CN118266588A - Probiotic crystal ball containing saliva combined with lactobacillus and bifidobacterium longum and preparation method thereof - Google Patents
Probiotic crystal ball containing saliva combined with lactobacillus and bifidobacterium longum and preparation method thereof Download PDFInfo
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- CN118266588A CN118266588A CN202211739970.4A CN202211739970A CN118266588A CN 118266588 A CN118266588 A CN 118266588A CN 202211739970 A CN202211739970 A CN 202211739970A CN 118266588 A CN118266588 A CN 118266588A
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
- A23L29/281—Proteins, e.g. gelatin or collagen
- A23L29/284—Gelatin; Collagen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P20/00—Coating of foodstuffs; Coatings therefor; Making laminated, multi-layered, stuffed or hollow foodstuffs
- A23P20/20—Making of laminated, multi-layered, stuffed or hollow foodstuffs, e.g. by wrapping in preformed edible dough sheets or in edible food containers
- A23P20/25—Filling or stuffing cored food pieces, e.g. combined with coring or making cavities
-
- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Zoology (AREA)
- Dispersion Chemistry (AREA)
- Mycology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a probiotic crystal ball preparation containing saliva and lactobacillus and bifidobacterium longum, which comprises a shell structure and an inner core structure, wherein the shell structure comprises gelatin, glycerol and water; the core structure comprises Lactobacillus salivarius, bifidobacterium longum and hydrogenated oil. The preservation number of the saliva combined lactobacillus is CCTCC NO: m2022172; the preservation number of the bifidobacterium longum is CCTCC NO: m2019780. The invention adopts a double-layer seamless capsule technology with gelatin as a main shell material and hydrogenated oil and fungus powder as inner cores, so that probiotics are not digested by gastric juice and enter intestinal tracts to have higher survival rate. In addition, the inner core of the invention uses the screened saliva combined lactobacillus VHProbi A strain to generate no hemolysin, can not dissolve blood cells, and has safety. It has scavenging effect on DPPH free radical and hydroxyl free radical; and has a wider antibacterial spectrum.
Description
Technical Field
The invention relates to the technical field of application of probiotics, in particular to a probiotic crystal ball containing saliva combined lactobacillus and bifidobacterium longum and a preparation method thereof.
Background
Probiotics are preparations of viable microorganisms that, when ingested in sufficient quantities, can confer a benefit to the host by improving and maintaining the balance of intestinal microbiota. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal tract, the effect of promoting nutrient absorption and maintaining intestinal health is achieved, so that single microorganism or mixed microorganism with definite composition beneficial to health is produced. The probiotic function of probiotics is widely studied in the fields of medicine, food and feed, and the like, and intestinal symbiotic microorganisms greatly influence the physiology of the gastrointestinal tract, as well as the extra-intestinal organs and systems through their metabolic activities and molecular interactions with host cells. More and more strains have proven to be probiotics and are used by humans, such as lactobacillus, bifidobacterium etc. However, the survival of intestinal probiotics is affected by many factors, such as temperature, pH, pressure, gastric acid, bile salts, etc. The probiotics can reach intestinal tracts only by being digested in the oral cavity and the stomach after being orally taken, wherein the pH value of gastric juice is generally between 0.8 and 3.0, the survival of the probiotics is a great test, and the probiotics are easy to lose activity through the strong acid environment of the gastric juice, so that the survival rate of the probiotics in the human body is small.
To perform a probiotic function, it is desirable that the probiotic bacteria increase their survival rate during storage and in the environment within the intestinal tract of the host. How to make these probiotics pass through stomach and smoothly enter intestinal tract to act is a difficult problem to solve.
