A novel antioxidant Lactobacillus paracasei subsp. paracasei TDM-2 from the plateau, its cell-free extract and applications

Abstract

The present invention provides a novel antioxidant Lactiplantibacillus paracasei TDM-2, its cell-free extract and applications, belonging to the technical field of lactic acid bacteria. The Lactiplantibacillus paracasei TDM-2 provided by the present invention is deposited in the China General Microbiological Culture Collection Center, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 20, 2023, and the deposit number is CGMCC NO. 26579. The Lactiplantibacillus paracasei TDM-2 and its cell-free extract provided by the present invention have good scavenging rates of DPPH free radicals and tolerance to H2O2, and also have good reducing ability and scavenging ability for OH ‑ ions.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid bacteria, and in particular to a novel antioxidant Lactobacillus paracasei subsp. tolerans TDM-2, its cell-free extract and applications. Background Art

[0002] Oxidative stress can lead to excessive production of highly reactive molecules in the body such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), with the degree of oxidation exceeding the scavenging of oxides, resulting in an imbalance between the oxidation system and the antioxidant system, thereby causing tissue damage. ROS include superoxide anion (O 2- ·), hydroxyl radical (OH·) and hydrogen peroxide (H 2 O 2 ), etc., among which hydrogen peroxide is an important oxygen free radical formed during the oxidative stress reaction; RNS include nitric oxide (NO·), nitrogen dioxide (NO 2 ·) and peroxynitrite (ONOO - ·), etc. ROS and RNS can be generated through various metabolic pathways, such as chemical toxicant and drug metabolism, cell respiration, radiation, light, etc. ROS / RNS have high reactivity and can easily react with macromolecules in cells such as DNA / RNA, membrane lipids, and proteins. Their oxidative damage is closely related to the aging, tumors and various diseases of the body. Lactic acid bacteria are considered to be one of the main sources of natural antioxidants.

[0003] Lactic acid bacteria include Lactococcus, Pediococcus, Leuconostoc, Lactobacillus and Bifidobacterium. When facing oxygen stress, lactic acid bacteria can produce various antioxidant enzyme substances, such as antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), etc., which can scavenge OH·, O 2- ·, DPPH free radical, etc., decompose or reduce ROS and its metabolites, catalyze ROS to generate substances with lower toxicity, and reduce the possibility of damage to body cells. On the other hand, lactic acid bacteria can also play an antioxidant role through mechanisms such as inhibiting lipid peroxidation, chelating metal ions, regulating the host cell antioxidant defense system and oxidative stress-related cell pathways, and regulating the oxidative damage repair system.

[0004] At the same time, lactic acid bacteria can also regulate intestinal flora. The intestines store a wide variety of microorganisms with different functions, forming an extremely complex micro-ecosystem that participates in regulating the host's energy harvest, nutrient metabolism and immune regulation, and is closely related to the health of the body. When the intestinal flora is abnormal and the bacteria that form endotoxins proliferate excessively, it will lead to increased endotoxin levels in the blood, which will cause oxidative stress effects in the system. Lactic acid bacteria can promote the proliferation of beneficial bacteria, inhibit endotoxin-producing bacteria, avoid increased endotoxin levels in the blood, and reduce oxidative stress and inflammatory damage by regulating the composition and structure of the intestinal flora. In addition, lactic acid bacteria can stimulate the intestinal mucosa to secrete more mucus, prevent pathogens from invading the intestinal mucosa, and reduce the generation of intestinal oxidative stress.

[0005] As people's living standards improve, their demand for functional foods is increasing. Different lactic acid bacteria species have different effects. Therefore, in recent years, the exploration of the efficacy of lactic acid bacteria of different species has also become a research hotspot, especially the in-depth study of the antioxidant properties of lactic acid bacteria, which is of great significance to the production and development of new functional foods.

