Application of lactobacillus rhamnosus TG013 in preparation of antioxidant product

Abstract

The invention relates to the technical field of microorganisms, in particular to application of lactobacillus rhamnosus TG013 in preparation of an antioxidant product, the preservation number of the lactobacillus rhamnosus TG013 is CCTCC (China Center For Type Culture Collection) NO: M 2023036, and the antioxidant product comprises at least one of pyrroloquinoline quinone, nicotinic acid and an enzymatic antioxidant.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to application of a Lactobacillus rhamnosus TG013 in the preparation of antioxidant products. Background Art

[0002] Antioxidation is an important mechanism for maintaining health and delaying aging, and pyrroloquinoline quinone (PQQ), niacin and antioxidant enzymes play a key role in this. PQQ is a powerful antioxidant, and its antioxidant capacity far exceeds that of vitamin C. PQQ can directly scavenge reactive oxygen species (ROS), reduce the production of free radicals, and thus reduce oxidative stress. In addition, PQQ can promote mitochondrial biogenesis and optimize mitochondrial function by activating pathways such as PGC-1α, further enhancing the antioxidant capacity of cells. PQQ can also enhance the activity of intracellular antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), to help maintain the redox balance in the body. Niacin can participate in NAD + The synthesis of NAD indirectly supports the cell's antioxidant defense system. + It is a coenzyme for various oxidoreductases and participates in cellular energy metabolism and antioxidant responses. Antioxidant enzyme activity is an important indicator of the body's antioxidant capacity. Niacin can effectively scavenge hydroxyl and DPPH free radicals, inhibit the formation of lipid peroxides, and exhibit the ability to reduce iron ions. Furthermore, niacin can synergize with other antioxidants (such as vitamin C, vitamin E, and glutathione) by donating electrons to help these antioxidants continue to function, thereby enhancing the overall antioxidant effect. This synergistic effect enables niacin to play a vital role in the antioxidant defense mechanism, helping to protect cells from oxidative stress damage.

[0003] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), a Gram-positive bacillus of the genus Lactobacillus. Lactobacillus rhamnosus is widely used as a probiotic to improve intestinal health and regulate immune function. CN117247873A discloses a strain of Lactobacillus rhamnosus TG013, deposited with the China Center for Type Culture Collection under the CCTCC No. M2023036 on January 5, 2023. Experiments have shown that Lactobacillus rhamnosus TG013 has a blood pressure-lowering effect, but its ability to produce pyrroloquinoline quinone, niacin, and enzymatic antioxidants has not been reported. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a strain of Lactobacillus rhamnosus TG013 for use in the preparation of antioxidant products, which has the function of producing pyrroloquinoline quinone, niacin and enzymatic antioxidants.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Application of TG013 in the preparation of antioxidant products, wherein the rhamnosus lactobacillus ( Lacticaseibacillus rhamnosus ) The deposit number of TG013 is CCTCC NO: M 2023036.

[0006] Furthermore, the antioxidant product comprises at least one of pyrroloquinoline quinone, niacin and enzymatic antioxidants.

[0007] Furthermore, the Lactobacillus rhamnosus TG013 has a 16s rDNA sequence as shown in SEQ ID No. 1.

[0008] Furthermore, the Lactobacillus rhamnosus TG013 is used as a raw material for preparing food, medicine or health care products.

[0009] Furthermore, the method for preparing an antioxidant product by using Lactobacillus rhamnosus TG013 is to ferment and culture the Lactobacillus rhamnosus TG013 in a culture medium to obtain the antioxidant product.

[0010] Furthermore, the method for producing pyrroloquinoline quinone by Lactobacillus rhamnosus TG013 is to ferment and culture the Lactobacillus rhamnosus TG013 in a culture medium to obtain pyrroloquinoline quinone; Preferably, the culture medium is selected from at least one of LB liquid culture medium and MRS-TG culture medium; Preferably, the culture medium contains 0.1-5 g / L ammonium sulfate and 0.1-5 g / L riboflavin.

[0011] Furthermore, the method for producing niacin by Lactobacillus rhamnosus TG013 is as follows: fermenting and culturing the Lactobacillus rhamnosus TG013 in a culture medium to obtain niacin; Preferably, the culture medium is a culture medium for niacin determination.

