CN117106648A - Lactobacillus rhamnosus, and fermented product and application thereof - Google Patents
Lactobacillus rhamnosus, and fermented product and application thereof Download PDFInfo
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- CN117106648A CN117106648A CN202311064880.4A CN202311064880A CN117106648A CN 117106648 A CN117106648 A CN 117106648A CN 202311064880 A CN202311064880 A CN 202311064880A CN 117106648 A CN117106648 A CN 117106648A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0043—Nose
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- 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
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses lactobacillus rhamnosus, a ferment and application thereof. Lactobacillus rhamnosus (Lactobacillus rhamnosus) ST is preserved in China general microbiological culture Collection center (CGMCC) in the 2 nd month 27 of 2023, and the preservation number is CGMCC NO:26684. the lactobacillus rhamnosus ST can be metabolized to produce protein or polypeptide with antibacterial effect, has high antibacterial activity to a plurality of gram-positive bacteria and gram-negative bacteria, and has broad-spectrum antibacterial property. Further, the antibacterial effect of the ferment obtained by fermenting and culturing lactobacillus rhamnosus ST in a culture medium containing hyaluronic acid is obviously improved. The lactobacillus rhamnosus ST and the ferment thereof can be applied to the production of antibacterial products or antibacterial functional products, such as antibacterial agents, metacomposite, antibacterial nasal spray or oral antibacterial spray, and the like, and have important application value.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus, a fermentation product and application thereof.
Background
Lactic acid bacteria are probiotics widely applied to the fields of food, chemical industry and the like, and metabolic products such as antibacterial peptides, acidic substances and the like have an inhibitory effect on the growth of partial pathogenic bacteria and spoilage bacteria in food, and pathogenic bacteria can be inhibited in the digestive tract in a space-occupying competition mode. Therefore, the lactobacillus fermentation product has better application value in the aspect of producing antibacterial products such as green preservatives or antibacterial functional products.
The prior art shows that the metabolic products and the antibacterial activity difference of the metabolic products generated by different types of lactic acid bacteria are large, so that the different types of lactic acid bacteria can be developed to be applied to antibacterial products or antibacterial functional products; for example, chinese patent publication No. CN104818232B discloses a lactobacillus plantarum AB-3 with antibacterial activity, which shows effective inhibition on phytophthora melo, fusarium oxysporum, penicillium roqueforti and apple anthracnose; the Chinese patent publication No. CN104611251A discloses a lactobacillus with broad-spectrum antibacterial activity, which has good antibacterial effect on various human and aquaculture biological pathogenic bacteria. Meanwhile, the same kind of lactobacillus can also produce fermentation products with obvious bacteriostasis or other functions because of different raw materials used in culture; for example, the Chinese patent publication No. CN104336416A discloses a lactobacillus plantarum strain and application thereof in alfalfa silage, wherein the lactobacillus plantarum strain can grow and produce acid in an environment with low soluble sugar, and has good antibacterial activity on pathogenic bacteria. It follows that the type of lactic acid bacteria, the type of fermentation substrate in the culture medium, and the nature of the substrate directly affect the growth and reproduction of lactic acid bacteria and the formation and accumulation of metabolites, and thus can be used for the development of new products with antibacterial activity.
Therefore, the lactobacillus strains with high antibacterial activity of the metabolites are screened, fermentation substrates and fermentation conditions of the lactobacillus strains are researched, and antibacterial products such as green preservatives and functional products such as metazoans are produced by the lactobacillus strains, so that the lactobacillus strains have important significance.
Disclosure of Invention
The invention aims at providing a lactobacillus strain with high antibacterial activity of a metabolite, and researching fermentation substrates and fermentation conditions of the antibacterial metabolite, thereby providing application of the lactobacillus strain in producing antibacterial products such as green preservative and functional products such as metaplasia.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides lactobacillus rhamnosus (Lactobacillus rhamnosus), which is named as ST, wherein the ST of the lactobacillus rhamnosus (Lactobacillus rhamnosus) is preserved in the common microorganism center (China General Microbiological Culture Collection Center, CGMCC) of the China general microbiological culture Collection center (CGMCC) in 2 months and 27 days of 2023, and the strain preservation address is as follows: beijing, chaoyang area, north Chenxi Lu No. 1, 3; the preservation number of the strain is CGMCC No.26684.
