CN113755357A - Lactobacillus preparation and application thereof - Google Patents

Lactobacillus preparation and application thereof Download PDF

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Publication number
CN113755357A
CN113755357A CN202010500756.8A CN202010500756A CN113755357A CN 113755357 A CN113755357 A CN 113755357A CN 202010500756 A CN202010500756 A CN 202010500756A CN 113755357 A CN113755357 A CN 113755357A
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lactobacillus
preparation
helveticus
powder
lactobacillus helveticus
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刘斌
吴超
马国文
张海斌
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Inner Mongolia Yili Industrial Group Co Ltd
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Inner Mongolia Yili Industrial Group Co Ltd
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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
    • 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
    • A23V2400/137—Delbrueckii
    • 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
    • A23V2400/143—Fermentum
    • 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
    • A23V2400/147—Helveticus
    • 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
    • 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
    • A23V2400/175—Rhamnosus

Abstract

The invention relates to the technical field of probiotic preparations, in particular to a lactobacillus preparation and application thereof. In the lactobacillus preparation provided by the invention, magnesium stearate and pregelatinized starch are used as auxiliary materials, the preparation can be stored at normal temperature, and the thallus can keep higher survival rate and has good capability of field planting in intestinal tracts. Based on the ACE inhibitory activity of lactobacillus, particularly lactobacillus helveticus, the lactobacillus preparation provided by the invention can have the effect of improving hypertension, can be used as a medicine for improving hypertension or a health-care product or special medical application food suitable for hypertension patients, and can also be used as a food additive. Experiments show that the survival rate of the lactobacillus reaches 72.28% after the lactobacillus preparation provided by the invention is stored for 12 months at room temperature.

Description

Lactobacillus preparation and application thereof
Technical Field
The invention relates to the technical field of probiotic preparations, in particular to a lactobacillus preparation and application thereof.
Background
Hypertension is one of the most common cardiovascular diseases, and is the most major causative factor of coronary heart disease, sudden brain death, and heart and kidney failure. Angiotensin Converting Enzyme (ACE) is a target enzyme for screening antihypertensive drugs, and angiotensin I can be converted into angiotensin II with effects of contracting blood vessels and increasing blood pressure through the action of ACE; meanwhile, ACE degrades bradykinin with the function of reducing blood pressure, so that the bradykinin loses the function of reducing blood pressure and causes the increase of blood pressure. The angiotensin inhibitor (ACEI) can inhibit ACE activity, and has blood pressure lowering effect. The antihypertensive drug has certain side effects on treating hypertension, and the probiotics are used as important bacterial strains of the microecological preparation and have the function of potentially improving the health of organisms. Such as: regulating intestinal flora, regulating immunity, lowering blood pressure, reducing serum cholesterol, and resisting oxidation.
The blood pressure lowering effect of probiotics is that the probiotics can be metabolized to produce protease, so that protein is degraded, and ACE activity inhibitory peptide is produced. Proteases produced by the metabolism of probiotics, including endopeptidase and aminopeptidase, restrictively hydrolyze proteins, mainly casein, under the continuous action of these enzyme systems to produce bioactive peptides with different molecular sizes and actions, including small peptides which have been found to inhibit ACE activity. The active peptide as ACEI has been successfully applied to the research field of hypertension resistance, and is a hotspot of functional food development.
A number of probiotics have been found to have a hypotensive effect, among which lactobacillus helveticus (lactobacillus helveticus) is the main. L.hellveticus PR4 separated from Italy cheese can be metabolized to generate a protease, the protease can hydrolyze 6 different milk proteins including cow milk, sheep milk, goat milk, pig milk, buffalo milk and human milk to generate small peptides, 41 small peptides are obtained through purification, the small peptides are detected by using Nakarnura and Vermeirssen methods to detect the ACE inhibition rate, the small peptides with the ACE inhibition rate can inhibit the ACE activity by 70-100%, and the small peptides 19 derived from the human milk casein also have an antibacterial effect.
