CN116121142A - Lactococcus lactis (Lactococcus lactis) SPL018, and acquisition method and application thereof - Google Patents
Lactococcus lactis (Lactococcus lactis) SPL018, and acquisition method and application thereof Download PDFInfo
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Abstract
The invention discloses a lactococcus lactis (Lactococcus lactis) SPL018, an acquisition method and application. The strain is obtained by separating Ningqiang selenium-rich soil in Ankang city, and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC NO.25808 in year 2022 and month 09 and 26. When the inorganic selenium content is lower than 50mg/L, lactococcus lactis SPL018 can convert selenium into organic selenium; when the inorganic selenium content is 100-10000 mg/L, the lactococcus lactis SPL018 can convert selenium into elemental selenium, and the size of the elemental selenium is 50-100 nm. The selenium-containing microbial agent is prepared by utilizing the fermentation of lactococcus lactis SPL018, and can increase the absorption of selenium by plants, so that the compound microbial agent is applied to the reinforcement of selenium enrichment of crops in the modes of selenium application to soil, selenium spraying to leaf surfaces, seed dressing and the like, and is a safe, reliable and low-cost mode.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to a lactococcus lactis (Lactococcus lactis) SPL018, an acquisition method and application thereof.
Background
Selenium is a trace element necessary for human body, and has biological activities of resisting tumor, resisting oxidation, enhancing human body immunity, etc. Compared with inorganic selenium, the organic selenium has high absorption and utilization rate, good nutritive value, safe use and no toxic and side effect, and is the first choice for supplementing selenium. In recent years, research on the production of organic selenium by a microbial transformation method has been rapidly developed, and the selenium-rich fermentation process conditions are matured.
Lactic acid bacteria (Lactic acid bacteria, LAB) are gram-positive bacteria that are facultative anaerobic and can produce more than 50% of lactic acid using glucose, and are a generic term for a class of bacteria that can produce lactic acid. As a group of probiotics, lactic acid bacteria are widely used in many industries such as food, light industry, medicine and feed industry. In recent years, selenium-enriched lactobacillus has received general attention from scholars because of its outstanding antioxidant, antibacterial, anticancer and antimutagenic properties. It was found that as an element-enriched probiotic, the present invention provides a strain of lactococcus lactis (Lactococcus lactis) SPL018, which is capable of adsorbing inorganic selenium and bioconverting it to organic selenium. The bacterial strain lactococcus lactis SPL018 is utilized for preparing the selenium-containing microbial inoculum by fermentation, so that the absorption of selenium by plants can be increased, and the method is applied to strengthening the selenium enrichment of crops by means of soil selenium application, foliar selenium spraying, seed dressing and the like, and is a safe, reliable and low-cost method.
There is currently no report on the transformation of the strain lactococcus lactis SPL018 with selenium. The lactococcus lactis SPL018 provided by the invention has good biological oxidation function on simple substance selenium and organic selenium, so that the lactococcus lactis SPL018 can more effectively oxidize the simple substance selenium or the organic selenium into soluble tetravalent selenium in selenium-rich or selenium-poor soil, has good application potential for promoting plant selenium enrichment, and can be used as a good material for researching selenium oxidation and biological selenium enrichment.
The invention also provides the protective content of the lactococcus lactis SPL01, and the selenium-enriched microbial inoculum prepared from the lactococcus lactis SPL01 and the application of the selenium-enriched microbial inoculum. Compared with the prior art, the invention has the following advantages:
the lactococcus lactis SPL018 has the capability of oxidizing selenite to generate organic selenium, and is a supplement to limited selenium oxidation strain resources. The lactococcus lactis SPL018 is a strain capable of simultaneously converting organic selenium and elemental selenium, and has scientific research significance and agricultural application potential of selenium conversion mode strains. In addition, the invention provides a novel method for promoting the selenium enrichment of plants by utilizing the beneficial microbial agent, and the method has the characteristics of safety and environmental protection.
