Application of lactobacillus helveticus ZJUIDS11 in relieving ozostomia

Abstract

The invention belongs to the field of microorganisms, and particularly provides application of Lactobacillus helveticus ZJUIDS11: relieving and improving ozostomia (breath), and also provides application of the Lactobacillus helveticus ZJUIDS11 as well as application of the Lactobacillus helveticus ZJUIDS11 as an anti-inflammatory agent. The preservation number of the lactobacillus helveticus ZJUIDS11 is CGMCC (China General Microbiological Culture Collection Center) NO.24761, and the preservation number of the lactobacillus helveticus ZJUIDS11 is CGMCC NO.24761. The lactobacillus helveticus ZJUIDS11 can be used for inhibiting hydrogen sulfide and methyl mercaptan.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to application of Lactobacillus helveticus ZJUIDS11 in relieving halitosis (bad breath). BACKGROUND

[0002] Halitosis, also known as breath malodor or oral malodor, is the emission of unpleasant breath odor from the oral cavity, which often brings inconvenience to people's social activities and causes psychological pressure. It can affect people's oral health and may be exacerbated by poor oral hygiene. Halitosis mainly originates from the oral cavity, and is mainly caused by volatile sulfides, including hydrogen sulfide, methyl mercaptan and dimethyl sulfide, which are produced by Fusobacterium nucleatum through the putrefactive digestion of food residues, saliva and exfoliated cells retained in the oral cavity. Among them, hydrogen sulfide and methyl mercaptan account for 90%, which are the main volatile sulfides causing halitosis. More and more studies have confirmed that oral inflammation not only causes damage to oral health, but also is related to the occurrence and development of many systemic diseases, such as colorectal cancer, chronic hepatitis, chronic kidney disease, heart disease, etc. Halitosis can further aggravate periodontitis, pulpitis, calculus, and even cause tooth loss, oral squamous cell carcinoma, pulp infection and other oral diseases.

[0003] For the treatment of halitosis, various compounds such as chlorine dioxide, chlorhexidine and metal salts such as zinc salts have certain effect on preventing halitosis, but the taste is irritating during rinsing, which is difficult to adapt. Chlorhexidine is another valuable antiseptic that can inhibit a variety of microorganisms and control plaque accumulation, thereby reducing halitosis. Its method of action is to penetrate the cell membrane of oral bacteria, causing cell leakage and disruption of bacterial metabolism, thereby inhibiting cell growth. Long-term use can cause taste abnormalities and cause reversible staining on the teeth and mucosal surfaces. Therefore, research has shifted to finding the effects of natural ingredients, including various probiotics. Probiotics are defined as "live microorganisms that confer health benefits on the host when administered in adequate amounts", and interest in probiotics for mild oral health has gradually developed into an attractive means of preventing oral diseases.

[0004] In recent years, probiotics have become a research hotspot for alleviating halitosis. Many studies have shown that probiotics can reduce the number of odor-causing bacteria in the oral cavity and regulate the oral microbiota, thereby reducing halitosis and playing a crucial role in preventing or treating halitosis. Compared with ordinary drugs, probiotics have the advantages of high safety, no side effects, no drug resistance, and low cost. Lactobacillus helveticus is a gram-positive bacterium belonging to the genus Lactobacillus. Lactobacillus helveticus has the function of regulating intestinal flora and balancing intestinal flora. By increasing the number of beneficial bacteria and reducing the number of harmful bacteria, it promotes the balance of intestinal flora and maintains intestinal health. Lactobacillus helveticus also has the effect of inhibiting cholesterol absorption, reducing blood lipids and blood pressure, and has a certain protective effect on cardiovascular health. It has many physiological functions that are beneficial to human health. Due to its recognized safety and antibacterial spectrum mainly targeting pathogenic bacteria, Lactobacillus helveticus has little interference with the homeostasis between the human body and the symbiotic microbiota, and has the potential to become a biological means of regulating the oral microenvironment.

[0005] The patent CN114703092A discloses Lactobacillus plantarum CCFM1214, which significantly inhibits the biofilm formation of Fusobacterium nucleatum, reduces the amount of biofilm by 68.61%, and reduces the generation of volatile sulfides, improving the effect of halitosis better than traditional strains. By inhibiting the expression of the key gene (fn1220) of Fusobacterium nucleatum producing H2S, it fundamentally reduces the root cause of halitosis. In clinical experiments, it can reduce the oral VSCs value of volunteers by 44.29%.

