A preparation method of a starch-based probiotic supplement

Abstract

The present invention relates to a preparation method of a starch-based probiotic supplement. In this preparation method, porous starch with a narrow distribution pore structure is used as a loading medium, and probiotics are quantitatively introduced according to a ratio. The pore size and pore type of the porous starch provide a suitable microenvironment for the probiotics. Under mild reaction conditions, the probiotics can fully bind to the multi-level sites of the porous starch to form a B-type complex. The supplement prepared by this method can achieve billions of probiotics per gram of starch. The simple process maximally retains the viable count and bioactive functions of the probiotics, significantly improving the utilization rate of the probiotics during the processing. Therefore, this method can successfully prepare a starch-based probiotic supplement.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional foods, and relates to a preparation method of a probiotic supplement. In particular, porous starch with micron-sized pores is used as a carrier, and the macropores of the porous starch provide multi-level binding sites for probiotics, enabling the probiotics to fully bind. The simple process improves the viable count of the supplement and the utilization rate of probiotics. 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, thus showing beneficial biological effects on host diseases. However, in food processing, the number of probiotics adsorbed on the surface of natural starch granules is limited and it is difficult to meet the requirements of probiotic foods. Porous starch prepared by general methods is difficult to achieve good probiotic loading effects due to reasons such as too small pore size and uneven pore distribution. Summary of the Invention

[0003] The purpose of the present invention is to provide a preparation method of a starch-based probiotic preparation in view of the deficiencies of the prior art, so as to solve problems such as low viable count and low utilization rate of probiotics in food processing. The present invention uses porous starch with a narrow distribution pore structure to load micron-sized probiotics. Herein, the narrow distribution of the present invention means that within the specified pore size range, the pore formation success rate reaches more than 90%; the concentration of the narrow distribution pore structure provides a protective microenvironment for the survival of probiotics. Compared with the wide distribution pore structure, the uniformly distributed pore structure has a higher probiotic utilization rate and can retain the biological activity function of probiotics to the greatest extent, improving the viable count of probiotics. And as the diameter range of the pore structure becomes larger, the more viable probiotics are loaded.

[0004] Specifically, the present invention forms a set of preparation methods of a starch-based probiotic preparation through technical processes such as physically sterilizing porous starch, homogenizing a porous starch suspension, compounding a porous starch suspension with a probiotic culture, and micro-gelatinizing and culturing.

[0005] The present invention adopts the following technical solution: A preparation method of a starch-based probiotic preparation, 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 0.5-20 wt%, and the ratio of the mass of starch to the viable count is 1 g: 10 11 CFU / mL; the mixed solution is subjected to micro-gelatinization oscillation reaction at 30-40 °C for 1-2.5 h, and centrifuged to obtain a starch-based probiotic preparation; the pore size of the porous starch is above 1 μm.

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

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

[0008] Further, the centrifugation speed is 5000 - 8000 rpm, and the centrifugation time is 5 - 10 min.

[0009] In certain embodiments of the present invention, after sterilizing the porous starch 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 oscillation reaction is carried out in a micro-gelatinized environment. The macroporous structure of the porous starch provides multiple levels of sites for loading probiotics, and a starch-based probiotic preparation is obtained after centrifugation. Among them, the porous starch is sterilized by ultraviolet irradiation for 20 - 40 min, dissolved and suspended in sterile MRS broth, the homogenization speed is 7000 - 8000 rpm, the homogenization time is 2 - 4 min, 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 5 - 30 wt%, and it is cultivated in a micro-gelatinized environment at 35 - 40 °C for 1 - 2.5 h, and a starch-based probiotic preparation is obtained after centrifugation.

[0010] In the present invention, after sterilizing the porous starch, it is homogenized and suspended in an MRS solution. After quantitatively introducing probiotic cultures, the micro-gelatinized environment causes the macroporous structure of the porous starch to provide multiple levels of sites for probiotics, and the probiotics specifically bind to the starch chains under suitable reaction conditions.

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

[0012] For the probiotics encapsulated by the present invention, the viable cell count of probiotics per gram of starch is as high as billions, and the utilization rate of probiotics is over 50%.

[0013] 1. In the present invention, based on the known negative correlation between the ratio of amylose to amylopectin and the pore size, porous starch with a narrow pore size distribution is prepared, reasonable process means are sought to load probiotics, a starch-based probiotic preparation with a high load of probiotics is prepared, and a set of process flows is established for the production practice of highly efficient probiotic loading.