The lactobacillus rhamnosus ST is derived from pickle, the strain is identified as lactobacillus rhamnosus (Lacticaseibacillus rhamnosus), the lactobacillus rhamnosus ST is named, the lactobacillus rhamnosus ST grows in a culture medium, and the colony characteristics are as follows: the colony diameter is 1-2 mm, the surface is milky white, smooth and moist, the liquid drop shape, round shape, raised shape and neat edge. The lactobacillus rhamnosus ST and the fermented product thereof have broad-spectrum antibacterial activity, and not only have antibacterial activity on gram-positive bacteria such as staphylococcus aureus, bacillus megatherium, acne bacillus, enterococcus faecium and the like, but also have antibacterial activity on gram-negative bacteria such as escherichia coli, salmonella, pseudomonas putida, serratia and the like.
The invention provides lactobacillus rhamnosus ST fermentation product which is obtained by fermenting lactobacillus rhamnosus ST in a culture medium.
The invention provides a preparation method of lactobacillus rhamnosus ST fermentation product, which comprises the following steps:
s1, inoculating lactobacillus rhamnosus in a culture medium, and fermenting for 12-50 hours at the temperature of 35-38 ℃, wherein the lactobacillus rhamnosus is lactobacillus rhamnosus ST;
s2, centrifuging the fermentation liquor, and taking supernatant for ultrafiltration, wherein the obtained filtrate is lactobacillus rhamnosus ST fermentation product.
Further, before lactobacillus rhamnosus is inoculated in step S1, lactobacillus rhamnosus needs to be activated.
Further, the culture medium in the step S1 is a lactobacillus culture medium.
Preferably, the lactobacillus culture medium contains the following components in mass concentration: 8-10 g/L peptone, 3-4 g/L beef extract powder, 3-5 g/L yeast powder, 18-22 g/L glucose, 1-3g/L dipotassium hydrogen phosphate, 1-3g/L triammonium citrate, 4-6 g/L sodium acetate, 0.1-0.3 g/L magnesium sulfate, 0.04-0.06 g/L manganese sulfate and 1-3g/L tween 80.
Still further, the lactobacillus rhamnosus ST in step S1 is inoculated in the medium in an amount of 0.1% to 10%.
Further, the medium in step S1 contains hyaluronic acid or hyaluronate.
Preferably, the hyaluronate can be one or more of hyaluronate sodium salt, hyaluronate potassium salt and hyaluronate magnesium salt.
Further, the molecular weight of the hyaluronic acid or hyaluronate in the step S1 is 1kDa to 1800kDa.
Preferably, the molecular weight of the hyaluronic acid or hyaluronate in step S1 is 1kDa to 190kDa.
Further, the mass concentration of the hyaluronic acid or hyaluronate in the culture medium in the step S1 is 0.1% -10%.
Preferably, the mass concentration of the hyaluronic acid or hyaluronate in the culture medium in the step S1 is 1% -2%.
Preferably, the ultrafiltration in step S2 employs a 10kDa filter membrane.
Furthermore, the invention claims lactobacillus rhamnosus ST fermentation prepared by the preparation method of lactobacillus rhamnosus ST fermentation.
Furthermore, the invention also provides application of the lactobacillus rhamnosus ST or lactobacillus rhamnosus ST fermentation product in the production of an antibacterial product or an antibacterial functional product.
The invention provides a metacomposite, which contains lactobacillus rhamnosus ST and/or lactobacillus rhamnosus ST fermented products.
The invention provides an antibacterial agent, which contains lactobacillus rhamnosus ST and/or lactobacillus rhamnosus ST fermentation products.
The invention provides a bacteriostatic nose spray, which comprises lactobacillus rhamnosus ST fermentation product and physiological saline, wherein the mass ratio of the lactobacillus rhamnosus ST fermentation product to the physiological saline is 1:40-60.
The invention provides an oral antibacterial spray which comprises the following components in parts by weight: 70-90 parts of sorbitol, 1-20 parts of lactobacillus rhamnosus ST fermentation product, 7-9 parts of sea salt, 2-4 parts of menthol, 10-20 parts of licorice extract, 10-20 parts of honeysuckle extract and 400-600 parts of water.
The beneficial effects of the invention are as follows:
(1) The lactobacillus rhamnosus ST provided by the invention can metabolize proteins or polypeptides with antibacterial effect, has an inhibitory effect on a plurality of gram-positive bacteria and gram-negative bacteria, has high antibacterial activity and has broad-spectrum antibacterial property.
