CN110951652A - Selenium-enriched lactobacillus preparation and preparation method thereof - Google Patents
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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 subjected to freeze preservation into an MRS liquid culture medium for anaerobic culture, and performing cyclic subculture for 2-3 times to obtain a thallus culture; (2) weighing 2g of selenium-rich tea sample, adding purified water, soaking to obtain supernatant, and filtering with sterile filter membrane to obtain selenium-rich tea extract; (3) preparing a tomato juice-TE culture medium (4), centrifuging to obtain a thallus culture, and collecting thallus; (5) washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the tomato juice-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃, and fermenting for 48-96 hours; (6) centrifuging the obtained fermented thallus, washing with sterile physiological saline for 3 times, centrifuging, and removing supernatant to obtain selenium-rich lactobacillus preparation. The invention has the advantage that the organic selenium is efficiently enriched from the selenium-enriched 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 selenium-enriched tea on the basis of lactobacillus plantarum ST-III; the lactobacillus plantarum ST-III strain is disclosed in the invention patent with the publication number of CN 1207382C, the invention name of 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) preparing selenium-rich tea extract: weighing 2g of selenium-rich tea sample, adding 100 ℃ pure water of 100mL, soaking for 10min, taking supernatant, and filtering by a sterile filter membrane to obtain selenium-rich tea extract;
(3) preparing a tomato juice-TE culture medium: the preparation process of the tomato juice comprises the following steps: cleaning ripe tomatoes, peeling, juicing, filtering to obtain juice, boiling for 10 minutes, centrifuging at 6000g for 10 minutes, and taking supernatant; sterilizing the tomato juice at 115 ℃ for 10min, cooling to room temperature, and adding the selenium-rich tea extract obtained in the step (2); wherein the mass concentration of the selenium-rich tea extract added into a tomato juice-TE culture medium is 0.06% -0.10%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) and collecting thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the tomato juice-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃, and fermenting for 48-96 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.
By adopting the technical scheme, the organic selenium is efficiently enriched from the selenium-enriched tea by using a lactobacillus plantarum fermentation means.
Further, the selenium-rich tea is Enshi selenium-rich tea.
Further, in the step (2), the pore size of the sterile filter membrane is 0.44 μm.
Further, in the step (3), the adding amount of the selenium-enriched tea extract is 30mL/L-50 mL/L.
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.
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 lactic acid bacteria to synthesize the culture medium for inorganic selenium (mainly sodium selenate Na) in chemical reagent2SeO3) The enrichment of (1). The invention adopts fermentation means to efficiently enrich organic selenium from green tea;
②, the selenium-rich Enshi tea has high selenium content, and can provide abundant selenium source for lactobacillus and increase selenium content in lactobacillus product;
③ the lactobacillus preparation prepared by the invention can convert selenium element to form organic selenium while enriching.
Drawings
FIG. 1 shows the appearance of a puree of selenium enriched lactic acid bacteria (ST-III: Lactobacillus plantarum ST-III; LGG: Lactobacillus rhamnosus GG; and (-and + denote pre-and post-enrichment, respectively).
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 and 100mL pure water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane to obtain green tea extract;
(3) preparing a tomato juice-TE culture medium: the preparation process of the tomato juice comprises the following steps: cleaning ripe tomatoes, peeling, juicing, filtering to obtain juice, boiling for 10 minutes, centrifuging at 6000g for 10 minutes, and taking supernatant; sterilizing the tomato juice at 115 ℃ for 10min, cooling 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 a tomato juice-TE culture medium is 0.10 percent;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) and collecting thallus;
(5) fermenting thalli: cleaning the thalli collected in the step (4) with normal saline (0.9% NaCl) for three times, then suspending the thalli in the tomato juice-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 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 13.5 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) 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 a tomato juice-TE culture medium: the preparation process of the tomato juice comprises the following steps: cleaning ripe tomatoes, peeling, juicing, filtering to obtain juice, boiling for 10 minutes, centrifuging at 6000g for 10 minutes, and taking supernatant; sterilizing the tomato juice at 115 ℃ for 10min, cooling 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 a tomato juice-TE culture medium is 0.06%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) and collecting thallus;
(5) fermenting thalli: cleaning the thalli collected in the step (4) with normal saline (0.9% NaCl) for three times, then suspending the thalli in the tomato juice-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 2 is 10.8 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 ℃ pure water and soaking for 10min to obtain supernatant, and filtering by using a sterile filter membrane to obtain Enshi selenium-enriched tea extract;
(3) preparing a tomato juice-TE culture medium: the preparation process of the tomato juice comprises the following steps: cleaning ripe tomatoes, peeling, juicing, filtering to obtain juice, boiling for 10 minutes, centrifuging at 6000g for 10 minutes, and taking supernatant; sterilizing the tomato juice at 115 ℃ for 10min, cooling to room temperature, and adding the extract of Enshi selenium-rich tea obtained in the step (2); wherein the mass concentration of the extract of Enshi selenium-rich tea added with tomato juice-TE culture medium is 0.08%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) and collecting thallus;
(5) fermenting thalli: cleaning the thalli collected in the step (4) with normal saline (0.9% NaCl) for three times, then suspending the thalli in the tomato juice-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 72 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 of the selenium-enriched lactobacillus preparation obtained in the embodiment 3 is 12.7 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 Enshi selenium-rich tea extract: weighing 2g of Enshi selenium-enriched tea sample, adding 100 ℃ pure water and soaking for 10min to obtain supernatant, and filtering by using a sterile filter membrane to obtain Enshi selenium-enriched tea extract;
(3) preparing a tomato juice-TE culture medium: the preparation process of the tomato juice comprises the following steps: cleaning ripe tomatoes, peeling, juicing, filtering to obtain juice, boiling for 10 minutes, centrifuging at 6000g for 10 minutes, and taking supernatant; sterilizing the tomato juice at 115 ℃ for 10min, cooling to room temperature, and adding the extract of Enshi selenium-rich tea obtained in the step (2); wherein the mass concentration of the extract of Enshi selenium-rich tea added with tomato juice-TE culture medium is 0.10%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) and collecting thallus;
(5) fermenting thalli: cleaning the thalli collected in the step (4) with normal saline (0.9% NaCl) for three times, then suspending the thalli in the tomato juice-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃ and fermenting for 96 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 of the selenium-enriched lactobacillus preparation obtained in the embodiment 4 is measured to be 13.9 mg/g.