[0006] The invention CN114917176B discloses a composite probiotic that helps improve halitosis. The composite probiotic includes Lactobacillus plantarum CCFM1214 and Ligilactobacillus salivarius CCFM1215. It can be used to prepare products for alleviating halitosis.

[0007] The invention CN114621895A discloses Ligilactobacillus salivarius CCFM1215, which can inhibit Fusobacterium nucleatum and improve oral odor.

[0008] Patent CN116200307A "Cat oral care probiotic composition and its application" discloses a probiotic composition, including animal bifidobacterium and lactic acid bacteria, the lactic acid bacteria are any one or more combinations of saliva lactobacillus, lactobacillus casei, enterococcus faecalis, lactobacillus rhamnosus, lactobacillus helveticus, lactobacillus acidophilus and lactobacillus reuteri, which can effectively improve the cat oral source of halitosis problem from the source, and also can reduce dental plaque formation and alleviate gingivitis and dental caries problems, and can be applied to prepare cat oral odor cleaner.

[0009] Patent CN118126896A "Saliva combined lactobacillus and its use in improving oral health" discloses that saliva combined lactobacillus has good inhibitory effect on common oral pathogenic bacteria such as streptococcus mutans, fusiform bacteria with nucleus and porphyromonas gingivalis, can coagulate with these pathogenic bacteria, and has anti-inflammatory effect, thus helping to improve oral health.

[0010] Lactobacillus helveticus R0052 has the effect of improving gastrointestinal function, thereby reducing halitosis caused by gastrointestinal problems; the invention "Probiotic composition and preparation method thereof" of CN113980844A discloses that the probiotic composition contains lactobacillus helveticus R0052, which has antagonistic effect on oral streptococcus mutans, so it can be reasonably expected to inhibit the colonization of streptococcus mutans in the oral cavity, thereby regulating the oral microbial environment, and achieving the effect of preventing and relieving dental caries.

[0011] Lactobacillus helveticus L10 has the effect of improving oral odor, and the invention "Vaginal health probiotic composition and its application" of CN112342175B discloses that the combination of lactobacillus plantarum L-137 and lactobacillus helveticus L10 can be used to prepare vaginal health health products, feed and feed additives.

[0012] The invention "Lactobacillus helveticus for improving oral health and probiotic composition" of CN118146972A discloses that the lactobacillus helveticus K6 with preservation number CGMCC No.15702 has the effect of improving oral health.

[0013] The invention "Use of heat-treated lactobacillus and composition for inhibiting adhesion of oral pathogenic bacteria" of CN107281228A is used to prepare a medicine for inhibiting the adhesion of oral pathogenic bacteria, and the lactobacillus is a lactobacillus casei GMNL-143, a lactobacillus helveticus GMNL-164 or a lactobacillus rhamnosus GMNL-464, and the invention further relates to a composition for inhibiting the adhesion of oral pathogenic bacteria.

[0014] The invention of CN110063374A, "Composite probiotic composition for improving breath, lozenge and preparation method thereof", discloses that the composite probiotic composition comprises Lactobacillus paracasei Z6, Lactobacillus plantarum Lp-6, Lactobacillus plantarum P9 and Lactobacillus rhamnosus M9. The above-mentioned bacteria must be used at the same time to remove volatile sulfur compounds that cause bad breath.

[0015] In summary, using probiotics to prevent and treat bad breath may be a potential approach, and screening lactic acid bacteria strains with special efficacy and using them for production can better utilize their value. However, there are still relatively few microecological products targeting bad breath on the market, and it is still urgently needed to develop probiotics targeting bad breath and provide more diverse probiotic solutions.

[0016] The invention of CN 116064286 B, "Lactobacillus helveticus ZJUIDS11 with improved non-alcoholic liver disease and its application", discloses a Lactobacillus helveticus ZJUIDS11 with a preservation number of CGMCC NO.24761. The application of the Lactobacillus helveticus ZJUIDS11 in preparing products for improving non-alcoholic liver disease is also disclosed. SUMMARY

[0017] The technical problem to be solved by the present application is to provide a new use of Lactobacillus helveticus ZJUIDS11 for relieving and improving bad breath (mouth odor), thereby solving the problem of oral health.

[0018] To solve the above technical problem, the present application provides the use of Lactobacillus helveticus ZJUIDS11 for relieving and improving bad breath (mouth odor); the preservation number of Lactobacillus helveticus ZJUIDS11 is CGMCC NO.24761.