(2) The antibacterial effect of lactobacillus rhamnosus ST fermentation obtained by fermenting and culturing lactobacillus rhamnosus ST in a culture medium containing hyaluronic acid is obviously improved, and the antibacterial effect is better.
(3) The lactobacillus rhamnosus ST and the ferment thereof can be applied to the production of antibacterial products or antibacterial functional products, such as antibacterial agents, metacomposite, antibacterial nasal spray or oral antibacterial spray, and the like, and have important application value.
Drawings
FIG. 1 shows a colony morphology of Lactobacillus rhamnosus ST isolated from the strain.
FIG. 2A morphological image of Lactobacillus rhamnosus ST under a microscope.
FIG. 3 phylogenetic tree of Lactobacillus rhamnosus ST.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples for the purpose of illustration and not limitation, and various modifications may be made within the scope of the present invention as defined by the appended claims.
Lactic acid bacteria culture Medium (MRS) is a general culture medium for culturing lactic acid bacteria, and the lactic acid bacteria culture mediums used in the following examples and comparative examples include the following components: 10g/L of peptone, 5g/L of beef extract powder, 4g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 2g/L of triammonium citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 1g/L of tween 80.
EXAMPLE 1 isolation and characterization of Lactobacillus rhamnosus ST
1. Experimental method
(1) Strain isolation and colony morphology
Sampling radix Raphani pickle water, placing into 500ml triangular flask containing glass beads and 90ml sterile water, and shaking at 30deg.C shaker for 30min at 110r/min to obtain bacterial suspension; then 1ml of the bacterial suspension is taken and diluted by 10 times of sterile physiological saline in gradient to 10 -3 -10 -6 The strain is subjected to gradient dilution and coated on a plate of a separation culture medium, single colonies of the strain are separated, and the colony morphology and the optimal culture temperature are observed, wherein the culture medium adopts a lactobacillus culture medium.
(2) Identification of strains
Culturing the strain into bacterial liquid, taking bacterial liquid for dyeing and microscopy; when the strain is cultured until the strain is in an exponential growth phase, taking fresh bacterial liquid in the exponential growth phase, centrifugally collecting bacterial cells, extracting genome DNA by adopting a bacterial genome extraction kit, adopting a universal primer to amplify PCR (polymerase chain reaction), carrying out 1.0% agarose gel electrophoresis separation and detection on amplified products to obtain the purification of the amplified products, carrying out 16SRNA gene full-length sequence sequencing on the purified genome, and completing the 16S rRNA gene full-length sequence sequencing by Shanghai biological technical bulletin. The test results were aligned in GeneBank to determine species.
(3) Antibacterial test
The antibacterial activity of the separated strains is measured by adopting an oxford cup agar diffusion method, indicator bacteria are acne bacillus and enterococcus faecium, escherichia coli, bacillus megatherium, salmonella, staphylococcus aureus, pseudomonas putida and serratia, wherein the acne bacillus, the enterococcus faecium and the escherichia coli are respectively inoculated in LB culture medium, the bacillus megatherium, salmonella, staphylococcus aureus, pseudomonas putida and serratia are respectively inoculated in beef extract peptone liquid culture medium, a layer of 5mL solid culture is respectively poured at the bottom of a culture dish, oxford cups are sequentially placed on a flat plate after solidification, after the temperature of the lower layer of the solid culture medium is reduced to 55-60 ℃, the indicator bacteria culture solutions are respectively inoculated in the respective culture mediums according to the inoculation amount of 1 per mill, 10mL of the solid culture medium is respectively poured after solidification, oxford cups are respectively taken out, the separated strains (in the exponential growth phase) are respectively inoculated in holes, lactobacillus LGG (ATCC 53013) is respectively used as a contrast, and physiological saline is respectively added as a blank refrigerator, and the culture dishes are respectively placed at the temperature of 2 ℃ for diffusion for 10h, and then the flat plate is subjected to bacteriostasis measurement, and the flat plate culture is placed for 37 h.
2. Experimental results
(1) Bacterial separation results
Isolated bacterial colonies are shown in FIG. 1, and the isolated bacterial colonies are characterized by: the diameter of the colony is 1-2 mm, and the colony is milky white, smooth and moist in surface, drop-shaped, round, raised and neat in edge; the strain grows faster at the temperature of 35-38 ℃ at the optimal culture temperature of 37 ℃ and is initially judged to be similar to lactobacillus rhamnosus.
