A multifunctional fermentative Lactobacillus mucilaginosus KD-1 and its application

Abstract

The present invention provides a multifunctional fermented mucus lactobacillus KD-1 and its application. The strain has been deposited in the China Center for Type Culture Collection on April 6, 2023, with a deposit number of CCTCC NO: M2023477. The fermented mucus lactobacillus KD-1 has multiple functions of converting and preparing nano-selenium, producing exopolysaccharides, and fermenting goat milk to produce antioxidant peptides, and can be used to prepare nano-selenium, selenium-enriched probiotic fermented milk, exopolysaccharides, and antioxidant fermented milk.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to a multifunctional fermentation mucus lactobacillus KD-1 and an application thereof. Background Art

[0002] Fermented Lactobacillus mucilaginosus is one of the lactic acid bacteria that the country allows to be used in food. It can metabolize lactose, galactose and other sugars to produce metabolites such as lactic acid, acetic acid, succinic acid, ethanol, etc. Fermented Lactobacillus mucilaginosus is widely distributed in traditional fermented foods. Since traditional fermented foods are eaten all year round, fermented Lactobacillus mucilaginosus as a fermentation agent has the characteristics of safety and genetic stability, and has a great contribution to the flavor formation of traditional foods.

[0003] Literature reports that fermented Lactobacillus mucosa has antibacterial, cholesterol-lowering, immune-enhancing, liver and intestinal damage relief, and anti-atherosclerotic activities. Fermented Lactobacillus mucosa with other functions needs to be further explored.

[0004] Selenium is an indispensable trace element for organisms. Selenium deficiency is accompanied by the occurrence of many diseases. In most selenium-deficient areas, selenium is added to the diet of humans and animals as a nutritional additive, but the safety threshold of selenium as a supplement in food is very small. Some lactic acid bacteria have the ability to enrich selenium and can also convert low-bioavailable and toxic forms of selenium into highly bioavailable and non-toxic forms of nano-selenium. Compared with inorganic selenium, nano-selenium has more significant effects in antioxidant, antibacterial, and immunomodulatory properties, and it has lower toxicity, is easy to absorb, and has high bioavailability. Péter et al. successfully used probiotics lactic streptococci, bifidobacterium BB-12, and Lactobacillus acidophilus in yogurt to biotransform selenite and obtain nano-selenium (100-500nm) after separation and purification. Chun et al. used Lactobacillus casei to reduce sodium selenite into elemental nano-selenium (50-80nm) and distribute it in cells. In vivo and in vitro experiments proved that drugs containing Lactobacillus casei nano-selenium can effectively resist intestinal dysfunction caused by toxin-producing Escherichia coli. At present, there are many studies on the preparation of nano-selenium by microbial transformation at home and abroad, but there are no reports on the use of fermented mucus Lactobacillus.

[0005] Extracellular polysaccharide (EPS) is a carbohydrate secreted by lactic acid bacteria during their growth and metabolism. It has unique rheological properties and can be used as a stabilizer, gelling agent, and thickener in fermented dairy products and other foods. Mende et al. isolated EPS from thermophilic Streptococcus and applied it to yogurt. It was found that EPS can significantly increase the apparent viscosity of yogurt. This is because the negatively charged EPS is electrostatically attracted to the positively charged casein particles in the milk, thereby enhancing the polysaccharide-protein network structure. Purwandari et al. also showed that EPS can make yogurt have better rheological properties. Zhang Tiehua et al. added 1% EPS produced by thermophilic Streptococcus ST1 to yogurt and found that it could significantly change the viscosity of skim milk. Girard et al. found that the formation of yogurt gel is closely related to the kinetics of strain acidification. It can be seen that under the action of EPS, the viscosity of the product can be increased. It is necessary to obtain excellent strains that produce extracellular polysaccharides.