[0019] Specifically, this invention provides a novel use for Lactobacillus helveticus ZJUIDS11 (CGMCC NO. 24761) in the preparation of products (including pharmaceuticals) that have the function of relieving and improving halitosis.

[0020] An improvement to the use of Lactobacillus helveticus ZJUIDS11 of the present invention: Lactobacillus helveticus ZJUIDS11 can inhibit hydrogen sulfide and methanethiol (hydrogen sulfide and methanethiol produced by Fusobacterium nucleatum).

[0021] As a further improvement to the use of Lactobacillus helveticus ZJUIDS11 of the present invention: Lactobacillus helveticus ZJUIDS11 can inhibit the hydrogen sulfide emission of Fusobacterium nucleatum.

[0022] This invention provides a strain of *Lactobacillus helveticus* ZJUIDS11 that can prevent and improve halitosis and its applications. The ability of *Lactobacillus helveticus* ZJUIDS11 to improve halitosis and oral health was investigated by detecting its inhibitory effect on the levels of hydrogen sulfide and methanethiol produced by *Fusobacterium nucleatum*. Note: Methanethiol and hydrogen sulfide are the main volatile sulfur compounds (VSCs) causing halitosis.

[0023] This invention also provides Lactobacillus helveticus. (Lactobacillus helveticus The method for culturing strain ZJUIDS11 or its pure culture includes inoculating it into MRS liquid medium and carrying out expansion culture to obtain a culture of proliferated Lactobacillus helveticus.

[0024] This invention also provides Lactobacillus helveticus. (Lactobacillus helveticus Application of ZJUIDS11 in the preparation of preparations (live bacteria preparations) with the function of preventing and improving halitosis; that is, application in the preparation of preparations with the function of inhibiting oral pathogenic bacteria.

[0025] This invention provides a strain of Lactobacillus helveticus ZJUIDS11 that can relieve halitosis. Compared with existing Lactobacillus helveticus strains, the Lactobacillus helveticus ZJUIDS11 of this invention has the advantage that this strain can inhibit the production of hydrogen sulfide and methanethiol to relieve halitosis. The Lactobacillus helveticus strain of this invention can provide a theoretical basis for the development of probiotic products.

[0026] This invention also provides Lactobacillus helveticus. (Lactobacillus helveticus Application of ZJUIDS11 in the preparation of products that improve halitosis. Lactobacillus helveticus ZJUIDS11 or its pure culture can be used to prepare probiotic tablets, live bacteria preparations, fermented milk products, bacterial powder, solid beverages, chewing gum, mouthwash, etc. Attached Figure Description

[0027] The specific embodiments of the present application will be further described below in conjunction with the accompanying drawings.

[0028] Figure 1 Figure for detecting hydrogen sulfide produced by Lactobacillus helveticus ZJUIDS11 itself in the present application.

[0029] Figure 2 Figure for detecting methyl mercaptan produced by Lactobacillus helveticus ZJUIDS11 itself in the present application.

[0030] Figure 3 Figure for effect of Lactobacillus helveticus ZJUIDS11 on inhibiting production of hydrogen sulfide by Fusobacterium nucleatum in the present application.

[0031] Figure 4 Figure for effect of Lactobacillus helveticus ZJUIDS11 on inhibiting production of methyl mercaptan by Fusobacterium nucleatum in the present application.

[0032] Figure 5 Figure for detecting discharge amount of hydrogen sulfide produced by Fusobacterium nucleatum inhibited by Lactobacillus helveticus ZJUIDS11 in the present application. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with specific examples, but the scope of protection of the present application is not limited to the following: Example 1, preparation of Fusobacterium nucleatum bacterial suspension and supernatant of Lactobacillus helveticus ZJUIDS11 according to conventional techniques: Preparation of Fusobacterium nucleatum bacterial suspension: Fusobacterium nucleatum ATCC25586 was selected, a preservation tube of Fusobacterium nucleatum was taken out from a-80℃ refrigerator, slightly shaken, and a porcelain strain was picked up with a gun head, and streaking operation was performed on a BHI blood agar plate culture medium, and anaerobic culture was performed at 37℃ for 24h, and after single colonies were obtained, single colonies were picked up and streaked on a BHI blood agar plate culture medium to obtain single colonies. Single colonies were inoculated in a BHI liquid culture medium for large-scale culture to obtain Fusobacterium nucleatum bacterial liquid. The bacterial liquid was centrifuged at 8000r, 4℃ for 10min to obtain Fusobacterium nucleatum bacterial bodies, which were washed twice with PBS solution, and the Fusobacterium nucleatum bacterial bodies were resuspended in PBS solution to 10 7 CFU / ml to obtain Fusobacterium nucleatum bacterial suspension.

