Pediococcus pentosaceus sc009 and applications thereof

Abstract

The application discloses Pediococcus pentosaceus SC009 and an application thereof, is preserved in the China General Microbiological Culture Collection Center, and has a preservation time of June 28, 2021 and a preservation number of CGMCC No.22796.The bacteria have the effects of promoting sleep and resisting oxidation, and can be used for preparing sleep-improving and oxidation-resisting food and sleep-disorder treating medicine.In animal experiments, the bacteria can significantly shorten the sleep latency of mice and prolong the sleep time length, improve the sleep quality of mice, significantly improve the total SOD and reduced glutathione content in serum of middle-aged and old rats, reduce the MDA and protein carbonyl content, and have significant antioxidant activity, and can prolong the survival time of old rats.Pediococcus pentosaceus SC009 has the effects of promoting sleep and resisting oxidation, is conducive to market popularization and utilization, and has a broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial product technology, specifically to a Pediococcus pentosaceus SC009 and its applications. Background Technology

[0002] Humans spend nearly one-third of their lives sleeping. Sleep is an essential physiological process for humans and higher animals, fundamental to maintaining physical strength and health. With the increasingly fast pace of modern life and escalating competitive pressures, the incidence of insomnia is rising year by year, seriously affecting human physical health and social functioning. It is reported that transient insomnia affects approximately 80% of the population, while chronic insomnia affects about 15%. In the United States, about 56% of the population suffers from insomnia; a survey of insomnia in the general population of six cities in China, including Beijing, showed that the prevalence of insomnia among adults in the past 12 months was 57% (44%-68%), and the proportion of "shallow sleep" was as high as 77.3%. Therefore, research and prevention of insomnia are urgently needed. Currently, the treatment of sleep disorders mainly relies on the use of synthetic sedative and hypnotic drugs, which are effective and fast-acting, but have adverse effects such as dependence, cognitive and motor impairment, and rebound effects upon withdrawal. Therefore, there is an urgent need to find safe and effective food supplements.

[0003] Oxidative stress refers to the increased levels of intracellular oxygen free radicals, leading to damage to lipids, proteins, and DNA. Highly reactive oxygen free radicals, including superoxide anion radicals (O2-), hydroxyl radicals (-OH), and hydrogen peroxide (H2O2), constitute reactive oxygen species (ROS) in the body. Numerous studies have shown that sufficient levels of ROS can lead to a variety of chronic diseases, including atherosclerosis, arthritis, diabetes, Alzheimer's disease, neurodegenerative diseases, and cardiovascular diseases.

[0004] Most organisms have evolved enzymatic and non-enzymatic antioxidant defense and repair systems to protect themselves from oxidative damage. Superoxide dismutase (SOD) is the most important and widely studied enzymatic antioxidant system in the body, primarily targeting superoxide anion free radicals. Malondialdehyde (MDA) is an indicator of lipid peroxidation in the body, reflecting the degree of cellular damage. MDA detection is usually combined with SOD measurement; the level of SOD activity indirectly reflects the body's ability to scavenge oxygen free radicals, while the level of MDA indirectly reflects the severity of free radical attack on cells. Protein carbonyl is an indicator of protein oxidative damage, reflecting the process of carbonylation of amino acids in the side chain of proteins after attack by H2O2 or superoxide anions. Although the body has various complex antioxidant systems, these inherent antioxidant systems are usually insufficient to counteract oxidative damage. Antioxidants can, to some extent, delay or prevent cellular oxidation, protecting the body from oxidative damage. Several synthetic antioxidants currently on the market are widely used to delay lipid oxidation, including butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). However, due to their potential to cause liver damage and cancer, the safety of these synthetic antioxidants is highly questionable. Therefore, there is an urgent need to find safe and effective natural antioxidants.

