Lactobacillus plantarum cadoni having an anti-fatigue effect and a ferment thereof

Abstract

The present application relates to the field of microorganism, specifically relates to a kind of lactobacillus plantarum CADONI with anti-fatigue effect and its ferment.The lactobacillus plantarum (Lactobacillus plantarum) CADONI of the present application itself and its metabolite have excellent antioxidant activity and anti-fatigue effect, can be used as probiotic to develop functional food, feed and probiotic preparation etc., can bring significant economic benefits and social benefits.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a plant lactobacillus CADONI with anti-fatigue properties and its fermentation products. Background Technology

[0002] Studies have shown a link between gut microbiota and neuronal transmission in the brain. Gut epithelial cells transmit signals bidirectionally to the brain through hormone secretion and immune expression. Gut microbiota adhering to the gut influence the secretion and expression of small intestinal epithelial cells, playing a regulatory role in the gut-brain axis. The gut produces many neurotransmitters similar to those in the brain, such as serotonin, dopamine, and gamma-aminobutyric acid (GABA), all of which play crucial roles in regulating mood. These neurotransmitters can influence the gut-brain axis through gut microbiota, thereby regulating fatigue levels. However, no related technologies have been reported yet.

[0003] While existing technologies such as CN105831644A and CN104543680A mention the application of *Lactobacillus plantarum* powder in anti-fatigue compositions, their anti-fatigue effects rely on the synergistic effect of multiple components, resulting in a relatively complex composition. CN110791451A mentions that *Lactobacillus plantarum* has a regulatory effect on GABA, but this is mainly used to increase GABA levels in insomniac rats to improve sleep quality, leading to the indirect conclusion that it can alleviate mental fatigue. However, the application of this strain does not actually produce a direct anti-fatigue effect. Summary of the Invention

[0004] This invention, through isolation and purification, unexpectedly screened a pure strain of *Lactobacillus plantarum* CADONI from kefir grains—the fermentation seed used in kefir yogurt consumed by generations of herders in Xinjiang. This strain and its metabolites can promote dopamine release, inhibit GABA synthesis, and improve physical fatigue. Furthermore, this invention unexpectedly discovered that using carob protein as a nitrogen source during fermentation significantly enhances the anti-fatigue effect of *Lactobacillus plantarum* CADONI. Based on these findings, this invention provides an anti-fatigue *Lactobacillus plantarum* CADONI and its fermentation products.

[0005] Specifically, this invention first provides Lactobacillus plantarum CADONI, which was deposited on September 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), and classified as Lactobacillus plantarum, with accession number CGMCC No. 23413.

[0006] The 16S RNA sequencing results of *Lactobacillus plantarum* CADONI are shown in SEQ ID No. 1. The cells are rod-shaped, occurring singly, sometimes in pairs, or in chains. Colonies are milky white, round, and have a raised, smooth surface. It is a Gram-positive, catalase-negative bacterium that can utilize glucose and lactose to produce acid. This strain and its metabolites can regulate the intestinal flora, thereby affecting the gut-brain axis and modulating fatigue levels.

[0007] The present invention further provides a microbial agent containing the aforementioned Lactobacillus plantarum CADONI.

[0008] The present invention further provides a fermentation product or fermentation extract, which is obtained by fermentation of the aforementioned Lactobacillus plantarum CADONI.

[0009] In this invention, the fermentation product is the supernatant and / or bacterial cells of the fermentation broth obtained by fermenting Lactobacillus plantarum CADONI; the fermentation extract is an organic solvent extract of the supernatant and / or bacterial cells of the fermentation broth obtained by fermenting Lactobacillus plantarum CADONI.

[0010] In addition, the present invention also provides a fermentation method for Lactobacillus plantarum CADONI, using one or more of tryptone, carob protein, hydrolyzed wheat protein, soybean peptone, and yeast powder as nitrogen sources.

[0011] Preferably, the nitrogen source is carob protein.

[0012] When the nitrogen source is carob protein, the fermentation effect of the strain in this invention (including cell growth effect and product anti-fatigue effect) is better.

[0013] As a preferred embodiment, the culture medium used during fermentation contains the following components in parts by weight:

[0014] 35-45 parts nitrogen source, 25-35 parts carbon source, 15-25 parts tomato juice, 10-15 parts carrot juice, and 3-7 parts oligosaccharides;

[0015] The carbon source is selected from one or more of glucose, ribose, lactose, galactose, fructose, sucrose, maltose, mannose, and trehalose.

[0016] The oligosaccharide is selected from one or more of fructooligosaccharides, xylooligosaccharides, galactooligosaccharides, stachyose, inulin, and polydextrose.

[0017] The present invention also found that after deep fermentation by the above method, not only is it beneficial to cell growth, but the anti-fatigue ability of the resulting fermentation broth is also significantly enhanced.

[0018] As a preferred embodiment, each 1L of culture medium used during fermentation contains the following components:

[0019] Nitrogen source 35-45g, carbon source 25-35g, tomato juice 15-25g, carrot juice 10-15g, oligosaccharides 3-7g.

[0020] As a preferred method, the fermentation temperature is 25-35℃.