Microencapsulation technology has been an important technology to protect the viability of probiotics under acidic conditions of processing, storage and stomach. The microcapsule technology is to encapsulate a target core material (solid, liquid or even gas) with an embedding material to form a microcapsule with a semipermeable or sealed capsule membrane with a diameter in the range of 1-5000 μm (typically 5-400 μm). The microcapsule structure for protecting probiotics mainly comprises a matrix structure, a crosslinking structure and an outer coating structure. Wherein, the matrix structure is a coating wall formed by the polymer through hydrogen bond, van der Waals force, hydrophobic force and the like, and is a typical structure of a product prepared by spray drying, ultrasonic vacuum spray drying, spray cooling, electrostatic spinning and supercritical technology. In crosslinked structures, the polymer chains are typically crosslinked by ions or enzymes, which are typical structures of samples prepared by extrusion, emulsification, enzymatic gelation, and impact aerosol techniques. The probiotics microcapsule with the two structures can enable a small amount of probiotics to be exposed in gastric juice, so that partial bacterial quantity loss can be caused, and the survival rate of the probiotics reaches 68% -78%. The outer wrapping structure is mainly used for wrapping the probiotics in the capsule shell or the crystal ball shell in a multi-layer manner, so that the probiotics are isolated from being contacted with gastric juice, and the probiotics are better protected.
Disclosure of Invention
The invention aims to provide a probiotic crystal ball preparation containing saliva combined lactobacillus and bifidobacterium longum and application thereof, wherein the used saliva combined lactobacillus VHProbi A (Lagilactobacillus salivarius VHProbi A) and bifidobacterium longum VHProbi Y (Bifidobacteriumlongum VHProbi Y) are both screened from infant feces, the bifidobacterium longum VHProbi Y08 can effectively reduce the genetic mutation of cancerogenic substances on human cells, the saliva combined lactobacillus VHProbi A17 can inhibit the growth of oral pathogenic bacteria, and the saliva combined lactobacillus VHProbi A and bifidobacterium longum VHProbi Y have strong artificial gastric juice tolerance. The probiotic crystal ball has stronger stress resistance and good stability, and can be widely applied to the fields of foods, health products and the like.
The invention firstly provides a probiotic crystal ball which comprises a shell structure and an inner core structure, wherein the shell structure comprises gelatin, glycerol and water; the inner core structure comprises saliva combined lactobacillus, bifidobacterium longum and hydrogenated oil;
Preferably, the mass parts of the gelatin, the glycerol and the water are as follows: 18-25 parts of gelatin, 6-8 parts of glycerol and 69-76 parts of water;
A specific description of the embodiment, wherein the mass part ratio of the gelatin, the glycerin and the water is 18 parts of the gelatin, 6 parts of the glycerin and 76 parts of the water; or 20 parts of gelatin, 8 parts of glycerol and 72 parts of water; or 25 parts of gelatin, 6 parts of glycerol and 69 parts of water;
the hydrogenated oil is hydrogenated oil with a melting point of 42 ℃.
Preferably, the saliva is combined with lactobacillus, and the preservation number is CCTCC NO: m2022172;
The preservation number of the bifidobacterium longum is CCTCC NO: m2019780.
The preparation method of the probiotic crystal ball comprises the following steps:
Preparing an inner core solution by uniformly mixing and dispersing saliva combined lactobacillus and bifidobacterium longum bacterial powder in hydrogenated vegetable oil, and uniformly stirring;
preparing a shell solution: dissolving gelatin in water, adding glycerol, continuously dissolving, stirring, and pouring into a shell solution storage tank of a capsule making machine;
Preparing a probiotic crystal ball: the outer shell solution storage tank and the inner core solution storage tank of the capsule machine are concentric nozzles. And then controlling and regulating the pump speed to 650mL/h, controlling the pressure of an inner core tank to 5KPa, enabling an outer shell solution to just wrap the inner core solution in the titration process, dripping the inner core solution into a liquid paraffin solution tank with the temperature controlled at 10 ℃ to enable the inner core solution to gel, forming continuous core-shell crystal balls, ventilating and drying the prepared crystal balls at the temperature of 25 ℃ in a room until the water content is below 3.0%, and obtaining the composite probiotics crystal balls with the diameter of 1.6 mm.
The invention adopts a double-layer seamless capsule technology with gelatin as a main shell material and hydrogenated oil and fungus powder as inner cores, so that probiotics are not digested by gastric juice and enter intestinal tracts to have higher survival rate. In addition, the inner core of the invention uses the screened saliva combined lactobacillus VHProbi A strain to generate no hemolysin, can not dissolve blood cells, and has safety. It has scavenging effect on DPPH free radical and hydroxyl free radical; has obvious inhibition effect on five oral pathogenic bacteria such as streptococcus mutans, porphyromonas gingivalis, actinomyces viscosus, actinobacillus conglomerates and clostridium nucleatum, has obvious inhibition effect on intestinal pathogenic bacteria such as escherichia coli and salmonella enteritidis, and has wider inhibition spectrum.
