CN111004753B - Lactobacillus paracasei capable of highly producing selenocysteine and application thereof - Google Patents
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Abstract
The invention discloses lactobacillus paracasei capable of highly producing selenocysteine and application thereof, belonging to the technical field of microorganisms. According to the invention, a lactobacillus paracasei CCFM1089 strain is screened out, the lactobacillus paracasei CCFM1089 strain can efficiently convert inorganic selenium to produce selenocysteine, the lactobacillus paracasei CCFM1089 strain is inoculated into a culture medium containing the inorganic selenium for fermentation for 8 hours, and the total selenium content in the lactobacillus paracasei CCFM1089 strain freeze-dried powder can reach 400.65mg/kg, the organic selenium content in the total selenium reaches 96%, and the selenocysteine content in the organic selenium reaches 89%.
Description
Technical Field
The invention relates to lactobacillus paracasei capable of highly producing selenocysteine and application thereof, belonging to the technical field of microorganisms.
Background
Selenium is one of the essential trace elements for human and animal survival and is usually present in the form of selenoprotein. The Chinese Nutrition society lists selenium as one of 15 nutrients essential to human body, and the recommended intake of selenium element for people over 18 years old is clearly suggested in the reference intake of dietary nutrients (DRIs) of Chinese residents formulated in 2013. Insufficient selenium intake may lead to cardiovascular disease, Kaschin-Beck disease, muscular and immune dysfunction. Therefore, the timely selenium supplement is very important for the health of human bodies.
At present, common selenium supplement products are mainly divided into inorganic selenium and organic selenium. Among them, inorganic selenium generally has a high toxicity, shows cytotoxic effects such as cell detachment, increase of intracellular vacuoles, cell membrane rupture, cell necrosis and acute lysis at a low concentration, and causes DNA damage and cell death, and has a low safety, and the bioavailability of inorganic selenium is low. Therefore, inorganic selenium is not recommended as a selenium supplement product for human use.
The organic selenium is a substance formed by combining inorganic selenium with organic nutritional ingredients such as amino acid, protein, active polysaccharide and the like, and mainly comprises three kinds of selenocysteine, selenomethylselenocysteine and selenomethionine. Compared with inorganic selenium, the organic selenium has greatly reduced toxic and side effects and high safety, is easier to digest and absorb in human bodies, and has a higher bioavailability than inorganic selenium, so that the intake of the organic selenium is a main selenium supplement form for people needing selenium supplement at present.
Among the three organic selenium, selenium methyl selenocysteine is mainly used as a nutrient supplement to play a role in enhancing immunity, but the selenium methyl selenocysteine can be absorbed by a human body only after being converted into methyl selenium and selenocysteine by beta-lyase of the human body, and the bioavailability is lower compared with other organic selenium; selenomethionine mainly plays a role in increasing the oxidation resistance of an organism, improving the immunity and the like, and because the selenomethionine enters a human body in a mode of replacing sulfur by selenium, the bioavailability is higher, but because the tRNA of the human body cannot identify methionine and the selenomethionine, when the intake of the methionine is limited, a large proportion of the selenomethionine replaces the methionine to be nonspecifically added into protein in the body, so that a large amount of selenium is accumulated in the body, and a certain risk also exists; selenocysteine mainly plays roles of reconstructing bone mass, enhancing immune system, regulating thyroid function, improving male fertility, reducing cancer risk, reducing aging, relieving fatigue and the like, and like selenomethionine, the selenocysteine enters a human body by replacing sulfur with selenium, but the selenocysteine does not randomly replace amino acid like the selenomethionine, but has a triplet codon UGA of the selenocysteine, and like other amino acids, the selenocysteine is involved in protein translation and can be regulated and controlled by genes, so that the selenocysteine has higher safety compared with the selenomethionine, and more biological activity is embodied. Therefore, the selenium supplement product containing selenocysteine has the widest market prospect.
