bacteria are related to the ability to adsorb heavy metals.

[0098] Example 9: Determination of probiotic properties of Lactobacillus paracasei FMBL L23249 FJX

[0099] 1. Acid and bile salt resistance

[0100] The activated lactobacilli cultured for 24 hours were centrifuged (10,000 rpm, 5 minutes, 4°C), the supernatant discarded, and the bacterial precipitate collected. The culture was then added to MRS liquid culture medium with different pH values ​​(2.5, 3, 3.5, 4) and different bile salt concentrations (0.05%, 0.1%, 0.5%). After 0 and 4 hours at 37°C, samples were taken for smearing. After 48 hours, the viable bacteria were counted and their survival rate under different pH conditions was calculated.

[0101] Table 9 Acid resistance determination of Lactobacillus paracasei FMBL L23249 FJX

[0102] Table 10 Bile salt tolerance determination of Lactobacillus paracasei FMBL L23249 FJX

[0103] As shown in Tables 9 and 10, Lactobacillus paracasei FMBL L23249 FJX can survive at pH 4 to pH 2.5. The survival rate was 40.55±1.47% at pH 4, and remained above 23±1.47% at pH 2.5. At a bile salt concentration of 0.05%, the survival rate reached 51.64±0.88%. This indicates that Lactobacillus paracasei FMBL L23249 FJX possesses excellent acid and bile salt tolerance, which enables the strain to colonize sites, thereby surviving, growing, and exerting its probiotic effects in the gastrointestinal tract.

[0104] 2. Antibacterial Experiment

[0105] The Oxford cup method was used to determine the antibacterial activity of the metabolites of Lactobacillus plantarum FMBL L23220 CLL, using Escherichia coli (10411), Escherichia coli (21530), Escherichia toxigenic (10421), Salmonella typhimurium (10420), Listeria monocytogenes (LS1), and Salmonella enteritidis (SM1) as indicator bacteria (see Table 6 for purchase sources). The strain was inoculated into MRS medium at a 2% inoculum, cultured at 37°C for 24 hours, and centrifuged at 8000 rpm for 10 minutes. The supernatant was filtered and sterilized to prepare a cell-free supernatant. The indicator bacteria were inoculated at 10 6 The CFU / mL concentration was spread on the corresponding solid culture medium. A sterile Oxford cup was placed upright on the culture dish coated with the pathogen. 0.2 mL of cell-free supernatant was added to the Oxford cup. The culture dish was placed at 4°C for 4 hours of diffusion and then incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured.

[0106] Table 11 Sources of indicator bacteria

[0107] Table 12 Antibacterial ability determination of Lactobacillus paracasei FMBL L23249 FJX

[0108] Note: ≤15+; 15<inhibition zone diameter≤20++; 20<inhibition zone diameter≤25+++; inhibition zone diameter>25+++; no inhibition zone -.

[0109] As shown in Table 12, Lactobacillus paracasei FMBL L23249 FJX has good antibacterial ability against enteropathogenic Escherichia coli (10411), enterotoxigenic Escherichia coli (10421), enterohemorrhagic Escherichia coli (21530), Salmonella enterica serovar Typhimurium (10420), and Salmonella enterica serovar Typhimurium (SM1).

[0110] 3. Drug sensitivity test

[0111] The disc diffusion method (KB method) was used. The drug sensitivity information was shown in Table 13. The strain was inoculated into sterilized liquid MRS medium at a 2% inoculum volume and cultured at 37°C for 24 h. The activated strain was diluted and spread (the bacterial concentration was 10 7 -10 8 CFU / mL) were measured. Sterile tweezers were used to evenly place drug-sensitive paper discs on the discs. The samples were incubated at 37°C for 24 hours. The diameter of the inhibition zone on the discs was recorded. The test was repeated three times and the average value was taken. The sensitivity of the strain to the discs was determined based on the diameter of the inhibition zone. The experimental results were determined according to the CISI data and drug sensitivity classification annotations developed by the US Committee for Clinical Laboratory Standards.

