Lactobacillus crispatus YYS-J3 with acrylamide degradation and lead adsorption functions and its application

Abstract

This invention relates to the field of microbial technology, providing a *Lactobacillus crispatus* YYS-J3 with acrylamide degradation and lead adsorption functions, and its applications. The preservation number of *Lactobacillus crispatus* YYS-J3 is CGMCC No. 28184. This *Lactobacillus crispatus* YYS-J3 can degrade or adsorb acrylamide, adsorb lead acetate, has good SOD enzyme production capacity, exhibits good aggregation properties, and has broad adaptability to carbon sources. This bacterium can provide a new probiotic source for the development of functional products such as acrylamide or heavy metal detoxification products and SOD enzyme products, and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of Lactobacillus curvatureus YYS-J3 with acrylamide degradation and lead adsorption functions and its applications. Background Technology

[0002] Acrylamide is a byproduct of the Maillard reaction between reducing sugars and aspartic acid during high-temperature processing of carbohydrates. It is commonly found in fried, baked, or roasted foods. Current research indicates that...

[0003] Acrylamide possesses potential carcinogenicity, neurotoxicity, genotoxicity, and reproductive toxicity, and is therefore classified as a Group 2A carcinogen by the World Health Organization. Acrylamide is water-soluble and can be absorbed by the human body through various routes, including the respiratory tract, digestive tract, and skin, affecting human health. Current methods for reducing acrylamide levels primarily involve lowering the sugar content of raw materials and adding organic acids to reduce some acrylamide formation. However, this can affect the sensory quality of food, and many small workshops producing fried foods do not pay attention to the content of these hidden harmful substances. Therefore, it is necessary to find a method for daily maintenance to promptly remove acrylamide from food or the body.

[0004] With the development of modern industry, lead is widely used in automobiles, fuels, paints, coatings, pesticides, batteries, printing, cosmetics, and other fields. This lead directly enters the atmosphere, soil, and water. Lead in the atmosphere is directly absorbed by the human body through the respiratory tract, while lead in the soil and water is accumulated and absorbed by plants and animals, entering the food chain and ultimately harming human health. Because lead is a highly accumulative heavy metal that cannot be degraded, its accumulation in the body can damage the nervous, immune, respiratory, digestive, urinary and reproductive, cardiovascular, and skeletal systems. Currently, lead poisoning is mainly treated with laxatives and emetics, which have significant side effects and are unsuitable as routine daily detoxification methods. Therefore, it is necessary to find an effective method for removing lead through daily detoxification.

[0005] Superoxide dismutase (SOD) is a multifunctional active enzyme system that integrates six functions: scavenging, activation, regeneration, repair, self-healing, and nutrient supply. It has anti-radiation, anti-cancer, antioxidant, and anti-aging effects. SOD can treat a variety of skin diseases such as myocardial infarction, vascular heart disease, edema, psoriasis, dermatitis, eczema, and pruritus, and has important clinical application value. However, due to its complex extraction process, its production is extremely limited, and related products are extremely rare and expensive.

[0006] Probiotics are a type of microorganism that are beneficial to human health. Studies have shown that probiotics can regulate the body's balance through various pathways. However, there are very few probiotics on the market that can remove acrylamide and adsorb heavy metal lead. How to develop a probiotic that can significantly degrade or adsorb acrylamide, adsorb heavy metal lead, and produce SOD enzymes is precisely the problem and challenge that those in the field are dedicated to solving. Summary of the Invention

[0007] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Lactobacillus curvatureii* YYS-J3, derived from infant feces, classified and named *Lactobacillus curvatureii*, with the Latin scientific name: Lactobacillus crispatus It was deposited on August 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28184.

[0008] The present invention also provides a composition containing Lactobacillus curvatureus YYS-J3 as described above.

[0009] Furthermore, the composition includes one of a microbial preparation or a pharmaceutical.

[0010] Furthermore, in the composition, the number of *Lactobacillus curvatureis* YYS-J3 is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g.

[0011] Preferably, in the composition, the number of *Lactobacillus curvatureis* YYS-J3 is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0012] Furthermore, the composition comprises one or more combinations of uninactivated Lactobacillus curvature YYS-J3 and freeze-dried Lactobacillus curvature YYS-J3.

[0013] This invention also provides the application of Lactobacillus curvature YYS-J3 as described above in the preparation of functional products;

[0014] The functional product includes at least one of the following functions:

[0015] (1) Degradation of acrylamide;

[0016] (2) Adsorption of heavy metal lead;

[0017] (3) Produces SOD enzyme.

