CN110951651A

CN110951651A - Selenium-enriched lactobacillus preparation and preparation method thereof

Info

Publication number: CN110951651A
Application number: CN201911395726.9A
Authority: CN
Inventors: 鄢明辉; 刘振民; 游春苹; 扶晓菲
Original assignee: Bright Dairy and Food Co Ltd
Current assignee: Bright Dairy and Food Co Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-04-03

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 green tea extract; (4) centrifuging the obtained thallus culture, and collecting thallus; (5) washing collected thalli with physiological saline for three times, then suspending in the obtained MRS-TE culture medium, fully shaking up, standing at 37 ℃ and fermenting; (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 of efficiently enriching the organic selenium from the green tea by adopting a fermentation method.

Description

Selenium-enriched lactobacillus preparation and preparation method thereof

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 Enshi 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 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 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 mass concentration of the Enshi selenium-rich tea extract added into an 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 obtained fermentation thallus is 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 damage of high-temperature sterilization to effective components in the green tea is reduced while the sterilization effect is achieved.

Further, in the step (5), the fermentation time was 48 hours.

Furthermore, the adding amount of the Enshi selenium-rich tea extract is 30mL/L-50 mL/L.

Through the technical scheme, the 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 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.

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) 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;

(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 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 of the selenium-enriched lactobacillus preparation obtained in the embodiment 1 is 11.2 mg/g.

Example 2

(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-rich tea extract is 50mL/L, and the mass concentration of the Enshi selenium-rich 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;

(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;

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 measured to be 19.3 mg/g.

Example 3

(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;

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 15.7 mg/g.

Example 4

(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 50 mL/L; adding the Liuan Guapian tea extract into an MRS-TE culture medium, wherein the mass concentration of the Liuan Guapian tea extract 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;

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

(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 40 mL/L; adding the Liuan Guapian tea extract into an MRS-TE culture medium, wherein the mass concentration of the Liuan Guapian tea extract 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;

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 2.7 mg/g.

Example 6

(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 30 mL/L; adding the Liuan Guapian tea extract into an MRS-TE culture medium, wherein the mass concentration of the Liuan Guapian tea extract 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;

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 6 is 2.1 mg/g.

Comparative example 1

The preparation method of this comparative example is the same as example 1 except that MRS medium was directly used instead of green tea extract. By the same operation, the selenium content in the obtained lactic acid bacteria preparation was not detected.

Comparative example 2

(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.2003May; 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;

(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;

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	6
							Concentration of selenium-enriched tea	0.06	0.10	0.08	-	-	-
Concentration of ordinary green tea	-	-	-	0.10	0.08	0.06
							Content after enrichment	11.2	19.3	15.7	3.5	2.7	2.1

Comparison of the effects of example 2 with the comparative examples, see table 2 below:

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

1. The preparation method of the selenium-enriched lactobacillus preparation is characterized by comprising the following steps:

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 the selenium-enriched lactic acid bacteria preparation as claimed in claim 1, wherein in the step (3), the amount of the extract of Enshi selenium-enriched tea added is 30mL/L to 50 mL/L.

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. 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.

7. 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 6.