Generally, the strain serving as a microecological preparation is required to be capable of being planted in intestinal tracts, has a good probiotic function and can keep a high survival rate in a storage process. The probiotics have slower metabolism and higher survival rate under the low temperature condition, so most of the current domestic and overseas microecological preparations are required to be stored under the low temperature condition, the survival rate of general lactic acid bacteria reaches 0 within about 30 days under the normal temperature condition, and the low temperature condition is not beneficial to the transportation, storage and use of the microecological preparations.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a lactobacillus preparation that can be stably stored at room temperature, a preparation method thereof, and use thereof in improving hypertension.
The lactobacillus preparation provided by the invention comprises the following components in percentage by mass:
0.25 to 10 percent of lactobacillus powder;
0.01 to 1.5 percent of magnesium stearate;
the balance being pregelatinized starch;
the lactobacillus is selected from lactobacillus helveticus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus fermentum or lactobacillus paracasei.
In some embodiments, the lactobacillus preparation consists of the following components in mass fraction:
0.5 percent of lactobacillus powder;
0.5% of magnesium stearate;
99% of pregelatinized starch.
In some embodiments, the lactobacillus preparation consists of the following components in mass fraction:
0.25 percent of lactobacillus powder;
0.1% of magnesium stearate;
pregelatinized starch 99.65%.
In some embodiments, the lactobacillus preparation consists of the following components in mass fraction:
5% of lactobacillus powder;
1% of magnesium stearate;
pregelatinized starch 94%.
In some embodiments, the lactobacillus preparation consists of the following components in mass fraction:
10% of lactobacillus powder;
1.5% of magnesium stearate;
88.5% of pregelatinized starch.
In the invention, the Lactobacillus paracasei is selected from Lactobacillus paracasei431, Lactobacillus paracasei 150DCU or Lactobacillus paracasei 10450;
in the present invention, the lactobacillus helveticus is selected from lactobacillus helveticus flav54, lactobacillus helveticus l1, lactobacillus helveticus l10, lactobacillus helveticus fm006, lactobacillus helveticus43524, lactobacillus helveticus lb, lactobacillus helveticus llb, lactobacillus helveticus43517, lactobacillus helveticus43490, lactobacillus helveticus lhh 12, or lactobacillus helveticus rg.
The Lactobacillus delbrueckii subspecies bulgaricus is Lactobacillus delbrueckii subsp. bulbgaricusl 7,
The lactobacillus plantarum is Lactobacillus plantarum LP 299V;
the Lactobacillus rhamnosus is Lactobacillus rhamnosus HN 001;
the lactobacillus fermentum CECT 5716.
The experiment of the invention shows that in 8 strains of lactobacillus, the lactobacillus helveticus exhibits stronger acid-resistant and bile salt-resistant capability, while in 11 different strains of lactobacillus helveticus, the lactobacillus helveticus FLAV54 exhibits stronger acid-resistant and bile salt-resistant capability, so that the strain is considered to have stronger colonization capability in human intestinal tracts. And the inhibiting effect of Lactobacillus helveticus FLAV54 on ACE is also obviously better than that of other strains.
The lactobacillus powder also comprises a freeze-drying protective agent; the freeze-drying protective agent comprises: skim milk, trehalose, and ascorbic acid;
wherein the mass ratio of the skim milk to the trehalose to the ascorbic acid is 6:1: 0.5.
The lyoprotectant consists of three aqueous solutions: 30 wt% of skim milk, 20 wt% of trehalose and 50 wt% of ascorbic acid, wherein the volume ratio of the 30 wt% skim milk solution to the 20 wt% trehalose solution to the 50 wt% ascorbic acid solution is 20:5: 1.
The lactobacillus preparation provided by the invention is in the form of tablets, powder or capsules.
A method of preparing a lactobacillus preparation comprising: fermenting and collecting lactobacillus bacterial mud, freeze-drying, mixing with pregelatinized starch, and adding magnesium stearate to obtain lactobacillus preparation.
