Vitamin ace antioxidant coating method for improving freeze-drying recovery rate, in vivo stability, and distribution stability of lactic acid bacteria

Abstract

The present invention relates to a vitamin ACE antioxidant coating method for improving the freeze-drying recovery rate, in vivo stability, and distribution stability of lactic acid bacteria. The coating composition according to the present invention has the excellent effect of improving the freeze-drying recovery rate and in vivo and distribution stability of lactic acid bacteria by applying an antioxidant material and protecting lactic acid bacteria vulnerable to oxidative stress.

Description

Vitamin ACE antioxidant coating method for improving the recovery rate, in vivo stability, and distribution stability of lactic acid bacteria freeze-dried products.

[0001] The present invention relates to a vitamin ACE antioxidant coating method for improving the recovery rate, in-vivo stability, and distribution stability of lactic acid bacteria freeze-dried.

[0002] Lactic acid bacteria (LAB) are diverse Gram-positive bacteria widely present in nature, including plants and animals. Probiotic strains of Lactobacillus and Bifidobacterium, in particular, are known to have various health-promoting effects. Public interest in the application of probiotic lactic acid bacteria in food and feed is growing due to their nutritional and functional benefits for humans and animals. However, during industrial processing and in the gastrointestinal tract, lactic acid bacteria are exposed to adverse environments, including high or low temperatures, low pH, bile acids, oxygen, and limited nutrients, which can cause stress.

[0003] These stress factors affect LAB survival during processing, shelf life during storage, and survival, proliferation, and function in the gastrointestinal tract. Therefore, it is crucial to isolate strains with high functionality and high stress tolerance to ensure sufficient viable bacteria in the final product and effective health-promoting effects in the host. Oxidative stress is a critical factor, as it significantly impacts viability and product quality. LAB are considered aerobic anaerobes, and their oxygen sensitivity is a major factor limiting their viability. Anaerobic bacteria lack the ability to synthesize active electron transport chains, making survival in aerobic environments difficult.

[0004] High oxygen levels produce superoxide anions (O 2　 -), leading to the formation of reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and highly reactive hydroxyl radicals (HO·). When ROS accumulate, they cause oxidative stress, damaging proteins, DNA, and lipids and even leading to cell death. Therefore, applying technologies to prevent oxidative stress in LAB is important for proper production and storage of LAB as well as ensuring high bacterial viability in the gastrointestinal tract.

[0005] Accordingly, the inventors of the present invention have completed the present invention by developing a lactic acid bacteria coating method to improve the lyophilized recovery rate, in vivo stability, and distribution stability of lactic acid bacteria after extensive efforts.

[0006] The purpose of the present invention is to provide a composition for coating lactic acid bacteria containing an antioxidant.

[0007] In addition, the present invention aims to provide a method for increasing the survival rate of lactic acid bacteria, which comprises a step of treating lactic acid bacteria with the above-described lactic acid bacteria coating composition.

[0008] In addition, the present invention aims to provide a freeze-drying protection agent coating method comprising the steps of (a) treating lactic acid bacteria with the above-described lactic acid bacteria coating composition; and (b) freeze-drying.

[0009] The present invention provides a composition for coating lactic acid bacteria comprising at least one of the group consisting of vitamins A, C and E.

[0010] At this time, the lactic acid bacteria coating composition preferably further includes trehalose, maltodextrin, and L-cysteine ​​HCl monohydrate.

[0011] The present invention comprises trehalose, maltodextrin and L-cysteine ​​HCl monohydrate, vitamins and the balance distilled water, wherein the vitamins include at least one selected from the group consisting of vitamins A, C and E, and the trehalose, maltodextrin and L-cysteine ​​HCl monohydrate are added in amounts of 2 to 8% (w / w), 1 to 3% (w / w) and 0.05 to 0.8% (w / w), respectively.

[0012] A composition for coating lactic acid bacteria is provided, wherein the above vitamins A, C and E are added in amounts of 0.01 to 0.05% (w / w), 0.1 to 0.5% (w / w) and 0.01 to 0.05% (w / w), respectively.

[0013] In the lactic acid bacteria coating composition according to the present invention, the lactic acid bacteria coating composition may further include a combination of at least one selected from the group consisting of, for example, fructooligosaccharide, skim milk powder, tris (hydroxymethyl) aminomethane, citric acid, fructose, kanamycin sulfate, glycerol, glucose, sucrose, ethylene glycol, propylene glycol, DMSO, dextran, 1,2-propandiol, hyaluronic acid, mannitol, arginine, and albumin.

[0014] The composition for coating lactic acid bacteria according to the present invention is preferably a cryoprotectant.

[0015] The present invention provides a method for increasing the survival rate of lactic acid bacteria, comprising the step of treating lactic acid bacteria with a composition for coating lactic acid bacteria according to the present invention.

[0016] The present invention provides a freeze-drying protection agent coating method comprising the steps of (a) treating lactic acid bacteria with a lactic acid bacteria coating composition according to the present invention; and (b) freeze-drying.

[0017] At this time, the composition can preferably be treated with a lactic acid bacteria culture solution or a lactic acid bacteria culture concentrate.

[0018] The freeze-drying protection agent coating method according to the present invention is a method for improving the survival rate, freeze-drying recovery rate, in-vivo stability, and distribution stability of lactic acid bacteria.

[0019] The coating composition according to the present invention has an excellent effect of improving the freeze-drying recovery rate and in-vivo and distribution stability of lactic acid bacteria by applying an antioxidant material to protect lactic acid bacteria that are vulnerable to oxidative stress.

[0020] Figure 1 shows the results of confirming the appearance of Bifidobacterium Spp. depending on whether it is coated with a cryoprotectant according to the present invention.

[0021] Figure 2 shows the results of confirming the appearance of Lactobacillus Spp. depending on whether it is coated with a cryoprotectant according to the present invention.

[0022] Figure 3 shows the results of evaluating the freeze-drying recovery rate of Bifidobacterium bifidum coated with a cryoprotectant according to the present invention.

[0023] Figure 4 shows the results of evaluating the freeze-drying recovery rate of Bifidobacterium longum coated with a cryoprotectant according to the present invention.

[0024] Figure 5 shows the results of evaluating the freeze-drying recovery rate of Bifidobacterium animalis sp. lactis coated with a cryoprotectant according to the present invention.

[0025] Figure 6 shows the results of evaluating the freeze-drying recovery rate of Bifidobacterium longum subspinfantis coated with a cryoprotectant according to the present invention.

[0026] Figure 7 shows the results of evaluating the freeze-drying recovery rate of Bifidobacterium breve coated with a cryoprotectant according to the present invention.

[0027] Figure 8 shows the results of evaluating the freeze-drying recovery rate of Lactiplantibacillus plantarum coated with a cryoprotectant according to the present invention.

[0028] Figure 9 shows the results of evaluating the freeze-drying recovery rate of Lactobacillus gasseri coated with a cryoprotectant according to the present invention.

[0029] Figure 10 shows the results of evaluating the freeze-drying recovery rate of Lacticaseibacillus paracasei coated with a cryoprotectant according to the present invention.

[0030] Figure 11 shows the results of evaluating the freeze-drying recovery rate of Ligilactobacillus salivarius coated with a cryoprotectant according to the present invention.

[0031] Figure 12 shows the results of evaluating the freeze-drying recovery rate of Lactobacillus helveticus coated with a cryoprotectant according to the present invention.

[0032] Figure 13 shows the results of evaluating the freeze-drying recovery rate of Limosilactobacillus fermentum coated with a cryoprotectant according to the present invention.

[0033] Figure 14 shows the results of evaluating the freeze-drying recovery rate of Lacticaseibacillus casei coated with a cryoprotectant according to the present invention.

[0034] Figure 15 shows the results of evaluating the freeze-drying recovery rate of Lactobacillus acidophilus coated with a cryoprotectant according to the present invention.

[0035] Figure 16 shows the results of evaluating the freeze-drying recovery rate of Lactobacillus delbrueckiissp.bulgaricus coated with a cryoprotectant according to the present invention.

[0036] Figure 17 shows the results of evaluating the freeze-drying recovery rate of Lacticaseibacillus rhamnosus coated with a cryoprotectant according to the present invention.