[0006] The increase in free radical levels leads to an imbalance between oxidation and antioxidant effects in the body, which in turn causes a series of diseases and threatens people's health. Studies have shown that some lactic acid bacteria can hydrolyze proteins during fermentation to produce antioxidant peptides. These antioxidant peptides not only have good antioxidant properties, but are also safe and non-toxic. Therefore, it is urgent to isolate and screen strains that produce antioxidant peptides from fermented milk protein. Summary of the invention

[0007] In order to solve the problems of the prior art, the present invention provides a multifunctional fermentation Lactobacillus mucosa KD-1 and its application. The fermentation Lactobacillus mucosa KD-1 has multiple functions of converting and preparing nano-selenium, producing extracellular polysaccharides and fermenting goat milk to produce antioxidant peptides.

[0008] The present invention is achieved through the following technical solutions:

[0009] A multifunctional fermentative mucus lactobacillus (Limosilactobacillus fermentum) KD-1, which was deposited in the China Center for Type Culture Collection on April 6, 2023, with the deposit number CCTCC NO: M 2023477.

[0010] A method for preparing nano-selenium probiotics, comprising:

[0011] 1) culturing the fermented mucus Lactobacillus KD-1 in MRS broth medium to the logarithmic phase, and centrifuging to obtain bacterial sludge;

[0012] 2) adding phosphate buffer to the obtained bacterial sludge to prepare a bacterial suspension, and adding sodium selenite solution to the bacterial suspension to carry out a conversion reaction to obtain a conversion solution;

[0013] 3) centrifuging the conversion solution, discarding the supernatant, and washing with sterile water to obtain nano-selenium probiotics;

[0014] Preferably, the concentration of fermented mucus lactobacillus KD-1 in the bacterial suspension is 0.02-0.06 g / mL, the concentration of sodium selenite in the bacterial suspension is 50-250 μg / mL, after adding the sodium selenite solution to the bacterial suspension, the pH value is adjusted to 6.4-7.5, the conversion reaction time is 26-46 h, and the centrifugation conditions are: 6000 rpm-800 rpm, 4-10° C., and centrifugation for 10-20 min.

[0015] The nano-selenium probiotics are obtained by the preparation method.

[0016] A method for preparing nano-selenium, comprising:

[0017] The fermented mucus lactobacillus KD-1 is cultured in MRS broth medium to a logarithmic phase, and centrifuged to obtain bacterial sludge;

[0018] A phosphate buffer solution is added to the obtained bacterial sludge to prepare a bacterial suspension, and a sodium selenite solution is added to the bacterial suspension to perform a conversion reaction to obtain a conversion solution;

[0019] Nano-selenium is extracted from the obtained conversion liquid.

[0020] A method for preparing extracellular polysaccharide comprises: inoculating activated fermentation mucus lactobacillus KD-1 into a sugar-producing culture medium, culturing at a constant temperature, centrifuging, collecting supernatant and adding trichloroacetic acid solution, centrifuging to remove precipitation, adding ethanol and standing, centrifuging, collecting to obtain crude polysaccharide precipitation, dissolving the crude polysaccharide precipitation in water, and dialyzing to obtain extracellular polysaccharide.

[0021] A selenium-enriched probiotic fermented milk, wherein the fermented mucus lactobacillus and sodium selenite are added into the fermentation raw materials.

[0022] Preferably, the fermentation raw material is raw cow's milk, raw goat's milk, reconstituted cow's milk or reconstituted goat's milk.

[0023] The invention discloses an antioxidant probiotic fermented milk, wherein the fermented mucus lactobacillus is added into the fermentation raw materials.

[0024] Preferably, the DPPH free radical scavenging rate is not less than 60%, the superoxide anion scavenging rate is not less than 70%, the ferrous ion chelating rate is not less than 65%, the peptide content is not less than 0.50 mg / L, and the viable bacterial count is not less than 1.0×10 8 CFU / g.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The fermented mucus lactobacillus KD-1 of the present invention has multiple functions of converting and preparing nano-selenium, producing extracellular polysaccharides, and fermenting goat milk to produce antioxidant peptides. The fermented mucus lactobacillus KD-1 can use bacterial cells to convert selenite to prepare nano-selenium. The conversion system is simple and does not need to be carried out in a culture medium, thereby avoiding the influence of culture medium components on the preparation of nano-selenium probiotic powder and the extraction of nano-selenium. The strain is used to prepare selenium-enriched probiotic fermented milk, which can significantly accelerate the fermentation of yogurt starter TW; the fermented mucus lactobacillus KD-1 can produce extracellular polysaccharides in MRS broth to improve its antioxidant properties, and can produce antioxidant peptides in bovine and goat milk fermentation to increase the functionality of fermented milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The morphology of milk kefir grains LF and the colony morphology of fermented Lactobacillus mucilaginosus KD-1 purchased from Fushun, Liaoning;