In the present invention, the fermentation comprises: strain activation, primary fermentation and secondary fermentation;
wherein the culture medium for the primary fermentation and the secondary fermentation comprises water and 1.5 wt% of glucose, 0.5 wt% of tyrosone, 0.7 wt% of beef powder, 0.5 wt% of anhydrous sodium acetate, 0.5 wt% of potassium acetate, 0.05 wt% of L-cysteine, 0.2 wt% of calcium chloride, 0.05 wt% of magnesium sulfate, 0.012 wt% of manganese sulfate and 800.06 wt% of tween;
in the invention, the condition of the primary fermentation is anaerobic culture at 38 ℃ for 11-12 hours;
the condition of the secondary fermentation is anaerobic culture at 38 ℃ for 8-9 hours. The inoculation amount of the secondary fermentation culture is 5%.
The strain activation adopts a solid culture medium streak culture method. The method specifically comprises the following steps: inoculating the strain to a solid culture medium for streaking, and performing anaerobic culture at 38 ℃ for 48 hours; the single colonies were streaked again and anaerobically cultured at 38 ℃ for 48 hours.
In the invention, the fermented fermentation liquor is centrifuged to prepare the lactobacillus bacterial sludge. The centrifugation condition is 10000rpm, 4 ℃ centrifugation for 20min, and supernatant is discarded.
The freeze-drying comprises the following steps: mixing the lactobacillus bacterial sludge with a freeze-drying protective agent solution, and freeze-drying;
the lyoprotectant solution includes water and skim milk, trehalose, and ascorbic acid.
Each 26mL of lyoprotectant solution contained 6g skim milk, 1g trehalose, and 0.5g ascorbic acid.
The volume ratio of the freeze-drying protective agent solution to the fermentation liquor is 20-30: 400. in some embodiments, the volume ratio of the lyoprotectant solution to the fermentation broth is 26: 400.
The freeze-drying procedure is that the material is frozen to-30 ℃, and the temperature of a clapboard of a freeze dryer is raised: -15 ℃ (1h) → -8 ℃ (1h) → -3 ℃ (1h) → 3 ℃ (1h) → 10 ℃ (1h) → 15 ℃ (1h) → 25 ℃ (2 h).
The lactobacillus preparation or the lactobacillus preparation prepared by the method is applied to the preparation of products for improving hypertension.
In the invention, the product is as follows: medicine, health product or food for special medical use.
The lactobacillus preparation disclosed by the invention or the lactobacillus preparation prepared by the method is applied as an additive of food;
the food is a dairy product, preferably yogurt, milk powder, cheese, lactobacillus beverage, ice cream and plant-based fermented product. The plant-based fermented product is fermented soymilk or fermented fruit juice.
In the lactobacillus preparation provided by the invention, magnesium stearate and pregelatinized starch are used as auxiliary materials, the preparation can be stored at normal temperature, and the thallus can keep higher survival rate and has good capability of field planting in intestinal tracts. Based on the ACE inhibitory activity of lactobacillus, particularly lactobacillus helveticus, the lactobacillus preparation provided by the invention can have the effect of improving hypertension, can be used as a medicine for improving hypertension or a health-care product or special medical application food suitable for hypertension patients, and can also be used as a food additive. Experiments show that the survival rate of the lactobacillus reaches 72.32 percent after the lactobacillus preparation provided by the invention is stored for 12 months at room temperature.
Drawings
FIG. 1 shows the growth of different lactobacilli in 0.3% bile acid salt MRS medium at pH 3.0;
FIG. 2 shows the growth of different lactobacilli in 0.3% bile acid salt MRS medium at pH 2.0;
FIG. 3 shows the growth of different Lactobacillus helveticus strains in 0.3% bile acid salt MRS medium at pH 3.0;
FIG. 4 shows the growth of different Lactobacillus helveticus strains in 0.3% bile acid salt MRS medium at pH 2.0.
Detailed Description
The invention provides a lactobacillus preparation and application thereof, and a person skilled in the art can realize the preparation by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Among them, Lactobacillus helveticus FLAV54 was purchased from Danisc (China) Co., Ltd.
Lactobacillus paracasei431 is available from Kehansen (China) Inc., Lactobacillus paracasei 150DCU and 10450, and Lactobacillus helveticus FLAV54, Lactobacillus rhamnosus HN001 are all commercial strains available from Danisco (China) Inc. The other Lactobacillus helveticus and Lactobacillus delbrueckii subspecies Bulgaria L7 were purchased from China center for Industrial culture Collection of microorganisms. LP299V was purchased from Shanghai Riegee Biotech, Inc., and Lactobacillus fermentum CECT5716 was purchased from Guangzhou Hui Jian Biotech, Inc.
