CN111088185A - Selenium-enriched lactobacillus preparation and preparation method thereof - Google Patents
Selenium-enriched lactobacillus preparation and preparation method thereof Download PDFInfo
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- 239000011669 selenium Substances 0.000 title claims abstract description 120
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 120
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 119
- 238000002360 preparation method Methods 0.000 title claims abstract description 61
- 241000186660 Lactobacillus Species 0.000 title claims abstract description 36
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 36
- 239000001963 growth medium Substances 0.000 claims abstract description 57
- 244000269722 Thea sinensis Species 0.000 claims abstract description 43
- 241001052560 Thallis Species 0.000 claims abstract description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229940094952 green tea extract Drugs 0.000 claims abstract description 22
- 235000020688 green tea extract Nutrition 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 18
- 238000005406 washing Methods 0.000 claims abstract description 18
- 238000009630 liquid culture Methods 0.000 claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 239000012528 membrane Substances 0.000 claims abstract description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 11
- 235000009569 green tea Nutrition 0.000 claims abstract description 10
- 238000001816 cooling Methods 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 238000002791 soaking Methods 0.000 claims abstract description 9
- 238000005303 weighing Methods 0.000 claims abstract description 9
- 239000000284 extract Substances 0.000 claims description 43
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 34
- 235000013616 tea Nutrition 0.000 claims description 33
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 32
- 230000001954 sterilising effect Effects 0.000 claims description 21
- 239000011572 manganese Substances 0.000 claims description 19
- 241000894006 Bacteria Species 0.000 claims description 18
- 239000004310 lactic acid Substances 0.000 claims description 17
- 235000014655 lactic acid Nutrition 0.000 claims description 17
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 16
- 108010080698 Peptones Proteins 0.000 claims description 16
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 16
- 235000015278 beef Nutrition 0.000 claims description 16
- 229940041514 candida albicans extract Drugs 0.000 claims description 16
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 16
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 16
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 16
- 239000012153 distilled water Substances 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 16
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 16
- 235000019319 peptone Nutrition 0.000 claims description 16
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 16
- 229920000053 polysorbate 80 Polymers 0.000 claims description 16
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 16
- 239000001632 sodium acetate Substances 0.000 claims description 16
- 235000017281 sodium acetate Nutrition 0.000 claims description 16
- 239000001509 sodium citrate Substances 0.000 claims description 16
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 16
- 229940038773 trisodium citrate Drugs 0.000 claims description 16
- 239000012138 yeast extract Substances 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 230000003213 activating effect Effects 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 229940099596 manganese sulfate Drugs 0.000 claims description 8
- 239000011702 manganese sulphate Substances 0.000 claims description 8
- 235000007079 manganese sulphate Nutrition 0.000 claims description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 8
- 239000002504 physiological saline solution Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 238000011146 sterile filtration Methods 0.000 claims 1
- 239000008213 purified water Substances 0.000 abstract description 5
- 238000005119 centrifugation Methods 0.000 abstract description 2
- 229940091258 selenium supplement Drugs 0.000 description 106
- 230000000052 comparative effect Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 6
- 239000013028 medium composition Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 4
- 241001462463 Lactobacillus plantarum ST-III Species 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000011781 sodium selenite Substances 0.000 description 3
- 235000015921 sodium selenite Nutrition 0.000 description 3
- PMYDPQQPEAYXKD-UHFFFAOYSA-N 3-hydroxy-n-naphthalen-2-ylnaphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(NC(=O)C3=CC4=CC=CC=C4C=C3O)=CC=C21 PMYDPQQPEAYXKD-UHFFFAOYSA-N 0.000 description 2
- 102000006587 Glutathione peroxidase Human genes 0.000 description 2
- 108700016172 Glutathione peroxidases Proteins 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000011655 sodium selenate Substances 0.000 description 2
- 229960001881 sodium selenate Drugs 0.000 description 2
- 235000018716 sodium selenate Nutrition 0.000 description 2
- 229960001471 sodium selenite Drugs 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000011845 Iodide peroxidase Human genes 0.000 description 1
- 108010036012 Iodide peroxidase Proteins 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229910003424 Na2SeO3 Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010039921 Selenium deficiency Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000001391 atomic fluorescence spectroscopy Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 150000004678 hydrides Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229940065287 selenium compound Drugs 0.000 description 1
- 150000003343 selenium compounds Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- Microbiology (AREA)
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention discloses a preparation method of a selenium-enriched lactobacillus preparation, which comprises the following steps: (1) inoculating the Lb.plantarum ST-III strain which is frozen and preserved into an MRS liquid culture medium, carrying out anaerobic culture and subculturing to obtain a thallus culture; (2) weighing 2g of green tea sample, adding purified water, soaking for 10min, taking supernatant, and filtering with sterile filter membrane to obtain green tea extract; (3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the obtained green tea extract; (4) collecting thalli from the thalli culture obtained by centrifugation; (5) washing the collected thalli with normal saline for three times, then suspending the thalli in the MRS-TE culture medium, shaking up, standing still and fermenting at 37 ℃; (6) centrifuging the fermented thallus, washing with sterile normal saline for 3 times, centrifuging, and removing supernatant to obtain selenium-rich lactobacillus preparation. The invention has the advantage of efficiently enriching the organic selenium from the green tea by adopting a fermentation method.