The production of selenocysteine by combining inorganic selenium with organic nutrients such as amino acid, protein, active polysaccharide and the like by utilizing the biotransformation function of microorganisms is a research hotspot in recent ten years. Compared with the existing plant extraction method and enzyme resolution method, the method has the advantages of low cost, simple process, high safety and the like. However, the conversion efficiency of the existing method for producing organic selenium by converting inorganic selenium through microorganisms is too low, so that the yield of the organic selenium produced by the method is always low, and the further development of the market of the organic selenium is undoubtedly hindered. For example, in the patent application with publication number CN103898163A, the screening of the drug by the drug; in the patent application publication No. CN110317757A, Tangxu et al screened a Lactobacillus plantarum CGMCC NO.17720, which enriched sodium selenite but had only 52.31% of the organic selenium.
Therefore, there is an urgent need to find microorganisms that can efficiently convert inorganic selenium to produce selenocysteine.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of providing Lactobacillus paracasei (Lactobacillus paracasei) capable of efficiently converting inorganic selenium to produce selenocysteine.
[ solution ]
In order to solve the technical problem, the invention provides a Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 strain, wherein the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 strain is preserved in Guangdong province microbial strain preservation center in 11 and 01 in 2019, the preservation number is GDMCC No.60880, and the preservation address is No. 59 building 5 of Mianzhou 100 institute of Mr. Zhonglu, Guangzhou city.
The Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 is obtained by separating a baby excrement sample from a more village in a Ruolaou pasture of Sichuan province, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, and the sequence obtained through sequencing is compared with the nucleic acid sequence in NCBI, so that the strain is Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
The bacterial colony of the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 on the MRS solid culture medium is round, white and smooth.
The invention also provides a method for producing organic selenium, which comprises the steps of inoculating the lactobacillus paracasei CCFM1089 into a fermentation culture medium for fermentation to obtain lactobacillus paracasei thalli, and then extracting the lactobacillus paracasei thalli to obtain the organic selenium; the fermentation culture medium contains inorganic selenium.
In one embodiment of the invention, the fermentation medium contains serine.
In one embodiment of the invention, the fermentation medium is a selenium-rich medium or a directional transformation medium;
the selenium-rich culture medium comprises 5-15 g/L of peptone, 5-15 g/L of yeast extract, 20-50 g/L of glucose, 5-15 g/L of beef extract, 1-5 g/L of anhydrous sodium acetate and 1-5 g/L, K of hydrogenous diamine citrate 2 HPO 4 ·3H 2 O 2～5g/L、MgSO 4 ·7H 2 O 0.25～0.58g/L、MnSO 4 ·H 2 0.05-0.30 g/L of O, 801g/L of Tween and 10-17 mg/L of sodium selenite pentahydrate;
the components of the directional transformation medium comprise 5-15 g/L of peptone, 5-15 g/L of yeast extract, 20-50 g/L of glucose and 5-15 g/L, K of beef extract 2 HPO 4 ·3H 2 O 2～5g/L、MgSO 4 ·7H 2 O 0.25～0.58g/L、MnSO 4 ·H 2 0.05-0.30 g/L of O, 801g/L of Tween, 10-17 mg/L of sodium selenite pentahydrate and 0.1-0.4 g/L of serine.
In one embodiment of the invention, the method comprises the steps of inoculating the lactobacillus paracasei CCFM1089 into a selenium-rich culture medium, fermenting at the temperature of 35-40 ℃, the rotating speed of 75-150 rpm and the pH of 5.0-6.0 to obtain lactobacillus paracasei thallus, and then extracting the lactobacillus paracasei thallus to obtain organic selenium;
in one embodiment of the invention, the method comprises the steps of inoculating the lactobacillus paracasei CCFM1089 into a directional transformation culture medium, fermenting at the conditions of the temperature of 35-40 ℃, the rotating speed of 75-150 rpm and the pH value of 5.0-6.0 to obtain lactobacillus paracasei thalli, and extracting the lactobacillus paracasei thalli to obtain the organic selenium.
