CN110257297B - Separated lactobacillus paracasei PC-01 for promoting oral health and application thereof - Google Patents

Separated lactobacillus paracasei PC-01 for promoting oral health and application thereof Download PDF

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CN110257297B
CN110257297B CN201910590646.2A CN201910590646A CN110257297B CN 110257297 B CN110257297 B CN 110257297B CN 201910590646 A CN201910590646 A CN 201910590646A CN 110257297 B CN110257297 B CN 110257297B
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lactobacillus paracasei
oral
bacteria
strain
culture
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CN110257297A (en
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张凌宇
马晨
马杰
杨慧娟
郭霄
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Inner Mongolia Mengniu Dairy Group Co Ltd
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Abstract

The application provides a Lactobacillus paracasei strain PC-01(Lactobacillus paracasei PC-01) separated from acid yak milk of Longren county of Dangxiang in the Lassa Tibet, wherein the microorganism preservation number of the Lactobacillus paracasei PC-01 is as follows: CGMCC No.17537, the strain can improve the unbalance of oral health flora, has probiotics with better acid tolerance and bile salt tolerance, has probiotic characteristics, can be directly used as a preparation for improving the oral micro-ecological environment, or is prepared from the lactobacillus paracasei strain serving as a raw material and used as a pathological state microorganism antagonist in the oral cavity, wherein the pathological state microorganism comprises at least one of streptococcus mutans, porphyromonas gingivalis, treponema denticola, prevotella denticola and fusobacterium nucleatum.

Description

Separated lactobacillus paracasei PC-01 for promoting oral health and application thereof
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a separated lactobacillus paracasei PC-01 for promoting oral health and application thereof.
Background
The oral cavity is a dynamic balance microbial environment with multi-flora symbiosis, the types of microbes planted in the oral cavity are complex, including bacteria, fungi, mycoplasma, protozoa, viruses and the like, and the microbes coexist, compete and antagonize at different parts of the oral cavity to form an oral cavity microecological system. At present, research proves that the known bacterial species in the oral cavity can reach more than 700 species, and the oral cavity can keep a healthy state only by keeping good ecological balance among microorganisms in the oral cavity and between the microorganisms and host oral cavity tissues. If the proportion of individual or a plurality of microbes inherent in oral cavity micro-ecology is obviously increased to become dominant bacteria of a colonization part, especially the proportion of pathogenic bacteria is increased due to the influence of external microbes or other factors, the original oral cavity micro-ecology balance is broken, flora imbalance can occur, oral cavity diseases can be caused, and vice versa. Therefore, the micro-ecological status of the host oral cavity is closely related to the health and disease of the oral cavity. Just because these different kinds of bacteria exist in a certain proportion, the external bacteria are destroyed once invading, and the oral cavity and the whole body are kept healthy.
Oral diseases include dental caries, periodontal disease, oral mucositis and oral health disorders of the elderly and pregnant women, wherein dental caries, periodontal disease and oral mucositis are associated with the microenvironment of the oral cavity. Periodontal inflammatory conditions are gingivitis and periodontitis, the pathogenesis of which is closely related to microorganisms, mainly induced by the formation of plaque, specifically the formation of biofilm on the tooth surface by the colonizing bacteria by means of food residues and the presence of salivary components, which can lead to deposited tartar, which is very difficult to remove, if not removed sufficiently in time at an early stage. If an increased number of bacteria are present at the gingival margin, this can lead to irritation of the gums, referred to as gingivitis. In susceptible individuals, gingivitis may progress to periodontitis and even to tooth loss, in particular, Lipopolysaccharide (LPS) present in gram-negative bacteria may elicit a non-specific immune response by LPS-stimulated macrophages, which releases prostaglandin E2(prostaglandin E2, PEG2) and pro-inflammatory mediators such as interleukins and TNF-alpha in the affected tissues. Among other things, pro-inflammatory mediators induce the release of more PGE2 and Matrix Metalloproteinases (MMPs) from resident fibroblasts, which destroy the extracellular matrix of the surrounding tissue. This allows bacteria to penetrate deeper into the tissue and promote the inflammatory process without relying on the outer layers of the epithelium and root, thereby promoting the formation of periodontal pockets, the alveolar bone that supports the tooth contracts in front of the advancing bacteria, causing the tooth to become unstable and, if left untreated, to lose tooth.
