CN110760456A

CN110760456A - Lactobacillus plantarum La1 for degrading cholesterol and application thereof

Info

Publication number: CN110760456A
Application number: CN201910616504.9A
Authority: CN
Inventors: 徐君飞; 张居作; 刘新路; 金媛悦
Original assignee: Huaihua University
Current assignee: Hebei Max Biotechnology Co ltd; Qingdao Fruit Science And Technology Service Platform Co ltd
Priority date: 2019-07-09
Filing date: 2019-07-09
Publication date: 2020-02-07
Anticipated expiration: 2039-07-09
Also published as: CN110760456B

Abstract

The invention discloses lactobacillus plantarum La1 for degrading cholesterol and application thereof, wherein the strain La1 is preserved, and the preservation unit is as follows: china center for type culture Collection, collection address: wuhan university in China, preservation date: 3, month and 4 in 2019, wherein the preservation number is CCTCC NO: m2019121. The strain has a cholesterol degradation rate of 75.31% in a semi-selective MRS culture medium, and has practical significance in promoting the development of sour meat industry, enriching the types of pork products and improving the value of the pork products.

Description

Lactobacillus plantarum La1 for degrading cholesterol and application thereof

Technical Field

The invention belongs to the technical field of food microorganism application, and particularly relates to lactobacillus plantarum La1 for degrading cholesterol and application thereof.

Background

Cholesterol is an important steroid substance in a human body, widely exists in various tissues and cells, plays an important physiological role in the human body, is an essential nutrient component of the human body, but excessive cholesterol accumulation in the body also brings various health problems, and cardiovascular and cerebrovascular diseases such as coronary heart disease, atherosclerosis, hyperlipidemia, cerebral apoplexy and the like which are related to overhigh serum cholesterol content seriously threaten the health of human beings at present. The intake standard of cholesterol in food is not more than 250 mg/d.person-300 mg/d.person recommended by nutrition and medical experts at home and abroad, but the intake of cholesterol is easy to exceed the standard along with the improvement of living standard of people and the increase of dietary nutrition.

The lactic acid bacteria are widely distributed in nature and have high application value in the fields of industry, agriculture, medicine and the like. The physiological functions of lactic acid bacteria in the body include: regulating intestinal flora balance, correcting intestinal dysfunction, inhibiting endotoxin, enhancing immunity, resisting aging, and resisting tumor. According to the reports, the lactobacillus has a degradation effect on the cholesterol content in human serum in various ways, not only can reduce the cholesterol content in food of people, but also can regulate the cholesterol metabolism of the body through enzyme, and further reduce the cholesterol content in the serum. In the last two decades, many foreign researchers have conducted extensive research on the reduction of serum cholesterol content by lactic acid bacteria, the prevention and treatment of cardiovascular and cerebrovascular diseases, and the like, and have obtained promising achievements. At present, the incidence of cardiovascular and cerebrovascular diseases in China is gradually increased, and the development of medicines and health care products with the function of reducing blood fat has very wide application prospect.

Pork is one of meat varieties generally eaten by people in China, but the pork is high in fat content, particularly fat, and higher in fat and cholesterol content, and the excessive intake of the fat and the cholesterol has obvious correlation with the morbidity of cardiovascular and cerebrovascular diseases, so that the health of a human body is seriously influenced. The fermented sour meat can degrade fat and cholesterol in pork under the action of microorganisms, so that the problem of overhigh fat and cholesterol in fat pork is solved, at present, few research reports on the aspect of researching and preparing specific strains of low-cholesterol food in China exist, and the preparation process of the fermented sour meat is relatively flexible and difficult to industrialize, so that lactic acid bacteria capable of degrading cholesterol are screened and separated from traditional fermented food fermented soya beans, the dominant strains for reducing cholesterol are obtained, and the dominant strains are further applied to the preparation of the fermented sour meat, so that novel functional fermented sour meat products with local characteristics are developed, the types of pork products are enriched, and the value of the pork products is improved.

Disclosure of Invention

The invention aims to provide lactobacillus plantarum La1 for degrading cholesterol.

The invention is realized by the following technical scheme:

the invention provides a strain La1, which is obtained by separating and purifying fermented soya beans, and the colony morphology of the strain is as follows: round, dark white, convex, unsmooth surface, irregular edge, diameter 1.5mm + -1 mm, and light pink color can be obtained after long-time storage.

