A method for encapsulating probiotics

Abstract

The present invention discloses a porous starch encapsulation method for efficiently protecting probiotics. This preparation method uses porous starch with nanoscale pore diameters as a loading medium, quantitatively introduces probiotics according to a ratio, and encapsulates the probiotics by utilizing the adsorption characteristics of the nanoscale pores. Under the reaction conditions of microgelatinization, the structure of the porous starch changes, and the probiotics are wrapped therein, and its starch shell provides a protective effect for the probiotics. This method can improve the retention rate of probiotics, reduce the loss of probiotics during food processing and transportation, and thus retain the biological functions of probiotics to a great extent. Therefore, this porous starch can become a good material for protecting probiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional foods and relates to a method for encapsulating probiotics. In particular, porous starch with nanoscale pore diameters is used as a loading carrier, and the probiotics are encapsulated by the adsorption characteristics of the nanoscale pore diameters of the porous starch. The micro-gelatinization environment promotes the structural change of the porous starch, and the probiotics are wrapped therein, thereby improving the retention rate of the probiotics in a complex environment. Background Art

[0002] Probiotics have been proven to be beneficial to gastrointestinal function. They can survive during the transportation in the oral cavity and stomach and multiply in large numbers in the intestine, thereby showing beneficial biological effects on host diseases, such as antibiotic-associated diarrhea, necrotizing enterocolitis, intestinal diseases, etc. However, in food processing, probiotics are easily exposed to environmental conditions such as heat, oxygen, and water activity, which affects their survival. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for encapsulating probiotics in view of the deficiencies of the prior art, so as to solve the problem of the survival rate of probiotics in a complex environment.

[0004] Specifically, the present invention uses porous starch with specific pore diameters to load probiotics, optimizes the loading process of probiotics, improves the retention rate of probiotics in a complex environment, and ensures the biological functions of probiotics. In a more preferred embodiment of the present invention, the pore diameters of the porous starch show a narrow distribution, and the narrow distribution means that within a specified pore diameter range, the pore-making success rate reaches more than 90%. For example, small pores with pore diameters in the range of 500 nm to 1000 nm account for more than 90% of the total amount, or small pores with pore diameters in the range of 500 nm to 1000 nm account for more than 90% of the total amount.

[0005] The present invention adopts the following technical solution: A method for encapsulating probiotics, characterized in that after sterilizing the porous starch, it is dispersed in a culture medium solution, and probiotics are added to obtain a mixed solution; in the mixed solution, the concentration of starch is 0.5 to 20 wt%, and the ratio of the mass of starch to the number of viable bacteria is 1 g: 10 11 CFU / mL; the mixed solution is subjected to micro-gelatinization oscillation reaction at 30 to 40 °C for 0.5 to 2 h, centrifuged, and freeze-dried to obtain starch-loaded probiotics; the pore diameter of the porous starch is within 1 μm.

[0006] Further, the culture medium solution is MRS broth.

[0007] Further, the starch is sterilized by ultraviolet light.

[0008] Further, the centrifugation speed is 3000 to 6500 rpm, and the centrifugation time is 10 min.

[0009] In certain embodiments of the present invention, after the porous starch is sterilized using ultraviolet light, MRS broth is used as a dissolution medium, and after homogenization, a uniform porous starch suspension is formed. Probiotic cultures are added in proportion, and an oscillating reaction is carried out in a micro-gelatinized environment to obtain porous starch particles loaded with probiotics. Among them, the porous starch is sterilized by ultraviolet irradiation for 10 to 30 minutes, dissolved and suspended in sterile MRS broth, the homogenization speed is 5000 to 10000 rpm, the homogenization time is 1 to 5 minutes, and probiotic cultures are added in proportion until the ratio of the mass of starch to the viable cell count is 1 g:10 11 CFU / mL, and the concentration of the porous starch suspension is 0.5 to 20 wt%. It is cultivated in a micro-gelatinized environment at 30 to 40 °C for 0.5 to 2 hours, the precipitate is taken by centrifugation, and freeze-dried for 48 hours to obtain porous starch particles encapsulating probiotics.