Detailed Description
The probiotics pellet is one kind of wrapping structure, and the probiotics is wrapped by wall materials, and the survival rate of the probiotics and the resistance to external reverse environment are determined by different wall materials. The choice of suitable wall materials has a decisive influence on the survival rate, pH adaptation, temperature tolerance, storage stability, etc. of the probiotics after microencapsulation. The selection of a suitable wall material is the first step in the preparation of the probiotic microcapsules, and the ideal probiotic microcapsule wall material should have the following conditions: (1) good biocompatibility and film forming property; (2) can protect and improve the activity of the core material; (3) non-reactive with the core material; (4) no toxic or side effects; (5) easy processing and good rheological properties; (6) Viscosity, permeability, hydrophilicity and solubility are satisfactory.
The invention mainly wraps functional bacterial strain for conditioning intestinal tracts, so the requirements on wall materials are gastric acid resistance, and probiotics can be rapidly dissolved and released in the intestinal tracts, so that the probiotics can be rapidly planted in the intestinal tracts and exert probiotic renting. The invention adopts a double-layer seamless capsule technology with gelatin as a main shell material and hydrogenated oil and fungus powder as inner cores, so that probiotics are not digested by gastric juice and enter intestinal tracts to have higher survival rate.
The functional strain wrapped by the invention is the saliva combined lactobacillus VHProbi A (Lagilactobacillus salivarius VHProbi A17) obtained by screening, and is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China at 3 months and 1 day in 2022: m2022172. The strain has remarkable inhibiting effect on streptococcus mutans and actinomyces viscosus which cause dental caries, and on Porphyromonas gingivalis, clostridium nucleatum and Actinobacillus semiactinomyces which cause periodontal diseases. But also can remove the biofilm formed by the streptococcus mutans and inhibit the growth and reproduction of the streptococcus mutans, actinomyces viscosus, porphyromonas gingivalis, clostridium nucleatum and actinomyces semiactinomyces.
The bifidobacterium longum used in the embodiment of the invention is bifidobacterium longum VHProbi Y (Bifidobacterium longum VHProbi Y) which has been preserved in the China center for type culture collection (CCTCC NO) of the university of Wuhan, china in 10 months and 8 days of 2019: m2019780.
The present invention will be described in detail with reference to examples.
Example 1: physicochemical Properties of Lactobacillus salivarius VHProbi A17
The saliva combined lactobacillus VHProbi A strain is obtained by screening healthy infant feces, and a blood cell plate experiment shows that the saliva combined lactobacillus VHProbi A strain does not produce hemolysin, can not dissolve blood cells, and has safety. The clearance rate of the catalyst to DPPH free radical is 20.9 percent, and the clearance rate of the catalyst to hydroxyl free radical is 10.6 percent.
The antibacterial effect of saliva combined with lactobacillus VHProbi A bacterial liquid and lysate on streptococcus mutans, actinomyces viscosus, porphyromonas gingivalis, actinobacillus conglomerates, clostridium nucleatum, escherichia coli and salmonella enteritidis was measured by using oxford cup method, and the diameters of the antibacterial circles are shown in table 1.
Table 1: inhibition effect of saliva combined with lactobacillus VHProbi A to pathogenic bacteria
| Oral pathogenic bacteria | A17 diameter of bacteria inhibition zone of fermentation broth | A17 diameter of inhibition zone of lysate |
| Streptococcus mutans | 27mm | 26mm |
| Porphyromonas gingivalis | 14.5mm | 10mm |
| Actinomycetes viscosus | 23.5mm | 22mm |
| Acinetobacter actinomyces | 11mm | 10mm |
| Clostridium nucleatum | 12.5mm | 11mm |
| Coli bacterium | >30mm | >30mm |
| Salmonella enteritidis | >30mm | >30mm |
From the results shown in Table 1, the saliva combined lactobacillus VHProbi A has obvious inhibition effect on five oral pathogenic bacteria such as streptococcus mutans, porphyromonas gingivalis, actinomyces viscosus, actinomyces conglomerate and clostridium nucleatum, has obvious inhibition effect on intestinal pathogenic bacteria such as escherichia coli and salmonella enteritidis, and has a wider inhibition spectrum.