Disclosure of Invention
The invention aims at providing a lactococcus lactis (Lactococcus lactis) SPL018, an acquisition method and application thereof,
the strain lactococcus lactis (Lactococcus lactis) SPL018 provided by the invention is a strain which is separated and screened from Ning strong selenium-rich soil in Ankang city of Shanxi province by the applicant, the strain SPL018 has microsphere shape and diameter of 0.2-0.4 mu m, and is cultured on an MRS culture medium at 33 ℃ for 48 hours, and the colony is milky, round, raised and complete and smooth in edge. The identification and sequence analysis of the 16S rDNA of the strain combined with physiological and biochemical detection show that the strain is lactococcus lactis (Lactococcus lactis). The strain is preserved in China general microbiological culture collection center (CGMCC) (address: north Xielu No.1, 3 of the North Chen West Lu of the Chaoyang area of Beijing city) for 26 days in the 09 month of 2022, and the preservation number is CGMCC No.25808, and is classified and named as lactococcus lactis (Lactococcus lactis) SPL018.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
an isolated lactococcus lactis (Lactococcus lactis) SPL018 is preserved in China general microbiological culture collection center (CGMCC) for culture Collection of microorganisms (CGMCC No. 25808) on the 09 and 26 th year of the 2022, and is classified and named as lactococcus lactis (Lactococcus lactis) SPL018, and the sequence of a 16S rDNA fragment of the lactococcus lactis is shown as SEQ ID No. 1.
The method for obtaining the lactococcus lactis (Lactococcus lactis) SPL018 comprises the following steps:
A. collecting root soil of crops such as cucumber, tea and the like planted in Ningqiang selenium-rich soil in Ankang city, shaking off the root soil collected in a sterile bag to serve as a separation sample source;
B. preparing a selenium-rich soil immersion medium: taking 10g of selenium-rich soil, adding 100mL of 0.2% hydrochloric acid, mixing and stirring at constant temperature of 40 ℃ for 30min, filtering and collecting filtrate with medium-speed filter paper, mixing with 900mL of pure water, adding 1.5% of sucrose, 1.2% of casein and MgSO 4 ·7H 2 O 0.06％,K 2 HPO 4 0.35％,CaCO 3 0.4%, pH6.0. Sub-packaging 100mL of the culture medium with 500mL triangular flask, and sterilizing at 121 ℃ for 32min for later use;
C. when the temperature of the culture medium is reduced to about 30-40 ℃ after sterilization, adding selenium-enriched soil according to the mass ratio of 5% under an ultra clean bench, and placing a triangular flask in a constant-temperature shaking table at 35 ℃ and 200rpm for enrichment culture for 48 hours;
D. and repeating the inoculation culture for 5 times, sampling, repeatedly scribing and separating single strains on a solid selenium-enriched soil immersion medium plate, inoculating pink or red strains into the selenium-enriched soil immersion medium for domestication, repeating the operation for a plurality of times, scribing and inoculating MRS medium for culture, selecting milky white, purifying to obtain a lactococcus lactis (Lactococcus lactis) strain, and naming the lactococcus lactis as SPL018.
The application of the lactococcus lactis SPL018 in selenium conversion is characterized in that after the lactococcus lactis SPL018 is activated, the lactococcus lactis SPL018 is inoculated into an MRS culture medium containing divalent inorganic selenium salt according to the volume ratio of 1-10%, and then nitrogen is introduced into the culture medium: carbon dioxide: the volume ratio of oxygen is 8:1:1, and the aeration rate of the mixed gas is 10NL/min, and fermentation conversion is carried out.
The lactococcus lactis SPL018 is characterized in that when the inorganic selenium content is lower than 20mg/L, the lactococcus lactis can convert selenium into organic selenium; when the inorganic selenium content is 100-8000 mg/L, the lactococcus lactis can convert selenium into elemental selenium, and the size of the elemental selenium is 100-200 nm.
The application of the lactococcus lactis SPL018 comprises that the preparation containing the lactococcus lactis SPL018 and active ingredients of a carrier is diluted by 100-1000 times and then is applied by means of soil application, foliar spraying, seed dressing and the like.