The prior art has several approaches to ameliorate the above problems, for example, mechanical removal of plaque and tartar, use of oral care products with strong antimicrobial properties. However, the above conventional methods are very harmful to the oral environment and cannot restore the original balance in a short time. Moreover, if bacteria in the oral cavity are killed blindly, the balance of normal microenvironment flora in the oral cavity is disrupted, which is detrimental to oral and general health. This requires that the balance of the indigenous flora in the oral cavity, i.e. the ecology of the oral cavity, be preserved in the prevention or treatment of common inflammatory diseases in the oral cavity.
Disclosure of Invention
One of the purposes of the application is to provide a lactobacillus paracasei strain separated from acid yak milk of Longren county, Dangchasa, Tibet.
The second purpose of the application is to prepare the lactobacillus paracasei strain into a microbial inoculum for improving the oral micro-ecological balance.
In order to achieve the purpose, the method is realized by the following technical scheme:
the method is characterized in that the collected sour yak milk is subjected to disinfection, inoculation, separation, purification and identification, and finally separated to obtain a Lactobacillus paracasei PC-01(Lactobacillus paracasei PC-01) capable of adjusting the oral microecological balance, the strain is submitted to a patent approved organization for preservation, the preservation time is 2019, 3 and 18 days, the microorganism preservation number is CGMCC No.17537, the strain is classified and named Lactobacillus paracasei, the preservation unit is the China general microorganism culture preservation center of the culture preservation management Committee, and the preservation address is West Lu No.1 Homew of the sunny region in Beijing, China academy of sciences microbial research institute.
The Lactobacillus paracasei strain Lactobacillus paracasei PC-01 separated by the application can improve the unbalance of oral health flora, has probiotics with better acid tolerance and cholate tolerance, and has the probiotic characteristic.
In the present application, the lactobacillus paracasei strain may be used as an agent for improving oral micro-ecological environment directly, or prepared from the lactobacillus paracasei strain as a raw material for use as an antagonist of microorganisms in pathological states in the oral cavity, wherein the microorganisms in pathological states include at least one of streptococcus mutans, porphyromonas gingivalis, treponema denticola, prevotella denticola, and fusobacterium nucleatum.
In one achievable form, the antagonist is in a solid, liquid or gaseous dosage form.
Alternatively, the antagonist comprises a powder, tablet, film formulation, solution, aerosol, granule, lozenge, pill, suspension, emulsion, capsule, syrup, liquid, elixir, extract, tincture, or fluid extract form or in a dosage form particularly suitable for oral administration.
It is another object of the present application to provide a method for preparing an oral micro-ecological environment improving agent using the aforementioned lactobacillus paracasei strain PC-01, the method comprising: the active or inactivated lactobacillus paracasei strain PC-01 is directly mixed with the remaining filling.
In the present application, the method for inactivating the lactobacillus paracasei strain PC-01 may be incubating the material obtained in step 2 at a temperature of 70 ℃ to 100 ℃ to thermally inactivate it, for example, the incubation may be performed at the temperature for 2 to 8 minutes.
Compared with the prior art, the lactobacillus paracasei PC-01 provided by the application shows various characteristics beneficial to oral health no matter an active strain or an inactivated strain thereof, and particularly has good antagonistic action on oral pathogenic bacteria and good proliferation capacity in an oral environment, so that the lactobacillus paracasei PC-01 has the function of adjusting the oral micro-ecological environment, and shows the functions of adjusting the flora balance in the oral cavity, cleaning the oral cavity, improving the breath and the like.
