Paracaseicillus casei, screening method and application thereof

Abstract

The present application relates to a kind of Lacticaseibacillus paracasei SMB10, which is preserved in China General Microbiological Culture Collection Center on November 14, 2022, and the preservation number is CGMCC No.26151.The present application also provides a method for screening Lacticaseibacillus paracasei as described above and application.The strain of the present application can be directly applied to selenite conversion, and can be further applied to food production and animal feed industries.In addition, the screening method of Lacticaseibacillus paracasei provided by the present application is simple, and the purity of the obtained strain is higher.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a type of Lactobacillus paracasei, its screening method, and its application. Background Technology

[0002] Selenium is an essential trace element, crucial for the normal development of animals and humans. Studies have shown that within safe limits, selenium not only possesses antioxidant, anti-aging, and cell-protecting and repairing activities, but also enhances the body's immune system and detoxification functions. However, outside these safe limits, selenium deficiency can lead to Keshan disease and Kashin-Beck disease, while excessive selenium intake can cause selenium poisoning. The nutritional value of selenium depends primarily on the type and concentration of selenium present in food or artificially supplemented. In nature, selenium mainly exists in inorganic forms, including selenate [Se(VI)], selenite [Se(IV)], elemental selenium [Se(O)], and [Se(-II)]. Organic forms include selenocysteine, selenomethionine, and selenoproteins. Compared to inorganic selenium, organic selenium and nano-selenium have higher bioavailability, lower toxicity, and are more readily absorbed and safely utilized by plants and animals. Organic selenium and nano-selenium can be synthesized through physical and chemical methods, but their production costs are high and their yields are low. However, by using microorganisms, inorganic selenium can be converted into organic and nano-selenium forms with high nutritional value. The biotransformation of selenium has gradually become a focus of attention. Summary of the Invention

[0003] (a) Technical problems to be solved

[0004] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a Lactobacillus paracasei that can efficiently convert selenite, a screening method thereon, and its application.

[0005] (II) Technical Solution

[0006] To address the aforementioned issues, this invention provides a strain of *Lacticaseibacillus paracasei* SMB10, which was deposited on November 14, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26151.

[0007] The present invention also provides a method for screening Lactobacillus paracasei as described above, comprising:

[0008] S1. Preparation of bacterial culture of small intestinal contents;

[0009] S2. The bacterial culture is inoculated into MRS liquid medium and cultured to obtain a small intestinal contents bacterial suspension.

[0010] S3. After diluting the bacterial suspension to different concentrations, spread it on MRS solid medium and incubate it statically to obtain colonies with different characteristics.

[0011] S4. The colonies with different characteristics are inoculated onto MRS solid medium using the four-zone streak method and incubated statically.

[0012] S5. Repeat step S4 until the colonies growing on the MRS solid medium are of a single type, and multiple pure colonies are obtained.

[0013] S6. Multiple pure colonies were inoculated onto MRS solid medium containing selenite and cultured statically. Strains with growth advantage and red color were selected to obtain the Lactobacillus paracasei, whose preservation number is CGMCC No. 26151.

[0014] Optionally, each liter of the MRS liquid culture medium comprises: S2, wherein each liter of the MRS liquid culture medium comprises: 9-12 g peptone, 8-10 g beef extract, 3-5 g yeast extract, 18-22 g glucose, 2-4 g anhydrous dipotassium hydrogen phosphate, 2-4 g anhydrous triammonium citrate, 4-6 g sodium acetate trihydrate, 0.2-0.3 g magnesium sulfate heptahydrate, 0.05-0.06 g manganese sulfate tetrahydrate, and 1-2 g Tween-80.

[0015] Optionally, 18-20g of agar is added to each liter of the MRS liquid culture medium to obtain the MRS solid culture medium.

[0016] Optionally, S1 specifically includes: taking a section of goat small intestine, rinsing the outer surface of the casing with sterile distilled water, cutting the casing with sterile scissors, scraping the contents of the small intestine into sterile distilled water with a sterile blade, stirring evenly, and then placing it on a shaker to obtain a bacterial solution of the contents of the small intestine.

[0017] Optionally, in S3, diluting the bacterial suspension to different concentrations specifically involves diluting the bacterial cell concentration in the bacterial suspension to 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 times.

[0018] Optionally, in S6, multiple pure colonies are inoculated onto MRS solid medium containing selenite and incubated statically using the four-zone streak method.

[0019] The present invention also provides the application of Lactobacillus paracasei as described above in selenite conversion.

[0020] Optionally, in the above applications, the initial selenite concentration is ≤300 mg·L⁻¹. -1 The selenite is a soluble selenite such as sodium selenite or potassium selenite.

