A kind of Pediococcus acidilactici YYS-CA3 with benzopyrene degradation and antibacterial effect and its application

Abstract

The invention relates to the technical field of microorganisms, and provides a lactic acid Pediococcus acidilactici YYS-CA3 having benzopyrene degradation and antibacterial effects and an application thereof; the preservation number of the lactic acid Pediococcus acidilactici YYS-CA3 is CGMCC No. 28612. The lactic acid Pediococcus acidilactici YYS-CA3 has good tolerance in artificial gastric juice and artificial intestinal juice, has excellent benzopyrene removal ability, has good antibacterial ability to some fungi (Candida albicans) and bacteria (Staphylococcus aureus, etc.), has good self-aggregation and other aggregation, has certain biofilm formation ability and hydrophobic interaction force, and can provide a new probiotic source for the development of functional products such as benzopyrene removal and antibacterial, and has good application value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Pediococcus acidilactici YYS-CA3 with benzopyrene degradation and antibacterial effects and applications thereof. Background Art

[0002] Benzopyrene is a polycyclic aromatic organic compound containing a benzene ring, and is listed as a Class I carcinogen by the International Agency for Research on Cancer under the World Health Organization. Benzopyrene mainly comes from coal tar, soot, tar, asphalt, and cigarette smoke; when cooking oil is heated to 270°C, compounds such as benzopyrene will be produced, so fried, stir-fried, and baked foods are common sources of benzopyrene. After entering the human body, benzopyrene will be converted into dozens of metabolites by enzymes in liver and lung cells until a substance called BPDE is formed. Once this substance binds to DNA, it will cause DNA mutations. Animal experiments have shown that long-term inhalation or consumption of foods containing high levels of benzopyrene can easily induce lung cancer, liver cancer, gastrointestinal cancer, etc. Since fried, stir-fried, and baked foods are delicious, it is extremely unrealistic to reduce the harm of benzopyrene by reducing or not eating benzopyrene.

[0003] Existing studies have found that microorganisms can use benzopyrene as a carbon source and degrade it directly or through co-metabolism with other organic matter. Therefore, it is extremely important to find probiotics that can efficiently degrade benzopyrene.

[0004] Candida albicans, Staphylococcus aureus and Escherichia coli are common pathogens in life. Candida albicans ) is a fungus that usually exists in the normal human mouth, upper respiratory tract, intestines and vagina. In the normal body, the number of Candida albicans is small and generally does not cause disease. However, when the body's immune function or general defense is reduced, or the mutual restraint of normal flora is out of balance, Candida albicans may multiply in large numbers and change its growth form (budding hyphae phase) to invade cells, thereby causing disease.

[0005] Staphylococcus aureus is a common bacterium that is widely distributed in nature and the human environment. This bacterium sometimes causes infections, including skin infections, pneumonia, food poisoning, pericarditis, etc.

[0006] Escherichia coli is a common intestinal bacterium that usually coexists with the host in the intestines, but can cause infection in some cases. It is usually associated with acute gastroenteritis and food poisoning, and sometimes leads to serious complications such as hemolytic uremic syndrome and thrombosis. Eating some antibacterial probiotics in daily life is helpful to reduce the harm of such pathogens to the human body and reduce the use of antibiotics, which is extremely beneficial to the body.

[0007] Based on this, there are some reports that publicly show that lactic acid bacteria have a certain ability to degrade benzopyrene; however, probiotics that have both high efficiency in degrading benzopyrene and can inhibit a variety of pathogens have not yet been developed. How to develop a probiotic that can efficiently degrade benzopyrene and inhibit a variety of pathogens is exactly the problem that this field is committed to solving. Summary of the invention

[0008] In order to solve the deficiencies of the prior art mentioned in the above background technology, the present invention provides a Pediococcus acidilactici YYS-CA3, and its technical solution is as follows:

[0009] Pediococcus acidilactici ( Pediococcus acidilactici ) YYS-CA3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 12, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 28612.

[0010] The present invention also provides a composition, the components of which include the Pediococcus acidilactici YYS-CA3 as described above.

[0011] In one embodiment, the composition includes one of a microbial preparation, a food, and a health product.

[0012] In one embodiment, in the composition, the number of the lactic acid bacteria YYS-CA3 is ≥ 1×10 6 CFU / mL or ≥1×10 6 In one embodiment, in the composition, the number of the lactic acid bacteria YYS-CA3 is ≥ 1×10 8 CFU / mL or ≥1×10 8 CFU / g.

[0013] In one embodiment, the composition comprises one or more combinations of non-inactivated bacteria of Pediococcus acidilactici YYS-CA3, inactivated bacteria of Pediococcus acidilactici YYS-CA3, metabolites of Pediococcus acidilactici YYS-CA3 strain, and freeze-dried strain of Pediococcus acidilactici YYS-CA3.