In one embodiment of the invention, the fermentation time is 6-8 h.
In one embodiment of the invention, the method comprises the steps of inoculating the lactobacillus paracasei CCFM1089 into a directional transformation medium, fermenting under the conditions of 37 ℃ of temperature, 100rpm of rotation speed and pH 6 to obtain lactobacillus paracasei thallus, and then extracting the lactobacillus paracasei thallus to obtain the organic selenium.
In one embodiment of the present invention, the inorganic selenium is sodium selenite, sodium selenate and/or elemental selenium.
In one embodiment of the present invention, the inorganic selenium is sodium selenite.
In one embodiment of the present invention, the organic selenium is selenocysteine.
The invention also provides the application of the lactobacillus paracasei CCFM1089 or the method in the production of organic selenium.
In one embodiment of the present invention, the organic selenium is selenocysteine.
[ advantageous effects ]
(1) The Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 is screened out, the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 can efficiently convert inorganic selenium to produce selenocysteine, the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 is inoculated into a culture medium containing the inorganic selenium for fermentation for 8 hours, so that the total selenium content in the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 thallus freeze-dried powder is up to 400.65mg/kg, the organic selenium content in the total selenium is up to 96%, and the selenocysteine content in the organic selenium is up to 89%, and therefore, the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 has an application prospect in the production of the selenocysteine.
(2) The invention provides a method for efficiently producing selenocysteine, which is characterized in that Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 capable of efficiently converting inorganic selenium to produce the selenocysteine is inoculated into a directional conversion culture medium for fermentation for 8 hours, so that the total selenium content in the freeze-dried powder of the Lactobacillus paracasei (Lactobacillus paracasei) strain CCFM1089 reaches 400.65mg/kg, the organic selenium content in the total selenium reaches 96 percent, and the selenocysteine content in the organic selenium reaches 89 percent.
Biological material preservation
A strain of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 is classified and named as Lactobacillus paracasei, is preserved in Guangdong province microorganism strain preservation center in 11 and 01 months in 2019, has the preservation number of GDMCC No.60880, and has the preservation address of No. 59 floor 5 of Michelia Torresiae No. 100 in Guangzhou city.
Drawings
FIG. 1: the colony characteristics of Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
FIG. 2: the bacterial characteristics of Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
Detailed Description
The invention is further elucidated with reference to a specific embodiment and a drawing.
Sodium selenite (product No. D16-1030015, CAS: 26970-82-1) referred to in the examples below was purchased from Shanghai Chungsai science and technology Co., Ltd; serine (product No. D23-A0108, CAS: 56-45-1) referred to in the following examples was purchased from Shanghai Chungsai science and technology Co., Ltd; protease XIV (product number: P5147, CAS: 9036-06-0) referred to in the following examples was purchased from Bailingwei technologies, Inc., Beijing; proteinase K (product No. 3375201, CAS: 39450-01-6) referred to in the following examples was purchased from Bailingwei science, Inc., Beijing; glucose referred to in the following examples was purchased from national pharmaceutical group chemical agents limited.
The media involved in the following examples are as follows:
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05 g/L, Tween 801mL/L, agar 20g/L, cysteine hydrochloride 0.5 g/L.
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05 g/L, Tween 801mL/L and cysteine hydrochloride 0.5 g/L.
Selenium-rich culture medium: 10g/L of peptone, 5g/L of yeast extract, 30g/L of glucose, 10g/L of beef extract, 2g/L of anhydrous sodium acetate and 2g/L, K of diammonium citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.5g/L、MnSO 4 ·H 2 0.25g/L of O, 801g/L of Tween and 2-5 mg/L of sodium selenite.
The detection methods referred to in the following examples are as follows:
the total selenium detection method comprises the following steps: refer to the method for analyzing total selenium in GB 5009.93-2017 national standard food for food safety.
The organic selenium detection method comprises the following steps: refer to the organic selenium analysis method in GB 1903.21-2016 national standard for food safety, food nutrition enhancer selenium-enriched yeast.