The invention is further illustrated by the following examples:
EXAMPLE 1 detection of acid and bile salt resistance of different bacterial species
1. Different lactobacilli (commercial strains) were selected for the detection of acid and bile salt resistance:
lactobacillus delbrueckii subsp.bulgaricus L7 (GL 01),
Lactobacillus paracasei431 (marked as GL02),
Lactobacillus plantarum LP299V (recorded as GL03),
Lactobacillus helveticus Lactobacillus plantarum FLAV54 (denoted as GL04),
Lactobacillus rhamnosus HN001 (recorded as GL05),
Lactobacillus fermenti CECT5716 (marked as GL06),
Lactobacillus paracasei 150DCU (denoted as GL07),
Lactobacillus paracasei 10450 (designated as GL08).
The method comprises the following steps: the rejuvenated strain was inoculated into MRS liquid medium of pH2.0 and pH3.0 added with 0.3% bile acid salt at 4% inoculum size, anaerobically cultured at 37 deg.C for 10 hr, and OD value was measured at 600nm wavelength with sterile MRS medium as control, the results are shown in FIGS. 1 and 2.
As can be seen from FIGS. 1 and 2, the OD values of GL02 and GL04 are highest and close to each other at pH3.0, and show the strongest acid and bile salt resistance, the OD values of GL07 and GL08 are second order, and also have stronger acid and bile salt resistance, and the growth density of the rest strains under the condition is lower, and the acid and bile salt resistance is poorer. Under the condition of pH2.0, the OD value of each strain is reduced to different degrees, the OD values of the strains GL02 and GL04 are slightly reduced and are still kept above 0.7, the OD value of the strain GL07 is above 0.5, the OD values of the other strains are relatively low, and the lactobacillus paracasei GL02 and the lactobacillus helveticus GL04 have strong acid resistance and bile salt resistance and good colonization in intestinal tracts.
2. Acid and bile salt resistance detection of different lactobacillus helveticus strains
Different lactobacillus helveticus (commercial strain) were selected for acid and bile salt resistance testing:
lactobacillus helveticus Lactobacillus plantarum FLAV54 (denoted as GL04),
Lactobacillus helveticus L1 (noted GL10),
Lactobacillus helveticus L10 (noted GL11),
Lactobacillus helveticus FM006 (noted GL12),
Lactobacillus helveticus43524 (noted as GL13),
Lactobacillus helveticus Lb (noted as GL14),
Lactobacillus helveticus LLB (noted GL15) and its preparation method,
Lactobacillus helveticus43517 (noted as GL16),
Lactobacillus helveticus43490 (noted GL17),
Lactobacillus helveticus Lh12 (noted as GL18),
Lactobacillus helveticus RG (noted GL19),
The method comprises inoculating the rejuvenated strain into MRS liquid culture medium (pH2.0 and pH3.0) added with 0.3% bile acid salt at 4% inoculation amount, performing anaerobic culture at 37 deg.C for 10 hr, and measuring OD value at 600nm with sterile MRS culture medium as control, with the results shown in FIGS. 3 and 4.
As can be seen from FIGS. 3 and 4, under the conditions of pH3.0, pH2.0 and 0.3% of bile salt, the OD values of the Lactobacillus helveticus GL04, GL14 and GL17 are close to and about 0.7, and the Lactobacillus helveticus GL12 has relatively low OD value, but can also reach more than 0.5, and has certain tolerance, and the OD values of the other strains are low. Generally, lactobacillus helveticus GL04, GL14 and GL17 have strong acid and bile salt resistance and have high survival rate in human intestinal tract.
3. In vitro assay for ACE inhibitory Activity of different strains
The ACE inhibitory activity of the lactic acid bacteria was measured by a visible spectrophotometer method. VERME IRSSEN et al improve the spectrophotometry established by HOLMQU IST et al with FAPGG as angiotensin I mimetic. FAPPG generates FA-phe and dipeptide G-G under the action of ACE, the degradation of FAPPG leads the light absorption value of a reaction system under 340nm to be linearly reduced, and the change of the light absorption value in unit time represents the enzyme activity or the reaction rate. The difference of enzyme activity between the added inhibitor and the non-added inhibitor is measured to indicate the magnitude of the inhibitory activity.
