CN112931617A - Mixed probiotic buccal tablet and preparation method thereof - Google Patents
Mixed probiotic buccal tablet and preparation method thereof Download PDFInfo
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- 239000006041 probiotic Substances 0.000 title claims abstract description 97
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 97
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- 229940046011 buccal tablet Drugs 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000000843 powder Substances 0.000 claims abstract description 49
- 241000218588 Lactobacillus rhamnosus Species 0.000 claims abstract description 33
- 241001134770 Bifidobacterium animalis Species 0.000 claims abstract description 29
- 229940118852 bifidobacterium animalis Drugs 0.000 claims abstract description 29
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 20
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 10
- 239000000600 sorbitol Substances 0.000 claims abstract description 10
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 claims abstract description 8
- 235000008939 whole milk Nutrition 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 7
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
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- 241000605862 Porphyromonas gingivalis Species 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
-
- 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
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/175—Rhamnosus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
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- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention belongs to the technical field of food, and discloses a mixed probiotic buccal tablet and a preparation method thereof, wherein the mixed probiotic buccal tablet is prepared by uniformly mixing sorbitol, whole milk powder, citric acid, lactobacillus rhamnosus X253 powder and bifidobacterium animalis subsp lactis i797 powder with magnesium stearate and tabletting. The mixed probiotic buccal tablet is prepared from lactobacillus rhamnosus X253 powder and bifidobacterium animalis subsp lactis i797 powder, the two probiotics have a synergistic effect, compared with the buccal tablet prepared from single probiotics, the prepared mixed probiotic buccal tablet has the functions of remarkably improving the planting rate of the probiotics and improving the defecation frequency of a human body, and has the advantages of high viable count, long bacterial activity maintaining time and the like. The preparation method is simple, and the prepared mixed probiotic buccal tablet is suitable for all kinds of people to eat.
Description
Technical Field
The invention belongs to the technical field of food, and relates to a buccal tablet and a preparation method thereof, in particular to a mixed probiotic buccal tablet and a preparation method thereof.
Background
There are a great number and variety of microorganisms including bacteria, fungi, viruses, etc. in the human body, and they are widely distributed in the body surface and the cavities and ducts of the human body, such as the reproductive tract, the digestive tract and the respiratory tract, etc., which are communicated with the outside. From the aspect of cell number, the total number of the somatic cells of various bacterial communities symbiotic with the human body even far exceeds the total number of the cells of the human body. The symbiotic floras maintain the stable environment of the colonized parts and even participate in the physiological activities of hosts, and the balance of floras of all parts in the human body is an important prerequisite for the health of the human body. The probiotics are active microorganisms which can exert beneficial effects on human bodies after being eaten, and the relation between the probiotics and human health becomes a research hotspot in recent years. After entering the human body, the probiotics can compete with opportunistic pathogens in the oral cavity and the intestinal tract for colonization sites, so that caries, oral inflammation, intestinal diseases and the like caused by opportunistic pathogens at the colonization sites are reduced, the flora structures of the oral cavity and the intestinal tract are changed, and the health of the human body is promoted.
At present, a plurality of probiotic products exist in the market, but the probiotic buccal tablets still belong to the small category due to weak functionality and bad taste, and meanwhile, the problems of single function, reduction of probiotic activity in the storage process, low planting rate after entering the human body and the like exist, and the probiotic buccal tablets are not widely accepted by consumers yet, so that the development of the probiotic buccal tablets which can adjust the oral and intestinal flora structure, have high probiotic activity and planting rate and are suitable for daily eating of the consumers is needed.
Disclosure of Invention
The invention aims to provide a mixed probiotic buccal tablet so as to achieve the purposes of regulating the flora structure of oral cavity and intestinal tract and improving the activity and colonization rate of probiotics;
the invention also aims to provide a preparation method of the mixed probiotic buccal tablet.
In order to achieve the purpose, the invention adopts the technical scheme that:
a mixed probiotic buccal tablet comprises the following raw materials of active ingredients in parts by weight: 75-80 parts of sorbitol, 10-20 parts of whole milk powder, 1.5-3 parts of magnesium stearate, 1.5-3 parts of citric acid, 1-1.5 parts of lactobacillus rhamnosus X253 powder and 1-1.5 parts of bifidobacterium animalis subsp.