[0034] Preparation of supernatant of Lactobacillus helveticus ZJUIDS11: The strain ZJUIDS11 stored in a glycerol tube was first streaked on a MRS agar plate for 2-3 times of activation, and then single colonies were picked up and cultured in a MRS liquid culture medium (MRS broth) at 37℃ for about 24h, and the concentration of the bacterial liquid reached about 10 10 CFU / mL, which was used as bacterial suspension.

[0035] The bacterial suspension was centrifuged at 8000g for 5 min at 4°C to collect the supernatant, which was filtered through a 0.22-micron filter to obtain the supernatant of Lactobacillus helveticus ZJUIDS11.

[0036] The following cases use the above Fusobacterium nucleatum bacterial suspension and the supernatant of Lactobacillus helveticus ZJUIDS11.

[0037] Example 2, Inhibition ability of Lactobacillus helveticus ZJUIDS11 on Fusobacterium nucleatum 1. Preparation of BHI blood agar plate medium: BHI broth powder, agar powder and distilled water were mixed in a ratio of 3.7g:2g:100ml, pH 7.2-7.4, autoclaved at 121°C for 15 minutes, and then 5% sterile defibrillated sheep blood was added to the medium after the medium was cooled to 55°C, mixed well, poured into a clean bacterial culture dish, cooled, and stored in a 4°C refrigerator for standby, thereby obtaining the BHI blood agar plate medium.

[0038] 2. Preparation of BHI liquid medium: 11.1g of BHI broth powder was weighed and added to 300ml of distilled water, pH 7.2-7.4, autoclaved at 121°C for 15 minutes, and cooled to room temperature for standby.

[0039] 3. Oxford cup inhibition method was used to determine the inhibition ability of Lactobacillus helveticus ZJUIDS11 on Fusobacterium nucleatum, and the specific steps were as follows: Oxford cups were placed on empty bacterial culture dishes, and the Fusobacterium nucleatum bacterial suspension was added to the BHI blood agar plate medium cooled to 55°C in a volume fraction of 1% (15ml / dish), mixed well, and then poured into the bacterial culture dish. After complete cooling, the Oxford cups were removed, and 200ul of the prepared supernatant of Lactobacillus helveticus ZJUIDS11 was added to the wells, and MRS liquid medium was used as a blank control. The culture was incubated at 37°C for 24h under anaerobic conditions, and then the size and diameter of the inhibition zone were measured with a vernier caliper. Whether there was a significant inhibition zone was observed, and the test was repeated three times.

[0040] The results are shown in Table 1. Lactobacillus helveticus ZJUIDS11 had a significant inhibitory effect on Fusobacterium nucleatum, with an inhibition zone diameter of 14.05mm, indicating that the probiotic bacteria could effectively inhibit the growth of Fusobacterium nucleatum and had certain potential.

[0041] Table 1 Inhibition effect of Lactobacillus helveticus ZJUIDS11 on Fusobacterium nucleatum

[0042] Example 3, Hydrogen sulfide and methanethiol detection test of Lactobacillus helveticus ZJUIDS11 1. Hydrogen sulfide detection test of Lactobacillus helveticus ZJUIDS11 Cut filter paper into 18 mm diameter circular paper pieces, pad on top of 15 mL disposable test tube cap, sterilize by high pressure steam at 121℃ for 30 min, dry, soak the filter paper with 0.3% lead acetate solution, air dry until no dripping, ready for use. Inoculate Lactobacillus helveticus ZJUIDS11 supernatant into fresh MRS liquid medium at 1% inoculation amount, tighten the test tube cap, use MRS liquid medium as blank control, use Fusobacterium nucleatum bacterial suspension (Fn) as positive control, after anaerobic culture at 37℃ for 5 d, take out the circular filter paper piece, observe the color change of the filter paper.

[0043] As shown in Figure 1 , neither Lactobacillus helveticus ZJUIDS11 nor the blank control group changed the color of the lead acetate test paper, while the positive control group with Fusobacterium nucleatum bacterial suspension changed the color of the lead acetate test paper to black, indicating that Lactobacillus helveticus ZJUIDS11 does not produce hydrogen sulfide gas.