The features of the present invention will be apparent from the following detailed description of the preferred embodiments, taken in conjunction with the accompanying drawings.
Drawings
FIG. 1 is a graph of colony growth of lactococcus lactis SPL018 in selenium enriched soil and MRS media.
Detailed Description
1. Isolation, acquisition and preservation of lactococcus lactis SPL018 strain
Collecting a 10cm tea root sample under the surface of a Ningqiang selenium-rich soil plantation in Ankang city, shaking off 50g of collecting root soil in a sterile bag to serve as a separation sample source; preparing a selenium-rich soil immersion medium: preparing a selenium-rich soil immersion medium: 10g of selenium-rich soil is taken, 100mL of 0.2% hydrochloric acid is added, the mixture is stirred for 30min at the constant temperature of 40 ℃, the filtrate is filtered and collected by medium-speed filter paper, and is mixed with 900mL of pure water, and 1.5% of sucrose, 1.2% of casein, 0.06% of MgSO4.7H2O, 0.35% of K2HPO, 0.4% of CaCO3 and pH6.0 are added. Sub-packaging 100mL of the culture medium with 500mL triangular flask, and sterilizing at 121 ℃ for 32min for later use; when the temperature of the culture medium is reduced to about 30-40 ℃ after sterilization, adding selenium-enriched soil according to the mass ratio of 5% under an ultra clean bench, and placing a triangular flask in a constant-temperature shaking table at 35 ℃ and 200rpm for enrichment culture for 48 hours; and repeating 5% inoculation culture for 5 times, sampling, repeatedly scribing on a solid selenium-enriched soil immersion culture medium plate to separate single bacterial strains, inoculating pink or red bacterial strains into the selenium-enriched soil immersion culture medium for domestication, repeating the operation for 3 times, scribing, inoculating MRS culture medium for culture, selecting milky white, purifying to obtain a lactococcus lactis (Lactococcus lactis) bacterial strain, and naming the lactococcus lactis as SPL018. The strain SPL018 cells are microspherical in shape and 0.2-0.4 μm in diameter, and cultured on MRS medium at 33deg.C for 48 hr to obtain colony with milky white, round, raised and smooth edges. The streaking pattern of the strain plates is shown in FIG. 1.
2. Identification and preservation of lactococcus lactis (Lactococcus lactis) SPL018 Strain
The isolated strain SPL018 was subjected to amplification of 16SrDNA sequences and second generation sequencing was aligned to a database. Genomic DNA of strain SPL018 was extracted using bacterial genome extraction kit (TIANGEN, beijing), and then 16S rDNA fragment of the strain was amplified using 16S rDNA fragment amplification and detection kit (TIANGEN, beijing) and sequenced using second generation sequencing technique (beijing aoke). The 16S rDNA sequence of strain SPL018 is shown in SEQ ID NO. 1. Comparison with NCBI database (http:// www.ncbi.nlm.nih.gov /), combined with strain physicochemical characterization results, shows that strain SPL018 is lactococcus lactis (Lactococcus lactis).
The strain lactococcus lactis (Lactococcus lactis) SPL018 was 16S rDNA series: GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTTGAGCGCTGAAGGTTGGTACTTGTACCGACTGGATGAGCAGCGAACGGGTGAGTAACGCGTGGGGAATCTGCCTTGAGCGGGGGACAACATTTGGAAACGAATGCTAATACCGCATAAAAACTTTAAACACAAGTTTTAAGTTTGAAAGATGCAATTGCATCACTCAAAGATGATCCCGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGATGATACATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGGTAGAGAAGAACGTTGGTGAGAGTGGAAAGCTCATCAAGTGACGGTAACTACCCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTATTAAGTCTGGTGTAAAAGGCAGTGGCTCAACCATTGTATGCATTGGAAACTGGTAGACTTGAGTGCAGGAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGCCTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGATGTAGGGAGCTATAAGTTCTCTGTATCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACTCGTGCTATTCCTAGAGATAGGAAGTTCCTTCGGGACACGGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAACGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACAGTGATGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTGGGAGTACCCGAAGTAGGTTGCCTAACCGCAAGGAGGGCGCTTCCTAAGGT
3. Application of lactic acid bacterium SPL018 in preparation of organic selenium and elemental selenium through fermentation and conversion
(1) The preserved lactococcus lactis SPL018 is inoculated into MRS culture media with different contents of bivalent inorganic selenium according to the volume ratio of 1-10% after being activated, and then nitrogen is introduced into the culture media comprising the bivalent inorganic selenium: carbon dioxide: the volume ratio of oxygen is 8:1:1, and the aeration rate of the mixed gas is 10NL/min, and the organic selenium can be obtained after fermentation is carried out for 36 hours.
