METHOD OF PRODUCING LACTIC ACID BACTERIA DUAL-COATED WITH PROTEIN AND POLYSACCHARIDE BY USING PROTEIN HYDROLYSATE

Abstract

The present disclosure relates to a method of producing lactic acid bacteria dual-coated with protein and polysaccharide by using a protein hydrolysate, and lactic acid bacteria having a dual coating, produced by the method. The lactic acid bacteria having a dual coating of protein and polysaccharide, produced according to the present disclosure, have very excellent dry-freezing viability, acid resistance and bile resistance. Accordingly, the lactic acid bacteria having a dual coating of protein and polysaccharide according to the present disclosure will be very useful for the production of fermented milk, processed milk, fermented soy products, processed foods, functional beverages, functional foods, common foods, etc.

Claims

1. A method of producing lactic acid bacteria having a dual coating of protein and polysaccharide coatings, the method comprising the steps of: (a) treating an aqueous protein solution with a protease, thereby preparing an aqueous protein hydrolysate solution having a protein hydrolysis rate of 45% to 95%; (b) adding a sugar component and a nitrogen source component for lactic acid bacteria culture to the prepared aqueous protein hydrolysate solution, followed by sterilization, and then inoculating and culturing the lactic acid bacteria in the sterilized aqueous solution; (c) recovering lactic acid bacteria cells from a fermented lactic acid bacteria culture obtained by the culturing; (d) adding an aqueous cryoprotectant solution and an aqueous polysaccharide solution to the recovered lactic acid bacteria cells, followed by mixing and homogenization; and (e) freeze-drying the homogenized aqueous lactic acid bacteria cell solution.

2. The method of claim 1, wherein the protein in step (a) is skim milk powder, isolated soy protein (ISP), or a mixture of skim milk powder and isolated soy protein (ISP).

3. The method of claim 2, wherein the hydrolysis rate of the skim milk powder is 59% to 93%.

4. The method of claim 2, wherein the hydrolysis rate of the skim milk powder is 75% to 92%.

5. The method of claim 2, wherein the hydrolysis rate of the skim milk powder is 80% to 90%.

6. The method of claim 2, wherein the hydrolysis rate of the isolated soy protein is 45% to 86%.

7. The method of claim 2, wherein the hydrolysis rate of the isolated soy protein is 61% to 80%.

8. The method of claim 2, wherein the hydrolysis rate of the isolated soy protein is 70% to 80%.

9. The method of claim 2, wherein the hydrolysis rate of the mixture of skim milk powder and isolated soy protein is 75% to 93%.

10. The method of claim 2, wherein the hydrolysis rate of the mixture of skim milk powder and isolated soy protein is 76% to 85%.

11. The method of claim 2, wherein the hydrolysis rate of the mixture of skim milk powder and isolated soy protein is 78% to 83%.

12. The method of claim 1, wherein the protein hydrolysis rate is a percentage value (%) obtained by dividing a OD value, which is a difference between the optical density (OD) value (start-point OD) of the aqueous protein solution, measured before treatment with the protease, and the optical density (OD) value (endpoint OD) of the aqueous protein solution, measured after treatment with the protease, by the start-point OD value.

13. The method of claim 1, wherein the protein hydrolysis rate is a percentage value (%) obtained by dividing a ppt value, which is a difference between the measured weight value (start-point ppt) of a precipitate, obtained by centrifuging the aqueous protein solution before treatment with the protease, and the measured weight value (endpoint ppt) of a precipitate, obtained by centrifuging the aqueous protein solution after treatment with the protease, by the start-point ppt value.

14. The method of claim 1, wherein the sugar component for lactic acid bacteria culture in step (b) is one or more selected from the group consisting of mixed lactose, fructose, sucrose and glucose.

15. The method of claim 1, wherein the nitrogen source component for lactic acid bacteria culture in step (b) is yeast extract or soy peptone.

16. The method of claim 1, wherein the cryoprotectant in step (d) is one or a mixture of two or more selected from the group consisting of trehalose, maltodextrin, mannitol, and skim milk powder.