[0014] The present invention also provides a fermented product, which is obtained by fermenting the Pediococcus acidilactici YYS-CA3 as described above.

[0015] The present invention also provides the use of the above-mentioned Pediococcus acidilactici YYS-CA3 and / or its fermentation product in the preparation of functional products.

[0016] In one embodiment, the functional products include food, health products, and toiletries.

[0017] In one embodiment, the Pediococcus acidilactici YYS-CA3 and / or its fermentation product in the functional product has at least one of the following effects:

[0018] (1) Degradation of benzopyrene;

[0019] (2) Inhibit Candida albicans fungi;

[0020] (3) Inhibit Staphylococcus aureus and Escherichia coli;

[0021] (4) It has both self-cohesion and other-cohesion;

[0022] (5) Ability to form biofilm;

[0023] (6) Has hydrophobic force

[0024] In one embodiment, the functional products include benzopyrene detoxification products, fungi and bacteria antibacterial products.

[0025] The present invention also provides use of the above-mentioned Pediococcus acidilactici YYS-CA3 or the above-mentioned composition in the preparation of fermented food.

[0026] In one embodiment, Pediococcus acidilactici YYS-CA3 is used as a probiotic to ferment fruits and Chinese herbs to prepare fermented food.

[0027] Based on the above, compared with the prior art, the lactic acid bacteria YYS-CA3 provided by the present invention has the following beneficial effects:

[0028] The lactic acid Pediococcus YYS-CA3 provided by the present invention has good tolerance in artificial gastric juice and artificial intestinal juice, can efficiently degrade benzopyrene, and has antibacterial ability against fungi Candida albicans, bacteria Staphylococcus aureus and Escherichia coli. The lactic acid Pediococcus YYS-CA3 can provide a new probiotic source for the development of functional products such as benzopyrene detoxification products and antibacterial products, and has important application value.

[0029] Other features and beneficial effects of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other beneficial effects of the present invention can be realized and obtained by the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. The positional relationships described in the drawings in the following description are based on the directions in which the components are drawn in the diagrams, unless otherwise specified.

[0031] Figure 1 This is a colony morphology diagram of Pediococcus acidilactici YYS-CA3 provided by the present invention.

[0032] Figure 2 This is the Gram staining diagram of Pediococcus acidilactici YYS-CA3 provided by the present invention.

[0033] Figure 3 This is a scanning electron microscope image of Pediococcus acidilactici YYS-CA3 provided by the present invention.

[0034] Figure 4 The present invention provides an agarose electrophoresis diagram of amplified 16S r DNA target fragment of Pediococcus acidilactici YYS-CA3.

[0035] Figure 5 The present invention provides a phylogenetic tree of the 16S rDNA gene of Pediococcus acidilactici YYS-CA3. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; the technical features designed in different implementation modes of the present invention described below can be combined with each other as long as they do not conflict with each other; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] In the description of the present invention, it should be noted that all terms (including technical terms and scientific terms) used in the present invention have the same meanings as those generally understood by ordinary technicians in the field to which the present invention belongs, and should not be understood as limitations on the present invention; it should be further understood that the terms used in the present invention should be understood to have the same meanings as these terms in the context of this specification and in the relevant fields, and should not be understood in an idealized or overly formal sense, unless explicitly defined in this invention.

[0038] The present invention also provides the following operation examples and embodiments:

[0039] The present invention provides Pediococcus acidilactici ( Pediococcus acidilactici )YYS-CA3 was isolated from the feces of a healthy child aged 36 months using anaerobic culture with MRS medium.

[0040] The present invention is to extract the bacteria from milk fan operation example is as follows:

[0041] Example 1 Screening and Isolation of Bacteria

[0042] Take 2g of feces from a healthy child aged 36 months, add 5ml of normal saline and mix well, then dilute to 10 -2 times, 10 -3 times, take 0.1 mL and spread it in MRS medium, culture it anaerobically at 37℃ for 48-72h, select several single colonies of suspected lactic acid bacteria, purify them three times, and then test the degradation effect of benzopyrene. Select the strain with good benzopyrene degradation effect, save it and name it YYS-CA3.

[0043] Example 2 Identification of bacterial species

[0044] 2.1 Morphological observation of YYS-CA3 bacteria

[0045] The colony morphology of YYS-CA3 is as follows Figure 1 As shown, the bacterial morphology is Figure 2 and 3 Its main morphological characteristics are as follows: on MRS medium, the colonies are round, convex, white and opaque; Gram staining is positive, the bacteria are spherical, and the diameter is 0.5-1μm.