[0018] Furthermore, the functional products include products that degrade acrylamide, products that adsorb heavy metal lead, and products that produce SOD enzymes.

[0019] Furthermore, Lactobacillus curvatureis YYS-J3, as a probiotic, is used to ferment fruits and traditional Chinese medicines to prepare fermented foods.

[0020] Based on the above, compared with the prior art, the *Lactobacillus curvatureii* YYS-J3 provided by the present invention has the following beneficial effects:

[0021] The *Lactobacillus curvatureii* YYS-J3 provided by this invention can degrade or adsorb acrylamide, has a good adsorption capacity for heavy metal lead, can produce SOD enzyme, has good coagulation ability, and can grow and utilize a variety of sugar water compounds. This bacterium can provide a new probiotic source for the development of functional products such as detoxification products (acrylamide and lead) and antioxidant products, and has important application value.

[0022] Other features and beneficial effects of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other beneficial effects of the invention can be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The colony morphology diagram of Lactobacillus curvature YYS-J3 provided in the embodiments of the present invention;

[0025] Figure 2 A scanning electron microscope image of Lactobacillus curvature YYS-J3 provided in an embodiment of the present invention;

[0026] Figure 3 Agarose gel electrophoresis image of the 16S rDNA target fragment amplified from Lactobacillus curvatureus YYS-J3 provided in this embodiment of the invention;

[0027] Figure 4 A phylogenetic tree diagram of the 16S rDNA gene of Lactobacillus curvature YYS-J3 provided in an embodiment of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0030] This invention relates to a type of curly lactobacillus ( Lactobacillus crispatus YYS-J3 was obtained from infant feces through anaerobic culture on MRS medium and is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 28184.

[0031] Example 1: Screening and isolation of YYS-J3 bacteria

[0032] Take approximately 1g of stool sample from a 9-month-old infant and dilute it to 10 g in physiological saline solution. -2 times, 10 -3 times, 10 -4 Take 0.1 mL of each diluted solution and place it in MRS medium for anaerobic culture. Select several colonies suspected to be lactic acid bacteria, purify them for three generations, and test their acrylamide removal and lead acetate adsorption effects. Select strains with better removal or adsorption effects, preserve them, and name them YYS-J3.

[0033] Example 2 Identification of YYS-J3 bacteria

[0034] 2.1 Morphological observation of YYS-J3 bacteria

[0035] The colony morphology of YYS-J3 is as follows: Figure 1 As shown, the bacterial cell morphology is as follows Figure 2 As shown. The main morphological characteristics of Lactobacillus curvature YYS-J3 are as follows: the colonies are round, milky white on MRS medium, smooth and translucent, the cells are rod-shaped, 0.6-0.7 μm wide and 1.5-8 μm long.

[0036] 2.2 Analysis of carbon source utilization of YYS-J3 bacteria

[0037] The biochemical experiments on lactic acid bacteria were conducted according to the standard method of GB4789.35. Specifically, a basal culture medium for lactic acid bacteria was prepared, containing esculin, cellobiose, maltose, mannitol, maltose, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% (w / v) sodium hippurate. Lactobacillus curvature YYS-J3 was inoculated into the medium at 1%. After the culture was completed, 0.2 mL of ninhydrin solution was slowly added along the wall of the test tube without shaking. The tubes were then incubated in a water bath at 36℃±1℃ for 10 minutes before the results were interpreted. The interpretation results are detailed in Table 1.

[0038] Table 1. Utilization of YYS-J3 saccharides

[0039]

[0040] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0041] Conclusion: According to the results in Table 1, YYS-J3 bacteria can utilize sugars such as maltose, sucrose, raffinose, lactose, aescin, cellobiose, mannitol, salicin, sorbitol, and inulin, exhibiting broad substrate adaptability. However, it cannot utilize sodium hippurate.

[0042] 2.3 Molecular biological identification of YYS-J3 bacteria

[0043] ① Extraction of YYS-J3 bacterial genomic DNA: Extraction was performed using a bacterial genomic DNA extraction kit from TIANGEN.

[0044] ② PCR amplification of the 16S rDNA sequence: The primers used to amplify the 16S rDNA gene sequence are:

[0045] F 9-27: 5'-GAGTTTGATCCTGGCTCAG-3';

[0046] R 1525-1542: 5'–AGAAAGGAGTGATCCAGCC-3';

[0047] PCR reaction system: 12.5 μL of 2×Mix, 1 μL each of primers and DNA, and 9.5 μL of ddH2O.