17. The method of claim 1, wherein the polysaccharide in step (d) is one or more selected from the group consisting of gums, water-soluble dietary fiber, indigestible maltodextrin, insoluble dietary fiber, starch, Levan, and resistant starch.

18. The method of claim 1, wherein the lactic acid bacteria are one or more strains selected from the group consisting of Streptococcus sp., Lactococcus sp., Enterococcus sp., Lactobacillus sp., Pediococcus sp., Leuconostoc sp., Weissella sp., and Bifidobacterium sp.

19. Lactic acid bacteria having a dual coating of protein and polysaccharide coatings, produced by the method of claim 1.

20. A food composition comprising lactic acid bacteria having a dual coating of protein and polysaccharide coatings, produced by the method of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 is a microscopic photograph of a culture of Streptococcus thermophilus CBT ST3 (6.0% skim milk powder and 0.003% protease).

[0072] FIG. 2 is a microscopic photograph of a culture of Bifidobacterium breve CBT BR3 (0.90% isolated soy protein and 0.015% protease).

[0073] FIG. 3 is a microscopic photograph of a culture of Lactobacillus acidophilus CBT LA1 (2.0% skim milk powder, 0.90% isolated soy protein, and 0.015% protease).

[0074] FIG. 4 is a SEM photograph of Streptococcus thermophilus CBT ST3 cells dual-coated according to a method of the present disclosure.

[0075] FIG. 5 is a SEM photograph of Bifidobacterium breve CBT BR3 cells dual-coated according to a method of the present disclosure.

[0076] FIG. 6 is a SEM photograph of Lactobacillus acidophilus CBT LA1 cells dual-coated according to a method of the present disclosure.

DETAILED DESCRIPTION

[0077] Specific examples described in the present specification are intended to represent exemplary embodiments or examples of the present disclosure, and the scope of the present disclosure is not limited thereby. It will be apparent to those skilled in the art that variations and other uses of the present disclosure do not depart from the scope of the present disclosure as defined in the appended claims of the present specification.

EXAMPLES

Example 1: Measurement of Protein Hydrolysis Rate

[0078] 1-1. Method for Measurement of Hydrolysis Rate

[0079] As protein sources that are used for dual coating of lactic acid bacteria, skim milk powder, isolated soy protein (ISP) and a mixture of skim milk powder and isolated soy protein were measured for their rate of hydrolysis induced by protease. Aqueous protein solutions containing different concentrations of each of skim milk powder and isolated soy protein were prepared. Each of the prepared aqueous protein solutions containing different concentrations of each of skim milk powder and isolated soy protein was placed in a reactor equipped with a stirrer, and then suspended and homogenized at 100 RPM at 60 C. Next, the prepared suspensions were adjusted to a pH of 8.20.2 by adding 1N NaOH thereto. Tables 1, 2 and 3 below show examples in which suspensions containing different concentrations of each of skim milk powder, isolated soy protein, and skim milk powder+isolated soy protein mixture were prepared.

TABLE-US-00001 TABLE 1 Skim milk powder suspension Concentration (%) Substrate amount (kg) Purified water (l) 1.0% 6 594 2.0% 12 588 3.0% 18 582 4.0% 24 576 5.0% 30 570 6.0% 36 564

TABLE-US-00002 TABLE 2 Isolated soy protein (ISP) suspension Concentration (%) Substrate amount (kg) Purified water (l) 0.15% 0.9 599.1 0.30% 1.8 598.2 0.45% 2.7 597.3 0.60% 3.6 596.4 0.75% 4.5 595.5 0.90% 5.4 594.6

TABLE-US-00003 TABLE 3 Skim milk powder + isolated soy protein (ISP) mixture suspension Skim milk powder Isolated soy protein (ISP) Concen- Substrate Concen- Substrate Purified tration (%) amount (kg) tration (%) amount (kg) water (l) 0.5% 3.0 0.10% 0.6 Purified water 1.0% 6.0 0.20% 1.2 added to 600 l 1.5% 9.0 0.30% 1.8 of aqueous 2.0% 12.0 0.40% 2.4 solution

[0080] Different concentrations of protease (Alcalase) were added to each of the pH-adjusted suspensions which were then subjected to a hydrolysis reaction at a pH of 6.8 or less for 2 hours. Using before pH adjustment of each suspension as a start point (before enzymatic treatment) and using after 2 hours of the enzymatic reaction as an endpoint, the hydrolysis rates of the protein sources were measured.