As a limitation, the lactobacillus rhamnosus X253 strain powder and the bifidobacterium animalis subsp lactis i797 strain powder are prepared by respectively inoculating the lactobacillus rhamnosus X253 strain and the bifidobacterium animalis subsp lactis i797 strain into an MRS liquid culture medium, carrying out enrichment culture at 37 ℃ for 22-26h, carrying out centrifugal separation and washing, carrying out vacuum freeze-drying after the obtained thalli are re-suspended by a freeze-drying protective agent solution, grinding into powder, and sieving with a 30-50-mesh sieve;
the Lactobacillus rhamnosus X253 is separated and screened from traditional fermented dairy products in Xinjiang in China, and the strain is preserved in the China general microbiological culture Collection center in 8-20 th month in 2019 with the preservation number of CGMCC No.18404, and the Latin article is named Lactobacillus rhamnosus which is disclosed for the first time in an online patent application document with the application number of 202010258772.0;
bifidobacterium animalis lactis i797 is isolated and screened from infant or infant intestinal flora, and has been deposited in the China general microbiological culture Collection center (CGMCC NO. 18403) at 20.8.2019 with the deposit number of CGMCC, and the Latin name is Bifidobacterium animalis subsp.lactis, which is disclosed for the first time in the online patent application with the application number of 202010259210.8.
As a further limitation, the inoculation amount of the Lactobacillus rhamnosus X253 or the Bifidobacterium animalis subsp.lactis i797 is 1.5-2.5% of the weight of the MRS liquid medium.
As a further limitation, the raw materials for preparing the effective components of the MRS liquid medium comprise, in parts by weight: 8-12 parts of casein peptone, 8-12 parts of beef extract, 4-6 parts of yeast extract, 16-24 parts of glucose, 4-6 parts of sodium acetate, 1.5-2.5 parts of diamine citrate, 800.8-1.2 parts of tween-E, K2HPO41.5-2.5 parts of MgSO4·7H20.1 to 0.3 portion of O and MnSO4·7H20.04-0.06 part of O and 1000 parts of distilled water.
As a further limitation, the temperature of the centrifugal separation is less than or equal to 4 ℃, the rotating speed is 6000-12000r/min, and the time is 10-15 min.
As a further limitation, the freeze-drying protective agent solution contains 115g/L of skim milk powder 105-.
The invention also provides a preparation method of the mixed probiotic buccal tablet, which is to uniformly mix all the raw materials and then tabletting to obtain the mixed probiotic buccal tablet.
As a limitation, before mixing, pulverizing sorbitol, whole milk powder, citric acid, Lactobacillus rhamnosus X253 bacteria powder and Bifidobacterium animalis subsp lactis i797 bacteria powder, and sieving.
As a further limitation, the screened mesh size is 60-70 mesh.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
the lactobacillus rhamnosus X253 strain powder and the bifidobacterium animalis subsp lactis i797 strain powder are reasonably mixed, the two probiotics have a synergistic effect, the prepared mixed probiotic buccal tablet has the functions of remarkably improving the planting rate of the probiotics and improving the defecation frequency of a human body compared with a buccal tablet prepared by adopting single probiotics, and the mixed probiotic buccal tablet has the advantages of high viable count, long maintenance time of the activity of the bacteria, long shelf life and the like;
in the mixed probiotic buccal tablet, the lactobacillus rhamnosus X253 and the bifidobacterium animalis subsp lactis i797 can compete with opportunistic pathogens in oral cavities and intestinal tracts for colonization sites and inhibit the proliferation of the opportunistic pathogens, so that the health of the oral cavities and the intestinal tracts is adjusted, the content of beneficial bacteria is improved, and the aim of adjusting the balance of oral cavity and intestinal flora is fulfilled;
the mixed probiotic buccal tablet can adjust the balance of oral flora, has the function of regulating the intestine, can effectively increase the defecation frequency of a human body, is beneficial to discharging metabolic wastes out of the body and promoting the metabolism of the human body, can improve the gingival bleeding condition, and can mutually promote the colonization rate of two probiotics in the oral cavity and increase the action effect of the two probiotics under the compounding condition.
The mixed probiotic buccal tablet has the advantages of good taste, low cost, simple preparation method and easily obtained raw materials; easy to produce and transport; the mixed probiotic buccal tablet is suitable for all kinds of people to eat.
