A Pediococcus acidilactici YYS-J2 capable of degrading acrylamide and producing high phenyllactic acid and its application

Abstract

The present invention relates to the field of microbial technology, and provides a Pediococcus acidilactici YYS-J2 that can degrade acrylamide and produce high levels of phenyllactic acid, and its application. Pediococcus acidilactici The preservation number of YYS-J2 is CGMCC No. 28183. This *Pediococcus lactis* YYS-J2 exhibits excellent acrylamide removal capacity, high efficiency in L-phenylalanine utilization and metabolism, high-quality phenyllactic acid production capacity, and excellent *Streptococcus mutans* antibacterial activity. It also produces superoxide dismutase (SOD) and shows good tolerance in simulated gastric and intestinal fluids. This *Pediococcus lactis* YYS-J2 can provide a new probiotic source for the development of functional products such as acrylamide removal products, antibacterial products, and SOD-producing 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 lactic acid cocci, YYS-J2, and its applications. Background Technology

[0002] Fried, baked, or roasted foods are popular and delicious. However, during high-temperature processing, the reducing sugars and aspartic acid in these foods undergo the Maillard reaction, producing byproducts such as acrylamide. Acrylamide is classified as a Group 2A carcinogen by the World Health Organization, and numerous studies have shown that it possesses potential carcinogenicity, neurotoxicity, genotoxicity, and reproductive toxicity. Acrylamide is water-soluble and can be absorbed by the human body through various routes, including the respiratory tract, digestive tract, and skin, thus affecting human health.

[0003] Existing methods for reducing acrylamide levels include reducing the sugar content of raw materials and adding organic acids to food to decrease acrylamide production. However, this can affect the sensory quality of food. Therefore, it is necessary to seek new ways to remove acrylamide from food or the body to maintain health.

[0004] Streptococcus mutans is a common pathogenic bacterium in the oral cavity. It initially adheres to the tooth surface by secreting adhesins, forming a biofilm. It then metabolizes carbohydrates such as sucrose in the mouth into acidic substances and insoluble glucans, which adhere to the tooth surface and damage and corrode the teeth, causing demineralization and leading to dental caries. According to the Fourth National Oral Health Epidemiological Survey conducted by the National Health and Family Planning Commission, the prevalence of dental caries in five-year-old and twelve-year-old children in my country is 71.9% and 34.5%, respectively. Therefore, it is extremely important to find a method to effectively inhibit Streptococcus mutans.

[0005] Phenylated acid is a natural antibacterial compound with broad-spectrum antibacterial properties. It can inhibit the growth of fungi and various bacteria, making it a high-quality natural biological preservative. Furthermore, phenyllactic acid is a derivative of tanshinone and possesses similar pharmacological effects, such as antiplatelet aggregation activity and regulation of human steroids. Phenylated acid is safe and non-toxic to humans and animals; therefore, phenyllactic acid produced by lactic acid bacteria can be considered a natural antibacterial substance.

[0006] Phenylalanine is an essential amino acid for the human body and a precursor to the synthesis of phenyllactic acid and tyrosine. However, some people with a deficiency in the phenylalanine hydroxylase gene cannot metabolize phenylalanine into tyrosine, leading to the accumulation of phenylalanine in the body and causing damage to the brain and nervous system. Therefore, consuming probiotics that can efficiently consume phenylalanine in food can not only reduce the accumulation of phenylalanine in the bodies of such individuals, but the phenyllactic acid produced also has antibacterial and conditioning effects similar to those of tanshinone.

[0007] Superoxide dismutase (SOD) is a multifunctional active enzyme system that integrates six functions: scavenging, activation, regeneration, repair, self-healing, and nutrient supply. It also has anti-radiation, anti-cancer, and antioxidant effects.

[0008] It has significant clinical applications; however, due to its complex extraction process, its yield is extremely limited.

[0009] The related products are extremely rare and expensive.

[0010] Currently, research on probiotic strains that can significantly remove acrylamide, efficiently utilize phenylalanine to produce phenyllactic acid, inhibit Streptococcus mutans, and secrete SOD is still lacking in this field. How to develop a probiotic that can significantly remove acrylamide, efficiently utilize phenylalanine to produce phenyllactic acid, inhibit Streptococcus mutans, and secrete SOD, for application in the removal of harmful substances such as acrylamide, oral care products, antibacterial products, and SOD-producing functional products, is precisely the problem that those skilled in the art are dedicated to solving. Summary of the Invention

[0011] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Pediococcus lactis* YYS-J2 strain. Pediococcus acidilactici YYS-J2 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. 28183.

[0012] The present invention also provides a composition comprising, as described above, Pediococcus lactis YYS-J2.