In addition, saliva combines lactobacillus VHProbi A and 17 to effectively bind common oral pathogenic bacteria, significantly inhibiting their adhesion to teeth or gums. And the saliva combined with lactobacillus VHProbi A has no obvious influence on the proliferation activity of normal gingival epithelial cells of human, and has good safety and no cytotoxicity.
Example 2: physicochemical Properties of Bifidobacterium longum VHProbi Y strain 08
The bifidobacterium longum VHProbi Y provided by the invention is screened from infant feces, is sensitive to common antibiotics such as erythromycin and the like, and has good biological safety. The strain has strong oxidation resistance, the clearance rate of DPPH free radical and HRS free radical respectively reaches 23.8% and 74.5%, the inhibition rate of lipid peroxidation resistance of supernatant fluid is 41.99%, and the inhibition rate of cell lipid peroxidation resistance is 43.17%. The strain can also effectively degrade cholesterol with degradation rate up to 20%. Meanwhile, bifidobacterium VHProbi Y with the same length can effectively relieve the gene mutation toxicity effect of 4NQO, IQ and other cancerogenic substances on human cells, has an effective tumor prevention mechanism and has a certain value in the aspect of preventing and treating cancers.
Example 3: preparation of bacterial powder
(1) Inoculating activated saliva combined lactobacillus VHProbi A and bifidobacterium longum VHProbi Y to MRS broth culture medium, and culturing at 37 ℃ for 24 hours to obtain seed solution;
(2) Inoculating the seed solution into MRS broth culture medium according to the volume ratio of 5%, and stopping fermentation after performing expansion culture for 24 hours at 37 ℃ to obtain fermentation liquor;
(3) The fermentation broth was centrifuged at 5000rpm for 10 minutes and the bacterial sludge was collected.
(4) Adding a freeze-drying protective agent into the bacterial mud according to the mass ratio of 10%, stirring uniformly, and then freeze-drying to obtain bacterial powder.
The viable bacteria amount in the bacterial powder is measured according to national standard GB 4789.35-2016-lactobacillus test for food microorganism detection.
The result shows that the live bacteria amount in the saliva combined lactobacillus VHProbi A and the bifidobacterium longum VHProbi Y bacterial powder prepared by the method is more than 2000 hundred million cfu/g.
Example 4 preparation of composite probiotic Crystal balls
The probiotics crystal ball provided by the embodiment comprises a shell structure and an inner core structure, wherein the shell structure comprises the following components in parts by weight: 18 parts of gelatin, 6 parts of glycerol and 76 parts of water.
The inner core structure comprises the following components in parts by weight: 3 parts of saliva combined lactobacillus powder, 2 parts of bifidobacterium longum powder and 95 parts of hydrogenated oil with the melting point of 42 ℃.
The preparation method comprises the following steps: the preparation method of the crystal ball comprises the steps of dissolving hydrogenated vegetable oil at 45 ℃, adding bacterial powder, stirring for 15min by using a stirrer at 200rpm, uniformly mixing and dispersing the bacterial powder in the hydrogenated vegetable oil, pouring the bacterial powder into an inner core solution storage tank of a capsule making machine, setting the stirring speed of the inner core storage tank at 10rpm and the temperature at 45 ℃. Preparing a shell solution: dissolving gelatin at 70deg.C, adding glycerol, dissolving at 70deg.C, stirring, and pouring into a shell solution storage tank of capsule making machine, wherein the temperature of the shell storage tank is 70deg.C. The outer shell solution storage tank and the inner core solution storage tank of the capsule machine are concentric nozzles. And then controlling and regulating the pump speed to 650mL/h, controlling the pressure of an inner core tank to 5KPa, enabling an outer shell solution to just wrap the inner core solution in the titration process, dripping the inner core solution into a liquid paraffin solution tank with the temperature controlled at 10 ℃ to enable the inner core solution to gel, forming continuous core-shell crystal balls, ventilating and drying the prepared crystal balls at the temperature of 25 ℃ at room temperature until the water content is below 3.0%, and obtaining the composite probiotics crystal balls with the diameter of 1.6 mm.
