CN112889925A - Inactivated probiotic milk tablet and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of food, and discloses a inactivated probiotic milk tablet and a preparation method thereof. The lactobacillus rhamnosus X253 inactivated probiotic powder is adopted, can effectively adhere to oral epithelial cells under the condition of heat inactivation, shows better adhesion performance than the lactobacillus rhamnosus X253, can be used as oral probiotic for permanent planting, competes with oral pathogenic bacteria for permanent planting sites, and regulates oral health; the inactivated probiotic milk tablet provided by the invention can inhibit oral pathogenic bacteria in vivo, and simultaneously improve the content of beneficial bacteria, so as to achieve the purpose of regulating the balance of oral flora. The inactivated probiotic milk tablet is suitable for various crowds to eat.

Description

Inactivated probiotic milk tablet and preparation method thereof

Technical Field

The invention belongs to the technical field of food, and relates to a milk tablet, in particular to a inactivated probiotic milk tablet and a preparation method thereof.

Background

A large number and variety of microbial populations live in the oral cavity of a human body, and the types of these microorganisms mainly include bacteria, fungi, and the like, and a complex micro-ecosystem is formed in the oral cavity. Under normal conditions, microorganisms in the oral cavity can form a relative equilibrium relationship through symbiosis, competition or antagonism and the like, and the microorganisms and the host can keep a dynamic equilibrium state. However, some opportunistic pathogens can destroy the relative balance relationship between the flora, and further destroy the dynamic balance state between the oral flora and the host, so that various oral diseases are caused, and even systemic diseases are caused.

Opportunistic pathogens originally present in the oral cavity or pathogenic microorganisms ingested from the outside can cause various oral diseases under certain conditions. Streptococcus mutans(s) is the leading causative bacterium of dental caries, secreting acidic substances and corroding enamel, causing demineralization of teeth and, in turn, caries. Porphyromonas gingivalis (p.gingivalis) is mainly inhabited in the gingival sulcus and periodontal pocket of the oral cavity, can cause rapid infection of the gingiva, and is a main pathogenic bacterium causing periodontitis. Bifidobacteria (bifidobacteria) are a kind of probiotic bacteria widely present in the habitats of human mouth, digestive tract, vagina, etc., and mainly exert their probiotic effects in the intestinal tract, but are also detected in the oral cavity of healthy people. However, with the wide use of antibiotics, the life and eating habits of modern people are continuously deteriorated, the balance relationship of flora in oral cavity is easily broken, the oral cavity problem is gradually highlighted, and the oral cavity problem becomes a frequent problem for modern people.

Disclosure of Invention

The invention aims to provide a milk tablet with inactivated probiotics to achieve the aim of regulating the balance of oral flora;

the invention also aims to provide a preparation method of the inactivated probiotic milk tablet.

In order to achieve the purpose, the invention adopts the technical scheme that:

the inactivated probiotics milk tablet comprises the following raw materials of active ingredients in parts by weight: 70-75 parts of sorbitol, 20-25 parts of whole milk powder, 2-4 parts of magnesium stearate, 0.5-2 parts of citric acid and 0.5-2 parts of inactivated probiotic powder.

As one limitation, the inactivated probiotic bacteria powder is lactobacillus rhamnosus X253 inactivated probiotic bacteria powder;

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 of the China Committee for culture Collection of microorganisms in 8 months and 20 days in 2019, has the preservation number of CGMCC No.18404 and is disclosed for the first time in an online patent application document with the patent number of 202010258772.0.

As a further limitation, the inactivated probiotic powder of the lactobacillus rhamnosus X253 is prepared by inoculating the lactobacillus rhamnosus X253 into an MRS liquid culture medium, performing anaerobic propagation culture at 35-40 ℃ for 22-26 h, performing centrifugal separation and probiotic washing, freeze-drying the obtained probiotic thallus under the protection of a freeze-drying protective agent solution, grinding, sieving with a 30-50-mesh sieve, and heating for inactivation.

As a further limitation, the inoculation amount of the lactobacillus rhamnosus X253 is 1-1.5% of the weight of the MRS liquid medium.