(1) Preparation of lactic acid bacteria ACE inhibitory peptide sample
Fermenting the purified strain in MRS culture medium, inoculating 5% of the strain into 10% skim milk (sterilizing at 115 ℃ for 20 min), fermenting at 37 ℃, centrifuging at 6000g for 10min after the skim milk is solidified to generate whey, taking supernatant, adjusting the pH to 8.0,10000g, centrifuging for 10min, taking supernatant, and placing in a refrigerator at 4 ℃ for later use.
(2) The reagents used before the test should be preheated at 37 ℃, the sample adding sequence is carried out according to the table 2, and because the temperature has great influence on the enzyme activity, the samples should be quickly added and uniformly mixed, and the absorbance is immediately measured under 340nm of an enzyme labeling instrument. Then placing the mixture into an environment at 37 ℃ to continue incubation for 30min, and measuring the absorbance of the system again.
TABLE 1 sample addition sequence, sample addition List, for ACE inhibitory activity assay
Reagent Control well (μ L) Sample well (μ L)
FAPGG(1.0mmoL/L) 50 50
Matrix buffer 40 0
Test sample 0 40
ACE(0.1U/mL) 10 10
Matrix buffer solution: prepared with double distilled water, 0.1mol/L borate buffer solution with pH value of 8.3 mol/L NaCl. FAPGG (1.0 mmoL/L): 3.994mg FAGG is added with matrix buffer solution to reach a constant volume of 10mL, dissolved and mixed, and placed at 4 ℃ in the dark.
Absorbance values at 0min for the control and sample wells were set as a1, b 1; setting as a2 and b2 after 30 min; the absorbance decrease value a of the control wells was a1-a 2; the absorbance decrease of the sample well, B-1-B2, ACE inhibition ratio (A-B)/A X100%
TABLE 2 determination of ACE inhibition of different lactic acid bacteria
Strain numbering GL08 GL05 GL04 GL02 GL03
ACE inhibitory ratio (%) 25.42 28.73 76.58 36.47 45.82
Performing in-vitro ACE enzyme activity inhibition test on the 8 strains of lactic acid bacteria, wherein 5 strains of lactic acid bacteria show ACE inhibition, the inhibition of strains GL04 and GL03 is obvious, the inhibition rates are both above 45%, and the inhibition rates of the other strains are lower; the inhibition rate of the strain GL04 reaches 76.58% at most, is obviously higher than that of other strains, and has obvious ACE inhibition effect.
TABLE 3 results of ACE inhibition assay of different Lactobacillus helveticus strains
Strain numbering GL04 GL10 GL12 GL13 GL14 GL17 GL18 GL19
ACE inhibitory ratio (%) 76.58 38.75 51.62 32.79 62.85 56.34 41.61 35.60
Performing in-vitro ACE enzyme activity inhibition test on 11 strains of Lactobacillus helveticus, wherein 8 strains show ACE inhibition, wherein the inhibition of strains GL04, GL12, GL14 and GL17 is obvious, the inhibition rate is over 55 percent, and the inhibition rate of the other strains is low; the highest inhibition rate of the strain GL04 reaches 76.58%, is obviously higher than that of other Lactobacillus helveticus strains, and has an obvious ACE inhibition effect.
Combining the above test results, lactobacillus helveticus GL04 has the strongest ACE inhibition rate compared with 7 different lactobacillus helveticus strains and 10 different lactobacillus helveticus strains, and therefore, the strain was selected to prepare the preparation.
