CN118285508A - Probiotic delivery method based on oxidized high amylose starch carrier - Google Patents

Abstract

The invention discloses a probiotic delivery method based on an oxidized high-amylose starch carrier, which is used for preparing a microcapsule delivery system by an inverse emulsion method and comprises microcapsule wall materials and microcapsule core materials, wherein the wall materials consist of sodium alginate and oxidized high-amylose starch, and the core materials consist of probiotics. The microcapsule has excellent protective effect, can effectively release core components in intestinal tracts, has good gastrointestinal slow release function, has good field planting effect and adhesion effect, and prolongs the residence time of probiotics in the intestinal tracts. The average grain diameter of the microcapsule prepared by the invention is about 300 mu m, the preparation method is simple and convenient, and the cost is low.

Description

A probiotic delivery method based on oxidized high-amylose starch carrier

Technical Field

The invention relates to a probiotic delivery method based on an oxidized high-amylose starch carrier, belonging to the technical field of biological delivery and sustained release.

Background technique

As people's health awareness and living standards improve, safe, healthy and nutritious functional products are gradually favored by consumers. Among them, the market acceptance of probiotic products has increased year by year, and it has developed into a sunrise industry, which is of great significance in promoting human health and improving the quality of human life. As researchers gradually explore the intestinal microbial flora and the important manifestation of flora imbalance in various diseases, we have found that supplementing probiotics can help restore the balance of the intestinal ecosystem and improve the body's immunity.

In order to promote host health, the minimum concentration of probiotics in the intestine must reach 10 ⁶ to 10 ⁷ cfu/mL. However, maintaining the activity of probiotics during processing, storage and digestion is a major challenge in the industry. Therefore, it is crucial to select suitable materials as carriers and perform a series of encapsulation and protection on probiotics. At present, microencapsulation technology is a mature and widely used new method. It uses natural or synthetic polymer materials to encapsulate bioactive substances to form semi-permeable or closed vesicle structures, so that it can provide good protection for the contents and reduce the impact of the external environment on its activity. Therefore, the use of this technology to encapsulate probiotics is considered to be one of the cost-effective protection strategies and is also a current research hotspot.

Natural starch is widely available and is the most abundant polysaccharide in plants. Its good compatibility and surface modifiability make it an ideal wall material for bioactive carriers. However, natural starch has disadvantages such as poor solubility and weak water holding capacity, which limits its application and development in the food industry. Many studies have found that chemical modification can control the structure of starch aggregates, improve their solubility, water absorption and other properties, so that they can better protect functional foods and promote their development and application in the food industry.

The encapsulation rate and survival rate can directly reflect the effect of probiotic encapsulation. The survival rate of encapsulated probiotics in the stomach and the release in the intestine are directly related to the survival and colonization of probiotics in the intestine. In addition, the slow release of probiotic microcapsules in the intestine is also conducive to the colonization of probiotics in the intestine. It is very important to improve the adhesion and colonization of probiotics in the intestine, because this plays an important role in maintaining intestinal health and overall health. First, the adhesion and colonization of probiotics can form a protective film to prevent harmful bacteria and toxins from invading the intestinal mucosa, thereby protecting intestinal health; secondly, the colonization of probiotics can competitively inhibit the growth of harmful bacteria in the intestine, maintain the balance of intestinal microecology, and reduce the invasion of harmful bacteria on the intestine; in addition, the colonization of probiotics in the intestine can promote the development and function of the immune system, enhance the barrier function of the intestinal mucosa, and improve the body's resistance; finally, probiotics adhering to the intestine can help decompose nutrients in food, promote their absorption and utilization, and improve the utilization rate of nutrients. Therefore, improving the adhesion and colonization of probiotics in the intestines can help maintain intestinal health and promote overall health.

Since different probiotic strains have specific properties, there is no uniform and fixed correlation between the probiotic encapsulation materials and the properties of the strains. Therefore, for specific probiotics, the effect of the encapsulation wall material on the encapsulation effect of the probiotics is unpredictable; whether the encapsulation wall material can simultaneously achieve a high encapsulation rate after encapsulation of the probiotic microcapsules, a high survival rate after storage and exposure to gastric juice, a sustained release effect in the intestine, and a high adhesion and colonization rate is also unpredictable. At the same time, it is very difficult to achieve a good encapsulation effect for multiple probiotic strains.

Summary of the invention

In view of this, the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing oxidized high-amylose starch-polysaccharide composite microcapsules. Another purpose of the present invention is to provide oxidized high-amylose starch-polysaccharide composite microcapsules prepared by the above preparation method. Another purpose of the present invention is to provide the application of the above oxidized high-amylose starch-polysaccharide composite microcapsules.

The oxidized starch-polysaccharide complex obtained by compounding oxidized high-amylose starch with polysaccharides in the present invention can adhere to the intestinal surface more easily, which is beneficial to the colonization of probiotics in the intestine. These new properties of the probiotic delivery system have not been reported yet.

To achieve the above object, the present invention is implemented by the following technical solutions:

The present invention provides a method for preparing probiotic microcapsules based on oxidized high-amylose starch carrier, comprising the following steps:

(1) oxidizing a high-amylose starch solution using an oxidant to obtain oxidized high-amylose starch;

(2) mixing the oxidized high-amylose starch solution with probiotics to obtain an oxidized starch-probiotic mixed solution, wherein 3×10 ⁸ to 3×10 ¹⁰ cfu of probiotics are mixed with every 100 mL of the oxidized high-amylose starch solution;

(3) dissolving the polysaccharide in water to obtain a polysaccharide solution with a concentration of 2 to 3 g/100 mL;

(4) mixing the oxidized high-amylose starch-probiotic mixed solution obtained in step (2) with the polysaccharide solution, maintaining the system temperature at 40 to 50° C., and adjusting the system pH to 4.5 to 4.8 to obtain an oxidized starch-probiotic-polysaccharide mixed solution (aqueous phase);

(5) Take 2-4 mL of the above-mentioned oxidized starch-probiotic-polysaccharide mixed solution, slowly add the oil phase under stirring to obtain a mixed solution, continue stirring for 10-20 minutes, add a curing agent, continue stirring for 10-20 minutes, centrifuge several times, wash with physiological saline 2-3 times, and obtain wet microcapsules;

(6) freeze-drying the wet microcapsules to obtain probiotic microcapsule powder.