As a further limitation, the temperature of the centrifugal separation is less than or equal to 4 ℃, the rotating speed is 6000-12000 r/min, and the time is 10-15 min.

As a further limitation, the heating inactivation temperature is 90-100 ℃ and the time is 4-6 h.

The freeze-drying protective agent solution further comprises 105-115 g/L of skim milk powder, 80-90 g/L of trehalose, 60-70 g/L of maltodextrin, 13-17 g/L of sodium glutamate and 8-12 g/L of sorbitol.

As a further limitation, toThe raw materials for preparing the effective components of the MRS liquid culture medium comprise, 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 MgSO (MgSO)₄·7H₂0.1 to 0.3 part of O and MnSO₄·7H₂0.04-0.06 part of O and 1000 parts of distilled water.

And (3) crushing and sieving the whole milk powder, the citric acid and the inactivated probiotic bacteria powder, mixing the sieved powder with magnesium stearate, and tabletting to obtain the inactivated probiotic bacteria milk tablet.

As a limitation, the aperture of the screened mesh is 60-70 meshes.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:

the invention adopts the inactivated probiotics powder, which has the same biological function as the active probiotics, and compared with the active probiotics, the inactivated probiotics has the advantages of high safety, no limit of bacteria amount, easy production and storage, and the like; the lactobacillus rhamnosus X253 inactivated probiotic powder preferably adopted by the invention can effectively adhere to oral epithelial cells under the condition of heat inactivation, shows better adhesion performance than the lactobacillus rhamnosus X253, can also be used as oral probiotic permanent planting to compete with the oral pathogenic bacteria for permanent planting sites, and regulates oral health; the inactivated probiotic milk tablet provided by the invention can inhibit oral pathogenic bacteria in vivo, and simultaneously improve the content of beneficial bacteria, so that the aim of regulating the balance of oral flora can be achieved;

the inactivated probiotic milk tablet provided by the invention has the advantages of good taste, low cost, simple preparation method and easily obtained raw materials; the lactobacillus rhamnosus X253 has the capability of inhibiting harmful strains in the oral cavity after being inactivated by heat, so that the oral environment is effectively improved, and oral diseases are prevented and treated; the inactivated probiotic milk tablet prepared by the invention has high safety, is not limited by the number of bacteria, and is easy to produce, transport and store.

The inactivated probiotic milk tablet is suitable for all kinds of people to eat.

Drawings

FIG. 1 is a graph showing a comparison of the adhesion indexes of inactivated bacteria X253 and live bacteria X253 to KB cells in example 7 of the present invention;

FIG. 2 shows the P.g gene expression levels of the blank control group and the groups C1-C6 in example 8 of the present invention;

FIG. 3 shows the S.m gene expression levels of the blank control group and the groups C1 to C6 in example 8 of the present invention;

FIG. 4 shows the Bif gene expression levels of the blank control group and the groups C1 to C6 in example 8 of the present invention.

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 1A method for preparing inactivated probiotic milk tablets

The preparation method of the inactivated probiotic milk tablet in the embodiment comprises the following steps of:

1) preparation of inactivated probiotic bacterium powder of lactobacillus rhamnosus X253

11) Taking 100g casein peptone, 100g beef extract, 50g yeast extract, 200g glucose, 50g sodium acetate, 20g diamine citrate, 10g tween-80 and 20g K₂HPO₄2g of MgSO 2₄·7H₂O, 0.5g MnSO₄·7H₂And mixing O and 10L of distilled water (namely 10kg of distilled water) uniformly to prepare the MRS liquid culture medium.