Drawings
FIG. 1 shows the growth of the bacterial colony of Lactobacillus paracasei PC-01 cultured in MRS agar medium at 37 deg.C under anaerobic condition for 72 h;
FIG. 2 shows an electron micrograph of Lactobacillus paracasei PC-01;
FIG. 3 shows the inhibition rate of the oral pathogenic bacteria S.mutans by the active probiotic bacteria Lactobacillus paracasei PC-01, Streptococcus salivarius K12 and Lactobacillus reuteri ATCC 55730;
FIG. 4 shows the inhibition rate of the active probiotic Lactobacillus paracasei PC-01, the active Streptococcus salivarius K12 and the active Lactobacillus reuteri ATCC55730 against the oral pathogen P.gingivalis;
FIG. 5 shows the inhibition rate of the oral pathogen F.nuclearum by the active probiotic Lactobacillus paracasei PC-01, the active Streptococcus salivarius K12 and the active Lactobacillus reuteri ATCC 55730;
FIG. 6 shows the inhibition rate of the oral pathogenic bacteria A. actinomycetes, by the active probiotic bacteria Lactobacillus paracasei PC-01, Streptococcus salivarius K12 and Lactobacillus reuteri ATCC 55730;
FIG. 7 shows the inhibition rate of the inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against the oral pathogen S.mutans;
FIG. 8 shows the inhibition rate of inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against the oral pathogen P.gingivalis;
FIG. 9 shows the inhibition rate of the inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against the oral pathogen F.tuberculosis;
FIG. 10 shows the inhibition rate of inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against oral pathogenic bacteria A.actinomycetes comomatics;
FIG. 11 shows the results of the viability of the probiotic Lactobacillus paracasei PC-01 against oral conditions;
FIG. 12 shows the oral breath changes of volunteers after three days of intake of live Lactobacillus paracasei PC-01 bacteria;
FIG. 13 shows the oral breath of volunteers three days after intake of viable Streptococcus salivarius K12;
FIG. 14 shows the oral breath of volunteers three days after intake of viable Lactobacillus reuteri ATCC 55730.
Detailed Description
The present application is further described below with reference to the following figures and examples, which are illustrative only and the present application is not limited to these examples.
The probiotic bacteria to which the present application relates will first be briefly described: probiotics (Probiotics) is a kind of active microorganisms beneficial to a host, and is a general term for active beneficial microorganisms which are planted in the intestinal tract and the reproductive system of a human body and can produce exact health effects so as to improve the microbial ecological balance of the host and play a beneficial role in the intestinal tract. A large number of researches prove that the probiotic taken in a proper amount also has the effects of improving the immunity of the organism, promoting the generation and metabolism of nutrient components of the organism, protecting the cardiovascular system, resisting tumors, resisting aging, resisting tumors and the like.
The lactobacillus paracasei is a common probiotic, can be planted in human intestinal tracts, and can improve the intestinal flora structure and adjust the micro-ecological environment in the intestinal tracts. After entering the intestinal tract of a human body, lactobacillus paracasei can generate metabolites which have an inhibiting effect on pathogenic bacteria, putrefying bacteria and other harmful microorganisms: organic acid, hydrogen peroxide, bacteriocin and the like, and play a role in regulating the balance of intestinal flora so as to promote digestion and absorption of a human body. At present, the effect of lactobacillus paracasei on the oral micro-ecological environment is not researched.
Examples
Example 1 growth form of bacterial cells and Electron micrograph
(I) Strain activation
1. Culture of viable Lactobacillus paracasei PC-01 bacteria
After the frozen tube preserved at minus 80 ℃ and storing the lactobacillus paracasei PC-01 is enriched and grown for 18h at 37 ℃ (logarithmic growth phase) in an MRS liquid culture medium (sterilized for 15min at 121 ℃), the frozen tube is transferred to the sterilized MRS culture medium again according to 2% V/V, finally, the viable count of the frozen tube is counted by adopting an MRS agar plate counting method, and microbial thalli are centrifugally collected for later use (logarithmic growth phase).
2. Preparation of killed Lactobacillus paracasei PC-01
Incubating the viable bacteria obtained from the culture of 1 for 3min at 85 ℃ to obtain inactivated Lactobacillus paracasei PC-01.
(II) growth morphology of cells and Electron micrograph
Culturing the obtained viable Lactobacillus paracasei PC-01 in MRS agar medium at 37 deg.C under anaerobic condition for 72h, wherein the growth condition of the colony is shown in FIG. 1, and the electron micrograph is shown in FIG. 2.