[0081] For measurement of the hydrolysis rate of skim milk power, the optical density at 610 nm was measured using a spectrophotometer (SHIMADZU, UV-1280).

Hydrolysis rate S(%)=[OD]/[start-point OD]100%

[OD]=start point ODendpoint OD

[0082] For measurement of the hydrolysis rate of isolated soy protein (ISP), first-step centrifugation was performed using a microcentrifuge (Hanil, micro-12) at 13,000 rpm for 15 minutes, the supernatant was discarded, second-step centrifugation for 15 minutes was performed, and then the weight of the precipitate (ppt) was measured.

Hydrolysis rate I(%)=[ppt]/[start-point ppt]100%

[ppt]=start point pptendpoint ppt

[0083] For measurement of the hydrolysis rate of a mixture of skim milk powder and isolated soy protein, the two characteristics (optical density and ppt) were measured and the average value of the measured values was calculated.

[0084] Hydrolysis rate S & I (%)=[hydrolysis rate S+hydrolysis rate 1]/2

[0085] 1-2. Results of Hydrolysis Rate Measurement

[0086] Table 4 below shows the results of measuring the hydrolysis rate of the skim milk powder depending on the concentration of the skim milk powder and the concentration of the enzyme.

TABLE-US-00004 TABLE 4 Results of measuring the hydrolysis rate of skim milk powder depending on the concentrations of skim milk powder and protease Skim Enzyme 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% 0.001% 90.9% 75.4% 73.2% 68.9% 65.4% 59.4% 0.002% 91.9% 78.7% 75.6% 72.1% 69.7% 66.7% 0.003% 92.1% 85.0% 80.6% 76.2% 73.9% 71.6% 0.004% 92.7% 88.2% 86.1% 85.3% 80.4% 76.4% 0.005% 93.4% 91.0% 87.9% 86.9% 84.2% 81.9% Concentration: based on skim milk powder solution, w/w %. Enzyme used: protease (product name: Alcalase 2.4L FG, manufactured by Novozymes A/S Denmark).

[0087] From the experimental results, it was confirmed that the hydrolysis rate of the skim milk powder at the same enzyme concentration decreased as the concentration of the skim milk powder increased, and the hydrolysis rate of the skim milk powder increased as the concentration of the enzyme increased.

[0088] Table 5 below shows the results of measuring the hydrolysis rate of the isolated soy protein depending on the concentration of the isolated soy protein and the concentration of the enzyme.

TABLE-US-00005 TABLE 5 Results of measuring the hydrolysis rate of isolated soy protein depending on the concentrations of isolated soy protein and protease ISP Enzyme 0.15% 0.30% 0.45% 0.60% 0.75% 0.90% 0.005% 81.6% 75.9% 68.1% 62.7% 53.3% 46.8% 0.010% 82.4% 77.3% 71.1% 65.4% 58.6% 50.3% 0.015% 83.7% 78.9% 74.0% 68.6% 62.7% 55.5% 0.020% 84.1% 80.0% 77.9% 72.6% 66.3% 60.7% 0.025% 85.6% 81.9% 79.1% 74.9% 69.9% 66.3% Concentration: based on isolated soy protein (ISP) solution, w/w % Enzyme used: protease (product name: Alcalase 2.4L FG, manufactured by Novozymes A/S Denmark).

[0089] It was confirmed that the hydrolysis rate of the isolated soy protein at the same enzyme concentration decreased as the concentration of the isolated soy protein increased, and the hydrolysis rate of the isolated soy protein increased as the concentration of the enzyme increased.

[0090] Table 6 below shows the results of measuring the hydrolysis rate depending on the concentration of the mixture of skim milk powder and isolated soy protein.

