CN111040973B - Lactobacillus rhamnosus capable of highly producing 2 forms of organic selenium and application thereof - Google Patents
Lactobacillus rhamnosus capable of highly producing 2 forms of organic selenium and application thereof Download PDFInfo
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Abstract
The invention discloses lactobacillus rhamnosus capable of highly producing 2 forms of organic selenium and application thereof, belonging to the technical field of microorganisms. The lactobacillus rhamnosus CCFM1090 is screened out, can efficiently convert inorganic selenium to produce 2 forms of organic selenium selenocysteine and selenomethylselenocysteine, is inoculated into a culture medium containing the inorganic selenium for fermentation for 8 hours, so that the total selenium content in the freeze-dried powder of the lactobacillus rhamnosus CCFM1090 can reach 570.34mg/kg, the organic selenium content in the total selenium reaches 94%, and the selenocysteine and selenomethylselenocysteine content in the organic selenium respectively reach 42% and 47%, therefore, the lactobacillus rhamnosus CCFM1090 has a very high application prospect in the production of the selenocysteine and/or the selenomethylselenocysteine.
Description
Technical Field
The invention relates to lactobacillus rhamnosus capable of highly producing 2 forms of organic selenium and application thereof, belonging to the technical field of microorganisms.
Background
Selenium (Se) is a trace element necessary for human and animal survival, is an essential component of the active centers of glutathione peroxidase, deiodinase and the like in the body of mammals, and has the physiological functions of protecting the structure and the function of a cell membrane from excessive oxidative damage, resisting cancer, detoxifying, protecting the liver and improving the immunity of the human body. Selenium generally exists in nature, and the content of selenium is different due to different geographical environments, so that the current 2/3 areas in China belong to low-selenium zones.
Research shows that selenium is very important for human health, and the lack of selenium can directly cause the reduction of the human immunity. Moreover, clinical medicine has proved that more than forty diseases threatening human health and life are related to selenium deficiency, such as cancer, cardiovascular diseases, liver diseases, cataract, pancreatic diseases, diabetes, reproductive system diseases, etc. Thus, one needs to ingest a sufficient amount of selenium as starch, protein and vitamins daily. In 2013 edition of reference intake of dietary nutrients (DRIs) of Chinese residents, which is formulated by the Chinese academy of nutrition, it is explicitly suggested that the recommended intake of selenium above 18 years old is 60 micrograms per day.
At present, the common and effective method for supplementing selenium is mainly to take various selenium supplementing products every day. The existing selenium supplement products mainly comprise inorganic selenium and organic selenium. Among them, inorganic selenium is mainly sodium selenite, which is mostly used in feed abroad due to its low absorption and utilization rate by human body and large toxic and side effects.
The organic selenium is a substance formed by combining inorganic selenium with organic nutrient components 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 organic selenium is the main selenium supplement form at present.
In the organic selenium, selenocysteine mainly enters a human body in a mode of replacing sulfur by selenium, and further has the functions of reconstructing bone mass, enhancing immune system, regulating thyroid function, improving male fertility, reducing the risk of cancer, reducing aging, relieving fatigue and the like; the selenium methyl selenocysteine is mainly converted into methyl selenium and selenocysteine by beta-lyase of a human body and then absorbed by the human body, thereby playing roles of enhancing immunity, supplementing selenium element and the like; selenomethionine enters a human body in a mode of replacing sulfur by selenium, the selenomethionine mainly plays roles of enhancing oxidation resistance, enhancing immunity and the like, but because the human tRNA can not identify methionine and selenomethionine, when the intake of methionine is limited, a large proportion of selenomethionine replaces methionine to be nonspecifically incorporated into protein in the body, so that a large amount of selenium is accumulated in the body, and certain risk also exists. Therefore, the selenium supplement product containing selenocysteine and/or selenomethylselenocysteine has wider market prospect.
The production of selenocysteine and/or selenomethylselenocysteine 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 microorganisms for converting inorganic selenium to produce selenocysteine and/or selenomethylselenocysteine is too low, the yield of the selenocysteine and/or selenomethylselenocysteine produced by the method is not high, and the further development of the organic selenium market 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, the microorganism which can efficiently convert inorganic selenium to produce the organic selenium with 2 forms of selenocysteine and selenomethylselenocysteine is urgently needed to be found.