[0046] 2.2 Analysis of carbon source utilization of YYS-CA3

[0047] The biochemical experiment of lactic acid bacteria was carried out according to the standard method of GB4789.35, specifically, the lactic acid bacteria basal medium was prepared with cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% (mass fraction w / v) sodium hippurate, and YYS-CA3 was inoculated at 1% (w / v);

[0048] Among them, 1% (w / v) sodium hippurate also needs to be added slowly along the wall of the test tube after the culture is completed. 0.2mL of ninhydrin solution (the dosage of 1% sodium hippurate is 2mL) should be added without shaking. Place it in a water bath at 36℃±1℃ for 10 minutes and then judge the results. The judgment results are detailed in Table 1.

[0049] Table 1 YYS-CA3 glycosylated compounds and main biochemical reactions

[0050] Sugar Name Cellobiose maltose Mannitol Salicin Sorbitol Interpretation of results + - - + - Sugar Name sucrose Raffinose Inulin lactose 1% Sodium Hippurate Interpretation of results - - - + -

[0051] Note: “+” indicates a positive test, and “-” indicates a negative test.

[0052] Conclusion: According to the results in Table 1, YYS-CA3 can only utilize cellobiose, salicin and lactose, but cannot utilize maltose, sucrose, raffinose, mannitol, salicin, sorbitol, inulin and sodium hippurate.

[0053] 2.3 Molecular biological identification of YYS-CA3

[0054] ① Extraction of YYS-CA3 bacterial genomic DNA: using TIANGEN's bacterial genomic DNA extraction kit;

[0055] ② PCR amplification of 16S rDNA sequence: Amplify the 16S rDNA gene sequence, the primers used are F 9-27: 5'-GAGTTT GAT CCT GGC TCA G-3'; R 1525-1542: 5'-AGA AAG GAG GTG ATC CAG CC-3'; PCR reaction system: 2×Mix 12.5 μL, primers and DNA 1 μL each, ddH 2 0 9.5μL.

[0056] PCR amplification program: 93℃ pre-denaturation for 4 min, then 94℃ denaturation for 30 s, 55℃ (16SrDNA), 72℃ extension for 90 s, a total of 30 cycles, and finally 72℃ full extension for 10 min, and stored at 4℃.

[0057] ③PCR product detection and sequencing analysis: Take 5ul of PCR product and separate and test it by gel electrophoresis in 1.0% agarose gel added with EB. The amplified 16SrDNA target fragment is 1511 bp long (see the agarose gel electrophoresis diagram of 16S rDNA target fragment amplification for details). Figure 4 ), where the 16SrDNA sequence of YYS-CA3 is as follows:

[0058]

[0059] ④ Phylogenetic analysis: Blast comparison analysis was performed on each 16S rRNA sequence in the NCBI data to obtain the sequence and Pediococcus acidilactici The sequence homology of the series of standard strains was greater than 99%, and the neighbor-joining method in MEGA 4 was used to construct a developmental tree (the results are shown in Figure 5 ).

[0060] Conclusion: Based on the morphological observation of YYS-CA3, HBI lactic acid bacteria biochemical identification, and homology analysis in the DNA phylogenetic tree, it was determined that YYS-CA3 was Pediococcus acidilactici ( Pediococcus acidilactici ) strains.

[0061] The performance characteristics of the Pediococcus acidilactici YYS-CA3 provided by the present invention are as follows:

[0062] Example 3 Removal effect of Pediococcus acidilactici YYS-CA3 on benzopyrene

[0063] The YYS-CA3 mother liquor of Pediococcus acidilactici was inoculated into MRS medium at 1% (w / v) and cultured anaerobically at 37°C for 24 hours to obtain YYS-CA3 fermentation liquid. An appropriate amount of benzopyrene was dissolved in dimethyl sulfoxide to prepare a 1 mg / ml benzopyrene mother liquor for later use.

[0064] Adsorption of benzopyrene by YYS-CA3:

[0065] Take 45 mL of YYS-CA3 fermentation broth and centrifuge at 4500 r / min for 15 min to obtain bacterial cells. Then, add physiological saline and use flow cytometry to adjust the total particle count of bacterial cells to (1.0±0.1)*10 9 cfu / mL, take 2 ml of the above bacterial suspension, add benzopyrene mother solution to the final concentration of benzopyrene is about 10μg / mL and 35μg / mL (calculated as treatment 1 and treatment 2), and use unsterile saline as control. After mixing, stand at 37℃ in the dark for 4h to adsorb, centrifuge and take the supernatant as the test sample for treatment 1 and treatment 2.

[0066] Furthermore, commercially available barbecued beef was taken, ground into powder, and mixed in a ratio of beef: YYS-CA3 bacterial suspension (w / v) = 1:2. 2 ml of each mixture was added with benzopyrene mother solution until the final benzopyrene concentration was about 10 μg / mL. After mixing, the mixture was allowed to stand at 37°C for 2 h, and the supernatant was taken by centrifugation to obtain the test sample of treatment 3. The control was an equal amount of uninfected beef and benzopyrene saline solution.