[0013] In one embodiment, the number of *Pediococcus lactis* YYS-J2 in the composition is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. In one embodiment, the number of *Pediococcus lactis* YYS-J2 in the composition is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0014] In one embodiment, the composition comprises one or more combinations of Pediococcus lactis YYS-J2 non-inactivated bacteria, Pediococcus lactis YYS-J2 inactivated bacteria, metabolites of Pediococcus lactis YYS-J2 strain, and freeze-dried Pediococcus lactis YYS-J2 strain.

[0015] The present invention also provides a fermentation product obtained by fermentation of *Pediococcus lactis* YYS-J2 as described above.

[0016] The present invention also provides the use of Pyorrhizococcus lactis YYS-J2 and / or its ferments as described above in the preparation of functional products.

[0017] In one embodiment, the functional product includes at least one of the following functions:

[0018] (1) Degradation or adsorption of acrylamide;

[0019] (2) Produces SOD;

[0020] (3) Utilize or metabolize L-phenylalanine;

[0021] (4) Produces phenyllactic acid;

[0022] (5) It has antibacterial ability against Streptococcus mutans and copolymerization ability against Streptococcus mutans.

[0023] In one embodiment, the functional products include acrylamide detox products, antibacterial products, and SOD-producing products.

[0024] Based on the above, compared with the prior art, the Lactococcus lactis YYS-J2 provided by the present invention has the following beneficial effects:

[0025] The *Pediococcus lactis* YYS-J2 strain provided by this invention can degrade or adsorb acrylamide in foods such as French fries, produce superoxide dismutase (SOD), efficiently utilize L-phenylalanine to produce phenyllactic acid, and has a strong antibacterial ability against oral pathogens such as *Streptococcus mutans*. Furthermore, it exhibits good tolerance in artificial gastric and intestinal fluids. This strain can provide a new probiotic source for the development of functional products such as acrylamide detoxification products, antibacterial products, and SOD-producing products, and has significant application value.

[0026] 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

[0027] 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. Unless otherwise specified, the positional relationships shown in the drawings in the following description are based on the direction in which the components are drawn in the figure.

[0028] Figure 1 This is a colony morphology diagram of Pietrococcus lactis YYS-J2.

[0029] Figure 2 This is a scanning electron microscope image of Pyotrophic Lateral Sclerosis YYS-J2.

[0030] Figure 3 Agarose gel electrophoresis image of the amplified 16S rDNA target fragment of Pediococcus lactis YYS-J2.

[0031] Figure 4 Phylogenetic tree diagram of the 16S rDNA gene of Pediococcus lactis YYS-J2.

[0032] Figure 5 This is a diagram showing the utilization of L-phenylalanine in the fermentation substrate by *Pediococcus lactis* YYS-J2.

[0033] Figure 6 Production of phenyllactic acid by *Pediococcus lactis* YYS-J2 in MRS media with different concentrations of L-phenylalanine.

[0034] Figure 7 The antibacterial effect of Pleurotus ostreatus YYS-J2 against Streptococcus mutans. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] The present invention also provides the following operation examples and embodiments:

[0038] The present invention provides Pyrococcus lactis YYS-J2 ( Pediococcus acidilactici YYS-J2 It is obtained by separating the pulp, sugar and honey from a mixture of bayberry pulp, sugar and honey after fermentation.

[0039] An example of the extraction process of this bacterium from a mixture of bayberry pulp, sugar, and honey is as follows:

[0040] Example 1: Screening and Isolation of Bacteria

[0041] Five bottles were prepared by mixing bayberry pulp, sugar, honey, and water in a mass ratio of 40:5:5:50 and allowing them to ferment at 37°C. One 1ml sample from the bottle with the best flavor was selected and diluted to a final volume of 10. -3 times, 10 -4 times, 10 -5 The diluted 0.1 mL fermentation broth was spread onto MRS medium containing CaCO3 and anaerobically cultured at 37℃ for 48-72 h. Several single colonies of suspected lactobacilli that could produce large lysate zones were selected, purified three times, and their acrylamide degradation effect was tested. The strain with better acrylamide degradation effect was selected, preserved, and named YYS-J2.

[0042] Example 2: Identification of bacterial strains

[0043] 2.1 Morphological observation of YYS-J2 bacteria

[0044] The colony morphology of YYS-J2 is as follows: Figure 1 As shown, the bacterial cell morphology is as follows Figure 2 As shown, the main morphological characteristics of YYS-J2 are as follows: the colonies are round, white and opaque on MRS medium; the cells are spherical, with a diameter of 0.6-1 μm.

[0045] 2.2 Physiological and biochemical analysis of YYS-J2 bacteria

[0046] 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, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% (w / v) sodium hippurate. YYS-J2 bacteria were inoculated at 1% (w / v).