[0048] PCR amplification program: 93℃ pre-denaturation for 4 min. Then denature at 94℃ for 30 s, 55℃ (16S rDNA), 72℃ extension for 90 s, for a total of 30 cycles, and finally extend at 72℃ for 10 min, and store at 4℃.

[0049] ③ PCR product detection and sequencing analysis: 5 μL of PCR product was separated and examined by gel electrophoresis in 1.0% (w / v) agarose gel containing EB. The amplified 16S rDNA target fragment was 1480 bp in length (see agarose gel electrophoresis image of 16S rDNA target fragment amplification). Figure 3 ),

[0050] ④ Phylogenetic analysis: Blast alignment analysis was performed on each 16S rRNA sequence in NCBI data to obtain the sequence and... Lactobacillus crispatus The sequence homology was greater than 99.7%, and the developmental tree was constructed using the Neighbor-joining method in MEGA 4 (results are shown in [link to results]). Figure 4 ).

[0051] The results of the 16S rDNA sequence determination of Lactobacillus curvature YYS-J3 are as follows:

[0052]

[0053] Conclusion: Based on morphological observation, biochemical identification of lactic acid bacteria, and homology analysis in the DNA phylogenetic tree, YYS-J3 was identified as *Lactobacillus curlis*. Lactobacillus crispatus ) bacterial strains.

[0054] The performance characterization of Lactobacillus curvature YYS-J3 provided by this invention is as follows:

[0055] Example 3: Analysis of the removal effect of Lactobacillus curvature YYS-J3 on acrylamide

[0056] The Lactobacillus curlis YYS-J3 stock solution was inoculated into MRS medium at a rate of 1% (v / v). After anaerobic culture at 37℃ for 24-48 h, the viable bacterial count was determined by flow cytometry to be 5.25*10⁻⁶. 8 CFU / mL was used to obtain the fermentation broth of *Lactobacillus curlis* YYS-J3. Acrylamide was added to the fermentation broth to a final concentration of 10 mg / L, and the mixture was incubated at 37℃ for 24 h. Using uninoculated MRS medium as a control, 2 mL of the fermentation broth was centrifuged, and the supernatant was filtered through a 0.22 μm microporous membrane and then injected into a brown liquid chromatography vial. Detection was performed using a Waters Alliance e1695 high-performance liquid chromatography (HPLC) system. The detection conditions were: SunFire C18 column (4.6 × 250 mm, 5 μm), injection volume: 5 μL, flow rate: 1 mL / min, mobile phase: acetonitrile:methanol:water = 1:3:96, injection temperature: 30℃, detection wavelength: 210 nm, elution time: 5.3–5.6 min. The control was uninoculated 10 mg / L acrylamide + MRS medium (CK). The acrylamide concentration of each treatment was measured, and the acrylamide removal rate (%) was calculated as (CK - treatment group) / CK * 100%. The removal rate of acrylamide by Lactobacillus curvature YYS-J3 in fermentation broth for 24 h was 56.72 ± 0.31%.

[0057] Example 4: Removal effect of Lactobacillus curvature YYS-J3 on acrylamide in French fry medium

[0058] Commercially available fried French fries were crushed, and 5g of the sample was added to 10 mL of the above-mentioned *Lactobacillus curvatureii* YYS-J3 fermentation broth and inactivated fermentation broth (fermentation broth boiled for 15 min). Using unfermented MRS medium as a control, acrylamide was added to a final concentration of 5 mg / L. After mixing, the mixture was allowed to stand at 37℃. Samples were taken at 2 h and 4 h, and the supernatant was centrifuged to determine the acrylamide content. The test results are shown in Table 2. Table 2 shows that after 2 h and 4 h of treatment, the removal rate of acrylamide from French fries by the *Lactobacillus curvatureii* YYS-J3 fermentation broth was 70.65% and 70.62%, respectively, while the removal rate by the inactivated *Lactobacillus curvatureii* YYS-J3 fermentation broth was 52.30% and 67.28%, respectively. This indicates that the *Lactobacillus curvatureii* YYS-J3 fermentation broth or its inactivated fermentation product can be used to remove acrylamide from French fries. Furthermore, compared with the effect of treatment in the fermentation broth for 24 hours (see Example 3), the removal rate after treatment in the French fry medium for 2 hours and 4 hours was higher than that in the fermentation broth. Moreover, the removal rate of acrylamide in the inactivated fermentation broth was greater than 50%, indicating that Lactobacillus curvatureis YYS-J3 is beneficial for the removal of acrylamide in foods such as French fries.