EXAMPLE 2 bacteriostatic test of Lactobacillus paracasei PC-01
Pathogenic bacteria activation
Streptococcus mutans(s): aerobic culture is carried out at 37 ℃ in 5mL of Tryptic soy Broth culture solution, and shake culture is carried out at 150rpm for 20 hours;
porphyromonas gingivalis (p. gingivalis): firstly carrying out anaerobic culture at 37 ℃ in 5mL of Tryptic soy Broth culture solution, and carrying out shake culture at 150rpm for 4 days;
nucleic acid bacterium (f.nucleolus): anaerobic culture at 37 ℃ in 5mL Brain-heart infusion medium Broth culture solution, and shake culture at 150rpm for 4 days;
actinobacillus actinomycetemcomitans group (a. actinomycetemecians): the culture was performed anaerobically at 37 ℃ in 5mL of Brain-heart infusion medium Broth medium, and the culture was performed with shaking at 150rpm for 20 hours.
(II) Co-culture experiment
Live bacteria and killed bacteria of Lactobacillus paracasei PC-01 are taken as experimental groups, live bacteria and killed bacteria of commercial oral probiotic strains Streptococcus salivarius K12 and live bacteria and killed bacteria of Lactobacillus reuteri ATCC55730 are taken as control groups, and the control groups are respectively co-cultured with the activated pathogenic bacteria in the experimental example (I) to carry out comparative experiments.
1. The experimental method comprises the following steps:
(1) preparing effective concentration pathogenic bacteria liquid
The activated culture solution for pathogenic bacteria of the experimental example (one) was prepared into a pathogenic bacteria solution, the culture solution for culturing each pathogenic bacteria was shown in table 1, and the concentration of pathogenic bacteria in each pathogenic bacteria solution was shown in table 2.
The preparation method of the bacterial liquid of each pathogenic bacterium comprises the following steps: the test strains were inoculated in a clean environment into a corresponding medium sterilized at 121 ℃ for 5min, and 5mL × 3 parts of each pathogenic bacteria solution was prepared in this example.
TABLE 1 culture solutions for the respective pathogenic bacteria
Figure BDA0002115912800000041
TABLE 2 concentration of each pathogenic bacterium solution
Figure BDA0002115912800000042
(2) The co-culture method comprises the following steps:
1) killing viable bacteria group: adding 1.00X 10 of the mixture into the bacterial liquid of each pathogenic bacterium9The CFU content of Lactobacillus paracasei PC-01, Streptococcus salivarius K12 and Lactobacillus reuteri ATCC55730 were all incubated with the corresponding viable bacteria at 85 ℃ for 3min to form 12 culture samples.
2) The viable bacteria group: adding 3.00X 10 to the culture medium of each pathogenic bacterium9CFU of live Lactobacillus paracasei PC-01, Streptococcus salivarius K12 and Lactobacillus reuteri ATCC55730 formed another 12 culture samples.
The specific conditions of co-cultivation are: incubate under anaerobic conditions at 37 ℃ and shaking at 150 rpm.
(III) bacteriostatic test
1. Sampling and detecting viable count of co-cultured samples at preset time points
(1) Sampling time for s.mutans pathogen bacterial liquid: 2h, 4h, 6h, 8h and 48 h;
(2) sampling time for a. actinomycetes comotians pathogenic bacteria liquid: 2h, 4h, 6h, 8h and 48 h;
(3) sampling time for p.gingivalis pathogenic bacteria liquid: 2h, 4h, 6h, 8h, 24h, 48h, 72h and 96 h;
(4) sampling time for f.nuclear pathogenic bacteria liquid: 2h, 4h, 6h, 8h, 24h, 48h, 72h and 96 h.
2. Count of surviving bacteria
After sampling of pathogenic bacteria, sequential dilution coating is carried out, and 3 dilution concentrations are selected and are respectively as follows: 10510 times of610 times of7And (5) diluting by times.