The molecular characteristics of the strain La1 provided by the invention, the DNA of the strain is extracted for PCR amplification to obtain the 16S rRNA gene of the strain, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The GenBank nucleic acid sequence library is subjected to homology comparison with the obtained sequences, and the strain La1 is found to have the closest relationship with the genus Lactobacillus, wherein the homology with Lactobacillus plantarum is the highest and reaches 99%. The strain was identified as Lactobacillus plantarum according to sequence homology and morphological analysis of 16S rDNA, and thus the resulting strain La1 of the present invention was named Lactobacillus plantarum La 1.

The strain La1 is preserved, and the preservation unit: china center for type culture Collection, collection address: wuhan university in China, preservation date: 3, month and 4 in 2019, wherein the preservation number is CCTCC NO: m2019121.

In another aspect of the present invention, the application of the strain La1 in degrading cholesterol is also within the protection scope of the present invention.

In another aspect of the present invention, the application of the strain La1 in the preparation of cholesterol-degrading drugs or health products is also within the protection scope of the present invention.

The invention has the beneficial effects that:

the cholesterol degradation rate of the strain La1 separated by the method reaches 75.31% in a semi-selective MRS culture medium, and the strain is used for obtaining a cholesterol-reducing dominant strain with an independent property right and further applied to the preparation of fermented sour meat so as to develop a novel functional fermented sour meat product with local characteristics, and has practical significance for promoting the development of sour meat industry, enriching the types of pork products and improving the value of the pork products.

Drawings

FIG. 1 is a graph showing the growth of strains SC1, DC1, La1, PC1 at 10 ℃;

FIG. 2 is a graph showing the growth of strains SC1, DC1, La1, PC1 at 45 ℃;

FIG. 3 is a growth curve of strains SC1, DC1, La1, PC1 under pH 9.6;

FIG. 4 is a standard curve for cholesterol;

FIG. 5 is a fermented sour meat.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 screening of strains

Weighing semen Sojae Preparatum, caulis et folium Brassicae Capitatae, radix Raphani, and chopped Capsici fructus 25.0g, adding 0.9% sterilized normal saline 225.0mL, crushing, mixing, standing, sucking supernatant 0.1mL, and performing gradient dilution (10)^-1-10^-8) And coating each gradient diluent on 20mL improved MRS agar culture media, performing 4 parallel culture at 37 ℃, performing anaerobic and aerobic constant-temperature culture for 72h, and observing the growth condition of colonies.

Selecting typical single colonies according to colony morphology and calcium dissolving rings, transferring the single colonies to a modified MRS culture medium (1mL/1.5mL centrifuge tube), performing static culture at a constant temperature of 37 ℃ for 1d, streaking the single colonies into the modified MRS agar culture medium, performing constant temperature culture at 37 ℃ for 72h, observing colony morphology characteristics of each strain, and numbering the single colonies respectively.

Gram staining was performed on the strains which were observed for colony morphology and numbered.

① slicing, smearing strain at logarithmic phase by conventional method, drying and fixing on alcohol lamp, wherein the strain cannot be too thick;

② primarily dyeing, dripping ammonium oxalate crystal violet dye liquor to cover the bacterium coating part, wherein the dyeing time is 1min, pouring out the dye liquor, and washing with water until the effluent water is colorless;

③ mordanting, washing with Lugol iodine solution to remove residual water stain, covering with iodine solution for 1min, pouring out iodine solution, and washing with water until the effluent is colorless;

④ decolorizing, sucking residual water on the slide, dripping 95% ethanol with a dropper to decolorize, and washing off ethanol with water when the effluent is colorless;

⑤ counterstaining, removing residual water on the slide, staining with safranin counterstaining solution for about 2min, washing with water, removing water, and air drying;

⑥ microscopic examination, and observing the gram staining result of each strain under a binocular biological microscope with an oil microscope.

The results of observing the colony morphology of 5 cholesterol-lowering strains isolated and screened from pickled radish, chopped hot pepper, fermented soya beans and pickled Chinese cabbage are shown in Table 1.