[0021] (III) Beneficial Effects

[0022] The *Lactobacillus paracasei* SMB10 of this invention is a selenium-enriched probiotic capable of converting selenite. As a probiotic, this strain, once alive in the intestines, can provide the human body with essential nutrients such as gastrointestinal motility, promote digestion and absorption, and enhance immunity. Furthermore, this strain exhibits high tolerance to inorganic selenium, including selenite, and can convert a large portion of selenite into nano-selenium with excellent conversion efficiency. This strain can accumulate selenium from the diet, thereby influencing the host's selenium intake and subsequently altering the expression of several selenoproteins in the host, directly providing a rich source of organic selenium (mainly selenoproteins). This strain can be directly applied to the bioconversion process of selenite and can also be further applied in food production and animal feed industries. In addition, the screening method for *Lactobacillus paracasei* provided by this invention is simple, and the obtained strain has high purity. Attached Figure Description

[0023] Figure 1 (a) is an electrophoresis diagram of PCR amplification of Lactobacillus paracasei SMB10 according to the present invention, and (b) is an electrophoresis diagram of DNA marker.

[0024] Figure 2 This invention provides a phylogenetic tree of Lactobacillus paracasei SMB10 based on its 16S rDNA sequence.

[0025] Figure 3 This is a selenium standard curve diagram of the present invention;

[0026] Figure 4 This is a graph illustrating the changes over time in the biotransformation of selenium by *Lactobacillus paracasei* in different concentrations of sodium selenite, as described in this invention. Detailed Implementation

[0027] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] This invention provides a Lacticaseibacillus paracasei SMB10, which was deposited on November 14, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26151. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] The present invention also provides a method for screening Lactobacillus paracasei as described above, comprising:

[0030] S1. Preparation of bacterial culture of small intestinal contents;

[0031] S1 specifically includes: taking a section of goat small intestine, rinsing the outer surface of the casing with sterile distilled water, cutting the casing open with sterile scissors, scraping the contents of the small intestine into sterile distilled water with a sterile blade, stirring evenly, and then placing it on a shaker to obtain a bacterial solution of the contents of the small intestine.

[0032] The obtained bacterial culture was cultured in a constant temperature incubator at 36℃ with shaking at 160 rpm for 20 min.

[0033] S2. The bacterial solution is inoculated into MRS liquid culture medium and cultured to obtain a small intestinal contents bacterial suspension.

[0034] Specifically, in one embodiment, the upper layer of the cultured bacterial solution is inoculated into MRS liquid medium at 5% (V / V) and cultured in a constant temperature incubator at 36°C with shaking at 160 rpm for 24 h to obtain a small intestinal contents bacterial suspension.

[0035] In S2, each liter of the MRS liquid culture medium comprises: 9-12 g peptone, 8-10 g beef extract, 3-5 g yeast extract, 18-22 g glucose, 2-4 g anhydrous dipotassium hydrogen phosphate, 2-4 g anhydrous triammonium citrate, 4-6 g sodium acetate trihydrate, 0.2-0.3 g magnesium sulfate heptahydrate, 0.05-0.06 g manganese sulfate tetrahydrate, and 1-2 g Tween-80.

[0036] S3. After diluting the bacterial suspension to different concentrations, spread it on MRS solid medium and incubate it statically to obtain colonies with different characteristics.

[0037] Specifically, the method for preparing MRS solid culture medium is to add 18-20g of agar to the above-mentioned MRS liquid culture medium.

[0038] Specifically, the cultured bacterial suspension is serially diluted with sterile water, which can be done at 10:10 ratios. -1 10-2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Dilute the bacterial suspension to a higher concentration. After dilution, take 50 μL of the bacterial suspension and spread it onto MRS solid medium. Perform three replicates for each dilution. Incubate the medium upside down in a 37°C incubator for about 48 h until obvious colonies are formed.

[0039] S4. Inoculate colonies with different characteristics into MRS solid medium and incubate statically;

[0040] S5. Repeat step S4 until the colonies growing on the MRS solid medium are of a single type, resulting in multiple pure colonies.

[0041] Specifically, single colonies with significantly different colony characteristics were selected and streaked onto MRS solid medium using a four-zone method. The medium was then incubated at 36°C for 48 hours. This step was repeated until multiple pure colonies were obtained.

[0042] Colony characteristics include colony morphology, color, surface texture, and edge shape.

[0043] S6. Multiple pure colonies were inoculated into MRS solid medium containing selenite and cultured statically. Strains with growth advantage and red color were selected to obtain the above-mentioned Lactobacillus paracasei, which has the ability to convert selenite.

[0044] The present invention also provides an application, specifically: applying *Lactobacillus paracasei* as described above in the conversion of selenite. Optionally, the selenite can be sodium selenite or potassium selenite.