(1) S. mutans group: sampling after the co-culture is finished, counting the number of bacteria by a Tryptic soy Agar (TS Agar) coating disc, respectively placing in an incubator at 37 ℃ for continuous aerobic culture (48 +/-0.5) h, and counting the number of the bacteria;
(2) gingivalis group: sampling after co-culture is finished, coating a flat plate with Tryptic soy + 5% sheet blood Agar (TS Agar), respectively placing in an incubator at 37 ℃ for continuous aerobic culture (96 +/-1) h, and counting the number of colonies;
(3) nuclear group: sampling after co-culture is finished, counting the number of bacteria by a Tryptic soy + 5% sheet blood Agar (TS Agar) coating disc, respectively placing in an incubator at 37 ℃ for continuous aerobic culture (96 +/-1) h, and counting the number of the bacteria;
(4) actinomycetes comomatics group: sampling after the co-culture is finished, and counting the number of bacteria by using a Tryptic soy + 5% sheet blood Agar (TS Agar) coating disc; respectively placing in an incubator at 37 ℃ for continuous aerobic culture (48 +/-0.5) h, and counting the number of colonies;
(5) blank control group (no sample to be tested was added, and 0.05g of sterile water was added instead of blank control).
Calculating the bacteriostatic rate by taking the blank control group as a reference, wherein the bacteriostatic rate can be calculated according to the following formula I:
the bacteriostasis rate is (number of bacteria in a control group-number of bacteria in an experimental group)/number of bacteria in a control group multiplied by 100 percent of formula I
3. The experimental results are as follows:
as shown in fig. 3 to 10:
FIG. 3 shows the inhibition rate of the oral pathogenic bacteria S.mutans by the active probiotic bacteria Lactobacillus paracasei PC-01, Streptococcus salivarius K12 and Lactobacillus reuteri ATCC 55730;
FIG. 4 shows the inhibition rate of the active probiotic Lactobacillus paracasei PC-01, the active Streptococcus salivarius K12 and the active Lactobacillus reuteri ATCC55730 against the oral pathogen P.gingivalis;
FIG. 5 shows the inhibition rate of the oral pathogen F.nuclearum by the active probiotic Lactobacillus paracasei PC-01, the active Streptococcus salivarius K12 and the active Lactobacillus reuteri ATCC 55730;
FIG. 6 shows the inhibition rate of the oral pathogenic bacteria A. actinomycetes, by the active probiotic bacteria Lactobacillus paracasei PC-01, Streptococcus salivarius K12 and Lactobacillus reuteri ATCC 55730;
FIG. 7 shows the inhibition rate of the inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against the oral pathogen S.mutans;
FIG. 8 shows the inhibition rate of inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against the oral pathogen P.gingivalis;
FIG. 9 shows the inhibition rate of the inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against the oral pathogen F.tuberculosis;
FIG. 10 shows the inhibition rate of inactivated probiotic Lactobacillus paracasei PC-01, inactivated Streptococcus salivarius K12 and inactivated Lactobacillus reuteri ATCC55730 against oral pathogenic bacteria A.
As can be seen from fig. 3-6, the oral pathogenic bacteria were significantly decreased in the culture broth co-cultured with the three active probiotics and the pathogenic bacteria in a certain period of time, wherein, the inhibition effect of Lactobacillus paracasei PC-01 group on four pathogenic bacteria s.mutans, p.gingivalis, f.tuberculosis and a.actinomycetecomomatics in 48h was most significant compared with that of the other two groups of commercial control bacteria, and the survival rates of the pathogenic bacteria at 48h were 10%, 12%, 47% and 18%, respectively, which were significantly lower than that of the control group.
From fig. 7-10, it can be seen that the oral cavity pathogenic bacteria were significantly reduced in the culture solutions co-cultured with the three inactivated probiotic bacteria and pathogenic bacteria for a certain period of time, and the survival rates for 48h were 15%, 11%, 42% and 12%, respectively, which were significantly lower than those of the control group.
As can be seen from the graphs of FIGS. 3 to 10, the inhibition rates of the active probiotic Lactobacillus paracasei PC-01 and the inactivated probiotic Lactobacillus paracasei PC-01 in the oral pathogenic bacteria all showed better inhibition abilities compared with those of the two commercial control groups.
In conclusion, both the active Lactobacillus paracasei PC-01 and the inactivated Lactobacillus paracasei PC-01 have better inhibition effect on oral pathogens, and the effect is obviously better than that of two commercial control groups.
Experimental example 3 Effect of Lactobacillus paracasei PC-01 on oral Microecological Environment
(first) measurement of the agglomeration Capacity
In this example, a comparative experiment was performed using Lactobacillus paracasei PC-01 as an experimental group and commercial oral probiotic strains Streptococcus salivarius K12 and Lactobacillus reuteri ATCC55730 as control groups.