Disclosure of Invention
[ problem ] to
The invention aims to solve the technical problem of providing Lactobacillus rhamnosus (Lactobacillus rhamnosus) which can efficiently convert inorganic selenium to produce selenocysteine and 2 forms of organic selenium such as selenomethylselenocysteine.
[ solution ]
In order to solve the technical problem, the invention provides a Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090, wherein the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 is preserved in Guangdong province microbial strain preservation center in 11 and 01 of 2019, the preservation number is GDMCC No.60881, and the preservation address is No. 59 building 5 of Miyaolu 100 of Guangzhou city.
The Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 is obtained by separating a fecal sample from a Rugosu village in a Ragladeshokes 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 subjected to nucleic acid sequence comparison in NCBI, so that the result shows that the strain is Lactobacillus rhamnosus and is named as Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM 1090.
The bacterial colony of the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 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 rhamnosus CCFM1090 into a fermentation culture medium for fermentation to obtain lactobacillus rhamnosus thalli, and then extracting the lactobacillus rhamnosus 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 and 5-10 g/L, K of beef extract2HPO4·3H2O 2～5g/L、MgSO4·7H2O 0.25～0.58g/L、MnSO4·H20.05-0.30 g/L of O, 801g/L of Tween and 7-12 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 5-10 g/L, K of beef extract2HPO4·3H2O 2～5g/L、MgSO4·7H2O 0.25～0.58g/L、MnSO4·H2O 0.05～0.30g/LThe pharmaceutical composition comprises 801g/L of tween, 8-17 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 rhamnosus CCFM1090 into a selenium-rich culture medium, fermenting at the temperature of 35-40 ℃, the rotating speed of 75-150 rpm and the pH value of 5.0-6.0 to obtain lactobacillus rhamnosus thalli, and extracting the lactobacillus rhamnosus thalli to obtain the organic selenium.
In one embodiment of the invention, the method comprises the steps of inoculating the lactobacillus rhamnosus CCFM1090 into a directional transformation 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 rhamnosus thalli, and extracting the lactobacillus rhamnosus 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 rhamnosus CCFM1090 into a directional transformation culture medium, fermenting at the conditions of 37 ℃ and 100rpm of rotation speed and pH of 6 to obtain lactobacillus rhamnosus thalli, and then extracting the lactobacillus rhamnosus thalli 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, selenomethionine and/or selenomethylselenocysteine.
In one embodiment of the present invention, the organic selenium is selenocysteine and/or selenomethylselenocysteine.
The invention also provides application of the lactobacillus rhamnosus CCFM1090 or the method in production of organic selenium.
In one embodiment of the present invention, the organic selenium is selenocysteine, selenomethionine and/or selenomethylselenocysteine.
In one embodiment of the present invention, the organic selenium is selenocysteine and/or selenomethylselenocysteine.
[ advantageous effects ]
(1) The Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 can efficiently convert inorganic selenium to produce 2 forms of organic selenium such as selenocysteine and selenomethylselenocysteine, the Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 is inoculated into a culture medium containing the inorganic selenium for fermentation for 8 hours, so that the total selenium content in the Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 thallus freeze-dried powder is 570.34mg/kg, the organic selenium content in the total selenium is 94%, and the selenocysteine and selenomethylselenocysteine contents in the organic selenium are 42% and 47% respectively, therefore, the Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 has a very high application prospect in the production of the selenocysteine and/or the selenomethylselenocysteine.
(2) The invention provides a method for efficiently producing selenocysteine and/or selenomethylselenocysteine, which is characterized in that Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 capable of efficiently converting inorganic selenium to produce selenocysteine and 2 forms of organic selenium of selenomethylselenocysteine is inoculated into a directional conversion culture medium for fermentation for 8 hours, so that the total selenium content in the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 thallus freeze-dried powder is up to 570.34mg/kg, the organic selenium content in the total selenium is up to 94%, and the contents of selenocysteine and selenomethylselenocysteine in the organic selenium are respectively up to 42% and 47%.
Biological material preservation
A strain of Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 is classified and named as Lactobacillus rhamnous, is preserved in Guangdong province microorganism strain preservation center in 11 and 01 months in 2019, has the preservation number of GDMCC No.60881, and has the preservation address of No. 59 floor 5 of Michelia Torresiae 100 of Guangzhou city.
Drawings
FIG. 1: colony characteristics of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM 1090.
FIG. 2: the thallus characteristics of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM 1090.