[0045] Specifically, preferably, the initial selenium concentration in sodium selenite is ≤300 mg·L⁻¹. -1 At this concentration, the conversion rate exceeds 90%.

[0046] The *Lactobacillus paracasei* SMB10 of this invention is a selenium-enriched probiotic capable of converting selenite. As a probiotic, this strain, once alive in the gut, can provide the human body with essential nutrients such as gastrointestinal motility, promote digestion and absorption, and enhance immunity. Furthermore, this strain exhibits high tolerance to inorganic selenium, including selenite, and can convert inorganic selenium into organic selenium. Therefore, this strain can accumulate selenium from the diet, influencing the host's selenium intake and thereby altering the expression of several selenoproteins in the host, directly providing a rich source of organic selenium (mainly selenoproteins). This strain can be directly applied to selenite conversion and can also be further applied in food production and animal feed industries. In addition, the screening method for *Lactobacillus paracasei* provided by this invention is simple, and the obtained strain has high purity.

[0047] Example

[0048] Example 1: Screening of Lactobacillus paracasei strains

[0049] (1) Take about 10 cm of goat small intestine and rinse the outer surface of the casing with sterile water. Cut open the casing with sterile scissors, and then scrape out all the contents of the small intestine with a sterile blade and place them in a conical flask containing sterile water. Add sterile glass beads and mix thoroughly. Incubate at 36℃ and shake at 160 rpm for 20 min.

[0050] (2) Take the upper layer of the cultured bacterial solution and inoculate it into MRS liquid medium at 5% (V / V), and culture it in a constant temperature incubator at 36℃ with shaking at 160 rpm for 24 h to obtain a small intestinal contents bacterial suspension.

[0051] (3) Dilute the cultured bacterial suspension with sterile water in a serial dilution series, with each dilution being 10:10. -5 10 -6 10 -7 10 -8 Spread 50 μL of the bacterial suspension onto MRS solid medium, with three replicates for each dilution. Incubate in an inverted 37°C incubator until obvious colonies are formed, which takes approximately 48 h. Select single colonies with significantly different colony characteristics and streak them onto MRS solid medium using a four-zone streak method. Incubate at 36°C for 48 h. Repeat this step until multiple pure colonies are obtained.

[0052] (4) The obtained pure colonies were inoculated onto MRS solid medium containing sodium selenite and cultured at 36°C until obvious colonies grew. During the growth process, the strain converted most of the sodium selenite into nano-selenium. Since nano-selenium is red, the converted selenium-enriched strain has red characteristics. Therefore, the red selenium-enriched strain with strong growth and obvious nano-selenium conversion effect was selected and retained to obtain the desired Lactobacillus paracasei.

[0053] Example 2: Strain Identification

[0054] The genus of *Lactobacillus paracasei* strain cultured by Sangon Biotech (Shanghai) Co., Ltd. was identified using 16S RNA. Genomic DNA was extracted using the SK8255 kit, and PCR amplification was performed using universal bacterial primers. The amplified bands were then detected by electrophoresis. (The electrophoresis results are shown in the image.) Figure 1 As shown in (a) and (b). Gel electrophoresis conditions were: 1% agarose gel electrophoresis, 150V, 100 mA, 20 min for observation. Figure 1 The results showed that after PCR amplification, the bacterial strain yielded a single band by gel electrophoresis. The fragment length compared with the DNA Marker was approximately 1500 bp, which was consistent with the length of the target product (1508 pb). Therefore, the identification results were reliable.

[0055] (1) The primers used are shown in Table 1.

[0056] Table 1: Primers used for strain identification

[0057]

[0058] (2) The PCR amplification reaction system is shown in Table 2.

[0059] Table 2: PCR amplification reaction system

[0060]

[0061] (3) The PCR reaction conditions are shown in Table 3.

[0062] Table 3 PCR reaction conditions

[0063]

[0064] After 16S rDNA gene sequencing, BLAST alignment analysis was performed on NCBI, and a phylogenetic tree was constructed. The results are as follows: Figure 2 As shown, the analysis results indicate that strain SMB10 has high homology with *Lactobacillus paracasei* subsp. *tolerans* strain H (MG818769.1) and is quite distant from other lactobacilli. Based on these characteristics, the strain screened in this example was named *Lactobacillus paracasei* SMB10. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2022, with accession number CGMCC No. 26151.

[0065] Gene sequencing of *Lactobacillus paracasei* yielded the following nucleotide sequence:

[0066] Lacticaseibacillus paracasei

[0067]

[0068] Example 3: Application of Lactobacillus paracasei SMB10 in the conversion of selenite

[0069] (1) Activation of strain: The selected Lactobacillus paracasei was inoculated into an Erlenmeyer flask containing MRS liquid culture medium and cultured at 36°C and 160 rpm for 12 h to activate the strain and serve as seed culture.