1. The experimental method comprises the following steps:
evaluated by monitoring the overnight co-culture liquid for a decrease in optical density at 620nm due to the formation of aggregates and precipitates, indicating that the proprietary probiotic of choice has a positive effect on oral pathogen reduction.
Percent aggregation capacity (% AC) values were obtained using the following equation II:
％AC＝{1-(ODtf/ODt0) 100 formula II
Wherein, ODtfAnd ODt0Respectively, the optical density at the final time and the optical density at the initial time.
The OD at the initial time was adjusted so that all the cultures contained the same amount of CFU/ml, i.e., the number of viable bacteria contained at the same concentration.
The embodiment shows that the probiotic Lactobacillus paracasei PC-01 has good cohesiveness, and the inventor believes that the mechanism of the good cohesiveness of the probiotic Lactobacillus paracasei PC-01 is that the bacterial strain inhibits or reduces dental plaque by interfering the formation of an oral pathogen biofilm, so that the aim of promoting the long-lasting health of the oral cavity is finally fulfilled. Reflecting to the fact, one of the effects of the probiotic Lactobacillus paracasei PC-01 provided by the application on oral health is that the probiotic Lactobacillus paracasei PC-01 has an encapsulation and aggregation effect on main harmful bacteria causing the problem of oral odor. After the bacteria are contacted with the oral cavity during the tooth brushing process, the harmful bacteria can be wrapped and condensed and are removed to leave the oral cavity, thereby maintaining the balance of oral flora and keeping fresh breath.
Preferably, the corresponding culture medium is selected to contain 5% CO at 37 deg.C2The probiotic Lactobacillus paracasei PC-01 was cultured for 48h in the environment of (1), and the results are shown in tables 3 and 4:
TABLE 3 comparison of the potency of addition of active microorganisms to a culture containing oral pathogens
Figure BDA0002115912800000061
TABLE 4 comparison of the energy concentrations of inactivated microorganisms added to the culture broth
Figure BDA0002115912800000071
As can be seen from tables 3 and 4: both active and inactivated microorganisms had significant ability to form aggregates, with Lactobacillus paracasei PC-01 group being significantly higher in both active and inactivated microorganism additions than the commercial control group.
(II) measurement of Balancing Effect of oral flora
1. Evaluation method of malodorous volatile compound generation: evaluation of the production when grown in simulated Medium similar to human diet by means of sensory evaluationMalodorous volatile compounds. Typically, during 48h, at 37 ℃ and 5% CO2Strains were grown in medium containing soy protein isolate (0.5% w/v) glucose (0.5% w/v), fructose (0.5% w/v), yeast extract (1% w/v), meat extract (1% w/v), eukaryotic cells (200 cells/ml) and pectin (0.5% w/v).
2. The evaluation method comprises the following steps: the strain is rated by a professionally trained, odor sensitive person for a rating score of malodour value between 1 and 5, wherein a score of 1 to 5 is representative of better to worse odor.
3. The experimental results are as follows:
generally, oral probiotics are not produced in conventional culture solutions with unpleasant odors. It is desirable that the probiotic Lactobacillus paracasei PC-01 does not produce unpleasant odor when it comes into contact with food or the like after oral ingestion. The results of this experiment, which was simulated in a medium similar to human diet, are shown in Table 5, where the strains of the present application did not produce unpleasant odors.
TABLE 5 malodor value production of different strains under equivalent conditions
Figure BDA0002115912800000072
(III) survival assays for oral conditions
Modern medical and pathological studies are increasingly proceeding with the treatment of oral diseases, and thus promoting oral health, starting from the idea that microorganisms alter the microenvironment of the oral cavity by antagonizing microorganisms involved in pathological conditions in the oral cavity, such as streptococcus mutans, porphyromonas gingivalis, treponema denticola, prevotella denticola, and fusobacterium nucleatum.
1. Survival evaluation methods for oral conditions:
the survival of the strain in the oral cavity was studied by subjecting it to simulated oral solution conditions.
Simulated oral solution was incubated at 37 ℃ and 5% CO2Incubate for 0, 2, 6, 8 and 24 hours, this time design comes from positiveThe normal human body takes in the food compartment.