Inoculating 0.1kg of lactobacillus rhamnosus X253 seed liquid into 10kg of MRS liquid culture medium (the inoculation amount of lactobacillus rhamnosus X253 is 1% of the weight of the MRS liquid culture medium and is marked as JZ1), carrying out anaerobic enrichment culture at 37 ℃ for 24h, centrifuging the obtained enrichment culture bacterial liquid at 4 ℃ at 12000r/min for 10min, removing supernatant, and washing probiotic bacteria with normal isovolumetric saline (which is isovolumetric with the culture medium, namely 10L) until the bacteria are white to obtain probiotic bacteria;

12) adding 220g of skimmed milk powder, 170g of trehalose, 130g of maltodextrin, 30g of sodium glutamate and 20g of sorbitol into water to prepare 2L of freeze-drying protective agent solution;

and (2) resuspending the probiotic bacteria in 2L of freeze-drying protective agent solution, uniformly mixing, adding into a freeze dryer for vacuum freeze drying, grinding the obtained freeze-dried product, sieving with a 40-mesh sieve, collecting the freeze-dried powder, and placing in a drying oven at 90 ℃ for heating and drying for 4h to obtain the lactobacillus rhamnosus X253 inactivated probiotic bacteria powder. Wherein, the collected freeze-dried powder can also be stored in a refrigerator below minus 80 ℃ for standby.

2) Preparation of inactivated probiotic milk tablet

21) Respectively pulverizing sorbitol, whole milk powder, citric acid, and Lactobacillus rhamnosus X253 inactivated probiotic bacteria powder, and sieving with 60 mesh sieve to obtain corresponding sorbitol powder, whole milk powder, citric acid powder, and Lactobacillus rhamnosus X253 inactivated probiotic bacteria powder;

22) taking 70kg of sorbitol powder, 22kg of whole milk powder, 0.8kg of citric acid powder and 1.2kg of lactobacillus rhamnosus X253 inactivated probiotic powder, uniformly mixing, adding 4kg of magnesium stearate, uniformly mixing, and tabletting to obtain the inactivated probiotic milk tablet, wherein the label is N1.

The inactivated probiotic milk tablet prepared by the embodiment is suitable for all kinds of people to eat.

Example 2-6 preparation method of inactivated probiotic milk tablet

Examples 2 to 6 are methods for preparing inactivated probiotic milk tablets, which have substantially the same steps as example 1, except for the difference in the amount of the raw materials, and are specifically shown in table 1:

table 1 raw material amounts and parameter schedule in inactivated probiotic milk tablets

The contents of the other portions of examples 2 to 6 are the same as those of example 1.

Example 7 determination of adhesion Properties of Lactobacillus rhamnosus X253 in two physiological states, live and inactivated

a1) Preparation of bacterial suspensions

Selecting a single colony of lactobacillus rhamnosus X253, inoculating the single colony into an MRS liquid culture medium, culturing at 37 ℃ overnight, inoculating the obtained seed bacterial liquid into 5mL of the MRS liquid culture medium (same as the example 1) according to the inoculation amount of 1 wt%, culturing at 37 ℃ for 24h, and transferring the obtained bacterial liquid into two 1.5mL Ep tubes, wherein 1mL of each tube is marked as bacterial liquid A and bacterial liquid B.

Centrifuging the bacterial liquid A at 6000rpm/min for 5min, then discarding the supernatant, and resuspending the obtained thallus precipitate in 1mL of RPMI 1640 culture medium (containing 15% of fetal bovine serum) to obtain bacterial liquid A1;

inactivating the bacterial liquid B by water bath at 65 ℃ for 30min, centrifuging at 6000rpm/min for 5min, removing supernatant, and resuspending the obtained thallus precipitate in 1mL of RPMI 1640 culture medium (containing 10% fetal calf serum) to obtain bacterial liquid B1;

adjusting the concentration of the bacterial liquid in the bacterial liquid A1 and the bacterial liquid B1 to 4.5 multiplied by 10 by adopting an ultraviolet spectrophotometry method⁹CFU/mL, respectively designated as bacterial suspension A2 (viable bacterium X253) and bacterial suspension B2 (inactivated bacterium X253).

a2) Oral KB cell culture

Oral epithelial cell line KB cells were cultured in 1640 cell culture medium (containing 10% heat-inactivated fetal bovine serum) and then cultured in a medium containing 5 v% CO₂And under the concentration condition, incubating in a constant temperature incubator at 37 ℃ until the growth state of the cells is observed to be good through microscopic examination, and obtaining the well-cultured KB cells.