[0028] Figure 2 Scanning electron micrographs of strain KD-1 ((a) is the strain before transformation, (b) is the strain after transformation, and (c) is the EDX analysis spectrum);

[0029] Figure 3 The effect of time on the production of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1;

[0030] Figure 4 Effect of temperature on the production of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1;

[0031] Figure 5 Effect of pH on the production of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1;

[0032] Figure 6 Effect of concentration on the production of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1;

[0033] Figure 7 The effect of the amount of bacteria added on the production of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1;

[0034] Figure 8 Nano-selenium freeze-dried powder prepared by fermentation of Lactobacillus mucilaginosus KD-1;

[0035] Fig. 9 Transmission electron microscopy image of nano-selenium;

[0036] Fig.10 Analysis of the surface chemical properties of nano-selenium particles prepared by fermentation of Lactobacillus mucilaginosus KD-1: XPS total spectrum (a) and Se3d spectrum (b);

[0037] Fig.11 Effect of fermentation time on pH value of selenium-enriched probiotic fermented milk. DETAILED DESCRIPTION

[0038] In order to further understand the present invention, the present invention is described below in conjunction with embodiments. These descriptions are only to further explain the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0039] Example 1 Isolation, property comparison and strain identification of fermented mucus Lactobacillus KD-1 of the present invention

[0040] 1. Obtaining fermented Lactobacillus mucilaginosus KD-1

[0041] The kefir grains LF (see Figure 1 (a) 5% was inoculated into sterilized and cooled goat milk, and fermented at room temperature for 24 hours. Elliker agar supplemented with sodium selenite was used as the separation medium. After repeated separation and screening, a lactic acid bacterium KD-1 with strong nano-selenium production ability was obtained by plate separation method. Its colony morphology is shown in Figure 1 (b).

[0042] 2. Identification of Lactobacillus mucilaginosus KD-1

[0043] 16S rDNA amplification and sequencing were used to determine the species of strain KD-1. Through denaturation, PCR amplification, PCR product purification and sequencing, the sequence was measured as shown in SEQ ID No. 1, and it was searched and homology analyzed in NCBI. The results of homology analysis showed that KD-1 was a fermentative mucus Lactobacillus, which was deposited in the China Center for Type Culture Collection (CCTCC) on April 6, 2023. The preservation location is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the preservation number is CCTCC NO: M 2023477.

[0044] SEQ ID No. 1 is as follows:

[0045]

[0046] Example 2 Preparation of Nano-Selenium by Fermentation of Lactobacillus mucilaginosus KD-1

[0047] The freeze-dried powder of fermented Lactobacillus mucilaginosus KD-1 was dissolved in MRS broth medium and cultured in a 37°C incubator for 24 h. After three generations of activation with an inoculum volume of 5% (v / v), the number of viable bacteria reached 10 8 , and placed it in a refrigerator at 4°C for standby use. Under sterile conditions, the activated fermented mucus lactobacillus KD-1 was inoculated into MRS broth medium with an inoculum amount of 5% (v / v) and placed in an incubator for 24 hours at 37°C. After the growth of the strain reached the logarithmic phase, the bacterial suspension was shaken, 50 mL of the bacterial suspension was taken and centrifuged at 8000r / min for 10 minutes to obtain bacterial mud, and then the bacterial mud was washed with phosphate buffer (pH=6.4). After repeated washing three times, the bacterial mud was obtained by centrifugation, 10 mL of phosphate buffer was added to prepare a bacterial suspension, and sodium selenite solution was added to the bacterial suspension to make its final concentration of 100μg / mL, and then it was placed in a 37°C incubator for transformation for 24 hours, the color of the transformation liquid was observed, and the changes in the bacterial surface before and after the transformation were observed by scanning electron microscopy and its components were analyzed by X-ray energy spectrum. The experimental results show that after 24 hours of conversion in a 100 μg / mL sodium selenite solution, a red precipitate is generated in the conversion liquid, which is nano-elemental selenium. This indicates that the fermented mucus lactobacillus KD-1 of the present invention can convert selenite into zero-valent nano-elemental selenium after reduction. The sodium selenite conversion rate of strain KD-1 reaches 85.12%, and the amount of nano-selenium converted per unit cell reaches 1.12 mg / g.