[0059] Table 2. Removal rate of acrylamide from French fries by Lactobacillus curvatureus YYS-J3

[0060]

[0061] Example 5: Adsorption capacity of Lactobacillus curvature YYS-J3 for lead acetate

[0062] Fermentation broth of *Lactobacillus curlifron* YYS-J3 after 24 hours of fermentation was mixed with lead acetate to a final concentration of approximately 25 mg / L. Using uninoculated MRS medium as a control, *Lactobacillus curlifron* YYS-J3 was inoculated and fermented for 24 hours. The lead content was then determined by centrifugation of the fermentation broth. Fermentation broth of *Lactobacillus curlifron* YYS-J3 after 24-48 hours of fermentation was collected by centrifugation at 4500 rpm for 10 minutes. Cells were added to PBS buffer and counted using flow cytometry. The cell concentration was adjusted to (1.0 ± 0.1) * 102. 9 The concentration of lead acetate was increased to approximately 1 mg / L, using PBS buffer without bacterial cells as a control. The solution was incubated at 37°C, and mixed every 30 minutes. After 4 hours, the solution was centrifuged, and the supernatant was collected. The analysis was performed according to the graphite furnace atomic absorption spectrometry method in GB5009.12-2017, using a PinAAcle900Z atomic absorption spectrometer. Lead adsorption rate (%) = (lead content...) 对照 - Lead content 处理组 ) / Lead content 对照*100%. The results showed that both the fermentation broth and suspension of Lactobacillus curvature YYS-J3 had good adsorption capacity for lead. The adsorption rate of lead by the fermentation broth of Lactobacillus curvature YYS-J3 was 76.56%, and the adsorption rate of lead by Lactobacillus curvature YYS-J3 cells was 81.21%.

[0063] Table 3. Adsorption rate of lead acetate by Lactobacillus curvature YYS-J3

[0064]

[0065] Example 6: Assay for SOD production by Lactobacillus curvature YYS-J3

[0066] SOD enzyme detection was performed using a superoxide dismutase (SOD) assay kit (Nanjing Jiancheng). Specifically, the supernatant of the *Lactobacillus curvatureis* YYS-J3 fermentation broth was obtained by centrifugation. The supernatant was mixed with water at a ratio of 2:3 (v / v), and the reaction system was prepared according to the kit instructions (Table 4). The mixture was thoroughly homogenized and incubated at 37°C for 20 minutes. The OD value was read at 450 nm using a microplate reader. One unit of SOD activity (U) was defined as the amount of enzyme corresponding to a 50% SOD inhibition rate in the reaction system. The formula for calculating SOD activity is as follows:

[0067]

[0068]

[0069] Among them, A 对照 A represents the OD value of the control group. 对照空白 A represents the OD value of the control group. 测定 A represents the OD value of the test group. 测定空白 This indicates the OD value of the blank control group.

[0070] Table 4. Reagent Kit Reaction System Operation Table

[0071]

[0072] The test results showed that the SOD enzyme activity in the fermentation supernatant of Lactobacillus curvature YYS-J3 was 51.33±0.95 U / mL, indicating that Lactobacillus curvature YYS-J3 has a good SOD enzyme production capacity.

[0073] Example 7: Autoagglutination rate and other agglutination rate test of Lactobacillus curvature YYS-J3

[0074] Fermentation broths of *Lactobacillus curlis* YYS-J3, *Streptococcus mutans*, and *Escherichia coli* were prepared separately. The YYS-J3 fermentation broth was prepared by inoculating *YYS-J3* stock solution at 1% (v / v) onto MRS medium and anaerobic culture for 48 h. The *Streptococcus mutans* fermentation broth was prepared by inoculating *Streptococcus mutans* (…) onto… Streptococcus mutans ATCC 25175) was inoculated into brain and heart extract medium at a rate of 1% (v / v) and fermented at 37°C and 150 rpm for 24 h. The E. coli fermentation broth was prepared by inoculating E. coli (… Escherichia coli ATCC35150 was inoculated into LB medium at a rate of 1% (v / v) and fermented at 37℃ and 150 rpm for 24 h. 2 mL or more of each bacterial fermentation broth was taken separately and centrifuged at 12000 rpm and 4℃ for 5 min to collect the bacterial sludge. The sludge was washed twice with sterile phosphate-buffered saline (PBS) at pH 7.0 (i.e., PBS was added to the sludge, the mixture was shaken to mix thoroughly, and then centrifuged at 12000 rpm and 4℃ for 5 min to collect the sludge). The bacterial suspension was adjusted to an absorbance of 0.6 ± 0.1 (A0) at 600 nm using sterile PBS. The bacterial suspensions of each bacterium were then calculated as A. O .