[0047] In addition, after the culture is completed, 0.2 mL of ninhydrin solution should be slowly added along the wall of the test tube for 1% sodium hippurate. The amount of 1% (w / v) sodium hippurate is 2 mL. Do not shake. After placing in a water bath at 36℃±1℃ for 10 min, the results are interpreted. The interpretation results are detailed in Table 1.

[0048] Table 1. Major physiological and biochemical reactions of YYS-J2

[0049]

[0050] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0051] Conclusion: According to the results in Table 1, YYS-J2 can only utilize cellobiose and salicin, and cannot utilize saccharides such as maltose, sucrose, raffinose, lactose, aescin, mannitol, salicin, sorbitol, inulin, and sodium hippurate.

[0052] 2.3 Molecular biological identification of YYS-J2

[0053] ① Extraction of YYS-J2 bacterial genomic DNA: Genomic DNA was extracted using a bacterial genomic DNA extraction kit from TIANGEN.

[0054] ②PCR amplification of the 16S rDNA sequence:

[0055] The primers used to amplify the 16S rDNA gene sequence were: F9-27: 5'-GAGTTT GAT CCT GGC TCA G-3'; R1525-1542: 5'-AGA AAG GAG GTG ATC CAG CC-3';

[0056] PCR reaction system: 12.5 μL of 2×Mix, 1 μL each of primers and DNA, and 9.5 μL of ddH2O.

[0057] PCR amplification program: 93℃ pre-denaturation for 4 min; then 94℃ denaturation for 30 s, 55℃ (16S rDNA), 72℃ extension for 90 s, for a total of 30 cycles, and finally 72℃ for a full extension for 10 min, and stored at 4℃.

[0058] ③ PCR product detection and sequencing analysis: 5 μL of PCR product was separated and examined by gel electrophoresis in 1.0% agarose gel containing EB. The amplified 16S rDNA target fragment was 1499 bp in length (see agarose gel electrophoresis image of 16S rDNA target fragment amplification). Figure 3 The YYS-J2 16S rDNA sequence obtained is as follows:

[0059]

[0060] ④ Phylogenetic analysis: Blast alignment analysis was performed on each 16S rRNA sequence in NCBI data to obtain the sequence and... Pediococcus acidilactici The sequence homology of the series of standard strains was greater than 99%. Developmental tree construction analysis was performed using the Neighbor-joining method in MEGA 4 (results are shown in...). Figure 4 ).

[0061] Conclusion: Based on morphological observation, physiological and biochemical identification, and homology analysis in the DNA phylogenetic tree, YYS-J2 was identified as Pediococcus lactis. Pediococcus acidilactici ) bacterial strains.

[0062] The performance characterization of the *Pediococcus lactis* YYS-J2 provided by this invention is as follows:

[0063] Example 3: Removal effect of Pyrococcus lactis YYS-J2 on acrylamide

[0064] The mother liquor of *Pediococcus lactis* YYS-J2 was inoculated into MRS medium at a rate of 1% (w / v) and anaerobic cultured at 37°C for 24 h to obtain the YYS-J2 fermentation broth (the same applies below). The viable count of the initial fermentation broth was determined by flow cytometry to be 5.7*10⁻⁶. 8 The removal efficiency was measured at cfu / mL for different times and concentrations.

[0065] The measurement process is as follows:

[0066] (1) Experimental group 1: Verifying the effect of different treatment times on the degradation of acrylamide:

[0067] Take 10 ml of fermentation broth after 24 h of fermentation, add acrylamide mother liquor until the final concentration of acrylamide in the solution is about 10 mg / L, take 2 mL of sample at different times (2 h, 4 h, 6 h, 24 h), centrifuge, and take the supernatant for testing;

[0068] (2) Experimental group two: Verifying the degradation effect of YYS-J2 on acrylamide at different concentrations:

[0069] Take 10 mL of fermentation broth after 24 hours of fermentation, add acrylamide mother liquor until the final concentration of acrylamide in the solution is approximately 1 mg / L, 10 mg / L, and 20 mg / L. After 6 hours of treatment, take 2 mL of sample, centrifuge and collect the supernatant.

[0070] (3) Control group: Acrylamide stock solution without inoculation + 10 ml MRS medium (CK), wherein the final concentration of acrylamide is 10 mg / L, and 2 mL of sample is centrifuged to obtain the supernatant.

[0071] All the above samples were filtered through a 0.22 μm microporous membrane and then placed in a brown liquid chromatography vial. The acrylamide concentration of each treated sample solution was determined using a Waters Alliance e1695 high-performance liquid chromatography system. The acrylamide removal rate results are shown in Table 2-3.