Comparative example 1
The preparation method of this comparative example was the same as that of example 1 except that green tea (Xinyang Maojian tea) having a low selenium content was used and the selenium content in the obtained preparation was 30. mu.g/g.
Comparative example 2
The preparation method of this comparative example is the same as example 1 except that MRS-Mn medium is directly used. In the same manner, the content of selenium in the lactic acid bacteria preparation obtained in this comparative example was not detected.
Comparative example 3
A method for preparing a selenium-enriched lactobacillus preparation comprises the following steps:
(1) activating strains: cryopreserved lb. rhamnosus GG strains (immunomodulating sequences of organism administration of Lactobacillus rhamnosus strain GG in nutritional volnterers. Schultz M, Linde HJ, Lehn N, Zimmermann K, Grossmann J, Falk W,
J.J Dairy Res.2003 May; 70(2) 165-73) is inoculated in MRS liquid culture medium, anaerobic culture is carried out for 18-22h at 37 ℃, and cell culture is obtained after 2-3 times of circulating subculture; 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 and 100mL pure water, soaking for 10min, taking supernatant, and filtering by using a sterile filter membrane to obtain green tea extract;
(3) preparing a tomato juice-TE culture medium: the preparation process of the tomato juice comprises the following steps: cleaning ripe tomatoes, peeling, juicing, filtering to obtain juice, boiling for 10 minutes, centrifuging at 6000g for 10 minutes, and taking supernatant; sterilizing the tomato juice at 115 ℃ for 10min, cooling 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 tomato juice-TE culture medium is 0.10%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) and collecting thallus;
(5) fermenting thalli: cleaning the thalli collected in the step (4) with normal saline (0.9% NaCl) for three times, then suspending the thalli in the tomato juice-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 3 is 9.3 mg/g.
Effect example 1
Comparison of the effects of the examples and comparative examples: units (mg/g), see Table 1 below:
| example 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Enriched selenium content | 13.5 | 0.03 | - (not detected) | 9.3 |
Conversion of lactic acid bacteria to selenium: in the prior art, selenium enriched by lactic acid bacteria is mainly expressed in a simple substance state and an organic state; the selenium in the elementary substance state can lead the prepared lactobacillus preparation to be red; the morphological observation of the lactobacillus rhamnosus GG (l.rhamnosus GG) bacterial mud in the embodiment shows that the selenium contained in the lactobacillus rhamnosus GG (l.rhamnosus GG) bacterial mud is mainly in an inorganic state, while the morphological observation of the lactobacillus plantarum ST-III bacterial mud in the invention shows that the selenium contained in the lactobacillus plantarum ST-III bacterial mud is in an organic state (the specific comparison is shown in the attached figure 1).
The invention adopts the lactobacillus fermentation method to effectively enrich selenium element from green tea and other plants; specifically, lactobacillus plantarum is activated conventionally, centrifuged to collect thalli, washed, inoculated in an MRS-TE culture medium (a certain amount of green tea extract is added), continuously cultured at constant temperature for 24 times, centrifuged, and washed with sterile normal saline for 3 times to obtain a lactobacillus preparation rich in organic selenium; compared with other selenium-enriched lactic acid bacteria, the preparation method can convert selenium element while enriching selenium under the synergistic effect of Lb.
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 (6)
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) preparing selenium-rich tea extract: weighing 2g of selenium-rich tea sample, adding 100 ℃ pure water of 100mL, soaking for 10min, taking supernatant, and filtering by a sterile filter membrane to obtain selenium-rich tea extract;
(3) preparing a tomato juice-TE culture medium: the preparation process of the tomato juice comprises the following steps: cleaning ripe tomatoes, peeling, juicing, filtering to obtain juice, boiling for 10 minutes, centrifuging at 6000g for 10 minutes, and taking supernatant; sterilizing the tomato juice at 115 ℃ for 10min, cooling to room temperature, and adding the selenium-rich tea extract obtained in the step (2); wherein the mass concentration of the selenium-rich tea extract added into a tomato juice-TE culture medium is 0.06% -0.10%;
(4) and (3) collecting thalli: centrifuging the thallus culture obtained in the step (1) and collecting thallus;
(5) fermenting thalli: washing the thalli collected in the step (4) with physiological saline for three times, then suspending the thalli in the tomato juice-TE culture medium obtained in the step (3), fully shaking up, standing at 37 ℃, and fermenting for 48-96 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.
2. The method for preparing a selenium-enriched lactic acid bacteria preparation as claimed in claim 1, wherein in the step (2), the selenium-enriched tea is enriches selenium-enriched tea.
3. 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.
4. The method for preparing a selenium-enriched lactic acid bacteria preparation as claimed in claim 1, wherein the selenium-enriched tea extract is added in an amount of 30mL/L to 50mL/L in step (3).
5. 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.
6. A selenium-enriched lactic acid bacteria preparation, which is directly prepared by the preparation method of the selenium-enriched lactic acid bacteria preparation according to any one of claims 1 to 5.
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