[0044] 2. Self-produced methanethiol test of Lactobacillus helveticus ZJUIDS11 Inoculate Lactobacillus helveticus ZJUIDS11 supernatant into MRS liquid medium at 1% inoculation amount, anaerobic culture at 37℃ for 24 h. Take 1 mL of bacterial solution, add 50 μL of 0.6% (w / v) L-methionine solution and 50 μL of 0.06% (w / v) DTNB solution, continue to place at 37℃ for 12 h. Centrifuge at 10000 r for 5 min. Use MRS liquid medium as blank control, use Fusobacterium nucleatum as positive control.

[0045] The principle of colorimetric detection of methanethiol is that DTNB reacts with the thiol group of methanethiol to synthesize yellow 5-thio-2-nitrobenzoic acid (salt) anion (TNB2-). As shown in Figure 2 , the supernatant of Lactobacillus helveticus ZJUIDS11 did not turn yellow after treatment with DTNB and L-methionine, while the supernatant of the positive control turned yellow obviously, indicating that Lactobacillus helveticus ZJUIDS11 does not produce methanethiol gas and does not cause halitosis.

[0046] DTNB: 5,5'-dithiobis (2-nitrobenzoic acid).

[0047] Example 4, Lactobacillus helveticus ZJUIDS11 inhibits the production of hydrogen sulfide and methanethiol by Fusobacterium nucleatum 1. Lactobacillus helveticus ZJUIDS11 inhibits the production of hydrogen sulfide by Fusobacterium nucleatum

[0048] F. nucleatum 50 μL, then add L. helveticus ZJUIDS11 supernatant 50 μL, 37 ℃ anaerobic incubation for 48 h, add 50 μl L-cysteine (0.06%, w / v) and 50 μl lead acetate (0.24%, w / v) to each well, and further anaerobic incubation for 12 h, and measure the H2S produced by spectrophotometry at 550 nm and observe the color change. The control group uses the same volume of blank MRS liquid medium instead of L. helveticus ZJUIDS11 supernatant, and the principle of this experiment is that lead acetate turns black when it meets hydrogen sulfide gas.

[0049] As shown in FIG. 1, the color of the 96-well plate in the L. helveticus ZJUIDS11 group did not change significantly, while the color of the solution in the control group turned black obviously; the OD550 of the L. helveticus ZJUIDS11 group was significantly different from that of the control group, indicating that L. helveticus ZJUIDS11 can inhibit the production of hydrogen sulfide gas by F. nucleatum. Figure 3 The hydrogen sulfide inhibition rate results are shown in Table 2. L. helveticus ZJUIDS11 has a significant inhibitory effect on the production of hydrogen sulfide by F. nucleatum, with an inhibition rate as high as 83.10%.

[0050] Hydrogen sulfide inhibition rate (%) = (blank control group OD550 - experimental group OD550) / blank control group OD550.

[0051] Table 2 Inhibitory effect of L. helveticus ZJUIDS11 on the production of hydrogen sulfide by F. nucleatum

[0052] 2. Test of L. helveticus ZJUIDS11 inhibiting the production of methanethiol by F. nucleatum

[0053] F. nucleatum 50 μL, then add L. helveticus ZJUIDS11 supernatant 50 μL, 37 ℃ anaerobic incubation for 48 h, add 50 μl L-cysteine (0.06%, w / v) and 50 μl lead acetate (0.24%, w / v) to each well, and further anaerobic incubation for 12 h, and measure the H2S produced by spectrophotometry at 550 nm and observe the color change. The control group uses the same volume of blank MRS liquid medium instead of L. helveticus ZJUIDS11 supernatant, and the principle of this experiment is that lead acetate turns black when it meets hydrogen sulfide gas. As shown in FIG. 1, the color of the 96-well plate in the L. helveticus ZJUIDS11 group did not change significantly, while the color of the solution in the control group turned black obviously; the OD550 of the L. helveticus ZJUIDS11 group was significantly different from that of the control group, indicating that L. helveticus ZJUIDS11 can inhibit the production of hydrogen sulfide gas by F. nucleatum.