In one embodiment, the oxidation degree of the oxidized high-amylose starch in step (1) is 8% to 12%.

In one embodiment, in the aqueous phase of step (4), the final concentration of oxidized high-amylose starch is 0.66 g/100 mL, the final concentration of polysaccharide is 0.83 g/100 mL, and the number of viable probiotics is 3×10 ⁸ cfu/mL.

In one embodiment, the probiotics include one or more of Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus bulgaricus and Streptococcus thermophilus.

In one embodiment, the polysaccharide is a plant polysaccharide, an animal polysaccharide or a polysaccharide of microbial origin; the polysaccharide of plant, animal or microbial origin includes one or more of chitosan, carrageenan, gum arabic and sodium alginate.

In one embodiment, the probiotics are obtained by centrifuging the probiotic culture at 4°C, removing the supernatant, washing the slurry with a physiological saline solution, and then centrifuging to remove the supernatant, repeating 2 to 3 times to obtain a probiotic slurry or suspension.

In one embodiment, the concentration of the high amylose starch solution in step (1) is 1 g/100 mL to 2 g/100 mL.

Preferably, the mass concentration of the high amylose starch solution in step (1) is 1.6 g/100 mL.

In one embodiment, the oxidant in step (1) is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and NaBr, the addition amount of the oxide is 0.8-0.9% and 35-40% of the high amylose content, respectively, the pH condition of the oxidation is 9.5-10, the temperature of the oxidation is 4-5°C, the stirring speed is 500-700rpm, and the stirring time is 20-30min;

Preferably, in step (1), the addition amounts of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and NaBr are 0.85% and 35% of the high amylose content, respectively, the oxidation pH is 10, the oxidation temperature is 4° C., the stirring speed is 600 rpm, and the stirring time is 20 min;

In one embodiment, the polysaccharide in step (3) is a plant polysaccharide, an animal polysaccharide or a polysaccharide of microbial origin; the polysaccharide of plant, animal or microbial origin includes one or more of chitosan, carrageenan, gum arabic and sodium alginate.

Preferably, the polysaccharide in step (3) is sodium alginate, and the concentration of sodium alginate is 2%.

In one embodiment, the curing agent described in step (5) is a 0.5-0.6M _CaCl2 solution, and the addition amount of the _CaCl2 solution is 40-50% of the entire mixed solution. The _CaCl2 solution should be added after the oxidized starch-probiotics-polysaccharide mixed solution is added to the soybean oil and stirred for 10-20 minutes.

Preferably, the curing agent in step (5) is a 0.5M _CaCl2 solution, and the amount of the _CaCl2 solution added is 40% of the entire mixed solution. The _CaCl2 solution should be added after the oxidized starch-probiotics-polysaccharide mixed solution is added to the soybean oil and stirred for 15 minutes.

In one embodiment, the oil phase in step (5) is a mixed oil phase of Span 80 and soybean oil, and the added amount of Span 80 in the mixed oil phase is 2.5-3% (w/v).

Preferably, the amount of Span 80 added to the mixed oil phase in step (5) is 2.5% (w/v).

In one embodiment, in the mixed solution of step (5), the volume ratio of the water phase to the oil phase is 1:3 to 3:1.

Preferably, the volume ratio of the water phase to the oil phase in step (5) is 1:1.

In one embodiment, after adding the curing agent in step (5), stirring is performed, the stirring speed is 700 to 800 rpm, and the stirring time is 10 to 20 minutes.

Preferably, the stirring speed in step (5) is 700 rpm and the stirring time is 10 min.

In one embodiment, the method for treating the wet microcapsules in step (6) is to pre-freeze the wet microcapsules at -80°C for 24 hours and then dry them using a freeze-drying method to obtain probiotic powder.

The invention also provides a probiotic microcapsule.

The present invention also provides an application of probiotic microcapsules in the preparation of gastric acid-resistant oral products, characterized in that the products include food, medicine, and health products; and the health products include probiotic preparations.

Beneficial effects:

(1) The present invention oxidizes high-amylose starch by oxidizing high-amylose starch through a TEMPO-mediated oxidation system, and compounding the oxidized high-amylose starch with sodium alginate to form an oxidized starch-polysaccharide complex, which is used as the shell of probiotic microcapsules, thereby optimizing the core-shell structure of the microcapsules. Compared with the emulsion prepared by oxidizing high-amylose starch, the microcapsules containing Lactobacillus rhamnosus prepared by the present invention have improved stability and sustained release effect, and the pH responsiveness is also increased;

(2) The probiotic microcapsules obtained by the present invention can effectively protect the activity of the embedded probiotics during storage. Compared with the unencapsulated probiotics, the survival rate of the probiotics in the probiotic microcapsules obtained by the present invention is increased by 2.5 times after storage for 28 days, and the survival rate in the gastric fluid environment is increased by 1 times, and the survival rate in the gastric environment can be stabilized at more than 90%; in addition, in the intestinal environment, compared with the probiotic microcapsules with high-amylose starch and ordinary oxidized starch as the wall material, the probiotic microcapsules obtained by the present invention have a better sustained release effect within 4 hours, thereby improving the colonization rate of probiotics in the intestine;

(3) The raw material of the probiotic microcapsules obtained by the present invention is an oxidized starch-polysaccharide complex. In addition to having pH responsiveness and sustained release effect, the complex can also adhere to the intestinal surface. Compared with the probiotic microcapsules with high amylose starch and ordinary oxidized starch as wall materials, the adhesion rates of the probiotic microcapsules prepared by the present invention are increased by 1.32 times and 1.74 times, respectively, which is more conducive to the colonization of probiotics in the intestine and improves the utilization rate of probiotics;

(4) The present invention uses oxidized high-amylose starch and polysaccharides from plants or microorganisms as microcapsule wall materials to encapsulate probiotics to prepare microcapsule powder. Since the present invention uses a reverse emulsion method, the conditions in the microencapsulation process are mild and do not involve high temperature, high-speed stirring, high-pressure homogenization and other process flows, which can effectively protect the activity of the probiotics. In addition, the process steps of the present invention are relatively simple, and the raw materials are cheap and easily available, which is beneficial for production enterprises to control production costs and improve production efficiency.