(2) Adding 500mg/L selenium sulfite solution into the fermentation broth according to the volume of 0.2-1.0%, and continuously fermenting and converting for 96 hours to obtain elemental selenium.
(3) During the period, sampling is carried out every 12 hours, and the content of organic selenium and simple substance selenium in the sample is detected by HPLC-HG-AFS by taking the inorganic selenium liquid culture medium without adding the strain SPL018 and with the same final concentration as a control.
(4) The detection result shows that the content of organic selenium in the fermentation supernatant of the lactococcus lactis SPL018 is continuously increased, and when the content of inorganic selenium is lower than 20mg/L, the lactococcus lactis can convert the selenium into the organic selenium; up to 36h to a maximum of 0.96mg/L, after which the course decreased. After 36h, the fermentation broth is supplemented with divalent selenium salt, and when the inorganic selenium content is 100-800 mg/L, the lactococcus lactis can convert selenium into elemental selenium, and the highest content of the elemental selenium can reach 101.23mg/L for 72 h. The results are specifically shown in the following table:
| time (h) | Organic selenium (mg/L) | Elemental selenium (mg/L) |
| 0 | 0 | 0 |
| 12 | 0.12 | 0 |
| 24 | 0.41 | 1.14 |
| 36 | 0.96 | 12.46 |
| 48 | 0.31 | 39.691 |
| 60 | 0.15 | 69.57 |
| 72 | 0 | 101.23 |
| 84 | 0 | 71.01 |
| 96 | 0 | 64.22 |
4. Preparation of selenium-containing microbial inoculum and application of lactococcus lactis SPL018 in selenium enrichment in crop cultivation
(1) Taking the lactococcus lactis SPL018 fermentation broth in the third embodiment, adding one or a mixture of more than 20% of cyclodextrin, kaolin, chitin and sodium carboxymethyl cellulose according to the mass ratio, then carrying out low-temperature air drying, crushing, and screening the obtained product by a 100-mesh sieve to obtain the compound microbial agent of the organic selenium, the elemental selenium and the lactococcus lactis with the selenium content of 100-1000 mg/Kg.
(2) The selenium-containing microbial agent prepared by the method has the following application method and application effect:
(3) After the selenium-containing microbial inoculum is diluted by 100-1000 times, soil application, foliar spraying, seed dressing and other modes are carried out to apply different crop application periods, and the crops are applied by adopting root irrigation, spraying or flushing methods, so that the selenium content of one mu of land is controlled to be 1000mg.
(4) Different modes of crop application
Rice, maize, wheat: applying 2 blocks in the ear alignment period or the grouting period
Melon and fruit: applied for 2-3 times in fruit swelling period
Tea leaves: spraying for 1-2 times in the vigorous growth period, and spraying again after picking
Vegetables: applied 1-2 times in long leaf period
Medicinal materials: applied for 2-3 times in the growing period
(5) Application effect
Selenium-containing microbial agent application experiments are carried out on Yunnan Kunming vegetable bases, spraying is carried out 2 times after the four-leaf period of the cabbages, and the dosage of the microbial agent is 200 g/mu at intervals of 10 days. The vegetable leaves after the Chinese cabbage is ripe are taken and detected according to the national standard GB5009.93-2017, and the result is 0.214mg/Kg. Selenium-containing microbial agent application experiments are carried out on apple plantation fields in the county of Xunyi of Shanxi province, and the application of the selenium-containing microbial agent is carried out three times in the fruit expanding period, and the consumption per mu is accumulated to be 500g. The apples are detected after being ripe and picked, the result is 0.614mg/Kg, and the standard requirements of selenium-enriched food standard DB61/T556-2018 are met.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, which is defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. An isolated lactococcus lactis (Lactococcus lactis) SPL018 is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) for a year 09 and a year 26, and has a preservation number of CGMCC NO.25808, and is classified and named as lactococcus lactis Lactococcus lactis SPL018.