[0072] The LC detection conditions were as follows: 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 acrylamide removal rate was calculated using the following formula:

[0073] Acrylamide removal rate / % = (CK-X) / CK×100%;

[0074] In the formula, CK represents the acrylamide concentration measured in the blank control group, and X represents the acrylamide concentration in the sample of experimental group one or experimental group two.

[0075] Table 2. Effects of different treatment times on the degradation of acrylamide

[0076]

[0077] Table 3. Degradation effect of YYS-J2 on acrylamide at different concentrations

[0078]

[0079] As shown in Table 2-3, Porphyromonas lactis YYS-J2 has a good removal effect on acrylamide.

[0080] Example 3: Removal effect of Pyrococcus lactis YYS-J2 on acrylamide in French fry medium

[0081] Commercially available fried French fries were crushed, and 5g of the sample was weighed and added to 10mL of YYS-J2 fermentation broth and inactivated fermentation broth (fermentation broth boiled for 15 min). With unfermented MRS medium as a control, acrylamide stock solution was added until the final acrylamide concentration was about 5 mg / L. After mixing, the mixture was allowed to stand at 37℃. Samples were taken at 2h and 4h, and the supernatant was centrifuged to determine the acrylamide content. The data obtained are shown in Table 4.

[0082] The YYS-J2 fermentation broth used was the fermentation broth in Example 3, and the inactivated fermentation broth was obtained by boiling the above YYS-J2 fermentation broth at 100°C for 15 minutes.

[0083] Table 4. Removal effect of YYS-J2 on acrylamide in French fries

[0084]

[0085] As can be seen from the data in Table 4:

[0086] After 2 hours and 4 hours of treatment, the removal rates of acrylamide in French fries by YYS-J2 fermentation broth were 44.08% and 78.30%, respectively. The removal rates of acrylamide in French fries by inactivated YYS-J2 fermentation broth were 22.00% and 76.78%, respectively. This indicates that YYS-J2 fermentation broth or its inactivated fermentation product can be used to remove acrylamide from French fries.

[0087] Compared to the treatment effect in the fermentation broth in Example 3, the acrylamide removal rate increased from 48.18% to 78.3% after treatment in the French fry medium for 4 hours, indicating that YYS-J2 can be applied to the removal of acrylamide from foods such as French fries. Further inactivation of the fermentation broth also significantly removed acrylamide from the French fries.

[0088] Example 4: The ability of Pediococcus lactis YYS-J2 to produce phenyllactic acid using L-phenylalanine

[0089] YYS-J2 yeast broth was inoculated into MRS medium containing different concentrations of L-phenylalanine (MRS+0: no L-phenylalanine added to MRS medium; MRS+0.5: exogenous L-phenylalanine added to MRS medium to a final concentration of approximately 0.5 g / L; MRS+1: exogenous L-phenylalanine added to MRS medium to a final concentration of approximately 1 g / L). Samples were taken before fermentation, and the mixture was cultured for 96 hours to obtain the YYS-J2 fermentation broth. The supernatant was centrifuged and filtered through a 0.22 μm microporous membrane. The contents of L-phenylalanine and phenyllactic acid were determined using a Waters Alliance e1695 high-performance liquid chromatography (HPLC). The results are shown in the figure below. Figure 5-6 ;

[0090] The HPLC detection conditions were as follows: column: SunFire C18 column (4.6 × 250 mm, 5 μm); column temperature: 30℃; injection volume: 10 μL; flow rate: 0.8 mL / min; mobile phase: phase A was 0.55% trichloroacetic acid methanol solution, and phase B was 0.05% trichloroacetic acid solution; elution program: 0–20 min, A:B linearly changed from 10% to 100%; 20–23 min, 100% phase A; 23–27 min, maintaining A:B at 10%; detection wavelength: 210 nm. L-phenylalanine eluted at approximately 9 min, and phenyllactic acid eluted between 12.1 and 13.4 min.

[0091] Among them, YYS-J2 yeast broth is the fermentation broth in Example 3.

[0092] YYS-J2's ability to produce phenyllactic acid from L-phenylalanine is... Figure 5 The data show that after fermentation with different amounts of L-phenylalanine using YYS-J2, the L-phenylalanine content in the culture medium decreased significantly. After 96 hours of fermentation, the L-phenylalanine content in the fermented product decreased from 0.24-1.10 (g / L) before fermentation to 0.049-0.117 (g / L), with a utilization and consumption rate of 79.49-93.35%. This indicates that YYS-J2 can significantly reduce the L-phenylalanine content in the fermented product.

[0093] After 96 hours of fermentation, the highest yield of phenyllactic acid detected in the culture medium reached 741.12 mg / L. Figure 6 This indicates that YYS-J2 has an extremely high phenyllactic acid production capacity.