[0037] Figure 18 shows the results of evaluating the freeze-drying recovery rate of Limosilactobacillus reuteri coated with a cryoprotectant according to the present invention.

[0038] Figure 19 shows the results of evaluating the freeze-drying recovery rate of Enterococcus faecalis coated with a cryoprotectant according to the present invention.

[0039] Figure 20 shows the results of evaluating the freeze-drying recovery rate of Pediococcus pentosaceus coated with a cryoprotectant according to the present invention.

[0040] Figure 21 shows the results of evaluating the freeze-drying recovery rate of Pediococcus acidilactici coated with a cryoprotectant according to the present invention.

[0041] Figure 22 shows the results of evaluating the freeze-drying recovery rate of Weissella confuse coated with a cryoprotectant according to the present invention.

[0042] Figure 23 shows the results of evaluating the freeze-drying recovery rate of Streptococcus thermophilus coated with a cryoprotectant according to the present invention.

[0043] To avoid confusion due to overlapping content, the description of redundant content has been omitted below. In other words, the content of the invention is not limited to the content described below, and should be interpreted based on the overall scope of the invention.

[0044]

[0045] Lactic acid bacteria are vulnerable to oxidative stress throughout the entire process, including cultivation, harvesting, freeze-drying, grinding, product manufacturing, and distribution.

[0046] Accordingly, the present invention provides a method for preventing oxidative stress caused throughout the entire process of culturing, harvesting, freeze-drying, crushing, product manufacturing, and distribution of lactic acid bacteria.

[0047] A preferred embodiment of the present invention for this purpose provides a composition for coating lactic acid bacteria, which comprises at least one selected from the group consisting of vitamins A, C and E, and the composition for coating lactic acid bacteria preferably further comprises trehalose, maltodextrin and L-cysteine ​​HCl monohydrate.

[0048] A more preferred embodiment of the present invention comprises trehalose, maltodextrin and L-cysteine ​​HCl monohydrate, vitamins and the balance distilled water, wherein the vitamins include at least one selected from the group consisting of vitamins A, C and E, and the trehalose, maltodextrin and L-cysteine ​​HCl monohydrate are added in amounts of 2 to 8% (w / w), 1 to 3% (w / w) and 0.05 to 0.8% (w / w), respectively.

[0049] The present invention provides a composition for coating lactic acid bacteria, wherein the above vitamins A, C and E are added in amounts of 0.01 to 0.05% (w / w), 0.1 to 0.5% (w / w) and 0.01 to 0.05% (w / w), respectively.

[0050] At this time, a more preferred embodiment of the present invention is that the composition is composed of 3 to 7% (w / w) of trehalose, 1 to 3% (w / w) of maltodextrin, 0.05 to 0.8% (w / w) of L-cysteine ​​HCl monohydrate, 0.01 to 0.05% (w / w) of vitamin A, 0.1 to 0.5% (w / w) of vitamin C, 0.01 to 0.05% (w / w) of vitamin E, and the remainder of distilled water.

[0051] The most preferred embodiment of the present invention is that the composition comprises 4 to 6% (w / w) of trehalose, 1.5 to 2.5% (w / w) of maltodextrin, 0.15 to 0.45% (w / w) of L-cysteine ​​HCl monohydrate, 0.01 to 0.03% (w / w) of vitamin A, 0.15 to 0.45% (w / w) of vitamin C, 0.01 to 0.03% (w / w) of vitamin E, and the remainder of distilled water.

[0052] This is because the composition described above can effectively prevent the decline in survival rate and functionality of lactic acid bacteria due to oxidative stress under various conditions including production, distribution, and application.

[0053] In addition, in the lactic acid bacteria coating composition according to the present invention, the lactic acid bacteria coating composition may further include a known cryoprotectant component, for example, it may include lipids, sugars, proteins, etc., and as a preferred example, fructo oligosaccharide, skim milk powder, tris (hydroxymethyl) aminomethane, citric acid, fructose, kanamycin sulfate, glycerol, glucose, sucrose, ethylene glycol, propylene glycol, DMSO, dextran, 1,2-propandiol, hyaluronic acid, mannitol, arginine, and It may further include one or more combinations selected from the group consisting of albumin, but is not limited thereto, and any cryoprotectant component known in the art may be further included without limitation in type.

[0054] According to one embodiment of the present invention, when using the composition for coating lactic acid bacteria according to the present invention, the survival rate, freeze-drying recovery rate, in-vivo stability, and distribution stability of lactic acid bacteria can be maximized for most known lactic acid bacteria.

[0055] Accordingly, the lactic acid bacteria coating composition according to the present invention can preferably be used as a cryoprotectant.

[0056]

[0057] Meanwhile, another preferred aspect of the present invention is to provide a method for increasing the survival rate of lactic acid bacteria, comprising the step of treating lactic acid bacteria with a composition for coating lactic acid bacteria according to the present invention.

[0058] At this time, the composition can effectively prevent oxidative stress resulting from the production, distribution and application of lactic acid bacteria, preferably by treating it with a lactic acid bacteria culture solution or a lactic acid bacteria culture concentrate.

[0059] That is, by treating the lactic acid bacteria coating composition according to the present invention to a lactic acid bacteria culture solution or a lactic acid bacteria culture concentrate, the yield of lactic acid bacteria can be increased by effectively preventing oxidative stress occurring during the process of forming the lactic acid bacteria regardless of the form in which the lactic acid bacteria are commercially sold, and thus it is economical and has the advantage of effectively promoting distribution and stability in the body.

[0060] At this time, the lactic acid bacteria can be manufactured into a formulation suitable for oral administration or parenteral administration (spray, inhalation, etc.).

[0061] Non-limiting examples of oral dosage forms include oily suspensions, troches, lozenges, tablets, aqueous suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs.

[0062] In addition, in order to formulate the lactic acid bacteria according to the present invention for oral administration, binders such as sorbitol, mannitol, starch, amylopectin, cellulose, lactose, saccharose or gelatin; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; and fragrances, syrups, sweeteners, etc. can also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be additionally used.

[0063] Non-limiting examples of parenteral formulations include powders for inhalation or aerosols for spraying. When formulated as powders or aerosols for inhalation, the dispersed concentrate or wet powder may be combined with additives such as propellants to facilitate dispersion.

[0064]

[0065] Meanwhile, the modern probiotics industry primarily utilizes the freeze-drying process to store and distribute beneficial microorganisms like lactic acid bacteria for extended periods. However, the oxidative stress generated during the freeze-drying process poses a significant problem, as it can lower cell viability.

[0066] Accordingly, another preferred embodiment of the present invention provides a freeze-drying protection agent coating method comprising the steps of (a) treating lactic acid bacteria with a lactic acid bacteria coating composition according to the present invention; and (b) freeze-drying.

[0067] At this time, the composition can preferably be treated with a lactic acid bacteria culture solution or a lactic acid bacteria culture concentrate.

[0068] The freeze-drying protection agent coating method according to the present invention is a method for improving the survival rate, freeze-drying recovery rate, in-vivo stability, and distribution stability of lactic acid bacteria.

[0069] According to one embodiment of the present invention, it was confirmed that the freeze-drying protective agent coating method according to the present invention can effectively increase the freeze-drying recovery rate (%), acid / bile resistance, and storage stability of lactic acid bacteria during the freeze-drying process of lactic acid bacteria by utilizing the antioxidant effects of vitamins A, C, and E. The method is economical because it can increase productivity by not having to add a larger amount of lactic acid bacteria by increasing the freeze-drying recovery rate during the manufacturing process of lactic acid bacteria for sale, and can effectively increase stability during the distribution process, and has the advantage of maintaining the effectiveness of probiotics in an optimal state even when used in the body.

[0070]

[0071] Hereinafter, the present invention will be described in more detail through the following examples or experimental studies. However, the scope of the rights of the present invention is not limited to the following examples or experimental studies, but also encompasses variations of technical concepts equivalent thereto.

