Application of lactobacillus helveticus Zhedu LBH-VI strain in selenium-rich products

Abstract

The invention provides an application of a lactobacillus helveticus Zhegu LBH-VI strain in a selenium-rich product, the selenium enrichment rate of the lactobacillus helveticus Zhegu LBH-VI strain with the preservation number of CCTCC NO: M 2023095 is greater than 97.90%, the inorganic selenium resistant concentration is greater than 70 [mu] g / mL, and the selenium-rich product prepared from the lactobacillus helveticus Zhegu LBH-VI strain has an obvious anti-oxidation effect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to application of Lactobacillus helveticus Zhegu LBH-VI strain in selenium-enriched products. Background Art

[0002] Selenium is a trace element necessary for human metabolism. It can protect the body from oxidative stress, regulate growth and development, help with anti-inflammatory, anti-infection and anti-viral effects, and has certain anti-cancer and anti-cancer functions. Selenium deficiency can lead to poor immune response, decreased cognitive ability, osteomalacia and Keshan disease. The Chinese Nutrition Society recommends a daily selenium intake of 50 to 250 μg per person, but the daily selenium intake of some people is far from the recommended intake. Selenium mainly exists in inorganic form in nature, but low concentrations of inorganic selenium have toxic side effects. Therefore, although selenium is an essential element for organisms, a slight excess of the necessary nutritional level will lead to poisoning, so its use must be strictly controlled. At the same time, the bioavailability of selenium is directly related to the chemical form. Inorganic selenium is metabolized quickly and is not easily absorbed. Therefore, it is crucial to explore and invent methods for converting inorganic selenium into organic selenium.

[0003] At present, the methods for converting inorganic selenium into organic selenium are mostly biotransformation methods, including plant enrichment method and microbial transformation method. The plant enrichment method mainly increases the selenium content in the soil, sprays selenium solution on plants, and uses the root tissue of plants to achieve selenium conversion; the microbial transformation method mainly adds selenium salts to the culture medium, so that microorganisms can metabolize inorganic selenium during growth and convert it into organic selenium such as selenoprotein, selenocysteine, methylselenocysteine, etc., thereby reducing the toxic side effects of inorganic selenium and improving the physiological activity and absorption rate of selenium. Because the microbial transformation method has the advantages of rapidity, high efficiency, and environmental protection, it is a method that is currently being studied and applied more. However, yeast is currently the most studied selenium-enriched microorganism. Selenium-enriched yeast has entered the industrialization stage, formed a complete production line and has been promoted to the market, while the research and invention on selenium-enriched lactic acid bacteria are still few and imperfect.

[0004] CN110317757A discloses a plant lactobacillus HJ-S2, which is isolated and screened from the intestine of beaked whales living below 300 meters in the deep sea, has good cholesterol degradation effect, and has certain selenium-enriching effect. However, the organic selenium conversion rate of the invented strain is low, and the selenium-enriching rate of the strain after the selenium-resistance acclimation experiment is only 52.31%, the yield of the product is low, and the selenium-enriching rate of the strain is low, indicating that there is more residual inorganic selenium in the fermentation liquid, which increases the production cost and sewage treatment cost, and is not conducive to industrial production.

[0005] CN 114686415 A studies Bacillus coagulans BC99 screened from a strain library. The strain has a good selenium-enriched effect. After selenium-resistant domestication, the strain's antioxidant capacity is also enhanced, and the DPPH free radical scavenging rate is significantly improved. However, the strain of the invention has a low tolerance to selenium, and its sodium selenite concentration range is 20-30 μg / mL. If it is used in actual production, the content of sodium selenite in the culture solution needs to be strictly controlled, otherwise it may cause the strain to grow slowly or poison. At the same time, to meet the production of a large number of selenium-enriched lactic acid bacteria, multiple batches of fermentation are required, which increases the difficulty of process control and production costs. Summary of the invention

[0006] The problem existing in the current prior art is that, in the research on converting inorganic selenium into organic selenium by microbial conversion, the research on selenium-rich lactic acid bacteria is still relatively small and incomplete. The existing selenium-rich strains have a low conversion rate for converting inorganic selenium into organic selenium, and have low tolerance to inorganic selenium, which increases the difficulty and cost of the fermentation process. The cost of fermentation liquid wastewater treatment is high, which is not conducive to industrial production.

[0007] The present invention isolates a strain of Lactobacillus helveticus that has the ability to efficiently convert organic selenium. The strain has excellent selenium resistance and selenium-enrichment characteristics and strong stress resistance, and has a wide range of applications. Specifically, the present invention proposes the following technical solutions:

[0008] In a first aspect, the present invention provides an application of Lactobacillus helveticus Zhegu LBH-VI strain in selenium-enriched products, wherein the Lactobacillus helveticus Zhegu LBH-VI strain is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2023095.

[0009] Preferably, the Lactobacillus helveticus Zhegu LBH-VI strain is used in selenium-enriched products, characterized in that the selenium-enrichment rate of the strain is greater than 97.90%, and the concentration of inorganic selenium resistant to the strain is greater than 70 μg / mL; preferably, the inorganic selenium includes sodium selenite.

[0010] Preferably, the use of the Lactobacillus helveticus Zhegu LBH-VI strain in selenium-enriched products is characterized in that the products include selenium-enriched bacterial powder, selenium-enriched bacterial agent, selenium-enriched food, selenium-enriched feed, selenium-enriched health products or selenium-enriched medicines.

[0011] In a second aspect, the present invention provides a selenium-enriched freeze-dried bacterial powder, characterized in that it comprises the Lactobacillus helveticus Zhegu LBH-VI strain with a preservation number of CCTCC NO: M 2023095 after selenium-enriched culture.

