CN103652891A - Lactobacillus casei embedding microcapsule and preparation method thereof - Google Patents

Abstract

The invention provides a microcapsule for embedding Lactobacillus casei, which is characterized in that: the microcapsule is a three-layer embedded multi-core structure, wherein Lactobacillus casei is embedded in the first hydrogel coating layer to form The microcore, the second coating layer outside the microcore is vegetable oil, and a plurality of microcores coated with vegetable oil are embedded in the third coating layer formed by water-soluble polymer materials, forming multi-core microcapsules. The microcapsule can significantly improve the survival rate of Lactobacillus casei during drying processing and storage, exhibits excellent preservation performance of live Lactobacillus casei, and has good application potential in the fields of food, medicine and feed.

Description

A kind of microcapsule of embedding Lactobacillus casei and preparation method thereof

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Technical field:

The invention relates to a microorganism embedding technology, in particular to the multi-core microencapsulation embedding technology of Lactobacillus casei, one of the three major probiotics.

Background technique:

Lactobacillus casei (Lactobacillus casei) is a kind of probiotics, which can survive in large quantities in the intestinal tract after entering the human body, regulate the balance of intestinal flora, promote digestion and absorption of the human body, etc. Functions such as immunity, cancer prevention and tumor growth inhibition have good application potential in the fields of food, medicine and feed. However, due to the increase of osmotic pressure during the drying process of microcapsules and the influence of factors such as oxygen stress during storage, Lactobacillus casei is very easy to die. A microencapsulated embedding system with oxygen resistance and stable osmotic pressure around the bacteria to improve the application performance of Lactobacillus casei.

After searching the prior art, it is found that the microencapsulation embedding method and embedding products for probiotics of Lactobacillus casei have the effect of improving the stress resistance of probiotics. The embedding wall materials and excipients of microcapsules are mainly sodium alginate, gelatin, hydroxymethyl cellulose, β-cyclodextrin, porous starch, pectin, acacia gum and prebiotics; the capsule structure type is mostly single core -Solid-core microcapsules, a few are single-core-liquid-core microcapsules; some microcapsules are embedded in two or more layers, but all are single-core microcapsules, and the outermost layer is embedded with oil or lecithin,

Invention content.

The present invention aims at the problem that Lactobacillus casei is not resistant to oxygen stress and osmotic pressure changes, and is prone to death during microencapsulation embedding and storage, and provides a microencapsulation embedding method with high activity Lactobacillus casei, which can effectively improve the heat resistance of Lactobacillus casei properties and storage properties.

The microcapsule of the present invention has a multi-core structure, the palm oil fat layer is the middle coating layer, and the outermost microcapsule wall material is a mixture of calcium carbonate, sodium alginate and xanthan gum.

On the one hand, the present invention provides a microcapsule for embedding Lactobacillus casei, which is characterized in that: the microcapsule is a three-layer embedded multi-core structure, wherein the Lactobacillus casei sludge or solid-state fermentation product is embedded in the first A microcore is formed in the hydrogel coating layer, and the second coating layer outside the microcore is vegetable oil, and a plurality of microcores coated with vegetable oil are embedded in the first water-soluble polymer material. Among the three coating layers, the said Lactobacillus casei sludge or solid-state fermentation product is produced by the Lactobacillus casei strain BFT-1 whose preservation number is CCTCC NO: M 2011424.

Preferably, the concentration of Lactobacillus casei is 10 ⁹ -10 ¹¹ cfu/ml. Preferably, the microcore structure is a hydrogel with a particle size of 10-100 μm, and the second coating layer is wrapped with a palm oil layer with a melting point of 24°C.

Preferably, the present invention relates to multi-core microcapsules for embedding Lactobacillus casei including: Lactobacillus casei, carrageenan, Span-80, palm oil, calcium carbonate, sodium alginate and xanthan gum, wherein: Lactobacillus casei Embedded in hydrogel microcores of carrageenan, dextrose, yeast extract, and lactic acid, which are further embedded in a mixture of Span-80 and palm oil to form an emulsion, which is further dispersed In the mixed solution of calcium carbonate, sodium alginate and xanthan gum, and drop into the solidification solution to form solidified multi-core microcapsules.

The particle size distribution of the Lactobacillus casei micro-core is 10-100 μm, and the particle size distribution of the multi-core microcapsule is 0.5-5 mm.

In the multi-core microcapsules, the encapsulation rate of Lactobacillus casei is 70-99%.