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 No: 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 citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L, Tween 801mL/L, agar 20g/L and 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 citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 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 and 2g/L of anhydrous sodium acetateg/L, 2g/L, K of citric acid hydrogen diamine2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.5g/L、MnSO4·H20.25g/L of O, 801g/L of Tween and 7-12 mg/L of sodium selenite pentahydrate.
The detection methods referred to in the following examples are as follows:
the total selenium detection method comprises the following steps: refer to the analysis method of 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, identification, observation and preservation of lactobacillus rhamnosus CCFM1090
1. Screening
Taking feces from Ruicha gorhami pasture of Sichuan province as a sample, and performing gradient dilution by 10 times to 10 times with sterile physiological saline-6Then 100 mul of the solution is respectively diluted by 10 times-4、10-5、10-6The diluted solution is coated on a MRS solid culture medium by a flat plate, is inversely 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, and after culturing for 48 hours at 37 ℃, selecting single colonies with different forms on the MRS solid culture medium again for streaking separation until obtaining pure single colonies with consistent forms; 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 ℃; taking 1mL of bacterial liquid in a sterile centrifuge tube, centrifuging for 3min at 8000r/min, discarding the upper layer culture medium, freeze-drying the obtained bacterial mud, detecting, freeze-dryingAnd (3) screening out a strain with high selenium enrichment to obtain a strain CCFM1090 according to the total selenium and organic selenium contents of the bacterial powder.
2. Identification
The genome of the strain CCFM1090 is extracted, the 16S rDNA of the strain CCFM1090 is amplified and sequenced (carried out by EnxWeijiji trading Co., Ltd., wherein the 16S rDNA amplified nucleotide sequence of the strain CCFM1090 is shown in SEQ ID NO. 1), and the sequence is subjected to nucleic acid sequence comparison in NCBI, so that the strain is Lactobacillus rhamnosus and is named as Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM 1090.
3. Observation of
A bacterial liquid dipped with Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 is streaked on an MRS solid culture medium, and after the bacterial liquid is cultured for 48 hours at 37 ℃, the bacterial colony is observed and is found to be round, white and smooth (see figure 1 specifically).
Streaking a bacterial liquid dipped with Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 on an MRS solid culture medium, and culturing at 37 ℃ for 48h to obtain a single 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 thalli 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 is carried out by a microscope to observe the thallus of the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090, and the thallus form is found to be in accordance with the typical characteristics of the Lactobacillus rhamnosus (see figure 2 in particular).
4. Preservation of
Selecting a single colony of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090, inoculating the single colony in an MRS liquid culture medium, and culturing at 37 ℃ for 18h 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 2: production of organic selenium (Lactobacillus rhamnosus CCFM1090+ selenium conversion)
The Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090, the Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, the Lactobacillus reuteri) P8-4, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei) GD41-6 obtained in example 1 are dipped in a glycerol tube in an inverted manner to obtain a strain liquid, and the strain liquid is obtained by culturing the strain liquid on a GD medium at 37 ℃ and streaking the strain liquid on a solid culture medium, wherein the strain liquid is obtained by culturing a single strain liquid of Lactobacillus strain GD 26-6, and the strain liquid is obtained by culturing the strain liquid at 37 ℃ in an inverted manner; selecting a single colony, inoculating the single colony into an MRS liquid culture medium, and culturing at 37 ℃ for 12h 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 deg.C to obtain thallus of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus reuteri P8-4, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4, and Lactobacillus casei (Lactobacillus casei) GD 41-6; washing pure water of Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus reuteri (Lactobacillus reuteri) P8-4, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4, Lactobacillus casei (Lactobacillus casei) GD41-6 for 2 times, removing supernatant, and freeze-drying to obtain Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 freeze-dried powder, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus reuteri (Lactobacillus reuteri) P8-4 freeze-dried powder, Lactobacillus (Lactobacillus casei) M2-06-F01-4-4 and Lactobacillus casei GD 41-GD 6;
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 citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.5g/L、 MnSO4·H2O0.25 g/L, Tween 801g/L and sodium selenite pentahydrate 8 mg/L;
lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, having a accession number of CGMCC No.6077, is described in patent application publication No. CN 102586148A; lactobacillus reuteri P8-4, Lactobacillus casei M2-06-F01-L4-1-4, and Lactobacillus casei GD41-6 are other lactic acid bacteria screened in the same batch as Lactobacillus rhamnosus CCFM1090 from stool samples derived from more villages in the Ragladesh pasture of Sichuan province.