[0070] (2) Preparation of sodium selenite stock solution: First, weigh 2.19 g of sodium selenite and dissolve it in 100 mL of deionized water to prepare a 10 g·L⁻¹ solution. -1 The sodium selenite stock solution was prepared by passing it through a sterile 0.22 μm nylon membrane using a sterile syringe, and then diluting the sterile sodium selenite stock solution to a selenium content of 100 mg·L⁻¹. -1 200 mg·L -1 300 mg·L -1 400 mg·L -1 500 mg·L -1 .

[0071] (3) Transformation of sodium selenite by *Lactobacillus paracasei* SMB10: The seed culture was inoculated into an Erlenmeyer flask containing MRS liquid medium at an inoculation rate of 3% (V / V), and cultured at 36℃ with shaking at 160 rpm for 8 h. Then, a sterile sodium selenite stock solution was added, and the culture was incubated at 36℃ with shaking at 160 rpm for 36 h. Since the sodium selenite solution is colorless while the biotransformed nano-selenium is red, the color of the bacterial suspension changed from colorless to red before and after culture. During the culture process, the bacterial suspension was sampled at different times, and the bacterial suspension samples obtained at different times were centrifuged. The supernatant was diluted to a certain factor, and the content of unconverted selenium in the supernatant after centrifugation was detected by atomic fluorescence spectrometry.

[0072] (4) Calculation of the conversion rate of sodium selenite by Lactobacillus paracasei

[0073] To plot the selenium standard curve: take 1000 mg∙L -1 1 mL of selenium standard solution was diluted to 100 mL with 10% dilute hydrochloric acid to obtain a 10 mg·L⁻¹ solution. -1 Selenium standard intermediate solution was further diluted to 1 μg∙L with 10% dilute hydrochloric acid. -1 2 μg∙L -1 5 μg∙L -1 10 μg∙L -1 20 μg∙L -1Different concentration gradients were investigated. Detection was performed using a hydride-based atomic fluorescence spectrometer, with a 15% potassium borohydride aqueous solution as the reducing agent. A standard curve was plotted with the concentration gradient on the x-axis and fluorescence intensity on the y-axis, as shown below. Figure 3 As shown.

[0074] The red bacterial suspension after biotransformation was centrifuged, and the supernatant was diluted to a certain factor and the content of unconverted selenium (C) was detected by atomic fluorescence spectrometry. t The reducing solution for the instrument is a 15% potassium borohydride aqueous solution.

[0075]

[0076] In the above formula:

[0077] C0 – Initial selenium concentration for the conversion of sodium selenite by *Lactobacillus paracasei*.

[0078] C t —Concentration of unconverted selenium in the supernatant after time t

[0079] like Figure 4 As shown, based on the changes in selenium biotransformation over time by *Lactobacillus paracasei* in different concentrations of sodium selenite, it can be seen that when the initial selenium concentration is ≤300 mg·L⁻¹, the biotransformation of selenium is significantly reduced. -1 At that time, the conversion efficiency of sodium selenite by *Lactobacillus paracasei* was basically stable after 24 hours, with conversion rates reaching 90% or higher. When the initial selenium concentration was 400 mg·L⁻¹, the conversion efficiency remained relatively stable. -1 At that time, the sodium selenite conversion rate of Lactobacillus paracasei was approximately 67.76% after 48 hours, when the initial selenium concentration was 500 mg·L⁻¹. -1 At that time, the sodium selenite conversion rate of *Lactobacillus paracasei* was only about 49.35% after 48 hours. Therefore, it can be concluded that when the initial selenium concentration is ≤300 mg·L⁻¹, the conversion rate of sodium selenite is relatively low. -1 After 24 hours, the conversion effect stabilized, with a conversion efficiency of 90% or higher. The *Lactobacillus paracasei* strain described in this application can be used for selenium concentrations ≤300 mg·L⁻¹. -1 In its conversion applications, the conversion conditions are mild, requiring only a weakly acidic environment with a pH of 5-6 and a temperature of around 36 degrees Celsius.

[0080] It should be understood that the above description of specific embodiments of the present invention is only for illustrating the technical approach and features of the present invention, and is intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. However, the present invention is not limited to the specific embodiments described above. All changes or modifications made within the scope of the claims of the present invention should be covered within the protection scope of the present invention.

Claims

1. A strain of *Lactaseibacillus paracasei* SMB10, characterized in that, The Lacticaseibacillus paracasei SMB10 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2022, with accession number CGMCC No. 26151.

2. The application of *Lactobacillus paracasei* as described in claim 1 in selenite conversion.

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