Example 2
1. Preparation of lactobacillus helveticus bacterial powder:
lactobacillus helveticus FLAV54, purchased from Danisco (China) Co., Ltd, was prepared by the following steps:
diluting a freeze-dried strain with normal saline, selecting a loop with an inoculating loop, streaking on an MRS solid culture medium, and then culturing in an anaerobic tank at 38 ℃ for 48 hours;
picking single colony, continuously performing streak purification on an MRS plate, and culturing for 48 hours in an anaerobic box at 38 ℃;
selecting 1 plate with good growth and colony morphology, scraping all lawn into a first-grade MRS liquid culture medium by using an inoculating loop, and performing anaerobic culture at 38 ℃ for 11-12 hours;
taking out the primary fermentation liquid, inoculating the primary fermentation liquid into a secondary MRS liquid culture medium at the temperature of 38 ℃ for anaerobic culture for 8-9 hours in an inoculation amount of 5%;
fifthly, centrifuging the secondary fermentation liquor at 10000rpm and 4 ℃ for 20min, removing supernatant, collecting bacterial sludge, adding 20mL of 30% skim milk, 5mL of 20% trehalose and 1mL of 50% ascorbic acid into every 400mL of bacterial sludge obtained from the fermentation liquor, uniformly stirring, and then pouring into a vacuum freeze dryer for freeze drying to obtain white freeze-dried bacterial powder GL 04.
The optimal component ratio of the fermentation medium (namely, the first-level MRS liquid medium and the second-level MRS liquid medium) is as follows: 1.5 percent of glucose, 0.5 percent of tyrosone, 0.7 percent of beef powder, 0.5 percent of anhydrous sodium acetate, 0.5 percent of potassium acetate, 0.05 percent of L-cysteine, 0.2 percent of calcium chloride, 0.05 percent of magnesium sulfate, 0.012 percent of manganese sulfate and 800.06 percent of Tween.
2. Preparation of lactobacillus preparation:
the lactobacillus helveticus GL04 bacterial powder, the pregelatinized starch and the magnesium stearate are prepared according to the following mass fractions: 0.5% of lactobacillus helveticus bacterial powder, 0.5% of magnesium stearate and the balance of pregelatinized starch.
The preparation method of the tablet comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and tabletting with a tabletting machine to obtain tablet with average weight of 0.5g and viable bacteria number of about 3.5 hundred million.
Example 3
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the pregelatinized starch and the magnesium stearate are prepared according to the following mass fractions: 0.5% of lactobacillus helveticus bacterial powder, 0.5% of magnesium stearate and the balance of pregelatinized starch. The preparation method of the capsule comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and filling into gelatin hollow capsules with a volume of 0.5mL and a viable count of about 3.5 hundred million per capsule.
Example 4
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the pregelatinized starch and the magnesium stearate are prepared according to the following mass fractions: 0.5% of lactobacillus helveticus bacterial powder, 0.5% of magnesium stearate and the balance of pregelatinized starch. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and about 7 hundred million viable bacteria.
Example 5
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the pregelatinized starch and the magnesium stearate are prepared according to the following mass fractions: 0.25% of lactobacillus helveticus bacterial powder, 0.1% of magnesium stearate and the balance of pregelatinized starch. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and about 3.5 hundred million viable bacteria.
Example 6
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the pregelatinized starch and the magnesium stearate are prepared according to the following mass fractions: 5% of lactobacillus helveticus bacterium powder, 1% of magnesium stearate and the balance of pregelatinized starch. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and 70 hundred million viable bacteria.
Example 7
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the pregelatinized starch and the magnesium stearate are prepared according to the following mass fractions: 10% of lactobacillus helveticus bacterium powder, 1.5% of magnesium stearate and the balance of pregelatinized starch. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and 140 hundred million viable bacteria.
Comparative example 1
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, dextrin and magnesium stearate are prepared according to the following mass fractions: 0.5% of lactobacillus helveticus bacterial powder, 0.5% of magnesium stearate and the balance of dextrin. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and about 7 hundred million viable bacteria.
Comparative example 2
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the lactose and the magnesium stearate are prepared according to the following mass fractions: 0.5 percent of lactobacillus helveticus bacterial powder, 0.5 percent of magnesium stearate and the balance of lactose. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and about 7 hundred million viable bacteria.
Comparative example 3
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the maltodextrin and the micropowder silica gel are prepared according to the following mass fractions: 0.5 percent of lactobacillus helveticus bacterial powder, 0.5 percent of micro-powder silica gel and the balance of maltodextrin. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and about 7 hundred million viable bacteria.