(2) Identification result of strain
The isolated strain was in the form of a rod under a microscope as shown in FIG. 2, and the strain was in the form of a Bacillus. The full-length sequence of the 16S rRNA gene is shown as SEQ ID NO. 1, the sequence of the full-length sequence of the 16S rRNA gene is compared in GeneBank to determine species, a phylogenetic tree based on isolated strains is shown as figure 3, the strain (shown as Lacticaseibacillus rhamnosus strain CGMCC 26684) belongs to lactobacillus rhamnosus Lacticaseibacillus rhamnosus, and the closest species is Lacticaseibacillus rhamnosus strain 2717.
The isolated and identified strain is preserved, the new strain is named as ST, and the lactobacillus rhamnosus (Lactobacillus rhamnosus) ST is preserved in the China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC) at the 2 nd month 27 of 2023, and the strain preservation address is: beijing, chaoyang area, north Chenxi Lu No. 1, 3; the preservation number of the strain is CGMCC No.26684.
(3) Antibacterial effect measurement
The lactobacillus rhamnosus ST fermentation product has broad-spectrum antibacterial property, has antibacterial effect on staphylococcus aureus Staphylococcus aureus, bacillus megatherium Bacillus megateriumde Bary, acnes Propionibacterium acnes and enterococcus faecium Enterococcus faecium in gram-positive bacteria, and also has antibacterial effect on Escherichia coli, salmonella Salmonella pullorum, pseudomonas putida Pseudomonas putida and serratia Serratia marcescens in gram-negative bacteria. The specific antibacterial test results are shown in table 1 below.
Table 1 results of bacteriostasis test of lactobacillus rhamnosus ST and lactobacillus rhamnosus in general
Example 2 (minimum temperature and minimum incubation time, no hyaluronic acid)
A preparation method of lactobacillus rhamnosus ST fermentation comprises the following steps:
s1, inoculating activated lactobacillus rhamnosus in a lactobacillus culture medium, wherein the inoculation amount of lactobacillus rhamnosus ST in the culture medium is 0.5%; fermenting for 12 hours at 35 ℃, wherein the lactobacillus rhamnosus is lactobacillus rhamnosus ST;
s2, centrifuging the fermentation liquor at 8000rpm for 20min, taking supernatant for ultrafiltration, wherein a 10kDa filter membrane is adopted for ultrafiltration, and the obtained filtrate is lactobacillus rhamnosus ST fermentation product.
Example 3 (highest temperature and longest incubation time, without hyaluronic acid)
A preparation method of lactobacillus rhamnosus ST fermentation comprises the following steps:
s1, inoculating activated lactobacillus rhamnosus in a lactobacillus culture medium, wherein the inoculation amount of lactobacillus rhamnosus ST in the culture medium is 0.5%; fermenting at 38deg.C for 50 hr, wherein the lactobacillus rhamnosus is lactobacillus rhamnosus ST;
s2, centrifuging the fermentation liquor at 8000rpm for 20min, taking supernatant for ultrafiltration, wherein a 10kDa filter membrane is adopted for ultrafiltration, and the obtained filtrate is lactobacillus rhamnosus ST fermentation product.
Example 4 (moderate temperature and incubation time, without hyaluronic acid)
A preparation method of lactobacillus rhamnosus ST fermentation comprises the following steps:
s1, inoculating activated lactobacillus rhamnosus in a lactobacillus culture medium, wherein the inoculation amount of lactobacillus rhamnosus ST in the culture medium is 0.5%; fermenting for 30 hours at 37 ℃, wherein the lactobacillus rhamnosus is lactobacillus rhamnosus ST;
s2, centrifuging the fermentation liquor at 8000rpm for 20min, taking supernatant for ultrafiltration, wherein a 10kDa filter membrane is adopted for ultrafiltration, and the obtained filtrate is lactobacillus rhamnosus ST fermentation product.
Example 5 (1 kDa,2% sodium hyaluronate was added based on example 4)
A preparation method of lactobacillus rhamnosus ST fermentation product, the steps are the same as those of example 4, except that sodium hyaluronate with molecular weight of 1kDa is added into the culture medium in the step S1, and the mass concentration of the sodium hyaluronate in the culture medium is 2%.