[0094] Example 5: Antibacterial activity of Pyotrophic Lactococcus YYS-J2 against Streptococcus mutans

[0095] The fermentation broth of *Pediococcus lactis* YYS-J2 was used to determine its resistance to *Streptococcus mutans* (using the Oxford cup method). Streptococcus mutans The antibacterial activity of ATCC 25175 was demonstrated, with YYS-J2 exhibiting an inhibition zone diameter of up to 35.00±5.20 mm against Streptococcus mutans (e.g., ATCC 25175). Figure 7 As shown in the figure, YYS-J2 has excellent antibacterial activity against oral pathogen Streptococcus mutans.

[0096] Among them, the fermentation broth of YYS-J2 is the fermentation broth in Example 3.

[0097] Example 6: Test of autoagglutination rate (%) and agglutination rate (%) of Pediococcus lactis YYS-J2

[0098] Fermentation broths of *Streptococcus mutans* and *YYS-J2* were prepared. An appropriate amount of fermentation broth was centrifuged at 12000 r / min and 4℃ for 5 min, and the bacterial sludge was collected. The sludge was washed twice with sterile phosphate-buffered saline (PBS) at pH 7.0 (i.e., PBS was added to the colonies, the mixture was shaken to mix thoroughly, and then centrifuged at 12000 r / min and 4℃ for 5 min to collect the bacterial cells). A suspension and a bacterial suspension with an absorbance of 0.6 ± 0.1 (A0) at 600 nm were then prepared using PBS.

[0099] Self-agglutination rate (%): YYS-J2 bacterial mud was prepared into a suspension and a bacterial suspension with an absorbance of 0.6±0.1 (A0) at a wavelength of 600 nm using sterile PBS. After standing for 24 h, the absorbance value Ax (x=24) was measured to obtain the self-agglutination rate. The results are detailed in Table 5.

[0100] Its agglutination rate (%): The absorbance of the suspension of YYS-J2 and Streptococcus mutans was adjusted to 0.6±0.1 (A0) at a wavelength of 600 nm using sterile PBS to obtain a mixed suspension (the ratio of the two bacteria is 1:1). After standing for different time periods (2, 4, 24) h, the absorbance value Ax (x=2, 4, 24) was measured to obtain the autoagglutination rate. The results are detailed in Table 5.

[0101] The formula for calculating cohesive force, or agglomeration rate, is as follows:

[0102] Cohesive strength R / % = (1-A) x / A0)×100%;

[0103] Where A0 represents the initial absorbance value, and Ax represents the absorbance value after X hours of processing.

[0104] Table 5. Determination of autoagglutination rate and agglutination rate against Streptococcus mutans of YYS-J2.

[0105]

[0106] The data in the table above shows that:

[0107] The autoagglutination rates of YYS-J2 at 2, 4, and 24 (h) were 9.75, 12.08, and 32.33 (%), respectively, which were relatively low. However, the agglutination rates with Streptococcus mutans were 37.91, 39.00, and 53.08 (%), respectively, which were significantly higher than the autoagglutination rate. This indicates that it has a good ability to agglutinate Streptococcus mutans, which will be beneficial for the removal of Streptococcus mutans.

[0108] Example 7: SOD production test of Pediococcus lactis YYS-J2

[0109] SOD detection was performed using a superoxide dismutase (SOD) assay kit (Nanjing Jiancheng). The specific testing procedure is as follows:

[0110] The supernatant was obtained by centrifuging the YYS-J2 fermentation broth. The supernatant was mixed with water at a volume ratio of 2:3 to prepare the sample. The reaction system was prepared according to the instructions of the kit (see Table 6 for details). The mixture was thoroughly mixed and incubated at 37°C for 20 minutes. The OD value was read by a microplate reader at 450 nm. The amount of enzyme corresponding to a 50% SOD inhibition rate in the reaction system in this study is one unit of SOD activity (U).

[0111] Among them, the fermentation broth of YYS-J2 is the fermentation broth in Example 3;

[0112] The formulas for calculating SOD activity and inhibition rate are as follows:

[0113]

[0114]

[0115] In the formula, A refers to the OD value.

[0116] Table 6: Enzyme Reaction System Operation Table in the Instructions

[0117]

[0118] The test results showed that the SOD enzyme activity in the fermentation supernatant of YYS-J2 was 51.98±0.49 U / mL, indicating that YYS-J2 has a good SOD production capacity.

[0119] Example 8: Survival analysis of *Pediococcus lactis* YYS-J2 in a simulated gastric juice environment.