TABLE 1 typical Strain colony morphology Observation

The 5 cholesterol-lowering strains SC1, LC1, DC1, La1 and PC1 obtained by separation were subjected to gram staining, and the results of gram staining revealed that the strains SC1, LC1, DC1, La1 and PC1 were G⁺。

Example 2 physiological and Biochemical assay identification

1.1 growth test

The lactic acid bacteria grew well between 10 ℃ and 45 ℃ and pH 9.6 was the growth limit pH of lactic acid bacteria, so it was preliminarily identified whether the selected strains were lactic acid bacteria by measuring the growth curve of each strain at 10 ℃, 45 ℃ and pH 9.6.

① 10 deg.C growth experiment, inoculating 5 strains into MRS liquid culture medium, 10 ℃Shaking culture at constant temperature, and measuring OD once every 3h_560nmAnd measuring for 42h continuously, and drawing a growth curve.

Growth experiment at ② 45 deg.C, inoculating 5 separated strains into MRS liquid culture medium, shake culturing at 45 deg.C, and measuring OD every 3 hr_560nmAnd measuring for 42h continuously, and drawing a growth curve.

③ pH 9.6 growth experiment, adjusting pH of MRS liquid culture medium to 9.6, inoculating 5 separated strains, shake culturing at 30 deg.C, measuring OD every 3h_560nmAnd measuring for 42h continuously, and drawing a growth curve.

1.2 Catalase assay

Dropping a drop of 3% hydrogen peroxide solution on a clean and sterile glass slide, picking a ring of purified single colony, placing the single colony in the hydrogen peroxide solution, observing for 30s, and determining the single colony to be positive if bubbles are generated and negative if the single colony is not generated.

1.3 liquefaction test of gelatin

Inoculating test strains in a gelatin culture medium, culturing at 30 ℃, taking out test tubes every 24h, and continuously observing for 7d, wherein the culture medium of the test tubes which are not inoculated is a blank control. And (3) placing the control group and the experimental group in a refrigerator at 4 ℃ until the control tube is solidified, and observing whether the test tube is solidified or not, wherein if the test tube is liquefied to be positive, the solidification is negative.

1.4 nitric acid reduction test

Inoculating test strains in a nitrate reducing liquid culture medium, culturing at 30 ℃ for 1d, 3d and 5d, and then determining. 0.5mL of the solution A and 0.5mL of the solution B are mixed, 0.1mL of the mixed reagent is added on the surface of the agar slant culture, and the test is positive if the mixed reagent is red within 10min, otherwise, the mixed reagent is negative.

1.5 production of H₂S test

Inoculating the test strain into a semi-solid ferrous sulfate culture medium, culturing for 2d at 30 ℃, and observing, wherein the culture medium turns black to be positive, otherwise, the culture medium turns negative.

1.6 acid and gas evolution test for glucose

To PY medium were added glucose 30g, Tween 800.5 mL, agar 6g, and bromocresol purple 1.4mL (1.6%), followed by sterilization at 112 ℃ for 30 min. Inoculating test strain in melted agar soft column at temperature not higher than 45 deg.C, mixing, covering with a layer of 2% agar with thickness of about 7mm, culturing at 30 deg.C, and indicating agent in culture medium to yellow to indicate acid production; the phenomenon of the agar in the test tube appearing topping indicates the gas production.

1.7 motility test

And (3) inoculating the cultured strain to a semisolid culture medium by puncturing an inoculating needle, culturing for 3d at 30 ℃, and observing whether the edge is clear or not by penetrating light. If the culture growth spreads from the puncture line to the surrounding cloud, it indicates motility, and is positive, otherwise it is negative.

1.8 litmus milk test

Test bacteria are inoculated in litmus milk culture medium and incubated for 24h at 35 ℃, and the observation result is as follows:

① acid production-lactose is fermented to produce acid, which turns the indicator into pink.

② producing gas by fermenting lactose and simultaneously producing gas, and can break away vaseline thereon.

③ coagulation casein in cow's milk coagulates due to too much acid production.

④ peptonization, the coagulated casein was hydrolyzed to peptone, the upper liquid of the medium became clear, and the bottom part could be left with casein that was not completely peptonized.

⑤ producing alkali, lactose is not fermented, and nitrogen-containing substances are decomposed to generate amine and ammonia, the culture medium is changed into alkali, and the indicator is changed into blue.

1.9 sugar fermentation experiments

Inoculating a proper amount of pure culture in a sugar fermentation tube, and culturing at 36 +/-1 ℃ for 48h, wherein the observed result shows that blue is negative and yellow is positive.