[0005] *Pediococcus pentosaceus*, a Gram-positive coccus belonging to the family Streptococceae and the genus *Pediococcus*, ferments glucose to produce lactic acid. It is found in wort and fermented plant materials such as pickles, kimchi, and silage. In 2014, *Pediococcus pentosaceus* was approved as a new food ingredient by the National Health and Family Planning Commission of China. As a lactic acid bacterium, the probiotic functions of *Pediococcus pentosaceus* have received increasing attention in recent years. Kwon et al. found that *Cordyceps militaris* fermented by *Pediococcus pentosaceus* could inhibit inflammatory responses and alleviate contact dermatitis. Three teams—Lv et al., Shi et al., and Dubey et al.—discovered that *Streptococcus pentosaceus* could reduce D-galactosamine, carbon tetrachloride, and cadmium-induced liver damage. Xu et al. found that *Streptococcus pentosaceus* could resist *Clostridium difficile* infection. Current research on the antioxidant effects of *Pediococcus pentosaceus* is mostly limited to in vitro experiments. Ayyash et al. found that Pediococcus pentosaceus M41 cleared DPPH and ABTS at rates of 76.5% and 48.9% respectively in vitro. However, there are currently no reports of Pediococcus pentosaceus promoting sleep or having antioxidant effects in in vivo experiments. Summary of the Invention

[0006] This invention provides a Pediococcus pentosaceus SC009 and its applications, which have sleep-improving (promoting sleep) and antioxidant effects, and can be used to prepare foods, health foods and drugs that improve sleep and have antioxidant effects.

[0007] This invention first provides a Pediococcus pentosaceus SC009, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 28, 2021, with accession number CGMCC No. 22796, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, postal code 100101.

[0008] The morphological characteristics of Pediococcus pentosaceus SC009 provided by this invention are: Gram-positive, non-spore-forming, non-motile, obligate anaerobic tetracoccus.

[0009] The Pediococcus pentosaceus SC009 provided by this invention has a survival rate of over 35% after treatment at pH 3.0 for 4 hours.

[0010] The survival rate of Pediococcus pentosaceus SC009 provided by this invention reached over 70% after 4 hours of treatment in bovine bile at a concentration of 0.3-1.0%.

[0011] The live and heat-inactivated Pediococcus pentosaceus SC009 bacteria provided by this invention can significantly shorten sleep latency and prolong sleep duration in mice induced by sodium pentobarbital, thereby improving sleep quality.

[0012] The Pediococcus pentosaceus SC009 provided by this invention can significantly increase the total SOD and reduced glutathione in the serum of middle-aged and elderly rats, and reduce the content of MDA and protein carbonyl groups, thus exhibiting significant antioxidant activity.

[0013] The Pediococcus pentosaceus SC009 provided by this invention can prolong the survival time of aged rats.

[0014] This invention provides a microbial product containing Pediococcus pentosaceus SC009, which can be any dosage form containing live or inactivated Pediococcus pentosaceus SC009.

[0015] This invention further provides a freeze-dried bacterial powder of *Pediococcus pentosaceus* SC009, prepared by inoculating *Pediococcus pentosaceus* SC009 into a solid culture medium for anaerobic culture to obtain seed culture, which is then inoculated into a liquid culture medium for fermentation. The supernatant after fermentation is the bacterial solution, which is then freeze-dried under vacuum to obtain the freeze-dried bacterial powder. Preferably, the liquid culture medium is any culture medium suitable for lactic acid bacteria; the solid culture medium is obtained by adding agar to the liquid culture medium; the fermentation temperature in the liquid culture medium is 35-37℃, and the fermentation time is 12-72 hours.

[0016] This invention provides the use of Pediococcus pentosaceus SC009 or its freeze-dried bacterial powder in the preparation of foods that improve and promote sleep. The foods can be in the form of dairy products, compressed candies, beverages, desserts, candies, sauces, and any other common or health foods.

[0017] This invention provides the use of the aforementioned Pediococcus pentosaceus SC009 in the preparation of a medicament for treating sleep disorders, specifically insomnia.

