Bad Breath Relief Probiotics

1. Product introduction

Product name: Oral flavor probiotic powder
Product specifications: 2g*30 pieces/box
Indications: The strain composition provided by this product can inhibit the growth of key pathogenic bacteria that cause bad breath, and can effectively relieve the symptoms of bad breath.
Instructions for use: 3 times a day, 1 tablet each time; it is recommended to take the dry powder orally directly after meals. People with chronic bad breath need to take it for a long time until their breath becomes fresh.
Preservation method: Store in a cool place, preferably refrigerated at 4°C.
Viable count: Probiotic mixture (50 billion CFU/g), Bacillus coagulans PB-BC 02 (20 billion CFU/g) and Lactobacillus salivarius HH-LS 17 (30 billion CFU/g).
Other ingredients: 200mg sorbitol, 200mg citric acid, 200mg peppermint powder, 200mg vitamin C, 100mg fructooligosaccharide, 200mg galactooligosaccharides, 100mg sucralose, 100mg food flavor.

2. Pathogenesis

Oral flora is a key component of the oral mucosal barrier and immune response, which can resist pathogen invasion and prevent and treat oral diseases. Oral microecological imbalance can destroy the oral mucosal barrier and provide conditions for the colonization of odor-producing bacteria, thereby causing oral diseases such as halitosis. Oral halitosis (IOH) accounts for 80% to 90% of halitosis and is mainly caused by the production of volatile sulfur compounds by oral microorganisms. Fusobacterium nucleatum can decompose sulfur-containing substrates in the oral cavity into volatile sulfur compounds. Volatile Sulfur Compounds (VSCs), and their metabolites can also produce substances such as skatole, cadaverine, indole, and putrescine, which can cause oral malodor.
At present, the conventional treatments for bad breath mainly include mechanical removal (brushing teeth, tongue scraping, etc.), chemical therapy (chlorhexidine mouthwash, chewing gum, etc.), and the use of traditional Chinese medicine and natural products (tea polyphenols, vitamin C, catechins, etc.). and the use of probiotics, etc. Traditional treatment methods have short-term effects, and long-term use can cause oral flora imbalance. Probiotics can regulate oral flora from the root and inhibit the growth of odor-producing bacteria. Therefore, the use of probiotics to treat halitosis has gradually become a research hotspot.

3. Mechanism of probiotics improving bad breath

Like diseases with microbial causes, such as dental caries and periodontitis, bad breath can be treated microbially by targeting the causative bacteria associated with it. Probiotics can inhibit the growth of oral pathogenic bacteria, regulate the oral flora, and maintain the health of the oral microecology. The main mechanisms by which probiotics work include the following aspects: (1) Compete for binding sites, reduce the colonization of pathogenic bacteria, and co-aggregate with pathogenic bacteria. ; (2) Compete for nutrients and growth factors and produce antibacterial substances; (3) Enhance the host's immune response and inhibit the production of pro-inflammatory cytokines induced by pathogens.

4. Hehe Biotech's probiotics for improving bad breath

The strain number of Bacillus coagulans is PB-BC02 and the name is Bacillus coagulans. It is deposited in the China Type Culture Collection Center at Wuhan University in Wuhan, China. The deposit date is December 12, 2018. The deposit number is CCTCC NO: M2018889.

5. Evaluation of the effect of improving bad breath

In this study, 5 probiotic strains were initially screened by evaluating the ability of probiotics to inhibit the production of H2S by Fusobacterium nucleatum, and further evaluated the ability of 5 probiotic strains to inhibit the production of volatile sulfide compounds (VSCs) and biofilm by Fusobacterium nucleatum. Bacillus coagulans PB-BC 02 and Lactobacillus salivarius HH-LS 17 were two strains of probiotics with good in vitro effects. Finally, the inhibitory rates of single-strain probiotics and compound probiotics on the growth of Fusobacterium nucleatum were compared. The results showed that the complex The inhibitory effect of probiotics on the growth of Fusobacterium nucleatum is better than that of single bacteria, indicating that the combination of Bacillus coagulans PB-BC 02 and Lactobacillus salivarius HH-LS 17 has the effect of improving bad breath. The test methods and results are as follows:

5.1 The ability of probiotics to inhibit H2S production by Fusobacterium nucleatum

Test principle: The reaction of H2S and FeSO4 to produce black ferrous sulfide FeS precipitate is used to evaluate the ability of different probiotics to inhibit H2S production by Fusobacterium nucleatum.
Specific operation: Make a solution of ferrous sulfate and sodium thiosulfate, filter it through a 0.22 μm sterile filter membrane, and add it to the BHI culture medium to make the final concentration of ferrous sulfate and sodium thiosulfate 0.2‰ and thiosulfate 0.3‰, Then, 2% 109CFU/mL Fusobacterium nucleatum bacterial suspension was added, followed by 5% probiotic bacterial solution, and cultured anaerobically at 37°C for 36 h.
Test results: Screen based on the amount of precipitation produced. "-" means no precipitation, indicating that probiotics have strong inhibitory ability against Fusobacterium nucleatum. "+" has a little precipitation, "++" has a lot of precipitation, and " +++" There is a lot of sediment. The results are shown in Table 1. Among the 31 probiotic strains, 11 strains had a little precipitation, 8 strains had a lot of precipitation, 7 strains had a large amount of precipitation, and 5 strains (Lactobacillus acidophilus HH-LA26, Lactobacillus paracasei HH-LP58, Lactobacillus salivarius HH-LS17, Lactobacillus bifidum HH-BL18 and Bacillus coagulans PB-BC 02) showed no precipitation, indicating their potential to improve halitosis.

Table 1 Ability of 31 probiotic strains to inhibit H2S production by Fusobacterium nucleatum

5.2 The ability of probiotics to inhibit the production of volatile sulfur compounds (VSCs) by Fusobacterium nucleatum

Test method: Five strains of probiotics were initially screened based on their ability to inhibit H2S production by Fusobacterium nucleatum, and the VSCs produced by Fusobacterium nucleatum were further quantified using a Halimeter instrument. A total of 2 mL of 107 CFU/mL Fusobacterium nucleatum bacterial suspension and 15% (v/v) probiotic bacterial solution were added to the Hungate test tube. After co-culture for 9 hours, the probiotics' ability to inhibit the production of Fusobacterium nucleatum was measured. capabilities of VSCs.
Test results: VSCs are the main culprit of bad breath. They not only reflect the degree of bad breath, but also damage gum tissue, which is a very important indicator. By measuring the inhibitory rate of the five strains obtained from the preliminary screening on VSCs produced by Fusobacterium nucleatum, the strains were further screened. The results are shown in Table 2. Among them, the inhibitory ability of 3 strains was greater than 80%, and the inhibitory rate of 2 strains was greater than 80%. Below 70%. Therefore, three probiotic strains including Lactobacillus salivarius HH-LS17, Lactobacillus bifidus longum HH-BL18, and Bacillus coagulans PB-BC02 were selected to study their effects on the biofilm formation of Fusobacterium nucleatum.

Table 2 Effect of probiotics on VSCs production by Fusobacterium nucleatum

5.3 The ability of probiotics to inhibit Fusobacterium nucleatum biofilm formation

Test method: Adjust the bacterial concentration of Fusobacterium nucleatum to 107 CFU/mL and let it stand for 9 hours. Then add it to a 96-well plate. Add 180 μL of Fusobacterium aggregates bacterial suspension and 20 μL of probiotic bacterial solution to each well. 37 Cultivation at ℃ for 48 h. In the negative control group, the same volume of MRS medium was added instead of the probiotic liquid, and in the blank control, the same volume of BHI was added instead of the probiotic liquid. After incubation, wash twice with PBS buffer, then fix with 99% methanol for 15 min, discard the supernatant, and dry at room temperature. After drying is complete, add 100 μL of 0.1% crystal violet solution to each well, stain for 5 minutes, wash twice with sterile water after staining, and then place at room temperature until completely dry. Finally, add 200 μL of 33% acetic acid solution for dissolution. After mixing with a pipette, pipet 175 μL from each well and add it to a new 96-well plate, and read the absorbance value at 570 nm with a microplate reader. The calculation method of biofilm-mediated reduction is inhibition rate (%) = (negative control biofilm - amount of supernatant intervention biofilm) / negative control biofilm.
Test results: Periodontal pockets and tongue coating are risk factors for bad breath because they provide an ideal growth environment for bacteria that produce VSCs and other odor molecules. Microorganisms usually exist in the form of biofilms in periodontal pockets and tongue coating. Fusobacterium nucleatum serves as an intermediate bridge bacteria in the formation of dental plaque. The formation of its biofilm is the key to causing bad breath. Therefore, inhibiting its biofilm is an important step in alleviating bad breath. Possibilities are provided. The crystal violet staining method was used to determine the ability of Lactobacillus to inhibit the formation of biofilm by Fusobacterium nucleatum. The results are shown in Table 3. When 5% probiotic bacterial solution was added, Bacillus coagulans PB-BC 02 and Lactobacillus salivarius HH-LS 17 has the best inhibition effect, exceeding 35%. Based on the results of inhibiting the production of VSCs and biofilms by Fusobacterium nucleatum, Bacillus coagulans PB-BC 02 and Lactobacillus salivarius HH-LS 17 have better in vitro effects. Next, their effects on the growth of Fusobacterium nucleatum are evaluated.