Detailed Description
The present invention is further illustrated by the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure.
Example 1 preparation method of mixed probiotic buccal tablet
The embodiment comprises the following steps which are carried out in sequence:
one) preparation of probiotic powder
I) Taking 240g of casein peptone, 240g of beef extract, 120g of yeast extract, 480g of glucose, 120g of sodium acetate, 50g of diamine citrate, 24g of tween-80 and 50g K2HPO、3g MgSO4·7H2O、1g MnSO4·7H2O and20kg of distilled water, and uniformly mixing to prepare the MRS liquid culture medium.
Inoculating 0.2kg of seed liquid of lactobacillus rhamnosus X253 or bifidobacterium animalis subsp lactis i797 into 10kg of MRS liquid culture medium (the inoculation amount of the lactobacillus rhamnosus X253 or the bifidobacterium animalis subsp lactis i797 is 2% of the weight of the corresponding MRS liquid culture medium and is marked as JZ1), after carrying out enrichment culture at 37 ℃ for 24 hours, respectively carrying out centrifugal separation on the obtained corresponding bacterial liquid at 4 ℃ for 10min at 12000r/min, removing supernatant, taking corresponding sediments (namely bacteria) and washing the bacteria with 10kg of normal saline until the bacteria are white, thus obtaining the corresponding lactobacillus rhamnosus X253 bacteria or bifidobacterium animalis subsp lactis i797 bacteria;
II) adding 2200g of skimmed milk powder, 1700g of trehalose, 1300g of maltodextrin, 300g of sodium glutamate and 200g of sorbitol into water, and performing constant volume to 20L to obtain a freeze-drying protective agent solution;
respectively taking lactobacillus rhamnosus X253 thalli or bifidobacterium animalis subsp lactis i797 thalli to be resuspended in 10L of the freeze-drying protective agent solution to be mixed uniformly, then placing the mixture into a freeze dryer to be subjected to vacuum freeze drying, grinding the obtained freeze-dried product, sieving the powder with a 40-mesh sieve, and collecting freeze-dried powder, namely lactobacillus rhamnosus X253 bacterial powder or bifidobacterium animalis subsp lactis i797 bacterial powder;
wherein, the collected freeze-dried powder can also be stored in a refrigerator at the temperature of minus 80 ℃ for later use.
II) preparation of mixed probiotic buccal tablets
Respectively taking sorbitol, whole milk powder, citric acid, lactobacillus rhamnosus X253 bacteria powder and bifidobacterium animalis subsp lactis i797 bacteria powder, crushing, and sieving by a 60-mesh sieve to obtain corresponding sorbitol powder, whole milk powder, citric acid powder, lactobacillus rhamnosus X253 bacteria powder and bifidobacterium animalis subsp lactis i797 bacteria powder;
and (3) uniformly mixing 75kg of sorbitol powder, 20kg of whole milk powder, 1.5kg of citric acid powder, 1kg of lactobacillus rhamnosus X253 powder and 1.5kg of bifidobacterium animalis subsp lactis i797 powder, adding 3kg of magnesium stearate, uniformly mixing, and tabletting to obtain the mixed probiotic buccal tablet, wherein the mark is N1.
EXAMPLES 2-6 preparation of Mixed probiotic lozenges
Examples 2 to 6 are methods for preparing mixed probiotic lozenges, which have substantially the same steps as in example 1, but differ only in the amount of raw materials and the process parameters, as detailed in table 1:
table 1 summary of raw material amounts and parameters in mixed probiotic lozenges
The contents of other portions of examples 2 to 6 are the same as those of example 1.
Example 7 functional evaluation of Mixed probiotic lozenges to regulate the balance of oral flora and promote the colonization of probiotics
In this embodiment, the mixed probiotic buccal tablets and the control buccal tablets are subjected to trial eating tests, and oral pathogenic bacteria and probiotic colonization conditions of a subject are detected, so that functional evaluation that the mixed probiotic buccal tablets regulate oral flora balance and promote probiotic colonization is performed.
(1) Preparing trial buccal tablets:
the trial eating buccal tablets comprise nine types: the mixed probiotic buccal tablets prepared in examples 1-6 are N1-N6, Lactobacillus rhamnosus X253 buccal tablet M1, Bifidobacterium animalis subsp lactis i797 buccal tablet M2 and blank control buccal tablet M3.