On the other hand, the present invention relates to the preparation method of the above-mentioned multi-core microcapsules of Lactobacillus casei embedded, comprising the following steps:

The first step is to prepare Lactobacillus casei hydrogel solution: the preparation pH is 3.5-5.0, the mass percentage of carrageenan is 0.1-2%, the content of glucose is 0.1-0.5%, the content of yeast extract is 0.1-0.5%, Lactic acid concentration is 1~5% mixed aqueous solution, and the temperature is adjusted to 38~45°C. Then add Lactobacillus casei sludge or solid-state fermentation product to form Lactobacillus casei hydrogel solution. Preferably, the most suitable temperature is 40°C, and the optimum concentration of Lactobacillus casei is 8×10 ¹⁰ cfu/mL.

The second step is to prepare the Lactobacillus casei hydrogel microcore coated with palm oil in the outer layer: the Lactobacillus casei hydrogel solution described in the first step is in a volume ratio of 1:1 to 5 (the best ratio is 1: 3) Mix with palm oil at the same temperature, wherein the palm oil includes Span-80 with a mass percentage concentration of 1-5%, form an emulsion under stirring at 1000-2000 rpm, and then cool to 25°C to form The Lactobacillus casei hydrogel microcore coated with palm oil has a structural feature of a solid gel microcore.

The third step is to prepare multi-core microcapsules embedded with Lactobacillus casei micro-core: the Lactobacillus casei micro-core emulsion coated with the outer palm oil described in the second step is in a volume ratio of 1:2～4 (optimum ratio 1:3) mixed with calcium carbonate-sodium alginate-xanthan gum mixed solution (the mass ratio of sodium alginate and xanthan gum is 20:1~5, the total mass percentage concentration is 1~3%, calcium carbonate The mass percentage content is 0.5~3%, the temperature is 22~26°C), and it is dropped into the 1~4% calcium chloride solution at the temperature of 0~20°C, and after half an hour, the solidification of embedded Lactobacillus casei microcores is formed. Multi-core microcapsules, in which Lactobacillus casei microcores, palm oil and sodium alginate are all solid structures.

All percentages in the present invention, unless otherwise specified, belong to mass percentages.

The present invention adjusts the particle size distribution of the Lactobacillus casei microcore, the encapsulation efficiency of the Lactobacillus casei, and the Lactobacillus survival rate and particle size distribution of finished multi-core microcapsules to meet various application requirements. For example, by increasing the concentration of carrageenan, Lactobacillus casei microcores with larger particle sizes can be obtained, and vice versa, microcores with smaller particle sizes are obtained, and the concentration of carrageenan is negatively correlated with the particle size of microcores; The volume ratio of Lactobacillus casei hydrogel solution can obtain higher encapsulation efficiency of Lactobacillus casei, otherwise the encapsulation efficiency decreases, and the volume ratio of palm oil and Lactobacillus casei hydrogel has a negative correlation with the encapsulation efficiency; The size of the exit pore can adjust the size of the final multi-core microcapsule. The smaller the extrusion pore size, the smaller the particle size, and the larger the extrusion pore size, the larger the particle size. The extrusion pore size is positively correlated with the size of the multi-core microcapsule.

The content of Lactobacillus casei in the multi-core microcapsules embedded with Lactobacillus casei prepared by the present invention is 10 ⁹ to 10 ¹¹ cfu/mL, and after heat treatment at 85°C for 3 minutes, the number of remaining viable bacteria is 10 ⁷ to 10 ¹⁰ cfu/mL ; Sealed storage at 25°C for 60 days, the number of remaining viable bacteria is 10 ⁸ ~ 10 ¹⁰ cfu/mL; 4 hours in artificially simulated gastric juice, the release of Lactobacillus casei is not higher than 20%; 4 hours in artificially simulated intestinal juice, Lactobacillus casei The release amount is not less than 90%. Compared with traditional products, the heat resistance and storage performance are significantly improved, and the performance of gastrointestinal dissolution is excellent.

Beneficial effect

The preparation and operation of the present invention are simple, and the obtained microcapsules have no adhesion, good spheroidity, rounded surface, slow release under artificially simulated gastric juice, and low release amount, and can quickly and massively release Lactobacillus casei under artificially simulated intestinal juice, which is beneficial to cheese milk Targeted release and efficacy of bacilli in the animal gut.