[0067] Take the supernatant sample of treatment 1-3 obtained by the above centrifugation (i.e., the sample to be tested in treatment 1-3), add 2 mL of chloroform, extract and shake for more than 10 min, let it stand overnight, take the lower organic phase and put it into a brown liquid phase bottle, perform liquid chromatography detection, and measure the removal rate of benzopyrene in treatment 1-3, see Table 2 for details.

[0068] Liquid chromatography detection conditions: The content of benzopyrene was detected by high performance liquid chromatography, with a SunFireC18 column (4.6×250 mm, 5 μm), a UV detection wavelength of 290 nm, a mobile phase of chromatographic grade pure ethanol, a column temperature of 39.9°C, a flow rate of 1 mL / min, and an injection volume of 20 μL. The elution time of benzopyrene was about 4.1 min. The calculation formula for the removal rate of benzopyrene is:

[0069] Removal rate of benzopyrene / % = (CK-X) / CK*100.

[0070] Wherein, CK represents the benzopyrene concentration measured for the blank control sample, and X represents the benzopyrene concentration measured for the treated sample.

[0071] Table 2 Removal effect of Pediococcus acidilactici YYS-CA3 on benzopyrene

[0072]

[0073] According to Table 2, YYS-CA3 has a good removal effect on benzopyrene. In the physiological saline medium, after adding YYS-CA3 for 4 hours, the concentration of benzopyrene was detected to be reduced from 12.18ug / mL and 34.11ug / mL in the control to 0.13ug / mL and 3.03ug / mL, respectively, with removal rates as high as 98.94% and 91.11%; in the physiological saline medium with barbecued beef added, the concentration of benzopyrene also decreased from 11.06ug / mL in the control to 1.30ug / mL, with a removal rate as high as 89.17%, indicating that YYS-CA3 has an excellent removal effect on benzopyrene.

[0074] Example 4 Analysis of biofilm formation ability of Pediococcus acidilactici YYS-CA3

[0075] Prepare MRS medium, add various oligosaccharides (fructose oligosaccharide, inulin) at 0.5 g / L, inoculate YYS-CA3 at 2% (w / v), ferment and culture at 37 °C for 24 h, use the corresponding medium as diluent, and use flow cytometry to adjust the number of bacterial particles in each fermentation liquid to (1.0 ± 0.1) * 10 8cfu / mL, 200ul of the adjusted fermentation dilution was taken and placed in a 96-well plate, cultured at 37℃ for 24h, then the culture medium was discarded, 0.2mL1% crystal violet was added for 30 min, rinsed with distilled water, and 0.2mL 95% ethanol was added to dissolve, and the absorbance of the obtained colored solution was measured at 590nm. Repeat the test 3 times and take the average value as OD X The value was calculated by taking the uninoculated culture medium as the control and taking the repeated value as the OD C The measurement results are shown in Table 3.

[0076] Table 3 Biofilm formation ability of Pediococcus acidilactici YYS-CA3

[0077]

[0078] When OD X :OD C When the ratio is greater than 1, it indicates that the bacteria have the ability to form biofilm. The larger the ratio, the stronger the ability. The results in Table 3 show that the OD of each treatment group X :OD C The values ​​were all greater than 1.5, indicating that YYS-CA3 had a certain ability to form biofilms, and the addition of oligofructose and inulin was beneficial to promoting its biofilm formation.

[0079] Example 5 Antibacterial ability of Pediococcus acidilactici YYS-CA3 against pathogenic bacteria

[0080] The fermentation broth of Pediococcus acidilactici YYS-CA3 (the fermentation broth is the YYS-CA3 fermentation broth used in Example 3) was taken and its activity against Candida albicans ( Canidia Albicans FSCC 129002), Staphylococcus aureus ( Staphylococcus Aureus ATCC25923) and Escherichia coli ( Escherichia coli ATCC35150), the specific data are shown in Table 4: Table 4 Antibacterial ability of Pediococcus acidilactici YYS-CA3 against pathogenic bacteria

[0081]

[0082] It can be seen that the diameter of the inhibition zone of YYS-CA3 against Candida albicans is 14.20mm, the diameter of the inhibition zone of Staphylococcus aureus is 13.00mm, and the diameter of the inhibition zone of Escherichia coli (also known as Escherichia coli) is 14.67mm, indicating that YYS-CA3 has a good antibacterial effect on the above pathogens.

[0083] Example 6 Test of the autoagglutination rate (%) and other agglutination rate (%) of Pediococcus acidilactici YYS-CA3

[0084] Preparation of YYS-CA3 and Staphylococcus aureus ( Staphylococcus Aureus ATCC25923) fermentation broth, take the fermentation broth of the adapted bacteria and centrifuge it at 4500r / min and 4℃ for 10 min, collect the bacterial sludge, wash the bacterial sludge twice with sterile phosphate buffer (PBS) at pH=6.8 (i.e. add PBS to the colony, shake and mix evenly, centrifuge it at 4500r / min and 4℃ for 10 min), collect the bacteria. Then use PBS to prepare the absorbance at a wavelength of 600nm to be 0.6±0.1 (A 0 ) of bacterial suspensions and bacterial suspensions.

