CN114806930B

CN114806930B - Probiotic composition and application thereof

Info

Publication number: CN114806930B
Application number: CN202210342926.3A
Authority: CN
Inventors: 赵仕凡; 崔云龙; 尚改双
Original assignee: Qingdao Eastsea Pharmaceutical Co ltd
Current assignee: Qingdao Eastsea Pharmaceutical Co ltd
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-11-22
Anticipated expiration: 2042-03-31
Also published as: CN114806930A

Abstract

The invention discloses a probiotic composition and application thereof, relating to the technical field of micro-ecology; the probiotic composition comprises bacillus coagulans (bacillus coagulans) TBC169, bifidobacterium infantis (bifidobacterium infantis) DH231 and lactobacillus rhamnosus (lactobacillus rhamnosus) DH-Lr-121. The probiotic composition disclosed by the invention has the function of improving dental caries, and has a better effect under a certain compatibility proportion. The probiotic composition prepared by the invention not only can inhibit the growth of cariogenic bacteria streptococcus mutans and improve the dental caries of children, but also can improve the constipation of children.

Description

Probiotic composition and application thereof

Technical Field

The invention relates to the technical field of microecology, in particular to a probiotic composition and application thereof.

Background

Dental caries is a common chronic oral disease caused by an imbalance in the homeostasis between the host and the microflora. It is estimated that 23 million people worldwide have permanent dental caries and more than 5.3 million children have deciduous dental caries. The proportion of schoolchildren affected by caries is 60% -90%, which is common in both developed and developing countries. Dental caries is a cause of pain and discomfort, and in addition to loss of aesthetics, sensitivity to hot and cold beverages is increased, and in addition, dental treatment is costly.

The formation of bacterial plaque biofilm is a prerequisite for caries to occur. The mature plaque biofilm is a three-dimensional micro-ecological environment comprised of a plurality of bacteria embedded in a matrix consisting essentially of water-insoluble polysaccharides having a certain thickness. In normal plaque biofilm, the oral flora is in a state of dynamic equilibrium. When the host, diet or microbial growth factors alter the ecological balance, the acidifying bacteria in the plaque will lower the pH of the biofilm environment. Subsequently, the demineralization-remineralization balance in the tooth shifts to mineral loss, resulting in tooth decay. Streptococcus mutans has a strong acid-producing and acid-fast capacity and has been recognized as the main type of cariogenic bacteria. The large number of exopolysaccharides synthesized by streptococcus mutans is important for the complex three-dimensional structure of dental plaque. In fact, many studies report a relationship between the presence of Streptococcus mutans, biofilm formation, and associated caries risk.

Functional constipation is also one of the most common gastrointestinal diseases in children, and is one of the important factors affecting the healthy growth of children. The global prevalence of constipation symptoms in children varies, estimated in the range of 0.3% to 28%. The imbalance of the intestinal flora is an important factor for inducing constipation.

Currently, several methods have been reported to prevent dental caries in children, the most prominent of which are fluoride (aqueous fluoridated and fluoridated toothpastes) treatment and plaque removal using antibacterial mouthwashes. Other methods of preventing dental caries include the use of pit and fissure sealants on vulnerable pits and crevices of the teeth, but these forms of treatment are expensive. A disadvantage of fluoride therapy is that excessive fluoride intake can lead to bone and plaque teeth. A disadvantage of antibacterial mouthwashes is that even beneficial bacteria in the oral cavity are killed, creating an open, noncompetitive surface for pathogens to re-enter the oral cavity; in addition, prolonged use can result in staining of teeth.

The use of products of traditional Chinese medicines or extracts thereof or enzyme preparations for preventing dental caries has been reported, but these methods or formulations are not well suited for children, and the side effects of traditional Chinese medicines on children are unknown; the method using the enzyme component cannot guarantee the effectiveness of the product because the enzyme is extremely easy to inactivate.

The advantage of probiotics over other prophylactic methods is that they are generally considered safe and have additional health benefits, such as maintaining a healthy gut. The mechanism of action of probiotics on oral health is believed to be antagonistic activity against pathogens, co-aggregation and growth inhibition of pathogens, production of bacteriocins, organic acids and hydrogen peroxide, and interaction with oral epithelium, thereby reducing the pathogenicity and cariogenic potential of biofilm microorganisms. Immunity also appears to be associated with innate and adaptive immune responses that combat oral disease.