[0006] Due to its special geographical and climatic environment, the Qinghai-Tibet Plateau in my country contains unique biological species resources, especially rich lactic acid bacteria resources, and the local special environment gives lactic acid bacteria biological characteristics and genetic diversity. Considering the preciousness and particularity of lactic acid bacteria germplasm resources in my country's high-altitude and cold regions, it is of great scientific significance to protect and develop and utilize high-quality lactic acid bacteria germplasm resources in my country's extreme environments, establish a database of lactic acid bacteria germplasm resources in my country's extreme environments, and screen lactic acid bacteria resources with obvious regional characteristics and special biological functions. Therefore, it is very necessary to screen plateau lactic acid bacteria with functional characteristics. Summary of the invention

[0007] The invention aims to provide a novel antioxidant plateau Lactobacillus paracasei TDM-2 and a cell-free extract thereof and application thereof.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a novel antioxidant plateau Lactobacillus paracasei TDM-2, which is deposited in the General Microbiology Center of China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of February 20, 2023 and a deposit number of CGMCCNO.26579.

[0010] The present invention also provides application of the novel antioxidant plateau Lactobacillus paracasei TDM-2 in preparing antioxidant functional foods.

[0011] The present invention also provides an antioxidant cell-free extract prepared from the novel antioxidant Lactobacillus paracasei subsp. paracasei TDM-2.

[0012] The present invention also provides a method for preparing an antioxidant cell-free extract, which is characterized by comprising the following steps:

[0013] Collect the cells in the activated solution of TDM-2, adjust the cell concentration and then perform ultrasonic disruption, and collect the supernatant as the cell-free extract.

[0014] Preferably, the method for collecting the cells in the activated solution of TDM-2 is centrifugation, the temperature of the centrifugation is 3-5 °C, the rotation speed is 7500-8500 g, and the time is 5-15 min.

[0015] Preferably, PBS is used to adjust the cell concentration, and the adjusted cell concentration is 1×10 9 CFU / mL to 1×10 10 CFU / mL.

[0016] Preferably, the power of the ultrasonic disruption is 320-400 W. During ultrasonic disruption, the cycle of ultrasonic treatment for 3-7 s and stopping for 3-7 s is adopted, and the total ultrasonic time is 20-40 min.

[0017] The present invention also provides the application of the antioxidant cell-free extract prepared from Lactobacillus paracasei subsp. paracasei TDM-2 in the preparation of antioxidant functional foods.

[0018] Lactobacillus paracasei subsp. paracasei TDM-2 can utilize its own antioxidant enzyme system to resist oxidative stress; it can also effectively stimulate the antioxidant system of the host and improve the activity of antioxidant enzymes. It can also produce various metabolites with antioxidant activity, such as butyric acid, folic acid, glutathione, etc.; at the same time, it can produce lactic acid, butyric acid, propionic acid, etc., reduce the intestinal pH, inhibit the growth of various pathogenic bacteria, maintain the balance of the intestinal flora, and help reduce oxidative stress. The cell-free extract prepared by centrifuging to collect TDM-2 cells and sonicating the cells in an ice bath and then collecting the supernatant in the present invention has a good scavenging rate of DPPH free radicals and a tolerance to H 2 O 2 , and also has good reducing ability and scavenging ability for OH - ions. Description of the Drawings

[0019] Figure 1 It is the phylogenetic tree of strain TDM-2 in Example 2;

[0020] Figure 2 It is the strain morphology of TDM-2 cultured in Example 2.

[0021] Depositing Description

[0022] Lacticaseibacillus paracasei TDM-2 with antioxidant property, this strain is deposited in the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposition date is February 20, 2023, and the deposition number is: CGMCC NO. 26579. Detailed Implementation Modes

[0023] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0024] Example 1

[0025] Obtaining of Lacticaseibacillus paracasei (TDM-2)

[0026] The milk sample collected from the plateau pasture was sucked into a sterilized test tube with a screw cap using a sterile pipette, frozen with an ice pack, and sent back to the laboratory as soon as possible. Take 1 mL of the milk sample and add it to 9 mL of sterile physiological saline, shake well to obtain a suspension of 10 -1 CFU / mL.

[0027] Take 1 mL of this suspension and serially dilute it to 1x10 -7 、1x10 -8 、1x10 -9 CFU / ml. Respectively pipette 100 μL of 3 different dilution degrees and evenly spread them on the MRS plate medium, and incubate anaerobically at 37 °C for 48 h. Select the typical lactic acid bacteria colonies that are round, medium-sized, convex, slightly white, moist, and have regular edges from the cultured bacteria. After multiple purifications, add 25% glycerol and store at -80 °C. A total of 103 strains were stored. Through further screening, a strain of lactic acid bacteria was obtained and named TDM-2.