Example 5: preparation of composite probiotics crystal balls
The probiotics crystal ball provided by the embodiment comprises a shell structure and an inner core structure, wherein the shell structure comprises the following components in parts by weight: 20 parts of gelatin, 8 parts of glycerol and 72 parts of water.
The inner core structure comprises the following components in parts by weight: 3 parts of saliva combined lactobacillus powder, 2 parts of bifidobacterium longum powder and 95 parts of hydrogenated oil with the melting point of 42 ℃.
The preparation method of the shell solution and the core solution is the same as above, then the pump speed is controlled and regulated to 650mL/h, the pressure of the core tank is 8KPa, the core solution is just wrapped by the shell solution in the titration process, the shell solution is dripped into a liquid paraffin solution tank with the temperature controlled at 10 ℃ to gel the shell solution, so as to form continuous core-shell crystal balls, and the prepared crystal balls are ventilated and dried at the temperature of 25 ℃ at room temperature until the water content is below 3.0%, so that the composite probiotic crystal balls with the diameter of 2.2mm are obtained.
Example 6: preparation of composite fungus crystal ball
The probiotics crystal ball provided by the embodiment comprises a shell structure and an inner core structure, wherein the shell structure comprises the following components in parts by weight: 25 parts of gelatin, 6 parts of glycerol and 69 parts of water.
The inner core structure comprises the following components in parts by weight: 5 parts of saliva combined lactobacillus powder, 2 parts of bifidobacterium longum powder and 93 parts of hydrogenated oil with the melting point of 42 ℃.
The preparation method of the shell solution and the core solution is the same as above, then the pump speed is controlled and regulated to 600mL/h, the pressure of the core tank is 8KPa, the core solution is just wrapped by the shell solution in the titration process, the shell solution is dripped into a liquid paraffin solution tank with the temperature controlled at 10 ℃ to gel the shell solution, so as to form continuous core-shell crystal balls, and the prepared crystal balls are ventilated and dried at the temperature of 25 ℃ at room temperature until the water content is below 3.0%, so that the composite probiotic crystal balls with the diameter of 2.5mm are obtained.
Example 7: preparation of composite fungus crystal ball
The probiotics crystal ball provided by the embodiment comprises a shell structure and an inner core structure, wherein the shell structure comprises the following components in parts by weight: 25 parts of gelatin, 8 parts of glycerol and 67 parts of water.
The inner core structure comprises the following components in parts by weight: 5 parts of saliva combined lactobacillus powder, 5 parts of bifidobacterium longum powder and 90 parts of hydrogenated oil with the melting point of 42 ℃.
The preparation method of the shell solution and the core solution is the same as above, then the pump speed is controlled and regulated to 600mL/h, the pressure of the core tank is 10KPa, the core solution is just wrapped by the shell solution in the titration process, the shell solution is dripped into a liquid paraffin solution tank with the temperature controlled at 10 ℃ to gel the shell solution, so as to form continuous core-shell crystal balls, and the prepared crystal balls are ventilated and dried at the temperature of 25 ℃ at room temperature until the water content is below 3.0%, so that the composite probiotic crystal balls with the diameter of 3.0mm are obtained.
Example 8: test for resistance to Artificial gastric juice
1. Preparation of artificial gastric juice
5G of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000ml of distilled water is added, pH 1.2 is adjusted by dilute hydrochloric acid, and then sterilization is carried out for 15min at 121 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly dissolved by shaking, and the pig mucosa pepsin is put into a water bath shaker at 37 ℃ for warm bath for 1 hour so as to simulate the temperature of a human body.
2. Artificial gastric juice tolerance test of probiotics pellet
2.1 Determination of the amount of Crystal coccus
38Ml of anaerobic diluent was incubated in a water bath at 45℃for 7min, 2g of the probiotic crystal balls of example 4, example 5, example 6 and example 7 were added respectively, incubated for 5min, homogenized at 45℃for 5min by a homogenizer, and the amount of enterococcus was determined according to GB4789.35-2016 "food microbiology test lactic acid bacteria test".