Description
Technical Field
The invention relates to the technical field of lactobacillus preparations, in particular to a selenium-rich lactobacillus preparation and a preparation method thereof.
Background
Selenium is a trace mineral element necessary for human bodies and is an important element for maintaining normal growth and metabolism of life; food and Agricultural Organization (FAO) and World Health Organization (WHO) of the United nations have commonly confirmed that an important trace element necessary for human bodies is closely related to the maintenance of normal physiological functions of the bodies and the occurrence and development of various diseases, is an essential component of many enzyme activity centers in mammals, such as glutathione peroxidase (GPx), deiodinase (ID) and the like, and has the physiological functions of protecting the structure and the function of cell membranes from excessive oxidative damage, resisting cancer, detoxifying, protecting the liver and improving the immunity of the human bodies. Selenium cannot be stored for a long time or synthesized in a human body, and the human body must continuously obtain selenium element from diet to supply needs of the human body. According to statistics, 2/3 people live in low-selenium or selenium-deficient areas, about 3 hundred million people are in a selenium-deficient state, so that the artificial selenium supplement method has been widely concerned by relevant experts at home and abroad.
The traditional method for supplementing selenium is to prepare an oral preparation by using inorganic sodium selenite, but the sodium selenite has strong toxic and side effects. Research shows that the organic selenium compound obtained by biotransformation of inorganic selenium has low toxicity and higher absorption rate, and has great significance for promotion in China and improvement of selenium deficiency state.
In recent years, selenium-enriched lactic acid bacteria become an important research direction in the field of lactic acid bacteria, and are expected to replace selenium-enriched yeast to become a preferred high-efficiency selenium-enriched microecological preparation. From the perspective of the related intellectual property layout, the research on selenium-enriched lactic acid bacteria is limited to a limited number of lactobacillus strains; more importantly, the existing research is the enrichment condition of the lactic acid bacteria in the synthetic culture medium for chemical inorganic selenium reagents (mainly sodium selenate), and the guidance significance of related results to the food industry is limited.
Disclosure of Invention
In order to solve the problems, the invention aims to disclose a selenium-enriched lactobacillus preparation and a preparation method thereof, which adopts a fermentation means to efficiently enrich organic selenium from green tea on the basis of lactobacillus plantarum ST-III; the lactobacillus plantarum ST-III strain is derived from the invention patent with the publication number of CN 1207382C, and is named as the lactobacillus plantarum ST-III strain and the application thereof in regulating blood fat.
The invention is realized by the following technical scheme:
specifically, on the one hand, the preparation method of the selenium-enriched lactobacillus preparation is provided, and comprises the following steps:
(1) activating strains: inoculating the Lb. plantarum ST-III strain in MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture;
(2) preparation of green tea extract: weighing 2g of green tea sample, adding 100 ℃ pure water and 100mL pure water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane to obtain green tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the green tea extract obtained in the step (2); wherein the mass concentration of the green tea extract added into the MRS-TE culture medium is 0.06% -0.10%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 24-48 h;
(6) and (4) centrifuging the fermentation thalli obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
By adopting the technical scheme, the prepared fermentation bacteria are rich in selenium element, and the selenium content can reach 19.3 mg/g.