At present, only few documents report probiotic methods, but the production cost is high, the process is complex, and no probiotic product capable of simultaneously improving dental caries and constipation of children exists.

Disclosure of Invention

The present invention aims to provide a probiotic composition and its use, which can improve dental caries and constipation of children at the same time, to solve the problems of the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a probiotic composition for simultaneously improving dental caries and constipation of children, which comprises Bacillus coagulans TBC169, bifidobacterium infantis (Bifidobacterium infantis) DH231 and Lactobacillus rhamnosus DH-Lr-121.

Further, the mass ratio of the Bacillus coagulans (Bacillus coagulousns) TBC169 to the Bifidobacterium infantis (Bifidobacterium infantis) DH231 to the Lactobacillus rhamnosus (Lactobacillus rhamnosus) DH-Lr-121 is (0.9-2.8): (0.5-2.5): (1.8-4.2).

Further, the probiotic composition further comprises fructo-oligosaccharide and polydextrose.

Preferably, the mass ratio of Bacillus coagulans (Bacillus coagulans) TBC169 to Bifidobacterium infantis (Bifidobacterium infantis) DH231 to Lactobacillus rhamnosus (Lactobacillus rhamnous) DH-Lr-121 is 2.2:2:3.

the invention also provides a product for simultaneously improving dental caries and constipation of children, which comprises the probiotic composition.

The invention also provides application of the probiotic composition in preparing a product for simultaneously improving dental caries and constipation of children.

The invention discloses the following technical effects:

the probiotic composition prepared by the invention comprises Bacillus coagulans (Bacillus coagulans) TBC169, bifidobacterium infantis (Bifidobacterium infantis) DH231 and Lactobacillus rhamnosus (Lactobacillus rhamnosus) DH-Lr-121, has the function of improving decayed teeth, and has better effect under a certain proportion. Meanwhile, the probiotic composition prepared by the invention not only can inhibit the growth of cariogenic bacteria streptococcus mutans and improve children dental caries, but also has a good improvement effect on children constipation.

Compared with traditional Chinese medicinal materials or traditional Chinese medicinal extracts, the probiotic composition prepared by the invention is safe and free of side effects, the raw materials are suitable for children groups, and the oral micro-ecological balance and the intestinal micro-ecological balance are improved; the preparation process is simple, the cost is low, and a natural bidirectional protection mode is provided for the tooth and intestinal health of children.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a score chart of Streptococcus mutans;

FIG. 2 is a graph of plaque mutans streptococci scores; * Indicates a significant difference (P < 0.01) from the control group; * Shows differences but is not significant (P < 0.05) compared with the control group;

FIG. 3 is a graph showing the growth of Streptococcus mutans; * Indicates a significant difference (P < 0.01) from the control group; * Shows differences but is not significant (P < 0.05) compared with the control group;

FIG. 4 is a graph showing the effect of different formulations on anaerobic bacterial count; * Indicates a significant difference (P < 0.01) from the control group; * Shows differences but is not significant (P < 0.05) compared with the control group;

FIG. 5 is a graph of the effect of different formulations on the number of facultative anaerobes; * Indicates a significant difference (P < 0.01) from the control group; * Shows differences but is not significant (P < 0.05) compared with the control group;

fig. 6 is a graph showing a change in defecation frequency.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

In the following examples, bacillus coagulans (Bacillus coaguluns) TBC169 is deposited in the general microbiological center of the china committee for culture collection of microorganisms with the deposit number of CGMCC No.1207, which is disclosed in patent ZL 200410098660.4;

bifidobacterium infantis (Bifidobacterium infantis) DH231 is separated from normal feces of human body, is identified as Bifidobacterium infantis (Bifidobacterium infantis) by the examination of institute of microbiology of Chinese academy of sciences, is preserved in China general microbiological culture Collection Center (CCM) at 8.6.2019, with the preservation address of No. 3 of West Lu 1 of the sunward area of Beijing, and the preservation number of CGMCC No.18372;

lactobacillus rhamnosus DH-Lr-121 is separated from normal excrement of human body, is tested by institute of microbiology of Chinese academy of sciences and identified as Lactobacillus rhamnosus, is preserved in the general microbiological center of China Committee for Culture Collection of Microorganisms (CCM) 30 th 2016 (9 th month), has the preservation address of No. 3 of Xilu 1 of North Chen of the sunward area in Beijing, and has the preservation number of CGMCC No.13076.