[0072]

[0073] [Example 1: Preparation of Vitamin ACE Antioxidant Protectant (Cryoprotectant)]

[0074] 1. Preparation of vitamin ACE antioxidant protection agent (cryoprotectant)

[0075] For the preparation of a vitamin ACE antioxidant protective agent, various combinations of ingredients as shown in Table 1 below were utilized. Here, trehalose 5% (w / v), maltodextrin 2% (w / v), and L-cysteine ​​HCl monohydrate 0.3% (w / v) were used as cryoprotective ingredients, and vitamin A 0.02% (w / v), vitamin C 0.30% (w / v), and vitamin E 0.02% (w / v) were added to prepare a cryoprotectant.

[0076] The specific composition of vitamin ACE antioxidant protection agent is shown in Table 1 below.

[0077] Raw material weight % Example 1-1. Control Distilled water 100.00 Total 100.00 Example 1-2. TrehaloseTrehalose 5.00 Distilled water 95.00 Total 100.00 Example 1-3. MaltodextrinMaltodextrin 2.00 Distilled water 98.00 Total 100.00 Example 1-4. Trehalose+MaltodextrinTrehalose 5.00Maltodextrin 2.00 Distilled water 93.00 Total 100.00 Example 1-5. Trehalose+Maltodextrin+ L-CysteineTrehalose 5.00Maltodextrin 2.00L-Cysteine ​​HCl monohydrate 0.30 Distilled water 92.70 Total 100.00 Example 1-6. Trehalose+Maltodextrin+ L-Cysteine+ Vitamin ATrehalose 5.00Maltodextrin 2.00L-Cysteine ​​HCl monohydrate 0.30Vitamin A 0.02Distilled water 92.68Total 100.00Example 1-7.Trehalose+Maltodextrin+L-Cysteine+Vitamin Crehalose 5.00Maltodextrin 2.00L-Cysteine ​​HCl monohydrate 0.30Vitamin C 0.30Distilled water 92.4Total 100.00Example 1-8.Trehalose+Maltodextrin+L-Cysteine+Vitamin Erehalose 5.00Maltodextrin 2.00L-Cysteine ​​HCl monohydrate 0.30Vitamin E 0.02Distilled water 92.68Total 100.00Example 1-9.Trehalose+Maltodextrin+ L-Cysteine+ Vitamin ACETrehalose5.00Maltodextrin2.00L-Cysteine ​​HCl monohydrate0.30Vitamin C0.30Vitamin A0.02Vitamin E0.02Distilled water92.36Total amount100.00

[0078]

[0079] 2. Coating method

[0080] Each strain was cultured at a high concentration according to Table 2 below, and then centrifuged at 7,000 rpm for 10 minutes to obtain a concentrated culture solution. The pH of the culture solution was adjusted to 6.50 using 4 M NaOH.

[0081] Thereafter, the cryoprotectant prepared in Example 1 was added to each culture concentrate at a ratio of 22% (v / v), and then mixed at 200 rpm for 5 minutes to coat each lactic acid bacteria with the cryoprotectant (Figs. 1 and 2). Fig. 1 shows the results of confirming the appearance of Bifidobacterium Spp. depending on whether it was coated with the cryoprotectant according to the present invention, and Fig. 2 shows the results of confirming the appearance of Lactobacillus Spp. depending on whether it was coated with the cryoprotectant according to the present invention.

[0082] BifidobacteriumSpp.BifidobacteriumbifidumBifidobacteriumlongumBifidobacteriumanimalisssp. lactisBifidobacteriumlongumsubspinfantisBifidobacteriumbreveLactobacillusSpp.LactiplantibacillusplantarumLactobacillusgasseriLacticaseibacillusparacaseiLigilactobacillussalivariusLactobacillushelveticusLimosilactobacillusfermentumLactobacilluscaseiLactobacillus acidophilusLactobacillusdelbrueckiissp.bulgaricusLacticaseibacillusrhamnosusLimosilactobacillusreuteriOther Lactic acid bacteriaSpp.EnterococcusfaecalisPediococcuspentosaceusPediococcusacidilacticiWeissellaconfuseStreptococcusthermophilus

[0083]

[0084] 3. Freeze-drying

[0085] The lactic acid bacteria culture concentrate coated with each cryoprotectant according to Table 2 above was dried in a freeze dryer to obtain a freeze-dried product.

[0086]

[0087] [Experimental Example 1: Evaluation of the recovery rate of lyophilized lactic acid bacteria]

[0088] The viable cell count (CFU / g) of each lactic acid bacteria in the freeze-dried product obtained in Example 1 was measured to evaluate the lyophilization recovery rate of lactic acid bacteria. The experiment was repeated three times, and the measured data were expressed as the mean value ± standard deviation. The statistical significance of the experimental results was tested using one-way analysis of variance (ANOVA) and Tukey HSD test using SPSS ver. 9.0 (IBM, USA), and a significant difference (p<0.05) was confirmed between the experimental groups.

[0089] As a result of the experiment, the conditions using cryoprotectants with added vitamins A, C, and E (Examples 1-5 to 1-7) showed higher recovery rates than the conditions with added trehalose and / or maltodextrin (Examples 1-1 to 1-3), and in particular, the conditions including all of vitamins A, C, and E (Example 1-9) showed a high average recovery rate of 43.41% for all lactic acid bacteria (Figs. 3 to 23).

[0090] Figures 3 to 23 are the results of evaluating the freeze-drying recovery rate of lactic acid bacteria coated with a cryoprotectant according to the present invention (Figure 3: Bifidobacterium bifidum, Figure 4: Bifidobacterium longum, Figure 5: Bifidobacterium animaliss sp. lactis, Figure 6: Bifidobacterium longum subspinfantis, Figure 7: Bifidobacterium breve, Figure 8: Lactiplantibacillus plantarum, Figure 9: Lactobacillus gasseri, Figure 10: Lacticaseibacillus paracasei, Figure 11: Ligilactobacillus salivarius, Figure 12: Lactobacillushelveticus, Figure 13: Limosilactobacillus fermentum, Figure 14: Lacticaseibacillus casei, Figure 15: Lactobacillus acidophilus, Figure 16:Lactobacillus delbrueckiissp.bulgaricus, Figure 17:Lacticaseibacillus rhamnosus, Figure 18:Limosilactobacillus reuteri, Figure 19:Enterococcus faecalis, Figure 20:Pediococcuspentosaceus, Figure 21:Pediococcusacidilactici, Figure 22:Weissella confuse, Figure 2 23:Streptococcusthermophilus).

[0091]

[0092] [Experimental Example 2: Acid and Bile Resistance Evaluation]

[0093] For this experiment, a probiotic powder obtained by crushing the freeze-dried product obtained in Example 1 was used.

[0094] Acid resistance was measured using the artificial gastric fluid conditions of the food disintegration test (sodium chloride 2.0 g, dilute hydrochloric acid 24.0 ml / L, pH 1.2), adjusted to a final pH of 2.0 and pH 2.5, and then exposed to the addition of probiotic powder at a concentration of 10%. After exposure for 2 hours, taking into account the time it takes to pass through the stomach, the number of viable cells was measured.

[0095] Bile resistance was measured using a medium containing 0.5% bile acid filtered and added aseptically, and probiotic powder was added at a concentration of 10%, reacted for 2 hours, and then the number of viable cells was measured.

[0096] The statistical significance of the experimental results was tested using one-way analysis of variance (ANOVA) and Tukey HSD test using SPSS ver. 9.0 (IBM, USA), and significant differences (p<0.05) between the experimental groups were confirmed.

[0097] The results are shown in Tables 3 to 23 below.