2.2 Artificial gastric juice digestion test
Weighing 2g of the probiotic crystal balls in example 4, example 5, example 6 and example 7, adding the probiotic crystal balls into 8ml of artificial gastric juice with constant temperature (10 min in a warm bath at 37 ℃) and shaking the mixture evenly by hand, and then placing the mixture into a water bath at 37 ℃ for digestion for 2 hours; the artificial gastric juice is removed by filtration with sterile filter paper, then the filtered crystal ball is placed in a sterile container and soaked in 18 ml of physiological saline for 10min, then the sterile filter paper is filtered, and the filter paper is washed twice with the sterile physiological saline, then the anaerobic diluent is added to ensure that the total weight of the crystal ball and the anaerobic diluent is about 40 g, and the real mass is recorded. Then the bacterial load is measured according to the method for measuring the bacterial load of the enterococcus. And comparing the data before and after the artificial gastric juice digestion of the probiotic crystal ball to obtain the survival rate of the probiotic crystal ball after the artificial gastric juice digestion.
2.3 Test of gastric juice tolerance by artificial fungus powder
0.04 G of saliva combined lactobacillus VHProbi A and bifidobacterium longum VHProbi Y powder are weighed and added into 9.96ml of artificial gastric juice which is kept at a constant temperature (10 min in a warm bath at 37 ℃), the mixture is shaken up manually and then put into a water bath at 37 ℃ for digestion for 2 hours, the viable bacteria amount before and after the digestion of the artificial gastric juice is respectively measured, and the survival rate of the powder after the digestion of the artificial gastric juice is obtained through the data comparison of the real gastric juice and the residual gastric juice.
Table 2: survival rate table of probiotics crystal ball viable bacteria after artificial gastric juice digestion
| Sample of | Survival rate of probiotics |
| Example 4 probiotic crystal ball | 91.11％±2.11％ |
| Example 5 probiotic crystal ball | 90.67％±3.04％ |
| Example 6 probiotic crystal ball | 91.25％±2.35％ |
| Example 7 probiotic crystal ball | 92.02％±4.06％ |
| Saliva combined lactobacillus VHProbi A and fungus powder | 1.00％±0.01％ |
| Bifidobacterium longum VHProbi Y and 08 powder | 0.80％±0.01％ |
The probiotic crystal balls prepared in the example 4, the example 5 and the example 6 can resist the artificial gastric juice with the pH value of 1.2 after being digested by the artificial gastric juice, and the bacterial load survival rate is as high as more than 90%, wherein the probiotic crystal ball in the example 7 has the best artificial gastric acid resistance effect. The result shows that the prepared crystal ball can well protect probiotics from being digested by artificial gastric juice and can smoothly reach intestinal tracts.
Example 9: probiotic crystal ball disintegration test
7.1 Preparation of Artificial gastric juice
The artificial gastric juice is prepared as above.
7.2 Preparation of artificial intestinal juice
5G of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH2PO4 and 3.0g of ox gall salt are respectively weighed, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized at 115 ℃ for 20min. Then adding 1g of pancreatin before using, shaking to dissolve, placing into a water bath shaker at 37 ℃ to warm bath for 1h so as to simulate the temperature of human body.
7.3 Disintegration test
According to the description method and the device of the Chinese pharmacopoeia 'disintegration time limit test', 6 pieces of probiotic crystal balls of the example 4, the example 5, the example 6 and the example 7 are respectively taken, the artificial gastric juice solution is checked for 2 hours without adding a baffle plate, each crystal ball is not disintegrated and cracked, a hanging basket is taken out, a small amount of physiological water is used for washing, each pipe is added with the baffle plate, the inspection is changed in the artificial intestinal juice according to the method, the complete dissolution time of the crystal balls is recorded, and the disintegration time is found to be 7min,6min,8min and 6min respectively, so that the crystal balls can be completely dissolved in the intestinal juice within 1 h.