a3) Cell adhesion experiment

Respectively connecting well-cultured KB cells to 10 6-well plates (divided into 2 groups and 5 in each group) with cover glass, culturing overnight until the KB cells are completely attached to the wall, removing the culture solution, rinsing with a brand-new culture solution twice, then respectively adding bacterial liquid A2 or bacterial liquid B2 to each group (namely adding bacterial liquid A2 to one group and adding bacterial liquid B2 to the other group), and adding 5 v% CO into the mixture₂Symbiotic culture at 37 deg.C under the condition of concentration, and taking 16 wells for each groupThe plate was terminated at 1h, 2h, 4h, 6h and 8 h. After the culture is terminated, respectively taking out the glass slides, washing the glass slides for three times by PBS liquid, fixing the glass slides by methanol and carrying out gram staining, randomly selecting 50 cells under an oil lens, calculating lactobacillus rhamnosus on the visible cell surfaces, and calculating the adhesion index by the following calculation formula:

adhesion index ═ number of adherent bacteria/number of cells × 100%.

See fig. 1 for specific calculation results. From the figure 1, it can be seen that within 1 hour to 6 hours, the adhesion index of the heat-inactivated lactobacillus rhamnosus X253 (inactivated bacteria X253) to KB cells is higher than that of the lactobacillus rhamnosus X253 (viable bacteria X253), and after 6 hours, the adhesion index of the lactobacillus rhamnosus X253 (viable bacteria X253) to KB cells is higher than that of the heat-inactivated lactobacillus rhamnosus X253 (inactivated bacteria X253), and the result shows that within the initial time, the adhesion ability of the heat-inactivated lactobacillus rhamnosus X253 (inactivated bacteria X253) to oral KB cells is stronger than that of the lactobacillus rhamnosus X253 (viable bacteria X253).

Example 8 functional evaluation of inactivated probiotic milk tablets for regulating oral flora balance

A blank control milk tablet was prepared as in the step of example 1, except that no powder of inactivated probiotic bacteria of lactobacillus rhamnosus X253 was added to the blank control milk tablet.

In this example, the inactivated probiotic milk tablets N1 to N6 prepared in examples 1 to 6 were subjected to a drinking test.

The trial test disclosed recruits 35 subjects over 18 years old, wherein the subjects need to meet the following requirements: male and female (female is in non-pregnant period); more than 20 primary teeth are reserved in the oral cavity; periodontal treatment, tooth whitening (porcelain veneering, cold light whitening, etc.), orthodontics, etc. have not been done in the last 6 months; local medicine (such as using gargle, watermelon frost, etc.) without oral cavity within 1 month; in 2-6 weeks, no tooth-filling plan is provided, and antibiotics (such as amoxicillin, cephalosporins, roxithromycin, oxyfloxacin and the like), hormones (such as glucocorticoid, thyroid hormone, insulin and the like) and immunosuppressants (such as dexamethasone, methylprednisolone, sirolimus and azathioprine) are not taken in the half month; no administration of antibiotics (such as amoxicillin, cephalosporins, roxithromycin, ofloxacin, etc.), hormones (such as glucocorticoid, thyroid hormone, insulin, etc.) and immunosuppressants (such as dexamethasone, methylprednisolone, sirolimus, azathioprine) is planned for 2-6 weeks; the test population is investigated without obvious abnormal occlusion relation (abnormal corresponding relation between the upper jaw dentition and the lower jaw dentition and low chewing efficiency), and 17 persons are found to have the oral odor.

The population participating in the test is randomly divided into 7 groups, wherein each group comprises 5 persons, namely a blank control group and C1-C6 groups, wherein the population with the oral odor condition is distributed as evenly as possible, 3 persons exist in the blank control group and the C1-C2 groups respectively, and 2 persons exist in the C3-C6 groups respectively.

