Selenium-enriched lactobacillus acidophilus and application thereof
By using Lactobacillus acidophilus JGSLA09 fermentation technology, inorganic selenium is converted into organic selenium, which solves the problems of high cost and low dissolution rate of traditional selenium-enriched foods, and realizes efficient and safe selenium supplementation and product diversification, while enhancing the antioxidant function of asparagus tea.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AGRI INST OF AGRI JIANGXI PROVINCE
- Filing Date
- 2026-04-10
- Publication Date
- 2026-06-05
AI Technical Summary
Traditional selenium-enriched foods rely on natural selenium-enriched raw materials, which have high production costs and low selenium dissolution and bioavailability, making it difficult to achieve stable and efficient dietary selenium supplementation goals.
Inorganic selenium was converted into organic selenium using Lactobacillus acidophilus JGSLA09, and then fermented with asparagus tea substrate to produce selenium-enriched asparagus tea tablets. By optimizing the strain culture conditions and the asparagus tea pretreatment process, the conversion rate and bioavailability of selenium were improved.
It significantly improves the bioavailability and stability of selenium, enhances the antioxidant properties of products, reduces raw material costs, expands product forms, and has good process adaptability and industrialization potential.
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Figure CN122146533A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and in particular to a selenium-enriched Lactobacillus acidophilus and its applications. Background Technology
[0002] Selenium is an essential trace element for the human body, participating in various physiological metabolic processes and playing a vital role in maintaining immune function, antioxidant defense, and thyroid hormone synthesis. The human body cannot synthesize selenium and must obtain it through diet. However, selenium intake in daily diets is often insufficient, and long-term selenium deficiency may lead to endemic diseases such as Keshan disease and Kashin-Beck disease, and increase health risks such as cardiovascular disease and immune dysfunction. Selenium exists in nature mainly in two forms: inorganic selenium and organic selenium. Inorganic selenium (such as sodium selenite) has high biotoxicity, while organic selenium (such as selenoamino acids) has better safety and higher bioavailability, making it more suitable as a dietary selenium source.
[0003] Currently, most selenium-enriched foods on the market rely on crops grown in selenium-rich soil or the direct addition of inorganic selenium salts. The former is geographically limited and costly, while the latter suffers from low absorption efficiency and potential toxicity. Therefore, developing efficient, safe, and cost-effective organic selenium conversion technologies has become a research hotspot in the functional food field. Lactic acid bacteria, as a recognized safe food-grade microorganism, possesses a strong ability to enrich and convert trace elements, enabling the conversion of inorganic selenium into organic selenium and nano-selenium, significantly improving the bioavailability and physiological activity of selenium, and providing a new technological pathway for the development of selenium-enriched functional foods.
[0004] Asparagus, a highly nutritious vegetable with both medicinal and edible properties, is rich in polyphenols, flavonoids, polysaccharides, and other active ingredients, earning it the title of "King of Vegetables." Asparagus tea, processed from asparagus, retains its nutritional characteristics while expanding its consumption options. However, traditional selenium-enriched asparagus tea relies heavily on selenium-enriched asparagus, resulting in high production costs and limited selenium dissolution during brewing, hindering the achievement of stable and efficient dietary selenium supplementation and limiting its widespread application. Furthermore, asparagus itself has a high purine content, further restricting its consumption by some groups. To address these issues, this study employed probiotic fermentation technology, which effectively reduced the purine content in the product while increasing the content of extracellular polysaccharides and γ-aminobutyric acid (GABA), and enhancing antioxidant properties. This strain, applied to asparagus tea fermentation, demonstrated multiple functional potentials, including sleep aid, purine reduction, and antioxidant effects, showing promising development prospects.
[0005] To address the aforementioned issues, this invention combines microbial selenium enrichment technology with the deep processing of asparagus tea. Utilizing a highly efficient selenium-enriching strain of *Lactobacillus acidophilus* JGSLA09 obtained through screening, inorganic selenium is converted into easily absorbed organic selenium and nano-selenium through a fermentation process. This technology not only effectively improves the conversion rate and bioavailability of selenium but also enriches the product forms of selenium-enriched asparagus tea. It overcomes the technical bottlenecks of traditional processes, such as strong dependence on raw materials and low selenium dissolution rates, providing a feasible solution for developing novel, efficient, and convenient selenium-enriched functional foods. Summary of the Invention
[0006] The purpose of this invention is to provide a selenium-enriched Lactobacillus acidophilus and its application. This invention solves the technical problems of traditional selenium-enriched tea products, such as reliance on natural selenium-enriched raw materials, high production costs, and low selenium dissolution rate and bioavailability during brewing. By utilizing the biotransformation capabilities of the highly efficient selenium-enriched Lactobacillus acidophilus JGSLA09, inorganic selenium is efficiently converted into organic selenium, and then coupled with asparagus tea substrate through fermentation to ultimately produce selenium-enriched asparagus tea tablets. This significantly improves the bioavailability, stability, and ease of consumption of selenium in the final product while reducing raw material costs.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of Lactobacillus acidophilus JGSLA09, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34272 and deposit date of April 21, 2025.