[0048] After the fermented mucus lactobacillus KD-1 was transformed into selenium-enriched, it was processed and observed by scanning electron microscope and X-ray energy spectrum analysis. The results are shown in Figure 2.

[0049] from Figure 2 It can be seen that the morphology of fermented Lactobacillus mucoides KD-1 without the addition of sodium selenite is full rod-shaped (Figure (a)), and after the reduction of sodium selenite, the fermented Lactobacillus mucoides KD-1 has inward cavities on the surface of the bacteria, and there are round nanoparticles on the surface of the bacteria (Figure (b)). EDX energy spectrum analysis is performed on the round nanoparticles in Figure (b), and from the energy spectrum analysis diagram in Figure (c), it can be found that there is a characteristic peak of selenium at 1.4KeV, indicating that fermented Lactobacillus mucoides KD-1 produces nano-selenium after reducing selenite, and it is distributed on the surface and outside of the bacteria.

[0050] Example 3 Process optimization for preparing nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1 and characterization of nano-selenium

[0051] 1. Analysis of the influence of time on the preparation of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1

[0052] The fermented Lactobacillus mucilaginosus KD-1 cultured in MRs broth for 24 h was centrifuged to obtain bacterial sludge and washed three times, then made into a bacterial suspension with a bacterial sludge concentration of 0.05 g / mL and a pH of 6.4 (the concentration of sodium selenite was 100 μg / mL), and was transformed at a constant temperature of 37°C for different times (12, 24, 36, 48 and 60 h), and the sodium selenite reduction rate and the conversion liquid a were determined. * Red value, the result is Figure 3 shown.

[0053] Depend on Figure 3 It can be seen that the conversion rate of sodium selenite first increases and then decreases with the increase of reaction time. When the time reaches 36h, the reduction rate of sodium selenite reaches a maximum value of 86.67%, and when the time is 48 and 60h, the conversion rate of sodium selenite is 86.57% and 86.42% respectively, which is close to the maximum value, indicating that during the reaction of fermentation of Lactobacillus mucilaginosus KD-1 and sodium selenite, sodium selenite has been basically reduced to nano-selenium at 36h. * The change of red value is basically consistent with the reduction rate of sodium selenite, reaching a maximum value of 22.62 at 48 hours and a * The red value of 22.32 is relatively close. Comprehensive considerations show that 36 h is the optimal time for fermentation of Lactobacillus mucilaginosus KD-1 to transform and prepare nano-selenium.

[0054] 2. Analysis of the influence of temperature on the preparation of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1

[0055] The temperature for the fermentation of Lactobacillus mucilaginosus KD-1 to prepare nano-selenium was set at 33°C, 35°C, 37°C, 39°C and 41°C, and the conversion time was 48h. The results are as follows Figure 4 shown.

[0056] Depend on Figure 4 It can be seen that the conversion rate of sodium selenite and a * The red values ​​increased first and then decreased with the increase of temperature. When the temperature was 31℃, the conversion rate of Lactobacillus mucilaginosus KD-1 was 77.92%, which is a * The red value is also relatively low at 17.2. When the temperature reaches 37°C, the conversion rate of sodium selenite reaches a maximum of 84.18%, and a * The red value also reached a maximum of 23.14. Therefore, it can be seen that the optimal temperature for fermenting Lactobacillus mucilaginosus to produce nano-selenium is 37°C.