Further, in the step (2), the pore size of the sterile filter membrane is 0.44 μm.
Through the technical scheme, the aseptic filter membrane achieves the sterilization effect and simultaneously reserves the effective components in the green tea.
Further, in the step (5), the fermentation time was 48 hours.
Further, in the step (3), the amount of the green tea extract added is 30mL/L to 50 mL/L.
Further, in the step (2), the green tea extract is enriches selenium-rich tea extract.
Through the technical scheme, the green tea extract contains selenium, so that a selenium source is provided for lactic acid bacteria.
Further, the MRS liquid medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water; the pH value of the MRS liquid culture medium is 6.5, the sterilization temperature is 121 ℃, and the sterilization time is 15 min.
Further, the MRS-Mn medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water; the pH value of the MRS-Mn culture medium is 6.5, the sterilization temperature is 121 ℃, and the sterilization time is 15 min.
On the other hand, the invention also provides a selenium-enriched lactobacillus preparation which is directly prepared by the preparation method of the selenium-enriched lactobacillus preparation.
Through the technical scheme, the prepared preparation is rich in organic selenium, and the health effect of the selenium can be better exerted.
Compared with the prior art, the invention has the following advantages:
①, the prior art only provides the enrichment condition of the inorganic selenium (mainly sodium selenate Na2SeO3) in the chemical reagent when the lactobacillus synthesizes the culture medium, but the invention adopts the fermentation method to efficiently enrich the organic selenium from the green tea;
② the lactobacillus preparation prepared by the invention can convert selenium element to form organic selenium while enriching.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The starting materials in the following examples were all commercially available unless otherwise specified.
Example 1
A method for preparing a selenium-enriched lactobacillus preparation comprises the following steps:
(1) activating strains: inoculating the Lb. plantarum ST-III strain in MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture; the MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(2) preparation of green tea extract: weighing 2g of green tea sample, adding 100 ℃ pure water of 100mL, soaking for 10min, taking supernatant, and filtering by a sterile filter membrane of 0.44 mu m to obtain green tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the green tea extract obtained in the step (2); wherein the addition amount of the green tea extract is 30mL/L, and the mass concentration of the green tea extract added into an MRS-TE culture medium is 0.06%; MRS-Mn medium composition is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 48 hours;
(6) and (4) centrifuging the fermentation thallus obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
The selenium content was determined as follows: the measurement is carried out according to hydride atomic fluorescence spectrometry in GB 5009.93-2017 determination of selenium in food. The specific selenium-rich amount is calculated according to the following formula:
selenium-rich (mg/g) thallus intracellular selenium content (mg)/dry cell weight (g) in thallus
The selenium content in the selenium-enriched lactobacillus preparation obtained in the embodiment 1 is 17.5 mg/g.
Example 2
A method for preparing a selenium-enriched lactobacillus preparation comprises the following steps:
(1) activating strains: inoculating the Lb. plantarum ST-III strain in MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture; the MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(2) preparing Enshi selenium-rich tea extract: weighing 2g of Enshi selenium-enriched tea sample, adding 100 ℃ and 100mL of purified water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane of 0.44 mu m to obtain Enshi selenium-enriched tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the Enshi selenium-rich tea extract obtained in the step (2); wherein the addition amount of the Enshi selenium-enriched tea extract is 30mL/L, and the mass concentration of the Enshi selenium-enriched tea extract added into the MRS-TE culture medium is 0.06%; MRS-Mn medium composition is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 24 hours;
(6) and (4) centrifuging the fermentation thallus obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
The method for measuring the selenium content is the same as that of example 1;
the selenium content in the selenium-enriched lactobacillus preparation obtained in the embodiment 2 is 11.2 mg/g.