[0010] In the present invention, after the sterilized porous starch is homogenized and suspended in an MRS solution, and probiotic cultures are quantitatively introduced, the nano-scale pores of the porous starch adsorb the probiotics. The micro-gelatinized environment promotes the structural transformation of the porous starch, and the probiotics are wrapped inside the porous starch. The starch shell improves the retention rate of the probiotics in a complex environment and provides effective protection for the probiotics.

[0011] The beneficial technical effects of the invention are as follows:

[0012] The probiotics encapsulated by the present invention have a retention rate of more than 15% under the freeze-drying condition of -50 °C and a retention rate as high as more than 95% under the heat treatment condition of 45 °C.

[0013] 1. The present invention, based on the known negative correlation between the ratio of amylose to amylopectin and the pore size, prepares porous starch with nano-scale pores, seeks reasonable technological means to load probiotics, prepares porous starch particles capable of providing protection for probiotics, and establishes a set of technological processes that can be used in the production practice of probiotic protection.

[0014] 2. The present invention provides a green, environmentally friendly, simple and efficient processing flow. The product is safe and edible and has certain functionality, verifying that the porous starch with nano-scale pores can be used as a good carrier for loading probiotics, providing good protection for probiotics in a complex environment, and improving the probiotic retention rate. Description of the Drawings

[0015] Figure 1 It is a scanning electron microscope (SEM) micrograph of the porous starch loaded with probiotics prepared by the present invention, which is used to directly prove the existence and protection effect of the probiotics prepared by the present invention.

[0016] Figure 2X-ray photoelectron spectroscopy (XPS) data image of probiotic-loaded porous starch prepared according to the present invention, which is used to directly prove the existence of probiotics.

[0017] Figure 3 Scanning electron microscopy (SEM) micrograph and X-ray photoelectron spectroscopy (XPS) data image of the comparative example in the present invention, which are used to prove the influence of the protective effect of porous starch on the retention rate of probiotics. Specific embodiments

[0018] Porous starch has a large specific surface area and excellent adsorption properties and is commonly used as an adsorbent. This is important for the ideal adsorption of active substances, which can be better protected in porous starch, extending the shelf life and stability. More importantly, the starch-based cytoskeleton exhibits unique curved channels and enzyme-triggered degradability in vivo, enabling the transportation and controlled release of guest objects.

[0019] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following will, in conjunction with the accompanying drawings and preferred embodiments, elaborate in detail on the specific implementation manners, structures, features, and effects according to the present invention.

[0020] The present invention will be further illustrated by the following examples. The following examples are for illustrative purposes and are not intended to limit the scope of the present invention.

[0021] Example 1

[0022] A porous starch for efficiently protecting probiotics and its encapsulation method, the steps are as follows:

[0023] (1) Preparation of nano-porous starch: Treat the compound starch suspension with ultrasonic waves, and then perform alcohol precipitation on the ultrasonic solution; after concentrating and culturing the ultrasonic-alcohol precipitation solution, centrifuge, and take the precipitate for drying at 50 °C, where the amylose in the starch suspension accounts for 80% of the mass of amylose / the total mass of amylose and amylopectin.

[0024] (2) It is measured that the porous starch prepared in step 1 is concentrated in the range of 1 - 500 nm, and the small pores with pore diameters in the range of 15 - 30 μm account for more than 90% of the total amount; after ultraviolet sterilization of the porous starch, it is suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension.

[0025] (3) Prepare a porous starch-probiotic complex: Add probiotic culture according to a ratio, and the ratio of the mass of starch to the number of viable bacteria is 1 g:10 11 CFU / mL, and cultivate in a slightly gelatinized environment at 37 °C for 1 h.