[0012] In a third aspect, the present invention provides a method for preparing selenium-enriched freeze-dried bacterial powder, comprising freeze-drying the Lactobacillus helveticus Zhegu LBH-VI strain with a preservation number of CCTCCNO: M 2023095 after selenium-enriched cultivation.

[0013] Preferably, the method for preparing the selenium-enriched freeze-dried bacterial powder comprises the following steps:

[0014] (1) activating the Lactobacillus helveticus Zhegu LBH-VI strain in a culture medium;

[0015] (2) subjecting the activated strain to high-density culture and fermentation, and adding sterile sodium selenite solution during the fermentation;

[0016] (3) centrifuging the fermented bacterial solution, discarding the supernatant, and obtaining selenium-enriched Lactobacillus helveticus bacterial sludge;

[0017] (4) Mixing the selenium-enriched Lactobacillus helveticus slurry with a freeze-drying protective agent and freeze-drying.

[0018] Preferably, in the method for preparing selenium-enriched freeze-dried bacterial powder, in step (4), the bacterial mud is 1-2 parts by weight, and the freeze-drying protective agent is 3-5 parts by weight.

[0019] Preferably, in the method for preparing selenium-enriched freeze-dried bacterial powder, in step (4), the freeze-drying protective agent includes one or more of skim milk powder, maltodextrin, and erythritol; preferably, in parts by weight, the freeze-drying protective agent includes 4-6 parts of skim milk powder, 2-4 parts of maltodextrin, and 1-3 parts of erythritol.

[0020] In a fourth aspect, the present invention provides selenium-enriched freeze-dried bacterial powder prepared by the method for preparing selenium-enriched freeze-dried bacterial powder.

[0021] In a fifth aspect, the present invention provides a probiotic solid beverage, characterized in that it comprises the selenium-rich freeze-dried bacterial powder; preferably, the probiotic solid beverage comprises, by weight, 35-50 parts of selenium-rich freeze-dried bacterial powder, 20-40 parts of nut powder, 5-15 parts of fruit freeze-dried powder, and 5-15 parts of dietary fiber; preferably, the nut powder comprises one or more of chia seed powder, lotus seed powder, and cashew powder; further preferably, the fruit freeze-dried powder comprises one or more of navel orange, lemon, yellow peach, blueberry, cherry tomato, and cranberry; more preferably, the dietary fiber comprises one or more of fructooligosaccharide, galacto-oligosaccharide, and β-glucan.

[0022] In a sixth aspect, the present invention provides a compressed candy, characterized in that it comprises the selenium-rich freeze-dried bacterial powder; preferably, the probiotic compressed candy comprises, by weight, 35-50 parts of selenium-rich freeze-dried bacterial powder, 15-30 parts of fruit freeze-dried powder, 10-30 parts of sweetener, 0.5-1.5 parts of Vc, 0.5-1.5 parts of Ve, and 0.1-0.25 parts of magnesium stearate; preferably, the fruit freeze-dried powder comprises one or more of navel orange, lemon, yellow peach, blueberry, cherry tomato, and cranberry; further preferably, the sweetener comprises one or more of sorbitol, erythritol, sucralose, and maltitol; more preferably, the sweetener comprises 5-15 parts of sorbitol and 5-15 parts of maltitol.

[0023] In a seventh aspect, the present invention provides a biological bacterial agent, characterized in that the selenium-enriched freeze-dried bacterial powder and / or the cell lysate of the Lactobacillus helveticus ZheguLBH-VI strain with a preservation number of CCTCC NO: M 2023095 after selenium-enriched culture.

[0024] In an eighth aspect, the present invention provides the use of the selenium-enriched freeze-dried bacterial powder or the biological bacterial agent in selenium-enriched foods, selenium-enriched medicines, selenium-enriched health products and selenium-enriched feeds.

[0025] The beneficial effects of the present invention include:

[0026] Lactobacillus helveticus Zhegu LBH-VI has excellent selenium tolerance and selenium enrichment characteristics. The selenium enrichment rate of the strain is greater than 97.90%, and the concentration of inorganic selenium tolerance is greater than 70μg / mL. At the same time, the strain has strong stress resistance, and has the physiological characteristics of being resistant to low temperature, acid and bile salts, and can survive in artificial gastrointestinal fluid.

[0027] The preparation method of the selenium-enriched freeze-dried bacterial powder of the present invention is simple to operate and has a clean formula. It can be applied to foods such as probiotic solid beverages to regulate the balance of flora in the human gastrointestinal tract. It is rich in a large amount of organic selenium, which can significantly improve the body's antioxidant capacity and enhance the body's own immunity, while avoiding the toxic and side effects of excessive selenium on the body, and has broad application prospects.

[0028] Strain deposit information

[0029] The Lactobacillus helveticus Zhegu LBH-VI strain provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with a deposit number of CCTCC NO: M 2023095, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown is the colony morphology of Lactobacillus helveticus Zhegu LBH-VI strain;

[0031] Figure 2 Shown is a microscopic examination image of Lactobacillus helveticus Zhegu LBH-VI strain;

[0032] Figure 3 Shown are the growth curves of Lactobacillus helveticus Zhegu LBH-VI strain in MRS broth medium containing sodium selenite at a concentration of 70 μg / mL and blank MRS broth medium;

[0033] Figure 4 Shown is the sodium selenite standard curve; DETAILED DESCRIPTION

[0034] The Lactobacillus helveticus provided by the present invention is isolated from homemade yak milk residues of herders in Zhegu grassland at an altitude of 4600 meters in Tibet. The species identification is Lactobacillus helveticus (Lactobacillus helveticus), which is a natural wild-type microbial strain, belonging to the "List of Bacteria that can be used for food" update announcement issued by the National Health Commission on August 25, 2022, and can be widely used in various fermented dairy products. The Lactobacillus helveticus provided by the present invention is named: Lactobacillus helveticus (Lactobacillus helveticus) Zhegu LBH-VI strain; Lactobacillus helveticus of the present invention has excellent selenium resistance and selenium-rich characteristics, and at the same time, it has strong stress resistance, and it has the characteristics of low temperature resistance, acid resistance and bile resistance. The selenium-enriched probiotic solid beverage and selenium-enriched functional food prepared by the strain of the present invention have a high organic selenium content, and compared with the solid beverage prepared by the unselenium-enriched strain, its antioxidant capacity is significantly improved.