Table 3 Inhibitory effects of probiotics on Fusobacterium nucleatum biofilm

5.4 Effect of probiotics on the growth of Fusobacterium nucleatum

Test method: Add 190 μL of 107 CFU/mL Fusobacterium nucleatum bacterial suspension to each well of a 96-well plate, then add 10 μL of probiotic bacterial solution, culture anaerobically at 37°C, and monitor at 600 nm every 3 hours. Measure the absorbance value and draw a growth curve.
Test results: Many oral diseases are caused by imbalance of oral flora. A certain antibacterial ability helps probiotics regulate the oral flora. The results are shown in Figure 1. The inhibitory effect of probiotics on Fusobacterium nucleatum is in the order of compound probiotics > Lactobacillus salivarius HH-LS 17 > Bacillus coagulans PB-BC 02 > MRS medium control group, indicating that both strains of probiotics are It can inhibit the growth of Fusobacterium nucleatum, and the compounding of the two strains has a better inhibitory effect on Fusobacterium nucleatum.

Figure 1 Effect of probiotics on the growth of Fusobacterium nucleatum

In summary, Bacillus coagulans PB-BC 02 and Lactobacillus salivarius HH-LS 17 can inhibit the production of H2S, VSCs and biofilm by Fusobacterium nucleatum, and the ability to inhibit the growth of Fusobacterium nucleatum after compounding is stronger than that of single strains, indicating that The combination of Bacillus coagulans PB-BC 02 and Lactobacillus salivarius HH-LS 17 can improve bad breath.

references:

[1] Karbalaei M, Keikha M, Kobyliak NM, Khatib Zadeh Z, Yousefi B, Eslami M. Alleviation of halitosis by use of probiotics and their protective mechanisms in the oral cavity. New Microbes New Infect. 2021 Apr 23;42:100887. doi: 10.1016/j.nmni.2021.100887. PMID: 34123388; PMCID: PMC8173312.
[2] Penala S, Kalakonda B, Pathakota KR, Jayakumar A, Koppolu P, Lakshmi BV, Pandey R, Mishra A. Efficacy of local use of probiotics as an adjunct to scaling and root planning in chronic periodontitis and halitosis: A randomized controlled trial. J Res Pharm Pract. 2016 Apr-Jun;5(2):86-93. doi: 10.4103/2279-042X.179568. PMID: 27162801; PMCID: PMC4843589.
[3] Jiang Zhentao. Screening of lactobacilli that inhibit Fusobacterium nucleatum and evaluation of their effectiveness in improving halitosis [D]. Jiangnan University, 2022. DOI: 10.27169/d.cnki.gwqgu.2022.000533.
[4] Huang Zhiqiang, Cheng Yongbo. Construction and evaluation of a rat halitosis model caused by oral flora imbalance [J]. Oral Medicine Research, 2023, 39(08): 745-750. DOI: 10.13701/j. cnki.kqyxyj.2023.08.015.
[5] Yang Wenjie, Ye Wei. In vitro study on the antibacterial effect of two kinds of probiotics on halitosis-causing bacteria [J]. Oral Medicine, 2015, 35(03):179-182.DOI:10.13591/j.cnki.kqyx .2015.03.005.

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