[0026] (4) Centrifugation after reaction: Centrifuge at a centrifugal force of 5000 rpm for 10 min, then carefully remove the supernatant to obtain a precipitate, which is freeze-dried for 48 h;

[0027] (5) Observation of probiotic morphology: Spread a small amount of starch granules passing through a 75-μm pore sieve evenly on a conductive adhesive and coat with gold. Observe the morphology under a scanning electron microscope (SEM) at an acceleration voltage of 3 kV and a magnification of ×10,000. It can be seen that most probiotics are wrapped in porous starch in a semi-embedded or fully-embedded form.

[0028] (6) X-ray photoelectron spectroscopy (XPS): Determine the elemental surface composition of unencapsulated and encapsulated Lactobacillus plantarum. When the pressure is less than 2.0×10 -7 mbar (12 kV, 6 mA), press the freeze-dried bacterial powder (20 - 30 mg) into a small plate and place it in the XPS chamber. Calculate the binding energy based on the C1s binding energy peak set at 284.8 eV. Perform a narrow scan within a binding energy range of 20 eV to determine the chemical functionality in O1s. The area under each peak is used to calculate the peak intensity, and the elemental surface concentration ratios of nitrogen and oxygen to carbon are obtained.

[0029] (7) Determination of cold and heat resistance of probiotics: To evaluate the protective effect of porous starch encapsulation on probiotics, measurements are carried out under freeze-drying (-48 °C) and heat treatment at 30 °C and 45 °C for 15 min respectively. Immerse 100 mg of the sample in 1 mL of PBS, heat and incubate in water for 15 min, and then cool to room temperature in an ice-water bath. Use the agar plate assay method to evaluate the viable cells surviving after heat treatment.

[0030] For the porous starch loaded with probiotics prepared in this example, the retention rate of probiotics after freeze-drying is 15%, and the retention rate after heat treatment is 95%; at the same time, the XPS results show that the addition of probiotics significantly increases the nitrogen element content. This method can successfully prepare porous starch particles loaded with probiotics.

[0031] Example 2

[0032] A porous starch for efficiently protecting probiotics and its encapsulation method are as follows:

[0033] (1) Preparation of nano-porous starch: Treat the compound starch suspension with ultrasonic waves, and then perform alcohol precipitation on the ultrasonic solution; after concentrating and culturing the ultrasonic-alcohol precipitation solution, centrifuge it, and take the precipitate for drying at a temperature of 50 °C, where the amylose in the starch suspension accounts for 50% of the mass of amylose / the total mass of amylose and amylopectin.

[0034] (2) It was measured that the porous starch prepared in step 1 was concentratedly distributed in the range of 500 nm to 1000 nm, and the small pores with pore diameters in the range of 500 nm to 1000 nm accounted for more than 90% of the total; after ultraviolet sterilization, the porous starch was suspended in MRS broth and homogenized at 5000 rpm for 1 min to prepare a 10 wt% porous starch suspension.

[0035] (3) Prepare the porous starch-probiotic complex: Add the probiotic culture according to the ratio. The ratio of the mass of starch to the number of viable bacteria is 1 g: 10 11 CFU / mL, and cultivate in a slightly gelatinized environment at 37 °C for 1 h.

[0036] (4) Centrifuge after reaction: Centrifuge at a centrifugal force of 5000 rpm for 10 min, then carefully remove the supernatant. After obtaining the precipitate, freeze-dry it for 48 h;

[0037] (5) Observation of probiotic morphology: Spread a small amount of starch particles passing through a 75-μm pore sieve evenly on the conductive adhesive and coat it with gold. Observe the morphology through a scanning electron microscope (SEM) at an accelerating voltage of 3 kV with a magnification of ×10,000. It can be seen that most probiotics are wrapped in the porous starch in a semi-embedded or fully embedded form.

[0038] (6) X-ray photoelectron spectroscopy (XPS): Determine the elemental surface composition of unencapsulated and encapsulated Lactobacillus plantarum. When the pressure is less than 2.0×10 -7 mbar (12 kV, 6 mA), press the freeze-dried bacterial powder (20 - 30 mg) into a small plate and place it in the XPS chamber. Calculate the binding energy according to the C1s binding energy peak set at 284.8 eV. Perform a narrow scan within a binding energy range of 20 eV to determine the chemical functionality in O1s. The area under each peak is used to calculate the peak intensity, and the elemental surface concentration ratios of nitrogen and oxygen to carbon are obtained.