Description of drawings

Fig. 1 is a microcapsule appearance and a photogram of the internal microcore structure, wherein. A, B, C and D are photos of the microcapsule appearance and internal microcore structure of Examples 1, 2, 3 and 4 in sequence (the upper row is the appearance, and the lower row is the corresponding internal structure).

Lactobacillus casei BFT-1, classification name: Lactobacillus casei BFT-1, Latin scientific name: Lactobacillus casei BFT-1, preservation number: CCTCC NO: M 2011424, preservation date is November 25, 2011, preservation unit is: China Typical Culture Collection Center, address: Wuhan University, Wuhan, China, zip code: 430072.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

The multi-core microcapsules related to embedding Lactobacillus casei of the following examples include: Lactobacillus casei BFT-1 ( Lactobacillus casei BFT-1) (preservation number: CCTCC NO: M2011424, preservation date is November 25, 2011, preservation The unit is China Typical Culture Collection Center, address: China. Wuhan. Wuhan University), Lactobacillus casei hydrogel micro-core, palm oil, sodium alginate and xanthan gum microcapsule wall, in which: Lactobacillus casei is embedded in The hydrogel micro-core is further coated on the outside by palm oil and sodium alginate-xanthan gum in turn, finally forming a multi-core microcapsule embedding Lactobacillus casei.

The Lactobacillus casei sludge or the Lactobacillus casei solid-state fermentation material in all the following examples is produced by the Lactobacillus casei strain BFT-1 with the preservation number of CCTCC No: M2011424.

Example 1

Step 1: Suspend 0.1g of carrageenan in 100mL of distilled water, heat to dissolve, add 0.1g of glucose, 0.1g of yeast extract and 1.0g of lactic acid, adjust the pH to 3.5 with sodium hydroxide, and cool to 38°C.

Step 2: Then add Lactobacillus casei sludge or Lactobacillus casei solid-state fermentation material to the solution formed in Step 1, and mix evenly (the concentration of Lactobacillus casei in the mixed solution is 6.3×10 ⁹ cfu/mL).

Step 3: Mix the above mixed solution obtained in step 2 with 100mL of palm oil at a temperature of 38°C, wherein the palm oil includes 1% Span-80 by mass percentage, and stir at a stirring speed of 1000 rpm to When the temperature dropped to 25° C., the stirring was continued for 30 minutes, at which point the Lactobacillus casei hydrogel micro-core emulsion was obtained, and the average particle diameter of the micro-core was 93.2 μm.

Step 4: Further mix the above-mentioned 200mL microcore emulsion formed in step 3 with 400mL calcium carbonate, sodium alginate and xanthan gum solution (the mass ratio of sodium alginate to xanthan gum is 20:1, and the total mass percentage concentration is 1 %, the mass percentage of calcium carbonate is 0.5%, and the temperature is 22°C), and then extruded into a 1% calcium chloride solution at 0°C to form solidified multi-core microcapsules with a particle size of about 0.5mm (Figure 1, A).

The microcapsules have no adhesion, good spheroidity, and rounded surface. The number of Lactobacillus casei is 1×10 ⁹ cfu/mL, and the encapsulation efficiency is 71.3%. After heat treatment at 85°C for 3 minutes, the number of remaining viable bacteria is 2.5×10 ⁷ cfu/mL, unembedded Lactobacillus casei sludge or solid-state fermentation material was diluted 1000 times, no viable bacteria were detected; when stored in a sealed container at 25°C for 60 days, the remaining viable bacteria were 4.1×10 ⁸ cfu/mL; 4 hours under artificial simulated gastric juice, the release amount of Lactobacillus casei was 19.8%, and under artificial simulated intestinal juice for 4 hours, the release amount of Lactobacillus casei was 95.7%.

Example 2

Step 1: Suspend 2g of carrageenan in 100mL of distilled water, heat to dissolve, add 0.5g of glucose, 0.5g of yeast extract and 5.0g of lactic acid, adjust the pH to 5.0 with sodium hydroxide, and cool to 45°C.

Step 2: Add Lactobacillus casei sludge or Lactobacillus casei solid-state fermentation material to the solution formed in Step 1, and mix evenly (the concentration of Lactobacillus casei in the mixed solution is 3.5×10 ¹² cfu/mL).

Step 3: Mix the above mixed solution formed in step 2 with 500mL of palm oil at a temperature of 45°C, wherein the palm oil includes 5% Span-80 by mass percentage, and stir at a stirring speed of 2000 rpm Stirring was continued for 30 minutes until the temperature dropped to 35° C., at which point a Lactobacillus casei hydrogel micro-core emulsion was obtained, and the average particle diameter of the micro-core was 10.4 μm.