[0035] The commercially available lactic acid bacteria solid beverage and commercially available lactic acid bacteria pressed candy in the comparative examples of the present invention were purchased online on a shopping platform.

[0036] The source information of the instruments and reagents used in the examples of the present invention is shown in Table 1 below.

[0037] Table 1

[0038] Reagents / Instruments Purity / Model Selling Manufacturers Disodium EDTA Analytical grade MacLean's reagent 3,3'-Diaminobenzidine Analytical grade Aladdin Biotech Sodium selenite Analytical grade Aladdin Biotech Toluene Analytical grade Xilong Chemical Spectrophotometer UVmini-1285 Shimadzu Corporation Automatic growth curve analyzer Bioscreen C The Group

[0039] Example 1 Screening of Lactobacillus helveticus

[0040] 1. Sample collection and bacterial enrichment

[0041] Take 25g of homemade yak milk residue from herders in Zhegu Grassland, Tibet, and mix it with 225mL of sterile saline to obtain a uniform sample solution. Dilute the sample solution in a gradient manner and take 10 -3 , 10 -4 , 10 -5 , 10 -6 The dilution of the concentration was spread on MRS medium plates for culture. After culture at 37°C for 48 hours, colonies grew on the MRS medium.

[0042] 2. Initial screening of bacterial strains

[0043] According to the standard colony characteristics of Lactobacillus helveticus, a single colony is picked for separation and purification and continued cultivation. Separation and purification are repeated for no less than three times to obtain purified colonies. Culture characteristics: The optimal growth temperature is 37°C, facultative anaerobic, and grown in MRS medium.

[0044] 3. Acid production experiment

[0045] The single colonies obtained from the initial screening of the strains were cultured on MRS medium plates containing 0.2% CaCO3. After culturing at 37°C for 48 hours, the colonies were observed to have no transparent zones around them. Single colonies with strong acid production ability and large transparent zones were selected for further separation and purification.

[0046] Morphological characteristics: The growth state in MRS agar medium is: the growth morphology in MRS agar medium is milky white colonies, round, smooth edges, raised bacteria, and rough surface. The colony morphology of the obtained strain is as follows Figure 1 shown.

[0047] 4. Gram staining

[0048] The strain obtained by repeated screening was subjected to Gram staining. Gram staining was typical positive, and the strain obtained was the target strain. The microscopic examination image is as follows: Figure 2 As shown. Under the microscope, the cells are short rod-shaped, without flagella, do not produce spores, and do not move.

[0049] 5. Catalase test: Use an inoculation loop to pick up a single colony of Gram-positive strains and place it on a glass slide. Add a small amount of 15% H2O2 solution and select catalase-negative bacteria that do not produce bubbles.

[0050] 6. Identification of strains

[0051] The isolated and purified strain is Gram-positive, H2O2 catalase-negative, acid-producing, and non-gas-producing. The 16S rDNA gene sequence was sequenced, and the results were compared with the NCBI GenBank database for homology analysis. The results showed that the strain was Lactobacillus helveticus. The strain was named Lactobacillus helveticus ZheguLBH-VI strain, and was deposited in the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with a deposit number of CCTCCNO: M2023095.

[0052] Example 2 Experiment on selenium-enriched and selenium-resistant strains

[0053] 1. Preparation of broth containing sodium selenite (Na2SeO3)

[0054] (1) Preparation of sodium selenite mother solution: Accurately weigh 0.10 g of sodium selenite and dissolve it in pure water. The volume is adjusted to 100 mL in a volumetric flask to prepare a sodium selenite solution with a concentration of 1 mg / mL.

[0055] (2) Preparation of MRS-Na2SeO3 culture medium: Dilute the sodium selenite stock solution to concentrations of 10 μg / mL, 30 μg / mL, 50 μg / mL, and 70 μg / mL, respectively. Take 5 mL of each solution and add it to 2.7 g of MRS broth culture medium to prepare MRS-Na2SeO3 culture medium containing different concentrations.

[0056] 2. Selenium resistance test of strains

[0057] Take the Lactobacillus helveticus Zhegu LBH-VI strain preserved in a glycerol tube, inoculate it into 5 mL of sterile MRS broth culture medium at a 2% inoculation rate, and culture it in a 37°C constant temperature incubator for activation for 24 h. Continue to inoculate the first-level activated seed liquid into 5 mL of sterile MRS broth culture medium at a 2% inoculation rate, and culture it at 37°C for 12 h to the logarithmic phase.