[0039] (7) Determination of the cold and heat resistance of probiotics: To evaluate the protective effect of porous starch encapsulation on probiotics, measurements were carried out under freeze-drying (-48 °C) and heat treatment at 30 °C and 45 °C for 15 min respectively. The agar plate assay was used to evaluate the viable cells surviving after heat treatment.

[0040] For the porous starch-loaded probiotics prepared in this example, the retention rate of probiotics after freeze-drying was 18%, and the retention rate after heat treatment was 99%; at the same time, the XPS results showed that the addition of probiotics significantly increased the nitrogen element content. This method can successfully prepare porous starch particles loaded with probiotics.

[0041] Comparative example 1: Use ordinary corn starch as a probiotic carrier

[0042] (1) Preparation of ordinary starch suspension: The ordinary corn starch after ultraviolet sterilization was dissolved and suspended in MRS broth, homogenized at 5000 rpm for 1 min to prepare a 10 wt% starch suspension.

[0043] (2) Preparation of starch-probiotic complex: Probiotic culture was added in proportion, and the ratio of the mass of starch to the number of viable bacteria was 1 g: 10 11 CFU / mL, and it was cultivated in a slightly gelatinized environment at 37 °C for 1 h.

[0044] (3) Centrifugation after reaction: Centrifuged at a centrifugal force of 5000 rpm for 10 min, and then the supernatant was carefully removed. After obtaining the precipitate, it was freeze-dried;

[0045] (4) Observation of probiotic morphology: A small amount of starch granules passing through a 75 μm pore sieve were evenly spread on the conductive adhesive and coated with gold. The morphology was observed by scanning electron microscopy (SEM) at an acceleration voltage of 3 kV and a magnification of ×10,000. It can be seen that the surface of the starch presented relatively scattered and sporadic probiotics.

[0046] (5) X-ray photoelectron spectroscopy (XPS): To determine the elemental surface composition of unencapsulated and encapsulated Lactobacillus plantarum. When the pressure was less than 2.0×10 -7 mbar (12 kV, 6 mA), the freeze-dried bacterial powder (20 - 30 mg) was pressed into a small plate and placed in the XPS chamber. The binding energy was calculated based on the C1s binding energy peak set at 284.8 eV. Narrow scans were performed in the binding energy range of 20 eV to determine the chemical functionality in O1s. The area under each peak was used to calculate the peak intensity, and the elemental surface concentration ratios of nitrogen, oxygen, and carbon were obtained.

[0047] (6) Determination of cold and heat tolerance of probiotics: To evaluate the protective effect of porous starch encapsulation on probiotics, the determination was carried out at freeze-drying (-48 °C) and heat treatment at 30 °C and 45 °C for 15 min, respectively. The agar plate assay was used to evaluate the viable cells surviving after heat treatment.

[0048] For the ordinary starch-loaded probiotics prepared in this example, the retention rate of the probiotics after freeze-drying was 0.24%, and the retention rate after heat treatment was 6.2%; the XPS results showed that with the addition of probiotics, the nitrogen element content increased, and the protection rate of the probiotics loaded in this example was significantly lower than that of the porous starch with nanoscale pores.

[0049] Comparative Example 2: Using porous starch with micron-sized pores as a probiotic carrier

[0050] (1) Preparation of micron - level porous starch: Use ultrasonic treatment on the compound starch suspension, and then perform alcohol precipitation on the ultrasonic solution; after concentrating and culturing the ultrasonic - alcohol precipitation solution, centrifuge it, and take the precipitate for drying at 50 °C. Among them, the amylose in the starch suspension accounts for 10% of the mass of amylose / the total mass of amylose and amylopectin.