[0120] (1) The survival rate test process is as follows:

[0121] Collect the YYS-J2 bacteria fermented for 24 hours, centrifuge at 12000 r / min for 5 min to collect the cells, add an equal volume of physiological saline (0.85%) and mix well for later use; prepare artificial gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3 and 3 g / L pepsin), adjust the pH to 2.0, 2.5 and 3.0, filter through a 0.22 μM microporous membrane and set aside; take 1 mL of the treated sample and add it to 9 mL of artificial gastric fluid with a pH of 2.75, incubate at 37℃, and take 0.9 mL of untreated (0 h) and treated samples for (1, 2, 3, 5) h each time, add 0.1 mL of the solution. PI was stained at 37℃ for 10 min, and 0.1 ml was taken into 0.9 mL of ultrapure water. The total bacterial count P1 / % and the number of dead bacteria P2 / % were detected by flow cytometry. The survival rate / % at different time points was calculated based on this. The survival rate of the untreated bacteria was used as a 100% control. The gastrointestinal tolerance of each treatment was calculated.

[0122] The formula for calculating bacterial survival rate at different treatment times is as follows:

[0123] Survival rate / % = [(P1) 处理组 - P2 处理组 ) / P1 处理组 ] / [ (P1 对照 - P2 对照 ) / P1 对照 ].

[0124] (2) The survival rate of bacteria YYS-J2 in different gastric juice environments is shown in Table 7 below:

[0125] Table 7 Survival rate of YYS-J2 in an artificial gastric fluid environment (%)

[0126]

[0127] According to the data, when the bacteria YYS-J2 were treated in a simulated gastric juice environment with a pH of 2.5 for 1-2 hours, the survival rate of YYS-J2 was 21.09-45.36%.

[0128] When treated in a simulated gastric fluid environment with a pH of 3.0 for 1-5 hours, the survival rate of YYS-J2 was 89.52-97.71%, indicating that YYS-J2 has good tolerance in the artificial simulated gastric fluid environment, which provides a good basis for its degradation of acrylamide in the gastric fluid environment.

[0129] In this context, P1 treatment group refers to the total number of bacterial particles in the treatment group, and P2 treatment group refers to the number of dead bacterial particles in the treatment group. P1 control refers to the total number of bacterial particles in the control group, and P2 control refers to the number of dead bacterial particles in the control group.

[0130] Example 9: Survival analysis of Pediococcus lactis YYS-J2 in a simulated artificial pancreatic juice environment

[0131] (1) Take the YYS-J2 bacteria fermented for 24 h, centrifuge at 12000 r / min for 5 min to collect the bacterial cells, add the same volume of physiological saline (0.85%) and mix well for later use; prepare protein pancreatic juice (0.1% pancreatin w / v, 0.15% bovine bile), adjust the pH to 7.5 and 8.0 respectively, filter through a 0.22 μM microporous membrane for later use, take 1 mL of the treated bacterial solution into 9 mL of protein pancreatic juice at different pH values, and incubate at 37℃. Take samples at 3 and 6 (h) of treatment, take 0.9 mL each time, add 0.1 mL of PI dilution solution, stain at 37℃ for 10 min, and detect the total bacterial count P1 / % and mortality P2 / % by flow cytometry, and calculate the survival rate / % at different time periods. The number of untreated survivors is used as 100% control to calculate the bacterial survival rate.

[0132] The formula for calculating bacterial survival rate is:

[0133] Survival rate / % = [(P1) 处理组 - P2 处理组 ) / P1 处理组 ] / [ (P1 对照 - P2 对照 ) / P1 对照 ].

[0134] In this context, P1 treatment group refers to the total number of bacterial particles in the treatment group, and P2 treatment group refers to the number of dead bacterial particles in the treatment group. P1 control refers to the total number of bacterial particles in the control group, and P2 control refers to the number of dead bacterial particles in the control group.

[0135] (2) The bacterial survival rate of strain YYS-J2 is shown in Table 8:

[0136] Table 8 Survival rate of YYS-J2 in artificial simulated pancreatic juice environment / %

[0137]

[0138] According to the data, the survival rate of strain YYS-J2 was 91.29% and 43.99% when treated in pancreatic juice environment at pH 7.5 for 3 and 6 (h), respectively, and the survival rate was 65.33% and 44.52% when treated in pancreatic juice environment at pH 8.0, respectively, indicating that it has good pancreatic juice tolerance.

[0139] This invention also provides the following application examples of Pediococcus lactis YYS-J2:

[0140] Example 12 Preparation of probiotic agent from Pediococcus lactis YYS-J2

[0141] Pediococcus lactis YYS-J2 is inoculated into a culture medium (e.g., MRS medium) and cultured at 0-38°C for more than 15 hours. The bacterial cells are collected by centrifugation and resuspended in, for example, physiological saline or PBS buffer to prepare a liquid bacterial agent containing Pediococcus lactis YYS-J2. Optionally, Pediococcus lactis YYS-J2 bacterial cells are resuspended in a cell protectant and a carrier, and then freeze-dried to obtain a solid bacterial powder preparation containing Pediococcus lactis YYS-J2.