Example 1

The matching proportion of the probiotic composition for optimally inhibiting the streptococcus mutans is determined by testing the streptococcus mutans level research in saliva and plaques:

1. population screening

This example screened 126 children, and 48 were selected based on inclusion and exclusion criteria. The mean age of the patients was 8.5 ± 1.6 years (range 5-15 years). 20 men (41.67%) and 28 women (58.33%). The children were divided into 6 groups by randomization (computer generated random numbers), 8 persons each, probiotic a, B, C, D, E and control group (control), with the ingredient ratios of each formula group shown in table 1 below. The sample size for each group of children was based on confidence level (95%) and error range (1%). No conjugate during the study; thus, all 48 children completed the study (fig. 1). The dosage and time of the probiotic formula group and the control group are the same, and the probiotic formula group and the control group are dissolved in water and then taken for 2 times a day (morning and evening) for 30 days. Selected 48 children were provided oral hygiene instructions after screening (screening day) and were asked to maintain oral health during treatment, prior to study initiation. Instructions are provided regarding the proper brushing regimen, as well as the necessity of brushing twice daily and forceful rinsing after meals. However, they were instructed not to brush their teeth 24 hours before the 1 st sampling test (day after screening and selection) and the 2 nd sampling test (after 14 days of treatment) in order to collect plaque easily.

Table 1 mass ratio of material components of each formulation group

2 selection criteria

2.1 inclusion criteria

The study included children of any sex (including constipated or non-constipated people) between 5 and 15 years of age with a higher caries risk (DMFS/defs ≧ 5). Other inclusion criteria: children who did not take any form of antibiotics or probiotics prior to the study three months, children who were willing to chew tablets and participate in the study, parents of the subjects were willing to provide written informed consent and follow the learning procedure.

2.2. Exclusion criteria

Exclusion criteria included children with severe infections, systemic disease, weakened immune systems, and congenital abnormalities; children receiving fluoride treatment; children using xylitol chewing gum, probiotic products, antibiotics or corticosteroids within three months after study initiation; and those who are unwilling to participate in the study.

3. Collection of saliva samples

Carious saliva samples were collected from 9 to 10 am (Lashley cups did not stimulate sublingual and submandibular gland fluid collection) to avoid any diurnal variation.

4. Collection of plaque samples

Supragingival plaque was collected from all teeth using a sterile toothpick. The samples were then transferred to sterile tubes and diluted at a ratio of 1.

5. Estimation of saliva and plaque Streptococcus mutans and Lactobacillus

The number of saliva and plaque mutans streptococci and lactobacilli was estimated by a chair-side test (CRT Bacteria, ivoclar Vivadent AG, schaan, liechtenstein). After colonies were identified and counted, the Streptococcus mutans scored a range of 1-4 based on the number of colony forming units (cfu/mL) (Table 2).

TABLE 2

6 statistical analysis

Using the t-test, the measurement data are expressed as mean values with SD. Values of P < 0.01 were considered significant; p < 0.05 is different but not significant.

7. As a result, the

7.1. Levels of Streptococcus mutans

As shown in fig. 1, there was a different reduction in the streptococcus salivarius levels (as indicated by the score) after 30 days of probiotic administration in each formula compared to the control group, and there was a significant change (P < 0.01) in formula C compared to the control group; while the E formula group was changed but not significant compared to the control group; compared with the control group, the streptococcus mutans of other formula groups is reduced to a certain extent, but has no significant difference compared with the control group. According to the change, the inhibition degrees of different formula groups on the salivary streptococcus mutans are different, and the experiment proves that the inhibition effect of the formula group C on the salivary streptococcus mutans is the best.