[0008] This invention provides a method for preparing selenium-enriched Lactobacillus acidophilus using Lactobacillus acidophilus JGSLA09. Lactobacillus acidophilus JGSLA09 is inoculated into a culture medium containing sodium selenite and cultured in a shaker for 24-72 hours to obtain selenium-enriched Lactobacillus acidophilus.
[0009] Preferably, the inoculum amount of Lactobacillus acidophilus JGSLA09 is 1%~6% (v / v); the concentration of sodium selenite in the culture medium is 10~50 μg / mL; and the sodium selenite is added when the strain reaches the logarithmic growth phase.
[0010] Preferably, the shaking incubator rotates at 120-180 rpm and the incubation temperature is 35-38℃.
[0011] This invention provides a selenium-enriched Lactobacillus acidophilus prepared by the method described above.
[0012] This invention provides a method for fermenting asparagus tea using selenium-enriched Lactobacillus acidophilus, comprising the following steps: (1) The asparagus tea (brand: Shandong Caoxian Juxinyuan asparagus tea) was crushed, pretreated with compound enzyme hydrolysis, and then extracted with water or ethanol, combined with ultrasonic-assisted treatment to obtain asparagus tea extract. (2) The selenium-enriched Lactobacillus acidophilus was centrifuged, washed and diluted to obtain a fermentation agent; (3) The fermentation agent obtained in step (2) is inoculated into the asparagus tea extract obtained in step (1) and fermented at a constant temperature to obtain selenium-enriched asparagus tea fermentation liquid.
[0013] Preferably, in step (1), the compound enzyme is cellulase and pectinase in a volume ratio of 1 to 3:1, and the amount added is 0.5% to 1.2% of the mass of asparagus tea powder; the enzymatic hydrolysis conditions are: pH 4.0 to 4.8, temperature 35 to 48°C, and time 1.0 to 1.5 h.
[0014] Preferably, in step (1), the conditions for water extraction are: temperature 80~100℃, time 10~30min; the conditions for ethanol extraction are: ethanol concentration 50%~60%, temperature 40~60℃, time 1.0~1.5h; and the power of ultrasonic-assisted treatment is 150~300 W, time is 10~30min.
[0015] Preferably, in step (3), the amount of fermentation agent added is 0.01% to 0.5% of the mass of asparagus tea extract; the conditions for constant temperature fermentation are: temperature 30 to 37°C, rotation speed 120 to 180 rpm, and time 1 to 3 days.
[0016] This invention provides a selenium-enriched fermented asparagus tea prepared by the method described above.
[0017] Furthermore, the source of Lactobacillus acidophilus strains is explained below: 1. Isolation and purification of bacterial strains Lactobacillus acidophilus strain JGSLA09 was isolated from an intestinal fecal sample of a healthy adult male (BMI 22.8) in Nanchang, China. The feces were collected using a sterile sampler and sent to the laboratory within 2 hours.
[0018] Take 1-2 g of fecal sample, add sterile physiological saline and vortex mix well to prepare 10 - ¹Diluent, then serially diluted 10-fold to 10. -5 Three parallel samples are used for each gradient. Take 10... - ²~10 -5Spread 100 μL of each graded bacterial suspension onto MRS solid plates containing 0.04% bromocresol purple, and incubate upside down at 37±1℃ for 24-48 h. Pick single colonies that are round, smooth, with regular edges and a yellow color ring, and number them with a sterile marker.
[0019] Pick numbered colonies using an inoculation loop and inoculate them onto MRS plates using the three-step streak method. Incubate at 37±1℃ for 24-48 h. Repeat the above purification steps 3-6 times until uniform, uncontaminated single colonies are obtained, which are the purified lactic acid bacteria strains.