Preparation of lactobacillus rhamnosus X253 buccal tablet M1: the preparation method and raw materials of the buccal tablet are basically the same as those of the buccal tablet in the embodiment 1, and the difference is only that no bifidobacterium animalis subsp lactis i797 bacterial powder is added;
preparation of bifidobacterium animalis subsp lactis i797 buccal tablet M2: the preparation method and the raw materials of the buccal tablet are basically the same as those of the embodiment 1, and the difference is that no lactobacillus rhamnosus X253 bacteria powder is added;
preparation of blank control buccal tablet M3: the preparation method and the raw materials of the buccal tablet are basically the same as those of the buccal tablet in the embodiment 1, and the difference is that no lactobacillus rhamnosus X253 bacterial powder and bifidobacterium animalis subsp lactis i797 bacterial powder are added;
(2) subject Condition
Males over 18 years of age or non-pregnant females were recruited as subjects who were asked to: no oral treatment is performed in nearly 6 months; antibiotics, hormones and immunosuppressants have not been administered for nearly 2 weeks; no oral cavity is used for local application in about 1 month.
(3) Grouping of test persons
Randomly dividing 45 subjects into 9 groups, each group comprises 5 persons, and respectively comprises a blank control group and a group C1-C8, wherein the blank control group trial eats blank control buccal tablet M3; C1-C6 groups correspondingly take mixed probiotics containing tablets N1-N6 in a one-to-one mode; trial taking of Lactobacillus rhamnosus X253 buccal tablet M1 in group C7; c8 group trial eaten animal Bifidobacterium lactis subspecies i797 buccal tablet M2.
The eating method comprises the following steps: taking 1 tablet (0.8 g/tablet) three times a day, placing the tablet in oral cavity, keeping in oral cavity for at least 2-3min, and then swallowing, taking once in the morning, in the middle of the day, and in the evening, and not brushing teeth, eating, and rinsing in half an hour, and keeping oral cavity sanitary according to original habit during taking, and not using fluorine-containing toothpaste during taking.
Sampling of tartar samples: before the experiment begins and two weeks after eating the mixed probiotic buccal tablet, tartar in the gap between the left and right lower teeth of the third molar teeth of all subjects is taken.
(4) Oral pathogen detection
Using a DNA extraction kit to extract tartar sample DNA, and performing fluorescence quantitative PCR detection on the contents of Streptococcus mutans (hereinafter, S.m) and Streptococcus gordonii (hereinafter, S.g) Porphyromonas gingivalis (hereinafter, P.g) in the oral cavity of all subjects to obtain ct values of each sample. Taking week 0 as control group and week 2 as experimental group, and adopting relative quantification method, i.e. 2–ΔΔctMethod, the gene expression level of the target bacteria at week 2 was calculated.
The calculation method comprises the following steps:
calculating the difference value of the ct values of the samples at 0 week and 2 weeks, taking the sample at 0 week as a control group, taking the sample at 2 weeks as an experimental group:
Δ ct (experimental group) ═ ct (target gene, experimental group) -ct (reference gene, experimental group)
Δ ct (control group) ═ ct (target gene, control group) -ct (reference gene, control group)
Calculating the difference value of two delta ct:
Δ Δ ct ═ Δ ct (experimental group) - Δ ct (control group)
Calculating the relative expression quantity of the gene:
2-ΔΔctrelative expression amount of the gene
The data were analyzed by GraphPad Prism 8 software, and the results were significantly different, expressed as P < 0.05, and the following results were obtained:
TABLE 2 Gene expression level of each pathogen at week 2
| Group of | S.m | S.g | P.g |
| Group C1 | 0.791* | 0.631* | 0.952* |
| Group C2 | 0.913* | 0.932* | 0.823* |
| Group C3 | 0.954* | 0.987* | 0.675* |
| Group C4 | 0.904* | 0.902* | 0.812* |
| Group C5 | 0.911* | 0.852* | 0.723* |
| Group C6 | 0.853* | 0.972* | 0.811* |
| Group C7 | 2.436 | 1.142 | 1.893 |
| Group C8 | 3.162 | 1.271* | 2.311 |
| Blank control group | 4.096 | 2.305 | 2.934 |
Note: p < 0.05 compared to placebo
The results show that after eating the mixed probiotic buccal tablet for 2 weeks, the expression levels of the genes S.m, S.g and P.g in the oral cavity of the subjects in the C1-C6 groups are in a remarkably reduced trend, compared with a blank control group, the expression levels of the genes of pathogenic bacteria in the C7 group and the C8 group are reduced, but only the expression level of the gene S.g in the C8 group is reduced remarkably, other reduction effects are not remarkable, and the inhibition effect of the Lactobacillus rhamnosus X253 buccal tablet in the C7 group on the pathogenic bacteria is superior to that of the Lactobacillus bifidus subsp lactis 79i 7 in the C8 group on the average value. The results show that the probiotic buccal tablets prepared by respectively and independently adding the lactobacillus rhamnosus X253 or the bifidobacterium animalis subsp lactis i797 can inhibit the colonization of oral pathogenic bacteria to a certain extent, but the action effect is not obvious on the whole; the result shows that the mixed probiotic buccal tablet prepared by reasonably proportioning the two probiotics provided by the invention has an inhibiting effect obviously stronger than the single strain effect, and shows that the two strains have the capability of mutually promoting and jointly inhibiting pathogenic bacteria.