In summary, the present invention uses oxidized high-amylose starch and polysaccharides from plants or microorganisms as microcapsule wall materials, uses an inverse emulsion method to prepare probiotic microcapsules, and uses _CaCl2 solution for solidification, so that probiotic microcapsules with higher protection ability can be prepared at a lower cost and with a simpler process.

Detailed ways

The present invention provides a method for preparing probiotic microcapsules based on oxidized high-amylose starch carrier, comprising the following steps:

(1) oxidizing high-amylose starch to obtain oxidized high-amylose starch;

(2) mixing the oxidized high-amylose starch solution with probiotics to obtain an oxidized starch-probiotic mixed solution, wherein 3×10 ⁸ to 3×10 ¹⁰ cfu of probiotics are mixed with every 100 mL of the oxidized high-amylose starch solution;

(3) dissolving the polysaccharide in water to obtain a polysaccharide solution with a concentration of 2.0 to 3.0%;

(4) after mixing the oxidized starch-probiotic mixed solution obtained in step (2) with the polysaccharide solution, maintaining the system temperature at 40-50° C., adjusting the system pH at 4.5-4.8, to obtain an oxidized starch-probiotic-polysaccharide mixed solution;

(5) Take 2-4 mL of the above oxidized starch-probiotic-polysaccharide mixed solution, slowly add the oil phase under stirring, continue stirring for 10-20 minutes, add the curing agent, and continue stirring for 10-20 minutes. Centrifuge several times and wash with physiological saline 2-3 times to obtain wet microcapsules;

(6) freeze-drying the wet microcapsules to obtain probiotic microcapsule powder;

In the present invention, high-amylose starch is added to boiling water, mixed evenly, cooled, and then oxidized by adding oxidants 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and NaBr to obtain an oxidized high-amylose starch solution. In the present invention, the mass concentration of the high-amylose starch solution is 1-2 g/100 mL, and more preferably 1.6 g/100 mL. The addition amount of the oxide is 0.8-0.9% and 35-40% of the high-amylose starch content, respectively, and more preferably 0.85% and 35%, respectively. The pH condition of the oxidation is 9.5-10, and further optimized to 10. The temperature of the oxidation is 4-5°C, and more preferably 4°C. The stirring speed is 500-700 rpm, and more preferably 600 rpm. The stirring time is 20-30 min, and more preferably 20 min.

After obtaining the oxidized starch solution, the present invention mixes the oxidized starch solution with probiotics to obtain an oxidized starch-probiotic mixed solution. In the present invention, the probiotics are obtained by centrifuging a frozen probiotic culture at 4°C and removing the supernatant, or by washing twice with a sterilized saline solution and then centrifuging to obtain a probiotic mud or suspension; after the oxidized high-amylose starch solution and the probiotics are mixed, a magnetic stirrer is used to mix the solution evenly.

In the present invention, polysaccharide is dissolved in water to obtain a polysaccharide solution with a concentration of 2.0-3.0%, and the concentration of the polysaccharide solution is optimized to be 2.0%.

After preparing the oxidized starch-probiotic mixed solution and the polysaccharide solution, the present invention mixes the two to obtain the oxidized starch-probiotic-polysaccharide mixed solution. In the present invention, the oxidized starch-probiotic mixed solution and the polysaccharide solution are mixed in equal volumes; stirring treatment is required after the mixing and after adjusting the pH of the system, and the stirring speed of the stirring treatment is preferably 600-800rpm, and more preferably 700rpm; the present invention does not specifically limit the stirring time after the oxidized starch-probiotic mixed solution and the polysaccharide solution are mixed, and it is advisable to mix evenly; the stirring time after adjusting the pH of the system is preferably 10-20min, and more preferably 15min; the temperature of the system is preferably maintained at 40-50°C, and more preferably 45°C; the pH of the adjustment system is preferably 4.5-5.0, and more preferably 4.7.

After preparing the oxidized starch-probiotic-polysaccharide mixed solution, the present invention adds the aqueous phase to the oil phase and continues to stir for 10 to 20 minutes, then adds the curing agent and continues to stir for 10 to 20 minutes. Wet microcapsules are obtained after multiple centrifugation and 2 to 3 times of washing with physiological saline. In the present invention, the oil phase is a mixed oil phase of Span80 and soybean oil, and the amount of Span80 added to the mixed oil phase is 2.5 to 3% (w/v); more preferably 2.5% (w/v). The ratio of the aqueous phase to the oil phase in the present invention is 1:3 to 3:1, and more preferably 1:1. The stirring after the aqueous phase of the present invention is added to the oil phase is a magnetic stirrer stirring, and the stirring speed is 700 to 800 rpm, more preferably 700 rpm; the stirring time is 10 to 20 minutes; more preferably 10 minutes.

After obtaining the wet microcapsules, the present invention freeze-dries the wet microcapsules to obtain probiotic powder. In the present invention, the freeze-drying treatment method for the wet microcapsules is to pre-freeze the wet microcapsules at -80°C for 12 to 36 hours, preferably 24 hours.

The present invention also provides probiotic microcapsules prepared by the above-mentioned preparation method. The probiotic microcapsules obtained have the characteristics of small product size, good product structural stability, easy to add in a variety of product forms, and a wide range of applications, and are particularly suitable for oral probiotic preparations to be added and used in medicines. The probiotic microcapsules prepared by the present invention can effectively protect the activity of the embedded probiotics during storage, and can achieve the protection of the embedded probiotics in the gastric fluid environment. When added to the medicine, the probiotics can be delivered to the intestine and slowly released, and the probiotic effect of the probiotics can be well exerted.

The present invention also provides the use of the probiotic microcapsules in preparing a gastric acid-resistant oral probiotic preparation. The present invention has no special restrictions on the dosage form and excipients of the gastric acid-resistant oral probiotic microcapsule preparation, and conventional probiotic dosage forms and excipients in the art can be used.

The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

Example 1 A method for preparing Lactobacillus rhamnosus microcapsules based on oxidized high-amylose starch carrier

The specific preparation method is as follows:

(1) Preparation of bacterial suspension: 100 μL of the frozen Lactobacillus rhamnosus LGG was thawed and added to MRS liquid culture medium. After incubation at 37°C for 36 h, 100 μL of the activated Lactobacillus rhamnosus LGG was placed in MRS liquid culture medium. After incubation at 37°C for 24 h, the culture was centrifuged at 4°C and the supernatant was removed to obtain a Lactobacillus rhamnosus suspension of 3×10 ⁸ cfu/mL.