7.2 changes in plaque Streptococcus mutans

As shown in fig. 2, after 30 days of probiotic administration in each formula group, there was a different reduction in the level of streptococcus mutans in plaque samples compared to the control group; compared with the control group, the C formula group and the E formula group, the inhibition effect on the streptococcus mutans in the plaque is obviously changed compared with the control group (P is less than 0.01); the inhibition effect of the formula A on plaque streptococcus mutans is also better, and compared with a control group, the inhibition effect is different but not significant (P is less than 0.05); the remaining groups B and D also showed inhibitory effects on Streptococcus mutans in the plaques, but were less effective than groups C and E.

Example 2 testing of different formulations for antibacterial Activity against Streptococcus mutans (bacterial biofilm, bacterial adhesion)

The experimental method comprises the following steps: to determine the antibacterial activity of the different formulations, streptococcus mutans was cultured overnight in BHI broth at 37 ℃. Streptococcus mutans cultures were diluted with BHI broth to turbidity equivalent to McFarland 0.5 (1.5X 10) ⁸ Individual cells/mL). Then, 100. Mu.L of the Streptococcus mutans suspension and 100. Mu.L of the supernatant treated by each formulation group were added to wells of a 96-well microtiter plate and repeated 6 times. The plates were then incubated anaerobically at 37 ℃ for 24 hours. In control wells, the culture fluid was sterile MRS broth. After incubation the OD (600 nm) was recorded using a microplate reader.

The experimental results are as follows: as shown in fig. 3, each formula group showed significant inhibition of the growth of streptococcus mutans, with no significant difference between formula groups; but the formula of the group C shows the most obvious inhibition effect compared with the control group (the significant difference P between the formula and the control group is less than 0.01); in addition, the inhibition effect of the A formula group and the E formula group on the streptococcus mutans is only slightly inferior to that of the C group, and the inhibition effect is different from that of the control group (p < 0.05).

Example 3 Effect of different formulation set products on the Total number of anaerobes and facultative anaerobes

Collecting dental bacteria samples of decayed children: and scraping the inner side and the outer side of each tooth of the children to be tested by using a cotton swab at 0, 3 and 5 weeks of the children to be tested taking the probiotic products of the formula groups and the commercial products and the products of the control group respectively to be used as tooth bacteria sampling samples. The number of anaerobes and facultative anaerobes in the culture medium is tested. The experimental results are shown in fig. 4 and 5. As can be seen from figures 4 and 5, the probiotic products of each formula can effectively increase the number of oral anaerobes and facultative anaerobes and help to restore the oral microecological balance. The formula C can obviously improve the quantity of oral anaerobes and facultative anaerobes compared with a product group sold in the market, and has obvious difference compared with a control group, and other formula groups can also obviously improve the quantity of oral flora compared with the control group and the product group sold in the market, but the effect is not better than that of the formula C.

From the experimental results of the above examples, it can be seen that, in the probiotic compositions of the formula groups, the probiotic composition of the formula group C has the best inhibition effect on streptococcus mutans, and can effectively improve the oral flora balance and restore the oral micro-ecological balance.

The preparation (BC, BE) of the probiotic product is carried out based on the C formula group with the best inhibition effect on the streptococcus mutans and the E formula group with the next best inhibition effect, and the improvement effect of the probiotic product on the functional constipation of children is further considered.

BC formula: 2.0g of bifidobacterium infantis DH231 bacterial powder, 3.0g of lactobacillus rhamnosus DH-Lr-121 bacterial powder,

2.2g of Bacillus coagulans TBC169 bacterial powder, 28g of fructo-oligosaccharide, 21g of polydextrose and 9.9g of acetylated distarch adipate.

BE formula: 2.4g of bifidobacterium infantis DH231 bacterial powder, 3.5g of lactobacillus rhamnosus DH-Lr-121 bacterial powder,

2.8g of Bacillus coagulans TBC169 bacterial powder, 17g of fructo-oligosaccharide, 24g of polydextrose and 8.8g of acetylated distarch adipate.