[0112] Table 13 Drug sensitive tablet information

[0113] Table 14 Lactobacillus paracasei FMBL L23249 FJX drug sensitivity test

[0114] Note: R: resistant; I: intermediately sensitive; S: sensitive

[0115] To ensure the safety of probiotics for human consumption, antibiotic resistance is an important indicator for in vitro safety assessment of probiotic strains. Table 14 shows that Lactobacillus paracasei FMBL L23249 FJX is sensitive to rifampicin, tetracycline, minocycline, penicillin, levofloxacin, ampicillin, cefotaxime, and chloramphenicol; and resistant to gentamicin, streptomycin, oxacillin, and cefoxitin.

[0116] 4. Antioxidant Capacity Determination

[0117] The cultured bacterial liquid was centrifuged (10,000 rpm, 10 min, 4° C.) to collect the cells, which were washed three times with PBS buffer and then prepared into a bacterial suspension. The cell-free supernatant was the supernatant filtered through a 0.22 μm microporous filter membrane.

[0118] (1) Determination of the ability to scavenge diphenyltrinitrophenylhydrazine free radical (DPPH)

[0119] Prepare a 0.2 mM DPPH-methanol solution. Mix the supernatant or bacterial suspension thoroughly with the 0.2 mM DPPH-methanol solution at a 1:1 ratio. Incubate in the dark for 60 minutes and measure the OD517 nm value of the supernatant. Perform three replicate experiments.

[0120] Where: D0: absorbance of 1 mL PBS buffer at OD517 nm; D1: absorbance of 1 mL sample solution + 1 mL DPPH-methanol solution at 517 nm; D2: absorbance of 1 mL sample solution + 1 mL anhydrous methanol.

[0121] (2) Determination of ABTS scavenging ability

[0122] Prepare a 7 mmol / L ABTS working solution: Accurately measure 15 mL of a 3.8 g / L ABTS solution (prepared with deionized water) and add 0.264 mL of potassium persulfate solution (140 mmol / L). Mix thoroughly and incubate in the dark at room temperature for 12-16 hours. Adjust the OD (734 nm) of the working solution to 0.7 ± 0.02 using anhydrous ethanol. Mix the sample to be tested with the adjusted ABTS working solution in a 1:6 ratio. Incubate at room temperature for 30 minutes and measure the absorbance at 734 nm. Perform the experiment in triplicate.

[0123] Where Ac is the absorbance of an equal volume of water; A is the absorbance of the sample solution.

[0124] Table 15 Antioxidant capacity of Lactobacillus paracasei FMBL L23249 FJX

[0125] As shown in Table 15, Lactobacillus paracasei FMBL L23249 FJX has good scavenging ability for DPPH and ABTS free radicals. The scavenging ability for DPPH free radical is bacterial suspension > supernatant, and the scavenging ability for ABTS is bacterial suspension > supernatant.

[0126] In the following examples, the inventors prepared the Lactobacillus paracasei FMBL L23249 FJX into different types of bacterial agents or foods.

[0127] Application Example 1: Preparation of Lactobacillus paracasei FMBL L23249 FJX bacterial agent

[0128] Culture medium preparation: water and culture medium raw materials were used to prepare a culture medium containing 20 g / L glucose, 8 g / L beef extract, 10 g / L yeast extract, 9 g / L NaCl, 1 mL / L Tween 80, 2 g / L K2HPO4, 5 g / L sodium acetate, 2 g diammonium hydrogen citrate, 0.58 g MgSO4·7H2O, and 0.25 g MnSO4·4H2O. The pH was adjusted to 6.8 to obtain the culture medium.

[0129] Preparation of protective agent: Use water and protective agent raw materials to prepare a protective agent containing 120 g / L skim milk powder, 20 mL / L glycerol, 22 g / L maltodextrin, 60 g / L trehalose, and 22 g / L galacto-oligosaccharide.

[0130] Lactobacillus paracasei FMBL L23249 FJX was inoculated at a 2% inoculum into the above-mentioned culture medium sterilized at 115°C for 15 minutes; the culture medium contained 20 g / L glucose, 8 g / L beef extract, 10 g / L yeast extract, 9 g / L NaCl, 1 mL / L Tween 80, 2 g / L K2HPO4, 5 g / L sodium acetate, 2 g diammonium hydrogen citrate, 0.58 g MgSO4·7H2O, and 0.25 g MnSO4·4H2O, and the pH was adjusted to 6.8. The cells were cultured at 37°C for 24 hours, washed twice with pH 7.2 PBS buffer, and resuspended in a protective agent to a concentration of 10 11 CFU / mL. The protective agent contains 120 g / L skim milk powder, 20 mL / L glycerol, 22 g / L maltodextrin, 60 g / L trehalose, and 22 g / L galacto-oligosaccharide. Next, the suspension is pre-cultured at 37°C for 60 minutes and then freeze-dried to obtain the Lactobacillus paracasei FMBL L23249 FJX inoculum.