[0085] Self-agglutination rate (%): The YYS-CA3 bacterial slurry was made into a slurry with sterile PBS and the absorbance at a wavelength of 600 nm was 0.6±0.1 (A 0 ) were placed for (2, 4, 24) h before the absorbance value Ax (x=2, 4, 24) was measured to obtain the autoagglutination rate. The results are shown in Table 5.

[0086] Agglutination rate (%): The YYS-CA3 and Staphylococcus aureus suspensions were adjusted to an absorbance of 0.6±0.1 at a wavelength of 600 nm using sterile PBS, and then the two suspensions were mixed in a ratio of 1:1 (v / v) to obtain a mixed suspension (A 0 ), after standing for different time periods (2, 4, 24) h, the absorbance value Ax (x=2, 4, 24) was measured to obtain its agglutination rate. The results are detailed in Table 5.

[0087] The calculation formula of cohesion, i.e. agglutination rate, is:

[0088] Cohesion R / %=(1-A x / A 0 )×100.

[0089] Among them, A 0 represents the initial absorbance value, and Ax represents the absorbance value after treatment for X hours.

[0090] Table 5 Determination of the self-agglutination rate and agglutination rate of YYS-CA3 against Staphylococcus aureus

[0091]

[0092] It can be seen that the self-agglutination rates of YYS-CA3 at 2, 4, and 24 (h) were 5.35%, 11.98%, and 74.46%, respectively. Its 24h self-agglutination rate was extremely high. Its agglutination rates with Staphylococcus aureus were 8.38%, 24.59%, and 36.77%, respectively. It has a certain agglutination force, indicating that it has a good ability to agglutinate Staphylococcus aureus, which will be beneficial to the removal of Staphylococcus aureus.

[0093] Example 7 Hydrophobic interaction test of Pediococcus acidilactici YYS-CA3

[0094] Take the lactic acid bacteria YYS-CA3 fermented for 24 hours, centrifuge to remove the supernatant, wash twice with PBS, and add PBS to adjust the OD 600nm The value was 0.4±0.05, that is, the YYS-CA3 bacterial suspension was obtained, and the actual OD 600nm The value is counted as A 0 ;

[0095] Take 3 mL of YYS-CA3 bacterial suspension, add 3 mL of xylene, mix thoroughly for 5 min, let stand for 1 h, take the lower aqueous phase, and measure OD 600nm The value is recorded as A t , the bacterial hydrophobic force was calculated, and the results are shown in Table 6. The calculation formula is:

[0096] Bacterial hydrophobic force / %=(A 0 -A T ) / A 0 *100.

[0097] Table 6 YYS-CA3 hydrophobic force test

[0098] project <![CDATA[A 0 ]]> <![CDATA[A t ]]> YYS-CA3 hydrophobic interaction <![CDATA[OD 600nm Value]]> 0.412±0.001 0.348±0.008 15.385±1.978

[0099] It can be seen that the calculated hydrophobic force of YYS-CA3 in xylene is 15.385%, indicating that YYS-CA3 has a certain hydrophobic force.

[0100] Example 8 Survival analysis of Pediococcus acidilactici YYS-CA3 in an artificial simulated gastric fluid environment

[0101] (1) The survival rate test process is as follows:

[0102] Take the YYS-CA3 strain that has been fermented for 15 hours, centrifuge at 12000 r / min for 5 minutes to collect the bacteria, add the same volume of physiological saline (0.85%) and mix well for later use;

[0103] Prepare artificial gastric juice (125 mM NaCl, 7 mM KCl, 45 mM NaHCO 3and 3g / L pepsin), adjust the pH to 2.0, 2.5, and 3.0, filter through a 0.22 μM microporous filter membrane and set aside; take 1 mL of the treated sample in 9 mL of artificial gastric juice with different pH values, and culture at 37°C. Take untreated (0h, i.e., control) and treated x (1, 2, 3, 5)h samples, take 0.9 mL each time, add 0.1mL PI, stain at 37°C for 10min, take 0.1ml in 0.9mL ultrapure water, and detect the bacterial viability rate P value by flow cytometry, with the control as P O , the processed sample is P X ;

[0104] The calculation formula of bacterial survival rate at different treatment times is:

[0105] Survival rate / %=P X / P 0 *100.

[0106] (2) The survival rates of strain YYS-CA3 in different gastric fluid environments are shown in Table 7 below.