[0018] The aforementioned Pediococcus pentosaceus SC009 can be used to prepare sleep-improving foods, sleep-improving health foods, or drugs for treating sleep disorders. The preparation method specifically includes:

[0019] Pediococcus pentosaceus SC009 is fermented and then freeze-dried under vacuum to obtain freeze-dried bacterial powder. The freeze-dried bacterial powder is added to food, health food or medicine to obtain sleep-improving food, sleep-improving health food or medicine for treating sleep disorders.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention discovers a novel *Pediococcus pentosaceus* SC009, which possesses sleep-promoting and antioxidant properties. It can be used to prepare foods, health foods, or drugs for treating sleep disorders that improve sleep and provide antioxidant effects. In animal experiments, *Pediococcus pentosaceus* SC009 significantly shortened sleep latency and prolonged sleep duration in mice, improving sleep quality. It also significantly increased the levels of total SOD and reduced glutathione in the serum of middle-aged and aged rats, while decreasing the levels of MDA and protein carbonyl groups, exhibiting significant antioxidant activity. Furthermore, it prolonged the survival time of aged rats. Due to its sleep-promoting and antioxidant effects, *Pediococcus pentosaceus* SC009 is suitable for market promotion and has broad application prospects. Attached Figure Description

[0022] Figure 1This is a comparative graph showing the effects of heat inactivation of SC009 on sleep latency (A) and sleep duration (B) in sodium pentobarbital-induced sleep mice. Figure 1 Figure A shows a comparison of the effects of heat inactivation of SC009 on sleep latency in sodium pentobarbital-induced sleep mice. Figure 1 Figure B shows a comparison of the effects of heat inactivation of SC009 on sleep duration in sodium pentobarbital-induced sleep mice. Detailed Implementation

[0023] The following embodiments are illustrative only, and the present invention is not limited to these embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0025] In the following examples, the reagents, materials, instruments and equipment used are all commercially available.

[0026] The Pentosacchariformis SC009 of the present invention can be first inoculated into a fixed culture medium suitable for the growth of lactic acid bacteria for anaerobic culture to obtain seeds, and then inoculated into a liquid culture medium suitable for the growth of lactic acid bacteria.

[0027] A suitable liquid culture medium for the growth of Pediococcus pentosaceus SC009, which is a prior art medium suitable for the culture of lactic acid bacteria, as is known to those skilled in the art, may specifically contain: hydrolyzed casein 1-10 g / L, plant peptone 1-5 g / L, yeast extract 1-2 g / L, glucose 1-5 g / L, L-cysteine ​​0.2-0.5 g / L, dipotassium hydrogen phosphate 0.5-2.0 g / L, magnesium chloride 0.2-0.5 g / L, zinc sulfate 0.10-0.25 g / L, calcium chloride 0.05-0.15 g / L, ferric chloride 0.0001-0.0005 g / L, Tween 80 1.0-2.0 g / L, pH=6.5-7.4.

[0028] A suitable immobilized culture medium for the growth of Pediococcus pentosaceus SC009 can be prepared by adding 10-30 g / L of agar to the liquid culture medium.

[0029] The culture conditions suitable for Pediococcus pentosaceus SC009 can be any existing conditions suitable for lactic acid bacteria culture, such as a temperature of 35-37°C and a culture time of 12-72 hours.

[0030] Example 1. Isolation and purification of Pediococcus pentosaceus SC009

[0031] Using pickled cabbage liquid from Zhejiang as a sample, the culture was inoculated into MRS solid medium and anaerobically cultured at 37°C for 48 h. Smooth white colonies were picked and Gram-stained; under a microscope, the bacteria were observed to be Gram-positive, non-spore-forming tetrad cocci. These colonies were then inoculated into MRS liquid medium and anaerobically cultured at 37°C for 48 h. The genome was extracted, and 16S rDNA was amplified by PCR, sequenced, and compared, identifying and preserving *Pediococcus pentosaceus* SC009.