The selenium form detection method comprises the following steps: putting the sample into a mortar, adding liquid nitrogen, grinding and crushing, putting 0.15g of the ground sample into a centrifuge tube, adding 6mL of Tris-HCl (50mmol/L, pH 7.2.2) for dissolving, and crushing for 5min by using an ultrasonic cell crusher; then adding protease K (10mg) and protease XIV (15mg) every 12h, performing oscillatory enzymolysis at 37 deg.C and 100r/min for 24h, centrifuging at 4000r/min for 10min, and filtering the supernatant with 0.22 μm filter membrane; morphological analysis was performed by HPLC-ICP-MS.
Example 1: screening and identification of lactobacillus paracasei CCFM1089
1. Screening
Taking the excrement of the infants from the ditch-changing village of the Ruolgai pasture in Sichuan province as a sample, and carrying out gradient dilution by 10 times to 10 times by using sterile physiological saline -6 Then 100 mul of the solution was diluted by 10 times -4 、10 -5 、10 -6 The diluted solution is plated on an MRS solid culture medium, inverted cultured for 48 hours at 37 ℃, and the colony morphology is observed and recorded; selecting colonies with different forms on an MRS solid culture medium for streaking separation, culturing at 37 ℃ for 48 hours, and selecting single colonies with different forms on the MRS solid culture medium again for streaking separation until pure single colonies with consistent forms are obtained; selecting a single colony on an MRS solid culture medium, inoculating the single colony in a 5mL selenium-enriched culture medium, and culturing for 18h at 37 ℃; and (3) putting 1mL of bacterial liquid into a sterile centrifuge tube, centrifuging for 3min at 8000r/min, removing an upper culture medium, freeze-drying the obtained bacterial sludge, detecting the total selenium and organic selenium content of the freeze-dried bacterial powder, and screening out a strain with high selenium enrichment to obtain a strain CCFM 1089.
2. Identification
The genome of the strain CCFM1089 was extracted, the 16S rDNA of the strain CCFM1089 was amplified and sequenced (by EnxAccigy Jieyi trading Co., Ltd., wherein the 16S rDNA of the strain CCFM1089 had the nucleotide sequence shown in SEQ ID NO. 1), and the nucleotide sequences of the sequences were aligned at NCBI, and the result showed that the strain was Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
Example 2: observation and preservation of Lactobacillus paracasei CCFM1089
1. Observation of
The liquid of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 obtained in example 1 was streaked on MRS solid medium, and after culturing at 37 ℃ for 48 hours, the colonies were observed and found to be round, white, and smooth (see fig. 1 in detail).
Streaking a bacterial liquid dipped with Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 on an MRS solid culture medium, and culturing for 48h at 37 ℃ to obtain a single bacterial colony; taking a clean glass slide, slightly heating the clean glass slide on flame, and cooling the clean glass slide; after cooling, dripping a small drop of sterile water at the central part of the clean glass slide, and picking a small amount of bacteria on an MRS solid culture medium beside flame by using an inoculating loop to mix with water; burning off redundant thalli on the inoculating loop, and forming the thalli into a uniform thin layer with the diameter of 1cm by using the inoculating loop; after the uniform thin layer on the clean glass slide is naturally dried, heating for 3-4 times on a slow fire for fixing; dripping 1-2 drops of crystal violet solution at the smear position, and dyeing for 1 min; washing off the dye solution with water, and sucking off water on the glass slide with water sucking paper; after drying, microscopic examination was carried out to observe the bacterial cells of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, and the shapes of the bacterial cells were found to conform to the typical characteristics of Lactobacillus paracasei (see FIG. 2 in particular).
2. Preservation of
Selecting a single colony of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, inoculating the single colony into an MRS liquid culture medium, and culturing at 37 ℃ for 18 hours to obtain a bacterial liquid; taking 1mL of bacterial liquid in a sterile centrifuge tube, centrifuging for 3min at 8000r/min, removing the upper culture medium, resuspending bacterial sludge in 30% glycerol solution, and preserving at-80 ℃.
