A kind of bacteria of genus Lactobacillus and its use

Abstract

The bacteria of the genus Lactobacillus provided by the invention include: Lactobacillus paracasei J3703, which has been deposited in the China General Microorganism Culture Collection Center with a deposit number of CGMCC No. 26348; and / or Lactobacillus pentosus K0802, which has been deposited in the China General Microorganism Culture Collection Center with a deposit number of CGMCC No. 26349. The invention finds through research that the bacteria of the genus Lactobacillus can efficiently degrade azo artificial pigments, with a decolorization rate of more than 90%, can be used to reduce the content of azo artificial pigments in the gastrointestinal tract of the human body, can be used as or prepare food or food additives or biological preparations, and has good application prospects in processing edible azo artificial pigments.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a bacterium of the genus Lactobacillus and a use thereof. Background Art

[0002] Edible coloring is often used as a food additive in food production and processing to increase visual appeal, thereby increasing the beauty of food and improving people's appetite. Edible azo artificial synthetic coloring is the most common type of food coloring, accounting for about 60% to 70% of synthetic coloring in the food industry. It is often used in dairy products, ice cream, soft drinks, flavors and many baked goods. Although edible azo artificial coloring has been widely used and is considered a safe food coloring, there are also some research reports on the potential health threats of edible azo artificial coloring. It is reported that there is an association between allura red and attention deficit hyperactivity disorder (ADHD). In addition, studies have found that allura red and sunset yellow, edible azo artificial colors widely used in food, can induce and accelerate the development of colitis.

[0003] There are a large number of microorganisms in the human intestine, and these intestinal microorganisms are closely related to human health. Studies have found that there are some active microorganisms in nature that can colonize in the human intestine and change the composition of the flora in a certain part of the human intestine, which are beneficial to human health. These microorganisms are collectively referred to as probiotics, such as lactobacilli. Lactobacilli are extremely widely distributed in nature, with rich species diversity, and most of them are essential flora for the human body and have important physiological functions. Lactobacilli can promote the effective conversion of nutrients such as protein, fat, and sugar in food into small molecule fragments, and promote the absorption of nutrients by the human body. In addition, lactobacilli can effectively inhibit harmful bacteria in the intestine to a certain extent, which is beneficial to improving intestinal flora and improving the body's immune function. Lactobacilli are gradually known to people because of their good health care functions, but there are no research reports on the degradation of edible azo artificial pigments by lactobacilli. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a bacterium of the genus Lactobacillus and a use thereof. The bacterium of the genus Lactobacillus can efficiently degrade azo artificial pigments and has good application prospects.

[0005] To this end, the present invention provides the following technical solutions:

[0006] A bacterium of the genus Lactobacillus, comprising:

[0007] Lactobacillus paracasei J3703 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit address at Institute of Biology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, with the deposit number CGMCC No.26348 and the deposit date December 27, 2022; and / or

[0008] Lactobacillus pentosus K0802 has been deposited in the General Microbiology Center of the China Culture Collection Administration, the deposit address is Institute of Biology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit number is CGMCC No.26349, and the deposit date is December 27, 2022.

[0009] Optionally, the Lactobacillus paracasei J3703 contains the nucleotide sequence shown in SEQ ID No. 1; and / or

[0010] The Lactobacillus pentosus K0802 contains the nucleotide sequence shown in SEQ ID No.2.

[0011] A microbial preparation contains the bacteria of the genus Lactobacillus or a culture thereof.

[0012] A method for preparing the microbial preparation comprises activating the bacteria of the genus Lactobacillus and enlarging the culture.

[0013] Optionally, the culture condition is anaerobic culture at 37±1°C for 24–48h.

[0014] The bacteria of the genus Lactobacillus, the microbial preparation or the microbial preparation prepared by the preparation method of the microbial preparation can be used to degrade azo artificial pigments.

[0015] Optionally, the azo artificial color is an edible azo artificial color;

[0016] Optionally, the edible azo artificial coloring material includes sunset yellow or allura red.

[0017] Optionally, the method has the purpose of reducing the content of azo artificial pigments in the human gastrointestinal tract.

[0018] A product for degrading azo artificial pigments comprises the bacteria of the genus Lactobacillus, the microbial preparation or the microbial preparation prepared by the preparation method of the microbial preparation.

[0019] Optionally, the product includes food, food additives or biological preparations.

[0020] A method for degrading azo artificial pigments comprises utilizing the bacteria of the genus Lactobacillus, the microbial preparation or the microbial preparation prepared by the preparation method of the microbial preparation.

[0021] Optionally, the method for degrading azo artificial pigments comprises the following steps: inoculating a bacterial solution of Lactobacillus bacteria into a culture medium containing azo artificial pigments for decolorization and degradation.