[0058] The activated Lactobacillus helveticus ZheguLBH-VI strains cultured to the logarithmic phase were inoculated into 5mL MRS broth medium containing 10μg / mL, 30μg / mL, 50μg / mL, and 70μg / mL of sodium selenite at a 2% inoculation rate, and the MRS broth medium without sodium selenite was used as a blank control. The inoculated broth medium was fully shaken and mixed, 350μL was spotted on a 96-well plate, and placed on a Bioscreen fully automatic microbial growth curve analyzer to determine the growth curve of 20h cultured at each sodium selenite concentration, set the temperature to 37°C, and measure once every 30min. According to the growth status of the strain, the optimal selenium resistance concentration was determined in combination with the color change of the bacteria after static culture at 37°C for 20h. Figure 3 This is the growth curve of Lactobacillus helveticus in MRS broth medium containing 70 μg / mL sodium selenite and blank MRS broth medium.

[0059] 3. Determination of Selenium-enriched Rate of Bacteria

[0060] Determination of selenium-enriched rate of bacteria by 3,3'-aminobenzidine colorimetry

[0061] (1) Drawing of standard curve

[0062] Preparation of sodium selenite standard solution: Take 1 mL of sodium selenite mother solution with a concentration of 1 mg / mL, dilute it to volume in a 1000 mL volumetric flask with pure water, and prepare a 1 μg / mL sodium selenite standard solution.

[0063] Preparation of 5% EDTA-2Na (disodium ethylenediaminetetraacetate) solution: Accurately weigh 5g EDTA-2Na and dilute it to 100mL pure water.

[0064] Preparation of 0.5% 3,3'-diaminobenzidine solution: Since 3,3'-diaminobenzidine is insoluble in water, 0.5 g of 3,3'-diaminobenzidine is weighed and dissolved in 100 mL of 1 mol / L hydrochloric acid.

[0065] Pipette 0, 2, 4, 6, 8, and 10 mL of 1 μg / mL sodium selenite standard solution, transfer them into a 125 mL separatory funnel and add distilled water to 35 mL. Add 1 mL of 5% EDTA-2Na solution and shake well. Add 4 mL of 0.5% 3,3'-diaminobenzidine and shake well. Adjust the pH to neutral, add 10 mL of toluene and shake vigorously for 1 min to allow the toluene to fully extract the inorganic selenium. Place in a dark place and let stand for 30 min to separate the layers. Discard the water layer, filter the toluene layer, collect the filtrate, and use a UV spectrophotometer to measure the absorbance of the toluene layer at a wavelength of 420 nm. Repeat each test three times and take the average value. Figure 4As shown, a standard curve was drawn with the sodium selenite content as the horizontal axis and the absorbance value as the vertical axis, and its linear regression equation was calculated to be y=0.0181x+0.0014, and the correlation coefficient was R2=0.9995.

[0066] (2) Determination of selenium-enriched rate of bacterial strains

[0067] The Lactobacillus helveticus Zhegu LBH-VI strain was inoculated into MRS broth medium at an inoculum size of 2% and cultured in a constant temperature incubator at 37°C. According to the strain growth curve, the early logarithmic phase, i.e., 4 hours, was determined to be the optimal time for adding selenium. After the strain was cultured for 4 hours, sodium selenite solutions of different contents were added to the culture medium until the concentrations were 10 μg / mL, 20 μg / mL, and 30 μg / mL, respectively. The strain after selenium addition was further cultured for 24 hours, the content of residual inorganic selenium in the culture medium was determined, and then the selenium enrichment rate of the strain was calculated.

[0068] Determination of residual inorganic selenium: Centrifuge the cultured sample at 4°C, 5000×g in a high-speed centrifuge for 10 minutes. Take 1mL of supernatant, add pure water to 35mL, add 1mL of 5% EDTA-2Na solution, and shake well. Add 4mL of 0.5% 3,3'-diaminobenzidine and shake well, adjust the pH to neutral, add 10mL of toluene and shake vigorously for 1min to allow toluene to fully extract inorganic selenium. The rest of the operation method is consistent with the determination method of the standard solution.

[0069] Selenium enrichment rate % = (organic selenium content / total selenium content) × 100%

[0070] Among them, organic selenium content = total selenium content - residual inorganic selenium content.

[0071] At a sodium selenite concentration of 30 μg / mL, the selenium enrichment rate and bacterial color results after 24 hours of culture are shown in Table 2. The LBH-V1 bacteria are not red, and the selenium enrichment rate reaches 97.90%. If the bacterial color is red, it means that the organic selenium content in the bacteria is low and the strain shows pathological growth.

[0072] Table 2 Selenium enrichment rate and bacterial color of Lactobacillus helveticus Zhegu LBH-VI strain in MRS medium containing 30 μg / mL sodium selenite

[0073] Strain number Selenium enrichment rate Bacteria color LBH-V1 97.90％ -

[0074] Note: ++ means the bacteria are red, + means the bacteria are slightly red, - means the bacteria are not red

[0075] 4. Determination of organic selenium content in Lactobacillus helveticus Zhegu LBH-VI

[0076] The selenium-enriched bacterial liquid was centrifuged at 4°C, 5000×g in a high-speed centrifuge for 10 min, the fermentation waste liquid was discarded, and the bacterial liquid was washed twice with physiological saline. 2 g of bacterial sludge was taken and the organic selenium content in the strain was determined by hydride generation-atomic fluorescence spectrometry (HG-AFS).

[0077] (1) Total protein extraction: 2 g of bacterial mud sample was incubated in 30 ml of 0.25 M NaOH solution at 50° C. for 4 hours, and the supernatant was obtained by centrifugation at 10,000×g for 10 minutes. The residue was washed twice in 10 mL of 0.25 M NaOH. The supernatants were then mixed and 95% saturated with ammonium sulfate at 4° C. The resulting precipitate was collected by centrifugation and then dissolved in 10.0 ml of 50 mM Tris-HCl (pH=8.0) solution. The solution was filtered and dialyzed three times with 1.0 L of 50 mM Tris-HCl (pH=8.0) at 4° C. to remove any other small molecules. Finally, the remaining solution was freeze-dried and stored at -20° C. for future use.