Before the start of the experiment, plaque (as a sample) in the third molar cleft between the left and right lower teeth of all the subjects was collected, and the contents of streptococcus mutans (hereinafter, referred to as s.m), porphyromonas gingivalis (hereinafter, referred to as P.g), and bifidobacterium (hereinafter, referred to as Bif) in the oral cavities of all the subjects were analyzed. Starting from the first day, test groups in groups C1-C6 eat inactivated probiotic milk tablets N1-N6 one by one, three times a day and 1 tablet (0.8 g/tablet) at a time. The administration method comprises putting the tablet into oral cavity, and keeping in oral cavity for at least 2-3min before swallowing. The food is taken once every day after breakfast, lunch and dinner for 30min, and is taken within half an hour without brushing teeth, eating and gargling. The oral hygiene is kept according to the original habit during the eating period, and no fluorine-containing toothpaste is used during the test period. After the second week (i.e. 2 weeks after taking the inactivated probiotic milk tablet), tartar (as a sample) in the gap between the third molars of the left and right lower teeth was removed with sterile dental floss and stored in a sterile petri dish at-80 ℃. The blank control group of human subjects eat the control milk tablets (without assisting in reducing the effect of oral pathogenic bacteria), and the other groups are completely consistent with the groups C1-C6. And respectively extracting DNA contained in each group of tartar samples, performing fluorescence quantitative PCR detection on target bacteria, and calculating the gene expression level of the target bacteria after 2 weeks finish by using a relative quantification method by taking an experimental result before the beginning of an experiment as a control.

The data of the gene expression level of the target bacteria were analyzed by GraphPad Prism 8 software, and P <0.05 indicated that the results were significantly different, and P <0.01 indicated that the results were significantly different, and the results are shown in fig. 2 to 4.

As can be seen from FIG. 2, after eating the inactivated probiotic milk tablet for 2 weeks, the P.g gene expression levels in the oral cavities of the tested persons in groups C1-C6 were all significantly reduced (P <0.01) compared with the blank control group; the experiments show that the inactivated probiotic milk tablet can remarkably inhibit gene expression of P.g in oral cavities of tested people, reduce the content of P.g in oral flora and further reduce the occurrence probability of oral diseases.

As can be seen from fig. 3, after eating the inactivated probiotic milk tablet for 2 weeks, compared with the blank control group, the gene expression level of s.m in the oral cavity of the tested persons in groups C1-C6 was significantly reduced (P <0.05), and the above experiments indicate that the inactivated probiotic milk tablet of the present invention can significantly inhibit the gene expression of s.m and reduce the content of s.m in the oral flora, thereby maintaining the balance of oral flora and inhibiting the occurrence and development of oral diseases.

As can be seen from FIG. 4, after eating the inactivated probiotic milk tablet for 2 weeks, compared with the blank control group, the gene expression level of Bif in the oral cavity of the tested persons in groups C1-C6 is significantly increased (P <0.05), and the experiments show that the inactivated probiotic milk tablet of the invention can significantly increase the expression level of Bif, increase the content of Bif in the oral flora, compete with pathogenic bacteria for colonization sites, and is beneficial to maintaining the balance of the oral flora.

Example 9 functional evaluation of inactivated probiotic milk tablets to improve oral malodour

In this example, a review survey was conducted on 17 persons with oral malodor among the subjects enrolled in example 8, and the use feelings of the 17 subjects were summarized, with the following specific results:

TABLE 2 results of oral malodour amelioration by inactivated probiotic milk tablets

As can be seen from table 2, none of the subjects with oral malodor in the blank control group improved their oral malodor after the end of the experiment, while 14 subjects with oral malodor in the C1-C6 group improved their oral malodor after the end of the experiment, 10 of them improved significantly, and 4 slightly improved them. The experiments prove that the inactivated probiotic milk tablet can obviously improve the peculiar smell of the oral cavity.