[0098] BifidobacteriumbifidumpH 2.0 (%)pH 2.5 (%)0.5% oxgallControl3.68±2.55a9.18±2.55a4.00±1.50aTrehalose9.61±2.40b17.51±2.40b5.00±2.00aMaltodextrin10.88±3.97a19.28±3.97b10.00±2.50bTrehalose + Maltodextrin14.58±3.77b24.48±3.77c15.00±2.80cTrehalose + Maltodextrin + L-Cysteine16.67±4.27b27.40±4.27c18.00±3.40cTrehalose + Maltodextrin + L-Cysteine + Vitamin A18.85±3.78c20.46±3.78b13.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin C17.59±4.78b28.70±4.78c14.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E16.12±3.33c26.63±3.33c12.00±2.70aTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E26.51±2.59d31.20±2.59d28.00±3.00d

[0099] BifidobacteriumlongumpH 2.0 (%)pH 2.5 (%)0.5% oxgallControl3.06±3.32a5.51±3.32a4.50±1.80aTrehalose8.12±1.33a15.41±1.33b4.00±2.10aMaltodextrin11.93±4.47b20.76±4.47b9.00±2.60aTrehalose + Maltodextrin14.18±3.32b23.91±3.32c14.00±2.90bTrehalose + Maltodextrin + L-Cysteine16.26±3.78b26.83±3.78c17.00±3.00bTrehalose + Maltodextrin + L-Cysteine + Vitamin A17.08±3.78b27.98±3.78c12.00±2.30bTrehalose + Maltodextrin + L-Cysteine + Vitamin C15.67±3.33b25.99±3.33c13.00±2.40bTrehalose + Maltodextrin + L-Cysteine + Vitamin E15.99±3.33b26.45±3.33c11.00±2.50bTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E28.06±2.59c43.38±2.59d26.00±2.80c

[0100] Bifidobacteriumanimalisssp.lactispH 2.0 (%)pH 2.5 (%)0.5% oxgallControl8.02±4.29a11.25±4.29a7.00±1.50aTrehalose16.29±5.45b22.85±5.45b13.00±2.00bMaltodextrin19.28±5.57b27.04±5.57c18.00±2.50bTrehalose + Maltodextrin23.72±5.39c33.27±5.39c23.00±2.80cTrehalose + Maltodextrin + L-Cysteine24.17±4.62c33.89±4.62c27.00±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A23.07±4.29c32.35±4.29c21.00±2.40cTrehalose + Maltodextrin + L-Cysteine + Vitamin C23.18±4.62c32.51±4.62c23.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E23.62±4.99c33.12±4.99c20.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E32.21±2.02d45.17±2.02d35.00±2.70d

[0101] Bifidobacteriumlongumsubsp.InfantispH 2.0 (%)pH 2.5 (%)0.5% oxgallControl10.79±3.71a15.13±3.71a6.00±1.60aTrehalose18.45±4.90b25.87±4.90b14.50±2.10bMaltodextrin18.04±5.03b25.30±5.03b18.50±2.40bTrehalose + Maltodextrin24.16±4.84c33.88±4.84c23.50±2.80cTrehalose + Maltodextrin + L-Cysteine25.42±4.05c35.65±4.05c27.50±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A25.97±3.71c36.42±3.71c22.50±2.30cTrehalose + Maltodextrin + L-Cysteine + Vitamin C28.41±4.05c39.85±4.05c24.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin E25.69±4.43c36.03±4.43c21.50±2.40cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E36.08±3.74d50.60±3.74d37.00±2.90d

[0102] BifidobacteriumbrevepH 2.0 (%)pH 2.5 (%)0.5% oxgallControl11.8±3.13a16.55±3.13a9.00±1.50aTrehalose17.83±4.36b25.00±4.36b15.00±2.00bMaltodextrin17.45±4.49b24.47±4.49b20.00±2.50cTrehalose + Maltodextrin24.39±4.30c34.20±4.30c25.00±2.80cTrehalose + Maltodextrin + L-Cysteine25.65±3.48c35.97±3.48c29.00±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A26.20±3.14c36.74±3.14c23.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin C27.95±3.48c39.19±3.48c24.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin E25.93±3.87c36.36±3.87c22.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E32.25±3.17d45.23±3.17d37.00±3.00d

[0103] LactiplantibacillusplantarumpH 2.0 (%)pH 2.5 (%)0.5% oxgallControl2.10±2.50a14.30±2.50a8.50±1.50aTrehalose7.45±2.40a18.78±2.40a14.50±2.00bMaltodextrin4.85±2.80a18.46±2.80a18.50±2.50bTrehalose + Maltodextrin15.50±3.00c28.7±3.00b22.50±2.80cTrehalose + Maltodextrin + L-Cysteine12.70±3.30b26.92±3.30b26.50±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A16.70±3.20c29.92±3.20b23.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin C18.50±3.40c33.72±3.40c24.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E13.70±3.10b25.92±3.10b22.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E31.80±2.70d46.32±2.70d35.50±3.00d

[0104] LactobacillusgasseripH 2.0 (%)pH 2.5 (%)0.5% oxgallControl5.15±2.50a16.35±2.50a9.00±1.50aTrehalose9.2±2.40a20.04±2.40a14.00±2.00aMaltodextrin13.7±2.80b24.90±2.80b18.00±2.50bTrehalose + Maltodextrin20.3±3.00b31.50±3.00c22.00±2.80cTrehalose + Maltodextrin + L-Cysteine22.5±3.30c33.70±3.30c26.00±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A23.5±3.20c36.70±3.20c23.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin C22.4±3.40c34.50±3.40c24.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E24.5±3.10c35.70±3.10c22.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E31.8±2.70d47.12±2.70d35.00±3.00d

[0105] LacticaseibacillusparacaseipH 2.0 (%)pH 2.5 (%)0.5% oxgallControl4.10±2.50a14.30±2.50a8.00±1.50aTrehalose10.05±2.40b23.38±2.40b14.00±2.00bMaltodextrin12.60±2.80b26.21±2.80b18.00±2.50cTrehalose + Maltodextrin20.15±3.00c33.35±3.00c22.00±2.80cTrehalose + Maltodextrin + L-Cysteine22.35±3.30c35.57±3.30c26.00±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A23.35±3.20c36.57±3.20c23.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin C22.35±3.40c34.57±3.40c24.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E25.15±3.10c30.37±3.10b22.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E47.65±2.70d37.97±2.70c34.50±3.00d

[0106] LigilactobacillussalivariuspH 2.0 (%)pH 2.5 (%)0.5% oxgallControl7.00±2.50a17.20±2.50a8.50±1.50aTrehalose14.10±2.40b27.43±2.40b14.50±2.00bMaltodextrin11.65±2.80a25.26±2.80b18.50±2.50bTrehalose + Maltodextrin20.2±3.00b33.4±3.00c22.50±2.80cTrehalose + Maltodextrin + L-Cysteine23.4±3.30c36.62±3.30c26.50±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A22.4±3.20c34.62±3.20c23.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin C28.4±3.40c31.62±3.40c24.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E28.2±3.10c39.62±3.10c22.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E30.7±2.70d44.02±2.70d35.50±3.00d

[0107] LactobacillushelveticuspH 2.0 (%)pH 2.5 (%)0.5% oxgallControl4.25±2.50a14.45±2.50a9.00±1.50aTrehalose7.00±2.40a16.33±2.40a14.00±2.00aMaltodextrin2.50±2.80a16.11±2.80a18.00±2.50bTrehalose + Maltodextrin20.10±3.00c33.30±3.00c22.00±2.80cTrehalose + Maltodextrin + L-Cysteine22.30±3.30c36.52±3.30c26.00±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A23.30±3.20c36.52±3.20c23.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin C24.10±3.40c39.32±3.40c24.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E22.30±3.10c34.52±3.10c22.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E32.60±2.70d42.92±2.70d35.00±3.00d

[0108] LimosilactobacillusfermentumpH 2.0 (%)pH 2.5 (%)0.5% oxgallControl5.35±4.00a15.55±4.00a11.50±1.70aTrehalose8.40±3.90a21.73±3.90b12.50±2.20aMaltodextrin11.40±4.30b23.40±4.30b21.50±2.70bTrehalose + Maltodextrin18.50±4.50b32.70±4.50c25.50±3.00cTrehalose + Maltodextrin + L-Cysteine20.70±4.80b33.92±4.80c29.50±3.20cTrehalose + Maltodextrin + L-Cysteine + Vitamin A22.70±4.70b35.92±4.70c26.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin C24.70±4.90c38.92±4.90c27.00±2.80cTrehalose + Maltodextrin + L-Cysteine + Vitamin E23.70±4.60c36.92±4.60c25.00±2.90cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E34.80±4.20d49.32±4.20d38.50±3.20d