[0028] Example 2

[0029] Species Identification of Lacticaseibacillus paracasei TDM-2

[0030] The observations of the colony and cell morphology of the strain TDM-2 obtained in Example 1 and the results of physiological and biochemical identification are as follows: The colony is milky white, round, convex, with relatively regular edges, smooth and moist on the surface, easy to pick, Gram-positive bacteria, showing long rod-shaped under the microscope, and non-spore-forming. Catalase-negative, oxidase-negative.

[0031] Clone the 16S rDNA sequence of TDM-2 and sequence the 16S rRNA sequence. Compare the sequencing results by BLAST in Genbank to determine the phylogenetic status of TDM-2. The phylogenetic tree is shown in Figure 1 . TDM-2 was further identified as Lactobacillus paracasei. The morphology of Lactobacillus paracasei strain TDM-2 is shown in Figure 2 .

[0032] Example 3

[0033] Preparation of liquid bacterial agent of TDM-2

[0034] Inoculate the TDM-2 bacterial solution stored at -80 °C in the laboratory into the sterilized MRS medium and culture overnight at 37 °C, with continuous passage for 3 times. Collect the culture solution of the last passage, centrifuge at 13,000 g for 10 min, collect the bacterial cells, wash 3 times with PBS, resuspend in PBS, and adjust the bacterial concentration to 10 8 CFU / mL.

[0035] Example 4

[0036] Preparation of cell-free extract of TDM-2 bacterial solution

[0037] After culturing the TDM-2 bacterial solution overnight at 37 °C, centrifuge (4 °C, 8000 g, 10 min) to collect the bacterial cells, wash 3 times with PBS, resuspend in PBS, and adjust the number of bacteria to 1×10 10 CFU / mL. Ultrasonically disrupt the cells in an ice bath (0 °C), ultrasonic for 5 s, stop for 5 s, with a total ultrasonic time of 30 min and an ultrasonic power of 360 W. After ultrasonic disruption, centrifuge (4 °C, 8000 g, 10 min), and collect the supernatant as the cell-free extract.

[0038] Example 5

[0039] Determination of the DPPH free radical scavenging rate of Lactobacillus rhamnosus LGG (CICC 6001) and Lactobacillus paracasei TDM-2

[0040] Weigh 0.008 g of DPPH and dissolve it in absolute ethanol, make up the volume to 100 mL to prepare 0.2 mmol / L DPPH. Respectively pipette 1.5 mL of the bacterial solutions of the two bacteria (the bacterial concentrations are both 1×10 8 CFU / mL, and TDM-2 is prepared in Example 3) and add 1.5 mL of 0.2 mmol / L DPPH. React in the dark at room temperature (25 °C) for 30 min, centrifuge at 8000 g, 4 °C for 10 min, take the supernatant, and measure the absorbance of the supernatant at 517 nm. The measurement results are shown in Table 1. It can be seen from the results that the DPPH free radical scavenging rate of TDM-2 is higher than that of the control strain LGG.

[0041] Table 1 DPPH free radical scavenging rate of Lactobacillus paracasei TDM-2

[0042]

[0043] Note: The control strain is LGG, the most stable commercial strain at present. Comparing with it can objectively reflect the antioxidant performance of strain TDM-2 (the same below).

[0044] Example 6

[0045] Tolerance determination of LGG (CICC 6001) and Lactobacillus paracasei TDM-2 to H 2 O 2

[0046] 100 μL of LGG (concentration of 1×10 8 CFU / mL) and TDM-2 bacterial solution (concentration of 1×10 8 CFU / mL, prepared in Example 3) were respectively inoculated into MRS broth containing 0 mmol·L -1 , 0.5 mmol·L -1 , 1 mmol·L -1 and 2.0 mol·L -1 H 2 O 2 . Anaerobic culture was carried out at 37 °C for 48 h, and its tolerance was observed. The measurement results are shown in Table 2. It can be seen from the results that the tolerance of TDM-2 to H 2 O 2 at each gradient is higher than that of the control strain LGG.