TABLE-US-00006 TABLE 6 Results of measuring the hydrolysis rate depending on the concentration of the mixture of skim milk powder and isolated soy protein and the concentration of the enzyme ISP Skim 0.10% 0.20% 0.30% 0.40% 0.5% 92.7% 91.8% 90.6% 88.8% 1.0% 88.4% 87.2% 86.3% 85.1% 1.5% 84.6% 83.7% 82.6% 80.4% 2.0% 80.1% 78.9% 77.6% 76.8% Concentration: based on the solution of the mixture of skim milk powder and isolated soy protein, w/w %; Enzyme used: 0.015% protease (product name: Alcalase 2.4L FG, manufactured by Novozymes A/S Denmark).

[0091] It was confirmed that the hydrolysis rate at the same enzyme concentration decreased as the concentration of the mixture of skim milk powder and isolated soy protein increased. In addition, at the same enzyme concentration, the hydrolysis rate of the isolated soy protein in the mixture was higher than when the isolated soy protein was used alone.

Example 2: Measurement of Culturability Depending on Hydrolysis Rate of Protein

[0092] 2-1: Experiment on Culturability Depending on Concentration-Dependent Hydrolysis Rate of Skim Milk Powder

[0093] In the aqueous protein hydrolysate solution prepared in Example 1, 30 kg of mixed lactose, 6 kg of soy peptone, 12 kg of yeast extract, 1.2 kg of potassium phosphate dibasic, 120 g of magnesium sulfate, 600 g of L-ascorbic acid, 240 g of L-glutamic acid and 600 g of polysorbate-80 were dissolved to a final volume of 1,200 L. The resulting solution was sterilized using a heat exchanger (Alfalaval, Sweden) at a temperature of 130 C. and a flow rate of 1,850 l/hr and transferred to a 1.2-KL-volume anaerobic fermentation tube. Next, 5 L of Streptococcus thermophilus CBT ST3 as a starter was inoculated into the solution and then fermented for 13 hours while maintaining the pH at 6.0 with ammonia. After fermentation, the culturability of the lactic acid bacteria depending on the concentration-dependent hydrolysis rate of the skim milk powder and on the concentration of protease was measured. For measurement of the culturability of the lactic acid bacteria, 1 ml of the culture was taken in 9 ml of dilution water and vortexed, and then the number of viable cells was analyzed by the decimal dilution method. The results of the measurement are shown in Table 7 below.

TABLE-US-00007 TABLE 7 Experimental results for the culturability of Streptococcus thermophilus CBT ST3 (KCTC 11870BP) depending on the concentration-dependent hydrolysis rate of skim milk powder and on the concentration of protease Skim Enzyme 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% 0.001% 4.9E+09 7.1E+09 7.4E+09 7.3E+09 7.0E+09 6.7E+09 0.002% 5.0E+09 7.4E+09 7.6E+09 7.5E+09 7.2E+09 6.9E+09 0.003% 5.2E+09 8.3E+09 8.2E+09 8.3E+09 7.7E+09 7.1E+09 0.004% 5.2E+09 8.2E+09 8.3E+09 8.2E+09 8.2E+09 7.4E+09 0.005% 5.3E+09 8.3E+09 8.4E+09 8.3E+09 8.3E+09 7.6E+09

[0094] From the experimental results shown in Table 7 above, it was confirmed that when the skim milk powder and the protease were used at concentrations of 2% or higher and 0.003% or higher, respectively, excellent culturability could be ensured. At the skim milk powder concentration that was increased to exceed a certain level, the culturability tended to decrease rather than increase, as the proportion of the skim milk powder not degraded by the enzyme increased. As can be seen in a microscopic photograph of FIG. 1, these results were presumed to be because the hydrophobicity of the culture was increased due to an increase in the amount of the non-degraded, non-covalently bonded hydrophobic water-semisoluble peptide, and thus the lactic acid bacteria agglomerated and were not easily divided.