[0020] 2. Preservation of strains
[0021] Select purified target lactic acid bacteria single colonies, inoculate them into MRS liquid medium, and anaerobic culture at 37±1℃ for 24-48h; take 0.5mL of the above bacterial solution and 0.5mL of 20%-30% sterile glycerol and add them to a sterilized strain preservation tube, mix thoroughly, and store in a -80℃ refrigerator.
[0022] 3. Morphological identification of strains
[0023] Take the purified strain and stain it according to the instructions of the Gram staining kit. After staining, observe it under an optical microscope to determine the Gram staining positivity, whether it has a capsule, whether it has flagella, and other morphological characteristics.
[0024] 4. Molecular identification of strains
[0025] DNA extraction and 16S rDNA PCR amplification were performed on suspected Lactobacillus acidophilus strains. The purified suspected target strains were inoculated into MRS broth medium and cultured at 37°C for 24 h. DNA was then extracted using a bacterial genomic DNA extraction kit. PCR amplification was performed using the extracted DNA as a template. The total reaction volume was 25 μL: 1 μL of 10 μM upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1), 1 μL of 10 μM downstream primer 1495R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID NO.2), 12.5 μL of 2×Taq plus Buffer, 1 μL of template DNA, and sterile ddH2O was added to a total volume of 25 μL. Sterile ultrapure water was used as a negative control instead of template DNA. The amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 29 cycles; and a final extension at 72℃ for 5 min. After amplification, the target DNA fragment was recovered and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.3. 5. Morphological and molecular identification results of the strain Morphological observation of the suspected Lactobacillus acidophilus colonies obtained by streak plating purification revealed that the strain appeared milky white, round, smooth, with regular edges, and opaque on the culture medium (see Figure 1). Gram staining and microscopic observation of the strain showed that it was a Gram-positive bacterium with rod-shaped cells, no capsule, and no flagella (see Figure 2).
[0026] 16S rDNA was amplified using a direct bacterial cell amplification method, and the resulting 16S rDNA sequence was compared using BLAST in the NCBIGenome database. The results showed that the 16S rDNA sequence of this strain had greater than 99% homology with known Lactobacillus acidophilus, thus identifying the strain as Lactobacillus acidophilus and naming it Lactobacillus acidophilus JGSLA09.
[0027] Compared with the prior art, the present invention has the following beneficial effects: Firstly, in terms of selenium conversion and enrichment, *Lactobacillus acidophilus* JGSLA09 exhibits excellent inorganic selenium conversion capabilities, achieving a conversion rate of up to 98.6% for sodium selenite to organic selenium, significantly improving the bioavailability and safety of selenium. After cultivation in selenium-containing media, the selenium content of this strain significantly increases, providing a highly active selenium-enriched inoculant for subsequent fermentation, overcoming the technological limitations of traditional reliance on naturally sourced selenium-rich raw materials.
[0028] Secondly, regarding the enhancement of nutrition and function in fermented products, the asparagus tea extract fermented by this strain yielded a high total selenium content in the resulting selenium-enriched asparagus tea fermented powder, with a DPPH free radical scavenging rate as high as 71.3%–78.5%, indicating that the product possesses excellent antioxidant activity. Simultaneously, the fermentation process also increased the content of functional components such as γ-aminobutyric acid, extracellular polysaccharides, total polyphenols, and total flavonoids, giving the product multiple health benefits including selenium supplementation, antioxidant properties, and sleep aid.
[0029] Finally, this technical solution provides a complete and controllable coupled process for microbial selenium enrichment and plant substrate fermentation. By optimizing the strain culture conditions and the pretreatment process for asparagus tea, it systematically solves the technical bottlenecks of low selenium dissolution rate, insufficient bioavailability, and limited functional components in traditional selenium-enriched tea products. This process is not limited to a specific end product form; the selenium-enriched fermented substrate produced can be directly applied to liquid beverages, solid powders, or as functional ingredients, demonstrating good process adaptability and industrial application potential. Attached Figure Description
[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0031] Figure 1 This is a colony morphology diagram of selenium-enriched Lactobacillus acidophilus.
[0032] Figure 2 This is a microscopic image of the morphology of selenium-enriched Lactobacillus acidophilus.
[0033] Figure 3 The effect of inoculum size on selenium enrichment in Lactobacillus acidophilus.
[0034] Figure 4 The effect of sodium selenite concentration on selenium enrichment in Lactobacillus acidophilus.
[0035] Figure 5 The effect of culture time on selenium enrichment of Lactobacillus acidophilus.