Example 3: production of organic selenium (Lactobacillus paracasei CCFM1089+ selenium-rich conversion)
The Lactobacillus paracasei (Lactobacillus paracasei) CCFM8610, the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei)34-3 are used as controls, and the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, the Lactobacillus plantarum (Lactobacillus plantaris) CCFM8610, the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei)34-3 are inversely dipped in a glycerin tube to obtain a single bacterial liquid culture medium, and the single bacterial liquid is obtained by culturing at 37 ℃ and 36 ℃ respectively; selecting a single colony, inoculating the single colony into an MRS liquid culture medium, and culturing for 12 hours at 37 ℃ to obtain a culture solution; inoculating the culture solution into an MRS liquid culture medium in an inoculation amount of 1% (v/v), and culturing at 37 ℃ for 12h to obtain a seed solution; inoculating the seed solution into a selenium-rich culture medium with an inoculation amount of 5% (v/v), and culturing for 8h under the conditions of 37 ℃ of temperature, 100rpm of rotation speed and 6.0 of pH to obtain a fermentation liquid; centrifuging the fermentation liquor at 8000r/min for 15min at 4 ℃ to obtain thallus of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei) 34-3; washing pure water of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei)34-3 for 2 times, removing supernatant, and freeze-drying to obtain freeze-dried powder of Lactobacillus paracasei (Lactobacillus paracasei) FM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus paracasei) FJSSZ4-L2, Lactobacillus paracasei (Lactobacillus paracasei) M2-06-F01-L26-34 and Lactobacillus paracasei (Lactobacillus paracasei) M2-3668-3-34;
wherein, the selenium-rich culture medium: 10g/L of peptone, 5g/L of yeast extract, 30g/L of glucose, 10g/L of beef extract, 2g/L of anhydrous sodium acetate and 2g/L, K of dihydrodiamine citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.5g/L、MnSO 4 ·H 2 O0.25 g/L, Tween 801g/L, and sodium selenite pentahydrate 8 mg/L;
the preservation number of Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 is CGMCC No.6077, which is described in patent application publication No. CN 102586148A; lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei)34-3 are other lactic acid bacteria screened from infant stool samples derived from the village of the Thanksguard pasture of Sichuan province in the same batch as Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
Detecting the total selenium content, the organic selenium content and the selenium form distribution in the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 freeze-dried powder, the Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 freeze-dried powder, the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2 freeze-dried powder, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 freeze-dried powder and the Lactobacillus casei (Lactobacillus casei)34-3 freeze-dried powder (the detection results are shown in Table 1).
As can be seen from Table 1, Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 can efficiently convert inorganic selenium to produce selenocysteine, and the total selenium content in the freeze-dried powder of Lactobacillus paracasei CCFM1089CCFM1089 reaches 398.64mg/kg, the organic selenium content in the total selenium reaches 81%, and the selenocysteine content in the organic selenium reaches 79% only by fermenting for 8 hours; and the transformation efficiency of the Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 (preservation number is CGMCC No.6077), the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei)34-3 for transforming inorganic selenium to produce selenocysteine is far lower than that of the Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
TABLE 1 Total selenium content, organic selenium content and selenium form distribution in lyophilized powder of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei)34-3
Example 4: production of organic selenium (Lactobacillus paracasei CCFM1089+ Directional transformation)
On the basis of the embodiment 2, the selenium-rich culture medium is replaced by a directional transformation culture medium (5-15 g/L of peptone, 5-15 g/L of yeast extract, 20-50 g/L of glucose and 5-10 g/L, K of beef extract) 2 HPO 4 ·3H 2 O 2～5g/L、MgSO 4 ·7H 2 O 0.25～0.58g/L、MnSO 4 ·H 2 0.05-0.30 g/L of O, 801g/L of Tween, 8-17 mg/L of sodium selenite pentahydrate and 0.1-0.4 g/L of serine), which are specifically as follows:
the Lactobacillus paracasei (Lactobacillus paracasei) CCFM8610, the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei)34-3 are used as controls, and the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, the Lactobacillus plantarum (Lactobacillus plantaris) CCFM8610, the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei)34-3 are inversely dipped in a glycerin tube to obtain a single bacterial liquid culture medium, and the single bacterial liquid is obtained by culturing at 37 ℃ and 36 ℃ respectively; selecting a single colony, inoculating the single colony into an MRS liquid culture medium, and culturing for 12 hours at 37 ℃ to obtain a culture solution; inoculating the culture solution into an MRS liquid culture medium in an inoculation amount of 1% (v/v), and culturing at 37 ℃ for 12h to obtain a seed solution; inoculating the seed solution into a directional transformation culture medium with an inoculation amount of 5% (v/v), and culturing for 8h under the conditions of 37 ℃ of temperature, 100rpm of rotation speed and 6.0 of pH to obtain a fermentation liquid; centrifuging the fermentation liquor at 8000r/min for 15min at 4 ℃ to obtain thallus of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei) 34-3; washing pure water of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei)34-3 for 2 times, removing supernatant, and freeze-drying to obtain freeze-dried powder of Lactobacillus paracasei (Lactobacillus paracasei) FM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus paracasei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus paracasei) M2-06-F01-4-5394 and Lactobacillus casei (Lactobacillus 3-34);
wherein, the directional transformation culture medium: 10g/L of peptone, 5g/L of yeast extract, 30g/L of glucose, 10g/L of beef extract, 2g/L of anhydrous sodium acetate and 2g/L, K of dihydrodiamine citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.5g/L、MnSO 4 ·H 2 0.25g/L of O, 801g/L of Tween, 10mg/L of sodium selenite pentahydrate and 0.2g/L of serine;
the preservation number of Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 is CGMCC No.6077, which is described in patent application publication No. CN 102586148A; the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei)34-3 are other lactic acid bacteria screened from infant fecal samples derived from village-more-ditched Ragladespasture in Sichuan province in the same batch as Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
Detecting the total selenium content, the organic selenium content and the selenium form distribution in the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 freeze-dried powder, the Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 freeze-dried powder, the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2 freeze-dried powder, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 freeze-dried powder and the Lactobacillus casei (Lactobacillus casei)34-3 freeze-dried powder (the detection results are shown in Table 2).
As can be seen from Table 2, Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 can efficiently convert inorganic selenium to produce selenocysteine, and the total selenium content in the freeze-dried powder of Lactobacillus paracasei CCFM1089CCFM1089 reaches 400.65mg/kg, the organic selenium content in the total selenium reaches 96%, and the selenocysteine content in the organic selenium reaches 89% only by fermenting for 8 hours; and the transformation efficiency of the Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 (preservation number is CGMCC No.6077), the Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei)34-3 for transforming inorganic selenium to produce selenocysteine is far lower than that of the Lactobacillus paracasei (Lactobacillus paracasei) CCFM 1089.
TABLE 2 Total selenium content, organic selenium content and selenium form distribution in lyophilized powder of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus casei (Lactobacillus casei) FJSSZ4-L2, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei)34-3
Comparative example 1: influence of sodium selenite concentration on organic selenium conversion efficiency (selenium-rich conversion)
On the basis of example 2, the concentration of sodium selenite in the selenium-enriched medium was replaced by 5mg/L and 20 mg/L.
The total selenium content, the organic selenium content and the selenium form distribution in the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 freeze-dried powder are detected (the detection results are shown in a table 3).
As can be seen from Table 3, when the concentration of sodium selenite is 5mg/L, the conversion efficiency of selenocysteine in the freeze-dried powder of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 is much lower than that when the concentration of sodium selenite is 8 mg/L; when the concentration of sodium selenite is 20mg/L, although the total selenium content in the freeze-dried powder of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 is improved, the conversion efficiency of selenocysteine is still far lower than that when the concentration of sodium selenite is 8 mg/L.