Bacterial growth values were obtained by quantifying bacterial growth by measuring optical density at 620 nm. The selected oral health pathogenic bacteria are a pathogenic bacteria collection culture solution which accounts for more than 80 percent, and the test strain preparation work comprises the following steps: dissolving standard strains of Streptococcus mutans (S.mutans), Porphyromonas gingivalis (P.gingivalis), Fusobacterium nucleatum (F.tuberculosis), Prevotella intermedia (P.intermedia), Graptospira periodontosis (C.periodontii), Treponema denticola (T.denticola), Actinomyces symbiota (A.actinomycetes comans) and Prevotella denticola (P.disiens)8 oral health problem indicator bacteria in a centrifuge tube filled with 10mL of BHI liquid culture medium, loosening a tube cover to make the bacteria fully contact with the anaerobic environment in the box, and carrying out anaerobic culture at 37 ℃ for 12-24h to obtain the enrichment of 8 standard strains, wherein the viable count is about 5 multiplied by 107CFU/mL. 10mL of each bacterium was added to 90mL of modified artificial saliva for use.
The artificial saliva is formulated as a modified biofilm culture medium BM-5, which may include, in particular: 2.5g/L of porcine gastric mucin; peptones 2 g/L; 1g/L of tryptic casein; 1g/L of yeast extract; 2.5g/L of potassium chloride (KCL); glucose 0.5 g/L; cystine hydrochloride 0.1 g/L; 1mg/L of hemin; dipotassium hydrogen phosphate (K)2HPO4·3H2O)0.114 g/L; potassium dihydrogen phosphate (KH)2PO4)0.2g/L。
Adding 10g/L sucrose into the above components, adjusting pH to 7.50 + -0.01 with 1mol/L NaOH solution, and sterilizing at 121 deg.C for 15 min.
The experimental design is divided into an experimental group of Lactobacillus paracasei PC-01 (experimental group), a commercial control group of Streptococcus salivarius K12 (control group 1) and Lactobacillus reuteri ATCC55730 (control group 2), and the primary administration amount of probiotics in the three experimental groups is 3 multiplied by 108CFU/mL. Counting the viable count of the three probiotics by adopting an agar plate counting method, and carrying out 72 hours at 37 ℃ in an anaerobic environment.
2. The experimental results are as follows:
the viability against oral conditions reflects another major feature of the probiotic Lactobacillus paracasei PC-01.
The results of the viability against oral conditions as shown in fig. 9, it can be seen from fig. 9 that all three probiotics exhibited good growth performance in the oral environment. After 24h, the probiotic Lactobacillus paracasei PC-01 has higher viable count, and shows that compared with other two commercial oral probiotics, the probiotic Lactobacillus paracasei PC-01 is more suitable for survival in the oral environment.
(IV) measurement of breath improvement Effect
1. Human test for halitosis removal
(1) Test objects: on the basis of the evaluation of the production of malodorous volatile compounds, a human test for the elimination of halitosis was performed. Wherein the volunteers: 82, 20-45 years old, all had subjective or objective symptoms of halitosis.
(2) Grouping:
1) blank control group: 0.85% physiological saline, 20 volunteers;
2) experimental groups: lactobacillus paracasei PC-01 group;
3) control group 1: streptococcus salivarius K12;
4) control group 2: lactobacillus reuteri ATCC55730,
wherein the number of volunteers in each of the experimental group, the control group 1 and the control group 2 is 22; adding 3X 108The probiotic bacteria of CFU are dissolved in physiological saline.
2. The test method comprises the following steps: the test period is 3d, each time of gargling is 3min, once a day (7: 30 in the morning every day), and the detection is carried out before and after gargling, and at 8h and 12h respectively; the concentration of VSCs, the main volatile sulfur compounds causing oral malodor (the main components are hydrogen sulfide, methyl mercaptan and dimethyl sulfide), in the oral cavity of volunteers was measured using a portable gas chromatograph, OralChroma.
3. The results of the experiment are shown in FIGS. 12-14:
FIG. 12 shows the oral breath changes of volunteers after three days of intake of live Lactobacillus paracasei PC-01 bacteria;
FIG. 13 shows the oral breath of volunteers three days after intake of viable Streptococcus salivarius K12;
FIG. 14 shows the oral breath of volunteers three days after intake of viable Lactobacillus reuteri ATCC 55730.