BC and BE are prepared by the same preparation process, and the preparation procedures are as follows:

(1) Weighing and mixing: weighing raw and auxiliary materials according to the formula; the materials which are checked to be correct are put into a mixer (HDA-800 type multidirectional motion mixer) one by one according to the sequence from more to less, the rotating speed of a motor is set to be 27 revolutions per minute (the rotating speed of a main shaft is 6 revolutions per minute), the materials are mixed for 20 minutes, and the mixing time is strictly controlled in the mixing process.

(2) Inner packaging: the composite film and the color ribbon are installed on the SK series strip-shaped packaging machine, the machine is started to preheat, the no-load machine adjustment is carried out, then the mixed powder is added into the hopper, and the loading amount is controlled to be stable in the inner packaging process.

(3) Outsourcing: and carrying out an outsourcing process on the internally wrapped product.

Example 4 Constipation-improving effects of the formulation and commercial products on Children

We performed a prospective, double-blind, placebo-controlled, randomized and parallel group trial. Pediatric constipation patients were collected from pediatric clinics (ages 1-8 years; constipation was at least 1 month, and included at least two of the following criteria: twice or less bowel movements per week, history of bowel retention, bowel pain or hard bowel movements; clinic a: n =2 b n =20 c. Subjects with known organic causes of constipation, such as hirschsprung, spina bifida, milk allergy and metabolic disease, were excluded. Patients were assessed for baseline constipation within two weeks using a bowel diary before they were included in the study. The above child patients were randomized into 4 groups: group a (n = 14) received the formula BC probiotic product; group B (n = 12) received the BE probiotic product; group C (n = 10) received commercial dietary fiber active probiotic powder I; group D (n = 10) received commercial dietary fiber active probiotic powder II. The taking method is as follows: the dose is 2g per person 2 times per day.

The defecation frequency was evaluated by the defecation diary. The caregiver evaluates the diary to check the defecation status (stool frequency, stool consistency, and stool volume). Stool consistency was scored according to the Bristol stool form scale and was of type 1-7 (type 1: discrete hard pieces such as nuts; type 2: sausage-like but lumpy; type 3: sausage-like or snake-like but with surface cracks; type 4: smooth and soft like sausage or snake; type 5: soft lumps with sharp edges; type 6: fluffy pieces, ragged edges, pasty stools; type 7: watery, no solid pieces). Patients went to an outpatient clinic two weeks prior to dosing and zero, two, and four weeks after inclusion in the study.

As can BE seen from fig. 6, the probiotic products BC and BE have better improvement effect on constipation of children than the commercial products, and the BC product has longer improvement benefit on constipation of children than the BE product, although the BC product has a better defecation promotion effect in a short period (2 weeks) than the BE and the commercial products, the defecation frequency is greatly improved after 4 weeks compared with the rest groups of products, which shows that the BC product improves the intestinal microecological balance by gradually improving the balance of intestinal flora, helps constipation children to recover the natural intestinal peristalsis defecation function, rather than realizing a rapid defecation effect through a short-term effect, and is more beneficial to the health of children.

In conclusion, the probiotics product prepared by the formula C has the best effect of improving the dental caries and the constipation of children.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A probiotic composition for simultaneously improving dental caries and constipation of children, which is characterized by comprising bacillus coagulans (Bacillus coagulans)Bacillus coagulans) TBC169, bifidobacterium infantis: (B)Bifidobacterium infantis) DH231 and Lactobacillus rhamnosus (Lactobacillus rhamnosus) DH-Lr-121; the bacillus coagulans TBC169 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.1207; the bifidobacterium infantis DH231 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.18372; the lactobacillus rhamnosus DH-Lr-121 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No.13076;

the mass ratio of the bacillus coagulans TBC169 to the bifidobacterium infantis DH231 to the lactobacillus rhamnosus DH-Lr-121 is 2.2:2:3.

2. the probiotic composition according to claim 1, characterized in that it further comprises fructooligosaccharides and polydextrose.

3. A product for simultaneously improving dental caries and constipation in children comprising the probiotic composition of claim 1 or 2.

4. Use of a probiotic composition according to claim 1 or 2, for the preparation of a product for simultaneously ameliorating dental caries and constipation in children.