Step 4: Further mix the above-mentioned 600mL microcore emulsion formed in step 3 with 2400mL calcium carbonate, sodium alginate and xanthan gum solution (the mass ratio of sodium alginate to xanthan gum is 20:5, and the total mass percentage concentration is 3 %, the mass percentage of calcium carbonate is 3%, the temperature is 26°C), and extruded into a 4% calcium chloride solution at a temperature of 20°C to form solidified multi-core microcapsules with a particle size of about 5 mm (Fig. 1, B ).

The microcapsules have no adhesion, good spheroidity, and rounded surface. The number of Lactobacillus casei is 1×10 ¹¹ cfu/mL, and the encapsulation efficiency is 99.1%. After heat treatment at 85°C for 3 minutes, the number of remaining viable bacteria is 1.2×10 ¹⁰ cfu/mL; sealed storage at 25°C for 60 days, the number of remaining viable bacteria is 4.8×10 ¹⁰ cfu/mL; 4 hours under artificial simulated gastric juice, the release of Lactobacillus casei is 12.5%, artificial simulated intestinal juice After 4 hours, the release amount of Lactobacillus casei was 90.5%.

Example 3

Step 1: Suspend 1g of carrageenan in 100mL of distilled water, heat to dissolve, add 0.25g of glucose, 0.25g of yeast extract and 2.5g of lactic acid, adjust the pH to 4.2 with sodium hydroxide, and cool to 41.5°C.

Step 2: Then add Lactobacillus casei sludge or Lactobacillus casei solid-state fermentation material to the solution formed in step 1, and mix evenly (the concentration of Lactobacillus casei in the mixed solution is 1.5×10 ¹¹ cfu/mL).

Step 3: Mix the above mixed solution with 250mL of palm oil at a temperature of 41.5°C, wherein the palm oil includes 2.5% Span-80 by mass percentage, and stir at a stirring speed of 1500 rpm until the temperature drops to 25 Stirring was continued for 30 minutes at a temperature of 0° C., at which point a Lactobacillus casei hydrogel micro-core emulsion was obtained, and the average particle diameter of the micro-core was 53.8 μm.

Step 4: further mix the above-mentioned 350mL microcore emulsion formed in step 3 with 1050mL calcium carbonate, sodium alginate and carrageenan solution (the mass ratio of sodium alginate to carrageenan is 20:3, and the total mass percentage concentration is 3%, The mass percentage of calcium carbonate is 2%, the temperature is 23 °C), and extruded into a 2.5% calcium chloride solution at a temperature of 10 °C to form solidified multi-core microcapsules with a particle size of about 3 mm (Figure 1, C).

The microcapsules have no adhesion, good spheroidity, rounded surface, the number of Lactobacillus casei is 1×10 ¹⁰ cfu/mL, the microcapsules are heat-treated at 85°C for 3 minutes, and the number of remaining viable bacteria is 3.2×10 ⁹ cfu/mL; sealed storage at 25°C for 60 days, the number of remaining viable bacteria is 5.6×10 ⁹ cfu/mL; 4 hours under artificial simulated gastric juice, the release of Lactobacillus casei is 16.3%, 4 hours under artificial simulated intestinal juice, The release rate of Lactobacillus casei was 94.3%.

Example 4

Step 1: Suspend 1 g of carrageenan in 100 mL of distilled water, heat to dissolve 0.4 g of glucose, 0.2 g of yeast extract and 1.8 g of lactic acid, adjust the pH to 4.0 with sodium hydroxide, and cool to 40°C.

Step 2: Then add Lactobacillus casei sludge or Lactobacillus casei solid-state fermentation material to the solution formed in Step 1, and mix evenly (the concentration of Lactobacillus casei in the mixed solution is 7.8×10 ¹¹ cfu/mL).

Step 3: Mix the above mixed solution with 150mL of palm oil at a temperature of 40°C, wherein the palm oil includes 4% Span-80 by mass percentage, and stir at a stirring speed of 2000 rpm until the temperature drops to 25 Stirring was stopped at ℃, and the Lactobacillus casei hydrogel micro-core emulsion was obtained at this moment, and the average particle diameter of the micro-core was 34.2 μm.