[0075] Self-agglutination rate test: The above-mentioned Lactobacillus curvatureus YYS-J3A0 bacterial suspension was allowed to stand for different time periods (2h, 4h, 24h) and the absorbance value A was measured. X .

[0076] Self-agglutination rate (%) = (1-A) x / A0)×100%;

[0077] Where x is 2, 4, or 24; A2 represents the absorbance value measured after the bacterial suspension has stood for 2 hours; A4 represents the absorbance value measured after the bacterial suspension has stood for 4 hours; and A24 represents the absorbance value measured after the bacterial suspension has stood for 24 hours. The same applies below.

[0078] Agglutination rate test: Take the above-mentioned YYS-J3 bacterial suspension, Streptococcus mutans bacterial suspension, and Escherichia coli suspension A respectively. O Take 1 ml of YYS-J3 bacterial suspension and 1 ml of Streptococcus mutans bacterial suspension to obtain the first mixed bacterial suspension, take 1 ml of YYS-J3 bacterial suspension and 1 ml of Escherichia coli suspension to obtain the second mixed bacterial suspension, and after standing for different time periods (2h, 4h, 24h), measure the absorbance A of the first mixed bacterial suspension and the second mixed bacterial suspension respectively.

[0079] Its aggregation rate (%) = (1-A) x / A0)×100%;

[0080] Where x is 2, 4, or 24.

[0081] The results are shown in Table 5 below: The autoagglutination rates of *Lactobacillus curvatureis* YYS-J3 at 2h, 4h, and 24h were 19.08%, 57.50%, and 77.17%, respectively, indicating a high autoagglutination rate and thus a good ability to produce extracellular polysaccharides. The agglutination rates of *Lactobacillus curvatureis* YYS-J3 with *Streptococcus mutans* were 43.75%, 55.08%, and 76.58%, respectively, while the agglutination rate with *Escherichia coli* ranged from 9.67% to 47.92%. This indicates that YYS-J3 has excellent pathogenic bacteria agglutination characteristics, which will be beneficial for the efficient removal of *Streptococcus mutans* and also has a certain aggregation effect on *Escherichia coli*.

[0082] Table 5. Autoagglutination rate and other agglutination rate of Lactobacillus curvatureus YYS-J3

[0083]

[0084] Example 8: Survival analysis of Lactobacillus curvature YYS-J3 in a simulated artificial gastric juice environment.

[0085] (1) The survival rate test process was as follows: YYS-J3 fermentation broth of *Lactobacillus curvatureensis* fermented for 24 h was collected by centrifugation at 12000 r / min for 5 min, and the same volume of 0.85% (w / v) physiological saline was added and mixed to obtain a bacterial suspension. Artificial gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3 and 3 g / L pepsin) was prepared, and the pH was adjusted to 2.0, 2.5, and 3.0. The suspension was then filtered through a 0.22 μM microporous membrane for later use. 1 mL of the bacterial suspension was added to 9 mL of artificial gastric fluid at different pH values ​​and incubated at 37℃ for x (1, 2, 3, 5) h. 0.9 mL of the treated sample was taken each time, and 0.1 mL of 20 ug / mL propidium iodide (PI) was added. After staining at 37℃ for 10 min, 0.1 mL of the treated sample was added to 0.9 mL of the PI solution. In mL of ultrapure water, the total bacterial count P1 and the number of dead bacteria P2 were detected by flow cytometry, and the survival rate / % at different time periods was calculated. The survival rate in the bacterial suspension without artificial gastric juice treatment was used as a 100% control to calculate the gastrointestinal tolerance of each treatment.

[0086] The formula for calculating bacterial survival rate at different treatment times is as follows:

[0087] Survival rate (%) = [(P1) 处理组 -P2 处理组 ) / P1 处理组 ] / [(P1 对照 -P2 对照 ) / P1 对照 ];

[0088] Among them, P1处理组 The total number of bacterial particles in the treatment group, P2 处理组 This refers to the number of dead bacterial particles in the treatment group, P1 对照 This refers to the total number of bacterial particles in the control group, P2. 对照 This refers to the number of dead bacterial particles in the control group.