[0095] 2-2: Experiment on Culturability Depending on Concentration-Dependent Hydrolysis Rate of Isolated Soy Protein

[0096] Like the case of the skim milk powder, in the hydrolysate solution obtained in Example 1, 24 kg of glucose, 6 kg of soy peptone, 18 kg of yeast extract, 1.2 kg of sodium acetate, 1.2 kg of potassium citrate, 120 g of magnesium sulfate, 1.8 kg of L-cysteine hydrochloride, 600 g of L-ascorbic acid and 1.2 kg of polysorbate-80 were dissolved to a final volume of 1,200 L. The resulting solution was sterilized using a heat exchanger (Alfalaval, Sweden) at a temperature of 130 C. and a flow rate of 1,850 t/hr and transferred to a 1.2-KL-volume anaerobic fermentation tube. Next, 5 L of Bifidobacterium breve CBT BR3 as a starter was inoculated into the solution, and then fermented for 14 hours while maintaining the pH at 6.5 with ammonia. After fermentation, the culturability of the lactic acid bacteria depending on the concentration-dependent hydrolysis rate of the isolated soy protein and on the concentration of protease was measured. For measurement of the culturability of the lactic acid bacteria, 1 ml of the culture was taken in 9 ml of dilution water and vortexed, and then the number of viable cells was analyzed by the decimal dilution method. The results of the measurement are shown in Table 8 below.

TABLE-US-00008 TABLE 8 Results of measuring the culturability of Bifidobacterium breve CBT BR3 (KCTC 12201BP) depending on the concentration-dependent hydrolysis rate of isolated soy protein and on the concentration of protease ISP Enzyme 0.15% 0.30% 0.45% 0.60% 0.75% 0.90% 0.005% 6.0E+09 8.0E+09 8.9E+09 9.3E+09 7.8E+09 6.9E+09 0.010% 6.1E+09 8.1E+09 9.0E+09 9.4E+09 8.3E+09 7.4E+09 0.015% 6.3E+09 8.2E+09 9.3E+09 9.4E+09 9.3E+09 8.2E+09 0.020% 6.3E+09 8.2E+09 9.3E+09 9.4E+09 9.1E+09 8.0E+09 0.025% 6.4E+09 8.4E+09 9.4E+09 9.5E+09 9.5E+09 7.9E+09

[0097] From the experimental results shown in Table 8 above, it was confirmed that when the isolated soy protein and the protease were used at concentrations of 0.45% or higher and 0.015% or higher, respectively, excellent culturability could be ensured. At the isolated soy protein concentration that was increased to exceed a certain level, the culturability tended to slightly decrease rather than increase, as the proportion of the isolated soy protein not degraded by the enzyme increased. As can be seen in a microscopic photograph of FIG. 2, these results were presumed to be because the hydrophobicity of the culture was increased due to an increase in the amount of the non-degraded water-semisoluble peptide, and thus the lactic acid bacteria agglomerated and were not easily divided.

[0098] 2-3: Experiment on Culturability Depending on Concentration-Dependent Hydrolysis Rate of Mixture of Skim Milk Powder and Isolated Soy Protein

[0099] In the hydrolysate solution of the mixture of skim milk powder and isolated soy protein, prepared in Example 1, 36 kg of crystalline fructose, 36 kg of yeast extract, 2.4 kg of potassium phosphate dibasic, 6 kg of sodium acetate, 1.2 kg of magnesium sulfate, 6 g of manganese sulfate, 1.2 kg of L-cysteine hydrochloride, 1.2 kg of L-ascorbic acid, 2.4 kg of polysorbate-80 and 7.2 kg of refined salt were dissolved to a final volume of 1,200 L. The resulting solution was sterilized using a heat exchanger (Alfalaval, Sweden) at a temperature of 130 C. and a flow rate of 1,850 l/hr and transferred to a 1.2-KL-volume anaerobic fermentation tube. Next, 5 L of Lactobacillus acidophilus CBT LA1 as a starter was inoculated into the solution, and then fermented for 20 hours while maintaining the pH at 5.5 with ammonia. After fermentation, the culturability of the lactic acid bacteria depending on the concentration-dependent hydrolysis rate of the mixture of skim milk powder and isolated soy protein was measured. For measurement of the culturability of the lactic acid bacteria, 1 ml of the culture was taken in 9 ml of dilution water and vortexed, and then the number of viable cells was analyzed by the decimal dilution method. The results of the measurement are shown in Table 9 below.