[0057] 3. Analysis of the effect of pH on the preparation of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1

[0058] The effects of different pH values ​​(5.6, 6.3, 7, 7.7, 8.4) on the preparation of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1 at 37°C for 36 hours were studied. Figure 5 shown.

[0059] Depend on Figure 5 It can be seen that the conversion rate of sodium selenite increases first and then decreases with the increase of pH. When the pH is 6, the conversion rate of sodium selenite is only 65.12%, while when the pH is 6.8, the conversion rate of sodium selenite reaches a maximum of 87.23%. * The red value also reached a maximum of 19.5. When the pH gradually increased, the conversion rate of sodium selenite gradually decreased, indicating that the fermentation of mucus lactobacillus KD-1 was more suitable for the conversion and preparation of nano-selenium under neutral conditions, and the appropriate conversion pH was 6.4-7.5.

[0060] 4. Analysis of the effect of sodium selenite concentration on the preparation of nano-selenium by fermentation of Lactobacillus mucilaginosus KD-1

[0061] The concentration of sodium selenite in the bacterial suspension of fermented Lactobacillus mucilaginosus KD-1 was adjusted to 50, 100, 150, 200 and 250 μg / mL respectively and the mixture was transformed at 37°C for 48 h. The results are as follows: Figure 6 shown.

[0062] Depend on Figure 6 It can be seen that with the increase of sodium selenite concentration, the conversion of sodium selenite first increases and then decreases, while a * The red value increases first and then becomes flat. When the concentration of sodium selenite is 100 μg / mL, the conversion rate of sodium selenite reaches 89.48% and reaches the maximum. * The red value also reached a maximum of 21.04. However, when the concentration of sodium selenite exceeded 100μg / mL, the conversion rate of sodium selenite gradually decreased. This is mainly because when the content of sodium selenite is low, the reductase in the bacteria can reduce sodium selenite faster, but when the concentration of sodium selenite increases, the toxicity of selenite ions may be the main reason for the decrease in the conversion rate of selenite in the strain at a higher selenium concentration. The appropriate concentration of nano-selenium is 50-150μg / mL.

[0063] 5. Analysis of the effect of the addition amount of fermented Lactobacillus mucilaginosus KD-1 on the preparation of nano-selenium

[0064] The amount of fermented mucus Lactobacillus KD-1 added to the bacterial suspension was adjusted to 0.02, 0.03, 0.04, 0.05 and 0.06 g / mL, respectively, and sodium selenite was added to a concentration of 100 μg / mL. The pH was adjusted to 7.2 and the transformation was carried out at a constant temperature of 37°C for 48 h. The results are as follows Figure 7 As shown. Figure 7 It can be seen that the conversion rate of sodium selenite and a *The red value changes relatively steadily after reaching a certain value as the amount of bacteria added increases. When the amount of bacteria added is 0.04 and 0.05 g / mL, the conversion rates of sodium selenite are 85.51% and 86.33%, which are close to each other. * The red values ​​are also relatively close to 21.84 and 21.31, indicating that when the content of sodium selenite is constant, the conversion rate of sodium selenite by bacteria gradually increases with the increase of bacterial content. However, after the sodium selenite is completely converted, the amount of nano-selenium no longer increases with the increase of bacteria.

[0065] 6. Effects of temperature, bacterial addition amount and conversion time on the conversion of Lactobacillus mucilaginosus KD-1 into nano-selenium

[0066] According to the results of the single factor test, the three factors that have a greater impact on the conversion and preparation of nano-selenium, namely, temperature, amount of bacteria added and time, were selected to optimize the process conditions. The sodium selenite conversion rate (%), the content of nano-selenium per unit cell (mg / g) and a * The red values ​​are the indicator optimization process conditions. The experimental design and results are shown in Table 1.