[0109] LacticaseibacilluscaseipH 2.0 (%)pH 2.5 (%)0.5% oxgallControl3.85±2.00a16.05±4.00a10.00±3.60aTrehalose6.90±3.90a22.23±3.90b15.70±4.10aMaltodextrin9.90±4.30b23.90±4.30b19.70±4.60bTrehalose + Maltodextrin17.00±4.50b33.20±4.50c23.70±4.90cTrehalose + Maltodextrin + L-Cysteine19.20±4.80c34.42±4.80c27.70±5.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A21.20±4.70c36.42±4.70c24.20±4.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin C23.20±4.90c39.42±4.90c25.20±4.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin E22.20±4.60c37.42±4.60c23.20±4.80cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E43.30±4.20d49.82±4.20d36.70±5.10d

[0110] Lactobacillus acidophiluspH 2.0 (%)pH 2.5 (%)0.5% oxgallControl2.70±1.00a16.65±5.70a7.00±3.60aTrehalose5.75±5.90a18.83±5.60a13.00±4.10bMaltodextrin8.75±6.30b24.50±6.00c17.00±4.60bTrehalose + Maltodextrin15.85±6.50c33.80±6.20c21.00±4.90cTrehalose + Maltodextrin + L-Cysteine18.05±6.80c35.02±6.50c25.00±5.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A20.05±6.70c37.02±6.40c21.50±4.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin C22.05±6.90c40.02±6.60c22.50±4.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin E21.05±6.60c38.02±6.30c20.50±4.80cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E30.30±6.20d40.42±5.90d34.00±5.10d

[0111] Lactobacillusdelbrueckiissp.bulgaricuspH 2.0 (%)pH 2.5 (%)0.5% oxgallControl3.85±4.00a9.45±4.00a4.00±2.60aTrehalose8.90±3.90a15.63±3.90b8.00±4.10aMaltodextrin6.90±4.30a17.30±4.30b14.00±4.60bTrehalose + Maltodextrin14.00±4.50b26.60±4.50c18.00±4.90cTrehalose + Maltodextrin + L-Cysteine16.20±4.80c27.82±4.80c22.00±5.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A18.20±4.70c29.82±4.70c18.50±4.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin C10.20±4.90b32.82±4.90c19.50±4.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin E19.20±4.60c30.82±4.60c17.50±4.80cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E30.30±4.20d43.22±4.20d31.00±5.10d

[0112] LacticaseibacillusrhamnosuspH 2.0 (%)pH 2.5 (%)0.5% oxgallControl6.20±2.50a16.40±2.50a8.00±1.50aTrehalose10.30±2.40a13.50±2.40a4.00±2.00aMaltodextrin15.10±2.80b15.30±2.80a8.00±2.50aTrehalose + Maltodextrin20.20±3.00c33.30±3.00c22.00±2.80cTrehalose + Maltodextrin + L-Cysteine22.50±3.30c36.72±3.30c26.00±3.00cTrehalose + Maltodextrin + L-Cysteine + Vitamin A23.70±3.20c36.92±3.20c23.00±2.50cTrehalose + Maltodextrin + L-Cysteine + Vitamin C24.30±3.40c39.62±3.40c24.00±2.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin E23.10±3.10c36.12±3.10c22.00±2.70cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E42.50±2.70d47.12±2.70d34.50±3.00d

[0113] LimosilactobacillusreuteripH 2.0 (%)pH 2.5 (%)0.5% oxgallControl5.85±2.00a12.30±5.70a6.50±3.60aTrehalose7.95±3.90a19.40±5.60b12.50±4.10bMaltodextrin10.75±3.30b21.20±6.00b16.50±4.60bTrehalose + Maltodextrin19.85±6.50c29.20±6.20c20.50±4.90cTrehalose + Maltodextrin + L-Cysteine22.15±6.80c32.62±6.50c24.50±5.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A23.35±6.70c32.82±6.40c21.50±4.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin C23.95±6.90c35.52±6.60c22.50±4.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin E22.75±6.60c32.02±6.30c20.50±4.80cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E42.15±6.20d43.02±5.90d37.00±5.10d

[0114] EnterococcusfaecalispH 2.0 (%)pH 2.5 (%)0.5% oxgallControl14.59±6.00a17.85±5.70a7.50±3.60aTrehalose17.64±5.90a24.13±5.60b13.50±4.10bMaltodextrin22.34±6.30b28.30±6.00b18.50±4.60bTrehalose + Maltodextrin30.84±6.50c36.90±6.20c23.50±4.90cTrehalose + Maltodextrin + L-Cysteine32.84±6.80c40.10±6.50c27.50±5.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A33.84±6.70c40.30±6.40c21.50±4.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin C34.34±6.90c42.00±6.60c22.50±4.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin E32.84±6.60c39.20±6.30c20.50±4.80cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E43.84±6.20d50.30±5.90d35.50±5.10d

[0115] PediococcuspentosaceuspH 2.0 (%)pH 2.5 (%)0.5% oxgallControl10.91±50a16.85±3.70a6.50±4.60aTrehalose13.96±4.90a23.13±3.60b12.05±5.10bMaltodextrin18.66±5.30b27.30±40b17.50±5.60bTrehalose + Maltodextrin27.16±5.50c35.90±4.20c22.05±5.90cTrehalose + Maltodextrin + L-Cysteine29.16±5.80c39.10±4.50c26.50±6.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A30.16±5.70c39.30±4.40c20.05±5.60cTrehalose + Maltodextrin + L-Cysteine + Vitamin C30.66±5.90c35.00±4.60c21.50±5.70cTrehalose + Maltodextrin + L-Cysteine + Vitamin E29.16±5.60c38.20±4.30c19.05±5.80cTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E40.16±5.20d49.30±3.90d34.05±6.10d

[0116] PediococcusacidilacticipH 2.0 (%)pH 2.5 (%)0.5% oxgallControl12.91±4.40a18.85±4.10a2.50±3.60aTrehalose15.96±4.30a25.13±4.00b6.50±4.10aMaltodextrin20.66±4.70b29.30±4.40b13.50±4.60bTrehalose + Maltodextrin29.16±4.90c37.90±4.60c18.50±4.90bTrehalose + Maltodextrin + L-Cysteine31.16±5.20c41.10±4.90c22.05±5.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A32.16±5.10c41.30±4.80c16.50±4.60bTrehalose + Maltodextrin + L-Cysteine + Vitamin C32.66±5.30c43.00±5.00c17.50±4.70bTrehalose + Maltodextrin + L-Cysteine + Vitamin E31.16±5.00c40.20±4.70c15.50±4.80bTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E42.16±4.60d51.30±4.30d30.50±5.10d

[0117] WeissellaconfusapH 2.0 (%)pH 2.5 (%)0.5% oxgallControl11.91±5.40a15.85±6.10a3.50±2.60aTrehalose14.96±5.30a22.13±6.00b7.50±4.10aMaltodextrin19.66±5.70a26.30±6.04b14.50±3.60bTrehalose + Maltodextrin28.16±5.90b34.90±6.60c19.50±3.90bTrehalose + Maltodextrin + L-Cysteine30.16±6.20b38.10±6.09c23.50±4.10cTrehalose + Maltodextrin + L-Cysteine + Vitamin A31.16±6.10b38.30±6.08c17.50±3.60bTrehalose + Maltodextrin + L-Cysteine + Vitamin C31.66±6.30b40.00±7.00c18.50±3.07bTrehalose + Maltodextrin + L-Cysteine + Vitamin E30.16±6.00b37.20±6.70c16.50±3.80bTrehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E41.16±5.60c48.03±6.30d31.50±4.10d

[0118] StreptococcusthermophiluspH 2.0 (%)pH 2.5 (%)0.5% oxgallControl8.41±5.40a11.61±6.10a7.16±2.60aTrehalose11.46±5.30a17.89±6.00b13.16±3.10bMaltodextrin16.16±5.70b22.06±6.40b18.16±3.60bTrehalose + Maltodextrin24.66±5.90c30.66±6.60c23.16±3.90cTrehalose + Maltodextrin + L-Cysteine26.66±6.20c33.86±6.90c27.16±4.10cTrehalose + Maltodextrin + L-Cysteine ​​+ Vitamin A27.66±6.10c34.06±6.80c21.16±3.60bTrehalose + Maltodextrin + L-Cysteine ​​+ Vitamin C28.16±6.30c35.76±7.00c22.16±3.70bTrehalose + Maltodextrin + L-Cysteine ​​+ Vitamin E26.66±6.00c32.96±6.07c20.16±3.80bTrehalose + Maltodextrin + L-Cysteine ​​+ Vitamins A, C, E37.66±5.06d44.06±6.30d35.16±4.10d

[0119]

[0120] As a result of the experiment, it was confirmed that the condition including all vitamins A, C and E (Example 1-9) showed the best acid resistance and bile resistance for all lactic acid bacteria, and was effective in maximizing the stability and efficacy of probiotics (increasing stability in the body).