[0089] (2) The survival rate of Lactobacillus curvature YYS-J3 in different gastric fluid environments is shown in Table 6. According to the data, the following can be seen:

[0090] The survival rate of *Lactobacillus curlifi* YYS-J3 was 53.13% after 1 hour of treatment in a simulated gastric fluid environment with a pH of 2.0. The survival rate remained stable between 17.26% and 19.88% after 2-5 hours of treatment. In a simulated gastric fluid environment with a pH of 2.5, the survival rate was 99.84%-66.99% after 1-5 hours of treatment. In a simulated gastric fluid environment with a pH of 3.0, the survival rate was greater than 100%. These results indicate that *Lactobacillus curlifi* YYS-J3 exhibits good tolerance in simulated gastric fluid environments and is unaffected by a pH of 3.0.

[0091] Table 6. Survival rate (%) of Lactobacillus curvatureis YYS-J3 in an artificial simulated gastric juice environment.

[0092]

[0093] Example 9: Survival analysis of Lactobacillus curvatureis YYS-J3 in a simulated artificial pancreatic juice environment.

[0094] (1) Take the fermentation broth of Lactobacillus curvature YYS-J3 fermented for 24 h, centrifuge at 12000 r / min for 5 min to collect the bacterial cells, add the same volume of 0.85% (w / v) physiological saline and mix well to prepare a bacterial suspension for later use; prepare protein pancreatic juice [0.1% (w / v) pancreatin, 0.15% (w / v) bovine bile], adjust the pH value to 7.5 and 8.0 respectively, filter through a 0.22 μM microporous membrane for later use, take 1 mL of the above bacterial suspension in 9 mL of protein pancreatic juice with different pH values, and incubate at 37℃. Take samples after 3 h and 6 h of treatment, take 0.9 mL of the treated sample each time, add 0.1 mL of PI dilution solution, stain at 37℃ for 10 min, and detect the total number of bacteria P1 and the number of dead bacteria P2 by flow cytometry, and calculate the survival rate at different time periods. The survival rate of bacteria is calculated with the survival number of untreated bacterial suspension as a control.

[0095] The formula for calculating bacterial survival rate is:

[0096] Survival rate (%) = [(P1) 处理组 -P2处理组 ) / P1 处理组 ] / [(P1 对照 -P2 对照 ) / P1 对照 ];

[0097] Among them, P1 处理组 This refers to the total number of bacterial particles in the treatment group, P2 处理组 This refers to the number of dead bacterial particles in the treatment group, P1 对照 This refers to the total number of bacterial particles in the control group, P2. 对照 This refers to the number of dead bacterial particles in the control group. The bacterial survival rate of *Lactobacillus curvatureis* YYS-J3 is shown in Table 7. Based on the data, we can see that:

[0098] The survival rates of Lactobacillus curvature YYS-J3 after treatment in pancreatic juice at pH 7.5 for 3 h and 6 h were 99.09% and 98.29%, respectively, while the survival rates in pancreatic juice at pH 8.0 were 101.10% and 101.05%, respectively, indicating that it is not affected by pancreatic juice.

[0099] Table 7 Survival rate (%) of Lactobacillus curvatureis YYS-J3 in an artificial simulated pancreatic juice environment

[0100]

[0101] This invention also provides the following application examples of Lactobacillus curvature YYS-J3:

[0102] Example 10: Preparation of a probiotic agent from Lactobacillus curvatureis YYS-J3

[0103] Lactobacillus curvature YYS-J3 was inoculated into a culture medium, such as MRS medium, and cultured at 0-38°C for more than 15 hours. The bacterial cells were collected by centrifugation and resuspended in, for example, physiological saline or PBS buffer to prepare a liquid bacterial preparation containing Lactobacillus curvature YYS-J3.

[0104] Optionally, Lactobacillus curvatureis YYS-J3 bacterial cells are resuspended in a cell protectant and a carrier, and then freeze-dried to obtain a solid bacterial powder preparation containing Lactobacillus curvatureis YYS-J3.

[0105] Optionally, Lactobacillus curlis YYS-J3 can be used as a raw material component in acrylamide detoxification products, heavy metal lead detoxification products, and SOD products. Lactobacillus curlis YYS-J3 can exist in the products in liquid or solid formulation form.