[0142] Optionally, Pediococcus lactis YYS-J2 can be used as a raw material component in the degradation or adsorption of acrylamide, reduction of L-phenylalanine, high production of phenyllactic acid, oral antibacterial products, and SOD production products. Pediococcus lactis YYS-J2 can exist in the products in liquid or solid formulation form.

[0143] Example 13: Preparation of fermented food from Pyrococcus lactis YYS-J2

[0144] To prepare a fermentation broth of Pseudococcus lactis YYS-J2, various fruits, Chinese herbal medicines, grains, and various sugars are used as auxiliary materials. Pseudococcus lactis YYS-J2 is inoculated and fermented for a certain period of time under certain temperature conditions (30-38℃) to prepare a fermented product. The fermented product is either inactivated or not inactivated. After dilution of the original liquid or in different proportions, common beverage auxiliary materials are added to prepare a fermented food.

[0145] Based on the results of the above embodiments, the Lactococcus lactis YYS-J2 provided by the present invention has the following properties and effects:

[0146] It can utilize carbon sources such as cellobiose and salicin, but cannot utilize sugar water compounds such as maltose, sucrose, raffinose, lactose, aescin, mannitol, salicin, sorbitol, inulin, and sodium hippurate. *Pediococcus lactis* YYS-J2 can degrade or adsorb acrylamide, with a removal rate of up to 70.41%, and can degrade or adsorb acrylamide in media such as French fries, with a removal rate as high as 78.30%. It can efficiently utilize L-phenylalanine, with a utilization rate of 79.49-93.35% for L-phenylalanine carried by *MRS*, and can completely absorb and utilize exogenous L-phenylalanine at concentrations of 0.5 g / L and 1 g / L. It can produce high levels of phenyllactic acid; in a culture medium containing 1.0 g / L phenylalanine, the yield of phenyllactic acid reaches as high as 741.12 mg / L. It is effective against *Streptococcus mutans* (…). Streptococcus mutans

[0147] ATCC 25175 exhibits excellent antibacterial activity, with an inhibition zone diameter as high as 35.00 mm. It also shows good agglutination activity against this bacterium, with an agglutination rate of 53.08% after 24 hours. It can produce SOD, with an enzyme activity of 51.98 ± 0.49 U / mL. Bacterium YYS-J2 survived for 1-2 hours in a gastric juice environment at pH 2.5 with a survival rate of 21.09-45.36%, and could survive for at least 5 hours in an artificially simulated gastric juice environment at pH 3.0, with a survival rate between 89.52-97.71%. Bacterium YYS-J2 survived for 3 and 6 hours in a pancreatic juice environment at pH 7.5 with survival rates of 91.29% and 43.99%, respectively, and for 3 and 6 hours in a pancreatic juice environment at pH 8.0 with survival rates of 65.33% and 44.52%, respectively. YYS-J2 bacteria, isolated from everyday foods, is highly safe for consumption. It can be used as an ingredient in functional products, possessing functions such as acrylamide degradation or adsorption, L-phenylalanine reduction, high phenyllactic acid production, oral antibacterial properties, and SOD production, showing broad application prospects. In summary, compared with existing technologies, the Lactococcus lactis YYS-J2 provided by this invention has the following beneficial effects:

[0148] This *Pediococcus lactis* YYS-J2 strain can provide a new probiotic source for the development of functional products such as acrylamide detoxification products, antibacterial products, and SOD-producing products, and has significant application value. For example:

[0149] (1) Lactococcus lactis YYS-J2 can be used as a raw material component of the composition to prepare a composition with the above-mentioned functions.

[0150] The bacterial strain present in the composition includes, but is not limited to, one or more combinations of the following: non-inactivated *Pediococcus lactis* YYS-J2, inactivated *Pediococcus lactis* YYS-J2, metabolites of *Pediococcus lactis* YYS-J2 strain, and freeze-dried *Pediococcus lactis* YYS-J2 strain. Preferably, in the composition, the number of *Pediococcus lactis* YYS-J2 is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. More preferably, the number of *Pediococcus lactis* YYS-J2 is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0151] (2) Various plants (such as fruits, Chinese herbal medicines, grains, etc.) can be used as raw materials, combined with various ingredients, and inoculated with Pyrococcus lactis YYS-J2 for fermentation treatment to prepare fermented products. These fermented products can be applied to products with functions such as degrading or adsorbing acrylamide, reducing L-phenylalanine, producing high levels of phenyllactic acid, oral antibacterial, and producing SOD.