TABLE-US-00009 TABLE 9 Results of measuring the culturability of Lactobacillus acidophilus CBT LA1(KCTC 11906BP) depending on the concentration- dependent hydrolysis rate of the mixture of skim milk powder and isolated soy protein (ISP) ISP Skim 0.10% 0.20% 0.30% 0.40% 0.5% 5.9E+09 6.1E+09 6.4E+09 6.3E+09 1.0% 6.2E+09 6.3E+09 7.2E+09 6.8E+09 1.5% 6.4E+09 7.0E+09 8.2E+09 7.2E+09 2.0% 6.8E+10 7.1E+09 8.1E+09 6.8E+09

[0100] From the experimental results shown in Table 9 above, it was confirmed that when the skim milk powder and the isolated soy protein were used as a mixture at concentrations of 1.5% or higher and 0.3% or higher, respectively, excellent culturability could be ensured. At the mixture concentration that was increased to exceed a certain level, the culturability tended to decrease rather than increase. As can be seen in a microscopic photograph of FIG. 3, these results were presumed to be because the hydrophobicity of the culture was increased due to an increase in the amount of the non-degraded water-semisoluble peptide, and thus the lactic acid bacteria agglomerated and were not easily divided.

Example 3: Measurement of Freeze-Drying Viability Depending on Protein Hydrolysis Rate

[0101] 3-1: Experiment on Freeze-Drying Viability Depending on Concentration-Dependent Hydrolysis Rate of Skim Milk Powder

[0102] The fermentation broth obtained in Example 2-1 above was centrifuged using a tubular type high-speed centrifuge (RPM: 15,000 or more, and G-force: 13,200 or more) at a flow rate of 4.0 l/min, and the cells coated with the remaining protein component were recovered by precipitation. 10 L of an aqueous cryoprotectant solution including 3 kg of trehalose, 1 kg of maltodextrin, 1 kg of mannitol and 1 kg of skim milk powder was sterilized by autoclaving and prepared, and 10 L of an aqueous polysaccharide solution obtained by dissolving 20 g of xanthan gum and 20 g of cellulose was sterilized by autoclaving and prepared. Then, the recovered cells and the prepared aqueous cryoprotectant solution and aqueous polysaccharide solution were homogenized by stirring at 200 RPM in a vertical mixer equipped with a whipper. Next, the homogenized mixture was frozen rapidly in a pre-freezer at 40 C., and then the freeze dryer shelf temperature was elevated stepwise from 0 C. by a rate of 10 C./2 hour, and then the mixture was finally freeze-dried at 37 C. In the process of hydrolysis with the protease, the remaining water-semisoluble protein component formed a protein coating on the cells, and the xanthan gum and cellulose polysaccharide components formed a cluster having a very dense structure by binding between the cells. As this time, as the amount of the water-semisoluble peptide was higher than a certain amount, the culturability of the lactic acid bacteria was reduced while the lactic acid bacteria tended to agglomerate. In addition, a suitable amount of the water-semisoluble peptide exhibited excellent freeze-drying viability, accelerated stability, acid resistance and bile resistance by protecting the cells from heat applied during the freeze-drying process. The results of the experiment are shown in Table 10 below.

TABLE-US-00010 TABLE 10 Experimental results for the freeze-drying viability of Streptococcus thermophilus CBT ST3 (KCTC 11870BP) depending on the concentration-dependent hydrolysis rate of skim milk powder and on the concentration of protease Skim Enzyme 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% 0.001% 57.7% 72.9% 63.8% 60.3% 59.0% 58.1% 0.002% 56.2% 73.4% 65.6% 62.9% 59.6% 58.8% 0.003% 55.9% 81.5% 67.1% 64.4% 60.7% 59.6% 0.004% 54.5% 71.5% 68.6% 67.0% 64.3% 61.1% 0.005% 53.1% 69.0% 70.3% 67.7% 65.2% 63.9%