[0054] Figure 4 ​As shown, the color of the 96-well plate in the Lactobacillus helveticus ZJUIDS11 group did not change significantly, while the solution in the control group turned significantly yellow. The OD430 of the Lactobacillus helveticus ZJUIDS11 group was significantly different from that of the control group, indicating that Lactobacillus helveticus ZJUIDS11 inhibits the production of methanethiol gas by Fusobacterium nucleatum.

[0055] The results of methanethiol inhibition rate are shown in Table 3. Lactobacillus helveticus ZJUIDS11 has a significant inhibitory effect on methanethiol produced by Fusobacterium nucleatum, with an inhibition rate as high as 86.88%.

[0056] Table 3. Inhibitory effect of Lactobacillus helveticus ZJUIDS11 on methanethiol production by Fusobacterium nucleatum.

[0057] Comparative experiment: Lactobacillus helveticus R0052, Lactobacillus helveticus Lafti® L10, Lactobacillus helveticus K6, and Lactobacillus helveticus GMNL-164 were subjected to an "inhibition of methanethiol production by Fusobacterium nucleatum" test according to Example 4 above. The results showed that the inhibition rate was less than 10%.

[0058] Example 5: Effect of Lactobacillus helveticus ZJUIDS11 on hydrogen sulfide emissions from Fusobacterium nucleatum To evaluate the headspace concentration of hydrogen sulfide, an Enkis portable gas detector was used to detect the concentration of hydrogen sulfide.

[0059] Lactobacillus helveticus ZJUIDS11 supernatant was inoculated into BHI liquid medium at a concentration of 15%, and Fusobacterium nucleatum suspension was inoculated into BHI liquid medium at a concentration of 1%. The media were sealed with sealing film and anaerobically cultured at 37°C for 24 hours. Hydrogen sulfide emissions were measured using an Enkiss portable gas detector. An equal volume of blank MRS liquid medium was used as a control group instead of the Lactobacillus helveticus ZJUIDS11 supernatant.

[0060] like Figure 5 As shown, the hydrogen sulfide emissions from the Lactobacillus helveticus ZJUIDS11 group were significantly lower than those from the MRS control group, indicating that Lactobacillus helveticus ZJUIDS11 significantly inhibits the emission of hydrogen sulfide gas from Fusobacterium nucleatum.

[0061] The results of hydrogen sulfide emission inhibition rate are shown in Table 4. Lactococcus lactis ZJUIDS-C004 has a significant inhibitory effect on hydrogen sulfide emission produced by Fusobacterium nucleatum, with an inhibition rate as high as 71.99%.

[0062] Table 4. Inhibitory effect of Pietrococcus lactis ZJUIDS-C004 on hydrogen sulfide emission from Fusobacterium nucleatum.

[0063] Example 6 Preparation of probiotic tablet using Lactobacillus helveticus ZJUIDS11 6.1. Process flow of probiotic tablet: Raw materials→weighing→sifting→premixing→dry mixing→tabletting→packaging→storage; 6.2. Operation points 1) Ingredients: weigh each raw material according to the weight component and mix evenly; in this example, the ratio of each active probiotic and the total number of viable bacteria per kilogram (kg) of tablet weight is: Lactobacillus helveticus: 3.0 x 10 12 CFU; fructooligosaccharides 400 g, xylooligosaccharides 40 g, and the rest is tablet excipient (microcrystalline cellulose and magnesium stearate), base powder water activity (Aw) 0.15-0.18; 2) Sifting: sift the ingredients through a 20-mesh sieve; 3) Premixing of probiotic powder and prebiotics (fructooligosaccharides, xylooligosaccharides); 4) Dry mixing of the remaining ingredients for 25-30 minutes; 5) Tabletting: adjust the tablet press pressure to 55 kN for tabletting; the resulting tablets are 15-25 mm in diameter and weigh about 0.75 g; 6) Packaging: PVDC film, aluminum foil film or aluminum foil bag, to obtain probiotic tablets. Ensure cold chain configuration (2-8°C) during transportation and sales.

[0064] After testing, the number of viable Lactobacillus fermentum in the tablets obtained in this example is about 1.2 x 10 11 CFU / kg.

[0065] Take one tablet with warm water after a meal every day to meet the needs.

[0066] Example 7 Preparation of Lactobacillus helveticus ZJUIDS11 starter with the effect of preventing halitosis Inoculate the original strain of Lactobacillus helveticus ZJUIDS11 into 11% skim milk (sterilized at 115°C for 20 min) and cultivate aerobically at 37°C for 18-24 h until coagulation, and continuously activate for two generations as the mother starter. Inoculate the mother starter into 11% skim milk (sterilized at 115°C for 20 min) at an inoculation amount of 3%-5%, and cultivate at 37°C for 18-24 h until coagulation, at which time the viable cell count can reach 10 9 ~10 10 CFU / mL, i.e. Lactobacillus helveticus ZJUIDS11 starter is obtained.