[0012] Furthermore, the method for producing an enzymatic antioxidant by Lactobacillus rhamnosus TG013 is as follows: fermenting and culturing the Lactobacillus rhamnosus TG013 in a culture medium to obtain the enzymatic antioxidant; Preferably, the culture medium is MRS medium.

[0013] Furthermore, during the fermentation culture, the culture temperature used is 30-40° C., and the culture time is 12-72 h.

[0014] Furthermore, before fermenting the Lactobacillus rhamnosus TG013 in a culture medium, the strain is taken out from a -80°C refrigerator, activated by streaking on an MRS agar plate using a disposable inoculation loop, and cultured in a 37°C anaerobic incubator until a single colony grows.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) A new use of TG013 in the production of pyrroloquinoline quinone. Experiments have confirmed that this strain has the ability to produce pyrroloquinoline quinone, and the pyrroloquinoline quinone yield can reach 98.35 μg / mL.

[0016] 2. The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) A new use of TG013 in niacin production. Experiments have confirmed that this strain has the ability to produce niacin, and the niacin yield can reach 23.40 μg / mL.

[0017] 3. The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) A new use of TG013 in the production of enzymatic antioxidants. Experiments have confirmed that the supernatant of this strain has a scavenging rate of 77.82% for DPPH free radicals, SOD enzyme activity is 121.00 U / mL, and GSH-Px enzyme activity is 302.01 U / mL. This strain has good antioxidant capacity.

[0018] 4. The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus TG013 can also produce pyrroloquinoline quinone, niacin and enzymatic antioxidants. Pyrroloquinoline quinone, niacin and enzymatic antioxidants can achieve synergistic antioxidant effects through different pathways, and have important research potential in maintaining health and delaying aging.

[0019] 5. This strain is a functional strain derived from the human body. It is originally a component of the human intestinal microbiome. It has a certain adhesion ability to the small intestinal epithelial cells and can better act and colonize. Moreover, this strain is derived from healthy donors. It is safe to use this strain as a raw material for the preparation of food, health products, and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The PQQ standard curve was determined by spectrometry in Example 1; Figure 2 The PQQ standard curve was determined by HPLC in Example 2; Figure 3 This is the chromatogram of the high performance liquid phase determination of PQQ in Example 2; Figure 4 This is an overlapping HPLC chromatogram of the niacin standard product in Example 3; Figure 5 For Example 3 Lacticaseibacillus rhamnosus TG013 nicotinic acid HPLC determination chromatogram; Figure 6 For Example 3 Lacticaseibacillus rhamnosus Chromatogram of nicotinic acid HPLC determination in JCM 1136. DETAILED DESCRIPTION

[0021] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. 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. The methods are all conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0022] The present invention relates to a Lactobacillus rhamnosus strain ( Lacticaseibacillus rhamnosus ) Application of TG013 in the preparation of antioxidant products, about Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The origin, biological identification, and physiological characteristics of TG013 are disclosed in CN117247873A and are incorporated herein by reference. The present invention Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus TG013 was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 2023036 on January 5, 2023, at the Wuhan University Collection Center. Lacticaseibacillus rhamnosus The 16s rDNA sequence of TG013 is as follows: The composition of the MRS medium used in the following examples is as follows: peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, sodium acetate 5.0 g / L, dimethyl phosphate 2.0 g / L, ammonium citrate 2.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, agar 15.0 g / L; Composition of the niacin assay medium (Haibo Biotechnology): acid-hydrolyzed casein 10.0 g / L, glucose 40 g / L, sodium acetate 20.0 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, L-tryptophan 0.1 g / L, L-cystine hydrochloride 0.4 g / L, magnesium sulfate 0.4 g / L, sodium chloride 0.02 g / L, ferrous sulfate 0.02 g / L, manganese sulfate 0.02 g / L, riboflavin 0.0004 g / L, calcium pantothenate 0.0002 g / L, and biotin 0.000001 g / L. LB liquid medium: tryptone 10 g / L, yeast extract powder 5.0 g / L, sodium chloride 10.0 g / L; MRS-TG medium: casein digest 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate tribasic 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.0 g / L, manganese sulfate 0.05 g / L, potassium hydrogen phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1.08 g / L, agar 15.0 g / L, riboflavin 1.0 g / L, L-cysteine 0.5 g / L.