[0067] Table 1 Experimental design and results of the process for preparing nano-selenium by fermenting Lactobacillus mucilaginosus KD-1

[0068]

[0069] As shown in Table 1, when the temperature is 32-42°C, the inoculation amount is 0.023-0.057%, and the conversion time is 26-46h, the conversion rate of sodium selenite to nano-selenium is 78.22-96.88%, and the nano-selenium content per unit cell is (0.806-1.759 mg) / g cell, a * The red values ​​are 17.73-23.70.

[0070] The fermented Lactobacillus mucilaginosus KD-1 transformation liquid was centrifuged (10000r / min, 4°C, 10min), the supernatant was discarded, the nano-selenium probiotic precipitate was collected, the nano-selenium therein was extracted, separated and freeze-dried to obtain nano-selenium freeze-dried powder. The results are as follows: Figure 8 The nano-selenium freeze-dried powder was characterized by transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). Fig. 9 and Fig.10 ,Depend on Fig. 9It can be seen that the nano-selenium particle size prepared by fermentation of Lactobacillus mucilaginosus KD-1 is about 200nm, and the nano-selenium has good dispersibility in water. Lampis et al. (Gigienal Sanitariia, 2000 (5): 32) prepared nano-selenium by reducing selenite with stenotrophomonasmaltophiliaSelTE02, and found that the nano-selenium was spherical, with a size range of 160nm to 250nm. The size and shape of the nano-selenium observed in this article are consistent. Fig.10 In (a), it is found that elements such as C, O, N, P, and S also exist on the surface of nano-selenium, and the intensity of these peaks is stronger than that of selenium, indicating that there are some biological macromolecules on the surface of nano-selenium that wrap the nano-selenium. Fig.10 In (b), there is a characteristic peak of zero-valent selenium near 55 eV, proving that zero-valent selenium exists on the surface of nano-selenium.

[0071] Example 4 Application of fermented Lactobacillus mucilaginosus KD-1 in selenium-enriched probiotic fermentation of goat milk

[0072] Goat milk powder and water were mixed into reconstituted milk in a ratio of 1:7 (w / v), sterilized at 90°C for 5 min, and then cooled to room temperature and divided into two groups. The first group was added with 0.05% (w / v) direct-injection fermentation agent TW and sodium selenite (7 μg / mL) as a control, and the second group was added with 0.03% (w / v) direct-injection fermentation agent TW, 0.02% (w / v) fermented mucus Lactobacillus KD-1 freeze-dried powder and sodium selenite (7 μg / mL), and then cultured at 42°C for different time periods (3, 4, 5, 6, 7h), and the pH value of selenium-enriched probiotic fermented goat milk was measured by sampling. The results are as follows: Fig.11 shown.

[0073] Depend on Fig.11 It can be seen that the pH of the two fermented goat milks first decreases and then tends to be stable with the increase of time, but the pH decreases faster when fermented with Lactobacillus mucus KD-1. After 4 hours of fermentation, the pH value of the fermented milk with Lactobacillus mucus KD-1 is 4.52±0.011, while the pH value of the control group is 4.91±0.013. After 6 hours of fermentation, the pH value reaches 4.52±0.013. In industrial production of fermented milk, fermentation is stopped when the pH value reaches 4.5 and then the milk is ripened. It can be seen that the addition of Lactobacillus mucus KD-1 promotes the fermentation of goat milk, reaching the fermentation end point 2 hours earlier than the control group, significantly shortening the fermentation time.

[0074] Example 5 Production of exopolysaccharides by fermentation of Lactobacillus mucilaginosus KD-1 and its antioxidant activity

[0075] The activated fermentation mucus Lactobacillus KD-1 was inoculated into the sugar-producing medium at an inoculation rate of 2%, cultured at a constant temperature of 37°C for 48 hours, and then centrifuged at 7500 r / min for 12 minutes at 4°C. The supernatant was collected and 10% trichloroacetic acid solution was added, and the precipitate was removed by centrifugation. After adding 3 times the volume of ethanol, it was placed at 4°C overnight, and then centrifuged at 7500 r / min for 12 minutes. The crude polysaccharide precipitate was collected and dissolved in distilled water, placed in a dialysis bag, and dialyzed for 24 hours to remove small molecules such as monosaccharides, pigments and peptides, and the EPS amount, DPPH free radical scavenging rate and ferrous ion chelating rate were measured.