(5) Oral probiotic detection
Extracting tartar sample DNA by using a DNA extraction kit, and performing fluorescent quantitative PCR detection on the contents of animal bifidobacteria (Bif) and lactobacillus (Lac) in the oral cavity of all subjects, wherein the detection and calculation method is the same as that of the oral pathogenic bacteria detection method in step (4).
The detection results are as follows:
TABLE 3 expression levels of genes of probiotics at week 2
| Group of | Bif | Lac |
| Group C1 | 1.218* | 1.236* |
| Group C2 | 1.183* | 1.292* |
| Group C3 | 1.117* | 1.312* |
| Group C4 | 1.173* | 1.301* |
| Group C5 | 1.192* | 1.308* |
| Group C6 | 1.184* | 1.289* |
| Group C7 | 0.763 | 1.035* |
| Group C8 | 0.971* | 0.992 |
| Blank control group | 0.650 | 0.876 |
Note: p < 0.05 compared to placebo
The results show that after eating the mixed probiotic buccal tablet for 2 weeks, the expression levels of Bif and Lac genes in the oral cavity of the subjects in the C1-C6 groups show a remarkable rising trend. Compared with a blank control group, the expression level of Bif gene of the C8 group is obviously improved, the expression level of Lac gene of the C7 group is obviously improved, and the rest genes have no obvious difference; the expression level of two genes in the C1-C6 groups is obviously improved. The results show that the probiotic buccal tablets prepared by respectively and independently adding lactobacillus rhamnosus X253 or bifidobacterium animalis subsp lactis i797 can improve the colonization rate of two probiotics to a certain extent, but can only show a remarkable effect on the content of one of the probiotics.
Example 8 functional evaluation of mixed probiotic lozenges to improve gum bleeding frequency
Gingival bleeding is one of the common symptoms in the department of stomatology, refers to spontaneous gingival bleeding or a small amount of bleeding caused by slight stimulation, and chronic inflammation is a common reason of gingival bleeding, so the bleeding frequency of the gingiva can reflect the health condition of the oral cavity and can also reflect the degree of oral inflammation specifically.