[0023] Example 1: Determination of PQQ production ability of Lactobacillus rhamnosus TG013 The Lactobacillus rhamnosus TG013 strain storage tube was taken out from -80℃, and 100 μL of bacterial liquid was inoculated into 10 mL LB liquid culture medium, and the strain seed liquid was obtained by resuscitation culture at 37℃ and 200 rpm for 24 hours. The seed liquid was inoculated into the LB liquid culture medium, and then placed at 37℃ and 200 rpm for 48 hours of fermentation culture to fully promote the growth and metabolism of the strain. After the culture, the strain culture liquid was transferred to a centrifuge tube and ultrasonically disrupted at 200 W for 10 minutes, and then centrifuged at 4℃ and 5000 rpm for 5 minutes to achieve effective separation of the bacteria and the supernatant, and the supernatant was collected to complete the preparation of the cell-free extract. The absorbance of the collected supernatant was measured at a wavelength of 326 nm using an enzyme marker. The PQQ standard was diluted to (4, 5, 10, 15, 20, 25 μg / mL) to construct the PQQ standard curve. The PQQ standard curve is as shown in the figure. Figure 1 As shown. Figure 1 As shown, the standard curve equation of PQQ is: Y = 0.01636X + 0.2254, R 2 =0.9999, which is consistent with the experimental design. The calculated PQQ concentration in the cell-free extract of Lactobacillus rhamnosus TG013 was 1.06 μg / mL, indicating that Lactobacillus rhamnosus TG013 is capable of producing PQQ.

[0024] Example 2: Determination of PQQ production ability of Lactobacillus rhamnosus TG013 Ammonium sulfate and riboflavin are used as fermentation substrates for the PQQ fermentation of the strain. Appropriate addition of these substances can increase the proliferation of the PQQ-producing strain. The above-mentioned Lactobacillus rhamnosus was cultured using MRS-TG medium. Lacticaseibacillus rhamnosus TG013, Lacticaseibacillus rhamnosus The JCM 1136 (NR_043408.1) strain collection tube was taken out from -80℃, 100 μL of the bacterial solution was aspirated and inoculated into 10 mL of LB liquid medium and cultured at 37℃, 200 rpm for 24 h to obtain the strain seed solution. Lacticaseibacillus rhamnosus TG013 and Lacticaseibacillus rhamnosus The JCM 1136 strain seed liquid was inoculated into MRS-TG medium and cultured at 37°C and 200 rpm for 48 h. The culture medium was then transferred to a centrifuge tube and ultrasonically disrupted at 200W for 10 min. The cell-free extract was then prepared by centrifugation at 4°C and 5000 rpm for 5 min.

[0025] Prepare a 1 mg / mL PQQ standard solution and dilute the standard to concentrations of 1000 μg / mL, 800 μg / mL, 500 μg / mL, 200 μg / mL, and 100 μg / mL.

[0026] Mobile phase preparation: water + 1‰ TFA (trifluoroacetic acid): acetonitrile + 1‰ TFA = 85:15 (V:V) Detection conditions: Use C 18 The chromatographic column was Waters, the detection wavelength was 330 nm, the column temperature was 40 °C, and the flow rate was 0.5 mL / min.

[0027] The concentration gradient of the standard curve is 100 μg / mL, 200 μg / mL, 500 μg / mL, 800 μg / mL, and 1000 μg / mL. The standard curve of PQQ determined by HPLC is as follows: Figure 2 ,from Figure 2 It can be seen that the retention time of PQQ is 3 min, Y=56.5X-1690, R 2 =0.9996, which is consistent with the linear relationship of the standard curve.

[0028] Will Lacticaseibacillus rhamnosus TG013 cell-free extract and Lacticaseibacillus rhamnosus JCM 1136 was filtered through a 0.22 μm organic filter membrane for detection. The chromatogram of PQQ liquid phase determination is shown in Figure 2. Figure 3 As shown. Figure 3 It can be seen that Lacticaseibacillus rhamnosus The peak area of TG013 cell-free extract was 3867, and the PQQ yield was 98.35 μg / mL; Lacticaseibacillus rhamnosus The peak area of the JCM 1136 cell-free extract was too low and the PQQ concentration was not detected.