[0032] Example 2. Acid resistance characteristics of Pediococcus pentosaceus SC009

[0033] Pediococcus pentosaceus SC009 was suspended in PBS at pH 1.0, 2.0, 3.0, 4.0, and 7.0, and incubated at 37°C for 1 h, 2 h, 3 h, and 4 h. The viability was determined by plate counting. The viability of Pediococcus pentosaceus SC009 at different pH conditions and time points is shown in Table 1.

[0034] Table 1. Viability of Pediococcus pentosaceus SC009 under acidic conditions

[0035]

[0036] Note: This experiment was conducted in 3 parallel trials, and the data are the mean ± SD.

[0037] Example 3. Bile resistance characteristics of Pediococcus pentosaceus SC009

[0038] Pediococcus pentosaceus SC009 was suspended in PBS containing 0%, 0.3%, 0.6%, and 1% bovine bile and incubated at 37°C for 1 h, 2 h, 3 h, and 4 h. Viability was determined using the plate count method. The viability of Pediococcus pentosaceus SC009 at different bile concentrations and time points is shown in Table 2. Pediococcus pentosaceus was resistant to 0.3%, 0.6%, and 1% bovine bile.

[0039] Table 2. Viability of Pediococcus pentosaceus SC009 after treatment with bovine bile

[0040]

[0041] Note: This experiment was conducted in 3 parallel trials, and the data are the mean ± SD.

[0042] Example 4. Biochemical reaction results of Pediococcus pentosaceus SC009

[0043] Biochemical identification experiments: Single colonies of *Pediococcus pentosaceus* grown on MRS plates were picked and subjected to biochemical reaction detection using the bioMérieux fully automated microbial biochemical identification system (VITEK 2 Systems Version). The main biochemical reactions of *Pediococcus pentosaceus* SC009 are shown in Table 3.

[0044] Table 3. Biochemical reaction results of Pediococcus pentosaceus SC009

[0045]

[0046] Example 5. Sleep-promoting properties of live and heat-inactivated Pediococcus pentosaceus SC009 bacteria.

[0047] (1) Take Pediococcus pentosaceus SC009 that has been anaerobic for 24 hours, centrifuge, wash three times with PBS, and dilute to 10⁻⁶. 9 A sample with approximately CFU / mL is considered a live bacteria sample.

[0048] (2) Take a portion of the live bacteria sample and boil it in boiling water for 15 minutes. This is the heat-inactivated bacteria sample after heat inactivation treatment.

[0049] (3) Take 20-24g of ICR mice and administer 2-4 × 10 mg per mouse per day. 8 Mice were administered live and heat-inactivated Pediococcus pentosaceus SC009 at CFU / kg via gavage for 14 consecutive days; mice in the blank control group were given the same volume of PBS for 14 consecutive days; mice in the positive control group were given the same volume of PBS for 13 consecutive days, and then given 2.5 mg / kg of diazepam on the fourteenth day.

[0050] (4) Intraperitoneally inject 50 mg / kg sodium pentobarbital and calculate the sleep latency and sleep duration of the mice. The sleep latency is defined as the period from the injection of sodium pentobarbital until the mouse's retrograde reflex disappears, and the sleep duration is defined as the period from the disappearance of the retrograde reflex until its recovery.

[0051] The results showed that both live and heat-inactivated *Pediococcus pentosaceus* SC009 significantly shortened the sleep latency and prolonged the sleep duration in mice, with a 200% increase in sleep duration. Figure 1 ). Figure 1 In the figure, (A) illustrates the effect of Pediococcus pentosaceus SC009 on the sleep latency of sodium pentobarbital-induced sleep in mice, and (B) illustrates the effect of Pediococcus pentosaceus SC009 on the sleep duration of sodium pentobarbital-induced sleep in mice; each group contained 7-8 mice. p < 0.05; p < 0.001.