Detecting the total selenium content, the organic selenium content and the selenium form distribution in Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 freeze-dried powder, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 freeze-dried powder, Lactobacillus reuteri (Lactobacillus reuteri) P8-4 freeze-dried powder, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 freeze-dried powder and Lactobacillus casei (Lactobacillus casei) GD41-6 freeze-dried powder (the detection result is shown in Table 1).
As can be seen from the table 1, the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 can efficiently convert inorganic selenium to produce selenocysteine and selenomethylselenocysteine, and only fermentation is carried out for 8 hours, so that the total selenium content in the Lactobacillus rhamnosus CCFM1090CCFM1090 freeze-dried powder can reach 489.21mg/kg, the organic selenium content in the total selenium reaches 70%, and the selenocysteine and the selenomethylselenocysteine content in the organic selenium respectively reach 42% and 43%; and the conversion efficiency of Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 (preservation number is CGMCC No.6077), Lactobacillus reuteri (Lactobacillus reuteri) P8-4, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei) GD41-6 for converting inorganic selenium to produce selenocysteine and selenomethylselenocysteine is far lower than that of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM 1090.
TABLE 1 Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 Freeze-dried powder, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 Freeze-dried powder, Lactobacillus reuteri (Lactobacillus reuteri) P8-4 Freeze-dried powder, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 Freeze-dried powder, and Lactobacillus casei (Lactobacillus casei) GD41-6
Total selenium content, organic selenium content and selenium form distribution in freeze-dried powder
Example 3: production of organic selenium (Lactobacillus rhamnosus CCFM1090+ 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)2HPO4·3H2O 2～5g/L、MgSO4·7H2O 0.25～0.58g/L、MnSO4·H20.05-0.30 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 rhamnosus (Lactobacillus rhamnosus) CCFM1090, the Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, the Lactobacillus reuteri) P8-4, the Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and the Lactobacillus casei (Lactobacillus casei) GD41-6 obtained in example 1 are dipped in a glycerol tube in an inverted manner to obtain a strain liquid, and the strain liquid is obtained by culturing the strain liquid on a GD medium at 37 ℃ and streaking the strain liquid on a solid culture medium, wherein the strain liquid is obtained by culturing a single strain liquid of Lactobacillus strain GD 26-6, and the strain liquid is obtained by culturing the strain liquid at 37 ℃ in an inverted manner; selecting a single colony, inoculating the single colony into an MRS liquid culture medium, and culturing at 37 ℃ for 12h 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 deg.C to obtain thallus of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus reuteri P8-4, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4, and Lactobacillus casei (Lactobacillus casei) GD 41-6; washing pure water of Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus reuteri (Lactobacillus reuteri) P8-4, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4, Lactobacillus casei (Lactobacillus casei) GD41-6 for 2 times, removing supernatant, and freeze-drying to obtain Lactobacillus rhamnosus (Lactobacillus rhamnous) CCFM1090 freeze-dried powder, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, Lactobacillus reuteri (Lactobacillus reuteri) P8-4 freeze-dried powder, Lactobacillus (Lactobacillus casei) M2-06-F01-4-4 and Lactobacillus casei GD 41-GD 6;
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 citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.5g/L、 MnSO4·H20.25g/L of O, 801g/L of Tween and 10mg/L of sodium selenite pentahydrate;
lactobacillus plantarum (Lactobacillus plantarum) CCFM8610, having a accession number of CGMCC No.6077, is described in patent application publication No. CN 102586148A; lactobacillus reuteri P8-4, Lactobacillus casei M2-06-F01-L4-1-4, and Lactobacillus casei GD41-6 are other lactic acid bacteria screened in the same batch as Lactobacillus rhamnosus CCFM1090 from stool samples derived from more villages in the Ragladesh pasture of Sichuan province.
Detecting the total selenium content, the organic selenium content and the selenium form distribution in Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 freeze-dried powder, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 freeze-dried powder, Lactobacillus reuteri (Lactobacillus reuteri) P8-4 freeze-dried powder, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 freeze-dried powder and Lactobacillus casei (Lactobacillus casei) GD41-6 freeze-dried powder (the detection results are shown in Table 2).