Step 4: Further mix the above-mentioned 250mL microcore emulsion formed in step 3 with 500mL calcium carbonate, sodium alginate and xanthan gum solution (the mass ratio of sodium alginate to xanthan gum is 20:3, and the total mass percentage concentration is 2 %, the mass percentage of calcium carbonate is 2.5%), and extruded into a 2% calcium chloride solution at a temperature of 20 °C to form solidified multi-core microcapsules with a particle size of about 2 mm (Fig. 1, D).

The microcapsules have no adhesion, good spheroidity, rounded surface, and the number of Lactobacillus casei is 1×10 ¹¹ cfu/mL. The microcapsules are heat-treated at 85°C for 3 minutes, and the number of remaining viable bacteria is 4.1×10 ¹⁰ cfu/mL; sealed storage at 25°C for 60 days, the number of remaining viable bacteria is 7.2×10 ¹⁰ cfu/mL; 4 hours under artificial simulated gastric juice, the release of Lactobacillus casei is 14.7%, 4 hours under artificial simulated intestinal juice, The release rate of Lactobacillus casei was 95.9%.

Claims (9)

1. A microcapsule for embedding Lactobacillus casei, characterized in that: the microcapsule is a multi-core structure embedded in three layers, wherein, Lactobacillus casei bacterium slime or solid-state fermentation product is embedded in the first hydrogel package A microcore is formed in the coating layer, and the second coating layer outside the microcore is vegetable oil, and a plurality of microcores coated with vegetable oil are embedded in a third coating formed by a water-soluble polymer material. In the layer, wherein, the said Lactobacillus casei sludge or solid-state fermentation product is produced by the Lactobacillus casei strain BFT-1 whose preservation number is CCTCC NO: M 2011424. 2 . The microcapsule according to claim 1 , wherein the concentration of the Lactobacillus casei is 10 ⁹ -10 ¹¹ cfu/mL. 3. The microcapsule according to claim 1, characterized in that, the micro-core structure is a hydrogel with a particle size of 10-100 μm, and the second coating layer is wrapped with a palm oil layer with a melting point of 24°C. 4. The microcapsule according to claim 1, wherein the water-soluble polymer material is sodium alginate-polyethylene glycol. 5. A method for preparing microcapsules comprising Lactobacillus casei, characterized in that the method comprises the following steps: Step 1: Prepare a pH of 3.5 to 5.0, a carrageenan mass percentage of 0.1 to 2%, and a glucose content of 0.1 ~0.5%, yeast extract content of 0.1~0.5%, lactic acid concentration of 1~5% mixed aqueous solution, and adjust the temperature to 38~45°C; then add Lactobacillus casei sludge or solid-state fermentation to the above solution The product is to form a Lactobacillus casei hydrogel solution, wherein said Lactobacillus casei sludge or solid-state fermentation product is produced by the Lactobacillus casei strain BFT-1 whose preservation number is CCTCC NO: M 20114244. 6. The method according to claim 5, characterized in that, and adjusting the temperature to 40°C, the optimum concentration of Lactobacillus casei is 8×10 ¹⁰ cfu/mL. 7. The method according to claim 5, characterized in that, the method also comprises step 2: preparing the Lactobacillus casei hydrogel microcore coated with outer palm oil: the Lactobacillus casei water prepared in step 1 The gel solution is mixed with Span-80 at the same temperature at a volume ratio of 1:1~5, wherein Span-80 includes palm oil with a mass percentage concentration of 1~5%, and is formed under stirring at 1000~2000 rpm The emulsion is then cooled to 25°C to form a Lactobacillus casei hydrogel microcore coated with palm oil, and the structure is characterized by a solid gel microcore. 8. method according to claim 7, is characterized in that, this method also comprises step 3: prepare the multi-core microcapsule of embedding Lactobacillus casei microcore: the cheese milk that the outer layer palm oil that step 2 forms is coated Bacillus micro-core emulsion is mixed with calcium carbonate-sodium alginate-xanthan gum mixed solution at a volume ratio of 1:2~4, and then dropped into 1~4% calcium chloride solution at a temperature of 0~20°C. Hours later, the solidified multi-core microcapsules embedding the Lactobacillus casei microcore are formed, and the Lactobacillus casei microcore, palm oil and sodium alginate-xanthan gum etc. are all solid structures in the capsule. 9. The method according to claim 8, wherein the mass ratio between sodium alginate and xanthan gum is 20:1-5, and the total mass percentage concentration of sodium alginate and xanthan gum is 1-3% , the mass percentage of calcium carbonate is 0.5~3%, and the temperature is 22~26°C.

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