[0014] 2. The present invention provides a green, environmentally friendly, simple and efficient processing flow. The product is safe for consumption and has certain functionality. It is confirmed that porous starch with a large pore diameter can be used as a good carrier for high-load probiotics, and the processing technology improves the viable cell count and utilization rate of probiotics. Description of the Drawings

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

[0017] Figure 3 Scanning electron microscope (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 pore size of porous starch and its preparation method on the probiotic loading amount and loading rate. Specific embodiments

[0018] As one of the most important carbohydrates in nature, starch is used as an economical encapsulating material due to its surface adhesion to probiotics, food safety, low cost, and high availability. The cell surface proteins of probiotics can specifically bind to β-1,4-linked glucose, and these sugars are involved in the adhesion of bacteria to starch granules. However, a high content of amylose may inhibit their binding. In addition, some small pores (nanoscale) present on the surface of starch granules also provide a rough environment with an increased surface area for loading probiotics. However, the amount of probiotics adsorbed on the surface of natural starch granules is limited, and the loading rate is low, unable to meet the requirements of probiotic foods.

[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, describe in detail the specific implementation manners, structures, features, and their effects according to the present invention.

[0020] The following further illustrates the present invention through examples. The following examples are for illustrative purposes and not for limiting the scope of the present invention.

[0021] Example 1

[0022] A preparation method of a starch-based probiotic preparation is as follows:

[0023] (1) Preparation of micron-scale 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 50 °C. Among them, the amylose in the starch suspension accounts for 30% of the mass of amylose / the total mass of amylose and amylopectin, the ultrasonic power is 30 W / ml, the ultrasonic frequency is 60 kHz, the ultrasonic time is 30 min, and the temperature of the ultrasonic solution is 80 °C.

[0024] (2) It is measured that the porous starch prepared in step 1 is concentrated in the range of 1 - 10 μm, and the small pores with a pore size in the range of 1 - 10 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 8000 rpm for 3 min to prepare a 20 wt% porous starch suspension.

[0025] (3) Equip with porous starch-probiotic complex: Add probiotic culture in proportion. The ratio of the mass of starch to the viable cell count is 1 g: 10 11 CFU / mL, and cultivate it in a slightly gelatinized environment at 37 °C for 2 h.

[0026] (4) Centrifuge after reaction: Centrifuge at a centrifugal force of 8000 rpm for 5 min, and then carefully remove the supernatant to obtain a precipitate.

[0027] (5) Observation of porous morphology: Evenly spread a small amount of starch granules passing through a 75-μm sieve on a 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 relatively dense probiotics are presented on the surface of the porous starch.

[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 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.

[0029] (7) Determination of probiotic loading: Evaluate the viability of Lactobacillus plantarum through the agar plate test. Hydrolyze the sample with α-amylase (100 mg, 10000 U / g) in phosphate buffer saline (PBS) (pH 7.2 - 7.4) to release the encapsulated Lactobacillus plantarum cells. Spread a series of diluted samples on MRS agar plates. Incubate the plates at 37 °C for 48 h. Count the colony-forming units (CFU). Determine the efficiency of probiotic encapsulation by porous starch by comparing the colonies obtained from different porous starches.

[0030] For the porous starch-based probiotic preparation prepared in this example, the viable cell count of probiotics per gram of starch is 5.3×10 10 CFU, and the utilization rate is 53%; meanwhile, the XPS results show that the addition of probiotics significantly increases the nitrogen element content. This method can successfully prepare a probiotic preparation with a high loading amount.

[0031] Example 2

[0032] A preparation method of a starch-based probiotic preparation is as follows:

[0033] (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, the ultrasonic power is 30 W / ml, the ultrasonic frequency is 60 kHz, the ultrasonic time is 30 min, and the temperature of the ultrasonic solution is 80 °C.

[0034] (1) It is measured that the porous starch prepared in step 1 is concentratedly distributed in the range of 10 - 15 μm, and the small holes with pore diameters in the range of 10 - 15 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 8000 rpm for 3 min to prepare a 20 wt% porous starch suspension.

[0035] (2) Preparation of 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 it in a slightly gelatinized environment at 37 °C for 2 h.

[0036] (3) Centrifugation after reaction: Centrifuge at a centrifugal force of 8000 rpm for 5 min, and then carefully remove the supernatant to obtain a precipitate.

[0037] (4) Observation of porous 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 the surface of the porous starch presents relatively dense probiotics.