Comparative example 4
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, lactose, microcrystalline cellulose and magnesium stearate are prepared according to the following mass fractions: 0.5% of lactobacillus helveticus bacterial powder, 10% of microcrystalline cellulose, 0.5% of magnesium stearate and the balance of lactose. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and about 7 hundred million viable bacteria.
Comparative example 5
The preparation method of the lactobacillus helveticus bacterium powder is the same as that of the embodiment 2, and the lactobacillus helveticus GL04 bacterium powder, the maltodextrin, the skim milk powder and the micro powder silica gel are prepared according to the following mass fractions: 0.5% of lactobacillus helveticus bacterial powder, 1.5% of skimmed milk powder, 0.5% of micro-powder silica gel and the balance of maltodextrin. The preparation method of the powder comprises the following steps: mixing the bacterial powder with the pregelatinized starch, adding magnesium stearate, mixing well, and packaging into aluminum plastic bags with 1g each bag and about 7 hundred million viable bacteria.
Effect detection
1. Water activity detection
Water is a substance necessary for the life activity of microorganisms, and if water is not involved, the microorganisms are in a complete dormant state and do not perform the life activity. Water activity, also known as water activity, refers to the degree to which free water in a substance can utilize water. The water activity represents the state of the presence of free water in the substance, and reflects the degree of binding ability between the substance and the free water, i.e., represents the degree of availability of the free water contained in the substance as the growth of microorganisms, and the smaller the value, the higher the degree of binding, the stronger the binding, and the more difficult the use by the microorganisms; the larger the value, the lower the degree of binding, and the easier the microorganism utilization. Water activity is an important factor affecting microbial stability. The lower the water activity, the better the survival of the thallus. The detection method refers to the measurement of the water activity of the food in GB5009.238-2016 national standard for food safety.
TABLE 4 Water Activity measurements for different materials
Material(s) Mushroom powder Pregelatinized starch Magnesium stearate Dextrin Lactose Maltodextrin Microcrystalline cellulose Silica gel micropowder Defatted milk powder
Water activity (aw) 0.038 0.092 0.251 0.174 0.340 0.208 0.245 0.263 0.237
TABLE 5 Water Activity measurements for various powders
Formulation number Example 2 Comparative example 1 Comparative example 2 Comparative example 3 Comparative example 4 Comparative example 5
Water activity (aw) 0.085 0.158 0.389 0.196 0.318 0.236
2. Formulation stability testing
TABLE 6 storage stability test results of the formulations at Normal temperature
Figure BDA0002524596710000111
Figure BDA0002524596710000121
The powders of example 2 and comparative examples 1-5 were prepared from materials of different water activities, with the water activity of the single material indicating: the pregelatinized starch has relatively low water activity less than 0.1, the lactose has water activity higher than 0.3 at most, and the bacteria powder is prepared by vacuum freeze drying, and has water activity of 0.034 at least. The water activity of the powder shows that most of the auxiliary materials in example 2 are pregelatinized starch, so the water activity is 0.085 at the lowest; the adjuvants of comparative examples 2 and 4 consist essentially of lactose and therefore have a higher water activity of more than 0.3; the water activities of the remaining comparative example powders were between 0.1 and 0.3.
Table 6 stability test results of different powders at ambient temperature for 12 months show that: under the conditions of the same bacterial powder and the same addition amount, the water activity of the auxiliary materials adopted in the embodiments is lower, so that the survival rate of the bacterial bodies of the embodiments after 12 months of storage can still reach more than 70 percent compared with the comparative examples; the water activity of the rest of the powder of the comparative example is relatively high, the survival rate of the thalli after 12 months is lower than 45 percent, particularly the survival rate of the thalli of the comparative example 2 and the comparative example 4 is only about 15 percent because the water activity of the preparation per se is high.