[0022] Optionally, the bacterial liquid of the Lactobacillus bacteria is prepared as follows: under sterile conditions, the Lactobacillus bacteria are inoculated into MRS liquid culture medium and anaerobically cultured at 37±1° C. for 24–48 hours.

[0023] Optionally, the decolorization conditions are: the pH of the culture medium is 6.0-7.0, the initial OD600 value of the bacterial solution is 0.3-0.8, the concentration of the azo artificial pigment is 0.06 g / L-0.3 g / L, and the culture is carried out anaerobicly at 37±1° C. for 48-72 h.

[0024] The technical solution of the present invention has the following advantages:

[0025] 1. The bacterium of the genus Lactobacillus provided by the present invention comprises: Lactobacillus paracasei J3703, which has been deposited in the China General Microorganism Culture Collection Center with a deposit number of CGMCC No.26348; and / or Lactobacillus pentosus K0802, which has been deposited in the China General Microorganism Culture Collection Center with a deposit number of CGMCC No.26349. The present invention has found that the bacterium of the genus Lactobacillus can efficiently degrade azo artificial pigments with a decolorization rate of more than 90%, can be used to reduce the content of azo artificial pigments in the human gastrointestinal tract, can be used as food or food additives or biological preparations, and has a good application prospect in processing edible azo artificial pigments.

[0026] The Lactobacillus paracasei J3703 provided by the present invention has been deposited in the General Microbiological Center of the China Microorganism Culture Collection Administration Committee, and has been deposited in the China General Microorganism Culture Collection Administration Center. The deposit address is the Institute of Biology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit number is CGMCC No. 26348, and the deposit date is December 27, 2022.

[0027] The Lactobacillus pentosus K0802 provided by the present invention has been deposited in the China General Microbiological Culture Collection Center and the General Microbiological Center of the China Microbiological Culture Collection Committee. The deposit address is Institute of Biology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit number is CGMCC No. 26349, and the deposit date is December 27, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 It is the full wavelength scanning diagram (temptation red) of the decolorized culture medium of different sampling points of Lactobacillus paracasei J3703 in Example 1;

[0030] Figure 2 It is a full wavelength scanning image (Allure Red) of the decolorized culture medium of Lactobacillus pentosus K0802 at different sampling points in Example 1;

[0031] Figure 3 This is a microscopic examination picture of Lactobacillus paracasei J3703;

[0032] Figure 4 This is a microscopic examination picture of Lactobacillus pentosus K0802;

[0033] Figure 5 is the phylogenetic tree of Lactobacillus paracasei J3703 in Example 1;

[0034] Figure 6 is the phylogenetic tree of Lactobacillus pentosus K0802 in Example 1;

[0035] Figure 7 It is the decolorization rate of different concentrations of allura red by Lactobacillus paracasei J3703 in Example 2;

[0036] Figure 8 is the decolorization rate of Lactobacillus pentosus K0802 to different concentrations of Allura Red in Example 2;

[0037] Fig. 9 The effect of different concentrations of allura red on the biomass of Lactobacillus paracasei J3703 in Example 3;

[0038] Fig.10The effect of different concentrations of Allura Red on the biomass of Lactobacillus pentosus K0802 in Example 3;

[0039] Fig.11 It is the full wavelength scanning diagram (sunset yellow) of the decolorized culture medium of different sampling points of Lactobacillus paracasei J3703 in Example 4;

[0040] Fig.12 This is a full wavelength scan of the decolorized culture medium of Lactobacillus pentosus K0802 at different sampling points in Example 4 (sunset yellow). DETAILED DESCRIPTION

[0041] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0042] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0043] The components of the MRS liquid culture medium in the present invention are: peptone 10.0g / L, yeast powder 5.0g / L, beef extract 10.0g / L, glucose 20.0g / L, Tween-80 1.0g / L, sodium acetate 5.0g / L, sodium citrate 2.0g / L, K2HPO4 2.0g / L, MgSO4·7H2O 0.2g / L, MnSO4·5H2O 0.054g / L, and the balance is water. Sterilize at 121°C for 15-20min before use and set aside. The difference between the MRS solid culture medium and the MRS liquid culture medium is that 15% agar is added to the formula.

[0044] The components of the decolorization medium are: tryptone 10.0 g / L, beef heart extract powder 17.5 g / L, glucose 2.0 g / L, NaCl 5.0 g / L, NaH2PO4 2.5 g / L, additional yeast powder 10.0 g / L, hemin chloride 0.001 g / L, L-cysteine ​​hydrochloride 0.6 g / L, NaCl 0.045 g / L, NaHCO3 0.4 g / L, K2HPO4 0.075 g / L, KH2PO4 0.177 g / L, (NH4)2SO4 0.45 g / L, MgSO4·7H2O 0.945 g / L, CaCl2 0.015 g / L.

[0045] The artificial edible colors Allura Red and Sunset Yellow are both commercially available products.