[0067] It is suitable for the preparation of dairy products (such as yogurt, cheese), functional foods or probiotic preparations. It is necessary to ensure that the viable cell count of the final product meets the standard (usually needs to reach 10 6~10 8 CFU / g or mL).

[0068] Example 8, Preparation of functional fermented milk with Lactobacillus helveticus ZJUIDS11 having the effect of preventing halitosis The conventionally pretreated cow milk was heated to about 60℃, 6% sucrose was added, and after being dissolved thoroughly, homogenization was performed under a pressure of 20 MPa. Then, after heat treatment at 95℃ for 5 min, it was cooled to 38℃. Lactobacillus helveticus ZJUIDS11 starter prepared according to the above example was inoculated at an inoculation amount of 5%, and fermentation was performed at 37℃ for 14-18 h until curdling, and after cooling, it was stored at 4℃, thereby obtaining functional fermented milk of Lactobacillus helveticus ZJUIDS11.

[0069] Example 9, Preparation of bacteria powder with Lactobacillus helveticus ZJUIDS11 having the effect of preventing halitosis Lactobacillus helveticus ZJUIDS11 was inoculated into 50 mL of MRS liquid medium using an inoculation loop, and was incubated in a 37℃ incubator for 18 h. Then, it was transferred to 250 mL of sterilized MRS liquid medium at an inoculation amount of 5%, and was incubated in a bacteria incubator at 37℃ for 24 h. The obtained fermentation broth was centrifuged, and then the supernatant was discarded and the bacterial pellet was collected, and the bacterial pellet was rinsed twice with sterile phosphate buffer solution (PBS) for 10 min each time at a centrifugal speed of 5000 r / min, thereby obtaining the bacterial pellet of Lactobacillus helveticus ZJUIDS11.

[0070] Trehalose 4.1 g, sodium glutamate 2.8 g, and sucrose 8 g were dissolved in 10 mL of 40℃ distilled water, sterilized by filtering through a 0.22 um microfiltration membrane, and then added to 90 mL of sterilized water, thereby obtaining a freeze-drying protective agent. 15 g of skim milk powder was dissolved in distilled water, thereby obtaining 100 mL of 15% skim milk powder solution, which was sterilized at 110℃, thereby obtaining a freeze-drying protective agent.

[0071] The bacterial pellet of Lactobacillus helveticus ZJUIDS11 prepared above was mixed with the freeze-drying protective agent at a ratio of 1 g:5 mL, thereby obtaining a bacterial suspension. 5 mL of the bacterial suspension was divided into 10 mL of sterilized glass bottles with stoppers, and was pre-frozen at -70℃ for 2 h, and then was placed in a vacuum freeze-drying machine for freeze-drying, and the freeze-drying conditions were a vacuum degree of 5 Pa, a shelf heating temperature of 20℃, and a cold trap temperature of -55℃. After freeze-drying for 30 h, the freeze-dried bacteria were broken, thereby obtaining bacteria powder of Lactobacillus helveticus ZJUIDS11 (probiotic bacteria powder).

[0072] Example 10, Preparation of probiotic solid beverage with Lactobacillus helveticus ZJUIDS11 having the effect of preventing halitosis The steps of preparing the probiotic solid beverage with Lactobacillus helveticus ZJUIDS11 are as follows: (1) Preparation of probiotic bacteria powder: Lactobacillus helveticus ZJUIDS11 probiotic bacteria powder can be prepared according to the above embodiment.

[0073] (2) The solid beverage formula is as follows: edible corn starch (balance), white granulated sugar (10%), fructo-oligosaccharide (6%), isomalto-oligosaccharide (9%), citric acid (according to the conventional amount of solid beverage), edible essence (according to the conventional amount of solid beverage), probiotic bacteria powder (2-5%).

[0074] After adding according to the formula, mix well to ensure that the viable count of Lactobacillus helveticus ZJUIDS11 is above 10 9 CFU / g.

[0075] (4) Pack according to 10 / 20 g per small package, 10-20 small packages per large package.