[0121]

[0122] [Experimental Example 3: Storage Stability Evaluation]

[0123] To evaluate storage stability, the lyophilized lactic acid bacteria raw material from Example 1 was divided into 10 g portions and sealed. The divided samples were stored at 40°C for 1 month, and samples were collected at weekly intervals.

[0124] Afterwards, the collected samples were serially diluted in sterile saline solution and spread on MRS and BL media. The spread media were cultured for 48–72 hours, and the viable cell count was measured to determine the survival rate under harsh conditions.

[0125] The statistical significance of the experimental results was tested using one-way analysis of variance (ANOVA) and Tukey HSD test using SPSS ver. 9.0 (IBM, USA), and significant differences (p<0.05) between the experimental groups were confirmed.

[0126] The results are shown in Tables 24 to 44 below.

[0127] BifidobacteriumbifidumWeeks1234Control32.80±5.7011.50±13.209.00±5.708.60±2.30Trehalose30.80±6.6013.70±5.7012.70±6.2010.70±1.30Maltodextrin32.80±7.0020.70±6.209.70±6.706.70±3.30Trehalose + Maltodextrin30.80±7.2013.70±6.709.70±7.009.70±1.30Trehalose + Maltodextrin + L-Cysteine32.80±7.5023.70±7.1012.70±7.204.70±0.80Trehalose + Maltodextrin + L-Cysteine + Vitamin A32.80±7.4016.70±6.508.70±6.707.70±1.10Trehalose + Maltodextrin + L-Cysteine + Vitamin C39.50±7.6031.80±6.6023.00±6.8012.10±3.30Trehalose + Maltodextrin + L-Cysteine + Vitamin E29.80±7.3021.80±6.409.00±6.905.90±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E55.60±6.9037.70±7.9030.20±7.2020.80±2.30

[0128] BifidobacteriumlongumWeeks1234Control33.70±6.7013.50±1.329.00±5.707.60±2.30Trehalose25.70±6.6015.70±1.9212.70±6.209.70±1.30Maltodextrin33.70±7.0022.70±2.429.70±6.705.70±3.30Trehalose + Maltodextrin21.70±7.2015.70±2.929.70±7.008.70±1.30Trehalose + Maltodextrin + L-Cysteine33.70±7.5025.70±3.3212.70±7.203.70±0.80Trehalose + Maltodextrin + L-Cysteine + Vitamin A23.70±7.4018.70±3.728.70±6.706.70±1.10Trehalose + Maltodextrin + L-Cysteine + Vitamin C20.40±7.6033.80±2.8223.00±6.8011.10±3.30Trehalose + Maltodextrin + L-Cysteine + Vitamin E30.70±7.3023.80±2.629.00±6.904.90±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E56.50±6.9039.70±4.1230.20±7.2019.8±2.30

[0129] Bifidobacteriumanimalisssp.lactisWeeks1234Control32.80±5.7011.50±3.209.00±5.708.60±2.30Trehalose24.80±6.6013.07±9.2012.7±6.2010.70±1.30Maltodextrin32.80±7.0020.70±4.209.70±6.706.70±3.30Trehalose + Maltodextrin20.80±7.2013.70±9.209.70±3.009.70±1.30Trehalose + Maltodextrin + L-Cysteine32.80±7.5023.70±3.2012.70±7.204.70±0.80Trehalose + Maltodextrin + L-Cysteine + Vitamin A22.80±7.4016.70±7.208.70±6.707.70±1.10Trehalose + Maltodextrin + L-Cysteine + Vitamin C19.50±7.6031.08±8.2023.00±6.8012.10±3.30Trehalose + Maltodextrin + L-Cysteine + Vitamin E29.80±7.3021.08±6.209.00±6.905.90±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E55.60±6.9037.07±1.2030.20±7.2020.80±2.30

[0130] Bifidobacteriumlongumsubsp. InfantisWeeks1234Control34.80±4.5012.38±10.208.10±2.907.80±2.00Trehalose26.80±4.4014.58±2.7011.8±3.409.90±1.00Maltodextrin34.80±4.8021.58±3.208.80±3.905.90±3.00Trehalose + Maltodextrin22.80±5.0014.58±3.708.80±4.208.90±1.00Trehalose + Maltodextrin + L-Cysteine34.80±5.3024.58±4.1011.80±4.403.90±0.50Trehalose + Maltodextrin + L-Cysteine + Vitamin A24.80±5.2017.58±3.507.80±3.906.90±0.80Trehalose + Maltodextrin + L-Cysteine + Vitamin C21.50±5.4032.68±3.6022.10±4.0011.30±3.00Trehalose + Maltodextrin + L-Cysteine + Vitamin E31.80±5.1022.68±3.408.10±4.105.10±1.80Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E57.60±4.7038.58±4.9029.30±4.4022.00±2.00

[0131] BifidobacteriumbreveWeeks1234Control27.80±3.8013.38±10.309.10±2.505.80±1.80Trehalose19.80±3.7015.58±2.8012.80±3.007.90±0.80Maltodextrin27.80±4.1022.58±3.309.80±3.503.90±2.80Trehalose + Maltodextrin15.80±4.3015.58±3.809.80±3.806.90±0.80Trehalose + Maltodextrin + L-Cysteine27.80±4.6025.58±4.2012.80±4.001.90±0.30Trehalose + Maltodextrin + L-Cysteine + Vitamin A17.80±4.5018.58±3.608.80±3.504.90±0.60Trehalose + Maltodextrin + L-Cysteine + Vitamin C14.50±4.7033.68±3.7023.10±3.609.30±2.08Trehalose + Maltodextrin + L-Cysteine + Vitamin E24.80±4.4023.68±3.509.10±3.703.10±1.60Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E50.60±4.0039.58±5.0030.30±4.0018.00±1.80

[0132] LactiplantibacillusplantarumWeeks1234Control25.00±6.6013.38±5.308.20±3.507.80±1.70Trehalose17.00±6.5015.58±3.8011.90±4.009.90±1.30Maltodextrin25.00±6.9022.58±4.308.90±4.505.90±2.70Trehalose + Maltodextrin13.00±7.1015.58±4.808.90±4.808.90±1.70Trehalose + Maltodextrin + L-Cysteine25.00±7.4025.58±5.2011.90±503.90±2.20Trehalose + Maltodextrin + L-Cysteine + Vitamin A15.00±7.3018.58±4.607.90±4.506.90±2.50Trehalose + Maltodextrin + L-Cysteine + Vitamin C11.70±7.5033.68±4.7022.20±4.6011.30±2.70Trehalose + Maltodextrin + L-Cysteine + Vitamin E22.00±7.2023.68±4.508.20±4.705.10±1.50Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E47.80±6.8039.58±6.0029.40±5.0020.00±1.70

[0133] LactobacillusgasseriWeeks1234Control21.00±4.6010.38±6.309.20±3.505.80±1.90Trehalose13.00±4.5012.58±4.8012.90±4.007.90±0.90Maltodextrin21.00±4.9019.58±5.309.90±4.503.90±2.90Trehalose + Maltodextrin9.00±5.1012.58±5.809.90±4.806.90±0.90Trehalose + Maltodextrin + L-Cysteine21.00±5.4022.58±6.2012.09±5.001.90±0.40Trehalose + Maltodextrin + L-Cysteine + Vitamin A11.00±5.3015.58±5.608.90±4.504.90±0.70Trehalose + Maltodextrin + L-Cysteine + Vitamin C7.70±5.5030.68±5.7023.20±4.609.30±2.90Trehalose + Maltodextrin + L-Cysteine + Vitamin E18.00±5.2020.68±5.509.20±4.703.10±1.70Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E43.80±4.8036.58±7030.40±5.0018.00±1.90