[0131] Application Example 2: Preparation of Fermented Milk Using Lactobacillus paracasei FMBL L23249 FJX of the Present Invention

[0132] After sugar is dissolved in fresh milk, the mixture is homogenized at 60° C. and 20 MPa, followed by heat preservation and sterilization at 90-95° C. for 5-8 minutes. When the temperature drops to 35° C., a mixed bacteria comprising the present invention's Lactobacillus paracasei FMBL L23249 FJX, a commercial dry powder starter Lactobacillus bulgaricus, and thermophilic Streptococcus is added, wherein the weight ratio of the mixed bacteria is 1:1:1 and the inoculation amount of the mixed bacteria is 0.03-2.0% of the weight of the fresh milk. The mixture is mixed, the mixture is heat preserved and fermented at 37° C., and after curdling, the mixture is refrigerated at 4° C. for 16 hours to obtain the fermented milk.

[0133] Application Example 3: Preparation of Fruit and Vegetable Beverages Containing Lactobacillus paracasei FMBL L23249 FJX

[0134] Fresh fruits and vegetables were washed and juiced, and then sterilized at high temperature, and then immediately cooled to about 37 ° C, and the Lactobacillus paracasei FMBL L23249 FJX bacterial agent prepared by the present invention was added to make its concentration reach 10 6 CFU / mL or above, and stored in a refrigerator at a temperature of 4° C. to obtain a fruit and vegetable beverage containing the live bacteria of Lactobacillus paracasei FMBL L23249 FJX of the present invention.

[0135] Application Example 4: Preparation of Microcapsules and Capsule Products Containing Lactobacillus paracasei FMBL L23249 FJX

[0136] The Lactobacillus paracasei FMBL L23249 FJX of the present invention was cultured in MRS medium for 24 h, centrifuged at 4°C and 8000 r / min for 10 min, and the cells were collected and washed twice with sterile saline. The cells were resuspended in sterile saline to a final cell concentration of 2×10 10 The bacterial suspension is added to a solution containing 2.5% sodium alginate and 1% pectin, and stirred thoroughly to uniformly disperse the cells in the sodium alginate solution. The mixture is then extruded into a solidifying solution containing 2% calcium chloride and chitosan to form micelles. The mixture is then allowed to solidify for 30 minutes, filtered to collect the micelles, and freeze-dried to obtain microcapsules containing Lactobacillus paracasei FMBL L23249 FJX of the present invention. The microcapsules have a particle size of 1.93-2.11 mm, an embedding efficiency of ≥93.16%, a strain survival rate of ≥92.57%, and are resistant to simulated gastrointestinal fluid and cold and heat stress. The microcapsules are then encapsulated into commercially available pharmaceutical capsules to obtain the capsule product.

[0137] Application Example 5: Preparation of plant milk containing Lactobacillus paracasei FMBL L23249 FJX using the present invention

[0138] Soybeans, almonds, and other raw materials are soaked at 80°C for 2 hours, peeled, and the soaking water is drained. The pulp is then ground with boiling water and kept at 80-85°C for 12-15 minutes. The resulting pulp is filtered through a 200-mesh screen to remove residue and then boiled. The solids are then removed by centrifugation. The resulting supernatant is cooled to about 37°C and inoculated with the present invention's Lactobacillus paracasei FMBL L23249FJX to a concentration of 10 6 CFU / mL or above, and stored in a refrigerator at a temperature of 4° C. to obtain a plant milk containing the viable bacteria FMBL L23249 FJX of the present invention.

[0139] Application Example 6: Preparation of probiotic milk tablets containing Lactobacillus paracasei FMBL L23249 FJX

[0140] 5.96 parts by weight of a Lactobacillus paracasei FMBL L23249 FJX agent prepared by a freeze-drying method, 65.0 parts by weight of whole milk powder, 7.92 parts by weight of white granulated sugar, 2.5 parts by weight of magnesium stearate, and 18.0 parts by weight of skim milk powder are respectively weighed, mixed with 1.0 part by weight of water, prepared into wet granules by a conventional method, and then tableted using a tablet press, dried in a dryer, and packaged to obtain the probiotic milk tablets of the present invention.

