KR100878339B1 - Methods of double microencapsulation of lactobacillus using prebiotic substrates - Google Patents

Abstract

A method for coating lactobacillus dually, and the dually coated lactobacillus prepared by the method are provided to improve acid resistance and heat resistance. A method for coating lactobacillus dually comprises the steps of culturing lactobacillus and preparing the freeze-dried strain; mixing the freeze-dried strain and a prebiotic substrate in a ratio of 7:1 to 11:1 by weight by using the hybridization system under the condition of the rotation velocity of 12,000-18,000 rpm, the temperature of 20-30 deg.C and the time of 2-4 min to coat the freeze-dried strain with a prebiotic substrate; and secondly coating the coated strain with an enteric coating material.

Description

Method for double microencapsulation of lactic acid bacteria using prebiotic substrates {Methods of double microencapsulation of Lactobacillus using prebiotic substrates}

The present invention relates to a double micro-coating method of lactic acid bacteria using a prebiotic substrate, more specifically, after culturing the lactic acid bacteria to create a freeze-dried cells, the freeze-dried cells hybridized with a prebiotic coating material ( performing a first coating process using a hybridisation system, and performing a second coating process on the first coated cells with an enteric coating material to improve acid resistance and heat resistance. It relates to a double microcoat method of lactic acid bacteria.

Microencapsulation has been widely used in the food, medicine and cosmetics industries. Microcoatings in the dairy industry have been applied to increase the viability of bacterial cultures and to facilitate delivery. Teixeira et al. (1995) reported that microcoating of lactic acid bacteria can enhance the stability and viability of lactic acid bacteria during storage, especially under poor conditions such as acid, alkali, heat and salt stresses occurring during food processing. Recently, several studies have reported the possibility of microcoated lactic acid bacteria using various coating techniques (Rao et al., 1989; Favaro-Trindade and Grosso, 2002), but in general, such as spray-drying methods. Dry coating technology used in food processing has shown a limitation in viability of bacteria associated with high temperature treatment and production of single size capsules (Park et al., 2002).

Dry coating techniques developed with hybridization systems have been developed to overcome these limitations. The hybridization system consists of six blades, an ultrafast rotating cylinder with stator and powder recirculation torque. The powder mixture (particulates of the host and guest), which is placed in the container, is filled tightly with the steam generated by the blades rotating at high speed, and during the process, the particles form an ordered mixture by coating the guest particles on the surface of the host particles. Has the principle of Compared to other microcoat techniques including spray-drying coatings, the hybridization system in the present invention exhibits a high microcoat capability and minimizes damage to bacteria caused by heat using a cooling system that maintains temperatures below 30 ° C. Have been reported (Takafumi et al., 1993).

Polysaccharides such as starch, alginate, carrageenan, and chitosan have been widely studied in microcoating materials of lactic acid bacteria (Koo et al., 2001). However, only a few studies have been studying the application of functional oligosaccharides as a source of coating material. Recently, theories have been developed that prebiotic oligosaccharides enhance the viability of probiotics in the upper digestive tract (Corcoran et al., 2005).

Prebiotic substrates are sugars in which two to eight monosaccharides such as glucose, galactose and fructose are bound. However, in recent years, prebiotics, which are functional foods, are mostly broken down into constituent monosaccharides by digestive enzymes in the body, whereas they are absorbed by the digestive enzymes and reach the large intestine. Say the sugar used. Indigestible major sugars can be divided into digestible and indigestible sugars. Since prebiotic substrates are indigestible, acetate, propionate and propionate can be prevented by beneficial bacteria such as bifidus in the large intestine, which are not degraded in the stomach It is converted to the same volatile fatty acids, so there are few calories.

All prebiotic substrates are reported to have different functionalities depending on chemical composition, binding type, and purity. These oligosaccharides are selectively used by beneficial indigenous intestinal microflora such as Bifidobacteria and Lactobacillus and may inhibit the growth of potential pathogens such as toxin producing Clostridia, bacteroids and Escherichia coli (de Vaux). Et al., 2002). In addition, the use of such oligosaccharides has been reported to affect the enteric point of beneficial bacteria such as lactic acid bacteria. Therefore, the use of prebiotics together with the prebiotic lactic acid bacteria may exhibit synergistic effects of improving the activity of probiotic strains in addition to the efficacy of prebiotics using prebiotic oligosaccharides as a sugar utilization source.

Lactobacillus acidophilus RP32 (ATCC 43121) showed excellent acid resistance and cholesterol-lowering effects in the host (Gilliland et al., 1985). We have established a method for the effect of seven prebiotic substrates on the viability of Lactobacillus ashidophilus RP32 and developed a new double dry-coating method for the production of effective probiotic formulations using a hybridization system. It was.

Accordingly, the present inventors have diligently researched to overcome the problems of the prior art, when the double micro-coating of the lactic acid bacteria using a prebiotic substrate, lactic acid bacteria used in the actual food without affecting the survival rate of the lactic acid bacteria It was confirmed that the acid resistance and heat resistance can be greatly improved, and the present invention was completed.

Therefore, the main object of the present invention is to provide a new concept double coating method using a prebiotic substrate having excellent physiological activity ability as a coating material in addition to the coating material used in the conventional to improve the acid resistance and heat resistance of lactic acid bacteria.

Another object of the present invention to provide a micro-coated lactic acid bacteria using the prebiotic substrate.

According to one aspect of the present invention, the present invention provides a method for dual microcoating of lactic acid bacteria for improving acid resistance and heat resistance, comprising the following steps:

a) culturing the lactic acid bacteria and then making the freeze-dried cells;

b) subjecting the freeze-dried cells to a prebiotic substrate using a hybridization system to perform a primary coating process; And

c) performing a secondary coating process on the primary coated cells in an enteric coating material.

In the present invention, micro-coating or microencapsulation is generally used to protect substances unstable under certain conditions such as nutrients and beneficial bacteria from gastric acid, heat, salinity, water, etc., or to improve solubility and dispersibility. It refers to a technique for fine packaging using a protective material, such as, in the present invention refers to a method of minimizing lactic acid bacteria killed by gastric acid using a lactic acid bacteria as a 'core', and a sugar as a coating agent. Dairy products containing the lactobacillus coated with the saccharide may reach the large intestine without being degraded by gastric acid and digestive enzymes, thereby improving the activation of the lactic acid bacteria in the large intestine.

In the method of the present invention, the lactic acid bacteria may be used as the intestinal beneficial bacteria, such as lactic acid bacteria mainly used in the production of dairy products, such as bifidus, rutheri, casei, Ashdophilus lactic acid bacteria, preferably Ashdophilus lactic acid bacteria ( Lactobacillus acidophilus ) is suitable.

In the method of the present invention, the pellets in which the lactic acid bacteria cultured in step a) are centrifuged are suspended in skim milk containing lactose and dried.

In the method of the present invention, the hybridization system in step b) has a weight ratio of the freeze-dried cells and the prebiotic coating material (prebiotic substrate) 7: 1 to 11: 1 (w / w), a rotor The speed of 1) is preferably 12,000 to 18,000 rpm, the running temperature is 20-30 ° C., and the running time is 2-4 minutes. More preferred hybridization conditions are as in the embodiment of the present invention, the weight ratio of freeze-dried cells and prebiotics coating material is 9: 1, the speed of the rotating cylinder is 15,000 rpm, the operating temperature is 25-30 ℃, and The run time is 3 minutes. In particular, when the running time of the hybridization system is more than 6 minutes, it was confirmed that lowering the activity of the live bacteria.

In the present invention, the prebiotics coating material is characterized in that the fructooligosaccharides (fructooligosaccharide, FOS), lactulose (lactulose) or raffinose (raffinose).

Prebiotics or prebiotic substrates of the present invention are intestinal health improving functional substances widely used in food and health-related businesses, dietary fiber, oligosaccharides, sugars that are not absorbed affect the growth of intestinal microorganisms. In particular, a component that helps the growth of gut microorganisms beneficial to the human body is called a prebiotic material. In the present invention, in order to improve the acid resistance and heat resistance of lactic acid bacteria, which are enteric beneficial bacteria, a method of double coating using a prebiotic material was developed. As the prebiotic material of the present invention, lactic acid bacteria can be firstly coated with inulin, xylitol, sorbitol, mannitol, FOS, lactulose or raffinose. Preference is given to using lactulose or raffinose.

In the method of the present invention, the enteric coating materials may be any coating material capable of maximizing the properties of the lactic acid bacteria, but preferably a commercially available dry coating agent, ie, an acid-soluble coating material. It is selected from the group consisting of Eudragit (Eudragit, methacrylate copolymer), Sureteric (poly-vinylacetate phthalate) and Compritol (Compritol, glyceryl behenate), more preferably as an acid soluble coating agent Polymethacrylate type polymer is used. Most preferably, the shrederics that dissolve at neutral pH are suitable.

According to another aspect of the present invention, the present invention provides a double micro-coated lactic acid bacteria improved acid resistance and heat resistance prepared by the above method.

The present invention provides a standard method for evaluating the prebiotic substrate capacity of lactic acid bacteria, a method for preparing a double microcapsules using a highly functional hybridization system and a method for determining the resistance to various stresses of a double dry coated Lactobacillus species. Provide basic data on activities and activities.

In the method of evaluating the resistance to various stresses of various types of dry coated Lactobacillus ashidophilus RP32, acidity, high salinity, high temperature, and viability at various temperatures were evaluated. Double dry-coated Lactobacillus ashidophilus RP32 of selected prebiotic and enteric coatings (reuteric) was found to outperform Lactobacillus ashidophilus RP32 without acid and heat resistance. The present invention also provides information on specific methods for evaluating prebiotics availability for growth of Lactobacillus ashidophilus RP32 and prebiotic substrates effective for growth of Lactobacillus ashidophilus RP32. . In the method for evaluating the prebiotics utilization of the present invention, seven prebiotic substrates were used, and they were sorbitol, mannitol, lactulose, kyitol, inulin, fructooligosaccharide, and raffinose. It was. Centrifugation and washing of bacteria and culturing and bacteriometry may be performed. Promoting growth of Lactobacillus ashdophyllus RP32, the effective biobial substrates of plutooligosaccharides, lactulose, and raffinose.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

As described above, according to the present invention, as a prebiotic substrate used for the growth of Lactobacillus ashidophilus RP32, it was selected as plutoolisaccharide, lactulose and raffinose and established by the present inventors. It was observed that by using a hybridization system, a double dry-coating method using various prebiotic substrates on Lactobacillus ashidophilus RP32 can be used to greatly improve acid and heat resistance. Therefore, it is expected to be applicable to the development of prebiotic and probiotic formulations that can effectively enhance the survival rate of lactic acid bacteria during oral administration.

Example 1 Lactic Acid Strains and Medium

Lactobacillus acidophilus RP32 (ATCC 43121) was obtained from a strain preservation solution stored in a food microbiology laboratory at Korea University, and placed in MRS broth (MRS broth; Difco, Sparks, MD, USA) at 37 ° C. Incubated once times. Strain preservation was stored at -80 ℃ and 20% glycerol was used as a cryoprotectant. Drying - in the fine coating process Lactobacillus ash also the filler's RP32 10% skim milk (skim milk), 0.3% of glucose (glucose), 0.3% Bacto peptone (Bacto ^TM peptone), Bacto proteosome's peptone No. 3 (BactoTM proteose peptone No.3; Difco, Franklin Lakes, NJ, USA), and cultured for 24 hours in a milk medium of pH 6.5 containing 0.3% yeast extract, the pH was 6.0 A pH automatic regulator (Jenco model 3671; Whatman Lab Sales, Hillsboro, OR, USA) was used to maintain. After culturing, the strain sample was centrifuged at 4 ° C. for 30 minutes, and the pellet was suspended in 10% skim milk containing 10% lactose and freeze-dried, and stored at −80 ° C. until use.

Example 2 Effect of Prebiotic Substrates on the Growth of Lactobacillus Ashdophilus RP32

Prebiotic substrates include sorbitol, mannitol, lactulose, xylitol (all obtained from the Sigma Chemical Co., St. Louis, MO, USA), inulin (inulin) (RAFILOSE ™ serif HP; Orafti, Tienen, Belgium), fructooligosaccharides (FOS; RAFTILOSE ™ serif P95; Orafti), and raffinose (Difco) were selected. The overnight culture of Lactobacillus ashdophyllus RP32 was centrifuged at 3000 × g for 30 minutes, after which the cell was washed twice with 0.85% NaCl. Thereafter, each prebiotic substrate containing various concentrations (0%, 0.50%, 0.75%, 1.00%, and 1.50%) was inoculated on the basic MRS medium containing no glucose and beef extracts. A positive control containing glucose and a negative control containing no prebiotic substrates were prepared. The culture was incubated for 24 hours under anaerobic conditions (BBL Gas-Pak system; Difco, Sparks, MD, USA). The viable cell count was measured by a standard plate method using MRS solid medium, and the pH change of the culture was measured using a pH meter (Beckman Instruments Inc., Fullerton CA, USA).

Table 1 below shows the growth ability of Lactobacillus ashdophyllus RP32 for various prebiotic substrates carried out in the present invention, and among these seven prebiotic substrates, fructooligosaccharide, lactulose In addition, it is possible to effectively use three substrates of raffinose, and it has been shown that the strain can be used after treatment of the culture for at least 24 hours. In addition, as the addition ratio of the prebiotic substrate was increased from 0% to 1.5%, it was determined that the growth rate was significantly increased and the pH was lowered with the growth of the strain.

TABLE 1 Review of growth capacity of Lactobacillus ashidophilus RP32 for various prebiotic formulations

¹ concentration of prebiotic formulation (substrate).

² Cell growth = log (cultured viable)-log (initial viable)

³ data were shown as mean ± standard deviation from three experiments.

^{a, b, c, d, e} Superscripts in the same row show significant differences (p <0.05).

Example 3 Double Dry Coating Process Using Hybridization System

In the present invention, the dual dry-coating process is a modification of the method established by the conventional foil et al. (2002) by a hybridization device (Nara Hybridization System, Model NHS-0 &NSH-1; Nara Machinerty Co. Ltd, Tokyo , Japan; Takafumi et al., 1993). Shuteric enteric coating systems based on polyphthalates (Sureteric ™ sans; Colorcon, Darford Kent, UK) were used as coating materials in the present invention. Selected prebiotics (fructooligosaccharides, lactulose, and raffinose) were subjected to powder processing of Lactobacillus ashidophilus RP32 ('core' particles) under an air pressure of 6 kg per cm ² in the first coating process. Preferred initial particle size was carried out to 100-200 μm. The ratio of the cells and the prebiotics coating material was carried out at 9: 1, 4: 1 and 2: 1 (w / w), and the rotational cylinder speed was fixed at 15,000 rpm (3 minutes). The temperature during the process was kept below 30 ° C.

Table 2 shows the number of viable cells after fixing the operating time to 3 minutes and increasing the rotor rotation speed of the hybridizer to the highest level of 15,000 rpm. The result was maintained at the same level as viewed it appears to be at least ¹⁰ 10 CFU / g of viable cells in both 5,000, 7,000, 10,000, 15,000 rpm compared with the number of live cells in the untreated control group 2.45 × ¹⁰ 10 CFU / g. These results were observed to mean that microencapsulation by the hybridization system did not significantly affect the probiotic activity.

TABLE 2 Survival Variation of Lactobacillus Ashdophyllus RP32 by Coating Process Conditions

^{In 1} hybridization system, the time is fixed at 3 minutes.

In addition, Table 3 below compares the activity of the strain according to the operating time while fixing the rotational speed of the rotor at 15,000 rpm in this coating process after mixing the strain with the coating material. The activity of lactic acid bacteria was not significantly affected at 15,000 rpm and 3 min treatment conditions, even after microencapsulation with the coating material, at about 10 ¹⁰ CFU / g, which is similar to that of the untreated control. However, when the microencapsulation was excessively performed at 15,000 rpm for 6 minutes, the vitality of the lactic acid cells was 3.45 × 10 ⁹ CFU / g, which was about 1/10 lower than that of the untreated control. Therefore, it is thought that excessive prolongation of treatment time may lower the activity of the probiotic.

TABLE 3 Survival Changes of Lactobacillus Ashdophyllus RP32 in Single Covering with Cartesic

^{1 The} rotor speed is fixed at 15,000 rpm in the hybridization system.

A second coating consisting of a male enteric coating was applied under the same conditions at 9: 1 (w / w) (the ratio of cell to prebiotics coating material). After coating treatment, the samples were dispersed with ethyl alcohol and analyzed for particle size with a particle analyzer (CILAS 106; CILAS, France). Single dry-coating was performed with samples using only maleic or prebiotic. After the coating process, the viable cell count and stability of the dry-coated cells were evaluated by incubating in anaerobic conditions at 37 ° C. for 72 hours using standard inoculation techniques, and then measuring the viable cell count. The uncoated Lactobacillus ashidophilus RP32 used as a control was prepared by the same coating process without coating material.

Therefore, in the microencapsulation conditions of lactic acid strain Lactobacillus ashdophyllus RP32 using the hybridization system used in the present invention, the activity of lactic acid bacteria was almost changed when compared to the untreated control at 15,000 rpm and 3 minutes or less. It was judged to be absent and finally set to the optimum condition of double dry coating.

TABLE 4 Survival Changes of Lactobacillus Ashdophyllus RP32 on Double Coating with Various Prebiotic Substrates

Example 4. Electron Microscopy

 The shape of the microcapsules was observed using a scanning electron microscope. For observation using scanning electron microscopy, prebiotic substrates, uncoated bacteria and dry-coated samples were adhered to the stub using double-sided tacky metal tape. Then, an ion-sputter coater was used to spray the gold-palladium layer for 60 seconds. The cell surface of Lactobacillus ashdophilus RP32 by coating treatment was observed using a scanning electron microscope (S-2380N; Hitachi, Tokyo, Japan) using a 15 kV voltage.

1 shows scanning electron micrographs (SEM) of single and double dry coated Lactobacillus ashidophilus RP32 (a: scanning electron micrograph of uncoded control Lactobacillus ashidophilus RP32, b: coating Lactobacillus ashidophilus RP32, encapsulated in a monolithic substance, c: fructooligosaccharide as a prebiotic substrate, d: lactulose as a prebiotic substrate, e: fructooligosaccharide as a single Dry-coated Lactobacillus Ashdophilus RP32, f: Lactobacillus single dry-coated Lactobacillus Ashdophyllus RP32, g: Fructo-oligosaccharides and sterically double dry-coated Lactobacillus Ashdophyllus RP32, h: Lactobacillus and hydrophobic double dry-coated Lactobacillus ashdophyllus RP32.).

First, in the single dry-coating of surretic used as coating material, uniform coating effect was observed in all treatments and showed smooth and uniform surface characteristics. In addition, in the case of a single dry-coated using a prebiotic substrate, the initial properties of the fructooligosaccharides were spherical in various sizes, which were not easily broken by cell complexes, and the cell particles surrounded the surface of the fructooligosaccharides. The shape was observed. Lactulose showed a lattice of lattice-like particles, and the degree of coating was similar to that of fructooligosaccharide. After treatment, the product was a non-uniform particle and a lattice protruding from the surface. Morphology was also observed after cell complexation. In order to achieve another object of the present invention, the surface is uniformly spherical after the secondary coating of the primary coated sample when the double coating is carried out using a wheteric to give functionality after the primary coating with a prebiotic substrate. I could confirm the change

Uncoated and dry-coated samples for transmission electron microscopy (TEM) observation were subjected to 4 ° C in a solution of 2% glutaaldehyde and 2% paraformaldehyde in 0.1 moles of glycine-hydrochloric acid buffer (pH 2.0). After fixing for 4 hours at 1% osmium tegoside was carried out again for 4 hours at 2 ℃. After washing, samples were dehydrated with ethanol and embedded in Polybead 812 resin (Polyscience, Warrington, Pa., USA). After an ultratin 60 nm section, it was finally stained with uranyl acetic acid and citric acid. Subsequently, the morphology of the cells was evaluated by Zeiss EM 912 TEM (LEO Electorn Microscope Ltd, Oberkochen, Germany).

FIG. 2 shows transmission electron micrographs (TEM) of dry coated Lactobacilli Ashdophilus RP32 (a: uncoated Lactobacilli Ashdophilus RP32, b: monolithic dry-coated) Lactobacillus ashidophyllus RP32, c: Fructo-oligosaccharides and hydrophobic double-coated Lactobacillus ashidophilus RP32, scale bar: 5 micrometers)

When the shape of the untreated control was observed, there was no change in the exterior of the lactobacillus strain, but when the cross-section was observed after encapsulating Lactobacillus ashdophilus RP32 using the coating material, the strains, The covering material surrounding was able to be confirmed visually. In addition, the cross-sectional view of the double dry coating strain showed a thicker portion surrounding the strains due to the double coating effect of the prebiotic substrate and the coating material, unlike the control and the single coating treatment.

Example 5 Determination of Resistance to Acids, Salts and Heat

The gastric juice was pretreated from 0.25M potassium chloride-hydrochloric acid buffer (pH 1.5) to which 1000 units / mL of pepsin was added according to a slightly modified method of the method previously described (Kobayashi et al., 1974). Lactobacillus ashidophilus RP32 (10 ⁹ CFU / g) with or without coating was inoculated into artificial gastric juice and then incubated at 37 ° C. for 0, 150, 300 minutes. Sequential dilutions of this culture (0.1% sterile peptone water) were inoculated into the MRS solid medium and viable counts were measured after 48 hours at 37 ° C. under anaerobic conditions. Flame resistance assessments have been carried out with some modification of previously established methods (Gardiner et al., 2000). Lactobacillus ashidophilus RP32 (10 ⁹ CFU g / L) by various coating treatments was inoculated in glycine-hydrochloric acid buffer (pH 1.5) added with 15% NaCl and 0, 60, 180 minutes at 37 ° C. Were incubated during. Viable counts were determined by the method described previously. In addition, for heat resistance experiments, Lactobacillus ashidophilus RP32 (10 ⁹ CFU g / L) was inoculated in glycine-hydrochloric acid buffer (pH 3.0) and 0, 40, 120, 180, and 240 at 55 ° C. The culture was vibrated in a room temperature bath for minutes. The coating material was removed in 0.1 M phosphate buffer (pH 7.0) and viable counts were measured by the previously described method.

Figure 3 shows the viability of dry-coated Lactobacillus ashidophilus RP32 after exposure to acidic conditions for various periods of time (black bars: 0 hours, gray bars 2.5 hours, black gray bars: 5 hours). No significant difference was observed up to 2.5 hours when compared to the control, single-coated, and double-coated treatments using three prebiotic substrates, but after five-hour incubation, the double-coated treatments were compared to the control and single-coated treatments. Significantly high resistance was observed.

Figure 4 shows the viability of dry coated Lactobacilli Ashdophyllus RP32 after exposure to saline conditions at various time periods (black bars: 0 hours, gray bars: 1 hour, black gray bars: 3 hours). At 15% salt concentration, no significant degradation of lactic acid bacteria was observed before and after coating. Therefore, the double coating conditions used in the experiment did not cause physical damage such as damage of lactic acid cell membrane.

On the other hand, Figure 5 shows the heat resistance of dry-coated Lactobacillus ashidophilus RP32 after exposure to 55 ° C. conditions (-●-: uncoated cells,-○-: single dry-coated cells). ,-▼-: cells double-coated with fructooligosaccharide,-□-: cells double-coated with lactulose,-■-: cells double-coated with raffinose). Interestingly, the reduction of viable cell counts compared to the initial cell number after heat treatment showed significant difference between control, single and double coated treatments after 120 minutes, and the thermal stability of double-coated strains was excellent. In case of long-term heat treatment up to 240 minutes, the number of bacteria between control and treatment was significantly different.

Example 6 Evaluation of Viability According to Various Storage Temperatures

For evaluation of the viability according to various storage temperatures of coated or uncoated Lactobacillus ashidophilus RP32, samples were placed in each sterile polyethylene tube and then stored at 4, 25, and 37 ° C. for 20 days. It was. Each sample was taken at planned storage intervals (0, 3, 7, 12, 16, and 20 days) and cell viability was measured using standard inoculation techniques on MRS solid medium.

FIG. 6 shows the survival of double dry-coated Lactobacillus ashidophilus RP32 and the effects of various prebiotic substrates at various storage temperature conditions. (a: Growth curve of single dry-coated Lactobacillus ashidophilus RP32, b: Dry-coated Lactobacillus ashidophyllus RP32, doubled with sureteric and fructooligosaccharides, c: Lactulose, d: Lactobacillus ashidophilus RP32 double-coated with raffinose). Interestingly, no differences were observed between treatments when stored at 4 and 25 ° C, but treatments double-coated with lactulose and raffinose at 37 ° C storage compared to 1-2 log ( log survival difference was observed.

[references]

1 shows scanning electron micrographs (SEM) of single and double dry coated Lactobacilli Ashdophilus RP32.

FIG. 2 shows transmission electron micrographs (TEM) of dry coated Lactobacilli Ashdophilus RP32.

3 shows the viability of dry-coated Lactobacillus ashidophilus RP32 after exposure to acidic conditions for various periods of time.

4 shows the viability of dry coated Lactobacilli Ashdophyllus RP32 after exposure to saline conditions at various time periods.

FIG. 5 shows the heat resistance of dry-coated Lactobacillus ashidophilus RP32 after exposure to 55 ° C. conditions.

FIG. 6 shows the survival of double dry-coated Lactobacillus ashidophilus RP32 and the effects of various prebiotic substrates at various storage temperature conditions.

Claims (7)

Double microcoating method of lactic acid bacteria to improve acid resistance and heat resistance comprising the following steps: a) culturing the lactic acid bacteria and then making the freeze-dried cells; b) the weight ratio of the freeze-dried cells to the prebiotic substrate is 7: 1 to 11: 1 (w / w) using a hybridization system, the speed of the rotating cylinder is 12,000 to 18,000 rpm, the operating temperature is 20-30 ° C., and a hybridization time of 2-4 minutes to perform the first coating process of the freeze-dried cells with a prebiotic substrate; And c) performing a secondary coating process on the primary coated cells in an enteric coating material. The method of claim 1, wherein the lactic acid bacteria is characterized in that the Ashdophilus lactic acid bacteria. The method according to claim 1, wherein the pellets obtained by centrifuging the lactic acid bacteria cultured in step a) are suspended in skim milk containing lactose and dried. delete The method of claim 1, wherein the prebiotics substrate is fructooligosaccharide (FOS), lactulose, or raffinose. The method of claim 1 wherein the enteric coating material is Sureteric. The double micro-coated lactic acid bacteria improved acid resistance and heat resistance prepared by the method according to any one of claims 1 to 3, 5 or 6.

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