[0029] Example 3: Lacticaseibacillus rhamnosus Determination of niacin production capacity of TG013 Lactobacillus rhamnosus TG013 and Lacticaseibacillus rhamnosus The JCM 1136 (NR_043408.1) strain collection tube was taken out from -80℃, 100 μL of the bacterial solution was aspirated and inoculated into 10 mL of LB liquid medium, and the strain seed solution was obtained by recovery culture at 37℃ and 200 rpm for 24 h. Lacticaseibacillus rhamnosus TG013, Lacticaseibacillus rhamnosus Fifty μL of JCM 1136 (NR_043408.1) strain seed solution was inoculated into 5 mL of niacin assay medium and cultured at 37°C and 200 rpm for 24 h. The culture medium was transferred to a centrifuge tube and ultrasonically disrupted at 200 W for 10 min. The tube was then centrifuged at 4°C and 5000 rpm for 10 min. The supernatant was collected to obtain a cell-free extract, which was filtered through a 0.22 μm organic filter membrane and set aside.

[0030] The niacin determination method used is as follows: Chromatographic conditions: C18 column (Waters), detection wavelength at 266 nm, column temperature at 25°C, flow rate at 1 mL / min, elution time at 10 min, injection volume at 10 μL.

[0031] Mobile phase configuration: 0.05 mol / L potassium dihydrogen phosphate solution: methanol = 90:10 (V / V).

[0032] Standard solution preparation: Accurately weigh 0.05 g of niacin standard into a 50 mL volumetric flask. Dissolve and dilute to volume with 0.01 mol / L hydrochloric acid. Shake well to prepare a 1 mg / mL standard solution. Dilute the standard solution to concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 1 μg / mL, and 0.5 μg / mL. Filter through a 0.22 μm microporous membrane, and aliquot 1 mL into a liquid phase injection vial for later use.

[0033] The formula for calculating niacin content is:

[0034] Note: V—nicotinic acid peak area of the strain supernatant; V0—niacin peak area of culture supernatant; b—Standard curve constructed based on the peak area and concentration of niacin standard: Y = aX+b; The concentration gradient of the standard curve was 0.5 μg / mL, 1 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 1 mg / mL. Figure 4 The HPLC overlapping chromatogram of niacin standard product shows that the retention time of niacin is 3.8 min, Y=483X+3120, and R2=0.995, which is consistent with the linear relationship of the standard curve.

[0035] Will Lacticaseibacillus rhamnosus TG013, Lacticaseibacillus rhamnosus The detection method of JCM1136 (NR_043408.1) cell-free extract was consistent with that of the standard. Figures 5 - 6 They are Lacticaseibacillus rhamnosus The chromatograms of nicotinic acid HPLC determination of TG013 and JCM 1136 show that Lacticaseibacillus rhamnosus The peak area of the TG013 cell-free extract was 32140. After deducting the kurtosis effect of the culture medium used for niacin determination, the niacin yield was 23.40 μg / mL. Lacticaseibacillus rhamnosus The peak area of the cell-free extract of JCM 1136 (NR_043408.1) was 23891. After deducting the influence of the abundance of the culture medium used for niacin determination, the niacin yield was 6.32 μg / mL.

[0036] Example 4:Lacticaseibacillus rhamnosus TG013 antioxidant function assay Will Lacticaseibacillus rhamnosus TG013, Lacticaseibacillus rhamnosus Remove the JCM1136 (NR_043408.1) strain from a -80°C freezer and activate it by streaking onto an MRS agar plate using a disposable inoculating loop. Incubate in an anaerobic incubator at 37°C until a single colony forms. Activate the seed solution by dipping a single colony from the MRS agar plate with a disposable inoculating loop, inoculating it into 5 mL of MRS medium, shaking it evenly, and incubating it in a shaker at 37°C, 200 rpm, for 24 hours.

[0037] Determination of DPPH free radical scavenging ability of Lactobacillus rhamnosus TG013 and JCM 1136 Preparation of DPPH sample solution: Accurately weigh 20.0 mg of DPPH powder and place it in a 250 mL volumetric flask. Dissolve it in anhydrous ethanol and dilute to the mark. Mix well to obtain a 0.2 mmol / L DPPH solution. Store at 4°C (use within 3.5 h).