[0078] (2) Determination of selenium content: Take 0.2g of the above-mentioned lyophilized protein sample and digest it in a mixture of 10ml of nitric acid and perchloric acid (4:1), place the sample at room temperature overnight, then heat it to 80℃ for 1h, then continue to heat it to 150-180℃ for 2h until the sample solution becomes clear and transparent; cool the sample container to 50℃, add 5ml of 6M HCl solution to reduce VI selenium to IV selenium, then heat the sample at 120℃ for 30min until the solution becomes clear and transparent; finally, cool the digested sample solution to room temperature and dilute it to 25ml with double distilled water. Take 5ml of the above diluted solution and add 1ml of concentrated HCl and 0.5ml of 10% K3(CN)6, and the selenium content is determined by HG-AFS method.

[0079] Determination conditions: The instrument is equipped with a high-performance hollow cathode selenium lamp, the measurement wavelength is 196nm, the negative high voltage is 320V, the lamp current is 60mA, the drying gas is nitrogen (3L / min), the carrier is 0.05M HCl (1ml / min) solution, NaBH4 1.2% (m / V) is used as the reducing liquid, the atomization temperature is 800℃, the reading method is peak area, the reading time is 10.0s, and the delay time is 1.0s.

[0080] The results showed that the content of organic selenium in total bacterial protein was 1498.6±32μg / g.

[0081] Example 3 Tolerance determination of Lactobacillus helveticus Zhegu LBH-VI

[0082] After screening of selenium-enriched and selenium-resistant experiments, Lactobacillus helveticus Zhegu LBH-VI was determined to be the best selenium-resistant and selenium-enriched strain.

[0083] 1. Determination of strains' low temperature resistance

[0084] The Lactobacillus helveticus Zhegu LBH-VI preserved in a glycerol tube was reactivated in MRS broth medium and inoculated into two 10 mL MRS broth mediums at a 2% inoculation rate. The two were placed in a 10°C and 15°C low-temperature constant temperature incubator for static culture, and the OD600 values ​​of the bacterial culture at 0h, 12h and 24h were measured.

[0085] 2. Determination of acid resistance of strains

[0086] The bacterial liquid after secondary activation was inoculated into 10 mL MRS broth medium with a pH value of 2.5 (pH adjusted with 1 mol / L hydrochloric acid) at a 2% inoculation rate. The bacterial liquid cultured for 0 h and 4 h was diluted and spread. After 24 h of plate culture, the colonies were counted and the survival rate of the strain was calculated.

[0087] Survival rate (%) = 4h viable bacteria count / 0h viable bacteria count

[0088] 3. Bile salt tolerance test

[0089] The bacterial suspension after secondary activation was centrifuged in a 5000×g high-speed centrifuge for 10 min, the supernatant was removed, and the suspension was washed twice with sterile saline. 10 mL of saline containing 0.3% ox bile was added and mixed thoroughly to prepare a bacterial suspension. The bacterial suspension at 0 h and 4 h was taken for dilution and coating, and the colonies were counted after 24 h of plate culture to calculate the survival rate of the strain.

[0090] 4. Determination of strain resistance to artificial gastric juice

[0091] The bacterial suspension after secondary activation was centrifuged in a 5000×g high-speed centrifuge for 10 min, the supernatant was removed, and the suspension was washed twice with sterile saline. 10 mL of sterile artificial gastric juice was added and mixed thoroughly to prepare a bacterial suspension. The bacterial suspension at 0 h and 4 h was taken for dilution and coating, and the colonies were counted after 24 h of plate culture to calculate the survival rate of the strain.

[0092] 5. Determination of strain resistance to artificial intestinal fluid

[0093] The bacterial suspension after secondary activation was centrifuged in a 5000×g high-speed centrifuge for 10 min, the supernatant was removed, and the suspension was washed twice with sterile saline. 10 mL of sterile artificial small intestinal fluid was added and mixed thoroughly to prepare a bacterial suspension. The bacterial suspension at 0 h and 4 h was taken for dilution and coating, and the colonies were counted after 24 h of plate culture to calculate the survival rate of the strain.

[0094] The low-temperature resistance of the strain is shown in Table 3. It can be seen that the strain can still grow in a low-temperature environment and has a strong low-temperature tolerance; the acid, bile salt and artificial gastrointestinal fluid resistance of the strain are shown in Table 4. It can be seen from the table that the strain has a high survival rate in various environments, indicating that the strain has good stress resistance and a certain intestinal colonization ability.

[0095] Table 3 Tolerance of Lactobacillus helveticus Zhegu LBH-VI under low temperature conditions

[0096] temperature <![CDATA[0h OD 600 ]]> <![CDATA[12h OD 600 ]]> <![CDATA[24h OD 600 ]]> 10℃ 0.094 0.656 1.470 15℃ 0.097 0.839 1.514

[0097] Table 4 Tolerance of Lactobacillus helveticus Zhegu LBH-VI under various environments

[0098]

[0099] Example 4 Preparation of freeze-dried powder of selenium-enriched Lactobacillus helveticus

[0100] 1. High-density culture and fermentation of selenium-rich Lactobacillus helveticus

[0101] The strains preserved in the glycerol tubes were activated. The first activation was inoculated into MRS broth medium at a 2% inoculation amount and cultured at 37°C for 24 hours. The second activation was inoculated into 2% inoculation amount and cultured at 37°C for 12 hours. The activated bacterial solution was inoculated into 2L MRS broth medium at a 10% inoculation amount, cultured and fermented at 37°C. When the fermentation reached 4 hours, sterile sodium selenite solution was added to a final concentration of 30 μg / mL for selenium enrichment, and the fermentation was continued for 20 hours.