[0036] Biological Preservation Instructions
[0037] Lactobacillus acidophilus JGSLA09 was deposited on April 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34272. Detailed Implementation
[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0039] Example 1
[0040] A method for preparing selenium-enriched Lactobacillus acidophilus, comprising the following steps: (1) Inoculate the Lactobacillus acidophilus JGSLA09 seed culture medium into MRS agar medium, and culture on a shaker at a speed of 120 rpm and a temperature of 36℃. The inoculation amount of the strain is 3.5% (v / v). (2) When the bacterial strain is in the logarithmic growth phase, add sodium selenite to a final concentration of 10 μg / mL and continue culturing for 12 h to obtain selenium-enriched Lactobacillus acidophilus.
[0041] Example 2
[0042] A method for preparing selenium-enriched Lactobacillus acidophilus, comprising the following steps: (1) Inoculate the Lactobacillus acidophilus JGSLA09 seed culture medium into MRS agar medium, and culture on a shaker at a speed of 180 rpm and a temperature of 38℃. The inoculation amount of the strain is 4.5% (v / v). (2) When the bacterial strain is in the logarithmic growth phase, add sodium selenite to a final concentration of 50 μg / mL and continue culturing for 72 h to obtain selenium-enriched Lactobacillus acidophilus.
[0043] Example 3
[0044] A method for preparing selenium-enriched Lactobacillus acidophilus, comprising the following steps: (1) Inoculate the Lactobacillus acidophilus JGSLA09 seed culture medium into MRS agar medium, and culture on a shaker at a speed of 150 rpm and a temperature of 37°C. The inoculation amount of the strain is 4% (v / v). (2) When the bacterial strain is in the logarithmic growth phase, add sodium selenite to a final concentration of 40 μg / mL, continue culturing for 60 h, and obtain selenium-enriched Lactobacillus acidophilus ( Figure 1 Image of selenium-enriched Lactobacillus acidophilus colony morphology; Figure 2 (Microscopic image of Lactobacillus acidophilus enriched with selenium).
[0045] The antioxidant capacity of selenium-enriched Lactobacillus acidophilus was analyzed, and the determination method was referenced in CN202411118189.4. The results showed that the selenium-enriched Lactobacillus acidophilus prepared in the examples had a DPPH free radical scavenging capacity of 67.94±5.02%, a hydroxyl free radical scavenging capacity of 43.51±1.54%, and a reducing power of 41.84±3.87%, demonstrating excellent antioxidant capacity.
[0046] Based on the optimized culture conditions, the applicant analyzed the Lactobacillus acidophilus JGSLA09 and found that after selenium-enriched culture, 95.5% of the selenium was organic selenium and 3.1% was nano-selenium. The organic selenium included selenomethionine, which accounted for more than 88% of the organic selenium content. This result indicates that Lactobacillus acidophilus JGSLA09 has a strong ability to convert inorganic selenium into organic selenium in a culture medium containing sodium selenite (inorganic selenium).
[0047] Example 4
[0048] A method for preparing asparagus tea using selenium-enriched Lactobacillus acidophilus fermentation, comprising the following steps: (1) Raw material preparation and processing: Asparagus tea was subjected to low-temperature ultra-fine grinding at -35℃ to 200 mesh. After pretreatment by enzymatic hydrolysis of cellulase and pectinase complex enzyme, it was extracted by water immersion and combined with ultrasonic assisted treatment to obtain asparagus tea extract. The enzymatic hydrolysis process parameters are as follows: the ratio of asparagus powder to water is 1:10, the ratio of cellulase to pectinase and the amount of compound enzyme added are 1:1 and 0.5% respectively (based on the mass of asparagus powder), the pH range is 4.0, the reaction temperature is 35℃, and the enzymatic hydrolysis time is 1.0h. The process parameters for water extraction are: extraction temperature 80℃, extraction time 10min, ultrasonic time 10min, and ultrasonic power 150w. (2) Preparation of selenium-enriched lactic acid bacteria fermentation agent: The selenium-enriched Lactobacillus acidophilus prepared in Example 3 was placed in a centrifuge tube, centrifuged, the supernatant was discarded, and the mixture was rinsed, diluted and mixed with sterilized skim milk to obtain the fermentation agent; (3) Fermentation of asparagus tea extract: Fermentation agent is inoculated into asparagus tea extract and fermented at constant temperature to obtain selenium-enriched asparagus tea fermentation liquid; the amount of fermentation agent added is 0.01%, the fermentation liquid is sterilized at 121℃ for 15 minutes, placed at 30℃, fermented at constant temperature for 1 day, rotating at 120 rpm, fermented in the dark, and the relative humidity is 60%.
