KR101927859B1 - Method for improving the stability and coating efficiency of probiotics using ultrasonic wave after freeze-drying and food composition containing freeze-dried powder of probiotics prepared thereby as effective component - Google Patents

Abstract

The present invention relates to a method for increasing the stability and coating efficiency of probiotics using ultrasound, and a food composition containing the probiotics lyophilized powder manufactured by the method as an active ingredient. The ultrasonic treatment is performed at the step after completing the cultivation of the probiotics or after mixing with the coating, thereby enhancing the stability of the probiotics and the coating efficiency. The survival rate at low or high temperature of the powder of the probiotics at step of the lyophilization and after the lyophilization is enhanced, and the survival rate in the digestive tracts is increased to increase the intestine reaching rate in vivo, thereby exhibiting the functionality as probiotics.

Description

TECHNICAL FIELD The present invention relates to a method for improving the stability and coating efficiency of probiotics using ultrasound, and a food composition containing the probiotics lyophilized powder prepared by the method as an effective ingredient. food composition containing freeze-dried powder of probiotics prepared thereby as effective component}

The present invention relates to a method for increasing the stability and coating efficiency of probiotics using ultrasonic waves and a food composition containing the freeze-dried powder of probiotics prepared by the method as an active ingredient. In the process of culturing and freeze-drying the probiotics, And a method of increasing the stability of the probiotics and the coating efficiency by treating the freeze-dried powder with a wavelength, and a food composition containing the freeze-dried powder of probiotics prepared by the method as an active ingredient.

Probiotics is a probiotic agent that improves intestinal microflora and thus has a beneficial effect on host animals. Fuller defines it in 1989 and has published in 2001 the 'Probiotics' FAO / WHO definition of 'living microorganisms' is widely used. In addition, Salminen et al. In 1999 defined "microbial agents or components of microorganisms that have beneficial effects on the host" and have extended the range of probiotics from live cells to dead cells. As research findings and scientific data suggest that human intestinal microorganisms, including probiotics, have a significant impact on human health, consumers are increasingly aware of probiotics and the demand for probiotic products is increasing. Lactobacillus 11 species ( L. acidophilus, L. casei, L. gasseri, L. delbruekii subsp. Bulgaricus, L. helveticus, L. fermentum, L. paracasei, L. plantarum, L Lactobacillus reuteri, L. rhamnosus and L. salivarius ), Lactococcus 1 ( Lc. lactis ), Enterococcus 2 ( E. faecium, E. faecalis ), Streptococcus 1 the S. thermophilus), Bifidobacterium (Bifidobacterium) 4 jong (B. bifidum, B. breve, B. longum, B. animalis subsp. was announced as probiotics with respect to the 19 kinds of strains to lactis) many companies are probiotics Research and sell products.

The functionalities of representative probiotics include various functions such as suppression of harmful bacteria, proliferation of beneficial bacteria, improvement of function of bowel movement, and various other researches and clinical results. However, these probiotics are also composed of proteins because the strains themselves contain proteins, and when they are put into the body, the membranes are damaged by stomach acid and bile acid, and thus they do not show the original functionality of probiotics. Therefore, in order to overcome these problems, in the industry producing probiotics, a high concentration of strains is added or the product is coated by an additional process. In particular, the coating technique by the injection of a high concentration of the strain increases the price of the product It is not only a great burden to consumers but also a problem that side effects can occur if more than one daily intake of live cells is consumed.

In addition, the additional coating process has the disadvantage that it is difficult to secure the stability of the probiotics as the cost of the product increases due to expensive facilities and coatings, and additional processes are added.

On the other hand, Korean Patent Registration No. 10-1605516 (Mar. 23, 2016) discloses a method of increasing the survival rate, storage stability, acid resistance and biliary cholesterol after lyophilization of lactic acid bacteria by adding proline during culturing of lactic acid bacteria or culturing of lactic acid bacteria There is a problem that the cost is increased or the taste is affected.

Accordingly, the inventors of the present invention found that when ultrasound-induced stress is applied to the probiotics in the course of culturing and freeze-drying the probiotics, survival rate and stability of the probiotics are enhanced, coating agents are uniformly dispersed, coating efficiency is improved, and distribution stability of probiotics is improved Thereby completing the present invention.

Korean Registered Patent No. 10-1605516 (Mar. 23, 201)

It is an object of the present invention to provide a method for increasing the stability and coating efficiency of probiotics by treating ultrasound during cultivation and lyophilization of probiotics.

Another object of the present invention is to provide a fermented milk containing a lyophilized powder of probiotics prepared by a method of increasing the stability and coating efficiency of probiotics by treating ultrasonic waves during cultivation and lyophilization of probiotics, And to provide a food composition such as a beverage.

In order to accomplish the above object, the present invention provides a method for culturing a probiotic culture, comprising the steps of: (a) (b) concentrating the ultrasound-treated probiotic culture solution of step (a) to prepare a probiotic concentrate; (c) adding a coating agent to the probiotic concentrate of step (b); And (d) lyophilizing the probiotic concentrate obtained in the step (c). The lyophilized powder of probiotics prepared by the method and the method for enhancing the stability of the probiotics and the coating efficiency A health functional food, a functional beverage, and the like.

The present invention also provides a method for producing a probiotic concentrate, comprising the steps of: (a) preparing a probiotic concentrate by concentrating the probiotic culture; (b) treating ultrasound while adding a coating agent to the probiotic concentrate of step (a); And (c) lyophilizing the ultrasound-treated probiotic concentrate of step (b) (see FIG. 2) and a lyophilized powder of probiotics prepared by the method The present invention provides a food composition comprising fermented milk, health functional food, functional beverage and the like as an effective ingredient.

The present invention also provides a method for producing a probiotic culture, comprising the steps of: (a) (b) concentrating the ultrasound-treated probiotic culture solution of step (a) to prepare a probiotic concentrate; (c) treating ultrasound while adding a coating agent to the probiotic concentrate of step (b); And (d) lyophilizing the ultrasound-treated probiotic concentrate obtained in step (c) (see FIG. 3) and a lyophilized powder of probiotics prepared by the method. The present invention provides a food composition comprising fermented milk, health functional food, functional beverage and the like as an effective ingredient.

Hereinafter, the present invention will be described in detail.

Probiotics according to the present invention are useful as Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp. Lactic acid bacteria, Lactobacillus sp. Lactobacillus plantarum , Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus gasseri , Lactobacillus delbrueckii ssp, Lactobacillus delbrueckii ssp, Lactobacillus sp. . bulgaricus), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus buffer momentum (Lactobacillus fermentum), Lactobacillus para casei (Lactobacillus paracasei), Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus ruteri (Lactobacillus reuteri), Lactobacillus Lactococcus lactis , Streptococcus thermophilus , Bifidobacterium longum , Bifidobacterium bifidum , Bifidobacterium breve , Bifidobacterium spp ., Bifidobacterium spp ., Bifidobacterium spp . And Bifidobacterium animalis ssp. Lactis strains.

The cultivation of the probiotics according to the present invention is carried out under conventionally known conventional lactic acid bacteria culture medium and culture conditions such as MRS broth medium.

The conventionally known conventional centrifugation method is used as a method for preparing the probiotic concentrate by concentrating the probiotic culture solution according to the present invention.

The method of lyophilizing the probiotic concentrate according to the present invention uses a conventionally known conventional freeze-drying method.

The coating agent according to the present invention can be used in combination with chitosan, malto-dextrin, indigestible dextrin, xanthan gum, XG, guar gum, carboxymethylcellulose, cellulose, CMC), hydroxyethylcellulose (HEC), polyvinylpyrrolidone (PVP), carbopol, sodium alginate, propylene glycol alginate, Alginate, polyethyleneglycol (PEG), triacetin, propylene glycol, acetyl triethyl citrate or triethyl citrate may be used, The cryoprotectant may be skimmed milk powder, fructooligosaccharide, trehalose, maltodextrin or glycerin.

The ultrasound treatment according to the present invention was performed by applying ultrasound waves of 100 Hz, 200 Hz, 300 Hz, 600 Hz, and 1000 Hz respectively for 1 minute, 2 minutes, 5 minutes, and 1 hour, respectively, in order to apply environmental stress to the extent that the probiotics are not killed. 7 minutes, 10 minutes.

In order to improve the survival rate and stability of probiotics by providing ultrasonic stress to the extent that the probiotics do not kill, and to improve the distribution stability of the probiotics by increasing the coating efficiency by dispersing the coating agent evenly, the ultrasonic treatment is performed by: i) Adding the coating agent to the probiotic concentrate, and iii) adding the coating agent to the probiotic culture solution and the probiotic concentrate.

Also, the food composition containing the lyophilized powder of probiotics prepared by the method of increasing the stability and coating efficiency of the probiotics by treating the ultrasonic wave in the culturing and lyophilizing process of the probiotics according to the present invention, , Drinks, beverage additives, fermented milk, health functional foods, and the like. When used as a food, a food additive, a beverage, a beverage additive, or a health functional food, it is possible to use various foods, fermented milk, meat, beverage, chocolate, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, But may be, but not limited to, a combination, a mainstream and other health functional food.

In particular, the fermented milk containing the lyophilized powder of probiotics prepared by the method of increasing the stability and coating efficiency of the probiotics by processing the ultrasound in the culturing and lyophilizing process of the probiotics according to the present invention is characterized in that the lyophilized powder of probiotics , Lactic acid bacteria culture solution and mixed juice syrup are mixed at a certain ratio, homogenized at 150 bar, cooled to below 10 ° C, and packaged in a container to prepare fermented milk.

The functional beverage containing the lyophilized powder of probiotics prepared by the method of increasing the stability of the probiotics and the coating efficiency by treating the ultrasound in the culturing and lyophilizing process of the probiotics according to the present invention can be used as a mixed nutritional syrup, The lyophilized powder of probiotics and water are mixed at a constant ratio, homogenized at 150 bar, cooled to below 10 ° C, and packaged in small containers such as glass bottles and plastic bottles to produce functional beverages.

In addition, the health functional food containing the lyophilized powder of probiotics prepared by the method of increasing the stability and coating efficiency of the probiotics by processing the ultrasonic wave in the culturing and lyophilizing process of the probiotics according to the present invention, In addition to containing dry powder, vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic amide, oligosaccharide, etc. may be added as nutritional supplement components and other food additives may be added.

The present invention relates to a method for culturing and freeze-drying a probiotic, comprising the steps of: i) adding a coating agent to a probiotic culture solution, ii) adding a coating agent to the probiotic concentrate, and iii) adding a coating agent to the probiotic culture solution and the probiotic concentrate, Dried fruit of the probiotic lyophilized powder as an active ingredient by increasing the coating efficiency, improving the survival rate at low or high temperature of the lyophilized powder of probiotics after freeze-drying and freeze-drying, and increasing the survival rate of the digestive tract, Health functional foods, and functional beverages.

1 is a schematic view showing a lyophilization process of a probiotic to be subjected to ultrasonic treatment in the culture medium of probiotics of the present invention.
2 is a schematic view showing a lyophilization process of a probiotic to be subjected to ultrasonic treatment in the process of adding a coating agent to the probiotic concentrate of the present invention.
FIG. 3 is a schematic view showing a lyophilization process of a probiotic to be subjected to ultrasonic treatment in a process of adding a coating agent to the probiotic culture solution and the probiotic concentrate of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following embodiments are not intended to limit the scope of the present invention, and ordinary variations by those skilled in the art are possible within the scope of the technical idea of the present invention.

≪ Example 1 >

1-1. After cultivation, lyophilized Lactobacillus plantarum HY7715 lyophilized powder was prepared.

Lactobacillus plantarum HY7715 was cultivated in an MRS broth medium and subjected to a constant wavelength of 100 Hz for 1 minute, 2 minutes, 5 minutes, 7 minutes, and 10 minutes, respectively, in order to apply primary environmental stress Was subjected to ultrasonic treatment. The Lactobacillus plantarum HY7715 culture supernatant was subjected to centrifugation at 8,000 rpm for 15 minutes. The Lactobacillus plantarum HY7715 concentrate was coated with 10 g of pressure-sterilized % Of skimmed milk powder was mixed at a weight ratio of 1: 1, and then freeze-dried powder was prepared by freezing at -70 ° C for 6 hours.

1-2. Preparation of freeze-dried powder of Bifidobacterium longum HY8001 ultrasonicated after incubation

Except that Bifidobacterium longum HY8001 was used in place of Lactobacillus plantarum HY7715 of Example 1-1.

1-3. Manufacture of freeze-dried powder of high-quality probiotic after ultrasonication after culturing

Each of the probiotics of Table 1 below was cultured in an MRS broth medium and subjected to ultrasonic treatment at a constant wavelength of 100 Hz for 5 minutes in order to apply primary environmental stress. Each of the probiotic cultures, which had been subjected to the ultrasonication treatment, were centrifuged at 8,000 rpm for 15 minutes, and each of the obtained probiotic concentrates was coated with an aqueous solution containing pressurized sterilized 10% by weight of skimmed milk powder as a coating agent and a cryoprotectant at a weight ratio of 1: , And then freeze-dried powder was prepared by freezing at -70 ° C for 6 hours.

≪ Example 2 >

2-1. Preparation of lyophilized Lactobacillus plantarum HY7715 lyophilized powder after coating

Lactobacillus Planta room (Lactobacillus plantarum) a Lactobacillus Planta room (Lactobacillus plantarum) Lactobacillus Planta room (Lactobacillus plantarum) obtained by centrifugation for 15 minutes at 8,000rpm HY7715 culture solution cultured in MRS broth medium HY7715 HY7715 For the concentrate, 1: 1, 2: 5, 5: 1, 1, 2, 3, 4 and 5 minutes were added to the concentrate in order to apply primary stress to the environment while mixing the coating solution and the aqueous solution containing pressurized sterilized 10% Min, 7 min, and 10 min with a constant wavelength of 100 Hz. Then, a lyophilized powder was prepared after freezing at minus 70 ° C for 6 hours.

2-2. Preparation of freeze-dried powder of Bifidobacterium longum HY8001 ultrasonically treated after coating

The procedure of Example 2-1 was repeated except that Bifidobacterium longum HY8001 was used instead of Lactobacillus plantarum HY7715 of Example 2-1.

2-3. Manufacture of lyophilized freeze-dried powder with ultrasonic treatment after coating

Each of the probiotics cultured in the MRS broth medium of each of the following probiotics shown in the following Table 1 was centrifuged at 8,000 rpm for 15 minutes, and each of the obtained probiotic concentrates was coated with 10% by weight of pressurized sterilized Ultrasonic treatment was performed at a constant wavelength of 100 Hz for 5 minutes in order to apply primary environmental stress while mixing the aqueous solution containing powdered milk at a weight ratio of 1: 1. Then, a lyophilized powder was prepared after freezing at minus 70 ° C for 6 hours.

≪ Example 3 >

3-1. Preparation of freeze-dried powder of Lactobacillus plantarum HY7715 complexed with ultrasound

Lactobacillus plantarum HY7715 was cultivated in an MRS broth medium and subjected to a constant wavelength of 100 Hz for 1 minute, 2 minutes, 5 minutes, 7 minutes, and 10 minutes, respectively, in order to apply primary environmental stress Was subjected to ultrasonic treatment. The Lactobacillus plantarum HY7715 culture supernatant was subjected to centrifugation at 8,000 rpm for 15 minutes. The Lactobacillus plantarum HY7715 concentrate was coated with 10 g of pressure-sterilized % Of an aqueous solution containing skimmed milk powder at a weight ratio of 1: 1 During the mixing, ultrasonic treatment was applied at a constant wavelength of 100 Hz for 1, 2, 5, 7, and 10 minutes to add secondary environmental stresses. Then, a lyophilized powder was prepared after freezing at minus 70 ° C for 6 hours.

3-2. Preparation of freeze-dried powder of Bifidobacterium longum HY8001 complex-treated with ultrasound

Was prepared in the same manner as in Example 3-1 except that Bifidobacterium longum HY8001 was used instead of Lactobacillus plantarum HY7715 of Example 3-1.

3-3. Manufacture of freeze-dried powder of high-quality probiotics combined with ultrasound

Each of the probiotics of the following Table 1 was cultured in an MRS broth medium, and each of the probiotic cultures was subjected to ultrasonic treatment at a constant wavelength of 100 Hz for 5 minutes to apply primary environmental stress. Each of the probiotic cultures, which had been subjected to the ultrasonication treatment, were centrifuged at 8,000 rpm for 15 minutes, and each of the obtained probiotic concentrates was coated with an aqueous solution containing pressurized sterilized 10% by weight of skimmed milk powder as a coating agent and a cryoprotectant at a weight ratio of 1: , Ultrasonic treatment was performed at a constant wavelength of 100 Hz for 5 minutes in order to apply secondary environmental stress during mixing. Then, a lyophilized powder was prepared after freezing at minus 70 ° C for 6 hours.

Test strain L. acidophilus HY7036 L. casei HY2782 L. gasseri HY7023 L. delbrueckii ssp. bulgaricus HY7901 L. helveticus HY7801 L. fermentum HY7301 L. paracasei HP7 L. rhamnosus HY1213 L. reuteri HY7501 Lc. lactis HY449 S. thermophilus HY9012 B. bifidum HY8308 B. breve HY8201 B. animalis ssp. lactis HY8002

<Example 4>

4-1. Preparation of fermented milk containing the freeze-dried powder of probiotic ultrasonicated after cultivation as an active ingredient

The lactic acid bacteria culture solution was prepared by stirring 95.36% by weight of crude oil and 4.6% by weight of skimmed milk powder (or mixed powdered milk) and having a specific gravity of 1.0473 to 1.0475 at 15 ° C, a titratable acidity of 0.200 to 0.220%, a pH of 6.55 to 6.70, (Brix ⁰ ) was about 16.3 to 16.5%. After mixing, the mixture was heat-treated with UHT (sterilized at 135 ° C. for 2 seconds) and cooled to an appropriate temperature. Streptococcus thermophilus and lactoseolytic enzyme (Valley laboratory, USA) were added at 0.02 wt% Was 1.0 X 10 ⁹ cfu / ml or more, the titratable acidity was 0.89 to 0.91%, and the pH was 4.55 to 4.65.

The mixed juice syrup was prepared by mixing 13 wt% of liquid fructose, 5 wt% of white sugar, 10.9 wt% of concentrated juice concentrate 56Brix ⁰ , 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence and 70 wt% of purified water, UHT heat treatment (sterilization at 135 캜 for 2 seconds) and cooling.

The 69% by weight of the lactic acid bacteria culture solution was homogenized at 150 bar in combination with 0.1% by weight of the freeze-dried powder of the probiotics of Example 1 and 30.4% by weight of the mixed fruit juice syrup and then cooled to 10 ° C or less to obtain a freeze- To prepare a fermented milk.

4-2. Preparation of fermented milk containing the freeze-dried powder of ultrasonic treated probiotics as an active ingredient after coating

The lyophilized powder of probiotics of the present invention was used as the active ingredient in the same manner as in Example 4-1 except that the lyophilized powder of probiotics of Example 2 was used instead of the lyophilized powder of probiotic of Example 1, .

4-3. Preparation of fermented milk containing freeze-dried powder of probiotic complex treated with ultrasound as an active ingredient

The lyophilized powder of probiotics of the present invention was used as the active ingredient in the same manner as in Example 4-1 except that the lyophilized powder of probiotics of Example 3 was used instead of the lyophilized powder of probiotic of Example 1, .

&Lt; Example 5 >

5-1. Preparation of a functional beverage containing an ultrasound-treated freeze-dried powder of probiotics as an active ingredient after culturing

Mixing juice syrup was 13% by weight of liquid fructose, white sugar, 2.5% by weight, 2.5% by weight brown sugar, mix juice concentrate 56Brix ⁰ 10.9% by weight of the pectin 1.0% by weight, fresh fruit mix Essence 0.1% by weight and pure water 70 wt% 35 ℃ Followed by UHT heat treatment (sterilization at 135 캜 for 2 seconds), followed by cooling.

Then, 30.4% by weight of the mixed fruit juice syrup prepared by the above method, 0.1% by weight of the lyophilized powder of Example 1, and 69.5% by weight of the remaining purified water were combined and homogenized at 150 bar, , PET bottles, etc., to prepare functional beverages containing the probiotics lyophilized powder of the present invention as an active ingredient.

5-2. Preparation of a functional beverage containing the lyophilized powder of ultrasonic treated probiotics as an active ingredient after coating

The lyophilized powder of probiotics of the present invention was used as the active ingredient and the lyophilized powder of the probiotics of Example 2 was used in the same manner as in Example 5-1 except that the lyophilized powder of probiotics of Example 2 was used instead of the lyophilized powder of probiotics of Example 1, A beverage was prepared.

5-3. Preparation of a functional beverage containing as an active ingredient a lyophilized powder of probiotics complex-treated with ultrasound

The lyophilized powder of probiotics of the present invention was used as the active ingredient in the same manner as in Example 5-1 except that the lyophilized powder of probiotics of Example 3 was used instead of the lyophilized powder of probiotic of Example 1, A beverage was prepared.

&Lt; Example 6 >

6-1. Preparation of Health Functional Foods Containing Ultrasonically Treated Frost-Dried Powder as an Active Ingredient After Cultivation

The nutritional supplement components (vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic acid amide) and oligosaccharide were added to 0.1% by weight of the lyophilized powder of the probiotics of Example 1 by lyophilization Was added in an amount of 10 parts by weight based on 100 parts by weight of the powder and mixed in a high-speed rotary mixer. 10 parts by weight of sterilized purified water was added to the mixture, mixed and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum dryer at 50 ° C and then passed through 12 to 14 mesh to uniformly produce granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.

6-2. Preparation of Health Functional Foods Containing Ultrasonically Treated Frost-Dried Powder as an Active Ingredient After Coating

The lyophilized powder of probiotics of the present invention was used as an active ingredient in the same manner as in Example 6-1 except that the lyophilized powder of probiotics of Example 2 was used instead of the lyophilized powder of probiotic of Example 1, Functional foods were prepared.

6-3. Preparation of Health Functional Foods Containing Probiotics Freeze-Dried Powder Combined with Ultrasound as an Active Ingredient

The lyophilized powder of probiotics of the present invention was used as an active ingredient in the same manner as in Example 6-1 except that the lyophilized powder of probiotics of Example 3 was used instead of the lyophilized powder of probiotics of Example 1, Functional foods were prepared.

&Lt; Test Example 1 >

Accelerated stability test of ultrasonic treated probiotics after incubation

100% weight of the lyophilized powder of probiotics of Examples 1-1 and 1-2 was packed in a stick and kept at a temperature of 40 ° C and a humidity of 70% in order to examine the optimal ultrasonic treatment conditions based on the storage stability of the probiotics during long- The survival rate of probiotics was calculated by accelerated test for 4 weeks.

Control groups were tested in the same manner as above using 100% by weight of lyophilized probiotic powder prepared in the same manner as in Example 1-1 and Example 1-2, except that the ultrasonic treatment was not performed after the cultivation of the probiotics.

The 'survival rate' means the percentage of viable cells counted after the accelerated stability test divided by the number of viable cells before the accelerated stability test.

The results are shown in Table 2 below.

As can be seen in the following Table 2, the survival rate of the control group Lactobacillus plantarum HY7715 was 25.3% and the survival rate of the control group Bifidobacterium longum HY8001 was 20.0% Showed a higher survival rate than the control group regardless of the time of ultrasound treatment.

The survival rate of Lactobacillus plantarum HY7715 in the experimental group with the highest survival rate was 5 minutes (survival rate: 55.1%), 7 minutes (survival rate: 54.1%), 10 minutes (survival rate: 54.5% And high survival rate. Experimental group treated with ultrasound for 5 minutes showed the highest survival rate under accelerated conditions.

The survival rate of Bifidobacterium longum HY8001 in the experimental group with the highest survival rate was 5 minutes (survival rate: 47.1%), 7 minutes (survival rate: 45.5%), 10 minutes (survival rate: 43.5% And high survival rate. Experimental group treated with ultrasound for 5 minutes showed the highest survival rate under accelerated conditions.

&Lt; Test Example 2 &

Freeze-dried survival rate test of ultrasonicated high-quality probiotics after culture

In order to confirm the survival rate of the probiotics under the ultrasound treatment conditions (5 min, 100 Hz) showing the highest survival rate in Test Example 1 during the freezing of the ultrasound-treated probiotics after culturing, the freeze-drying of the probiotics prepared by the method of Example 1 Survival rate was calculated.

The 'survival rate' means the percentage of viable cells after lyophilization divided by the number of viable cells before lyophilization.

The control group was a freeze-dried powder of probiotic prepared in the same manner as in Example 1, except that the ultrasonic treatment was not performed after the cultivation of the probiotics.

The results are shown in Table 3 below.

As can be seen in Table 3, the survival rate of Lactobacillus plantarum HY7715 was about 16% higher than that of the control group, and the survival rate of Bifidobacterium longum HY8001 was about 4 %.

In addition, survival rates of other high-quality probiotics (experimental group) showed a survival rate of at least 9% and up to 28%, and survival rates were improved compared to control groups showing at least 22% and up to 45% survival rate.

Strain name Control group Experimental group L. acidophilus HY7036 63% 85% L. casei HY2782 74% 86% L. gasseri HY7023 69% 80% L. delbrueckii ssp. bulgaricus HY7901 55% 91% L. helveticus HY7801 68% 77% L. fermentum HY7301 70% 72% L. paracasei HP7 75% 91% L. plantarum HY7715 70% 86% L. rhamnosus HY1213 72% 79% L. reuteri HY7501 63% 80% Lc. lactis HY449 66% 81% S. thermophilus HY9012 69% 84% B. bifidum HY8308 78% 82% B. breve HY8201 66% 86% B. longum HY8001 72% 76% B. animalis ssp. lactis HY8002 70% 88%

&Lt; Test Example 3 >

Accelerating stability test of ultrasonic treated probiotics after coating

100% weight of the lyophilized probiotics of Examples 2-1 and 2-2 was packed in a stick pack, and the temperature was 40 ° C and the humidity was 70%. In order to examine the optimal ultrasonic treatment conditions based on the storage stability of the probiotics during long- The survival rate of probiotics was calculated by accelerated test for 4 weeks.

The control group was prepared and used in the same manner as in Examples 2-1 and 2-2 except that the ultrasonic treatment was not performed after the cultivation of the probiotics.

The 'survival rate' means the percentage of viable cells counted after the accelerated stability test divided by the number of viable cells before the accelerated stability test.

The results are shown in Table 4 below.

As shown in the following Table 4, the survival rate of the control group Lactobacillus plantarum HY7715 was 24.1% and the survival rate of the control group Bifidobacterium longum HY8001 was 18.9% Showed a higher survival rate than the control group regardless of the time of ultrasound treatment.

The survival rate of Lactobacillus plantarum HY7715 in the experimental group with the highest survival rate was 5 minutes (survival rate: 63.3%), 7 minutes (survival rate: 60.2%) and 10 minutes (survival rate: 60.3% And high survival rate. Experimental group treated with ultrasound for 5 minutes showed the highest survival rate under accelerated conditions.

The survival rate of Bifidobacterium longum HY8001 in the experimental group with the highest survival rate was 5 minutes (survival rate: 55.3%), 7 minutes (survival rate: 52.7%) and 10 minutes (survival rate: 52.5% And high survival rate. Experimental group treated with ultrasound for 5 minutes showed the highest survival rate under accelerated conditions.

<Test Example 4>

Freeze-dried survival rate test of ultrasonic treated high quality probiotics after coating

In order to confirm the survival rate of the probiotics under the ultrasound treatment conditions (5 min, 100 Hz) showing the highest survival rate in Test Example 3 in the freezing process of the ultrasound-treated probiotics after coating, the freeze- Survival rate was calculated.

The 'survival rate' means the percentage of viable cells after lyophilization divided by the number of viable cells before lyophilization.

The control group was a freeze-dried powder of probiotic prepared by the same method as in Example 2, except that the coating was not subjected to ultrasonic treatment.

The results are shown in Table 5 below.

As can be seen in Table 5, the survival rate of Lactobacillus plantarum HY7715 was about 23% higher than that of the control group, and the survival rate of Bifidobacterium longum HY8001 was about 20 %.

In addition, survival rates of other high-quality probiotics (experimental group) showed a survival rate of at least 2% and up to 17%, indicating that the survival rate was improved compared to the control group showing a survival rate of at least 21% to 47%.

Strain name Control group Experimental group L. acidophilus HY7036 66% 87% L. casei HY2782 78% 93% L. gasseri HY7023 67% 86% L. delbrueckii ssp. bulgaricus HY7901 53% 92% L. helveticus HY7801 71% 98% L. fermentum HY7301 72% 92% L. paracasei HP7 79% 95% L. plantarum HY7715 74% 97% L. rhamnosus HY1213 71% 85% L. reuteri HY7501 62% 83% Lc. lactis HY449 62% 86% S. thermophilus HY9012 73% 89% B. bifidum HY8308 75% 88% B. breve HY8201 63% 89% B. longum HY8001 71% 91% B. animalis ssp. lactis HY8002 70% 90%

&Lt; Test Example 5 >

Accelerating stability test of ultrasound-combined probiotics

100% weight of the lyophilized probiotics of Examples 3-1 and 3-2 was packed in a stick and kept at a temperature of 40 ° C and a humidity of 70% in order to examine the optimal ultrasonic treatment conditions based on the storage stability of the probiotics during long- The survival rate of probiotics was calculated by accelerated test for 4 weeks.

The control group was prepared and used in the same manner as in Examples 3-1 and 3-2 except that the ultrasonic treatment was not performed after the cultivation of the probiotics.

The 'survival rate' means the percentage of viable cells counted after the accelerated stability test divided by the number of viable cells before the accelerated stability test.

The results are shown in Table 6 below.

As can be seen in the following Table 6, the survival rate of Lactobacillus plantarum HY7715 in the control group was 23.7% and the survival rate of the control group Bifidobacterium longum HY8001 was 18.6% Showed a higher survival rate than the control group regardless of the time of ultrasound treatment.

The survival rate of Lactobacillus plantarum HY7715 in the experimental group with the highest survival rate was 5 minutes (survival rate: 60.1%), 7 minutes (survival rate: 58.1%), 10 minutes (survival rate: 57.3% And high survival rate. Experimental group treated with ultrasound for 5 minutes showed the highest survival rate under accelerated conditions.

The survival rate of Bifidobacterium longum HY8001 in the experimental group with the highest survival rate was 5 minutes (survival rate: 52.1%), 7 minutes (survival rate: 51.5%), 10 minutes (survival rate: 49.9% And high survival rate. Experimental group treated with ultrasound for 5 minutes showed the highest survival rate under accelerated conditions.

&Lt; Test Example 6 >

Freeze-dried survival rate test of high-quality probiotics combined with ultrasound

In order to confirm the survival rate of probiotics under ultrasound treatment conditions (5 min, 100 Hz) showing the highest survival rate in Test Example 1 and Test Example 3 in the freezing process of the ultrasonic treated probiotics after culturing and coating, Freeze-dried survival rate of probiotics was calculated.

The 'survival rate' means the percentage of viable cells after lyophilization divided by the number of viable cells before lyophilization.

The control group was a freeze-dried powder of probiotic prepared by the same method as in Example 3, except that after the incubation and after the coating, the ultrasonic treatment was not performed.

The results are shown in Table 7 below.

As can be seen in Table 7, the survival rate of Lactobacillus plantarum HY7715 was about 10% higher than that of the control group, and the survival rate of Bifidobacterium longum HY8001 was about 10 %, And the survival rate of other prognostic probiotics was higher than the survival rate of the control group.

Strain name Control group Experimental group L. acidophilus HY7036 64% 81% L. casei HY2782 76% 85% L. gasseri HY7023 65% 86% L. delbrueckii ssp. bulgaricus HY7901 53% 91% L. helveticus HY7801 69% 82% L. fermentum HY7301 69% 88% L. paracasei HP7 74% 87% L. plantarum HY7715 73% 83% L. rhamnosus HY1213 75% 77% L. reuteri HY7501 66% 80% Lc. lactis HY449 63% 86% S. thermophilus HY9012 67% 89% B. bifidum HY8308 75% 82% B. breve HY8201 64% 86% B. longum HY8001 74% 84% B. animalis ssp. lactis HY8002 66% 90%

&Lt; Test Example 7 >

Distribution stability test of probiotics finished product

In order to confirm the stability in the circulation process of the finished probiotics, 35 parts by weight of dietary fiber, 10 parts by weight of oligosaccharide, 25 parts by weight of flavor powder and 10 parts by weight of lactose mixed powder 10 were mixed with 10 parts by weight of lyophilized powder of each of the probiotics of Examples 1 to 3, (25 ° C., humidity: 40 to 60%) and accelerated (40 ° C., humidity: 70 ° C., humidity: 40% or less) %) For 6 months. The survival rate of probiotics was calculated.

The 'survival rate' means the percentage of viable cell count after distribution stability test divided by viable cell count before circulating stability test.

Control groups were tested in the same manner as described above, except that the final product prepared using the freeze-dried powder of probiotics prepared in the same manner as in Examples 1 to 3 was used, except that the ultrasonic treatment was not performed.

The results are shown in Table 8 below (refrigeration conditions), Table 9 (room temperature conditions) and Table 10 (acceleration conditions).

As can be seen from the following Tables 8 to 10, the survival rate in the case of refrigerated storage was not significantly different from that in the control group and in Examples 1 to 3. However, in the case of storage at room temperature, the survival rate of Lactobacillus plantarum HY7715 was about 13 to 15% higher than that of the control group, and the survival rate of Bifidobacterium longum HY8001 was about 13 to 15% higher than that of the control group. The survival rate of other probiotics was also higher than that of the control group And the survival rate of Lactobacillus plantarum HY7715 was about 15 to 16% higher than that of the control group in the case of accelerated storage and the survival rate of Bifidobacterium longum HY8001 was higher than that of the control group Survival rate was about 15 to 16% higher than other survival rates, and the survival rate of other prognostic probiotics was also higher in the survival rate Respectively.

<Test Example 8>

Probiotics survival rate test of probiotics

8-1. Preparation of probiotic samples

The lyophilized powder of probiotics of Examples 1 to 3 was dissolved in PBS buffer to a final concentration of 1 x ^{10 9} cfu / ml.

8-2. Preparation of Electrolyte Solution

A standardized static in vitro digestion method suitable for food? an international consensus (M. Minekus et al., Food Funct. 2014 (5): 1113-1124) was prepared as shown in Table 11 below.

division Simulated Salivary Fluid (SSF, mmol / L) Simulated Gastric Fluid
(SGF, mmol / L) Simulated Intestinal Fluid
(SIF, mmol / L) KCl 15.1 6.9 6.8 KH ₂ PO ₄ 3.7 0.9 0.8 NaHCO ₃ 13.6 25 85 NaCl - 47.2 38.4 MgCl ₂ (H ₂ O) ₆ 0.15 0.1 0.33 (NH _{4) 2} CO ₃ 0.06 0.5 - CaCl ₂ (H ₂ O) ₂ 1.5 (0.75) 0.15 (0.075) 0.6 (0.3)

8-3. Measurement of survival rate of digestive tract

In the oral phase, SSF (Simulated Salivary Fluid) electrolyte solution of Table 11 and α-amylase of saliva derived from human body were added to the probiotic sample prepared in Example 8-1 and reacted at 37 ° C for 2 minutes .

In the stomach, the SGF (Simulated Gastric Fluid) electrolyte solution and the pepsin-derived pepsin of Table 11 were added to the digested sample at the oral stage, and the pH was adjusted to 3.0, followed by reaction at 37 ° C for 2 hours.

In the small intestine stage, porcine derived pancreatin and bile acid were added to digested samples in the stomach, adjusted to pH 7.0, and reacted at 37 ° C for 2 hours.

In the final absorption step, Brush Border Membrane Vesicles were added to the digested samples in the small intestine, adjusted to pH 7.0, and reacted at 37 ° C for 4 hours.

The viable counts of the probiotics remaining after the digestion and absorption process were analyzed according to a conventional method for measuring viable count of probiotics. The survival rate of the strains was expressed as survival rate (%) as compared with the initial number of bacteria before digestion and absorption.

The control group was tested in the same manner as above except that the lyophilized powder of probiotics prepared in the same manner as in Examples 1 to 3 was used, except that the lyophilized powder was not subjected to ultrasonic treatment.

The results are shown in Table 12 below.

As shown in Table 12 below, the survival rate of the digestive tract of Lactobacillus plantarum HY7715 was 4% (Example 1), 7% (Example 2) and 9% (Example 3). The survival rate of the digestive tract of Bifidobacterium longum HY8001 was higher than that of the control group by 8% (Example 1), 14% (Example 2) and 13% (Example 3) The survival rate of the gut probiotics was higher than that of the control group.

Claims (7)

(a) treating ultrasound in a culture medium of Probiotics for 5 minutes at a condition of 100 Hz;
(b) concentrating the ultrasound-treated probiotic culture solution of step (a) to prepare a probiotic concentrate;
(c) adding a coating agent to the probiotic concentrate of step (b); And
(d) lyophilizing the probiotic concentrate of step (c)
The probiotics may be selected from the group consisting of Lactobacillus plantarum , Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus gasseri , Lactobacillus delbrueckii , eseueseu blood Bulgaria kusu (Lactobacillus delbrueckii ssp. bulgaricus), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus buffer momentum (Lactobacillus fermentum), Lactobacillus para casei (Lactobacillus paracasei), Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus reuteri , Lactococcus lactis , Streptococcus thermophilus , Bifidobacterium longum , Bifidobacterium bifidum , Bifidobacterium bifidum , Bifidobacterium bifidum , Bifidobacterium bifidum , Te Solarium breve (Bifidobacterium breve), BP gambling Terry Enimeol less eseueseu blood lactis (Bifidobacterium animalis ssp. Lactis) a method of increasing the reliability, and any one of the coating efficiency of the probiotics, characterized in that one.
(a) preparing a probiotic concentrate by concentrating a culture of probiotics;
(b) treating ultrasound for 5 minutes at 100 Hz condition while adding a coating agent to the probiotic concentrate of step (a); And
(c) lyophilizing the ultrasound-treated probiotic concentrate of step (b)
The probiotics may be selected from the group consisting of Lactobacillus plantarum , Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus gasseri , Lactobacillus delbrueckii , eseueseu blood Bulgaria kusu (Lactobacillus delbrueckii ssp. bulgaricus), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus buffer momentum (Lactobacillus fermentum), Lactobacillus para casei (Lactobacillus paracasei), Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus reuteri , Lactococcus lactis , Streptococcus thermophilus , Bifidobacterium longum , Bifidobacterium bifidum , Bifidobacterium bifidum , Bifidobacterium bifidum , Bifidobacterium bifidum , Te Solarium breve (Bifidobacterium breve), BP gambling Terry Enimeol less eseueseu blood lactis (Bifidobacterium animalis ssp. Lactis) a method of increasing the reliability, and any one of the coating efficiency of the probiotics, characterized in that one.
(a) treating ultrasound in a culture medium of Probiotics for 5 minutes at a condition of 100 Hz;
(b) concentrating the ultrasound-treated probiotic culture solution of step (a) to prepare a probiotic concentrate;
(c) treating ultrasound for 5 minutes at 100 Hz while adding a coating agent to the probiotic concentrate of step (b); And
(d) lyophilizing the ultrasound-treated probiotic concentrate of step (c)
The probiotics may be selected from the group consisting of Lactobacillus plantarum , Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus gasseri , Lactobacillus delbrueckii , eseueseu blood Bulgaria kusu (Lactobacillus delbrueckii ssp. bulgaricus), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus buffer momentum (Lactobacillus fermentum), Lactobacillus para casei (Lactobacillus paracasei), Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus reuteri , Lactococcus lactis , Streptococcus thermophilus , Bifidobacterium longum , Bifidobacterium bifidum , Bifidobacterium bifidum , Bifidobacterium bifidum , Bifidobacterium bifidum , Te Solarium breve (Bifidobacterium breve), BP gambling Terry Enimeol less eseueseu blood lactis (Bifidobacterium animalis ssp. Lactis) a method of increasing the reliability, and any one of the coating efficiency of the probiotics, characterized in that one.
delete delete A food composition comprising a lyophilized powder of Probiotics prepared by the method of any one of claims 1 to 3 as an active ingredient.
The method according to claim 6,
Wherein the food composition is any one of a fermented milk, a health functional food, and a functional beverage.

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