[0076] In actual use, the method and amount are to take half an hour after a meal, 1-2 times a day, 1 small package each time, and take with warm water to avoid high temperature from destroying the activity of the bacterial population.

[0077] Example 11, preparation of probiotic bacteria chewing gum using Lactobacillus helveticus ZJUIDS11 powder The purpose of this embodiment is to provide a plate-type chewing gum containing high-activity Lactobacillus gasseri, which is composed of Lactobacillus helveticus ZJUIDS11 powder described in the above embodiment and gum base, powdered sugar, sucrose, calcium carbonate, glucose, flavoring agents, etc. Except for the gum base and the bacteria powder, the components necessary during the preparation of the plate-type chewing gum are all adjuvants, which are the same as in the prior art. The formula includes the following components in the weight percentage: Lactobacillus helveticus ZJUIDS11 powder 0.1%, gum base 29.9%, sugar and other components 70%.

[0078] (1) Modulation of the resin base: modulate the chewing gum base by adding vinyl acetate, ester gum, butyl phthalate butyl glycol ester, and sugar gum gum into a kneader, while adding emulsifiers and plasticizers, and strictly control the kneading temperature within the range of 80-120°C to make it into an integrated material; (2) Mixing: use a mixing machine, and mix in a certain order, first add the heated and dissolved chewing gum base, then add granulated sugar, glucose, sucrose, and flavoring agents, and stir evenly; (3) Shaping and packaging: the mixed raw materials are uniform in texture and have a moisture content of 2.1%, then cut into 8x25x5 cm rectangular small pieces using a cutting machine, then put into an extruder for extrusion, then pass through a multi-stage roller machine to press into a certain thickness of plate pieces, then cut into appropriate shapes using a cutting machine, and then keep in a temperature-controlled room with a temperature of 21-24°C and a relative humidity of 45-50% for 6 hours before packaging.

[0079] (4) The slab chewing gum containing highly active Bacillus coagulans provided in this embodiment has a live bacteria count of 5.0 x 10 cfu / g. The slab chewing gum containing highly active Bacillus coagulans provided in this embodiment has health care functions such as cleaning the oral cavity, freshening breath, antagonizing Streptococcus mutans, and preventing tooth decay.

[0080] In actual use, the method and dosage are as follows: take 1 tablet each time and place it in your mouth, chew it thoroughly until it is tasteless (about 10-15 minutes), and use it half an hour after meals.

[0081] Example 12: Preparation of probiotic mouthwash using Lactobacillus helveticus ZJUIDS11, which has the effect of preventing halitosis. A probiotic mouthwash composition for preventing halitosis comprises the following ingredients in parts by weight: Phase A: Water 87.96%, Glycerin 8%, Sucralose 0.03%, Cetylpyridinium chloride 0.05%; Phase B: 2% propylene glycol, 0.25% preservative, 0.05% methyl diisopropyl propionamide, 0.05% fragrance; Phase C: 1% Japanese cherry blossom extract, 0.5% fermentation supernatant of Lactobacillus fermentum ZJUIDS11, 0.1% xylitol, and 0.01% glucose oxidation.

[0082] The fragrance is a flavoring, and the preservative is PEG-40 hydrogenated castor oil.

[0083] A method for preparing a probiotic mouthwash composition with anti-haze effect includes the following steps: Add phase A to the emulsifying pot, heat to 80-85℃, stir evenly, and then cool down; pre-mix and stir phase B until evenly dissolved, and set aside; cool phase A in the emulsifying pot to 45℃, add phase B to the emulsifying pot, and stir until completely dissolved; add phase C raw material to the emulsifying pot, and stir until completely dissolved; inspect and discharge the material, allow it to age and stand, and after passing inspection, fill and package it, and after passing inspection, put it into storage.

[0084] In actual use, take about 10-15ml of mouthwash and hold it in your mouth to rinse thoroughly for 30 seconds to 1 minute.

[0085] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The uses of Lactobacillus helveticus ZJUIDS11, characterized by: It can relieve and improve bad breath; its accession number is CGMCCNO.24761.

2. The use of Lactobacillus helveticus ZJUIDS11 according to claim 1, characterized in that: Lactobacillus helveticus ZJUIDS11 can inhibit hydrogen sulfide and methanethiol.

3. The use of Lactobacillus helveticus ZJUIDS11 according to claim 2, characterized in that: Lactobacillus helveticus ZJUIDS11 can inhibit hydrogen sulfide emissions from Fusobacterium nucleatum.

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