[0038] Take 1 mL of the strain supernatant, add 1 mL of 0.2 mmol / L DPPH, mix well, let it stand at room temperature for 30 min, use anhydrous ethanol as a blank control, and measure the absorbance change at 517 nm.

[0039] The absorbance of the bacterial supernatant plus DPPH solution at a wavelength of 517 nm was measured as A1. A mixture of 1.0 mL of DPPH solution and 1.0 mL of anhydrous ethanol was used as a negative control to measure the absorbance as A2. A blank control was obtained by adding 1.0 mL of anhydrous ethanol solution to 1.0 mL of the bacterial supernatant solution and measuring its absorbance as A0. The clearance rate was calculated according to the following formula:

[0040] Determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme activities by fermentation of Lactobacillus mucilaginosus TG013 and JCM 1136 The SOD and GSH-Px enzyme activities in the supernatant of fermented Lactobacillus mucilaginosus TG013 culture were determined using a kit (Elabscience).

[0041] Lacticaseibacillus rhamnosus TG013 and Lacticaseibacillus rhamnosus The seed liquid of JCM1136 (NR_043408.1) strain was inoculated into MRS medium and cultured anaerobically at 37°C on a shaking incubator for 24 h. The supernatant of the strain was then collected for antioxidant index detection.

[0042] As shown in Table 1, Lacticaseibacillus rhamnosusThe scavenging rate of DPPH free radicals of the supernatant of TG013 bacterial culture was 77.82%, the SOD enzyme activity was 121.00 U / mL, and the GSH-Px enzyme activity was 302.01 U / mL, indicating that this strain has good antioxidant capacity.

[0043] Table 3 Antioxidant capacity of bacterial supernatant

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solutions claimed for protection by the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) The application of TG013 in the preparation of antioxidant products is characterized in that: The Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) The deposit number of TG013 is CCTCC NO: M 2023036.

2. The use according to claim 1, characterized in that The antioxidant product comprises at least one of pyrroloquinoline quinone, niacin and an enzymatic antioxidant.

3. The use according to claim 1 or 2, characterized in that The Lactobacillus rhamnosus TG013 has a 16s rDNA sequence as shown in SEQ ID No.

1.

4. The use according to claim 1 or 2, characterized in that The Lactobacillus rhamnosus TG013 is used as a raw material for preparing food, medicine or health care products.

5. The use according to claim 1, characterized in that The method for preparing an antioxidant product by using Lactobacillus rhamnosus TG013 comprises fermenting and culturing the Lactobacillus rhamnosus TG013 in a culture medium to obtain the antioxidant product.

6. The use according to claim 1, characterized in that The method for producing pyrroloquinoline quinone by Lactobacillus rhamnosus TG013 is as follows: fermenting and culturing the Lactobacillus rhamnosus TG013 in a culture medium to obtain pyrroloquinoline quinone; Preferably, the culture medium is selected from at least one of LB liquid culture medium and MRS-TG culture medium; Preferably, the culture medium contains 0.1-5 g / L ammonium sulfate and 0.1-5 g / L riboflavin.

7. The use according to claim 1, characterized in that The method for producing niacin by Lactobacillus rhamnosus TG013 is as follows: fermenting and culturing the Lactobacillus rhamnosus TG013 in a culture medium to obtain niacin; Preferably, the culture medium is a culture medium for niacin determination.

8. The use according to claim 1, characterized in that The method for producing an enzymatic antioxidant by using Lactobacillus rhamnosus TG013 comprises fermenting and culturing the Lactobacillus rhamnosus TG013 in a culture medium to obtain the enzymatic antioxidant; Preferably, the culture medium is MRS medium.

9. The use according to claim 6, 7 or 8, characterized in that During the fermentation culture, the culture temperature used is 30-40°C and the culture time is 12-72 h.

10. The use according to claim 6, 7 or 8, characterized in that Before fermenting the Lactobacillus rhamnosus TG013 in a culture medium, the strain is taken out from a -80°C refrigerator, activated by streaking on an MRS agar plate using a disposable inoculation loop, and cultured in a 37°C anaerobic incubator until a single colony grows.

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