[0049] (4) Preparation of selenium-enriched asparagus tea fermentation powder: The asparagus tea fermentation liquid was subjected to cell wall breaking, homogenization and concentration treatment in sequence, and then vacuum freeze-drying to obtain selenium-enriched asparagus tea fermentation powder. Before vacuum concentration, because the fermentation broth contains protein substances, foam is easily generated during concentration. 0.01%-0.05% of food-grade defoamer (polyglycerol fatty acid ester) can be added. Vacuum concentration is used, and its process parameters are: vacuum degree: 0.085 MPa, concentration temperature: 40℃, and rotation speed: 40 rpm.
[0050] Example 5
[0051] A method for preparing asparagus tea using selenium-enriched Lactobacillus acidophilus fermentation, comprising the following steps: (1) Raw material preparation and processing: Asparagus tea was subjected to low-temperature ultra-fine grinding at -35℃ to 400 mesh. After pretreatment by enzymatic hydrolysis of cellulase and pectinase complex enzyme, it was extracted with edible ethanol and combined with ultrasonic-assisted treatment to obtain asparagus tea extract. The enzymatic hydrolysis process parameters are as follows: the ratio of asparagus powder to water is 1:15, the ratio of cellulase to pectinase and the amount of compound enzyme added are 3:1 and 1.2% respectively (based on the mass of asparagus powder), the pH range is 4.8, the reaction temperature is 48℃, and the enzymatic hydrolysis time is 1.5h. The process parameters for ethanol extraction are: ethanol concentration 60%, extraction temperature 60℃, extraction time 1.5h, ultrasonic time 30min, and ultrasonic power 300w. (2) Preparation of selenium-enriched lactic acid bacteria fermentation agent: The selenium-enriched Lactobacillus acidophilus prepared in Example 3 was placed in a centrifuge tube, centrifuged, the supernatant was discarded, and the mixture was rinsed, diluted and mixed with sterilized skim milk to obtain the fermentation agent; (3) Fermentation of asparagus tea extract: Fermentation agent is inoculated into asparagus tea extract and fermented at constant temperature to obtain selenium-enriched asparagus tea fermentation liquid; the amount of fermentation agent added is 0.5%, the fermentation liquid is sterilized at 121℃ for 20 minutes, placed at 37℃, fermented at constant temperature for 3 days, the rotation speed is 120-180 rpm, fermented in the dark, and the relative humidity is 75%.
[0052] (4) Preparation of selenium-enriched asparagus tea fermentation powder: The asparagus tea fermentation liquid was subjected to cell wall breaking, homogenization and concentration treatment in sequence, and then spray dried to obtain selenium-enriched asparagus tea fermentation powder. Before vacuum concentration, because the fermentation broth contains protein substances, foam is easily generated during concentration. 0.01%-0.05% of food-grade defoamer (polyglycerol fatty acid ester) can be added. Vacuum concentration is used, and its process parameters are: vacuum degree: 0.095 MPa, concentration temperature: 50℃, and rotation speed: 60 rpm.
[0053] Example 6
[0054] A method for preparing asparagus tea using selenium-enriched Lactobacillus acidophilus fermentation, comprising the following steps: (1) Raw material preparation and processing: Asparagus tea was subjected to low-temperature ultrafine grinding at -35℃ to 300 mesh. After pretreatment by enzymatic hydrolysis of cellulase and pectinase complex enzyme, it was extracted by water immersion and combined with ultrasonic assisted treatment to obtain asparagus tea extract. The enzymatic hydrolysis process parameters are as follows: the ratio of asparagus powder to water is 1:10, the ratio of cellulase to pectinase and the amount of compound enzyme added are 1:1 and 1% respectively (based on the mass of asparagus powder), the pH range is 4.4, the reaction temperature is 40℃, and the enzymatic hydrolysis time is 1.0h. The process parameters for water extraction are: extraction temperature 90℃, extraction time 20min, ultrasonic time 20min, and ultrasonic power 200w. (2) Preparation of selenium-enriched lactic acid bacteria fermentation agent: The selenium-enriched Lactobacillus acidophilus prepared in Example 3 was placed in a centrifuge tube, centrifuged, the supernatant was discarded, and the mixture was rinsed, diluted and mixed with sterilized skim milk to obtain the fermentation agent; (3) Fermentation of asparagus tea extract: Fermentation agent is inoculated into asparagus tea extract and fermented at constant temperature to obtain selenium-enriched asparagus tea fermentation liquid; the amount of fermentation agent added is 0.2%, the fermentation liquid is sterilized at 121℃ for 18 minutes, placed at 35℃, fermented at constant temperature for 2 days, rotating at 150 rpm, fermented in the dark, and the relative humidity is 70%.