[0152] The fermentation raw materials can be various commonly used plant fermentation raw materials, including but not limited to the fermentation raw material selection in the above scheme.

[0153] In summary, based on its characteristics, *Pediococcus lactis* YYS-J2 and / or its ferments can be used in functional products that include at least one of the following functions:

[0154] (1) Degradation or adsorption of acrylamide;

[0155] (2) Produces SOD;

[0156] (3) Utilize or metabolize L-phenylalanine;

[0157] (4) Produces phenyllactic acid;

[0158] (5) It has antibacterial ability against Streptococcus mutans and copolymerization ability against Streptococcus mutans.

[0159] Among them, products with the above (1)-(5) functions include, but are not limited to, detoxification products, SOD production products and antibacterial products (such as oral antibacterial agents, etc.), which have the effects of detoxification, SOD production and antibacterial; they can also have other obvious effects of inhibiting the occurrence or development of diseases based on the correlation between the effects of (1)-(5) and the occurrence or development of diseases, including but not limited to the effects of detoxification, SOD production and antibacterial.

[0160] For example, acrylamide has potential carcinogenicity, neurotoxicity, genotoxicity, and reproductive toxicity. Based on the ability of bacterium YYS-J2 to reduce or adsorb acrylamide levels, its application in functional products can not only produce detoxification effects but is also expected to have potential cancer risk prevention effects. Similarly, phenyllactic acid not only has antibacterial functions but also pharmacological effects similar to tanshinone, such as antiplatelet aggregation activity and regulation of human steroids. Based on the principle that bacterium YYS-J2 has a highly efficient phenyllactic acid production mechanism, its application in functional products can produce antibacterial effects and is also expected to have antiplatelet aggregation activity and steroid regulation effects. Furthermore, L-phenylalanine accumulation in the body can cause damage to the brain and nervous system. Based on the ability of bacterium YYS-J2 to efficiently utilize L-phenylalanine, its application in functional products is expected to produce potential effects such as preventing damage to the brain and nervous system.

[0161] It should be noted that:

[0162] (1) Definition:

[0163] The term "food" as used herein is used in a broad sense, encompassing both human food and drink. In some embodiments, the food product is suitable for and designed for human consumption.

[0164] The presence of *Pediococcus lactis* YYS-J2 in the composition includes, but is not limited to, non-inactivated bacteria, inactivated bacteria, metabolites, lyophilized strains, etc. It is anticipated that *Pediococcus lactis* YYS-J2 may also exist in the composition in other forms.

[0165] (2) The relevant prior art means or prior art terms involved in this application:

[0166] "SOD" is short for superoxide dismutase.

[0167] In this article, "has the ability to coagulate against Streptococcus mutans" refers to the fact that strain YYS-J2 has an agglutinating effect on Streptococcus mutans.

[0168] "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.

[0169] 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.

[0170] The biochemical experiments of lactic acid bacteria were conducted according to the standard method of GB4789.35. This is the existing technology, and its principle and method will not be described here.

[0171] The Oxford cup method is a method for determining antibiotic potency. It can generally be divided into two-dose method and three-dose method. This is an existing technology, and its principle and method will not be elaborated here.

[0172] (3) The formulations of the culture media used in the examples are as follows:

[0173] MRS medium (g / L): Casein peptone 10, beef extract 10, yeast extract 5, glucose 5, sodium acetate 5, K2HPO4 2, diammonium citrate 2, MgSO4·7H2O 0.2, MnSO4·H2O 0.05, Tween 80 1; pH 6.2. For solid medium, add 2% agar and 2% CaCO3 to the above and sterilize at 121℃ for 15 min.

[0174] 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.

[0175] 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.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 lactic acid cocci ( Pediococcus acidilactici YYS-J2, characterized in that: Its accession number is CGMCC No.28183.

2. A composition, characterized in that: Its components include the *Pediococcus lactis* YYS-J2 as described in claim 1.

3. The composition according to claim 2, characterized in that: The composition comprises one or more combinations of Pediococcus lactis YYS-J2 non-inactivated strain and Pediococcus lactis YYS-J2 freeze-dried strain.

4. The application of *Pediococcus lactis* YYS-J2 in the preparation of functional products, characterized by: The *Pediococcus lactis* YYS-J2 strain described in claim 1 is used; the functional product includes at least one of the following functions: (1) Degradation of acrylamide; (2) Produces SOD; (3) Utilizing L-phenylalanine; (4) Produces phenyllactic acid; (5) It has antibacterial ability against Streptococcus mutans and copolymerization ability against Streptococcus mutans.

5. The application according to claim 4, characterized in that: The functional products include products that inhibit Streptococcus mutans, products that degrade acrylamide, and products that produce SOD.

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