[0047] Table 2 Tolerance of Lactobacillus paracasei TDM-2 to H 2 O 2

[0048]

[0049]

[0050] Example 7

[0051] Determination of scavenging ability of LGG (CICC 6001) and Lactobacillus paracasei TDM-2 to OH - ions

[0052] ​​0.5 mL of the cell-free extract of LGG (prepared in the same manner as the cell-free extract of TDM-2 in Example 4) and the cell-free extract obtained in Example 4 were respectively added to 1 mL of o-phenanthroline (concentration 0.1%), then 1 mL of PBS and 1 mL of 2.5 mmol / L FeSO 4 , 1 mL of 20 mmol / L H 2 O 2 . After reacting in a constant temperature water bath at 37 °C for 1.5 h, the absorbance was measured at 536 nm. The measurement results are shown in Table 4. It can be seen from the results that the scavenging ability of TDM-2 for OH - ions was higher than that of the control strain LGG.

[0053] Table 4 Hydroxyl radical scavenging rate of Lactobacillus paracasei TDM-2 - Ion scavenging rate

[0054]

[0055] Example 8

[0056] Determination of the reducing ability of LGG (CICC 6001) and Lactobacillus paracasei TDM-2.

[0057] 0.5 mL of the cell-free extract of LGG (prepared in the same manner as the cell-free extract of TDM-2 in Example 4) and the cell-free extract obtained in Example 4 were respectively added with 0.5 mL of potassium ferricyanide (mass fraction 1%), 0.5 mL of 0.2 M PBS, mixed well, and water-bathed at 50 °C for 20 min. Rapidly cooled and 0.5 mL of trichloroacetic acid (TCA, mass fraction 10%) was added, and centrifuged at 4500 g and 4 °C for 10 min. 1 mL of the supernatant was reacted with 1 mL of ferric chloride (FeCl 3 , mass fraction 0.1%) for 10 min, and the absorbance was measured at 700 nm. The measurement results are shown in Table 5. It can be seen from the results that the reducing ability of TDM-2 was higher than that of the control strain LGG.

[0058] Table 5 Reducing ability of Lactobacillus paracasei

[0059]

[0060] Example 9

[0061] Determination of the total antioxidant capacity of LGG (CICC 6001) and Lactobacillus paracasei TDM-2.

[0062] The cell-free extracts of LGG (prepared in the same way as the cell-free extract of TDM-2 in Example 4) and the cell-free extract obtained in Example 4 were respectively tested using the Total Antioxidant Capacity Kit from Nanjing Jiancheng according to the kit instructions. The measurement results are shown in Table 6. It can be seen from the results that the total antioxidant capacity of TDM-2 is higher than that of the control strain LGG.

[0063] Table 6 Total Antioxidant Capacity of Lactobacillus paracasei TDM-2

[0064]

[0065] As can be seen from the above examples, the scavenging rate of DPPH free radicals, the tolerance to H 2 O 2 , the scavenging ability of OH - ions, the reducing ability and the total antioxidant capacity of Lactobacillus paracasei TDM-2 provided by the present invention are all superior to those of the most stable existing commercial strain LGG.

[0066] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A novel antioxidant Lacticaseibacillus paracasei TDM-2, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit date of February 20, 2023 and the deposit number of CGMCC NO. 26579.

2. Use of the novel antioxidant Lacticaseibacillus paracasei TDM-2 according to claim 1 in the preparation of antioxidant functional foods.

3. A method for preparing an antioxidant cell-free extract, characterized in that, it comprises the following steps: Collect the cells in the activated solution of TDM-2 in claim 1, adjust the cell concentration and then perform ultrasonic disruption, and collect the supernatant as the cell-free extract.

4. The preparation method according to claim 3, characterized in that, the method for collecting the cells in the activated solution of TDM-2 is centrifugation, the temperature of the centrifugation is 3-5 °C, the rotation speed is 7500-8500 g, and the time is 5-15 min.

5. The preparation method according to claim 4, characterized in that, Adjust the cell concentration with PBS, and the adjusted cell concentration is 1×10 9 CFU / mL to 1×10 10 CFU / mL.

6. The preparation method according to claim 5, characterized in that, the power of the ultrasonic disruption is 320-400 W. During ultrasonic disruption, a cycle of ultrasonic treatment for 3-7 s and then stopping for 3-7 s is adopted, and the total ultrasonic time is 20-40 min.

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