[0055] (4) Preparation of selenium-enriched asparagus tea fermentation powder: The asparagus tea fermentation liquid was subjected to cell wall breaking, homogenization and concentration treatment in sequence, and then dried by heat pump to obtain selenium-enriched asparagus tea fermentation powder. Before vacuum concentration, because the fermentation broth contains protein substances, foam is easily generated during concentration. 0.03% food-grade defoamer (polyglycerol fatty acid ester) can be added. Vacuum concentration is adopted, and its process parameters are: vacuum degree: 0.09 MPa, concentration temperature: 45℃, and rotation speed: 50 rpm.
[0056] Experimental Example 1
[0057] The total selenium content of the selenium-enriched asparagus tea fermented powder prepared by the methods in Examples 4 to 6 was determined. The determination method was based on the method described in the reference "Chen Yongbo, Liu Shuqin, Wang Yiling, et al. Evaluation of leaching rate and effectiveness of selenium in selenium-enriched tea [J]. Trace Elements and Health Research, 2016, 33(5):2." The blank control group was asparagus tea powder.
[0058] Specifically, 1g of selenium-enriched asparagus tea fermentation powder was placed in a small beaker and extracted three times with boiling ultrapure water, adding 100ml of water each time, with an extraction time of 10min. The three extracts were combined, and the total selenium content in the extracts was determined. The average value of the three measurements was taken, and the results are shown in Table 1.
[0059] Table 1 Results of total selenium content determination
[0060] The data show that the selenium content in the three examples is at a similar level, but the selenium content in Example 6 is slightly higher than that in Examples 4 and 5. This indicates that the selenium enrichment efficiency varies in the yeast powder prepared under different process parameters, which may be related to fermentation conditions, extraction methods, or subsequent processing. Further analysis shows that Example 6 used water extraction combined with ultrasonic-assisted treatment, with appropriate enzymatic hydrolysis conditions and fermentation time. These factors may have collectively promoted the effective conversion and retention of selenium during fermentation.
[0061] Overall, all three embodiments successfully enriched selenium in asparagus tea fermentation powder, and the selenium content reached levels suitable for functional foods. The process combination in Example 6 showed certain advantages in selenium retention, providing a reference direction for subsequent process optimization.
[0062] Experiment Example 2
[0063] DPPH free radical scavenging activity was determined spectrophotometrically. The principle is as follows: DPPH free radicals contain one unpaired electron and exhibit strong absorption (deep purple) near 525 nm. When a free radical scavenger is present in the system, the hydrogen atom or electron provided by the scavenger will pair with the DPPH free radical, causing a decrease in absorbance. The degree of fading is quantitatively related to the number of electrons received by the scavenger. Therefore, the antioxidant capacity of the sample can be assessed by changes in absorbance; a higher scavenging rate indicates stronger antioxidant activity.
[0064] 1. Sample Preparation
[0065] Take 1.0 g of the selenium-enriched asparagus tea fermentation powder from Examples 4-6, add 30 mL of 98℃ deionized water, and extract in a 98℃ water bath for 30 min. Filter while hot through a 400-mesh filter cloth, and bring the filtrate to a final volume of 50 mL to obtain the sample solution. The blank control group is asparagus tea powder.
[0066] 2. Measurement Procedure
[0067] Reagents: 0.1 mM DPPH-methanol solution, 0.05 mol / L Tris-HCl buffer (pH 7.4).
[0068] Sample tube: Add 2.0 mL of DPPH-methanol solution, 0.9 mL of Tris-HCl buffer, and 0.5 mL of sample solution in sequence.
[0069] Control tube: Add 2.0 mL DPPH-methanol solution, 0.9 mL Tris-HCl buffer, and 0.5 mL methanol sequentially.
[0070] Measurement: After adding samples to each tube and mixing them thoroughly, react in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm (zeroed with distilled water).
[0071] 3. Calculations and Results
[0072] The formula for calculating DPPH free radical scavenging rate is:
[0073] Each sample was measured in triplicate, and the average value was taken. The results are shown in Table 2.