[0141] In summary, the present invention provides a Lactobacillus paracasei FMBL L23249 FJX suitable for chickpea fermentation. The viable cell count reaches 8.79±0.011 g (CFU / mL) after 8 hours of fermentation and reaches a balance of 9.17±0.011 g (CFU / mL) after 12 hours of fermentation. The system can efficiently utilize chickpea extract to achieve rapid proliferation. After 16 hours of fermentation, the pH value is 3.76 and the titratable acidity is 56°, making it more suitable for chickpea extract systems. The viable bacterial count of the Lactobacillus paracasei FMBL L23249 FJX can reach 9.17±0.01 lg (CFU / mL) after fermentation for 12 hours, and the viable bacterial count of the Lactobacillus paracasei FMBL L23249 FJX in chickpea extract can be maintained at above 8.32±0.03 lg (CFU / mL) after storage at 4°C for 21 days. The Lactobacillus paracasei FMBL L23249 FJX can be used to prepare chickpea yogurt. The invention also provides the use of the Lactobacillus paracasei FMBL L23249 FJX in adsorbing heavy metals or in preparing medicines, foods, health products or cosmetics for preventing and treating heavy metal poisoning. The Lactobacillus paracasei FMBL L23249 FJX can be used to absorb heavy metals or in preparing medicines, foods, health products or cosmetics for preventing and treating heavy metal poisoning. FJX has high adsorption capacity for heavy metals lead and cadmium, with the adsorption rate for lead reaching 99.87% and the adsorption rate for cadmium reaching 82.74%; it has good acid and bile salt resistance, drug resistance, self-aggregation and hydrophobicity; the strain has inhibitory effects on enteropathogenic Escherichia coli (10411), enterotoxigenic Escherichia coli (10421), enterohemorrhagic Escherichia coli (21530), Salmonella enterica subspecies Typhimurium serovar (10420), and Salmonella enterica subspecies enterica (SM1); it is also resistant to gentamicin, streptomycin, oxacillin, and cefoxitin; it has antioxidant capacity; it can be used to prepare adsorbents with high adsorption capacity for heavy metals lead and cadmium, drugs that alleviate the toxicity of lead and / or cadmium and inhibit pathogenic bacteria, fermented foods, health products, food additives, feed or microbial preparations, etc., and has broad application prospects.

Claims

1. A Lactobacillus paracasei FMBL L23249 FJX, characterized in that: The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

2. A bacterial agent, characterized in that Contains the Lactobacillus paracasei FMBL L23249 FJX according to claim 1 or its fermentation liquid.

3. A fermented product, characterized in that The method comprises the Lactobacillus paracasei FMBL L23249 FJX according to claim 1.

4. Application of Lactobacillus paracasei FMBL L23249 FJX as claimed in claim 1, the bacterial agent as claimed in claim 2 or the fermented product as claimed in claim 3 in preparing a leavening agent, a fermented food, a health product, a food additive or a dietary supplement.

5. Application of Lactobacillus paracasei FMBL L23249 FJX as claimed in claim 1, the bacterial agent as claimed in claim 2 or the fermented product as claimed in claim 3 in removing heavy metals.

6. Application of Lactobacillus paracasei FMBL L23249 FJX as claimed in claim 1, the bacterial agent as claimed in claim 2 or the fermented product as claimed in claim 3 in preparing medicines, health products and cosmetics for preventing and treating heavy metal poisoning.

7. The use according to claim 6, characterized in that The heavy metal poisoning is chronic heavy metal poisoning.

8. Use of the Lactobacillus paracasei FMBL L23249 FJX as claimed in claim 1, the bacterial agent as claimed in claim 2 or the fermentation product as claimed in claim 3 in the preparation of an antioxidant product.

9. Use of Lactobacillus paracasei FMBL L23249 FJX as claimed in claim 1, the bacterial agent as claimed in claim 2 or the fermented product as claimed in claim 3 in preparing an antibacterial product.

10. A dairy product, characterized in that The method is prepared by using the Lactobacillus paracasei FMBL L23249 FJX described in claim 1, the bacterial agent described in claim 2 or the fermentation product described in claim 3.

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