As shown in FIG. 1, under the condition of 10 ℃, the logarithmic growth phase of the strain La1 is 21-30 h, the logarithmic growth phase of the strain SC1 is 18-24 h, the logarithmic growth phase of the strains DC1 and PC1 is 15-39 h, and relatively speaking, the growth vigor of the strains DC1 and PC1 is higher than that of the strains La1 and SC 1.

As shown in FIG. 2, the logarithmic growth phase of strains La1 and PC1 was 9 to 24 hours, the logarithmic growth phase of strain DC1 was 6 to 24 hours, and the SC1 strain grew slowly, the logarithmic growth phase appeared 24 hours later, under the condition of 45 ℃.

As shown in FIG. 3, under the condition of pH 9.6, the strains SC1, DC1, La1 and PC1 have similar growth vigor and the logarithmic growth phase is 6-15 h, wherein the growth vigor of the strains La1 and PC1 is relatively better.

The growth test results of strains SC1, DC1, La1 and PC1 under the conditions of 10 ℃, 45 ℃ and pH 9.6 show that the screened strains SC1, DC1, La1 and PC1 can be well adapted to the extreme growth environment of lactic acid bacteria, and grow vigorously about 24 hours of culture, wherein the strains La1 and PC1 have the best adaptability to the growth environment.

The results of other physiological and biochemical tests of strains SC1, DC1, La1 and PC1 are shown in Table 2.

TABLE 2 results of other physiological and biochemical tests

According to the growth test and other physiological and biochemical test results of strains SC1, DC1, La1 and PC1, the strains SC1, DC1, La1 and PC1 can be preliminarily determined to be lactic acid bacteria, strains La1 and PC1 are the same lactobacillus, and strains DC1 and SC1 are different lactobacillus.

Example 3 Cholesterol Standard Curve

Drawing a cholesterol standard curve: accurately sucking 0mL, 0.5mL, 1.0 mL, 1.5mL and 2.0mL of cholesterol working solution, placing in a 10mL test tube, and adding glacial acetic acid into each tube to make the total volume reach 4 mL. Adding 1mL concentrated sulfuric acid along the tube wall, mixing, and measuring OD_560nm. Concentration of the standard solution as abscissa, OD_560nmFor the ordinate, a standard curve (fig. 4) was plotted, and a regression equation was established.

As can be seen from FIG. 4, the concentration of the cholesterol solution was 0 mg/mL-0.25 mg/mL, and the OD_560nmHas better correlation, the regression equation is that y is 2.4592x-0.0018, R²＝0.9958。

Sample preparation: inoculating strains SC1, LC1, DC1, La1 and PC1 into a semi-selective liquid MRS culture medium, shaking and culturing at 37 ℃ for 1d, sucking 0.5mL of fermentation liquor, adding 3.0mL of absolute ethyl alcohol and 2.0mL of 50% KOH, uniformly mixing by a shaker, and saponifying in a constant-temperature water bath at 65 ℃ for 1 h. Shaking every 30min to completely saponify; after saponification, taking out the test tube and cooling; adding 3mL of 5% sodium chloride and 10mL of n-hexane, tightly covering a glass plug, shaking for 2min, and standing for layering.

And (3) measuring the degradation rate of cholesterol in the strain fermentation liquor: taking 2mL of upper-layer n-hexane, placing the upper-layer n-hexane in a 10mL colorimetric test tube with a plug, evaporating to dryness in a constant-temperature water bath at 65 ℃, adding 4mL of glacial acetic acid, preparing the subsequent steps according to the standard curve, and measuring OD (optical density) of fermentation liquor of different strains_560nmSubstituting the standard curve regression equation of cholesterol to calculate the cholesterol content in the fermentation liquor of different strains, and calculating the degradation rate of the cholesterol in the fermentation liquor of different strains according to the formula (1).

The cholesterol degradation rate (%) — cholesterol degradation amount/initial cholesterol content of the medium in formula (1). The results are shown in Table 3.

TABLE 3 Cholesterol content and Cholesterol degradation Rate in fermentation broths of different strains

As can be seen from Table 3, the cholesterol-lowering effect of the aerobic strain LC1 extracted from the chopped hot pepper is not obvious, and the cholesterol-lowering capability of the four strains, such as SC1, DC1, La1, PC1 and the like is relatively strong.