[0102] The bacterial liquid after fermentation was transferred into a sterile centrifuge bottle, and centrifuged at 5000×g, 4°C for 10 min. The supernatant was discarded, and the bacteria were washed twice with sterile physiological saline to obtain selenium-enriched Lactobacillus helveticus mud.

[0103] 2. Preparation of Lyoprotectant

[0104] The weight proportions of the freeze-dried protective agent components are as follows: 10 parts of skim milk powder, 6 parts of maltodextrin, and 4 parts of erythritol. Each protective agent is dissolved in pure water, stirred and mixed thoroughly, and kept in a constant temperature water bath at 90°C for 30 minutes for sterilization and disinfection.

[0105] 3. Preparation of Freeze-dried Bacterial Powder

[0106] The cleaned selenium-enriched Lactobacillus helveticus mud and lyophilization protective agent are prepared in a ratio of 1:3, the mud is added to the sterilized lyophilization protective agent, mixed thoroughly in a sterile lyophilization bottle, placed in a -30°C refrigerator for pre-freezing for 12 hours, and the pre-frozen bacterial milk is placed in a vacuum freeze dryer for lyophilization for 48 hours to obtain selenium-enriched Lactobacillus helveticus lyophilized bacterial powder.

[0107] Example 5 Application of selenium-enriched Lactobacillus helveticus in probiotic solid beverages

[0108] 1 kg of probiotic solid beverage raw materials were prepared, and the formula and mass proportion were as follows: 35 parts of selenium-enriched Lactobacillus helveticus freeze-dried powder, 20 parts of walnut powder, 10 parts of navel orange freeze-dried powder, and 5 parts of oligofructose. The above formulas were crushed and mixed to obtain mixed raw materials; the raw materials were mixed and packaged to obtain bagged probiotic solid beverages, each bag of probiotic solid beverage was 10 g, and the organic selenium contained was about 150 μg. One bag a day can meet the daily needs of the human body and avoid the toxic side effects caused by excessive selenium.

[0109] Example 6 Application of selenium-enriched Lactobacillus helveticus in selenium-enriched probiotic tablet candies

[0110] 1 kg of raw materials for probiotic tablet candy are prepared, and the formula and mass proportion are as follows: 50 parts of selenium-enriched Lactobacillus helveticus powder, 15 parts of navel orange freeze-dried powder, 5 parts of sorbitol, 5 parts of maltitol, 0.5 parts of Vc, 0.5 parts of Ve, and 0.15 parts of magnesium stearate. Vc and Ve freeze-dried powder are dissolved in sorbitol and maltitol, and mixed magnesium stearate is slowly added to make a soft material that is easily dispersed by light pressing, and the prepared soft material is granulated and dried; selenium-enriched Lactobacillus helveticus powder and navel orange freeze-dried powder are evenly mixed with the above-mentioned dry particles, and tablets are compressed by direct tableting to make selenium-enriched probiotic tablet candy, each piece of which has a mass of about 3.5 g and contains about 110 μg of organic selenium. Eating two pieces 30 minutes before going to bed every day can meet the daily selenium intake recommended by the Chinese Nutrition Society.

[0111] Example 7 Application of selenium-enriched Lactobacillus helveticus in probiotic solid beverages

[0112] 1 kg of probiotic solid beverage raw materials were prepared, and the formula and mass proportion were as follows: 50 parts of selenium-enriched Lactobacillus helveticus freeze-dried powder, 40 parts of walnut powder, 15 parts of navel orange freeze-dried powder, and 15 parts of oligofructose. The above formulas were crushed and mixed to obtain mixed raw materials; the raw materials were mixed and packaged to obtain bagged probiotic solid beverages, each bag of probiotic solid beverage was 10 g, and the organic selenium contained was about 150 μg. One bag a day can meet the daily needs of the human body and avoid the toxic side effects caused by excessive selenium.

[0113] Example 8 Application of selenium-enriched Lactobacillus helveticus in selenium-enriched probiotic tablet candies

[0114] 1 kg of raw materials for probiotic tablet candy are prepared, and the formula and mass proportion are as follows: 35 parts of selenium-rich Lactobacillus helveticus powder, 30 parts of navel orange freeze-dried powder, 15 parts of sorbitol, 15 parts of maltitol, 1.5 parts of Vc, 1.5 parts of Ve, and 0.25 parts of magnesium stearate. Vc and Ve freeze-dried powder are dissolved in sorbitol and maltitol, and mixed magnesium stearate is slowly added to make a soft material that is easily dispersed by light pressing, and the prepared soft material is granulated and dried; selenium-rich Lactobacillus helveticus powder and navel orange freeze-dried powder are evenly mixed with the above-mentioned dry particles, and tablets are compressed by direct tableting to make selenium-rich probiotic tablet candy, each piece of which has a mass of about 3.5 g and contains about 110 μg of organic selenium. Eating two pieces 30 minutes before going to bed every day can meet the daily selenium intake recommended by the Chinese Nutrition Society.

[0115] Example 9 Application of selenium-enriched Lactobacillus helveticus in probiotic solid beverages

[0116] The formula and mass proportion of the probiotic solid beverage are as follows: 4.5g of selenium-enriched Lactobacillus helveticus freeze-dried powder, 3g of walnut powder, 2g of navel orange freeze-dried powder, and 0.5g of oligofructose. The above formulas are crushed and mixed to obtain mixed raw materials; the raw materials are mixed and packaged to obtain bagged probiotic solid beverages, each bag of probiotic solid beverage is 10g, and contains about 150μg of organic selenium. One bag a day can meet the daily needs of the human body and avoid the toxic side effects caused by excessive selenium.