[0107] Table 7 Survival rate of YYS-CA3 in artificial simulated gastric fluid environment (unit, %)

[0108]

[0109] According to the data:

[0110] When the bacteria YYS-CA3 was treated in a simulated gastric fluid environment with a pH value of 2.5 for 1-2 hours, the survival rate of YYS-CA3 was greater than 81%. When treated in a simulated gastric fluid environment with a pH value of 3.0 for 1-5 hours, the survival rate of YYS-CA3 was 96.58-99.38%, indicating that YYS-CA3 has good tolerance in the artificial simulated gastric fluid environment, which provides a good basis for its degradation of benzopyrene in the gastric fluid environment.

[0111] Example 9 Analysis of the viability of Pediococcus acidilactici YYS-CA3 in a simulated artificial pancreatic juice environment

[0112] (1) Take YYS-CA3 fermented for 15 h, centrifuge at 12000 r / min for 5 min to collect the cells, add the same volume of physiological saline (0.85%) and mix well for later use;

[0113] Prepare pancreatic protein juice (0.1% pancreatin w / v, 0.15% ox bile), adjust the pH to 7.5 and 8.0 respectively, filter through 0.22 μM microporous filter membrane and set aside, take 1 mL of treated bacterial liquid in 9 mL of pancreatic protein juice with different pH values, and culture at 37℃. Take untreated (0h, i.e. control) and treated x (3, 6)h samples, take 0.9 mL each time, add 0.1ml PI diluent, stain at 37℃ for 10min, and detect the bacterial viability rate P value by flow cytometry, with the control as P O , the processed sample is P X

[0114] The calculation formula of bacterial survival rate at different treatment times is:

[0115] Survival rate / %=P X / P 0 *100.

[0116] (2) The bacterial survival rate of strain YYS-CA3 is shown in Table 8. According to the data, it can be seen that:

[0117] Table 8 Survival rate of YYS-CA3 in artificial simulated pancreatic juice environment (unit, %)

[0118]

[0119] It can be seen that when the bacteria YYS-CA3 was treated in the pancreatic juice environment with a pH of 7.5 for 3 and 6 (h), the survival rates were 94.72% and 73.81%, and in the pancreatic juice environment with a pH of 8.0, the survival rates were 95.31% and 41.42%, indicating that it has good tolerance to pancreatic juice.

[0120] The present invention also provides the following application examples of Pediococcus acidilactici YYS-CA3:

[0121] Example 10 Preparation of probiotics using Pediococcus acidilactici YYS-CA3

[0122] Inoculate Pediococcus acidilactici YYS-CA3 in a culture medium, such as MRS culture medium, and culture at 0-38°C for more than 15 hours, collect bacterial cells by centrifugation, and resuspend them in, for example, physiological saline or PBS buffer to prepare a liquid bacterial preparation containing Pediococcus acidilactici YYS-CA3. Alternatively, resuspend the bacterial cells of Pediococcus acidilactici YYS-CA3 in a cell protectant and a carrier, and freeze-dry to obtain a solid bacterial powder preparation containing Pediococcus acidilactici YYS-CA3.

[0123] Optionally, Pediococcus acidilactici YYS-CA3 can be used as a raw material component in antibacterial products (such as food and health products) that degrade benzopyrene, fungi and bacteria, and Pediococcus acidilactici YYS-CA3 can be present in the product in the form of a liquid or solid preparation.

[0124] Example 11 Preparation of fermented food using Pediococcus acidilactici YYS-CA3

[0125] The fermented product is prepared by inoculating the fermented product with various fruits, Chinese herbal medicines, cereal raw materials and various sugars as auxiliary materials, and fermenting the product under certain temperature conditions (30-38°C) for a certain period of time to prepare a fermented product. The fermented product is inactivated or not inactivated, and after diluting the original solution or in different proportions, common beverage auxiliary materials are added to prepare a fermented food.

[0126] According to the results of the above examples, the lactic acid bacteria YYS-CA3 provided by the present invention has the following properties and effects:

[0127] a. It can utilize carbon sources such as cellobiose, salicin and lactose, but cannot utilize carbohydrates such as maltose, sucrose, raffinose, mannitol, salicin, sorbitol, inulin and sodium hippurate;

[0128] b. It can degrade or adsorb benzopyrene. In saline medium, the removal rate of benzopyrene is as high as 98.94%. It can degrade or adsorb benzopyrene in roast beef medium, and the removal rate of benzopyrene is as high as 89.17%.

[0129] c. It has a certain ability to form biofilm. Adding oligofructose and inulin to MRS can significantly promote the formation of biofilm;

[0130] d. It has antibacterial effect on both fungi and bacteria, especially on harmful bacteria Candida albicans ( Canidia Albicans FSCC 129002 ) The diameter of the inhibition zone is 14.2 mm, which is effective against the harmful bacteria Staphylococcus aureus ( Staphylococcus Aureus ATCC25923 ) and Escherichia coli (Escherichia coli ATCC35150) The diameters of the inhibition zones were 13.00 mm and 14.67 mm, respectively;

[0131] e. It has good self-aggregation ability (the self-aggregation rate in 24 hours is 74.46%), and has certain alloaggregation ability to Staphylococcus aureus (the alloaggregation rate in 24 hours is 36.77%).