[0074] Table 2 Results of Antioxidant Determination
[0075] Table 2 shows the DPPH free radical scavenging rates of the selenium-enriched asparagus tea fermentation powders prepared in Examples 4 to 6. All three examples exhibited strong antioxidant activity, with Example 6 showing the highest scavenging rate at 78.5%, indicating optimal antioxidant capacity. This result is largely consistent with the trend of selenium content in Table 1, suggesting a positive correlation between selenium enrichment and improved antioxidant performance of the product.
[0076] In summary, the fermentation of asparagus tea with selenium-enriched Lactobacillus acidophilus not only achieved the organic conversion and enrichment of selenium but also significantly enhanced the product's antioxidant activity. Example 6 showed the best performance in both selenium content and antioxidant activity, providing experimental evidence for its use as a highly efficient functional food ingredient. Future research can further explore the relationship between selenium forms, fermentation metabolites, and antioxidant mechanisms to optimize product performance.
[0077] Experimental Example 3
[0078] The functional components of asparagus tea prepared by fermentation of selenium-enriched Lactobacillus acidophilus were determined. In this case, Example 6 was used as the fermentation group; the fermentation agent in step (2) of Example 6 was replaced with an equal amount of water as the fermentation control group.
[0079] Table 3. Indicators of various functional components after fermentation
[0080] "-" indicates that it was not detected. Table 4. Changes in purine content after fermentation
[0081] Table 5 Survey on the Decline
[0082] The results showed that although asparagus is rich in nutrients, it is high in purines, which mainly include adenine, guanine, hypoxanthine and xanthine.
[0083] After fermentation, the total purine content of asparagus powder decreased by 85.79%, with hypoxanthine and xanthine showing the largest decreases, exceeding 94%, indicating that the fermentation process of this strain can effectively reduce the purine content in asparagus.
[0084] Selenium-enriched culture of bacteria in Experiment Example 4
[0085] 1. Effect of inoculum size on selenium enrichment of Lactobacillus acidophilus JGSLA09
[0086] The *Lactobacillus acidophilus* JGSLA09 strain was inoculated into sterilized MRS liquid medium and cultured at 37°C with a shaker at 150 r / min for 24 h, activating for two generations. The optical density (OD) of the bacterial suspension at 600 nm was measured using 0.9% sterile physiological saline. 600 Adjust the pH to 0.6-0.8 and prepare the seed solution for later use.
[0087] Seed culture was inoculated into MRS liquid medium at an initial pH of 6 at inoculum rates of 1%, 2%, 3%, 4%, and 5% (v / v), and cultured until the logarithmic growth phase (6–8 h). Then, sterilized sodium selenite solution was added to bring the final sodium selenite concentration to 40 μg / mL, and the culture was incubated at 37°C and 150 r / min for 24 h. The cultured bacterial solution was centrifuged, and the bacterial precipitate was collected. The precipitate was washed three times with sterile 0.9% physiological saline and then used for further analysis. The selenium content in the samples was determined using hydride generation-atomic fluorescence spectrometry (AFS), and the selenium enrichment level and rate of *Lactobacillus acidophilus* JGSLA09 were calculated.
[0088] Figure 3 The results show the selenium enrichment performance of Lactobacillus acidophilus at different inoculum amounts. The results indicate that Lactobacillus acidophilus JGSLA09 exhibits the highest selenium enrichment at an inoculum amount of 2% (v / v), therefore the optimal inoculum amount is 2%.
[0089] 2. Effect of sodium selenite concentration on selenium enrichment of Lactobacillus acidophilus JGSLA09
[0090] Selenium-enriched Lactobacillus acidophilus JGSLA09 was prepared according to method 1 above, with an inoculum size of 2% (v / v). Aseptically treated sodium selenite stock solution was added to MRS liquid medium to make the final concentrations of sodium selenite in the system 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively. The culture was carried out at 37℃ for 24 h, and the selenium enrichment rate and selenium enrichment amount of the strain were determined.
[0091] Figure 4 The effect of different concentrations of sodium selenite on the selenium enrichment capacity of Lactobacillus acidophilus JGSLA09 was investigated. The results showed that the highest selenium enrichment capacity of Lactobacillus acidophilus JGSLA09 was achieved at a sodium selenite concentration of 40 μg / mL, therefore the optimal concentration was 40 μg / mL.
[0092] 3. Effect of culture time on selenium enrichment of Lactobacillus acidophilus JGSLA09
[0093] Selenium-enriched Lactobacillus acidophilus JGSLA09 was prepared according to method 2 above, with an inoculum amount of 2% (v / v) and a sodium selenite concentration of 40 μg / mL. The bacteria were incubated at 37℃ for 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, respectively, and the selenium enrichment rate and selenium enrichment amount of the strain were determined.