[0046] Example 1 Isolation, observation and identification of strains

[0047] 1. Isolation of strains

[0048] The bacteria of the genus Lactobacillus involved in the present invention are separated from traditional fermented foods in Yunnan. After collecting traditional fermented foods from various regions in Yunnan, 0.5g or 0.5mL of sample is added to 4.5mL of sterile physiological saline, and a 10-fold gradient dilution is performed. A suitable dilution is applied on an MRS solid culture medium, and anaerobically cultured at 37°C for 48h, and a single colony is picked for purification on the same MRS solid culture medium. After a single colony is grown, the specific morphology of each single colony is observed, and then Gram staining is performed, and the strain is observed under an electron microscope to ensure that the strain is a single strain, and the strain morphology is observed at the same time. The purified single colony is picked and added to an MRS liquid culture medium for enrichment culture, and a decolorization culture medium is added to a 20% (v / v) inoculum to screen the decolorization strain. By observing the color change of the decolorization culture medium and performing full wavelength scanning on the decolorization culture medium 0h without allure red, the decolorization culture medium containing allure red (the concentration of allure red is 0.1g / L) 0h, and the decolorization culture medium after 12h and 24h of culture, it is judged whether the strain can metabolize allure red. The full wavelength scanning images of the decolorized culture medium of the screened strain J3703 at different sampling points are shown in the figure below. Figure 1 As shown ( Figure 1 The line of J3703-12h overlaps with that of J3703-24h, so only three lines are seen in the figure). The full wavelength scanning images of the decolorized culture medium of the screened strain K0802 at different sampling points are shown in Figure 2 shown.

[0049] 2. Observation of strains and colony morphology

[0050] Strain J3703 is a rod-shaped, Gram-positive, facultative anaerobic bacterium. Its colonies are milky white, round, with neat edges, opaque, raised, and sticky. Strain K0802 is a rod-shaped, Gram-positive, facultative anaerobic bacterium. Its colonies are milky white, round, with neat edges, opaque, raised, and sticky. The microscopic examination of strain J3703 is shown below. Figure 3 As shown, the microscopic examination of strain K0802 is as follows Figure 4 shown.

[0051] 3. 16S rRNA Identification of Two Strains

[0052] The bacterial genomic DNA was extracted using a kit, and PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', see SEQ ID NO.3 in the sequence table) and 1492R (5'-GGTTACCTTGTTACGACTT-3', see SEQID NO.4 in the sequence table). The conditions for PCR amplification were: 94°C pre-denaturation for 5 min, 94°C denaturation for 1 min, 58°C annealing for 1 min, 72°C extension for 2 min, a total of 30 cycles, and finally 72°C end extension for 10 min. The PCR amplification system (total system 50 μL) was: 10×buffer 5 μL, dNTP 4 μL, 27F 1.5 μL, 1492R 1.5 μL, r-Taq 0.5 μL, double distilled water (Watsons) 35.5 μL, DNA template 2 μL. 16S rRNA was obtained and then sent for testing. The sequencing sequences are shown in SEQ ID No. 1 and SEQ ID No. 2. The sequences were compared by BLAST in NCBI, and it was found that the similarity between strain J3703 and Lactobacillus paracasei TMPC46K2A reached 98.53%; the relative degree between strain K0802 and Lactiplantibacillus pentosus HBUAS58338 reached 98.79%, so J3703 was identified as Lactobacillus paracasei and named as Lactobacillus paracasei J3703. The phylogenetic tree of the strain is shown in Figure 5 ; K0802 is Lactobacillus pentosus, and it is named Lactobacillus pentosus K0802. The phylogenetic tree of the bacterium is shown in Figure 6 The above two strains were deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and they are:

[0053] Lactobacillus paracasei J3703 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is Institute of Biology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit number is CGMCC No.26348, and the deposit date is December 27, 2022;

[0054] Lactobacillus pentosus K0802 has been deposited in the General Microbiology Center of the China Culture Collection Administration, the deposit address is Institute of Biology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit number is CGMCC No.26349, and the deposit date is December 27, 2022.

[0055] Example 2 Application of Lactobacillus paracasei J3703 and Lactobacillus pentosus K0802 in Degrading Edible Artificial Pigment Allura Red

[0056] Allura red was added to the decolorization medium to adjust the concentration of allura red to 0.06 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.2 mg / mL or 0.3 mg / mL. 150 mL of the above medium was added to a 250 mL conical flask and sterilized at 121°C for 20 min. Then, Lactobacillus paracasei J3703 or Lactobacillus pentosus K0802 was inoculated and the OD 600 The value was adjusted to 0.5, and then placed in an anaerobic incubator (37 ± 1 ° C) to monitor the change of the concentration of Allura Red over time. Take 5 mL of culture solution at 0h, 3h, 6h, 9h, 12h, 18h, 24h, 36h, 48h, 60h, and 72h, respectively, centrifuge at 10000rpm for 10min, take the supernatant, and use a spectrophotometer to measure its absorbance value at the characteristic absorption wavelength of Allura Red (504nm). The decolorization rate is calculated using the following formula:

[0057] Decolorization rate (%) = ((A0-A1) / A0) × 100%

[0058] In the formula, A0 represents the absorbance value of the culture medium containing allura red at 0 h, and A1 represents the absorbance value of the culture medium containing allura red after a period of culture.