[0117] Example 10 Application of selenium-enriched Lactobacillus helveticus in selenium-enriched probiotic tablet candies

[0118] The formula and mass proportion of the probiotic tablet candy are as follows: 2g of selenium-rich Lactobacillus helveticus freeze-dried powder, 0.9g of navel orange freeze-dried powder, 0.25g of sorbitol, 0.25g of maltitol, 0.03g of Vc, 0.03g of Ve, and 0.005g of magnesium stearate. Vc and Ve freeze-dried powder are dissolved in sorbitol and maltitol, and mixed magnesium stearate is slowly added to make a soft material that is easily dispersed by light pressing, and the prepared soft material is granulated and dried; selenium-rich Lactobacillus helveticus powder and navel orange freeze-dried powder are evenly mixed with the above-mentioned dry particles, and tablets are compressed by direct tableting to make selenium-rich probiotic tablet candies, each of which weighs about 3.5g and contains about 110μg of organic selenium. Eating two tablets 30 minutes before going to bed every day can meet the daily selenium intake recommended by the Chinese Nutrition Society.

[0119] Example 11 Functional Determination of Selenium-Enriched Lactobacillus Helveticus Solid Beverage and Compressed Candy

[0120] 1. Determination of Antioxidant Capacity

[0121] (1) Sample processing

[0122] 1 g of the Lactobacillus helveticus solid beverage of Example 5 or the compressed candy of Example 6 was taken respectively, dissolved in 9 mL of physiological saline and thoroughly mixed, and then centrifuged at 5000×g for 10 min. The supernatant of the samples was taken respectively for determination of DPPH free radical scavenging ability and ABTS scavenging ability. The selenium-enriched probiotic solid beverage was used as experimental group 1, and the selenium-enriched probiotic compressed candy was used as experimental group 2.

[0123] (1) DPPH free radical scavenging ability assay

[0124] Take 100 μL of the sample supernatant and add it to a 96-well ELISA plate. Add 100 μL of 2×10 -4 mol / L DPPH ethanol solution was used as the experimental group; 100 μL of sample supernatant and 100 μL of anhydrous ethanol were mixed as the blank group; 100 μL of anhydrous ethanol and 2×10 -4 mol / L DPPH ethanol solution was mixed as the control group; the experimental group, blank group, and control group were reacted at room temperature in the dark for 30 minutes, and the absorbance at 517nm was measured and recorded as A1, A2, and A3. As ascorbic acid (Vc) has a strong antioxidant function, 15μg / mL Vc was used instead of the sample as a positive control.

[0125]

[0126] Where:

[0127] B——DPPH free radical scavenging rate, %;

[0128] A1——absorbance value of the experimental group at 517 nm;

[0129] A2——absorbance value of blank group at 517nm;

[0130] A3——Absorbance value of the control group at 517nm.

[0131] (2) ABTS scavenging ability assay

[0132] Prepare 7mmol / L ABTS+ solution and 2.45mmol / L potassium persulfate solution, mix the two solutions in the same volume ratio, and place them in the dark at room temperature for 16 hours for use. Dilute the mixture to an absorbance of 0.70±0.02 at 734nm with 5mmol / L phosphate buffer with a pH value of 7.4 for use. Treat the sample according to the method of Example 7.1.(1), take 0.4mL of the sample supernatant and mix it with 0.4mL of the diluted ABTS solution, react at room temperature in the dark for 10 minutes, and then measure the absorbance A4 at 734nm. Use anhydrous ethanol as a blank control, and measure the absorbance A0 after the same reaction.

[0133] 15 μg / mL Vc was used as a positive control.

[0134]

[0135] Where:

[0136] C——ABTS clearance rate, %;

[0137] A4——absorbance value at 734nm after the sample is mixed with ABTS solution;

[0138] A0——Absorbance value of blank control at 734nm.

[0139] Comparative Example 1 Functional Determination of Solid Beverage Not Containing Selenium-Enriched Lactobacillus Helveticus

[0140] 1. Preparation of solid beverage without selenium-enriched Lactobacillus helveticus

[0141] The solid beverage was prepared according to the method of Example 5, except that the selenium-enriched Lactobacillus helveticus freeze-dried powder was not added and other formulas remained unchanged.

[0142] 2. Determination of Antioxidant Capacity

[0143] The experimental method was the same as that in “1. Determination of antioxidant capacity” in Example 11, and was set as comparison group 1.

[0144] Comparative Example 2 Functional Determination of Selenium-Free Lactobacillus Helveticus Tablet Candy

[0145] 1. Preparation of Selenium-free Lactobacillus helveticus Tablet Candy

[0146] The compressed candy was prepared according to the method of Example 6, without adding the freeze-dried powder of selenium-enriched Lactobacillus helveticus, and other formulas and processes remained unchanged.

[0147] 2. Determination of Antioxidant Capacity

[0148] The experimental method was the same as “1. Antioxidant capacity determination” in Example 11, and the control group 2 was set.

[0149] Comparative Example 3 Functional Determination of Commercially Available Lactobacillus Solid Beverage

[0150] Antioxidant capacity assay

[0151] The experimental method was the same as “1. Antioxidant capacity determination” in Example 11, and was set as comparison group 3.

[0152] Comparative Example 4 Functional Determination of Commercially Available Lactic Acid Bacteria Tablet Candies

[0153] Antioxidant capacity assay

[0154] The experimental method was the same as that in “1. Determination of antioxidant capacity” in Example 11, and the comparison group 4 was set.