2. The method for obtaining SPL018 of lactococcus lactis (Lactococcus lactis) according to claim 1, comprising the steps of:
A. collecting root soil of crops such as cucumber, tea and the like planted in Ningqiang selenium-rich soil in Ankang city, shaking off the root soil collected in a sterile bag to serve as a separation sample source;
B. preparing a selenium-rich soil immersion medium: taking 10g of selenium-rich soil, adding 100mL of 0.2% hydrochloric acid, mixing and stirring at constant temperature of 40 ℃ for 30min, filtering and collecting filtrate with medium-speed filter paper, mixing with 900mL of pure water, adding 1.5% of sucrose, 1.2% of casein and MgSO 4 ·7H 2 O 0.06％,K 2 HPO 4 0.35％,CaCO 3 0.4%, pH6.0. Sub-packaging 100mL of the culture medium with 500mL triangular flask, and sterilizing at 121 ℃ for 32min for later use;
C. when the temperature of the culture medium is reduced to about 30-40 ℃ after sterilization, adding selenium-enriched soil according to the mass ratio of 5% under an ultra clean bench, and placing a triangular flask in a constant-temperature shaking table at 35 ℃ and 200rpm for enrichment culture for 48 hours;
D. and then taking and repeatedly inoculating and culturing 5% of enrichment culture solution for 5 generations, sampling, repeatedly scribing and separating single bacterial strains on a solid selenium-enriched soil immersion culture medium plate, inoculating pink or red bacterial strains into the selenium-enriched soil immersion culture medium for domestication, repeatedly carrying out the operation for a plurality of times, scribing and inoculating MRS (MRS) culture medium for culture, selecting milky white, and purifying to obtain a lactococcus lactis (Lactococcus lactis) bacterial strain, and naming the lactococcus lactis as SPL018.
3. Use of lactococcus lactis SPL018 according to claim 1 for selenium oxidation, characterized in that after activation of lactococcus lactis SPL018 is inoculated in a volume ratio of 1-10% in an MRS medium containing divalent inorganic selenium salts, followed by aeration with a medium comprising nitrogen: carbon dioxide: the volume ratio of oxygen is 8:1:1, and the aeration rate of the mixed gas is 10NL/min, and fermentation conversion is carried out.
4. A method according to claim 3, wherein the lactococcus lactis is capable of converting selenium into organic selenium when the inorganic selenium content is below 20 mg/L; when the inorganic selenium content is 100-8000 mg/L, the lactococcus lactis can convert selenium into elemental selenium, and the size of the elemental selenium is 100-200 nm.
5. The use of lactococcus lactis SPL018 according to claim 1, comprising a formulation comprising lactococcus lactis SPL018 and a carrier active ingredient, diluted 100-1000 fold, followed by soil application, foliar spraying, seed dressing and the like.
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| CN108753670A (en) * | 2018-05-28 | 2018-11-06 | 西北工业大学 | Multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method |
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| CN108753670A (en) * | 2018-05-28 | 2018-11-06 | 西北工业大学 | Multi-functional compound micro-ecological preparation nanometer selenium-recombinant expression vasoactive intestinal peptide-Lactococcus lactis and preparation method |
Non-Patent Citations (1)
| Title |
|---|
| 齐睿婷，等: "乳酸乳球菌富硒及其硒多糖抗氧化活性研究", 食品工业科技, vol. 33, no. 11, 15 June 2012 (2012-06-15), pages 67 - 70 * |
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