[0038] (5) 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] (6) Determination of probiotic loading: The viability of Lactobacillus plantarum was evaluated by the agar plate test. The sample was hydrolyzed with α-amylase (100 mg, 10,000 U / g) in phosphate-buffered saline (PBS) (pH 7.2 - 7.4) to release the encapsulated Lactobacillus plantarum cells. A series of diluted samples were spread on MRS agar plates. The plates were incubated at 37 °C for 48 h. Colony-forming units (CFUs) were counted. The efficiency of probiotic encapsulation by porous starch was determined by comparing the colonies obtained from different porous starches.

[0040] For the porous starch-based probiotic preparation obtained in this example, the viable count of probiotics per gram of starch was 6.4×10 10 CFU, and the utilization rate was 64%; at the same time, XPS results showed that the addition of probiotics significantly increased the nitrogen element content. This method can successfully prepare a probiotic preparation with a high loading amount.

[0041] Example 3

[0042] A preparation method of a starch-based probiotic preparation is as follows:

[0043] (1) Preparation of micron-sized porous starch: The composite starch suspension was treated by ultrasonic wave, and then the ultrasonic solution was subjected to alcohol precipitation treatment; after the ultrasonic-alcohol precipitation solution was concentrated and cultured, centrifuged, and the precipitate was dried at 50 °C. Among them, the amylose in the starch suspension accounted for 5% of the mass of amylose / the total mass of amylose and amylopectin, the ultrasonic power was 30 W / ml, the ultrasonic frequency was 60 kHz, the ultrasonic time was 30 min, and the temperature of the ultrasonic solution was 80 °C.

[0044] (2) It was measured that the porous starch prepared in step 1 was concentrated in the range of 15 - 30 μm, and the small holes with pore diameters in the range of 15 - 30 μm accounted for more than 90% of the total amount; the porous starch was sterilized by ultraviolet light and then suspended in MRS broth, and homogenized at 8000 rpm for 3 min to prepare a 20 wt% porous starch suspension.

[0045] (3) Preparation of porous starch-probiotic complex: Probiotic culture was added in proportion, and the ratio of the mass of starch to the viable count was 1 g:10 11 CFU / mL, and cultured in a slightly gelatinized environment at 37 °C for 2 h.

[0046] (4) Centrifugation after reaction: Centrifuge at a centrifugal force of 8000 rpm for 5 min, and then carefully remove the supernatant to obtain a precipitate.

[0047] (5) Observation of porous morphology: A small amount of starch granules passing through a 75-μm sieve were evenly spread on a 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 was found that the surface of the porous starch presented relatively dense probiotics.

[0048] (6) 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 and oxygen to carbon were obtained.

[0049] (7) Determination of probiotic loading: The viability of Lactobacillus plantarum was evaluated by the agar plate test. The sample was hydrolyzed with α-amylase (100 mg, 10,000 U / g) in phosphate-buffered saline (PBS) (pH 7.2 - 7.4) to release the encapsulated Lactobacillus plantarum cells. A series of diluted samples were spread on MRS agar plates. The plates were incubated at 37 °C for 48 h. The colony-forming units (CFU) were counted. The encapsulation efficiency of the porous starch for probiotics was determined by comparing the colonies obtained from different porous starches.

[0050] For the porous starch-based probiotic preparation obtained in this example, the viable count of probiotics per gram of starch was 1.2×10 11 CFU, and the utilization rate was 80%; at the same time, the XPS results showed that the addition of probiotics significantly increased the nitrogen element content, indicating that this method could successfully prepare a probiotic preparation with a high loading amount.

[0051] Comparative Example 1: Using ordinary corn starch as a probiotic carrier

[0052] (1) Preparation of ordinary starch suspension: Ultraviolet-sterilized ordinary corn starch was dissolved and suspended in MRS broth, and homogenized at 8000 rpm for 3 min to prepare a 20 wt% ordinary starch suspension.

[0053] (2) Preparation of starch-probiotic complex: Probiotic culture was added in proportion until the volume / mass ratio with the porous starch was 1 g:10 11 CFU / mL, and cultured in a slightly gelatinized environment at 37 °C for 2 h.

[0054] (3) Centrifugation after reaction: After centrifugation at a centrifugal force of 8000 rpm for 5 min, the supernatant was carefully removed to obtain a precipitate.

[0055] (4) Observation of probiotic morphology: A small amount of starch granules passing through a 75-μm pore sieve were evenly spread on a 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 was visible that the surface of the starch presented relatively scattered and sporadic probiotics.

[0056] (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. A narrow scan was performed within a 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 and oxygen to carbon were obtained.