Example 6 (1 kDa,2% Potassium hyaluronate added based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the lactobacillus culture medium containing sodium hyaluronate is replaced by lactobacillus culture medium containing potassium hyaluronate.
Example 7 (1 kDa,0.01% sodium hyaluronate was added based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the concentration of sodium hyaluronate in the lactobacillus culture medium was 0.01%.
Example 8 (1 kDa,0.1% sodium hyaluronate was added based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the concentration of sodium hyaluronate in the lactobacillus culture medium was 0.1%.
Example 9 (1 kDa,10% sodium hyaluronate was added based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the concentration of sodium hyaluronate in the lactobacillus culture medium was 10%.
Example 10 (1 kDa,15% sodium hyaluronate was added based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 4, except that: the concentration of sodium hyaluronate in the lactobacillus culture medium was 15%.
Example 11 (addition of sodium hyaluronate 0.2kDa,2% based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the molecular weight of sodium hyaluronate is 0.2kDa.
Example 12 (addition of sodium hyaluronate 10kDa,2% on the basis of example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the molecular weight of sodium hyaluronate is 10kDa.
Example 13 (100 kDa,2% sodium hyaluronate added based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the molecular weight of sodium hyaluronate is 100kDa.
Example 14 (sodium hyaluronate 190kDa,2% based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 5, except that: the molecular weight of sodium hyaluronate is 190kDa.
Example 15 (addition of sodium hyaluronate 2000kDa,2% based on example 5)
Lactobacillus rhamnosus ST ferment, the preparation method is basically the same as example 4, except that: the molecular weight of sodium hyaluronate is 2000kDa.
Example 16
A lactic acid bacterium fermented product was produced in substantially the same manner as in example 4 except that sodium hyaluronate was added to the fermented product in the same amount as in example 5 after the fermented product was produced.
Example 17 (example of a fungus-corresponding product nasal spray, only protocol range, minimum value is supported)
An antibacterial nasal spray comprises lactobacillus rhamnosus ST fermentation product obtained in example 5 and physiological saline, wherein the mass of lactobacillus rhamnosus ST fermentation product is 10g, the mass of physiological saline is 400g, the osmotic pressure and pH of the nasal spray accord with the applicable range of human body, the nasal spray does not stimulate nasal mucosa, and the pH is preferably 5.
Example 18 (examples of nasal spray for fungus-corresponding products, only protocol range, optimum values are supported)
An antibacterial nasal spray comprises lactobacillus rhamnosus ST fermentation product obtained in example 5 and physiological saline, wherein the mass of lactobacillus rhamnosus ST fermentation product is 10g, the mass of physiological saline is 500g, the osmotic pressure and pH of the nasal spray accord with the applicable range of human body, the nasal spray does not stimulate nasal mucosa, and the pH is preferably 7.
Example 19 (examples of nasal spray for the corresponding products of bacteria, only protocol range, maximum value is supported)
An antibacterial nasal spray comprises lactobacillus rhamnosus ST fermentation product obtained in example 5 and physiological saline, wherein the mass of lactobacillus rhamnosus ST fermentation product is 10g, the mass of physiological saline is 600g, and the osmotic pressure and pH of the nasal spray accord with the application range of human body, and the preferred pH is 8.
Example 20 (example of oral bacteriostatic spray for the corresponding product of bacteria, only support protocol range, minimum)
An oral bacteriostatic spray comprises the following components: 70g of sorbitol, 1g of lactobacillus rhamnosus ST fermentation product obtained in example 5, 7g of sea salt, 2g of menthol, 10g of licorice extract, 10g of honeysuckle extract and 600g of water, and mixing the above components according to a certain proportion to obtain the oral antibacterial spray. Wherein the licorice extract and the honeysuckle extract are solid powder products obtained by a water extraction method which is commercially available.
Example 21 (examples of oral bacteriostatic spray for bacteria corresponding product, only support protocol range, optimum value)
An oral bacteriostatic spray comprises the following components: 80g of sorbitol, 10g of lactobacillus rhamnosus ST fermentation product obtained in example 5, 8g of sea salt, 3g of menthol, 15g of licorice extract, 15g of honeysuckle extract and 500g of water, and the components are mixed according to a proportion to obtain the oral antibacterial spray.