[0132] f. It has a certain hydrophobic force, and the hydrophobicity rate using xylene as the organic phase is 15.385%;

[0133] b. They can survive for 1-2 hours in a gastric fluid environment with a pH of 2.5, with a survival rate of more than 80%. They can survive for at least 5 hours in an artificial simulated gastric fluid environment with a pH of 3.0, with a survival rate between 96.58-99.38%;

[0134] c. The survival rates were 94.72% and 73.81% after being treated in a pancreatic juice environment with a pH of 7.5 for 3 and 6 hours, and 95.31% and 41.42% after being treated in a pancreatic juice environment with a pH of 8.0 for 3 and 6 hours;

[0135] j. Bacteria YYS-CA3 was isolated from the feces of healthy children aged 36 months. It is a natural beneficial bacterium with high food safety. It can be used in functional products that adsorb or degrade benzopyrene and inhibit harmful fungi and bacteria, and has good application prospects.

[0136] In summary, compared with the prior art, the Pediococcus acidilactici YYS-CA3 provided by the present invention has the following beneficial effects:

[0137] The lactic acid Pediococcus YYS-CA3 can provide a new probiotic source for the development of functional products such as benzopyrene detoxification products and antibacterial products, and has important application value, for example:

[0138] (1) Pediococcus acidilactici YYS-CA3 can be used as a raw material component of a composition to prepare a composition having the above functions; wherein the composition includes but is not limited to microbial preparations, foods, health products, etc.

[0139] The bacterial strains present in the composition include, but are not limited to, one or more combinations of non-inactivated strains of Pediococcus acidilactici YYS-CA3, inactivated strains of Pediococcus acidilactici YYS-CA3, metabolites of Pediococcus acidilactici YYS-CA3 strains, and freeze-dried strains of Pediococcus acidilactici YYS-CA3. Preferably, in the composition, the number of Pediococcus acidilactici YYS-CA3 is ≥ 1×10 6 CFU / mL or ≥1×10 6 CFU / g. Further preferably, the number of the lactic acid bacteria YYS-CA3 is ≥1×10 8 CFU / mL or ≥1×10 8 CFU / g.

[0140] (2) Various plants (such as fruits, Chinese herbal medicines, grains, etc.) can be used as raw materials, combined with various ingredients, inoculated with Pediococcus acidilactici YYS-CA3 for fermentation to prepare fermented products, which can be used to prepare products with benzopyrene detoxification, antibacterial and other functions;

[0141] The fermentation raw materials can be various types of conventional plant fermentation raw materials, and the auxiliary materials can also be conventional auxiliary materials, including but not limited to the above-mentioned schemes. The fermented product is not limited to being used for preparing fermented food, but can also be a health product and other products;

[0142] In summary, Pediococcus acidilactici YYS-CA3 and / or its fermentation products include at least one of the following functional effects, and based on the above effects, can be applied to functional products with related functions:

[0143] (1) Degradation of benzopyrene;

[0144] (2) Inhibit Candida albicans fungi;

[0145] (3) Inhibit Staphylococcus aureus and Escherichia coli;

[0146] (4) It has high self-cohesion and other-cohesion;

[0147] Among them, Pediococcus acidilactici YYS-CA3 and / or its fermented products have the above-mentioned (1)-(4) effects, and can be applied to functional products such as benzopyrene detoxification and antibacterial, and have obvious effects such as removing benzopyrene and inhibiting bacteria; they can also be applied to products with other obvious effects based on the effects of (1)-(4); wherein the functional products include but are not limited to food, health care products, etc.

[0148] In addition, Pediococcus acidilactici YYS-CA3 also has: (5) biofilm formation ability, which can promote its biofilm formation; (6) a certain hydrophobic interaction;

[0149] The hydrophobic force of bacteria refers to the ability of the bacterial surface to resist the adsorption force caused by the viscosity of water molecules. This force is mainly controlled by the cell membrane structure, the surface characteristics of biomolecules and the chemical composition, and has an important influence on their vitality. Studies have found that the hydrophobicity of bacterial surfaces can promote their reproduction, protect them from damage by environmental pollutants, and adapt to environmental changes. Therefore, Pediococcus acidilactici YYS-CA3 has good hydrophobic force, which is beneficial to its application and has good reproduction ability and environmental adaptability.

[0150] Bacteria YYS-CA3 has the ability to form biofilms and can promote the formation of biofilms. Specifically, it has the following effects:

[0151] The biofilm has a barrier function: water-soluble substances on both sides of the membrane cannot pass freely, which can prevent antibiotics, antibodies, complement, immune cells, etc. from entering the membrane, thereby protecting bacteria from attack.