[0103] Freeze-drying viability (%): the viability after the freeze-drying process was determined in view of the viable cell number and weight before and after freeze-drying. [Number of viable cells per g after freeze-dryingweight (g) after freeze-drying]/[number of viable cells per g before freeze-dryingweight (g) before freeze-drying]100%

[0104] As shown in Table 10 above, a skim milk powder concentration of 2% and a protease concentration of 0.003% showed the best freeze-drying viability (81.5%). As the skim milk powder concentration increased, the hydrolysis rate tended to decrease and the freeze-drying viability tended to decrease. It was confirmed that a certain amount of the non-degraded water-semisoluble peptide reduced protein denaturation caused by a physical factor applied to the cells and improved the freeze-drying viability. The results of measurement of the culturability and the freeze-drying viability indicated that both the culturability and the freeze-drying viability were excellent when treated with 2.0% skim milk powder and 0.003% enzyme.

[0105] 3-2: Experiment on Freeze-Drying Viability Depending on Concentration-Dependent Hydrolysis Rate of Isolated Soy Protein

[0106] According to the experimental method described in Example 3-1, an experiment on freeze-drying viability depending on the concentration-dependent hydrolysis rate of isolated soy protein was performed using the fermentation broth obtained in Example 2-2. The results of the experiment are shown in Table 11 below.

TABLE-US-00011 TABLE 11 Experimental results for the freeze-drying viability of Bifidobacterium breve CBT BR3(KCTC 12201BP) depending on the concentration-dependent hydrolysis rate of isolated soy protein and on the concentration of protease ISP Enzyme 0.15% 0.30% 0.45% 0.60% 0.75% 0.90% 0.005% 55.3% 64.9% 77.1% 63.2% 60.4% 58.6% 0.010% 57.9% 67.1% 78.9% 69.7% 63.2% 61.1% 0.015% 62.3% 68.1% 85.2% 70.4% 67.6% 64.8% 0.020% 65.9% 69.6% 76.1% 69.6% 65.8% 63.1% 0.025% 69.4% 70.1% 75.4% 68.4% 64.1% 61.1%

[0107] Freeze-drying viability (%): the viability after the freeze-drying process was determined in view of the viable cell number and weight before and after freeze-drying.

[number of viable cells per g after freeze-dryingweight (g) after freeze-drying]/[number of viable cells per g before freeze-dryingweight (g) before freeze-drying]100%

[0108] As shown in Table 11 above, an isolated soy protein concentration of 0.45% and a protease concentration of 0.015% showed the best freeze-drying viability (85.2%). As the isolated soy protein concentration increased, the hydrolysis rate tended to decrease and the freeze-drying viability tended to decrease. It was confirmed that a certain amount of the non-degraded water-semisoluble peptide reduced protein denaturation caused by a physical factor applied to the cells and improved the freeze-drying viability. The results of measurement of the culturability and the freeze-drying viability indicated that both the culturability and the freeze-drying viability were excellent when treated with 0.45% isolated soy protein and 0.015% enzyme.

[0109] 3-3: Experiment on Freeze-Drying Viability Depending on Concentration-Dependent Hydrolysis Rates of Skim Milk Powder and Isolated Soy Protein

[0110] According to the experimental method described in Example 3-1, an experiment on freeze-drying viability depending on the concentration-dependent hydrolysis rates of skim milk powder and isolated soy protein was performed using the fermentation broth obtained in Example 2-3. The results of the experiment are shown in Table 12 below.

TABLE-US-00012 TABLE 12 Freeze-drying viability of Lactobacillus acidophilus CBT LA1 (KCTC 11906BP) depending on the concentration-dependent hydrolysis rates of skim milk powder and isolated soy protein ISP Skim 0.10% 0.20% 0.30% 0.40% 0.5% 57.7% 59.9% 66.2% 62.9% 1.0% 63.8% 65.3% 71.6% 67.6% 1.5% 70.1% 73.0% 83.1% 70.3% 2.0% 68.8% 70.1% 74.2% 68.6% * Freeze-drying viability (%): the viability after the freeze-drying process was determined in view of the viable cell number and weight before and after freeze-drying.