Example 22 (example of oral bacteriostatic spray for the corresponding product of bacteria, only support the protocol range, maximum value)
An oral bacteriostatic spray comprises the following components: 90g of sorbitol, 20g of lactobacillus rhamnosus ST fermentation product obtained in example 5, 9g of sea salt, 4g of menthol, 20g of licorice extract, 20g of honeysuckle extract and 400g of water, and mixing the above components according to a proportion to obtain the oral antibacterial spray.
Comparative example 1 (modification of another Lactobacillus rhamnosus based on example 4)
Lactobacillus rhamnosus ferment, the preparation method is basically the same as example 4, except that: lactobacillus rhamnosus ST was exchanged for an equivalent amount of lactobacillus rhamnosus LGG (ATCC 53013).
Comparative example 2 (modification of another Lactobacillus rhamnosus on the basis of example 5)
A lactic acid bacterium fermented product was prepared in substantially the same manner as in example 5 except that: lactobacillus rhamnosus ST was exchanged for an equivalent amount of lactobacillus rhamnosus LGG (ATCC 53013).
EXAMPLE 23 antibacterial Capacity detection and comparison of Lactobacillus rhamnosus ST fermentation
1. Experimental method
The antibacterial ability of lactobacillus rhamnosus ST ferments of examples 2 to 16 and ferments of comparative examples 1 to 2 was tested. The testing method comprises the following steps: determining the bacteriostatic activity of a fermented product by adopting an oxford cup agar diffusion method, inoculating escherichia coli as an indicator bacterium to an LB (liquid) culture medium, inoculating salmonella and staphylococcus aureus to beef extract peptone liquid culture mediums, respectively pouring a layer of solid culture based on the bottom of a culture dish, placing oxford cups on a flat plate in sequence after solidification, respectively inoculating the indicator bacterium culture solutions to respective culture mediums according to 1 per mill inoculum size after the temperature of the lower layer of solid culture medium is reduced to 55-60 ℃, respectively pouring 10mL of solid culture medium as an upper layer, taking out the oxford cups after solidification, taking the fermented products of examples 1-7 and comparative examples 1-7, adding the fermented products into a hole site, placing the flat plate in a 37 ℃ incubator for culturing for 10 hours after 2 hours of diffusion in a refrigerator at 4 ℃, and measuring a bacteriostasis ring.
2. Experimental results
The test results are shown in table 2: as can be seen from table 2 by comparing the detection results of comparative examples 4 and 5, the addition of hyaluronic acid to the culture medium of lactobacillus rhamnosus ST gave a better antibacterial effect of the supernatant obtained by fermentation, the lactobacillus rhamnosus ST ferment obtained in example 5 had an antibacterial effect of 2.4 times as large as that of the lactobacillus rhamnosus ST ferment obtained in example 4 without hyaluronic acid, 2.1 times as large as that of the salmonella bacteria, and 2.7 times as large as that of the staphylococcus aureus bacteria.
Further, as can be seen from the comparison of the detection results of examples 7 to 15, when hyaluronic acid is added into the culture medium of lactobacillus rhamnosus ST, the degree of improvement of the antibacterial effect of the fermented product has a certain influence on the concentration and molecular weight of hyaluronic acid, and when the concentration and molecular weight of hyaluronic acid are too high or too low, the antibacterial effect of lactobacillus rhamnosus ST fermented product is slightly improved compared with that of lactobacillus rhamnosus ST fermented product without hyaluronic acid, but the improvement is not as remarkable as the range of 1-2000 kDa of molecular weight of hyaluronic acid and 0.1-10% of mass concentration of hyaluronic acid.
The results of examples 5 and 6 show that sodium hyaluronate and potassium hyaluronate both increase the bacteriostatic activity of lactobacillus rhamnosus ST ferments.
As can be seen from the detection results of example 5 and example 16, the antibacterial effect of the fermented product obtained by physically mixing the filtrate and the hyaluronic acid is inferior to that obtained by metabolizing hyaluronic acid by adding hyaluronic acid into a culture medium, and it is proved that hyaluronic acid needs to be added into the culture medium for metabolism, so that lactobacillus rhamnosus ST can be promoted to produce more antibacterial metabolites in the fermentation process.
As can be seen from the test results of example 5 and comparative examples 1-2, the antibacterial effect of the fermented product of lactobacillus rhamnosus ST was better than that of the fermented product of the existing lactobacillus rhamnosus LGG, which could not produce a better antibacterial effect with hyaluronic acid, probably because the lactobacillus rhamnosus ST produced a specific metabolite when using hyaluronic acid, thereby synergistically increasing the antibacterial effect.