3. Detection result of viable count of bacterial powder
The commercially available freeze-dried powder of Lactobacillus helveticus FLAV54 and the powder of GL04 prepared in example 2 of the present invention were stored under the same conditions and the viable cell count was checked, the results of which are shown in Table 7
TABLE 7 comparison of viable count test results of two kinds of bacteria powder
FLAV54 bacterial powder/batch Viable count (cfu/g) GL04 bacterial powder/batch Viable count (cfu/g)
20190325 5.03×1010 20190425 1.52×1011
20190620 5.56×1010 20190714 1.45×1011
20190816 6.72×1010 20190923 1.58×1011
The results show that: the viable count of the bacterial powder GL04 is obviously improved compared with that of the freeze-dried bacterial powder of the Lactobacillus helveticus FLAV54 which is not subjected to a specific treatment process.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (10)

1. A lactobacillus preparation is characterized by comprising the following components in percentage by mass:
0.25 to 10 percent of lactobacillus powder;
0.01 to 1.5 percent of magnesium stearate;
the balance being pregelatinized starch;
the lactobacillus is selected from lactobacillus helveticus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus fermentum or lactobacillus paracasei.
2. A Lactobacillus preparation according to claim 1, characterized in that,
the Lactobacillus paracasei is selected from Lactobacillus paracasei431, Lactobacillus paracasei 150DCU or Lactobacillus paracasei 10450;
the lactobacillus helveticus is selected from lactobacillus helveticus FLAV54, lactobacillus helveticus L1, lactobacillus helveticus L10, lactobacillus helveticus FM006, lactobacillus helveticus43524, lactobacillus helveticus Lb, lactobacillus helveticus LLB, lactobacillus helveticus43517, lactobacillus helveticus43490, lactobacillus helveticus Lh12, or lactobacillus helveticus RG;
the Lactobacillus delbrueckii subspecies bulgaricus is Lactobacillus delbrueckii subsp. bulbgaricusl 7,
The lactobacillus plantarum is Lactobacillus plantarum LP 299V;
the Lactobacillus rhamnosus is Lactobacillus rhamnosus HN 001;
the lactobacillus fermentum CECT 5716.
3. A lactobacillus preparation according to claim 1 or 2, wherein the lactobacillus powder further comprises a lyoprotectant; the freeze-drying protective agent comprises: skim milk, trehalose, and ascorbic acid;
wherein the mass ratio of the skim milk to the trehalose to the ascorbic acid is 6:1: 0.5.
4. A method for preparing a Lactobacillus preparation according to any of claims 1 to 3, comprising: fermenting and collecting lactobacillus bacterial mud, freeze-drying, mixing with pregelatinized starch, and adding magnesium stearate to obtain lactobacillus preparation.
5. The method of claim 4, wherein the fermenting comprises: strain activation, primary fermentation and secondary fermentation;
wherein the culture medium for the primary fermentation and the secondary fermentation comprises water and 1.5 wt% of glucose, 0.5 wt% of tyrosone, 0.7 wt% of beef powder, 0.5 wt% of anhydrous sodium acetate, 0.5 wt% of potassium acetate, 0.05 wt% of L-cysteine, 0.2 wt% of calcium chloride, 0.05 wt% of magnesium sulfate, 0.012 wt% of manganese sulfate and 800.06 wt% of tween.
6. The production method according to claim 4,
the condition of the primary fermentation is anaerobic culture at 38 ℃ for 11-12 hours;
the condition of the secondary fermentation is anaerobic culture at 38 ℃ for 8-9 hours.
7. The method according to any one of claims 4 to 6, wherein the fermented fermentation broth is centrifuged to obtain a sludge of Lactobacillus; the freeze-drying comprises the following steps: mixing the lactobacillus bacterial sludge with a freeze-drying protective agent solution, and freeze-drying;
the volume ratio of the freeze-drying protective agent solution to the fermentation liquor is 26:400, respectively;
the lyoprotectant solution includes water and skim milk, trehalose, and ascorbic acid.
8. The method according to claim 7, wherein 6g of skim milk, 1g of trehalose, and 0.5g of ascorbic acid are contained in 26mL of the lyoprotectant solution.
9. Use of a lactobacillus preparation according to any of claims 1 to 3, or prepared by a method according to any of claims 4 to 8, for the preparation of a product for ameliorating hypertension; the product is as follows: medicine, health product or food for special medical use.
10. Use of a lactobacillus preparation according to any of claims 1 to 3, or prepared by a method according to any of claims 4 to 8, as an additive in food products; the food is a dairy product, preferably a yoghurt, milk powder, cheese, lactic acid bacteria drink, ice cream or plant-based fermented product.
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