As can be seen from fig. 12 to 14, the three days after the mouth was rinsed with Lactobacillus paracasei PC-01, compared with the control group 1 and the control group 2, the three sulfide removing effects were significantly higher than those of the control group 1 and the control group 2, which indicates that Lactobacillus paracasei PC-01 can remove the volatile sulfur compounds, which are the main components causing halitosis, and the halitosis removing purpose can be achieved by regular use for a certain period of time.
Experimental example 4 antibiotic susceptibility test
The antibiotics are applied to various aspects of human life, and play an important role in the aspects of protecting human health and prolonging the human life. However, antibiotic resistance is one of the problems that ensues, and this experiment was conducted on the Lactobacillus paracasei PC-01 for resistance analysis.
1. The experimental method comprises the following steps:
the antibiotic susceptibility test method described in the present application proves the antibiotic susceptibility of the related patented strain Lactobacillus paracasei PC-01 according to the technical guidance given by the European Food Safety Agency (EFSA), and the culture conditions for the strain are as follows: colonies were recorded by growth (72. + -.2) h in 37 ℃ anaerobic culture on MRS agar plates containing the EFSA recommended antibiotic concentration.
2. The results of the experiment are shown in table 6:
TABLE 6 growth of Lactobacillus paracasei PC-01 resistant to common antibiotics
Figure BDA0002115912800000091
As can be seen from Table 6, the probiotic Lactobacillus paracasei PC-01 has no antibiotic resistance.
According to the experiment, the lactobacillus paracasei PC-01 provided by the application has the effects of promoting oral health and adjusting the balance of oral flora no matter live bacteria or dead bacteria. In addition, the living bacteria or the inactivated bacteria of the lactobacillus paracasei PC-01 have various same or similar physiological functions, and the inactivated lactobacillus paracasei PC-01 is not influenced by antibiotics, so that the content of the living bacteria in the oral cavity is not changed, and the quality stability is higher.
Further, the applicant believes that the capacity of inactivated lactobacillus paracasei PC-01 to combat pathogenic microorganisms is mainly due to two aspects: firstly, the immunity of the organism is effectively enhanced; secondly, more adsorption sites are exposed on the cell surface after inactivation, and the cell surface can be adsorbed on the surface of the mucosa of the organism to realize competitive exclusion of pathogenic bacteria. The applicant also finds that the inactivated lactobacillus paracasei PC-01 has cell extract with reducing capability and SOD enzyme activity, can remove active oxygen free radicals to play an antioxidation role, can achieve the same action effect as the active lactobacillus paracasei PC-01, and also performs a large amount of activity and inactivated probiotic contrast tests, and the result shows that the two effects can be achieved.
The present application has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to limit the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the presently disclosed embodiments and implementations thereof without departing from the spirit and scope of the present disclosure, and these fall within the scope of the present disclosure. The protection scope of this application is subject to the appended claims.

Claims (6)

1. The Lactobacillus paracasei strain PC-01(Lactobacillus paracasei PC-01) is characterized in that the microorganism preservation number of the Lactobacillus paracasei PC-01 is as follows: CGMCC No. 17537.
2. Use of a lactobacillus paracasei strain according to claim 1 for the preparation of an agent for improving the oral micro-ecological environment.
3. Use of a lactobacillus paracasei strain according to claim 1 for the preparation of a microbial antagonist of pathological conditions in the oral cavity; the microorganism in pathological state is at least one of streptococcus mutans, porphyromonas gingivalis, treponema denticola, prevotella denticola and fusobacterium nucleatum.
4. Use according to claim 3, wherein the antagonist is in solid, liquid or gaseous form.
5. Use according to claim 4, wherein the antagonist is in the form of a powder, tablet, film formulation, solution, aerosol, granule, lozenge, pill, suspension, emulsion, capsule, syrup, elixir, extract, tincture or fluid extract.
6. A method for preparing an oral micro-ecological environment improving agent using the lactobacillus paracasei strain PC-01 of claim 1, comprising: the active or inactivated lactobacillus paracasei strain PC-01 is directly mixed with the remaining filling.
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