[0057] (6) Determination of probiotic loading: The viability of Lactobacillus plantarum was evaluated by the agar plate test. The sample was hydrolyzed with α-amylase (100 mg, 10,000 U / g) in phosphate-buffered saline (PBS) (pH 7.2 - 7.4) to release the encapsulated Lactobacillus plantarum cells. A series of diluted samples were spread on MRS agar plates. The plates were incubated at 37 °C for 48 h. The colony-forming units (CFU) were counted. The encapsulation efficiency of common corn starch for Lactobacillus plantarum was determined by counting the colonies obtained.

[0058] For the common starch-based probiotic preparation obtained in this example, the viable count of probiotics per gram of starch was 9.7×10 8 CFU, and the utilization rate was 3%; the XPS results showed that with the addition of probiotics, the nitrogen element content increased. The viable count and utilization rate of the probiotics loaded in this example were significantly lower than those of the porous starch with a macroporous structure.

[0059] Comparative Example 2: Porous starch (wide pore size distribution) prepared by enzymatic hydrolysis was used as a probiotic carrier

[0060] (1) Preparation of porous starch: Common corn starch was enzymatically hydrolyzed with α-amylase, shaken in a water bath for 2 h, boiled at high temperature for 10 min, and then centrifuged to obtain a precipitate. The precipitate was washed 2 - 3 times with deionized water, and porous starch was obtained after centrifugation. The pore size of the porous starch was measured to be evenly distributed in the range of 1 - 1000 μm, without showing a concentrated narrow distribution.

[0061] (2) Preparation of a porous starch suspension: The porous starch sterilized by ultraviolet light was dissolved and suspended in MRS broth, and homogenized at 8000 rpm for 3 min to prepare a 20 wt% porous starch suspension.

[0062] (3) Equipped with porous starch-probiotic complex: Add probiotic culture in proportion until the volume / mass ratio with porous starch is 1 g:10 11 CFU / mL, and cultivate in a slightly gelatinized environment at 37 °C for 2 h.

[0063] (4) Centrifuge after reaction: Centrifuge at a centrifugal force of 8000 rpm for 5 min, and then carefully remove the supernatant to obtain a precipitate.

[0064] (5) Observation of porous morphology: Evenly spread a small amount of starch granules passing through a 75-μm sieve on a conductive adhesive and coat 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 the surface of the porous starch presents relatively dense probiotics.

[0065] (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.

[0066] (7) Determination of probiotic loading: Evaluate the viability of Lactobacillus plantarum through an agar plate test. Hydrolyze the sample with α-amylase (100 mg, 10000 U / g) in phosphate buffered saline (PBS) (pH 7.2 - 7.4) to release the encapsulated Lactobacillus plantarum cells. Spread a series of diluted samples on MRS agar plates. Incubate the plates at 37 °C for 48 h. Count the colony forming units (CFU). Determine the efficiency of probiotic encapsulation by porous starch by comparing the colonies obtained from different porous starches.

[0067] For the porous starch-based probiotic preparation obtained by enzymatic hydrolysis in this example, the viable probiotic count per gram of porous starch is 1.6×10 8 CFU, and the utilization rate is 0.48%; the XPS results show that with the addition of probiotics, the nitrogen element content increases. The viable probiotic count and utilization rate loaded in this example are significantly lower than those of the porous starch prepared by ultrasonic-alcohol precipitation, and the probiotic loading effect of wide distribution pore size is also far less good than that of narrow distribution pore size.

Claims

1. A preparation method of a starch-based probiotic supplement, 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 5-30 wt%, and the ratio of the mass of starch to the viable cell count is 1 g: 10 11 CFU / mL; the mixed solution is subjected to a micro-gelatinization oscillation reaction at 20-40 °C for 1-2.5 h, and then centrifuged to obtain a starch-based probiotic supplement; wherein the pore diameter of the said porous starch is above 1 μm, and it 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 taken and dried at a temperature of 35-75 °C to obtain the porous starch with uniform pore size distribution; the compound starch comprises at least amylose and amylopectin, and the mass of amylose accounts for 5-50% of the total mass of amylose and amylopectin; The ultrasonic power is 30 W / ml, the ultrasonic frequency is 60 kHz, the ultrasonic time is 30 min, and the temperature of the ultrasonic solution is 80 °C.

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

3. The preparation method according to claim 1, characterized in that, The porous starch is sterilized by ultraviolet rays.

4. The preparation method according to claim 1, characterized in that, The centrifugation speed is 5000-8000 rpm, and the centrifugation time is 5-10 min.

5. The preparation method according to claim 1, characterized in that, 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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