[0021] Furthermore, the present invention provides a fermentation product obtained by the above-described fermentation method.

[0022] Furthermore, the present invention also provides the application of the aforementioned Lactobacillus plantarum CADONI, or bacterial agent, or fermentation product, or fermentation extract, or fermentation product in the preparation of food additives, food, feed additives, animal feed, and pharmaceuticals.

[0023] Preferably, the food additive, food, feed additive, animal feed, or drug is used for at least one of the following purposes: 1) anti-fatigue; 2) anti-oxidation.

[0024] Furthermore, the present invention also provides a food additive or food (such as a functional food) containing the aforementioned Lactobacillus plantarum CADONI, or a bacterial agent, or a fermentation product, or a fermentation extract, or a fermentation product.

[0025] Furthermore, the present invention also provides a medicine (such as a probiotic preparation) containing the aforementioned Lactobacillus plantarum CADONI, or a bacterial agent, or a ferment, or a fermentation extract, or a fermentation product.

[0026] Furthermore, the present invention also provides a feed additive or animal feed containing the aforementioned Lactobacillus plantarum CADONI, or a microbial agent, or a fermentation product, or a fermentation extract, or a fermentation product.

[0027] Based on the above technical solution, the beneficial effects of the present invention include:

[0028] The Lactobacillus plantarum CADONI of the present invention and its metabolites have excellent antioxidant activity and anti-fatigue effects. They can be used as probiotics to develop functional foods, feeds and probiotic preparations, which can bring significant economic and social benefits. Attached Figure Description

[0029] Figure 1 The value represents the dopamine content in the serum of mice after exercise.

[0030] Figure 2 The value represents the serum γ-aminobutyric acid (GABA) content in mice after exercise. Detailed Implementation

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0032] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0033] Example 1: Isolation and Identification of Lactobacillus plantarum CADONI

[0034] Kefir granules, the starter culture for kefir yogurt passed down through generations in Xinjiang herder families, were rinsed with 0.9% sterile saline solution and inoculated into sterilized raw milk for activation and culture at 37°C for 24 hours. The activated milk was then transferred to sterilized selection medium using a sterile pipette and purified.

[0035] In the isolation culture, the streak plate method was used. After static incubation at 37℃ for 24 hours, colonies that were milky white in color, round in shape, and had a raised and smooth surface were picked.

[0036] Strain identification:

[0037] Morphology: The strains are rod-shaped, single, sometimes in pairs or chains.

[0038] Physiological and biochemical characteristics: Gram-positive, catalase-negative, capable of producing acid from glucose and lactose.

[0039] The 16sRNA sequencing results are shown in SEQ ID No. 1. Analysis revealed that the strain is Lactobacillus plantarum.

[0040] The strain was named Lactobacillus plantarum CADONI and deposited on September 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China). Its classification name is Lactobacillus plantarum, and its accession number is CGMCC No. 23413.

[0041] Example 2: Fermentation of Lactobacillus plantarum CADONI

[0042] A bacterial suspension was prepared from the Lactobacillus plantarum CADONI strain isolated and purified in Example 1, and inoculated into the fermentation culture medium. The suspension was fermented at 25°C for 48 hours.

[0043] The composition of the fermentation broth is as follows: each 1L of fermentation broth contains 25g carob protein, 20g glucose, 10g lactose, 25g tomato juice, 10g carrot juice, and 5g fructooligosaccharides.

[0044] After fermentation, the viable cell count was performed, and the result was 5.3 × 10⁻⁶. 11 cfu / mL.

[0045] Example 3: Fermentation of Lactobacillus plantarum CADONI

[0046] A bacterial suspension was prepared from the Lactobacillus plantarum CADONI strain isolated and purified in Example 1, and inoculated into the fermentation culture medium. The suspension was fermented at 25°C for 48 hours.

[0047] The composition of the fermentation broth is as follows: each 1L of fermentation broth contains 15g carob protein, 20g glucose, 10g lactose, 25g tomato juice, 10g carrot juice, and 5g fructooligosaccharides.

[0048] After fermentation, the viable cell count was performed, and the result was 4.7 × 10⁻⁶. 11 cfu / mL.

[0049] Example 4: Fermentation of Lactobacillus plantarum CADONI

[0050] A bacterial suspension was prepared from the Lactobacillus plantarum CADONI strain isolated and purified in Example 1, and inoculated into the fermentation culture medium. The suspension was fermented at 35°C for 48 hours.

[0051] The composition of the fermentation broth is as follows: each 1L of fermentation broth contains 25g carob protein, 20g glucose, 10g maltose, 15g tomato juice, 15g carrot juice, and 2g fructooligosaccharides.

[0052] After fermentation, the viable cell count was 6.2 × 10⁻⁶. 11 cfu / mL.

[0053] Example 5: Fermentation of Lactobacillus plantarum CADONI

[0054] A bacterial suspension was prepared from the Lactobacillus plantarum CADONI strain isolated and purified in Example 1, and inoculated into the fermentation culture medium. The suspension was fermented at 35°C for 48 hours.