Example 10: accelerated stability test
The probiotics acceleration test refers to that under the premise of ensuring that the failure mechanism of the probiotics product is not changed, the tested probiotics are accelerated to fail through strengthening temperature test conditions, so that the necessary probiotic stability is obtained in a short time, and the reliability or life index of the probiotics under normal conditions is evaluated. Since the shelf life of the pellet was relatively long, we used the stability of the probiotic pellet to be measured at 37 ℃ temperature extremes.
The composite probiotic crystal balls prepared in example 4, example 5, example 6 and example 7 are placed in a 37 ℃ incubator in a sealing manner, meanwhile, bacterial powder is taken as a comparison, bacterial amount is measured by sampling once every 5 days, and continuous detection is carried out for one month.
Table 2: bacterial load stability test table (LOG CFU/g) at 37 DEG C
From the results, the viable count of the bacterial powder gradually decreased with time. After 30 days of preservation, the bacterial amount of the probiotics pellet is not reduced, and the viable bacterial amount of the freeze-dried bacterial powder is respectively reduced by 0.89 and 1.2 orders of magnitude; therefore, compared with the pure freeze-dried powder, the stability of the bacteria in the probiotics crystal balls is better, and the decrease is slower. The saliva combined lactobacillus not only can inhibit oral pathogenic bacteria, but also can inhibit enteropathogenic bacteria such as escherichia coli and salmonella, and the bifidobacterium longum can relieve the effect of cancerogenic substances on human body cell gene mutation, and the two strains are combined for use, so that the intestinal tract can be better regulated and the immunity of the organism can be improved.
The probiotic crystal ball can be singly matched with prebiotics or other components to prepare a probiotic solid beverage, and can be directly taken orally, or can be added into various foods and beverages, such as various fruit and vegetable juices, yogurt and the like to prepare a probiotic composite beverage. The probiotics pellet can protect probiotics from reaching the intestinal tract smoothly and play a probiotic role in the intestinal tract.
Claims (8)
1. The probiotics crystal ball is characterized by comprising a shell structure and an inner core structure, wherein the shell structure comprises gelatin, glycerol and water; the core structure comprises Lactobacillus salivarius, bifidobacterium longum and hydrogenated oil.
2. The probiotic crystal ball according to claim 1, wherein the mass fraction of gelatin, glycerol and water is as follows: 18-25 parts of gelatin, 6-8 parts of glycerol and 69-76 parts of water.
3. The probiotic crystal ball according to claim 2, wherein the mass fraction of gelatin, glycerol and water is as follows: 18 parts of gelatin, 6 parts of glycerol and 76 parts of water.
4. The probiotic crystal ball according to claim 2, wherein the mass fraction of gelatin, glycerol and water is as follows: 25 parts of gelatin, 6 parts of glycerol and 69 parts of water.
5. The probiotic pellet of claim 1 wherein said hydrogenated oil is a hydrogenated oil having a melting point of 42 ℃.
6. The probiotic crystal ball of claim 1, wherein the saliva is associated with lactobacillus having a preservation number of CCTCC NO: m2022172.
7. The probiotic crystal ball of claim 1, wherein the bifidobacterium longum has a preservation number of cctccc NO: m2019780.
8. The probiotic crystal ball according to claim 1, wherein the preparation method comprises the following steps:
(1) Preparing an inner core solution by uniformly mixing and dispersing saliva combined lactobacillus and bifidobacterium longum bacterial powder in hydrogenated vegetable oil, and uniformly stirring;
(2) Preparing a shell solution: dissolving gelatin in water, adding glycerol, continuously dissolving, stirring, and pouring into a shell solution storage tank of a capsule making machine;
(3) Preparing a probiotic crystal ball: the pump speed of the capsule machine is 650mL/h, the pressure of the inner core tank is 5KPa, the inner core solution is just wrapped by the outer shell solution in the titration process, the inner core solution is dripped into the liquid paraffin solution tank with the temperature controlled at 10 ℃ to gel the inner core solution, so as to form a continuous crystal ball with a core-shell structure, and the prepared crystal ball is ventilated and dried at the temperature of 25 ℃ until the water content is below 3.0%, so that the composite probiotics crystal ball with the diameter of 1.6-3.0mm is obtained.
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