Example 3
A method for preparing a selenium-enriched lactobacillus preparation comprises the following steps:
(1) activating strains: inoculating the Lb. plantarum ST-III strain in MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture; the MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(2) preparing Enshi selenium-rich tea extract: weighing 2g of Enshi selenium-enriched tea sample, adding 100 ℃ and 100mL of purified water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane of 0.44 mu m to obtain Enshi selenium-enriched tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the Enshi selenium-rich tea extract obtained in the step (2); wherein the addition amount of the Enshi selenium-enriched tea extract is 50mL/L, and the mass concentration of the Enshi selenium-enriched tea extract added into the MRS-TE culture medium is 0.10%; MRS-Mn medium composition is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 48 hours;
(6) and (4) centrifuging the fermentation thallus obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
The method for measuring the selenium content is the same as that of example 1;
the selenium content in the selenium-enriched lactobacillus preparation obtained in the embodiment 3 is measured to be 19.3 mg/g.
Example 4
A method for preparing a selenium-enriched lactobacillus preparation comprises the following steps:
(1) activating strains: inoculating the Lb. plantarum ST-III strain in MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture; the MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(2) preparing Liuan Guapian tea extract: weighing 2g of Liuan Guapian tea sample, adding 100 ℃ pure water and 100mL pure water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane of 0.44 mu m to obtain Liuan Guapian tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the Liuan Guapian tea extract obtained in the step (2); wherein the addition amount of the Liuan Guapian tea extract is 50mL/L, and the mass concentration of the Liuan Guapian tea extract added into an MRS-TE culture medium is 0.10%; MRS-Mn medium composition is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 48 hours;
(6) and (4) centrifuging the fermentation thalli obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
The method for measuring the selenium content is the same as that of example 1;
the selenium content in the selenium-enriched lactobacillus preparation obtained in the embodiment 4 is 3.5 mg/g.
Example 5
A method for preparing a selenium-enriched lactobacillus preparation comprises the following steps:
(1) activating strains: inoculating the Lb. plantarum ST-III strain in MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture; the MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(2) preparing Enshi selenium-rich tea extract: weighing 2g of Enshi selenium-enriched tea sample, adding 100 ℃ and 100mL of purified water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane of 0.44 mu m to obtain Enshi selenium-enriched tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the Enshi selenium-rich tea extract obtained in the step (2); wherein the addition amount of the Enshi selenium-enriched tea extract is 40mL/L, and the mass concentration of the Enshi selenium-enriched tea extract added into the MRS-TE culture medium is 0.08%; MRS-Mn medium composition is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 24 hours;
(6) and (4) centrifuging the fermentation thallus obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
The method for measuring the selenium content is the same as that of example 1;
the selenium content in the selenium-enriched lactobacillus preparation obtained in the embodiment 5 is 15.7 mg/g.
Comparative example 1
The preparation method of this comparative example is the same as example 1 except that MRS-Mn medium was directly used without adding green tea extract. The same method as in example 1 was used to determine the selenium content, and the lactic acid bacteria preparation of this comparative example 1 was determined to have no detectable selenium content.
Comparative example 2
A method for preparing a selenium-enriched lactobacillus preparation comprises the following steps:
(1) activating strains: inoculating the frozen Lb.rhamnosus GG strain into MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture; the MRS liquid culture medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(2) preparing Enshi selenium-rich tea extract: weighing 2g of Enshi selenium-enriched tea sample, adding 100 ℃ and 100mL of purified water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane of 0.44 mu m to obtain Enshi selenium-enriched tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the Enshi selenium-rich tea extract obtained in the step (2); wherein the addition amount of the Enshi selenium-enriched tea extract is 50mL/L, and the mass concentration of the Enshi selenium-enriched tea extract added into the MRS-TE culture medium is 0.10%; MRS-Mn medium composition is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 48 hours;
(6) and (4) centrifuging the fermentation thallus obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
The method for measuring the selenium content is the same as that of example 1;
through determination, the selenium content in the selenium-enriched lactobacillus preparation obtained in the comparative example 2 is 9.2 mg/g; the preparation process of this comparative example is the same as that of example 2 except that lb. By the same operation, the selenium content in the obtained lactobacillus preparation is lower than that in the example 2, and the product obtained in the comparative example 2 is mainly inorganic selenium and has low organic selenium content.