In this example, all the subjects enrolled in example 7 were evaluated for their ability to improve the number of gingival bleeding by a buccal tablet, and during the trial taking period of the buccal tablet, the number of gingival bleeding was counted every two days, and the average results of each group were as follows:
table 4 results of improving gum bleeding frequency by mixed probiotic lozenges
| Group of | 0d | 2d | 4d | 6d | 8d | 10d | 12d | 14d |
| Group C1 | 3.8 | 3.2 | 3.7 | 2.9 | 2.4 | 1.5 | 1.3 | 1.3 |
| Group C2 | 4.4 | 3.2 | 3.9 | 2.4 | 2.7 | 1.8 | 1.3 | 0.8 |
| Group C3 | 4.2 | 4.1 | 3.2 | 3.5 | 3.1 | 2.2 | 1.4 | 0.7 |
| Group C4 | 4.5 | 3.1 | 2.9 | 2.5 | 2.8 | 2.3 | 1.2 | 0.9 |
| Group C5 | 3.9 | 3.8 | 3.9 | 3.6 | 3.1 | 2.4 | 1.5 | 1.2 |
| Group C6 | 3.7 | 3.6 | 3.6 | 3.1 | 2.6 | 1.5 | 1.4 | 1.1 |
| Group C7 | 3.5 | 3.2 | 3.4 | 3.5 | 4.1 | 3.2 | 3.2 | 3.3 |
| Group C8 | 4.9 | 4.4 | 4.2 | 4.2 | 3.7 | 3.5 | 4.2 | 4.5 |
| Blank control group | 4.3 | 4.2 | 4.6 | 4.2 | 4.1 | 4.2 | 4.1 | 4.5 |
The experimental result shows that compared with the test before the beginning of the experiment, the gingival bleeding times of the subjects in the C1-C6 groups are obviously reduced after the subjects eat the mixed probiotic buccal tablets for two weeks, wherein the subjects in the C2 group and the C3 group are more obvious; compared with the prior art, the mixed probiotic buccal tablets of the C7 group and the C8 group have a trend of improvement, but the improvement effect is not obvious, and the result further shows that the mixed probiotic buccal tablets have the capability of improving the gingival bleeding which is obviously better than that of single-strain probiotic buccal tablets.
Example 9 functional evaluation of mixed probiotic lozenges to improve the number of bowel movements
The defecation frequency can reflect the health condition of the intestinal tract of a human body, after the human body eats, a large amount of metabolites and food residues are generated by digestion and absorption of food, if the food stays in the body for more than 24 hours, a large amount of toxins are further metabolized under the action of intestinal microorganisms, the health of the intestinal tract is threatened, and the external expression form of the stay time of excrement in the intestinal tract is the defecation frequency.
In this example, the blank control group and the subjects in the groups C1-C8 enrolled in example 7 were evaluated for the functional improvement of defecation frequency of buccal tablets, during the trial eating period of buccal tablets, data of the number of times of defecation per week of the subjects were collected, the results were counted in weeks with the control before the start of the experiment, and the results of the number of times of defecation per week of each group are expressed as average values, and the results are as follows:
TABLE 5 improved results of the mixed probiotic lozenges on the number of defecation
The results show that the times of the peripheral defecation of the subjects in the group C1-C6 are close to the times of the normal defecation at the end of week 1, namely 7 times/week, and the times of the peripheral defecation exceed the times of the normal defecation at the end of the second week, which shows that the mixed probiotic buccal tablets can improve the times of the peripheral defecation of the subjects, promote the excretion of feces, reduce the generation of harmful substances, improve the health degree of intestinal tracts and have a certain intestinal function; the buccal tablets of group C7 and group C8 have slight improvement effect on the frequency of the peripheral defecation of a subject, do not show obvious difference, and show that the single probiotic buccal tablet does not have the function of promoting the defecation; the buccal tablets of the blank control group did not exhibit the effect of improving the number of stools.
Example 10 detection of probiotic Activity in Mixed probiotic lozenges
The number of viable bacteria in the mixed probiotic buccal tablets N1-N6 prepared in examples 1-6, the Lactobacillus rhamnosus X253 buccal tablet M1 prepared in example 7 and the Bifidobacterium animalis subsp.
The detection method comprises the following steps: weighing 1g of buccal tablet N1-N6, buccal tablet M1 and buccal tablet M2 respectively, grinding, dissolving the powder into 10mL of sterile water respectively to obtain corresponding buccal tablet suspensions for later use, adding 1mL of buccal tablet suspensions respectively by adopting an inverted plate method, sterilizing to an MRS solid culture medium, culturing for 48h, then carrying out colony counting, and detecting the number of viable bacteria in the buccal tablets;
the detection results are as follows:
TABLE 6 buccal tablet change in viable count within 8 weeks
The results show that the buccal tablet N1-N6 has viable count capable of maintaining viable count within 6 weeks at normal temperature>109CFU/g level and maintained at bacterial count for 8 weeks>108The CFU/g level shows that the mixed probiotic buccal tablet provided by the invention has high viable count and long maintenance time of the activity of the bacteria; the buccal tablet M1 and M2 can only maintain the number of bacteria within 1 week>109CFU/g level, only maintained bacterial count within 8 weeks>108The CFU/g level further shows that the mixed probiotic buccal tablet can ensure that the buccal tablet can maintain high bacterial count at normal temperature, but the single probiotic buccal tablet can hardly maintain high bacterial activity within 8 weeks, and the result shows that the mixed probiotic buccal tablet can promote the buccal tablet to maintain high activity after two probiotics are mixed.