[0155] The experimental results are as follows:

[0156] The free radical scavenging rates of the six groups of samples are shown in Table 5. It can be seen that the free radical scavenging rates of the solid beverages and compressed candies (experimental group 1 and experimental group 2) prepared with freeze-dried powder of selenium-enriched Lactobacillus helveticus are significantly better than those of the products prepared without powder (comparison group 1 and comparison group 2), and are also better than the commercially available non-selenium-enriched probiotic products of the same type (comparison group 3 and comparison group 4), and are comparable to or even better than the antioxidant capacity of Vc.

[0157] Table 5 Comparison of antioxidant capacity of different products

[0158]

[0159] The above description is only a preferred embodiment adopted by the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Application of Lactobacillus helveticus Zhegu LBH-VI strain in selenium-enriched products, wherein: The Lactobacillus helveticus Zhegu LBH-VI strain is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M2023095.

2. The Lactobacillus helveticus Zhegu LBH-VI strain according to claim 1 is used in selenium-enriched products, characterized in that: The selenium-enriched rate of the strain is greater than 97.90%, and the concentration of inorganic selenium resistant to the strain is greater than 70 μg / mL; preferably, the inorganic selenium includes sodium selenite.

3. The application of Lactobacillus helveticus Zhegu LBH-VI strain in selenium-enriched products according to claim 1 or 2, characterized in that, The products include selenium-rich bacterial powder, selenium-rich bacterial agent, selenium-rich food, selenium-rich feed, selenium-rich health products or selenium-rich medicines.

4. A selenium-enriched freeze-dried bacterial powder, characterized in that: The invention comprises a Lactobacillus helveticus Zhegu LBH-VI strain with a preservation number of CCTCC NO: M2023095 after selenium-enriched culture.

5. A method for preparing the selenium-enriched freeze-dried bacterial powder according to claim 4, comprising freeze-drying the Lactobacillus helveticus Zhegu LBH-VI strain with a deposit number of CCTCC NO: M2023095 after selenium-enriched cultivation.

6. The method for preparing selenium-enriched freeze-dried bacterial powder according to claim 5, comprising the following steps: (1) activating the Lactobacillus helveticus Zhegu LBH-VI strain in a culture medium; (2) subjecting the activated strain to high-density culture and fermentation, and adding sterile sodium selenite solution during the fermentation; (3) centrifuging the fermented bacterial solution, discarding the supernatant, and obtaining selenium-enriched Lactobacillus helveticus bacterial sludge; (4) Mixing the selenium-enriched Lactobacillus helveticus slurry with a freeze-drying protective agent and freeze-drying.

7. The method for preparing selenium-enriched freeze-dried bacterial powder according to claim 6, wherein: In step (4), the bacterial sludge is 1-2 parts by weight and the lyophilization protective agent is 3-5 parts by weight.

8. The method for preparing selenium-enriched freeze-dried bacterial powder according to claim 6 or 7, wherein: In step (4), the lyophilization protective agent comprises one or more of skim milk powder, maltodextrin, and erythritol; preferably, by weight, the lyophilization protective agent comprises 4-6 parts of skim milk powder, 2-4 parts of maltodextrin, and 1-3 parts of erythritol.

9. Selenium-enriched freeze-dried bacterial powder prepared by the method for preparing selenium-enriched freeze-dried bacterial powder according to any one of claims 5 to 8.

10. A probiotic solid beverage, characterized in that: Contains the selenium-enriched freeze-dried bacterial powder according to claim 4 or 9.

11. The probiotic solid beverage according to claim 10, characterized in that: In parts by weight, the invention comprises 35-50 parts of selenium-enriched freeze-dried bacteria powder, 20-40 parts of nut powder, 5-15 parts of fruit freeze-dried powder, and 5-15 parts of dietary fiber; preferably, the nut powder comprises one or more of chia seed powder, lotus seed powder, and cashew powder; further preferably, the fruit freeze-dried powder comprises one or more of navel orange, lemon, yellow peach, blueberry, cherry tomato, and cranberry; more preferably, the dietary fiber comprises one or more of fructo-oligosaccharide, galacto-oligosaccharide, and β-glucan.

12. A compressed candy, characterized in that: Contains the selenium-enriched freeze-dried bacterial powder according to claim 4 or 9.

13. The compressed candy according to claim 12, characterized in that: In parts by weight, the invention comprises 35-50 parts of selenium-enriched freeze-dried bacteria powder, 15-30 parts of fruit freeze-dried powder, 10-30 parts of sweetener, 0.5-1.5 parts of Vc, 0.5-1.5 parts of Ve and 0.1-0.25 parts of magnesium stearate; preferably, the fruit freeze-dried powder comprises one or more of navel orange, lemon, yellow peach, blueberry, cherry tomato and cranberry; further preferably, the sweetener comprises one or more of sorbitol, erythritol, sucralose and maltitol; more preferably, the sweetener comprises 5-15 parts of sorbitol and 5-15 parts of maltitol.

14. A biological bacterial agent, characterized in that: The invention comprises the selenium-enriched freeze-dried bacterial powder according to claim 4 or 9, and / or a cell lysate of Lactobacillus helveticus Zhegu LBH-VI strain with a preservation number of CCTCC NO: M 2023095 after being cultured with selenium.

15. Use of the selenium-enriched freeze-dried bacterial powder according to claim 4 or 9 or the biological bacterial agent according to claim 14 in selenium-enriched foods, selenium-enriched medicines, selenium-enriched health products and selenium-enriched feed.

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