[0051] (2) It is measured that the porous starch prepared in step (1) is concentratedly distributed in the range of 15 - 30 μm, and the small holes with pore diameters in the range of 15 - 30 μm account for more than 90% of the total amount; after ultraviolet sterilization of the porous starch, suspend it in MRS broth and homogenize it at 5000 rpm for 1 min to make a 10 wt% starch suspension.

[0052] (3) Prepare the starch - probiotic complex: Add probiotic culture according to a ratio, and the ratio of the mass of starch to the number of viable bacteria is 1 g: 10 11 CFU / mL, and cultivate it in a slightly gelatinized environment at 37 °C for 1 h.

[0053] (4) Centrifuge after reaction: Centrifuge at a centrifugal force of 5000 rpm for 10 min, then carefully remove the supernatant, and freeze - dry after obtaining the precipitate;

[0054] (5) Observation of probiotic morphology: Evenly spread a small amount of starch particles passing through a 75 - μm sieve on the conductive adhesive and coat it with gold. Observe the morphology through a scanning electron microscope (SEM) at an acceleration voltage of 3 kV with a magnification of ×10,000. It can be seen that most probiotics adhere to the surface of the porous starch.

[0055] (6) X - ray photoelectron spectroscopy (XPS): Determine the elemental surface composition of unencapsulated and encapsulated Lactobacillus plantarum. When the pressure is less than 2.0×10 - 7 mbar (12 kV, 6 mA), press the freeze - dried bacterial powder (20 - 30 mg) into a small plate and place it in the XPS chamber. Calculate the binding energy based on the C1s binding energy peak set at 284.8 eV. Perform a narrow scan within a binding energy range of 20 eV to determine the chemical functionality in O1s. The area under each peak is used to calculate the peak intensity, and the elemental surface concentration ratios of nitrogen, oxygen, and carbon are obtained.

[0056] (7) Determination of cold and heat tolerance of probiotics: To evaluate the protective effect of porous starch encapsulation on probiotics, measure it respectively under freeze - drying (-48 °C) and heat treatment at 30 °C and 45 °C for 15 min. Use the agar plate assay method to evaluate the viable cells surviving after heat treatment.

[0057] The probiotic-loaded porous starch prepared in this example has a retention rate of 0.06% after freeze-drying and 1.5% after heat treatment. The XPS results show that the addition of probiotics increases the nitrogen element content, and the retention rate of the probiotics loaded in this example is significantly lower than that of the porous starch with nanoscale pores.

Claims

1. A method for encapsulating probiotics, characterized in that, After subjecting the porous starch to sterilization treatment, it is homogenously dispersed in a culture medium solution, and probiotics are added to obtain a mixed solution; in the said mixed solution, the concentration of starch is 0.5 to 20 wt%, and the ratio of the mass of starch to the number of viable bacteria is 1 g:10 11 CFU / mL; the mixed solution is subjected to a micro-gelatinization oscillation reaction at 30 to 40 °C for 0.5 to 2 h, centrifuged, and freeze-dried to obtain probiotics loaded on starch; the pore diameter of the said porous starch is within 1 μm and is prepared by the following method: The compound starch suspension is treated with ultrasonic waves, and then the ultrasonic solution is subjected to alcohol precipitation treatment; after the ultrasonic-alcohol precipitation solution is concentrated and cultured, centrifuged, and the precipitate is dried at a temperature of 35-75 °C to obtain the porous starch; the compound starch comprises at least amylose and amylopectin, and the mass of amylose accounts for 50-80% of the total mass of amylose and amylopectin.

2. The encapsulation method according to claim 1, characterized in that, The culture medium solution is MRS broth.

3. The encapsulation method according to claim 1, wherein The porous starch is sterilized by ultraviolet rays.

4. The encapsulation method according to claim 1, characterized in that, The centrifugation speed is 3000-6500 rpm, and the centrifugation time is 10 min.

5. The encapsulation method according to claim 1, wherein After the porous starch is dispersed in the culture medium solution, homogenization treatment is carried out to form a uniform porous starch suspension.

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