Example 10 organoleptic evaluation of inactivated probiotic milk tablets

In this example, 30 subjects of groups C1-C6 in example 8 were used to perform a sensory evaluation survey on the inactivated probiotic milk tablet, wherein the specific evaluation criteria are shown in Table 3, and the results are shown in Table 4:

TABLE 3 evaluation criteria table for sensory evaluation

Scoring item	Sensory description criteria	Full mark
			Tissue state	Uniform color, moderate size, and no powder on surface	1
Taste of the product	Moderate hardness and easy melting	1
			Taste of the product	The milk has pure and moderate flavor and no peculiar smell	1

TABLE 4 sensory evaluation results Table

Group of	Organization state (score)	Mouthfeel (score)	Flavor (score)
				C1 group (n ═ 5)	5	5	5
C2 group (n ═ 5)	5	5	5
				C3 group (n ═ 5)	5	5	5
C4 group (n ═ 5)	5	5	5
				C5 group (n ═ 5)	5	5	5
C6 group (n ═ 5)	5	5	5

As can be seen from Table 4, the inactivated probiotic milk tablet of the invention has good tissue state, good taste and flavor, is suitable for daily eating by common consumers, and has the outstanding advantages of regulating the balance of oral flora and improving the peculiar smell of the oral cavity.

In conclusion, the lactobacillus rhamnosus X253 strain adopted by the invention still has the capability of inhibiting harmful bacteria in the oral cavity after heat inactivation. On the basis, the inactivated probiotic milk tablet can inhibit the colonization of pathogenic bacteria in the oral cavity, increase the colonization of beneficial bacteria, adjust the balance of oral flora, effectively improve the oral peculiar smell, adjust the oral environment and prevent and treat oral diseases.

In addition, the milk tablet prepared by the invention has good taste, low cost, simple preparation method and easily obtained raw materials, and the probiotics used by the invention is heat inactivated probiotic powder, so that the milk tablet has high safety, is not limited by the number of bacteria, and is easy to produce, transport and store.

Claims (10)

1. The inactivated probiotic milk tablet is characterized in that the raw materials for preparing the effective components comprise the following components in parts by weight: 70-75 parts of sorbitol, 20-25 parts of whole milk powder, 2-4 parts of magnesium stearate, 0.5-2 parts of citric acid and 0.5-2 parts of inactivated probiotic powder.

2. The inactivated probiotic milk tablet of claim 1, wherein the inactivated probiotic powder is lactobacillus rhamnosus X253 inactivated probiotic powder.

3. The inactivated probiotic milk tablet according to claim 2, wherein the inactivated probiotic powder of lactobacillus rhamnosus X253 is prepared by inoculating lactobacillus rhamnosus X253 into an MRS liquid medium, performing anaerobic proliferation culture at 35-40 ℃ for 22-26 h, performing centrifugal separation and probiotic washing, freeze-drying the obtained probiotic thallus under the protection of a freeze-drying protective agent solution, and then performing grinding, sieving and heating inactivation.

4. The inactivated probiotic milk tablet according to claim 3, wherein the amount of lactobacillus rhamnosus X253 inoculum is 1-1.5% by weight of the MRS liquid medium.

5. The inactivated probiotic milk tablet according to claim 3 or 4, wherein the temperature of the centrifugal separation is not more than 4 ℃, the rotation speed is 6000 to 12000r/min, and the time is 10 to 15 min.

6. The inactivated probiotic milk tablet according to claim 3 or 4, wherein the temperature of the heating inactivation is 90-100 ℃ for 4-6 hours.

7. The inactivated probiotic milk tablet according to claim 3 or 4, wherein the freeze-drying protective agent solution contains 105-115 g/L of skimmed milk powder, 80-90 g/L of trehalose, 60-70 g/L of maltodextrin, 13-17 g/L of sodium glutamate and 8-12 g/L of sorbitol.

8. The inactivated probiotic milk tablet according to claim 3 or 4, wherein the raw materials for preparing the active ingredients 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, K₂HPO₄1.5-2.5 parts of MgSO (MgSO)₄·7H₂0.1 to 0.3 part of O and MnSO₄·7H₂0.04-0.06 part of O and 1000 parts of distilled water.

9. A process for preparing the milk tablet of inactivated probiotics according to any one of claims 1 to 8, wherein the milk tablet of inactivated probiotics is prepared by taking sorbitol, whole milk powder, citric acid and powder of inactivated probiotics, crushing, sieving, mixing with magnesium stearate, and tabletting.

10. The method for preparing the inactivated probiotic milk tablet according to claim 9, wherein the mesh size of the sieve is 60-70 mesh.

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