(2) Preparation of oxidized high-amylose starch: 8.1 g of high-amylose starch (Shanghai Yuanye Biotechnology Co., Ltd.; product number: S27326; amylose content: 90%) used as microcapsule wall material was dissolved in 500 mL of water at 90°C, stirred evenly in a heated magnetic stirrer, and then cooled to room temperature. 0.0648 g of TEMPO and 3.24 g of NaBr were weighed and added to the above solution; the entire reaction system was placed in a constant temperature magnetic stirrer, the reaction temperature was maintained at 4°C, the pH was adjusted to 10, the stirring speed was 600 rpm, the reaction was stirred for 20 min, and the reaction was completed. A 3500Da dialysis bag was used for dialysis for 3 days. After freeze drying, an oxidized high-amylose starch with an oxidation degree of 10% was obtained.

(3) Preparation of aqueous phase

The above-mentioned oxidized high-amylose starch and sodium alginate powder (Qingdao Mingyue Seaweed Group Co., Ltd.; Article No.: MY-BCT-HZSN-1KG; Purity: 90%) were weighed and dissolved in sterile water to prepare an oxidized high-amylose starch solution with a concentration of 1.6 g/100 mL and a sodium alginate solution with a concentration of 2 g/100 mL. First, 0.5 mL of the oxidized high-amylose starch solution was mixed with 200 μL of the bacterial suspension, stirred with a magnetic stirrer at a speed of 700 rpm for 30 min to prepare an oxidized high-amylose starch-probiotic mixed solution, and then 0.5 mL of the sodium alginate solution was added thereto, stirred with a magnetic stirrer at a stirring speed of 700 rpm for 30 min to obtain an oxidized starch-probiotic-polysaccharide mixed solution, the system temperature was maintained at 45 ° C, the system pH was adjusted to 4.7, the magnetic stirrer was stirred at a stirring speed of 700 rpm for 30 min to obtain an oxidized starch-probiotic-polysaccharide mixed solution (aqueous phase). In the aqueous phase, the final concentration of oxidized high-amylose starch was 0.66 g/100 mL, the final concentration of polysaccharide was 0.83 g/100 mL, and the number of viable probiotics was 5×10 ⁷ cfu/mL.

(4) Preparation of oil phase

0.5 g of Span80 was mixed into 20 mL of soybean oil and stirred to obtain an oil phase.

(5) Preparation of probiotic microcapsules: The aqueous phase was slowly added to the oil phase under gentle stirring, and the mixture was stirred with a magnetic stirrer at a speed of 700 rpm for 10 min at room temperature. Subsequently, 20 mL of 0.5 M CaCl ₂ solution was added and stirring was continued for 10 min. Wet probiotic microcapsules were obtained by multiple centrifugation (2500 rpm, 5 min) and washing with physiological saline 2 to 3 times. The wet probiotic microcapsules were pre-frozen at -80°C for 24.0 h and then dried by freeze drying to obtain Lactobacillus rhamnosus microcapsule powder.

Example 2 A method for preparing Lactobacillus plantarum microcapsules based on oxidized high-amylose starch carrier

The Lactobacillus rhamnosus in Example 1 was replaced by Lactobacillus plantarum subspecies SGBCC D14056 to prepare Lactobacillus plantarum microcapsule powder. The remaining steps were the same as in Example 1.

Example 3 A method for preparing Lactobacillus casei microcapsules based on oxidized high-amylose starch carrier

The Lactobacillus rhamnosus in Example 1 was replaced by Lactobacillus casei JYLC-374 to prepare Lactobacillus casei microcapsule powder. The remaining steps were the same as in Example 1.

Example 4 A method for preparing bifidobacterium microcapsules based on oxidized high-amylose starch carrier

The Lactobacillus rhamnosus in Example 1 was replaced by Bifidobacterium lactis HN019 to prepare Bifidobacterium microcapsule powder. The remaining steps were the same as in Example 1.

Comparative Example 1 Omitting the oxidation operation of high amylose starch

Specific implementation method Refer to Example 1, except that the oxidized high-amylose starch in Example 1 is replaced with unoxidized high-amylose starch (Shanghai Yuanye Biotechnology Co., Ltd.; Product No.: S27326; amylose content is 90%) as the raw material for microcapsule wall material to prepare Lactobacillus rhamnosus microcapsule powder, and the remaining steps are the same as Example 1.

Comparative Example 2 Reducing the amylose content of oxidized high amylose starch

Specific implementation method Referring to Example 1, the difference is that the oxidized high-amylose starch (amylose content of 90%) in Example 1 is replaced by ordinary oxidized starch (amylose content of 5%) as the raw material of microcapsule wall material to prepare Lactobacillus rhamnosus microcapsule powder, and the remaining steps are the same as Example 1.

Comparative Example 3: Changing the degree of oxidation of oxidized high-amylose starch

Specific implementation method Refer to Example 1, the difference is that the oxidized high-amylose starch (amylose content is 90%) with an oxidation degree of 10% in Example 1 is replaced by an oxidized high-amylose starch with an oxidation degree of 4% as the raw material of the microcapsule wall material to prepare the rhamnosus Lactobacillus microcapsule powder, and the remaining steps are the same as Example 1. Among them, the oxidation degree is expressed as the mass fraction of the carboxyl group, and the oxidation degree of the oxidized high-amylose starch is controlled by adjusting the amount of TEMPO added. The oxidation degree measurement method is specifically as follows: weigh 0.12g of oxidized high-amylose starch and dissolve it in 20mL of boiling water, add 2 drops of phenolphthalein indicator while hot, titrate with 0.1mol/L NaOH solution to light pink, and do not change color within half a minute, and record the consumption of NaOH solution (V1, mL). Weigh the same mass of oxidized high-amylose starch and repeat the above operation, and record the consumption of NaOH solution (V2, mL). The oxidation degrees of different oxidized high-amylose starches are calculated according to the following formula.

Oxidation degree (%) = (((V_1-V_2)×C×0.045)/m)×100

Where m is the sample mass (g), C is the concentration of NaOH solution, mol/L.

Comparative Example 4: Omitting the addition of oxidized high-amylose starch

Specific implementation method Referring to Example 1, the difference is that the oxidized high-amylose starch (amylose content of 90%) in Example 1 is removed, and only sodium alginate is used as the raw material of microcapsule wall material to prepare Lactobacillus rhamnosus microcapsule powder. The remaining steps are the same as Example 1.

Comparative Example 5: Replacing oxidized high-amylose starch with phosphorylated high-amylose starch

Specific implementation method Refer to Example 1, the difference is that the oxidized high-amylose starch (amylose content of 90%) in Example 1 is replaced by phosphorylated high-amylose starch as the raw material of microcapsule wall material to prepare Lactobacillus rhamnosus microcapsule powder, and the remaining steps are the same as Example 1. Among them, the preparation method of sodium phosphate high-amylose starch is to dissolve 80g of high-amylose starch in 120mL of deionized water, add mixed phosphate (2.8g of sodium phosphate, 14.5g of sodium dihydrogen phosphate, 1.2g of disodium hydrogen phosphate) and 5g of stearic acid polyoxyethylene ether, adjust pH 5.5, stir at 80rpm/min at 53°C for 10h, centrifuge and air flow dry to obtain phosphorylated high-amylose starch.

Example 5 Structural Characterization of Microcapsules

The microcapsules obtained in Examples 1 to 4 of the present invention are single-layer embedding structures, and the average particle size of the microcapsules is about 300 μm. The microcapsules are formed by cross-linking oxidized high-amylose starch and sodium alginate.

1. Encapsulation rate detection

The probiotic microcapsule powders prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were dissolved in physiological saline to prepare a solution with a final concentration of 5%, and potassium dihydrogen phosphate buffer (0.05M, pH 6.8) was mixed with the above solutions at a volume ratio of 1:4, stirred at room temperature for 0.5 h, and then diluted and coated on MRS solid culture medium, and counted after incubation at 37°C for 2 days. The encapsulation efficiency (EE, %) of the probiotic microcapsule was calculated as follows:

EE(%)＝W2/W1×100％

Where W1 represents the total number of live LGG before encapsulation (CFU/mL), and W2 represents the number of live LGG released by the microspheres after encapsulation (CFU/mL). The results are shown in Table 1:

Table 1: Encapsulation efficiency of different Lactobacillus rhamnosus microcapsule powders

Group Encapsulation efficiency Example 1 93.11±0.21% Example 2 92.32±0.15% Example 3 91.27±0.27% Example 4 92.69±0.41% Comparative Example 1 49.43±0.34% Comparative Example 2 46.35±0.25% Comparative Example 3 51.73±0.32% Comparative Example 4 45.41±0.11% Comparative Example 5 72.13±0.19%

The results showed that using oxidized high-amylose starch as microcapsule wall material can effectively improve the encapsulation rate of several common probiotics including Lactobacillus rhamnosus. Compared with using unoxidized high-amylose starch as microcapsule wall material, the encapsulation rate is increased by 2 times; compared with ordinary oxidized starch as microcapsule wall material, the encapsulation rate is increased by 2 times; compared with low-oxidized oxidized starch as microcapsule wall material, the encapsulation rate is increased by 1.8 times; compared with not containing oxidized high-amylose starch and only using sodium alginate as microcapsule wall material, the encapsulation rate is increased by 2 times; compared with phosphorylated high-amylose starch as microcapsule wall material, the encapsulation rate is increased by 1.3 times. The experimental results show that the probiotic microcapsules with oxidized high-amylose starch as the wall material prepared by the present invention are suitable for the encapsulation of general probiotics. Compared with the oxidized high-amylose starch in the present invention, the encapsulation rates of unoxidized high-amylose starch, ordinary oxidized starch, oxidized high-amylose starch with low oxidation degree, sodium alginate and phosphorylated high-amylose starch as microcapsule wall materials are significantly lower than those of oxidized high-amylose starch. The probiotic microcapsules prepared by the present invention have excellent encapsulation rates.

2. Survival rate detection

The probiotic microcapsule powders prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were stored in an environment of 25° C., and the survival rate (%) within 28 days was detected.

The calculation method of survival rate: (number of surviving bacteria/total number of bacteria) × 100%;

Method for determination of viable bacteria count: flow cytometry was used to determine the number of viable bacteria before and during storage, so as to calculate the survival rate of probiotic microcapsules at different times in an environment of 25°C;

The results are shown in Table 2:

Table 2: Survival rate of different probiotic microcapsule powders at 25°C

Group 7-day survival rate 14-day survival rate 21-day survival rate 28-day survival rate Example 1 97.21±0.32% 93.43±0.21% 90.42±0.23% 86.58±0.27% Example 2 92.33±0.45% 86.27±0.31% 83.96±0.18% 78.68±0.35% Example 3 91.86±0.29% 85.71±0.17% 82.64±0.24% 77.73±0.31% Example 4 91.76±0.26% 84.36±0.22% 80.73±0.16% 75.92±0.11% Comparative Example 1 76.61±0.84% 67.26±0.61% 58.58±0.14% 51.38±0.52% Comparative Example 2 69.29±0.44% 60.86±0.19% 52.54±0.47% 46.36±0.78% Comparative Example 3 78.57±0.33% 69.45±0.27% 61.82±0.14% 55.57±0.29% Comparative Example 4 66.84±0.31% 57.44±0.29% 50.63±0.17% 43.33±0.19% Comparative Example 5 80.89±0.11% 72.46±0.78% 68.81±0.18% 61.56±0.35% Unencapsulated probiotics 58.78±0.73% 50.21±0.47% 43.53±0.55% 34.67±0.64%

The results showed that the survival rates of the probiotic microcapsules prepared according to Example 1 at 7 days, 14 days, 21 days and 28 days were 1.05 times, 1.08 times, 1.08 times and 1.10 times higher than those of the probiotic microcapsules prepared according to Example 2; 1.06 times, 1.09 times, 1.09 times and 1.11 times higher than those of the probiotic microcapsules prepared according to Example 3; and 1.05 times, 1.10 times, 1.12 times and 1.14 times higher than those of the probiotic microcapsules prepared according to Example 4. This indicates that when oxidized high-amylose starch is used as the wall material of the probiotic microcapsules, the survival rate after encapsulating Lactobacillus rhamnosus is higher than that after encapsulating other probiotics. Therefore, the microcapsule material based on oxidized high-amylose starch prepared by the present invention is more suitable for encapsulating Lactobacillus rhamnosus.