[0152] Biofilms are beneficial to nutrient acquisition: biofilms are semi-permeable and can selectively allow certain small molecules to pass through, thus facilitating bacteria to obtain nutrients.

[0153] Biomembrane substance transport function: Biomembrane has substance transport function, which enables substance exchange between cells and the surrounding environment. For example, cell membrane can realize substance exchange between cells and the surrounding environment through the transport function.

[0154] Biofilms protect bacteria from drug attacks: When bacteria form biofilms, their surface area increases, making it difficult for drugs to penetrate into the biofilm, thereby protecting the bacteria from drug attacks.

[0155] Therefore, the biofilm-forming ability of strain YYS-CA3 is beneficial to its application, and it can have good reproduction ability, survival ability and environmental adaptability.

[0156] It should be noted that:

[0157] (1) Definition:

[0158] The term "food" as used herein is broad and includes food and drink for humans. In certain embodiments, the food product is suitable for and designed for human consumption. The present application can be used to prepare solid preparations such as powders and tablets, and can also be dispersed in liquids to prepare liquid preparations and other preparations suitable for oral administration to humans, including but not limited to powders.

[0159] The composition includes but is not limited to microbial preparations, foods, and health products. The composition containing Pediococcus acidilactici YYS-CA3 can be used in other forms of products.

[0160] The presence of the Pediococcus acidilactici YYS-CA3 in the composition includes, but is not limited to, non-inactivated bacteria, inactivated bacteria, metabolites, freeze-dried strains, etc. It is expected that the Pediococcus acidilactici YYS-CA3 may also be present in the composition in other forms.

[0161] (2) Relevant prior art means or prior art terms involved in this application:

[0162] "OD" is the abbreviation of optical density, also known as absorbance. When light passes through the object, the energy difference before and after is the energy absorbed by the object. At a specific wavelength, there is a quantitative relationship between the concentration of the same object and the absorbed energy. This quantitative relationship can be used to determine the concentration of the object. x " is the optical density value measured when the wavelength is set to Xnm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate bacterial cell density. Among them, the "OD" value determination method is a prior art, and its principles and methods are not repeated here.

[0163] The use of flow cytometry to determine the total bacterial count P1 and the number of dead bacteria P2 is an existing technology, and its principles and methods will not be repeated here.

[0164] The biochemical experiment of lactic acid bacteria was carried out according to the standard method of GB4789.35, which is the existing technology, and its principle and method will not be repeated here.

[0165] The Oxford cup method is a method for determining the potency of antibiotics, which can generally be divided into a two-dose method and a three-dose method. This is an existing technology, and its principles and methods will not be repeated here.

[0166] (3) The formulas of the culture media used in the examples are as follows:

[0167] MRS medium (g / L): casein peptone 10, beef extract 10, yeast extract 5, glucose 5, sodium acetate 5, K 2 HPO 4 2. Diammonium citrate 2. MgSO 4 .7H 2 O 0.2, MnSO 4 .H 2 O 0.05, Tween 80 1; pH 6.2, solid medium plus 2% agar and 2% CaCO 3 , sterilize at 121℃ for 15min.

[0168] Unless otherwise specified, the experimental operation methods involved in the embodiments of the present invention are conventional experimental operation methods in the art, and the reagents or instruments involved can be purchased from regular channels.

[0169] In addition, those skilled in the art should understand that, although there are many problems in the prior art, each embodiment or technical solution of the present invention can be improved in only one or several aspects, without having to solve all the technical problems listed in the prior art or background technology at the same time. Those skilled in the art should understand that the content not mentioned in a claim should not be used as a limitation on the claim.

[0170] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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 Pediococcus acidilactici YYS-CA3, characterized in that: Its deposit number is CGMCC No.28612.

2. A composition, characterized in that: Its components include the Pediococcus acidilactici YYS-CA3 as claimed in claim 1.

3. The composition according to claim 2, characterized in that: The composition comprises one of a microbial preparation, a food and a health product.

4. The composition according to claim 2, characterized in that: The composition comprises one or more combinations of non-inactivated bacteria of Pediococcus acidilactici YYS-CA3, inactivated bacteria of Pediococcus acidilactici YYS-CA3, and freeze-dried strains of Pediococcus acidilactici YYS-CA3.

5. Application of Pediococcus acidilactici YYS-CA3 in the preparation of functional products, characterized in that: The Pediococcus acidilactici YYS-CA3 is the Pediococcus acidilactici YYS-CA3 as claimed in claim 1; The functional product is used for at least one of the following purposes: (1) Degradation of benzopyrene; (2) Inhibit Candida albicans fungi; (3) Inhibit Staphylococcus aureus and Escherichia coli.

6. The use according to claim 5, characterized in that: The functional products include food, health products, and toiletries.

7. The use according to claim 5, characterized in that: The functional products are benzopyrene detoxification products and antibacterial products.

Images