[0055] The fermentation medium used differed from that in Example 4 only in that carob protein was replaced with an equal amount of tryptone.

[0056] After fermentation, the viable cell count was performed, and the result was 3.4 × 10⁻⁶. 9 cfu / mL.

[0057] Experimental Example

[0058] 1. Antioxidant Experiment - Determination of DPPH Free Radical Scavenging Rate in Fermentation Broth

[0059] Take 2 mL of each of the fermentation broths from Examples 2, 3, and 4; take 2 mL of the fermentation broth from Example 5, and adjust its viable cell count to be the same as in Example 2. A 10 mg / L vitamin C solution (prepared with distilled water) was used as a positive control. Commercially available *Lactobacillus plantarum* was used as a control group; the fermentation culture medium components were the same as in Example 4. DPPH was prepared with anhydrous ethanol to a concentration of 2 × 10⁻⁶. -4A mol / L solution. Accurately pipette 2 mL each of the sample test solution and DPPH solution, mix well, and let stand at room temperature for 10 min, 20 min, and 30 min. Use 2 mL of anhydrous ethanol and 2 mL of DPPH reaction solution as blanks, and measure the absorbance at a wavelength of 517 nm. Each sample is measured in triplicate, and the average value is taken. Calculate the scavenging rate of DPPH free radicals for each test sample according to formula (1), and plot the time-scavenging rate curve.

[0060] (1) DPPH clearance rate % = (A blank - A sample) / A blank × 100%.

[0061] Experimental Results: Table 1 shows that the higher the viable cell count in the fermentation broth, the stronger the antioxidant capacity, i.e., the higher the DPPH scavenging rate. From the DPPH scavenging rates of Examples 2 and 5, it can be seen that even with the same viable cell count, the antioxidant capacity of the fermentation broth varies significantly depending on the culture medium composition.

[0062] Table 1 DPPH removal rate of fermentation broth

[0063]

[0064]

[0065] Note: * represents *p < 0.01 relative to Example 2; # represents #p < 0.01 relative to the positive control VC group; △ represents △p > 0.05 relative to the positive control VC group.

[0066] 2. Weighted Swimming Experiment

[0067] Principle: Improved exercise endurance is the most direct manifestation of enhanced fatigue resistance.

[0068] Methods: Refer to the "Technical Specifications for Inspection and Evaluation of Health Foods"

[0069] Laboratory animals: Adult mice, weighing 18-22 grams.

[0070] Grouping: Take 60 mL of fermentation broth from Examples 2, 3, and 4; take 60 mL of fermentation broth from Example 5, and adjust its viable cell count to be the same as in Example 2. Mice were orally administered 0.1 mL / 10 g bw of the test substance daily. The blank group received the same dose of distilled water, and the control group received the same number of viable cells of commercially available *Lactobacillus plantarum*. The fermentation culture medium composition was the same as in Example 4. Exercise endurance was measured after 30 days of continuous gavage.

[0071] Experimental results: See Table 2. Examples 2, 3, and 4 significantly increased the weight-bearing swimming time of mice compared with the blank control group, while Example 5 showed no significant difference in weight-bearing swimming time between the mice and the blank control group.

[0072] Table 2. Effects of fermentation broth on weight-bearing swimming time in mice.

[0073]

[0074]

[0075] Note: * represents p < 0.01 relative to the blank group; # represents p > 0.05 relative to Examples 2, 3, and 4; △ represents p > 0.05 relative to the blank group.

[0076] After the mice finished exercising, the levels of dopamine and γ-aminobutyric acid in their serum were measured. The results are shown in the table below. Figures 1-2 .

[0077] from Figure 1 It can be seen that there is a positive correlation between the swimming time of mice and the dopamine content in their bodies. The dopamine content of the control group mice was not significantly different from that of the blank group, while the dopamine content of the example group was significantly higher than that of the blank group. Figure 2 This indicates a negative correlation between swimming time and GABA (γ-aminobutyric acid) levels in mice. The GABA levels in the control group and the blank group were not significantly different, while the GABA levels in the example group were significantly lower than those in the blank group. The strain itself and its metabolites significantly increased swimming time and delayed fatigue in mice by promoting dopamine release and inhibiting GABA synthesis.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Lactobacillus plantarum ( Lactobacillus plantarum The application of CADONI in the preparation of anti-fatigue products is characterized by, The application preparation method includes the step of fermenting the *Lactobacillus plantarum* CADONI using a culture medium with carob protein as the nitrogen source; The *Lactobacillus plantarum* CADONI is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 23413.

2. The application according to claim 1, characterized in that, The culture medium used during fermentation contains the following components in parts by weight: 35-45 parts nitrogen source, 25-35 parts carbon source, 15-25 parts tomato juice, 10-15 parts carrot juice, and 3-7 parts oligosaccharides; The carbon source is selected from one or more of glucose, ribose, lactose, galactose, fructose, sucrose, maltose, mannose, and trehalose. The oligosaccharide is selected from one or more of fructooligosaccharides, xylooligosaccharides, galactooligosaccharides, and stachyose.

3. The application according to claim 1, characterized in that, The fermentation temperature is 25-35℃.

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