[0106] Example 11: Preparation of fermented food using Lactobacillus curvature YYS-J3

[0107] To prepare Lactobacillus curlis YYS-J3 yeast broth, various fruits, Chinese herbal medicines, grain raw materials, and various sugars are used as auxiliary materials. Lactobacillus curlis YYS-J3 is inoculated and fermented for a certain period of time under certain temperature conditions (30-38℃) to prepare fermented products. The fermented products are either inactivated or not inactivated. After the original liquid or different proportions are diluted, common beverage auxiliary materials are added to prepare fermented food.

[0108] Based on the results of the above embodiments, the *Lactobacillus curvatureis* YYS-J3 provided by the present invention has the following properties and effects:

[0109] a. It can utilize a variety of carbon sources such as aescin, cellobiose, maltose, mannitol, maltose, salicin, sorbitol, sucrose, raffinose, inulin, and lactose, and has a wide range of carbon source adaptability.

[0110] b. It can degrade or adsorb acrylamide, achieving a removal rate of 56.72% in MRS media. The removal effect is even better in media containing French fries, with removal rates exceeding 70% after 2 hours and 4 hours of treatment. Its inactivated fermentation broth can also remove acrylamide, with removal rates of 52.30% and 67.28% after 2 hours and 4 hours of treatment, respectively.

[0111] c. It can adsorb heavy metal lead, with adsorption rates of 81.21% and 76.56% in lead-containing PBS medium (lead content 1 mg / L) and MRS medium (lead content 25 mg / L), respectively.

[0112] d. It can produce SOD enzyme, and the SOD enzyme activity of its fermentation supernatant is 51.33 U / mL.

[0113] e. It has a good agglutination rate, with the highest self-agglutination rate of 77.17%, and the agglutination rate against Streptococcus mutans is as high as 76.58% after 24 hours, while the agglutination rate against Escherichia coli is 47.92%.

[0114] f. In an artificial gastric juice environment with pH 2.0, the survival rate is 53.13% after 1 hour. In an artificial gastric juice environment with pH 2.5, the survival rate is between 66.99% and 99.94% after at least 5 hours. In an artificial gastric juice environment with pH 3.0, the survival rate is not affected.

[0115] g. When treated in pancreatic juice at pH 7.5 for 3-6 hours, the survival rate is as high as 98% or more. When treated in pancreatic juice at pH 8.0 for 3-6 hours, the survival rate is not affected.

[0116] h. Lactobacillus curvature YYS-J3 was isolated from the feces of healthy human infants. It is highly safe for consumption and can be used as a detoxifier for acrylamide and lead, as well as an SOD enzyme. The product has broad application prospects in pharmaceuticals.

[0117] In summary, compared with the prior art, the *Lactobacillus curlis* YYS-J3 provided by this invention has the following beneficial effects:

[0118] The *Lactobacillus curlis* YYS-J3 can degrade or adsorb acrylamide, has good adsorption capacity for heavy metal lead, can produce SOD enzyme, has good aggregation ability, and has a wide range of carbon source utilization capabilities. This bacterium provides a new probiotic source for the development of functional products and has important application value, for example:

[0119] (1) Lactobacillus curvature YYS-J3 can be used as a raw material component of the composition to prepare a composition with the above-mentioned functions; wherein, the composition includes, but is not limited to, microbial preparations, drugs, etc.

[0120] The bacterial strain present in the composition includes, but is not limited to, one or more combinations of *Lactobacillus curlifi* YYS-J3 (non-inactivated), *Lactobacillus curlifi* YYS-J3 (inactivated), metabolites of *Lactobacillus curlifi* YYS-J3 strain, and freeze-dried *Lactobacillus curlifi* YYS-J3 strain. Preferably, the number of *Lactobacillus curlifi* YYS-J3 in the composition is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. More preferably, the number of *Lactobacillus curvatureis* YYS-J3 is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0121] (2) Various plants (such as fruits, Chinese herbal medicines, grains, etc.) can be used as raw materials, combined with various ingredients, and inoculated with Lactobacillus curvulariae YYS-J3 for fermentation treatment to prepare fermented products. These fermented products can be used to prepare products with hangover relief, antioxidant or weight loss functions.

[0122] The fermentation raw materials can be various conventionally used plant fermentation raw materials, and the excipients can also be existing conventional excipients, including but not limited to the above-mentioned scheme selections. The fermented products are, but not limited to, those used in the preparation of pharmaceuticals and other products.

[0123] In summary, Lactobacillus curlis YYS-J3 and / or its ferments, based on their characteristics, can be used in functional products that include at least one of the following functions:

[0124] (1) Degradation or adsorption of acrylamide;

[0125] (2) Produces SOD enzyme;

[0126] (3) Adsorption of lead acetate;

[0127] (4) Good aggregation properties;

[0128] (5) Good carbon source utilization capability.