[0076] Table 2 Extracellular polysaccharide production and antioxidant capacity of fermented Lactobacillus mucilaginosus KD-1

[0077] strain Extracellular polysaccharide yield mg / L DPPH free radical scavenging rate / % Ferrous ion chelation rate / % Fermented Lactobacillus mucilaginosus KD-1 210.25±1.58 70.32±1.86 48.39±2.12

[0078] As shown in Table 2, the extracellular polysaccharide production of fermented Lactobacillus mucilaginosus KD-1 is 210.25±1.58, the DPPH free radical scavenging rate is 70.32±1.86%, and the ferrous ion chelating rate is 48.39±2.12%, indicating that the fermented Lactobacillus mucilaginosus KD-1 of the present invention has the functions of producing extracellular polysaccharides and anti-oxidation.

[0079] Example 6 Preparation of Antioxidant Fermented Milk by Fermenting Bovine and Sheep Milk with Lactobacillus mucilaginosus KD-1

[0080] Fresh cow milk and fresh goat milk were sterilized at 90℃ for 10min respectively. After cooling to 40℃, activated fermentation Lactobacillus mucilaginosus KD-1 was inoculated with 5% and cultured at 37℃ for 15h. Three parallels were collected for each group to measure DPPH free radical scavenging rate, superoxide anion scavenging rate, peptide content, sensory evaluation, viable count, acidity and pH. The average results are shown in Table 3.

[0081] Table 3 Antioxidant activity of bovine and goat milk fermented by Lactobacillus mucilaginosus KD-1

[0082]

[0083] As shown in Table 3, after the bovine and goat milk was fermented by Lactobacillus mucilaginosus KD-1, its DPPH free radical scavenging rate was not less than 60%, superoxide anion scavenging rate was not less than 70%, ferrous ion chelating rate was not less than 65%, peptide content was not less than 0.50 mg / L, and viable bacterial count was not less than 1.0×10 8 CFU / g, the sensory evaluation and live bacteria count of cow's milk were higher than those of goat's milk, but the antioxidant activity of goat's milk was higher than that of cow's milk, indicating that the two types of fermented milks fermented with Lactobacillus mucus KD-1 have their own characteristics, and fermented Lactobacillus mucus KD-1 can be used to prepare antioxidant probiotic fermented milk.

Claims

1. A method for preparing nano-selenium probiotics, characterized in that: include: 1) Cultivating Limosilactobacillus fermentum KD-1 in MRS broth medium to the logarithmic phase, and centrifuging to obtain bacterial sludge; 2) adding a phosphate buffer solution to the bacterial sludge to prepare a bacterial suspension, and adding a sodium selenite solution to the bacterial suspension to perform a conversion reaction to obtain a conversion solution; 3) centrifuging the conversion solution, discarding the supernatant, and washing with sterile water to obtain nano-selenium probiotics; Among them, the fermented mucus lactobacillus (Limosilactobacillus fermentum) KD-1 has been deposited in the China Center for Type Culture Collection on April 6, 2023, with the deposit number of CCTCC NO: M 2023477; The concentration of the fermented mucus lactobacillus KD-1 in the bacterial suspension is 0.05 g / mL, and the concentration of sodium selenite in the bacterial suspension is 100 μg / mL; after adding the sodium selenite solution to the bacterial suspension, the pH value is adjusted to 6.8, and the conversion reaction time is 36 hours.

2. The nano-selenium probiotics obtained by the preparation method according to claim 1.

3. A selenium-enriched probiotic fermented milk, characterized in that: The fermentation raw material comprises the fermentation lactobacillus fermentum KD-1 and sodium selenite as claimed in claim 1; the fermentation raw material is raw cow's milk, raw goat's milk, reconstituted cow's milk or reconstituted goat's milk.

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