In conclusion, the mixed probiotic buccal tablet provided by the invention can inhibit pathogenic bacteria colonization in oral cavity, and has better effect than single probiotic buccal tablet; in addition, the mixed probiotic formula buccal tablet provided by the invention can improve the colonization rate of two probiotics in the oral cavity to a certain extent, and the colonization effect of the mixed probiotic formula buccal tablet is obviously superior to that of a single probiotic buccal tablet; particularly, compared with the single probiotic buccal tablets, the mixed probiotic buccal tablets have more outstanding capability of regulating intestinal flora and can effectively promote the balance and health of oral flora.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms, and any person skilled in the art may use the above-mentioned technical content as a teaching to make changes or modifications to the equivalent embodiments with equivalent changes, but all those simple changes, equivalent changes and modifications made to the above-mentioned embodiments without departing from the technical spirit of the present invention, and still all those embodiments are within the scope of the present invention as claimed in the claims.
Claims (9)
1. The mixed probiotic buccal tablet is characterized in that the raw materials for preparing the effective components of the mixed probiotic buccal tablet comprise the following components in parts by weight: 75-80 parts of sorbitol, 10-20 parts of whole milk powder, 1.5-3 parts of magnesium stearate, 1.5-3 parts of citric acid, 1-1.5 parts of lactobacillus rhamnosus X253 powder and 1-1.5 parts of bifidobacterium animalis subsp.
2. The mixed probiotic buccal tablet according to claim 1, wherein the lactobacillus rhamnosus X253 bacteria powder and bifidobacterium animalis subsp lactis i797 bacteria powder are prepared by respectively inoculating lactobacillus rhamnosus X253 bacteria powder and bifidobacterium animalis subsp lactis i797 bacteria powder into an MRS liquid culture medium, carrying out enrichment culture at 37 ℃ for 22-26h, carrying out centrifugal separation and washing, carrying out heavy suspension on the obtained thalli by using a freeze-drying protective agent solution, carrying out vacuum freeze-drying, grinding, and sieving.
3. The mixed probiotic buccal tablet according to claim 2, wherein the inoculation amount of lactobacillus rhamnosus X253 or bifidobacterium animalis subsp lactis i797 is 1.5-2.5% by weight of the MRS liquid medium.
4. The mixed probiotic buccal tablet according to claim 2 or 3, wherein the raw materials for preparing the effective components of the MRS liquid culture medium comprise, in parts by weight: 8-12 parts of casein peptone, 8-12 parts of beef extract, 4-6 parts of yeast extract, 16-24 parts of glucose, 4-6 parts of sodium acetate, 1.5-2.5 parts of diamine citrate, 800.8-1.2 parts of tween-E, K2HPO 41.5-2.5 parts, MgSO4·7H20.1 to 0.3 portion of O and MnSO4·7H20.04-0.06 part of O and 1000 parts of distilled water.
5. The mixed probiotic buccal tablet according to claim 2 or 3, wherein the temperature of the centrifugal separation is less than or equal to 4 ℃, the rotating speed is 6000-.
6. The mixed probiotic buccal tablet according to claim 2 or 3, characterized in that the freeze-drying protectant solution contains skim milk powder 105-115g/L, trehalose 80-90g/L, maltodextrin 60-70g/L, sodium glutamate 13-17g/L and sorbitol 8-12 g/L.
7. A preparation method of the mixed probiotic buccal tablet according to any one of claims 1 to 6, characterized in that the mixed probiotic buccal tablet is obtained by taking all raw materials, mixing uniformly and tabletting.
8. The preparation method of the mixed probiotic buccal tablet according to claim 7, wherein before the mixing, the sorbitol, the whole milk powder, the citric acid, the lactobacillus rhamnosus X253 bacteria powder and the bifidobacterium animalis subsp lactis i797 bacteria powder are crushed and sieved.
9. The method for preparing the mixed probiotic buccal tablet according to claim 8, wherein the sieving is performed by adopting a sieve with the aperture of 60-70 meshes.
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