[0134] LacticaseibacillusparacaseiWeeks1234Control34.00±3.6013.80±4.109.30±2.604.90±3.60Trehalose26.00±3.5016.00±2.6013.00±3.107.00±2.60Maltodextrin34.00±3.9023.00±3.1010.00±3.603.00±4.06Trehalose + Maltodextrin22.00±4.1016.00±3.6010.00±3.906.00±2.60Trehalose + Maltodextrin + L-Cysteine34.00±4.4026.00±4.0013.00±4.101.00±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamin A24.00±4.3019.00±3.409.00±3.604.00±2.40Trehalose + Maltodextrin + L-Cysteine + Vitamin C20.70±4.5034.10±3.5023.30±3.708.40±4.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E31.00±4.2024.10±3.309.30±3.802.20±3.40Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E56.80±3.8040.00±4.8030.50±4.1017.10±3.60

[0135] LigilactobacillussalivariusWeeks1234Control28.00±5.6012.80±6.108.30±4.606.90±3.60Trehalose20.00±5.5015.00±4.6012.00±5.109.00±2.60Maltodextrin28.00±5.9022.00±5.109.00±5.605.00±4.60Trehalose + Maltodextrin16.00±6.1015.00±5.609.00±5.908.00±2.60Trehalose + Maltodextrin + L-Cysteine28.00±6.4025.00±6.0012.00±6.103.00±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamin A18.00±6.3018.00±5.408.00±5.606.00±2.40Trehalose + Maltodextrin + L-Cysteine + Vitamin C14.70±6.5033.10±5.5022.30±5.7010.40±4.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E25.00±6.2023.10±5.308.30±5.804.20±3.40Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E50.80±5.8039.00±6.8029.50±6.1019.10±3.60

[0136] LactobacillushelveticusWeeks1234Control33.00±4.6012.80±5.108.30±3.606.90±4.60Trehalose25.00±4.5015.00±3.6012.00±4.109.00±3.60Maltodextrin33.00±4.9022.00±4.109.00±4.605.00±5.60Trehalose + Maltodextrin21.00±5.1015.00±4.609.00±4.908.00±3.60Trehalose + Maltodextrin + L-Cysteine33.00±5.4025.00±5.0012.00±5.103.00±3.10Trehalose + Maltodextrin + L-Cysteine + Vitamin A23.00±5.3018.00±4.408.00±4.606.00±3.40Trehalose + Maltodextrin + L-Cysteine + Vitamin C19.70±5.5033.10±4.5022.30±4.708.40±5.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E30.00±5.2023.10±4.308.30±4.804.20±4.40Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E55.80±4.8039.00±5.8029.50±5.1018.10±4.60

[0137] LimosilactobacillusfermentumWeeks1234Control29.00±3.608.80±4.104.30±2.607.90±3.60Trehalose21.00±3.5011.00±2.608.00±3.1010.00±2.60Maltodextrin29.00±3.9018.00±3.105.00±3.606.00±4.60Trehalose + Maltodextrin17.00±4.1011.00±3.605.00±3.909.00±2.60Trehalose + Maltodextrin + L-Cysteine29.00±4.4021.00±4.008.00±4.104.00±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamin A19.00±4.3014.00±3.404.00±3.607.00±2.40Trehalose + Maltodextrin + L-Cysteine + Vitamin C15.70±4.5029.10±3.5018.30±3.709.40±4.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E26.00±4.2019.10±3.304.30±3.805.20±3.40Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E51.80±3.8035.00±4.8025.50±4.1020.10±3.60

[0138] LacticaseibacilluscaseiWeeks1234Control28.00±3.709.80±4.206.30±2.708.90±3.70Trehalose20.00±3.6012.00±2.7010.00±3.2011.00±2.70Maltodextrin28.00±4.0019.00±3.207.00±3.707.00±4.70Trehalose + Maltodextrin16.00±4.2012.00±3.707.00±4.0010.00±2.70Trehalose + Maltodextrin + L-Cysteine28.00±4.5022.00±4.1010.00±4.205.00±2.20Trehalose + Maltodextrin + L-Cysteine + Vitamin A18.00±4.4015.00±3.506.00±3.708.00±2.50Trehalose + Maltodextrin + L-Cysteine + Vitamin C14.70±4.6030.10±3.6020.30±3.8010.40±4.70Trehalose + Maltodextrin + L-Cysteine + Vitamin E25.00±4.3020.10±3.406.30±3.906.20±3.50Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E49.80±3.9036.00±4.9027.50±4.2021.10±3.70

[0139] Lactobacillus acidophilusWeeks1234Control31.00±4.0010.80±4.506.30±3.004.90±4.00Trehalose23.00±3.9013.00±3.0010.00±3.507.00±3.00Maltodextrin31.00±4.3020.00±3.507.00±4.005.30±1.20Trehalose + Maltodextrin19.00±4.5013.00±4.007.00±4.306.00±3.00Trehalose + Maltodextrin + L-Cysteine31.00±4.8023.00±4.4010.00±4.505.00±2.50Trehalose + Maltodextrin + L-Cysteine + Vitamin A21.00±4.7016.00±3.806.20±2.004.00±2.80Trehalose + Maltodextrin + L-Cysteine + Vitamin C17.70±4.9031.10±3.9020.30±4.106.40±5.00Trehalose + Maltodextrin + L-Cysteine + Vitamin E28.00±4.6021.10±3.706.30±4.202.20±3.80Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E53.80±4.2037.00±5.2027.50±4.5017.10±4.00

[0140] Lactobacillusdelbrueckiissp. bulgaricusWeeks1234Control27.00±4.006.80±4.508.30±3.005.90±4.00Trehalose19.00±3.909.00±3.0012.00±3.509.00±3.00Maltodextrin27.00±4.3016.00±3.509.00±4.007.00±2.00Trehalose + Maltodextrin15.00±4.509.00±4.009.00±4.308.00±1.50Trehalose + Maltodextrin + L-Cysteine27.00±4.8019.00±4.4012.00±4.505.00±2.50Trehalose + Maltodextrin + L-Cysteine + Vitamin A17.00±4.7012.00±3.808.00±4.006.00±2.80Trehalose + Maltodextrin + L-Cysteine + Vitamin C13.70±4.9027.10±3.9022.30±4.108.40±5.00Trehalose + Maltodextrin + L-Cysteine + Vitamin E24.00±4.6017.10±3.708.30±4.204.20±3.80Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E49.80±4.2033.00±5.2029.50±4.5019.10±4.00

[0141] LacticaseibacillusrhamnosusWeeks1234Control33.00±5.0012.80±5.508.30±4.006.90±1.20Trehalose25.00±4.9015.00±4.0012.00±4.509.00±2.40Maltodextrin33.00±5.3022.00±4.509.00±5.005.00±2.60Trehalose + Maltodextrin21.00±5.5015.00±5.009.00±5.308.00±1.40Trehalose + Maltodextrin + L-Cysteine33.00±5.8025.00±5.4012.00±5.503.00±1.50Trehalose + Maltodextrin + L-Cysteine + Vitamin A23.00±5.7018.00±4.808.40±3.206.00±3.80Trehalose + Maltodextrin + L-Cysteine + Vitamin C19.70±5.9033.10±4.9022.30±5.108.40±1.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E30.00±5.6023.10±4.708.30±5.204.20±4.80Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E55.80±5.2039.00±6.2029.50±5.5018.10±5.00

[0142] LimosilactobacillusreuteriWeeks1234Control14.00±3.607.80±4.104.30±2.607.90±3.60Trehalose21.00±3.5011.00±2.608.00±3.105.00±2.60Maltodextrin19.00±3.908.00±3.105.00±3.606.00±4.60Trehalose + Maltodextrin17.00±4.1011.00±3.605.00±3.909.00±2.60Trehalose + Maltodextrin + L-Cysteine19.00±4.4021.00±4.008.00±4.104.00±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamin A19.00±4.3014.00±3.404.00±3.607.00±2.40Trehalose + Maltodextrin + L-Cysteine + Vitamin C15.70±4.509.10±3.5018.30±3.709.40±4.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E16.00±4.209.10±3.304.30±3.805.20±3.40Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E32.80±3.8025.00±4.8015.50±4.1010.10±3.60

[0143] EnterococcusfaecalisWeeks1234Control25.00±6.6013.38±5.308.20±3.507.80±1.70Trehalose17.00±6.5015.58±3.8011.90±4.009.90±1.30Maltodextrin25.00±6.9022.58±4.308.90±4.505.90±2.70Trehalose + Maltodextrin13.00±7.1015.58±4.808.90±4.808.90±0.70Trehalose + Maltodextrin + L-Cysteine25.00±7.4025.58±5.2011.90±5.003.90±0.20Trehalose + Maltodextrin + L-Cysteine + Vitamin A15.00±7.3018.58±4.607.90±4.506.90±0.50Trehalose + Maltodextrin + L-Cysteine + Vitamin C11.70±7.5033.68±4.7022.20±4.6011.30±2.70Trehalose + Maltodextrin + L-Cysteine + Vitamin E22.00±7.2023.68±4.508.20±4.705.10±1.50Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E47.80±6.8039.58±6029.40±5.0020.00±1.70

[0144] PediococcuspentosaceusWeeks1234Control21.00±4.6010.38±6.309.20±3.505.80±1.90Trehalose13.00±4.5012.58±4.8012.90±4.007.90±0.90Maltodextrin21.00±4.9019.58±5.309.90±4.505.90±1.00Trehalose + Maltodextrin9.00±5.1012.58±5.809.90±4.806.90±0.90Trehalose + Maltodextrin + L-Cysteine21.00±5.4022.58±6.2012.90±5.001.90±0.40Trehalose + Maltodextrin + L-Cysteine + Vitamin A11.00±5.3015.58±5.608.90±4.504.90±0.70Trehalose + Maltodextrin + L-Cysteine + Vitamin C7.70±5.5030.68±5.7023.20±4.609.30±2.90Trehalose + Maltodextrin + L-Cysteine + Vitamin E18.00±5.2020.68±5.509.20±4.703.10±1.70Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E43.80±4.8036.58±7.0030.40±5.0018.00±1.90

[0145] PediococcusacidilacticiWeeks1234Control34.00±3.6013.80±4.109.30±2.604.90±1.60Trehalose26.00±3.5016.00±2.6013.00±3.107.00±2.60Maltodextrin34.00±3.9023.00±3.1010.00±3.603.00±1.20Trehalose + Maltodextrin22.00±4.1016.00±3.6010.00±3.906.00±2.60Trehalose + Maltodextrin + L-Cysteine34.00±4.4026.00±4.0013.00±4.105.00±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamin A24.00±4.3019.00±3.409.00±3.604.00±1.20Trehalose + Maltodextrin + L-Cysteine + Vitamin C20.70±4.5034.10±3.5023.30±3.708.40±4.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E31.00±4.2024.10±3.309.30±3.806.20±3.40Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E56.80±3.8040.00±4.8030.00±4.1017.1±3.60

[0146] WeissellaconfusaWeeks1234Control28.00±5.6012.80±6.108.30±2.606.90±3.60Trehalose20.00±5.5015.00±4.6012.00±5.109.00±2.60Maltodextrin28.00±5.9022.00±5.109.00±5.605.00±1.60Trehalose + Maltodextrin16.00±6.1015.00±5.609.00±1.008.00±2.60Trehalose + Maltodextrin + L-Cysteine28.00±6.4025.00±6.0012.00±6.106.00±2.10Trehalose + Maltodextrin + L-Cysteine + Vitamin A18.00±6.3018.00±5.408.00±2.606.00±2.40Trehalose + Maltodextrin + L-Cysteine + Vitamin C14.70±6.5033.10±5.5022.30±5.7010.40±4.60Trehalose + Maltodextrin + L-Cysteine + Vitamin E25.00±6.2023.10±5.308.30±2.804.20±1.00Trehalose + Maltodextrin + L-Cysteine + Vitamins A, C, E50.80±5.8039.00±6.8029.50±6.1019.10±3.60

[0147] StreptococcusthermophilusWeeks1234Control33.00±4.6012.80±5.108.30±3.606.90±2.60Trehalose25.00 ±4.5015.00±3.6012.00±4.109.00±3.60Maltodextrin33.00±4.9022.00±4.1010.00±4.605.00±1.60Trehalose + Maltodextrin21.00±5.1015.00±4.6010.00±4.908.00±3.60Trehalose + Maltodextrin + L-Cysteine33.00±5.4025.00±5.0012.00±5.105.00±2.00Trehalose + Maltodextrin + L-Cysteine ​​+ Vitamin A23.00±5.3018.00±4.408.00±4.606.00±3.40Trehalose + Maltodextrin + L-Cysteine ​​+ Vitamin C19.70±5.5033.10±4.5022.30±4.708.40±5.60Trehalose + Maltodextrin + L-Cysteine ​​+ Vitamin E30.00±5.2023.10±4.3013.30±4.807.20±2.40Trehalose + Maltodextrin + L-Cysteine ​​+ Vitamins A, C, E55.80±4.8039.00±5.8029.5±5.1018.10±4.60

[0148]

[0149] As a result of the experiment, it was confirmed that the condition including all vitamins A, C, and E (Example 1-9) maintained the highest survival rate over time for all lactic acid bacteria, and was effective in maximizing the storage stability of probiotics (increasing distribution stability).

[0150]

[0151] In summary of the above results, it is judged that the antioxidant effect of the cryoprotectant according to Example 1-9 is effective in improving the survival rate of bifidobacteria by reducing oxidative stress generated during the freeze-drying process.

[0152] Therefore, by using the cryoprotectant according to Example 1-9 as a freeze-drying protectant for lactic acid bacteria, the freeze-drying recovery rate during production, stability during distribution, and functional stability in the body can be improved.

Claims

A composition for coating lactic acid bacteria comprising at least one of the group consisting of vitamins A, C and E. In claim 1, the composition for coating lactic acid bacteria further comprises trehalose, maltodextrin, and L-cysteine ​​HCl monohydrate. Contains trehalose, maltodextrin and L-cysteine ​​HCl monohydrate, vitamins and the remainder distilled water. The above vitamins include one or more of the group consisting of vitamins A, C and E, The above trehalose, maltodextrin and L-cysteine ​​HCl monohydrate are added in amounts of 2 to 8% (w / w), 1 to 3% (w / w) and 0.05 to 0.8% (w / w), respectively. A composition for coating lactic acid bacteria, wherein the above vitamins A, C and E are added in amounts of 0.01 to 0.05% (w / w), 0.1 to 0.5% (w / w) and 0.01 to 0.05% (w / w), respectively. A composition according to claim 2 or 3, wherein the lactic acid bacteria coating composition further comprises at least one combination selected from the group consisting of fructooligosaccharide, skim milk powder, tris (hydroxymethyl) aminomethane, citric acid, fructose, kanamycin sulfate, glycerol, glucose, sucrose, ethylene glycol, propylene glycol, DMSO, dextran, 1,2-propandiol, hyaluronic acid, mannitol, arginine, and albumin. In any one of the first to fourth paragraphs, The above lactic acid bacteria coating composition is, A composition which is a cryoprotectant. A method for increasing the survival rate of lactic acid bacteria, comprising the step of treating lactic acid bacteria with a composition according to any one of claims 1 to 4. Step (a) of treating lactic acid bacteria with a composition according to any one of claims 1 to 4; and A method for coating a freeze-drying agent, comprising a freeze-drying step (b); In paragraph 6, The above composition, A method of treating a lactic acid bacteria culture solution or a lactic acid bacteria culture concentrate. In paragraph 8, The above method, A method for improving the survival rate, freeze-drying recovery rate, in-vivo stability and distribution stability of lactic acid bacteria.

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