[0094] Figure 5The effect of different culture times on the selenium enrichment capacity of Lactobacillus acidophilus JGSLA09 was investigated. The results showed that the selenium enrichment of Lactobacillus acidophilus JGSLA09 was highest after 48 h of culture, therefore the optimal culture time was 48 h.
[0095] 4. Process optimization of selenium-enriched culture of Lactobacillus acidophilus JGSLA09
[0096] To determine the optimal culture process for selenium-enriched Lactobacillus acidophilus JGSLA09, this invention investigated inoculum size, culture time, and sodium selenite concentration, setting three levels for each factor and employing the L9(3)4 method. 4 Orthogonal experimental design was conducted using an orthogonal array (see Tables 6 and 7), with selenium enrichment rate and selenium enrichment amount as evaluation indicators. The results showed that the primary and secondary factors affecting selenium enrichment performance were: cultivation time (B) > sodium selenite concentration (C) > inoculum size (A), and the optimal combination was a cultivation time of 48 h, a sodium selenite concentration of 45 μg / mL, and an inoculum size of 2%, under which the selenium enrichment rate was 96.8%.
[0097] Table 6. Factors and Levels in Orthogonal Experiments
[0098] Table 7. Orthogonal Experiment Scheme and Results
[0099] Analysis of the optimized culture conditions revealed that after selenium-enriched culture, Lactobacillus acidophilus JGSLA09 contained 95.5% organic selenium and 3.1% nano-selenium. The organic selenium included selenomethionine, which accounted for more than 88% of the organic selenium content. This result indicates that Lactobacillus acidophilus JGSLA09 has a strong ability to convert inorganic selenium into organic selenium in a culture medium containing sodium selenite (inorganic selenium).
[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A strain of Lactobacillus acidophilus JGSLA09, characterized in that, The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34272 and deposit date of April 21, 2025.
2. A method for preparing selenium-enriched Lactobacillus acidophilus using Lactobacillus acidophilus JGSLA09 as described in claim 1, characterized in that, Lactobacillus acidophilus JGSLA09 was inoculated into a culture medium containing sodium selenite and cultured on a shaker for 12-72 hours to obtain selenium-enriched Lactobacillus acidophilus.
3. The method according to claim 2, characterized in that, The inoculum amount of Lactobacillus acidophilus JGSLA09 is 1%~6% (v / v); the concentration of sodium selenite in the culture medium is 10~50 μg / mL; the sodium selenite is added when the strain reaches the logarithmic growth phase.
4. The method according to claim 2 or 3, characterized in that, The shaking incubator operates at a speed of 120-180 rpm and a temperature of 35-38℃.
5. A selenium-enriched Lactobacillus acidophilus prepared by the method of any one of claims 2 to 4.
6. A method for fermenting asparagus tea using selenium-enriched Lactobacillus acidophilus, characterized in that, Includes the following steps: (1) The asparagus tea is crushed, pretreated by compound enzyme hydrolysis, and then extracted with water or ethanol, combined with ultrasonic-assisted treatment to obtain asparagus tea extract. (2) The selenium-enriched Lactobacillus acidophilus described in claim 5 is centrifuged, washed and diluted to obtain a fermentation agent; (3) The fermentation agent obtained in step (2) is inoculated into the asparagus tea extract obtained in step (1) and fermented at a constant temperature to obtain selenium-enriched asparagus tea fermentation liquid.
7. The method according to claim 6, characterized in that, In step (1), the compound enzyme is cellulase and pectinase in a volume ratio of 1 to 3:1, and the amount added is 0.5% to 1.2% of the mass of asparagus tea powder; the enzymatic hydrolysis conditions are: pH 4.0 to 4.8, temperature 35 to 48°C, and time 1.0 to 1.5 h.
8. The method according to claim 6, characterized in that, In step (1), the conditions for water extraction are: temperature 80~100℃, time 10~30min; the conditions for ethanol extraction are: ethanol concentration 50%~60%, temperature 40~60℃, time 1.0~1.5h; the power of ultrasonic assisted treatment is 150~300 W, and the time is 10~30min.
9. The method according to claim 6, characterized in that, In step (3), the amount of fermentation agent added is 0.01% to 0.5% of the mass of asparagus tea extract; the conditions for constant temperature fermentation are: temperature 30 to 37°C, rotation speed 120 to 180 rpm, and time 1 to 3 days.
10. The selenium-enriched fermented asparagus tea prepared by the method according to any one of claims 6 to 9.