[Number of viable cells per g after freeze-dryingweight (g) after freeze-drying]/[number of viable cells per g before freeze-dryingweight (g) before freeze-drying]100%

[0111] As shown in Table 12 above, when a mixture of 1.5% skim milk powder and 0.3% isolated soy protein was used, the best freeze-drying viability (83.1%) could be obtained. As the mixture concentration increased, the hydrolysis rate tended to decrease and the freeze-drying viability tended to decrease. It was confirmed that a certain amount of the non-degraded water-semisoluble peptide reduced protein denaturation caused by a physical factor applied to the cells and improved the freeze-drying viability. The results of measurement of the culturability and the freeze-drying viability indicated that both the culturability and the freeze-drying viability were excellent when treated with a mixture of 1.5% skim milk powder and 0.3% isolated soy protein.

Example 4: Experiment on Accelerated Stability, Acid Resistance and Bile Resistance by Dual Coating

[0112] An experiment was performed on the accelerated stability, acid resistance and bile resistance of non-coated lactic acid bacteria, lactic acid bacteria coated with protein alone, and dual-coated lactic acid bacteria. The results of the experiment are shown in Table 13 below.

TABLE-US-00013 TABLE 13 Experimental results for accelerated stability, acid resistance and bile resistance depending on non-coating, protein coating and polysaccharide coating Strain CBT ST3 CBT BR3 Protein hydrolysis conditions 2% skim milk powder + 0.45% ISP + Not used 0.003% enzyme Not used 0.015% enzyme Protein coating X X X X Polysaccharide X CMC-Na, X CMC-Na, X CMC-Na, X CMC-Na, coating (dual XG XG XG XG coating) Accelerated 19.2% 38.3% 54.2% 85.2% 12.5% 33.2% 44.1% 65.2% stability Acid resistance 35.2% 52.8% 70.0% 90.7% 23.6% 51.2% 69.5% 88.6% Bile resistance 30.7% 50.5% 70.7% 91.6% 26.6% 51.8% 56.8% 83.1% CMC-Na: cellulose; XG: xanthan gum;

[0113] Accelerated stability: 1 g of each sample was taken in 9 ml of dilution water and vortexed, and then the initial number of viable cells was analyzed by the decimal dilution method. Additionally, after each sample was incubated at 40 C., the number of viable cells was analyzed once a week for 4 weeks, and the viability relative to the initial number of viable cells was examined.

[0114] Acid resistance: 0.1 g of each sample was dissolved in 9.9 ml of MRS broth solution (pH 2.1) corrected with 1M HCl solution, and then the number of viable cells at 0 hour and 2 hours was analyzed while maintaining a temperature of 37 C., and the viability relative to the viability at 0 hour was examined.

[0115] Bile resistance: 0.1 g of each sample was dissolved in 9.9 ml of 0.5% oxgall-containing MRS broth solution, and then the number of viable cells at 0 hour and 2 hours was analyzed while maintaining a temperature of 37 C., and the viability relative to the viability at 0 hour was examined.

[0116] The results of comparative analysis of non-coated lactic acid bacteria, protein-coated lactic acid bacteria and dual-coated lactic acid bacteria indicated that the dual-coated lactic acid bacteria could exhibit better accelerated stability, acid resistance and bile resistance than the non-coated or single (protein)-coated lactic acid bacteria. Since the rate of death caused by a physical factor applied to the cells is low and the physiologically active function of the lactic acid bacteria cannot be lost, the dual-coated lactic acid bacteria exhibit improved accelerated stability, acid resistance and bile resistance.

[0117] As described above, the present disclosure relates to a method of producing lactic acid bacteria dual-coated with protein and polysaccharide by using a protein hydrolysate, and lactic acid bacteria having a dual coating, produced by the method. The lactic acid bacteria having a dual coating of protein and polysaccharide, produced according to the present disclosure, have very excellent dry-freezing viability, acid resistance and bile resistance. Accordingly, the lactic acid bacteria having a dual coating of protein and polysaccharide according to the present disclosure will be very useful for the production of fermented milk, processed milk, fermented soy products, processed foods, functional beverages, functional foods, common foods, etc.

[0118] Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present disclosure. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereof.