COATED PROBIOTIC, FOOD COMPOSITION CONTAINING THE SAME AND METHOD FOR PRODUCING THE SAME

Abstract

The present disclosure relates to a coated probiotic having enhanced acid tolerance, bile tolerance, gastrointestinal survivability, cold storage stability and room temperature storage stability by including milk-derived phospholipid and Aloe vera gel as a coating agent, a food composition containing the same, and a method for producing the same. As the coated probiotic includes the Aloe vera gel and the milk-derived phospholipid as the coating agent, it may have increased stabilities against external environmental stress, such as lyophilization stability and storage stability of the probiotic itself, and may have significantly enhanced acid tolerance and bile tolerance which are the indices of the gastrointestinal stability of the probiotic after taking.

Claims

1. A coated probiotic comprising: a probiotic; and a coating agent composed of milk-derived phospholipid and Aloe vera gel.

2. The coated probiotic of claim 1, wherein the probiotic is at least one selected from the group consisting of a Lactobacillus strain, a Lactococcus strain, an Enterococcus strain, a Streptococcus strain, and a Bifidobacterium strain.

3. The coated probiotic of claim 1, wherein the coating agent is obtained by mixing the Aloe vera gel and the milk-derived phospholipid together at a weight ratio of 1:0.1 to 2.

4. The coated probiotic of claim 1, comprising 30 to 99 parts by weight of the probiotic and 0.005 to 5 parts by weight of the coating agent.

5. The coated probiotic of claim 1, further comprising a cryoprotectant.

6. A food composition containing the coated probiotic of claim 1.

7. A method for producing a coated probiotic, the method comprising steps of: a) preparing a mixture by mixing a probiotic with a coating agent composed of milk-derived phospholipid and Aloe vera gel; and b) lyophilizing the mixture.

Description

DETAILED DESCRIPTION

[0055] Hereinafter, the present disclosure will be described in more detail. However, this description is provided only for illustrative purposes to help understand the present disclosure, and the scope of the present disclosure is not limited by this illustrative description.

Example 1. Production of Coated Probiotics—Different Types of Phospholipids

[0056] The types of probiotics used in this Example are shown in Table 1 below, and the composition of the cryoprotectant used in this Example is shown in Table 2 below.

TABLE-US-00001 TABLE 1 No. Probiotics 1 L. acidophilus HY7036 2 L. casei HY2782 3 L. gasseri HY7023 4 L. delbrueckii ssp. bulgaricus HY7901 5 L. helveticus HY7801 6 L. fermentum HY7301 7 L. paracasei HP7 8 L. plantarum HY7715 9 L. reuteri HY7501 10 L. rhamnosus HY1213 11 S. thermophilus HY9012 12 B. bifidum HY8308 13 B. breve HY8201 14 B. longum HY8001 15 B. animalis ssp. lactis HY8002

TABLE-US-00002 TABLE 2 Raw material name Content (%) Maltodextrin powder 7 Powdered skim milk 5 Purified water 88 Total 100

[0057] Probiotics were prepared by culturing in an edible medium suitable for each strain according to the strain culture manual known in the art. After each of the cultured media was centrifuged at 8,000 rpm for 15 minutes, only the strains were collected. Each of the strains, a cryoprotectant and a coating agent were mixed together, and the mixture was maintained under rapid freezing conditions (−40° C. or lower) for 4 to 24 hours, and then lyophilized, thus preparing probiotic powders. As the coating agent, 1% of milk-derived phospholipid, 1% of soybean-derived phospholipid, or 1% of egg yolk-derived phospholipid was mixed was mixed with the cryoprotectant and used. 90 parts by weight of each strain and 10 parts by weight of the mixture of the cryoprotectant and the coating agent were mixed together. As a control, probiotic powder was prepared by mixing each strain and the cryoprotectant together without the coating agent.

[0058] The compositions of the milk-derived phospholipid, the soybean-derived phospholipid and the egg yolk-derived phospholipid are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Milk-derived Soybean-derived Egg yolk-derived phospholipid phospholipid phospholipid Phosphatidylcholine (PC) 26 to 28 23 73 to 75 Phosphatidylethanolamine (PE) 23 to 25 22 18 Phosphatidylinositol (PI) 7 to 8 8 to 14 2 to 3 Phosphatidic acid (PA) ≤2  6 ≤1 Phosphatidylserine (PS)  8 to 12 ≤1 — Sphingomyelin (SPM) 25 to 26 —  3

[0059] 1-1. Measurement of Survival Rates of Coated Probiotics—Accelerated Test

[0060] The prepared probiotic powders were individually packaged, and the survival rates thereof were measured through an accelerated test at 45° C., and the bacterial survival rates of the probiotic powders are shown in Table 4 below in comparison with to the number of bacteria that were not lyophilized.

TABLE-US-00004 TABLE 4 Survival rates (%) before and after coating Egg yolk- Milk-derived Soybean-derived derived Test strain name Untreated phospholipid phospholipid phospholipid L. acidophilus HY7036 71 90 85 71 L. casei HY2782 65 89 82 66 L. gasseri HY7023 75 94 83 73 L. delbrueckii ssp. bulgaricus HY7901 67 91 84 66 L. helveticus HY7801 88 93 81 83 L. fermentum HY7301 71 92 78 84 L. paracasei HP7 81 91 83 85 L. plantarum HY7715 85 97 79 86 L. reuteri HY7501 83 87 71 85 L. rhamnosus HY1213 71 86 73 72 S. thermophilus HY9012 73 86 74 75 B. bifidum HY8308 69 84 71 76 B. breve HY8201 82 92 75 83 B. longum HY8001 86 90 85 85 B. animalis ssp. lactis HY8002 65 83 69 66

[0061] As shown in Table 4 above, as a result of comparing the survival rates of the untreated strains and the strains treated with different types of phospholipids applicable to food, it was confirmed that the survival rates of the strains coated with the soybean-derived phospholipid or the egg yolk-derived phospholipid as the coating agent were similar to those of the untreated strains lyophilized without using the coating agent, but the survival rates of the strains coated with the milk-derived phospholipid increased compared to those of the untreated strains or the strains coated with the soybean-derived phospholipid or the egg yolk-derived phospholipid as the coating agent.

[0062] 1-2. Measurement of Intestinal Survival Rates of Coated Probiotics

[0063] Each of the prepared probiotic powders was dissolved in PBS (phosphate buffer saline) to a final concentration of 1×10.sup.9 cfu/ml, thus preparing samples.

[0064] With reference to M. Minekus et al. (Food Funct. 2014 (5): 1113-1124), electrolyte solutions for use in digestion models were prepared. The compositions of the electrolyte solutions are shown in Table 5 below.

TABLE-US-00005 TABLE 5 Simulated Simulated Simulated Salivary Fluid Gastric Fluid Intestinal Fluid (SSF, mmol/L) (SGF, mmol/L) (SIF, mmol/L) KCl 15.1 6.9 6.8 KH.sub.2PO.sub.4 3.7 0.9 0.8 NaHCO.sub.3 13.6 25 85 NaCl — 47.2 38.4 MgCl.sub.2(H.sub.2O).sub.6 0.15 0.1 0.33 (NH.sub.4).sub.2CO.sub.3 0.06 0.5 — CaCl.sub.2(H.sub.2O).sub.2 1.5 (0.75) 0.15 (0.075) 0.6 (0.3)

[0065] In the oral stage, the SSF electrolyte solution and α-amylase of human saliva were added to each of the prepared samples and allowed to react at 37° C. for 2 minutes, and then the SGF electrolyte solution and porcine pepsin were added to each sample which was then adjusted a pH of 3.0, followed by reaction at 37° C. for 2 hours. In the small intestine stage, porcine pancreatin and bile acid were added to each of the above digested samples, which was then adjusted to a pH of 7.0, followed by reaction at 37° C. for 2 hours. In the final absorption step, brush border membrane vesicles were added to each of the above digested samples, which was then adjusted to a pH of 7.0, followed by reaction at 37° C. for 4 hours. To determine the number of probiotic bacteria remaining after all the digestion and absorption processes, the viable cell counts of the probiotics were analyzed according to a conventional viable lactic bacterial cell counting method, and the intestinal survival rate of each of the probiotic strains was expressed as a survival rate (%) compared to the initial bacterial cell count before digestion and absorption. The results of the measurement are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Survival rates (%) before and after coating Soybean- Egg yolk- Milk-derived derived derived Test strain name Untreated phospholipid phospholipid phospholipid L. acidophilus HY7036 56 58 56 55 L. casei HY2782 51 55 52 51 L. gasseri HY7023 46 49 47 46 L. delbrueckii ssp. bulgaricus HY7901 45 50 46 44 L. helveticus HY7801 35 35 31 33 L. fermentum HY7301 42 44 43 39 L. paracasei HP7 39 42 40 38 L. plantarum HY7715 52 60 53 43 L. reuteri HY7501 43 46 44 42 L. rhamnosus HY1213 42 43 41 40 S. thermophilus HY9012 23 25 22 21 B. bifidum HY8308 36 39 35 18 B. breve HY8201 32 37 31 20 B. longum HY8001 21 32 22 18 B. animalis ssp. lactis HY8002 45 50 46 39

[0066] As shown in Table 6 above, it was confirmed that the survival rates of the strains coated with the soybean-derived phospholipid or the egg yolk-derived phospholipid were similar to those of the untreated strains lyophilized without using the coating agent, but the survival rates of the strains coated with the milk-derived phospholipid increased compared to those of the untreated strains or the strains coated with the soybean-derived phospholipid or the egg yolk-derived phospholipid.

[0067] 2. Production of Coated Probiotics—Different Types of Polysaccharides

[0068] Coated probiotics were produced in the same manner as in Example 1, except that 1% of whole Aloe vera leaf, xanthan gum, locust bean gum, gum Arabic or Aloe vera gel was used instead of the milk-derived phospholipid, the soybean-derived phospholipid or the egg yolk-derived phospholipid as the coating agent.

[0069] The whole Aloe vera leaf is prepared by drying and powdering Aloe vera after removing non-edible parts (thorns, etc.) from Aloe vera. According to the standards and specifications for health functional food, the whole Aloe vera leaf contains 2.0 to 50.0 mg/g of anthraquinone-based compounds (as anhydrous barbaloin).

[0070] The Aloe vera gel is prepared by removing the non-edible parts and outer skin from Aloe vera, and then separating, drying and powdering only the gel. According to the standards and specifications for health functional food, the Aloe vera gel contains 30 mg/g or more of polysaccharides in solid state and 0.005% or less of anthraquinone-based compounds (as anhydrous barbaloin).

[0071] 2-1. Measurement of Survival Rates of Coated Probiotics—Accelerated Test

[0072] The bacterial survival rates of the probiotic powders were measured in the same manner as the accelerated test of Example 1-1, and the results of the measurement are shown in Table 7 below.

TABLE-US-00007 TABLE 7 Survival rates (%) before and after coating Whole aloe Xanthan Locust Gum Aloe Test strain name Untreated vera leaf gum bean gum Arabic vera gel L. acidophilus HY7036 71 71 74 72 75 91 L. casei HY2782 65 66 68 65 66 90 L. gasseri HY7023 75 72 76 77 73 95 L. delbrueckii ssp. bulgaricus 67 65 71 68 70 92 HY7901 L. helveticus HY7801 88 86 86 84 85 94 L. fermentum HY7301 71 72 74 73 74 93 L. paracasei HP7 81 77 75 76 80 92 L. plantarum HY7715 85 84 86 83 89 98 L. reuteri HY7501 83 82 81 80 84 88 L. rhamnosus HY1213 71 71 73 72 73 87 S. thermophilus HY9012 73 74 76 74 70 88 B. bifidum HY8308 69 70 70 72 72 85 B. breve HY8201 82 80 83 81 80 93 B. longum HY8001 86 85 84 84 81 91 B. animalis ssp. lactis 91 90 92 89 86 98 HY8002

[0073] As shown in Table 7 above, as a result of comparing the survival rates of the untreated probiotic strains and the probiotic strains coated with different types of polysaccharides applicable to food, it was confirmed that the survival rates of the probiotic strains coated with the whole Aloe vera leaf, xanthan gum, locust bean gum or gum Arabic as the coating agent were similar to those of the untreated strains lyophilized without using the coating agent, but the survival rates of the probiotic strains coated with the Aloe vera gel significantly increased compared to those of the untreated strains or the probiotic strains coated with the whole Aloe vera leaf, xanthan gum, locust bean gum or gum Arabic.

Example 3. Production of Coated Probiotics

[0074] The survival rates of probiotic strains treated with a mixture of the milk-derived phospholipid and the Aloe vera gel, which showed a high probiotic survival rate in Examples 1 and 2, were compared with the survival rates of probiotic strains treated with the milk-derived phospholipid or the Aloe vera gel alone.

[0075] Probiotic powders were produced in the same manner as in Example 1, except that the milk-derived phospholipid alone, the Aloe vera gel alone or 1 wt % of the mixture (0.5 wt % of the milk-derived phospholipid and 0.5 wt % of the Aloe vera gel) was used instead of the milk-derived phospholipid, the soybean-derived phospholipid or the egg yolk-derived phospholipid as the coating agent.

[0076] 3-1. Measurement of Survival Rates of Coated Probiotics—Accelerated Test

[0077] The bacterial survival rates of the probiotic powders were measured in the same manner as the accelerated test of Example 1-1, and the results of the measurement are shown in Table 8 below.

TABLE-US-00008 TABLE 8 Survival rates (%) before coating and after coating Milk-derived Aloe Treated with Test strain name Untreated phospholipid vera gel mixture L. acidophilus HY7036 71 90 91 90 L. casei HY2782 65 89 90 89 L. gasseri HY7023 75 94 95 94 L. delbrueckii ssp. bulgaricus HY7901 67 91 92 91 L. helveticus HY7801 88 93 94 93 L. fermentum HY7301 71 92 93 92 L. paracasei HP7 81 91 92 91 L. plantarum HY7715 85 97 98 97 L. reuteri HY7501 83 87 88 87 L. rhamnosus HY1213 71 86 87 86 S. thermophilus HY9012 73 86 88 91 B. bifidum HY8308 69 84 85 84 B. breve HY8201 82 92 93 92 B. longum HY8001 86 90 91 90 B. animalis ssp. lactis HY8002 65 83 84 83

[0078] As shown in Table 8 above, it was confirmed that the survival rates of the probiotic strains treated with the mixture of the milk-derived phospholipid and the Aloe vera gel as the coating agent were similar to those of the probiotic strains treated with the milk-derived phospholipid alone or the Aloe vera gel alone.

[0079] 3-2. Measurement of Intestinal Survival Rates of Coated Probiotics

[0080] The bacterial survival rates of the probiotic powders were measured in the same manner as the intestinal survival rate measurement described in Example 1-2, and the results of the measurement are shown in Table 9.

TABLE-US-00009 TABLE 9 Survival rates (%) before coating and after coating Milk-derived Aloe Treated with Test strain name Untreated phospholipid vera gel mixture L. acidophilus HY7036 56 58 59 61 L. casei HY2782 51 55 58 59 L. gasseri HY7023 46 49 47 50 L. delbrueckii ssp. bulgaricus HY7901 45 50 48 51 L. helveticus HY7801 35 35 36 38 L. fermentum HY7301 42 44 46 47 L. paracasei HP7 39 42 44 45 L. plantarum HY7715 52 60 59 62 L. reuteri HY7501 43 46 45 48 L. rhamnosus HY1213 42 43 46 47 S. thermophilus HY9012 23 25 26 28 B. bifidum HY8308 36 39 40 42 B. breve HY8201 32 37 36 39 B. longum HY8001 21 32 35 36 B. animalis ssp. lactis HY8002 45 50 52 53

[0081] As shown in Table 9 above, it was confirmed that the survival rates of the probiotic strains treated with the mixture of the milk-derived phospholipid and the Aloe vera gel as the coating agent slightly increased compared to those of the probiotic strains treated with the milk-derived phospholipid alone or the Aloe vera gel alone.

[0082] 3-3. Measurement of Survival Rates of Coated Probiotics—Different Temperature Conditions

[0083] The produced probiotic powders were stored for 1 month under cold conditions (temperature: 10° C. or lower, and humidity: 40% or less), room temperature conditions (temperature: 25° C., humidity: 40 to 60%) and accelerated conditions (temperature: 40° C., and humidity: 70%), and then the viable cell counts thereof were analyzed according to a conventional probiotic viable cell counting method, and the intestinal survival rate of each of the probiotic strains was expressed as a survival rate (%) compared to the viable cell count immediately after lyophilization.

TABLE-US-00010 TABLE 10 Survival rate (%) under cold conditions after lyophilization Milk-derived Aloe Treated with Test strain name Untreated phospholipid vera gel mixture L. acidophilus HY7036 90 97 96 99 L. casei HY2782 93 95 94 97 L. gasseri HY7023 92 97 96 99 L. delbrueckii ssp. bulgaricus HY7901 89 94 95 97 L. helveticus HY7801 91 92 97 97 L. fermentum HY7301 90 92 96 96 L. paracasei HP7 89 96 95 98 L. plantarum HY7715 93 95 99 99 L. reuteri HY7501 91 92 97 97 L. rhamnosus HY1213 84 91 90 93 S. thermophilus HY9012 82 89 88 91 B. bifidum HY8308 93 95 99 99 B. breve HY8201 88 95 94 97 B. longum HY8001 81 88 87 90 B. animalis ssp. lactis HY8002 83 90 89 92

TABLE-US-00011 TABLE 11 Survival rate (%) under room temperature conditions after lyophilization Milk-derived Aloe Treated with Test strain name Untreated phospholipid vera gel mixture L. acidophilus HY7036 63 77 76 79 L. casei HY2782 64 78 77 80 L. gasseri HY7023 63 78 79 81 L. delbrueckii ssp. bulgaricus HY7901 63 80 78 81 L. helveticus HY7801 64 79 81 82 L. fermentum HY7301 66 81 83 84 L. paracasei HP7 62 77 75 78 L. plantarum HY7715 61 76 74 77 L. reuteri HY7501 59 74 76 77 L. rhamnosus HY1213 62 76 74 77 S. thermophilus HY9012 56 71 73 74 B. bifidum HY8308 61 76 74 77 B. breve HY8201 66 80 83 84 B. longum HY8001 61 76 74 77 B. animalis ssp. lactis HY8002 70 86 88 89

TABLE-US-00012 TABLE 12 Survival rate (%) under accelerated conditions after lyophilization Milk-derived Aloe Treated with Test strain name Untreated phospholipid vera gel mixture L. acidophilus HY7036 61 68 70 71 L. casei HY2782 54 69 71 72 L. gasseri HY7023 53 71 70 73 L. delbrueckii ssp. bulgaricus HY7901 55 70 72 73 L. helveticus HY7801 54 69 71 72 L. fermentum HY7301 56 74 73 76 L. paracasei HP7 52 70 69 72 L. plantarum HY7715 51 70 68 71 L. reuteri HY7501 52 64 66 67 L. rhamnosus HY1213 51 66 68 69 S. thermophilus HY9012 53 61 63 64 B. bifidum HY8308 51 66 68 69 B. breve HY8201 55 70 72 73 B. longum HY8001 55 66 68 69 B. animalis ssp. lactis HY8002 61 76 78 79

[0084] As shown in Tables 10 to 12 above, it was confirmed that, when the probiotic powders were cold-stored, the survival rates of the probiotic strains treated with the mixture of the milk-derived phospholipid and the Aloe vera gel as the coating agent slightly increased compared to those of the untreated strains lyophilized without using the coating agent. In addition, it was confirmed that, when the probiotic powders were stored under the room temperature or accelerated conditions, the survival rates of the strains generally decreased, but the survival rates of the probiotic strains treated with the mixture of the milk-derived phospholipid and the Aloe vera gel as the coating agent were maintained at higher levels than those of the strains treated with the milk-derived phospholipid alone or the Aloe vera gel alone.

[0085] These results suggest that the use of the mixture of the milk-derived phospholipid and the Aloe vera gel as the coating agent helps to increase the survival rates of the strains under the cold, room temperature or accelerated conditions.

[0086] As described above, as the coated probiotic according to the present disclosure includes the Aloe vera gel and the milk-derived phospholipid as the coating agent, it may have increased stabilities against external environmental stress, such as lyophilization stability and storage stability of the probiotic itself, and may have significantly enhanced acid tolerance and bile tolerance which are the indices of the gastrointestinal stability of the probiotic after taking.

[0087] So far, the present disclosure has been described with reference to the embodiments thereof. Those of ordinary skill in the art to which the present disclosure pertains will appreciate that the present disclosure may be embodied in modified forms without departing from the essential characteristics of the present disclosure. Therefore, the disclosed embodiments should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present disclosure is defined by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present disclosure.