[0059] The decolorization rate of Lactobacillus paracasei J3703 on different concentrations of allura red Figure 7 As shown in the figure, for low concentration (0.06-0.1 mg / mL) of Allura Red, the decolorization rate of Lactobacillus paracasei J3703 can reach about 97%, and the decolorization time is only 12 hours. For high concentration (0.2-0.3 mg / mL) of Allura Red, the decolorization rate of Lactobacillus paracasei J3703 can also reach about 90%.

[0060] The decolorization rate of Lactobacillus pentosus K0802 on different concentrations of allura red Figure 8 As shown, for low concentrations (0.06–0.1 mg / mL) of Allura red, the decolorization rate of Lactobacillus pentosus K0802 can reach about 95%.

[0061] Example 3

[0062] The difference between this embodiment and embodiment 2 is that after centrifugation at 10000 rpm for 10 min, the supernatant is discarded, the centrifuge tube containing the bacterial sludge is placed in a 60°C oven, dried to constant weight (about 24 hours), and then weighed and the cell dry weight is calculated. The calculation formula is as follows:

[0063] Cell dry weight (g / L) = (B1-B0) / S1

[0064] Wherein B1 is the weight of the centrifuge tube containing the bacterial sludge dried to constant weight, and B0 is the weight of the empty centrifuge tube at constant weight. S1 is the sampling volume. The cell dry weight changes of Lactobacillus paracasei J3703 under different concentrations of tempura red are shown in Figure 2. Fig. 9 As shown, the cell dry weight changes of Lactobacillus pentosus K0802 are as follows Fig.10 As shown, it can be seen that different concentrations of allura red have a basic effect on the growth of the two strains.

[0065] Example 4 Application of Lactobacillus paracasei J3703 and Lactobacillus pentosus K0802 in Degrading Edible Artificial Pigment Sunset Yellow

[0066] Single colonies of Lactobacillus paracasei J3703 or Lactobacillus pentosus K0802 were selected for enrichment culture until the bacterial solution OD 600 The value was 0.3–0.8, and the inoculation amount was 20% (v / v) and added to the decolorization medium. The concentration of sunset yellow was adjusted to 0.08 mg / mL, and the culture was carried out anaerobically at 37°C. A decolorization medium without sunset yellow was set as a control. The color change of the decolorization medium was observed and the decolorization medium without sunset yellow at 0h, the decolorization medium with sunset yellow at 0h, and the decolorization medium after 12h and 24h of culture was scanned at full wavelength. The full wavelength scan of Lactobacillus paracasei J3703 is shown in the figure below. Fig.11 As shown, the full wavelength scanning image of Lactobacillus pentosus K0802 is as follows Fig.12 From the above two figures, it can be seen that Lactobacillus paracasei J3703 and Lactobacillus pentosus K0802 can degrade sunset yellow.

[0067] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Lactobacillus species ( Lactobacillus ) is characterized in that The bacteria is Lactobacillus pentosus ( Lactobacillus pentosus )K0802, has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 26349.

2. A microbial preparation, characterized in that: A bacterium comprising the Lactobacillus bacteria according to claim 1 or a culture thereof.

3. A method for preparing a microbial preparation as claimed in claim 2, characterized in that: The bacteria of the genus Lactobacillus are activated and cultured in an expanded manner.

4. The method for preparing the microbial preparation according to claim 3, characterized in that: The culture conditions are anaerobic culture at 37±1°C for 24-48 hours.

5. Use of the bacteria of the genus Lactobacillus as claimed in claim 1 or the microbial preparation as claimed in claim 2 for degrading azo artificial pigments; the azo artificial pigments are sunset yellow or allura red.

6. A product for degrading azo artificial pigments, characterized in that: It comprises the bacteria of the genus Lactobacillus as claimed in claim 1 or the microbial preparation as claimed in claim 2; the azo artificial pigment is sunset yellow or allura red.

7. The product for degrading azo artificial pigments according to claim 6, characterized in that: The product includes a food, a food additive or a biological preparation.

8. A method for degrading azo artificial pigments, characterized in that: The method comprises the following steps: using the bacteria of the genus Lactobacillus according to claim 1 or the microbial preparation according to claim 2 to decolorize and degrade in a culture medium containing an azo artificial pigment; the azo artificial pigment is sunset yellow or allura red.

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