In addition, the survival rate of the probiotic microcapsules prepared according to Example 1 at 7 days, 14 days, 21 days and 28 days was 1.25 times, 1.35 times, 1.53 times and 1.64 times higher than that of the probiotic microcapsules prepared according to Comparative Example 1, which indicates that as a wall material for probiotic microcapsules, oxidized high-amylose starch has a stronger protective effect on probiotics than unoxidized high-amylose starch; the probiotic microcapsules prepared according to Example 1 at 7 days, 14 days, 21 days and 28 days were 1.25 times, 1.35 times, 1.53 times and 1.64 times higher than that of the probiotic microcapsules prepared according to Comparative Example 1, which indicates that as a wall material for probiotic microcapsules, oxidized high-amylose starch has a stronger protective effect on probiotics than unoxidized high-amylose starch; The survival rates of the probiotic microcapsules prepared according to Example 1 at 7 days, 14 days, 21 days and 28 days were 1.37 times, 1.51 times, 1.71 times and 1.82 times higher than those of the probiotic microcapsules prepared according to Comparative Example 2, which indicates that oxidized high-amylose starch has a stronger protective effect on probiotics than ordinary oxidized starch as a wall material for probiotic microcapsules; the survival rates of the probiotic microcapsules prepared according to Example 1 at 7 days, 14 days, 21 days and 28 days were 1.24 times, 1.34 times and 1.6 times higher than those of the probiotic microcapsules prepared according to Comparative Example 3. times, 1.47 times and 1.56 times, which shows that as a wall material for probiotic microcapsules, oxidized high-amylose starch with a high oxidation degree has a stronger protective effect on probiotics than oxidized high-amylose starch with a low oxidation degree; the survival rate of the probiotic microcapsules prepared according to Example 1 at 7 days, 14 days, 21 days and 28 days is 1.46 times, 1.63 times, 1.80 times and 2.0 times higher than that of the probiotic microcapsules prepared according to Comparative Example 4, which shows that as a wall material for probiotic microcapsules , the composite material of oxidized high-amylose starch and sodium alginate has a better protective effect on probiotics than sodium alginate; the survival rate of the probiotic microcapsules prepared according to Example 1 at 7 days, 14 days, 21 days and 28 days is 1.21 times, 1.29 times, 1.32 times and 1.40 times higher than that of the probiotic microcapsules prepared according to Comparative Example 5, which shows that as a wall material for probiotic microcapsules, oxidized high-amylose starch has a better protective effect on probiotics than phosphorylated high-amylose starch. The probiotic microcapsules prepared according to Example 1 have a survival rate of 1.67 times, 1.86 times, 2.07 times and 2.52 times higher than that of the unembedded probiotic microcapsules at 7 days, 14 days, 21 days and 28 days. The experimental results show that the probiotic microcapsules prepared by the present invention can ensure the survival rate of probiotics under normal temperature conditions, and the oxidized high-amylose starch can effectively improve the protection ability of the microcapsules to probiotics.

3. Gastric juice tolerance test

The probiotic microcapsules prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were added to sterile artificial gastric juice (Shanghai Yuanye Biotechnology Co., Ltd., pH = 2.0) to prepare a solution with a probiotic capsule content of 0.1 g/ml, and incubated at 37°C, 150 rpm in a water bath for 2 hours to simulate the gastric environment. Subsequently, the solution was diluted with physiological saline and coated on MRS solid culture medium, and counted after incubation at 37°C for 2 days. The survival rate (%) of the probiotics was calculated, and the results are shown in Table 3:

Table 3: Survival rates of different probiotic microcapsule powders in artificial gastric juice

Group 10-minute survival rate 20-min survival rate 30-min survival rate 40-min survival rate Example 1 99.26±0.57% 98.84±0.41% 98.51±0.52% 97.89±0.42% Example 2 98.77±0.23% 95.91±0.16% 90.27±0.33% 82.27±0.36% Example 3 98.69±0.41% 96.03±0.27% 91.11±0.19% 81.58±0.22% Example 4 98.61±0.28% 95.88±0.26% 90.42±0.15% 82.79±0.42% Comparative Example 1 98.14±0.26% 92.35±0.19% 85.37±0.26% 72.63±0.21% Comparative Example 2 97.91±0.14% 90.16±0.12% 80.39±0.37% 69.48±0.42% Comparative Example 3 98.55±0.28% 93.77±0.34% 87.63±0.35% 76.94±0.27% Comparative Example 4 96.78±0.13% 89.91±0.18% 77.36±0.34% 67.85±0.11% Comparative Example 5 98.15±0.37% 95.21±0.17% 90.28±0.26% 81.98±0.23% Unencapsulated probiotics 76.83±0.78% 69.97±0.32% 57.13±0.63% 50.81±0.17%

The results show that the survival rates of the probiotic microcapsules prepared according to Example 1 in artificial gastric juice at 10min, 20min, 30min and 40min are 1.01 times, 1.03 times, 1.08 times and 1.18 times higher than those of the probiotic microcapsules prepared according to Example 2; 1.01 times, 1.02 times, 1.07 times and 1.19 times higher than those of the probiotic microcapsules prepared according to Example 3; 1.01 times, 1.03 times, 1.08 times and 1.18 times higher than those of the probiotic microcapsules prepared according to Example 4. Obviously, in artificial gastric juice, the survival rate of Lactobacillus rhamnosus is higher than that of the other three probiotics, which indicates that in the probiotic microcapsules prepared based on oxidized high-amylose starch, the protective effect of oxidized high-amylose starch on Lactobacillus rhamnosus is higher than that of the other three probiotics, so oxidized high-amylose starch is more suitable as a microcapsule material for Lactobacillus rhamnosus and can better protect Lactobacillus rhamnosus.

In addition, the survival rates of the probiotic microcapsules prepared according to Example 1 in artificial gastric juice at 10 min, 20 min, 30 min and 40 min were 1.01 times, 1.06 times, 1.15 times and 1.34 times higher than those of the probiotic microcapsules prepared according to Comparative Example 1, which indicates that in artificial gastric juice, oxidized high-amylose starch has a more effective protective effect on Lactobacillus rhamnosus than unoxidized high-amylose starch; ... The survival rates of the probiotic microcapsules prepared according to Example 1 at 10 min, 20 min, 30 min and 40 min in artificial gastric juice were 1.02 times, 1.08 times, 1.22 times and 1.4 times higher than those of the probiotic microcapsules prepared according to Comparative Example 2, which indicates that in artificial gastric juice, compared with ordinary oxidized starch, oxidized high-amylose starch has a more effective protective effect on Lactobacillus rhamnosus; the survival rates of the probiotic microcapsules prepared according to Example 1 at 10 min, 20 min, 30 min and 40 min in artificial gastric juice were 1.01 times, 1.08 times, 1.22 times and 1.4 times higher than those of the probiotic microcapsules prepared according to Comparative Example 3. .05 times, 1.12 times and 1.27 times, which shows that in artificial gastric juice, compared with oxidized high-amylose starch with low oxidation degree, oxidized high-amylose starch with high oxidation degree has a more effective protective effect on Lactobacillus rhamnosus; the survival rate of the probiotic microcapsules prepared according to Example 1 in artificial gastric juice at 10min, 20min, 30min and 40min is 1.03 times, 1.10 times, 1.27 times and 1.44 times higher than that of the probiotic microcapsules prepared according to Comparative Example 4, which shows that in artificial gastric juice, compared with Sodium alginate, oxidized high-amylose starch and sodium alginate complex have more effective protective effects on Lactobacillus rhamnosus; the survival rates of the probiotic microcapsules prepared according to Example 1 in artificial gastric juice at 10min, 20min, 30min and 40min are 1.01 times, 1.03 times, 1.08 times and 1.19 times higher than those of the probiotic microcapsules prepared according to Comparative Example 5, which indicates that in artificial gastric juice, oxidized high-amylose starch has a more effective protective effect on Lactobacillus rhamnosus than phosphorylated high-amylose starch. The survival rates of the probiotic microcapsules prepared according to Example 1 in artificial gastric juice at 10min, 20min, 30min and 40min are 1.29 times, 1.42 times, 1.71 times and 1.94 times higher than those of the unencapsulated probiotic microcapsules. The experimental results show that in a simulated gastric fluid environment at 37°C, the wall material layer of the probiotic microcapsule prepared by the present invention can provide more effective protection for the probiotics within 40 minutes, which means that the probiotics have better tolerance to artificial gastric fluid.

4. Characterization of release performance in artificial intestinal fluid

The probiotic microcapsules prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were added to sterile artificial intestinal fluid (Shanghai Yuanye Biotechnology Co., Ltd., pH = 6.8) to prepare a solution with a probiotic microcapsule content of 0.1 g/ml, and incubated at 37°C, 150 rpm in a water bath for 3 h to simulate the intestinal environment. 500 μL of samples were taken at 1, 2, 3, and 4 h, respectively, diluted with physiological saline and coated on MRS solid culture medium, and counted after incubation at 37°C for 2 days. The release performance of Lactobacillus rhamnosus (calculated as viable count logcfu/g) was investigated, and the results are shown in Table 4:

Table 4: Release performance of different probiotic microcapsule powders in artificial intestinal fluid (release amount per hour)

The results show that the probiotic microcapsules in Examples 1 to 4 show a slowly increasing probiotic release rate per hour within 1h to 4h in the simulated intestinal fluid. In the simulated intestinal fluid environment, oxidized high-amylose starch as the wall material layer of the microcapsule can achieve the slow release of the fixed probiotics, which is helpful for the colonization of the probiotics in the intestine. This is because the slower the release rate of the probiotic microcapsules, the longer the colonization time of the probiotics in the intestine, so that the function of the probiotics can be better exerted. This shows that the probiotic microcapsules prepared based on oxidized high-amylose starch have a slow release effect in artificial intestinal fluid, which can help the colonization of probiotics in the intestine. In the simulated intestinal fluid, the probiotic microcapsules in Comparative Examples 1 to 3 show a state of first increasing and then decreasing in the hourly probiotic release rate within 1h to 4h; the probiotic microcapsules in Comparative Example 4 show a state of gradually decreasing in the hourly probiotic release rate within 1h to 4h in the simulated intestinal fluid. However, the probiotic microcapsules have the disadvantages of first fast and then slow release and gradual decrease. First, the release rate is fast at first and then slow, which will lead to uneven colonization of probiotics in the intestine. Some probiotics will be released prematurely or excessively in the intestine, while other probiotics cannot be fully released and colonized. This uneven release rate will affect the survival and function of probiotics; secondly, the gradually decreasing release rate will lead to insufficient residence time of probiotics in the intestine, and it is impossible to achieve a long-term and stable colonization effect. Therefore, probiotics need to stay in the intestine for a certain period of time to effectively play the role of regulating intestinal flora, and the gradually decreasing release rate may cause some probiotics to stay in the intestine for too short a time, affecting their growth and reproduction. This shows that the probiotic microcapsules based on the complex of oxidized high-amylose starch and sodium alginate have a better release effect on probiotics in artificial intestinal fluid than the probiotic microcapsules based on unoxidized high-amylose starch, ordinary starch, low-oxidized oxidized high-amylose starch and sodium alginate, and are more conducive to the colonization of probiotics in the intestine. Although the probiotic microcapsules in Comparative Example 5 also showed a gradually increasing probiotic release rate within 1 h to 4 h in the simulated intestinal fluid, the release rate was too slow, which would result in insufficient probiotic release or require a longer time to release, thereby affecting the effect and stability of the probiotics. Therefore, the Lactobacillus rhamnosus microcapsules based on oxidized high-amylose starch were more suitable for the release and colonization of Lactobacillus rhamnosus in the intestine than the Lactobacillus rhamnosus microcapsules based on phosphorylated high-amylose starch as the wall material.

5. In vitro intestinal mucus adhesion characterization

The probiotic microcapsules described in Examples 1 to 4 and Comparative Examples 1 to 5 were subjected to an in vitro intestinal mucus adhesion experiment. The specific method was as follows: on a sterile operating table, the intestines of male rats were cut open, and 4 portions of 1.5 cm×1.5 cm×2 mm of the intestines were taken on a sterile glass slide, and 50 μL of Lactobacillus rhamnosus suspension (blank group), probiotic microcapsules of Examples 1 to 4, and probiotic microcapsules of Comparative Examples 1 to 5 were added dropwise, respectively, and placed in sterilized centrifuge tubes, placed in a 37°C water bath oscillator for 2 hours, and then taken out, rinsed with 300 μL of physiological saline, the rinse fluid was collected for gradient dilution, and the number of viable bacteria was counted. The adhesion rate was calculated according to the following formula, and the results are shown in Table 5.

Adhesion rate (%) = (total number of probiotics - number of unadhered probiotics) / total number of probiotics × 100%

Table 5 Adhesion rate of different probiotic microcapsules in the intestine in vitro

Group Adhesion rate (%) Blank group 13.27±0.57% Example 1 83.78±0.69% Example 2 72.36±0.29% Example 3 71.81±0.18% Example 4 72.57±0.34% Comparative Example 1 63.19±0.81% Comparative Example 2 47.92±0.64% Comparative Example 3 67.61±0.14% Comparative Example 4 45.39±0.22% Comparative Example 5 69.76±0.39%

The results show that the adhesion rate of the Lactobacillus rhamnosus microcapsules (Example 1) prepared by the present invention in the intestine reaches 83.78%, which is 1.15 times higher than that of Example 2, 1.16 times higher than that of Example 3, and 1.15 times higher than that of Example 4. Obviously, the adhesion rate of the Lactobacillus rhamnosus microcapsules based on oxidized high-amylose starch in the intestine is higher than that of the other three probiotic microcapsules with the same shell, which indicates that the oxidized high-amylose starch is more suitable as a wall material for encapsulating Lactobacillus rhamnosus.

The adhesion rate of the Lactobacillus rhamnosus microcapsules (Example 1) prepared by the present invention in the intestine is 1.32 times higher than that of Comparative Example 1, indicating that compared with unoxidized high-amylose starch, oxidized high-amylose starch as the shell of probiotic microcapsules will have a higher adhesion rate in the intestine. The adhesion rate of the Lactobacillus rhamnosus microcapsules (Example 1) prepared by the present invention in the intestine is 1.74 times higher than that of Comparative Example 2, indicating that compared with ordinary oxidized starch, oxidized high-amylose starch as the shell of probiotic microcapsules will have a higher adhesion rate in the intestine. The adhesion rate of the Lactobacillus rhamnosus microcapsules (Example 1) prepared by the present invention in the intestine is 1.23 times higher than that of Comparative Example 3, indicating that compared with low-oxidized oxidized high-amylose starch, high-oxidized oxidized high-amylose starch as the shell of probiotic microcapsules will have a higher adhesion rate in the intestine. The adhesion rate of the Lactobacillus rhamnosus microcapsules (Example 1) prepared by the present invention in the intestine is 1.84 times higher than that of Comparative Example 4, indicating that compared with sodium alginate, the complex of oxidized high-amylose starch and sodium alginate as the shell of probiotic microcapsules will have a higher adhesion rate in the intestine. The adhesion rate of the Lactobacillus rhamnosus microcapsules (Example 1) prepared by the present invention in the intestine is 1.20 times higher than that of Comparative Example 5. It is shown that compared with phosphorylated high-amylose starch, oxidized high-amylose starch as the shell of probiotic microcapsules will have a higher adhesion rate in the intestine. The experimental results show that the probiotic microcapsules with oxidized high-amylose starch as the wall material have better adhesion effect in the intestine. Furthermore, the comprehensive intestinal simulation experiment results show that the probiotic microcapsules with oxidized high-amylose starch as the wall material are more conducive to the sustained release and colonization of probiotics in the intestine.

Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (10)

1. A method for preparing probiotic microcapsules based on oxidized high-amylose starch carrier, characterized in that it comprises the following steps: (1) preparing oxidized high-amylose starch: adding high-amylose starch to boiling water and mixing evenly, adding an oxidant after cooling, stirring, removing impurities and drying to obtain oxidized high-amylose starch; (2) preparing an aqueous phase: mixing the oxidized high-amylose starch prepared in step (1) with the polysaccharide and probiotic suspension to obtain an aqueous phase; (3) preparing the oil phase: adding 2.5-3 g/100 ml Span 80 to soybean oil and stirring to obtain the oil phase; (4) Preparing probiotic microcapsules: mixing the aqueous phase prepared in step (2) and the oil phase prepared in step (3) to obtain a mixed solution, stirring, adding a curing agent, stirring, centrifuging, washing, and drying to obtain probiotic microcapsules. 2. The method according to claim 1, characterized in that the degree of oxidation of the oxidized high-amylose starch in step (1) is 8% to 12%. 3. The method according to claim 1, characterized in that in the aqueous phase of step (2), the concentration of oxidized high-amylose starch is 1-2 g/100 mL, the concentration of polysaccharide is 2-3 g/100 mL, and the number of viable probiotics is 3×10 ⁸ to 3×10 ¹⁰ cfu/mL. 4. The method according to claim 1, characterized in that the polysaccharide in step (2) is a plant polysaccharide, an animal polysaccharide or a polysaccharide of microbial origin; the polysaccharide of plant, animal or microbial origin comprises one or more of chitosan, carrageenan, gum arabic and sodium alginate. 5. The method according to claim 1, characterized in that in the mixed solution of step (4), the volume ratio of the water phase to the oil phase is 1:3 to 3:1. 6. The method according to claim 1, wherein the probiotics in step (1) include one or more of Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus bulgaricus and Streptococcus thermophilus. 7. The method according to claim 1, characterized in that the oxidant in step (1) includes but is not limited to TEMPO. 8. The method according to claim 1, characterized in that the curing agent in step (4) is a _CaCl2 solution. 9. Probiotic microcapsules prepared by the method according to any one of claims 1 to 8. 10. Use of the probiotic microcapsules according to claim 9 in the preparation of gastric acid-resistant oral products, characterized in that the products include food, medicine, and health products; and the health products include probiotic preparations.