[0052] Example 6. Antioxidant effect of Pediococcus pentosaceus SC009 on middle-aged and aged rats

[0053] (1) Take Pediococcus pentosaceus SC009 that has been anaerobic for 24 hours, centrifuge, wash three times with PBS, and dilute to 10⁻⁶. 9 Approximately CFU / mL;

[0054] (2) Take 14-month-old SD rats and administer 1-3 × 10⁻⁶ doses per rat per day.9 CFU-containing Pediococcus pentosaceus SC009 was administered by gavage for 28 consecutive days; the blank control group rats were given the same volume of PBS for 28 consecutive days.

[0055] (3) After the rats were sacrificed, blood samples were collected to detect antioxidant indicators (total SOD, MDA, protein carbonyl and reduced glutathione).

[0056] The results showed that Pediococcus pentosaceus SC009 significantly increased total SOD and reduced glutathione in the serum of middle-aged and elderly rats, and reduced the content of MDA and protein carbonyl groups, exhibiting significant antioxidant activity (Table 4).

[0057] Table 4. Antioxidant indices of Pediococcus pentosaceus SC009 in aged rats

[0058]

[0059] Note: Each group in this experiment contained 8 SD rats, and the data are mean ± SD. p < 0.05; p < 0.01; p < 0.001

[0060] Example 7 Preparation of bacterial culture

[0061] The Pentosacchariformis SC009 of the present invention can be first inoculated into a suitable solid culture medium for anaerobic culture to obtain seeds, and then inoculated into a suitable fermentation broth for fermentation. The supernatant after fermentation is the bacterial culture.

[0062] Example 8: Preparation of freeze-dried bacterial powder

[0063] The Pentosacchariformis SC009 of the present invention can be first inoculated into a suitable solid culture medium for anaerobic culture to obtain seeds, and then inoculated into a suitable fermentation broth for fermentation. The supernatant after fermentation is the bacterial solution, which is then dried in a low-temperature vacuum freeze dryer to obtain freeze-dried bacterial powder.

[0064] Example 9 Preparation of Capsules

[0065] The freeze-dried bacterial powder of Pediococcus pentosaceus SC009 of the present invention is placed in capsules to form a capsule preparation.

[0066] Example 10 Preparation of tablets

[0067] The lyophilized bacterial powder of Pediococcus pentosaceus SC009 of the present invention is compressed into tablets.

[0068] Example 11 Preparation of Granules

[0069] The freeze-dried bacterial powder of Pediococcus pentosaceus SC009 of the present invention is mixed evenly with an appropriate amount of sugar powder, granulated with an appropriate amount of ethanol, dried, granulated, and packaged.

[0070] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A type of Pediococcus pentosaceus SC009, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 22796.

2. A fermentation method for Pediococcus pentosaceus SC009 according to claim 1, characterized in that, The process includes the following steps: Pediococcus pentosaceus SC009 is first inoculated into a solid culture medium for anaerobic culture to obtain seed culture, and then inoculated into a liquid culture medium for fermentation. The supernatant after fermentation is the bacterial culture.

3. The fermentation method for Pediococcus pentosaceus SC009 according to claim 2, characterized in that, The liquid culture medium is any culture medium suitable for lactic acid bacteria cultivation; the solid culture medium is obtained by adding agar to the liquid culture medium; the fermentation temperature in the liquid culture medium is 35-37℃, and the fermentation culture time is 12-72h.

4. A freeze-dried bacterial powder of Pediococcus pentosaceus SC009, characterized in that, The preparation method is as follows: the bacterial solution obtained by the method described in claim 2 or 3 is subjected to vacuum freeze-drying to obtain freeze-dried bacterial powder.

5. The application of Pediococcus pentosaceus SC009 as described in claim 1 in the preparation of food products that improve sleep and have antioxidant properties.

6. The use of Pediococcus pentosaceus SC009 as described in claim 1 in the preparation of a medicament for treating sleep disorders.

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