[0129] Among them, products with the above-mentioned functions (1)-(5) include, but are not limited to, acrylamide or heavy metal detox products, SOD enzyme products, and functional products with detoxification, anti-oxidation and other directional effects; they may also be products with other directional effects based on the functions (1)-(5); among them, the functional products include, but are not limited to, pharmaceuticals.

[0130] It should be noted that:

[0131] (1) Definition:

[0132] The term "food" as used herein is used in a broad sense, including food and drink for humans. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders and tablets, as well as to disperse in liquids to prepare liquid dosage forms and other dosage forms suitable for oral administration to humans, including but not limited to powders.

[0133] The composition includes, but is not limited to, microbial preparations and pharmaceuticals, and the composition containing Lactobacillus curlis YYS-J3 can be used in other forms of products.

[0134] The form in which the *Lactobacillus curvatureis* YYS-J3 exists in the composition includes, but is not limited to, non-inactivated bacteria, inactivated bacteria, metabolites, freeze-dried strains, etc. It is anticipated that the *Lactobacillus curvatureis* YYS-J3 may also exist in the composition in other forms.

[0135] The functional product includes the aforementioned functions of degrading or adsorbing acrylamide, adsorbing heavy metal lead, producing SOD enzyme, and high aggregation. Based on the above functions, it is applied to the functional product to degrade or adsorb acrylamide, adsorb heavy metal lead, and produce SOD enzyme. Because it has the above-mentioned functions of degrading or adsorbing acrylamide, adsorbing heavy metal lead, and producing SOD enzyme, the efficacy of its application product includes, but is not limited to, detoxification, anti-oxidation, and other effects on the human body that can be achieved through the above functions.

[0136] (2) The relevant prior art means or prior art terms involved in this application:

[0137] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. x"OD" is the optical density value measured when the wavelength is set to X nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0138] “SOD” stands for superoxide dismutase.

[0139] The determination of total bacterial count P1 and dead bacterial count P2 using flow cytometry is an existing technology, and its principles and methods will not be elaborated here.

[0140] Carbon source utilization analysis was performed using biochemical identification strips for lactic acid bacteria. This is an existing technology, and its principles and methods will not be elaborated here.

[0141] (3) The formulations of the culture media used in the examples are as follows:

[0142] MRS medium (g / L): 10g casein peptone, 10g beef extract, 5g yeast extract, 5g glucose, 5g sodium acetate, 2g K2HPO4, 2g diammonium citrate, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 1mL Tween 80, pH=6.2. For solid medium, add 2% agar and 2% CaCO3 to the above and sterilize at 121℃ for 15min.

[0143] Brain and heart extract culture medium (g / L): brain extract powder 5.0, heart extract powder 12.5, peptone 5.0, tryptone 10.0, sodium chloride 5.0, glucose 2.0, disodium hydrogen phosphate 2.5, agar 20.0 (not included in liquid culture medium), pH 7.4±0.2, sterilized at 121 ℃ for 15 min.

[0144] LB medium (g / L): tryptone 10.0, sodium chloride 10.0, yeast extract 5.0, pH 7.5; (solid medium with 20.0 g agar powder), sterilize at 121℃ for 15 min.

[0145] Unless otherwise specified, the experimental procedures involved in the embodiments of the present invention are conventional experimental procedures in the art, and the reagents or instruments involved can be obtained from legitimate channels.

[0146] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of the present invention can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or the background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of *Lactobacillus curvatureii* with acrylamide degradation and lead adsorption functions (… Lactobacillus crispatus YYS-J3, characterized in that: Its accession number is CGMCC No.28184.

2. A composition, characterized in that: It contains Lactobacillus curvatureus YYS-J3 as described in claim 1.

3. The composition according to claim 2, characterized in that: The composition includes one of microbial preparations and pharmaceuticals.

4. The composition according to claim 2, characterized in that: The composition contains one or more combinations of uninactivated Lactobacillus curvature YYS-J3 and freeze-dried Lactobacillus curvature YYS-J3.

5. The application of Lactobacillus curlis YYS-J3 as described in claim 1 in the preparation of functional products; The functional product includes at least one of the following functions: (1) Degradation of acrylamide; (2) Adsorption of heavy metal lead; (3) Produces SOD enzyme.

6. The application according to claim 5, characterized in that: The functional products include products that degrade acrylamide, products that adsorb heavy metal lead, and products that produce SOD enzymes.

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