Effect example 1
Comparison of the effects of the different examples, see table 1 below:
| examples | 1 | 2 | 3 | 4 | 5 |
| Concentration of selenium-enriched tea | 0.06 | 0.06 | 0.10 | - | 0.08 |
| Concentration of ordinary green tea | - | - | - | 0.10 | - |
| Content after enrichment | 17.5 | 11.2 | 19.3 | 3.5 | 15.7 |
Comparison of the effects of example 1 and comparative example, see table 2 below:
| example 1 | Comparative example 1 | Comparative example 2 | |
| Enriched selenium content | 17.5 | - | 9.2 |
| Forms of selenium | Organic state | - | Inorganic state |
It can be known from the above table that the invention utilizes the fermentation means of lactic acid bacteria to enrich selenium element from green tea and other plants with high efficiency, namely lactobacillus plantarum is activated according to the routine, and inoculated in MRS-TE culture medium (adding a certain amount of green tea extract) after the thalli are collected by centrifugation and washed, and then centrifuged and washed for 3 times by aseptic normal saline after the constant temperature culture is continued for 24 times, thus obtaining the lactic acid bacteria preparation rich in selenium; compared with other selenium-enriched lactic acid bacteria (Lb.rhamnosus GG strains), the selenium-enriched lactobacillus strain can convert selenium element while enriching selenium under the synergistic action of the preparation method and the Lb.plantarum ST-III strains to form organic selenium.
The above-described embodiments are merely illustrative of one or more embodiments of the present invention, which are described in more detail and detail, but are not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (8)
1. The preparation method of the selenium-enriched lactobacillus preparation is characterized by comprising the following steps:
(1) activating strains: inoculating the Lb. plantarum ST-III strain in MRS liquid culture medium, anaerobically culturing at 37 deg.C for 18-22h, and circularly subculturing for 2-3 times to obtain thallus culture;
(2) preparation of green tea extract: weighing 2g of green tea sample, adding 100 ℃ pure water and 100mL pure water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane to obtain green tea extract;
(3) preparing an MRS-TE culture medium: naturally cooling the MRS-Mn culture medium to room temperature, and adding the green tea extract obtained in the step (2); wherein the mass concentration of the green tea extract added into the MRS-TE culture medium is 0.06% -0.10%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) to obtain thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the MRS-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 24-48 h;
(6) and (4) centrifuging the fermentation thalli obtained in the step (5), washing for 3 times by using sterile normal saline, centrifuging, and removing supernatant to obtain the selenium-enriched lactobacillus preparation.
2. The method for preparing a selenium-enriched lactic acid bacterium preparation according to claim 1, wherein in the step (2), the pore size of the sterile filtration membrane is 0.44 μm.
3. The method for preparing a selenium-enriched lactic acid bacteria preparation according to claim 1, wherein the fermentation time in step (5) is 48 hours.
4. The method for preparing a selenium-enriched lactic acid bacterium preparation according to claim 1, wherein the green tea extract is added in an amount of 30 to 50mL/L in step (3).
5. The method of preparing the selenium-enriched lactic acid bacteria preparation of claim 1, wherein in the step (2), the green tea extract is enriches selenium-enriched tea extract.
6. The method for preparing the selenium-enriched lactic acid bacteria preparation according to claim 1, wherein the MRS liquid medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate, 54mg of manganese sulfate and 1000mL of distilled water; the pH value of the MRS liquid culture medium is 6.5, the sterilization temperature is 121 ℃, and the sterilization time is 15 min.
7. The method for preparing the selenium-enriched lactic acid bacteria preparation according to claim 1, wherein the MRS-Mn medium comprises the following components: 10g of peptone, 10g of beef extract, 5g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1g of Tween 80, 200mg of magnesium sulfate and 1000mL of distilled water; the pH value of the MRS-Mn culture medium is 6.5, the sterilization temperature is 121 ℃, and the sterilization time is 15 min.
8. A selenium-enriched lactic acid bacteria preparation, which is directly prepared by the preparation method of the selenium-enriched lactic acid bacteria preparation as claimed in any one of claims 1 to 7.
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