As can be seen from Table 2, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 can efficiently convert inorganic selenium to produce selenocysteine and selenomethylselenocysteine, and only fermentation is carried out for 8 hours, so that the total selenium content in the Lactobacillus rhamnosus CCFM1090CCFM1090 freeze-dried powder can reach 570.34mg/kg, the organic selenium content in the total selenium reaches 94%, and the selenocysteine and selenomethylselenocysteine content in the organic selenium respectively reach 42% and 47%; and the conversion efficiency of Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 (preservation number is CGMCC No.6077), Lactobacillus reuteri (Lactobacillus reuteri) P8-4, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 and Lactobacillus casei (Lactobacillus casei) GD41-6 for converting inorganic selenium to produce selenocysteine and selenomethylselenocysteine is far lower than that of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM 1090.
TABLE 2 Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 Freeze-dried powder, Lactobacillus plantarum (Lactobacillus plantarum) CCFM8610 Freeze-dried powder, Lactobacillus reuteri (Lactobacillus reuteri) P8-4 Freeze-dried powder, Lactobacillus casei (Lactobacillus casei) M2-06-F01-L4-1-4 Freeze-dried powder, and Lactobacillus casei (Lactobacillus casei) GD41-6
Total selenium content, organic selenium content and selenium form distribution in freeze-dried powder
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.
Detecting the total selenium content, the organic selenium content and the selenium form distribution in the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 freeze-dried powder (the detection result is 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 and selenomethylselenocysteine in the freeze-dried powder of Lactobacillus rhamnosus CCFM1090 is far 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 Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 freeze-dried powder is improved, the conversion efficiency of selenocysteine and selenomethylselenocysteine 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 rhamnosus CCFM1090 Freeze-dried 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.
Detecting the total selenium content, the organic selenium content and the selenium form distribution in the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1090 freeze-dried powder (the detection result is shown in a table 4).
As can be seen from Table 4, at pH 5.5, the total selenium content in the lyophilized powder of Lactobacillus rhamnosus CCFM1090 was improved, but the conversion efficiency of selenocysteine and selenomethylselenocysteine was much lower than that at pH 6.
TABLE 4 Total selenium content, organic selenium content and selenium morphology distribution in Lactobacillus rhamnosus CCFM1090 Freeze-dried 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> university of south of the Yangtze river
<120> lactobacillus rhamnosus capable of highly producing 2 forms of organic selenium and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1448
<212> DNA
<213> Lactobacillus rhamnosus
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Claims (8)
1. Lactobacillus rhamnosus (A) strainLactobacillus rhamnosus) The lactobacillus rhamnosus is preserved in Guangdong province microorganism culture collection center in 2019, 11 and 01, and the preservation number is GDMCC No. 60881.
2. A method for producing organic selenium is characterized in that the method comprises the steps of inoculating the lactobacillus rhamnosus of claim 1 into a fermentation culture medium for fermentation to obtain lactobacillus rhamnosus thalli, and then extracting the lactobacillus rhamnosus thalli to obtain the organic selenium; the fermentation culture medium contains inorganic selenium; the inorganic selenium is sodium selenite; the organic selenium is selenocysteine, selenomethylselenocysteine and/or selenomethionine.
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 and 5-10 g/L, K of beef extract2HPO4·3H2O 2~5 g/L、MgSO4·7H2O 0.25~0.58 g/L、MnSO4·H20.05-0.30 g/L of O, 801g/L of Tween and 7-12 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 5-10 g/L, K of beef extract2HPO4·3H2O 2~5 g/L、MgSO4·7H2O 0.25~0.58 g/L、MnSO4·H20.05-0.30 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.
5. The method for producing organic selenium according to claim 4, wherein the method comprises the steps of inoculating the lactobacillus rhamnosus of claim 1 into a selenium-rich culture medium, fermenting at the temperature of 35-40 ℃ and the pH of 5.0-6.0 to obtain lactobacillus rhamnosus thalli, and extracting the lactobacillus rhamnosus thalli to obtain the organic selenium.
6. The method for producing organic selenium according to claim 4, wherein the method comprises the steps of inoculating the lactobacillus rhamnosus according to claim 1 into a directional transformation culture medium, fermenting at the temperature of 35-40 ℃ and the pH of 5.0-6.0 to obtain lactobacillus rhamnosus thalli, and extracting the lactobacillus rhamnosus thalli to obtain the organic selenium.
7. The method for producing organic selenium according to claim 4 or 6, wherein the fermentation time is 6-8 h.
8. Use of lactobacillus rhamnosus according to claim 1 or the process according to any of claims 2 to 7 for the production of organic selenium being selenocysteine, selenomethylselenocysteine and/or selenomethionine.
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