TABLE 3 Total selenium content, organic selenium content and selenium morphology distribution in Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 lyophilized powder
Comparative example 2: effect of pH on organic selenium conversion efficiency (selenium conversion)
On the basis of example 2, the pH of the fermentation was replaced by 5.5.
The total selenium content, the organic selenium content and the selenium form distribution in the Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 freeze-dried powder are detected (the detection results are shown in table 4).
As can be seen from Table 4, when the pH value is 5.5, the total selenium content in the lyophilized powder of Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 is improved, but the selenocysteine conversion efficiency is far lower than that when the pH value is 6.
TABLE 4 Total selenium content, organic selenium content and selenium morphology distribution in Lactobacillus paracasei (Lactobacillus paracasei) CCFM1089 lyophilized powder
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Suzhou selenium Tech Biotech Co., Ltd
<120> lactobacillus paracasei capable of highly producing selenocysteine and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 424
<212> DNA
<213> Lactobacillus paracasei
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ttgtgcaatt cgtcaacggc agccttagtt gctttttcaa tccctgtgcg aatgccaacc 420
ggga 424
Claims (8)
1. The Lactobacillus paracasei (Lactobacillus paracasei) is characterized by being preserved in Guangdong province microorganism culture collection center in 2019 at 11 and 01 months, and the preservation number is GDMCC No. 60880.
2. A method for producing organic selenium, which is characterized in that the method comprises the steps of inoculating the lactobacillus paracasei of claim 1 into a fermentation culture medium for fermentation to obtain lactobacillus paracasei thalli, and then extracting the lactobacillus paracasei thalli to obtain the organic selenium; the fermentation medium contains sodium selenite.
3. The method of claim 2, wherein the fermentation medium comprises serine.
4. The method for producing organic selenium according to claim 2, wherein the fermentation medium is a selenium-rich medium or a directional transformation medium;
the selenium-rich culture medium comprises 5-15 g/L of peptone, 5-15 g/L of yeast extract, 20-50 g/L of glucose, 5-15 g/L of beef extract, 1-5 g/L of anhydrous sodium acetate and 1-5 g/L, K of hydrogenous diamine citrate 2 HPO 4 ·3H 2 O 2～5g/L、MgSO 4 ·7H 2 O 0.25～0.58g/L、MnSO 4 ·H 2 0.05-0.30 g/L of O, 801g/L of Tween and 10-17 mg/L of sodium selenite pentahydrate;
the components of the directional transformation medium comprise 5-15 g/L of peptone, 5-15 g/L of yeast extract, 20-50 g/L of glucose and 5-15 g/L, K of beef extract 2 HPO 4 ·3H 2 O 2～5g/L、MgSO 4 ·7H 2 O 0.25～0.58g/L、MnSO 4 ·H 2 0.05-0.30 g/L of O, 801g/L of Tween, 10-17 mg/L of sodium selenite pentahydrate and 0.1-0.4 g/L of serine.
5. The method for producing organoselenium according to claim 4, wherein the method comprises inoculating the Lactobacillus paracasei according to claim 1 into a selenium-rich culture medium, fermenting at a temperature of 35-40 ℃ and a pH of 5.0-6.0 to obtain Lactobacillus paracasei cells, and extracting the Lactobacillus paracasei cells to obtain the organoselenium.
6. The method for producing organoselenium according to claim 4, wherein the method comprises inoculating the Lactobacillus paracasei according to claim 1 into a directional transformation medium, fermenting at a temperature of 35-40 ℃ and a pH of 5.0-6.0 to obtain Lactobacillus paracasei cells, and extracting the Lactobacillus paracasei cells to obtain the organoselenium.
7. The method for producing organic selenium according to claim 5 or 6, wherein the fermentation time is 6-8 h.
8. Use of lactobacillus paracasei according to claim 1 for the production of organic selenium.
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