TABLE 2 antibacterial test results for examples 2-16 and comparative examples 1-2
ND in Table 2 indicates undetected, no zone diameter.
EXAMPLE 24 protease sensitivity test of Lactobacillus rhamnosus ST fermentum
1. Experimental method
Protease susceptibility testing was performed using lactobacillus rhamnosus ST ferment of example 3 as an example. The testing method comprises the following steps: determining antibacterial activity of the fermented product by adopting oxford cup agar diffusion method, inoculating Escherichia coli as indicator bacteria into LB culture medium, inoculating salmonella and staphylococcus aureus into beef extract peptone liquid culture medium, pouring a layer of 5ml solid culture medium into a culture dish, placing oxford cups on a flat plate in sequence after solidification, respectively pouring 10ml solid culture medium as upper layer after the lower layer solid culture medium is cooled to 55-60 ℃ according to 1%o inoculum size of the indicator bacteria culture medium into the respective culture medium, taking out oxford cups after solidification, adding proteinase K into filtrate of example 3, performing enzymolysis at 35 ℃ for 10min to obtain a contrast solution 1, adding into a hole site, placing an equal amount of sterile normal saline as blank in a refrigerator respectively, placing the flat plate into a 37 ℃ incubator for culture for 10h after diffusion, and measuring a bacteriostasis ring.
2. Experimental results
As shown in table 3, it can be seen from table 3 that after the antibacterial peptide in lactobacillus rhamnosus ST ferment is digested with proteinase K, the antibacterial activity is significantly reduced, and the results of comparative example 3 and comparative liquid 1 show that the substance having antibacterial activity in the ferment liquid is a protein or polypeptide, which is easily acted on by proteinase K, and thus the antibacterial effect is reduced.
TABLE 3 antibacterial Activity after proteinase K enzymatic hydrolysis of antibacterial peptides in Lactobacillus rhamnosus ST ferments
It is to be understood that the above examples of the present invention are provided by way of illustration only and are not intended to limit the scope of the invention. It will be appreciated by persons skilled in the art that other variations or modifications may be made in the various forms based on the description above. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. Lactobacillus rhamnosus (Lactobacillus rhamnosus) ST, wherein the lactobacillus rhamnosus ST is deposited with the China general microbiological culture Collection center (CGMCC NO) at 2/27/2023: 26684.
2. a method for preparing lactobacillus rhamnosus ST ferment, which is characterized in that lactobacillus rhamnosus ST ferment is obtained by fermenting lactobacillus rhamnosus ST of claim 1 in a culture medium.
3. The method for producing lactobacillus rhamnosus ST ferment according to claim 2, comprising the steps of:
s1, inoculating lactobacillus rhamnosus ST of claim 1 into a culture medium, and fermenting for 12-50 hours at the temperature of 35-38 ℃;
s2, centrifuging the fermentation liquor, and taking supernatant for ultrafiltration, wherein the obtained filtrate is lactobacillus rhamnosus ST fermentation product.
4. A method for producing lactobacillus rhamnosus ST ferment according to claim 3, wherein the medium in step S1 is a lactobacillus medium.
5. A method for preparing lactobacillus rhamnosus ST ferment according to claim 3, wherein the inoculum size of lactobacillus rhamnosus ST in the culture medium in step S1 is 0.1% -10%.
6. The method for producing lactobacillus rhamnosus ST fermentum according to any one of claims 3 to 5, wherein the medium in step S1 contains hyaluronic acid or hyaluronate.
7. The method for producing lactobacillus rhamnosus ST ferment according to claim 6, wherein the molecular weight of the hyaluronic acid or hyaluronate in step S1 is 1kDa to 2000kDa.
8. The method for producing lactobacillus rhamnosus ST ferment according to claim 6, wherein the mass concentration of the hyaluronic acid or hyaluronate in the culture medium in the step S1 is 0.1% -10%.
9. Lactobacillus rhamnosus ST ferment prepared by the preparation method according to any one of claims 2-8.
10. Use of lactobacillus rhamnosus ST of claim 1 or lactobacillus rhamnosus ST ferment of claim 9 for the production of an antibacterial product or an antibacterial functional product.
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