Example 4 different lactic acid bacteria fermented sour meat and analysis of its cholesterol degradation rate

According to the results of comprehensive growth test, cholesterol degradation rate determination and physiological and biochemical test, the lactic acid bacteria La1 and PC1 are selected as ideal strains for preparing fermented sour meat, and subsequent tests are carried out.

1.1 preparation of the leavening agent

Through the tests, the lactic acid bacteria La1 and PC1 with strong cholesterol-lowering capability are selected, inoculated in an improved MRS culture medium, and shake-cultured at constant temperature of 30 ℃ to logarithmic phase for later use.

1.2 treatment of pork

After purchase of pork, the following treatments were performed:

① scalding fat pork, scraping, filtering to remove water, and cutting into blocks with length of 7cm and width of 15cm, each block weighing 100 g;

② pickling meat pieces with 15g of refined salt and 7g of fructus Zanthoxyli powder for 5 h;

③ adding corn flour 100g, refined salt 15g and pork, and stirring;

④ mixing all the flavoring with pork, and placing into a sealed jar.

1.3 fermentation culture of sour meat

And respectively spraying a leaven and a blank modified MRS culture medium on the treated pork according to the inoculation amounts of 0mL, 1mL, 1.5mL, 2mL, 2.5mL and 3mL, and pickling for 15d to prepare the sour meat.

1.4 determination of Cholesterol degradation Rate in sour meat

Taking 1g of each sample after fermentation, respectively, according to the cholesterol degradation rate determination method, taking the sample inoculated with blank modified MRS culture medium with corresponding volume as blank control, and determining the OD_560nmSubstituting the standard curve regression equation of cholesterol to calculate the cholesterol content in each sour meat product and further calculate the cholesterol degradation rate.

The fermented sour meat prepared by inoculating the lactobacillus plantarum and fermenting for 15d is shown in fig. 5, and as can be seen from fig. 5, after the fresh pork is subjected to the lactobacillus plantarum anaerobic fermentation for 15d, the lean meat changes from bright red to dark red, the fat meat is relatively dark in color, and the fat meat is hard in texture and has weak sour taste.

The results of analysis of the cholesterol content and the degradation rate of cholesterol in sour meat prepared with different lactobacillus plants at different inoculum sizes are shown in table 4.

TABLE 4 Cholesterol content in different fermented sour meats and their cholesterol degradation rate

As can be seen from Table 4, the cholesterol lowering effects of strains La1 and PC1 are generally comparable. Inoculating lactobacillus plantarum La1 into pork according to the proportion of 2.0mL/g, and fermenting for 15 days until the degradation rate of cholesterol in the sample is maximum and reaches 77.2%; secondly, plant lactic acid bacteria PC1 is inoculated into pork according to the proportion of 2.5mL/g, and after fermentation for 15 days, the degradation rate of cholesterol in the sample is 69.7%. The La1 strain with the inoculation amount of 2.0mL had the best effect of reducing cholesterol, and the effect was 77.2%.

Example 5 molecular characterization

The molecular characteristics of the strain La1, extracting the DNA of the strain to carry out PCR amplification to obtain the 16S rRNA gene of the strain, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The homology comparison of the GenBank nucleic acid sequence library and the obtained sequences shows that the strain La1 has the closest relationship with the genus Lactobacillus, wherein the homology with the Lactobacillus plantarum is the highest and reaches 99 percent. The strain was identified as Lactobacillus plantarum according to sequence homology and morphological analysis of 16S rDNA, and thus the resulting strain La1 of the present invention was named Lactobacillus plantarum La 1.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

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ggtagcaaac aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt 840

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Claims

1. Lactobacillus plantarum La1 for degrading cholesterol, which has been deposited in the China center for type culture Collection in 3 and 4 months in 2019 with the collection number of CCTCC NO: m2019121.

2. The lactobacillus plantarum La1 for degrading cholesterol according to claim 1, wherein the nucleotide sequence of 16S rRNA of lactobacillus plantarum La1 is shown in SEQ ID No. 1.

3. The use of the strain La1 according to claim 1 or 2 for degrading cholesterol.

4. The application of the strain La1 of claim 1 or 2 in preparing medicines or health products for degrading cholesterol.