Manufacturing method for Multi layer coated probiotics using LMA Pectin and Multi layer coated probiotics therefrom

Abstract

The present invention relates to a method for manufacturing multi-coated lactic acid bacteria using amidated lowmethoxyl pectin, comprising the steps of: mixing a water-soluble polymer with lactic acid bacteria to form a first-coated lactic acid bacteria; mixing amidated lowmethoxyl pectin with the first-coated lactic acid bacteria to form a second-coated lactic acid bacteria; mixing a water-soluble sugar with the second-coated lactic acid bacteria to form a third-coated lactic acid bacteria; and coating the third-coated lactic acid bacteria with a protein substance that does not cause allergies to form a fourth-coated lactic acid bacteria.

Description

Technology Field

[0001] The present invention relates to a method for producing multi-coated lactic acid bacteria using amidated lowmethoxyl pectin and multi-coated lactic acid bacteria produced therefrom. Background Technology

[0002] Lactic acid bacteria refer to bacteria that produce lactic acid through fermentation. They are known to have a regulating effect on the digestive system by inhibiting the proliferation of harmful bacteria when colonizing the digestive tract. As health functional foods, lactic acid bacteria are primarily isolated from fermented foods such as kimchi, doenjang, cheese, and yogurt. Through research on their specific functionalities, they are recognized as individually approved ingredients by the Ministry of Food and Drug Safety. For example, it has been proven that lactic acid bacteria can help inhibit the growth of harmful bacteria, improve immune hypersensitivity, reduce body fat, and alleviate menopausal symptoms when they proliferate in the intestines. However, there are cases where lactic acid bacteria fail to reach the intestines alive due to death caused by gastric acid (stomach) and bile acids (small intestine) as they pass through the digestive system.

[0003] Various studies are being conducted to address these issues, and a representative method involves coating probiotics to ensure they survive reaching the intestines. However, conventional coating technologies required additional freeze-drying protective agents to ensure product safety. Furthermore, improvements are needed as coating agents such as skim milk powder, whey protein, and isolated soy protein are classified as allergens. Prior art literature

[0004] Korean Patent Publication No. 2009-0082035 (Published July 29, 2009) The problem to be solved

[0005] The present invention aims to provide a method for producing multi-coated lactic acid bacteria using bio-amidated lowmethoxyl pectin and multi-coated lactic acid bacteria produced therefrom. means of solving the problem

[0006] The objective of the present invention is achieved by a method for manufacturing multi-coated lactic acid bacteria using amidated lowmethoxyl pectin, comprising the steps of: mixing a water-soluble polymer with lactic acid bacteria to form a first-coated lactic acid bacteria; mixing amidated lowmethoxyl pectin with the first-coated lactic acid bacteria to form a second-coated lactic acid bacteria; mixing a water-soluble sugar with the second-coated lactic acid bacteria to form a third-coated lactic acid bacteria; and coating the third-coated lactic acid bacteria with a protein substance that does not cause allergies to form a fourth-coated lactic acid bacteria.

[0007] The above water-soluble polymer may include at least one of carboxymethyl cellulose (CMC), hydroxyethylcellulose (HEC), xanthan gum (XG), guar gum (GG), polyvinylpyrrolidone (PVP), carbopol, sodium alginate, and propylene glycol alginate.

[0008] The above water-soluble sugar may include at least one of sucrose, trehalose, maltose, lactose, and fructo-oligosaccharide.

[0009] The above protein material may include at least one of pea peptone, rice protein, and yeast extract.

[0010] In the first coating lactic acid bacteria formation step, the concentration of the water-soluble polymer may be 0.02 to 0.08 wt%, in the second coating lactic acid bacteria formation step, the concentration of the amidated lowmethoxyl pectin may be 0.05 to 0.3 wt%, in the third coating lactic acid bacteria formation step, the concentration of the water-soluble sugar may be 3 to 13 wt%, and in the fourth coating lactic acid bacteria formation step, the concentration of the protein may be 0.2 to 1.5 wt%.

[0011] The above water-soluble polymer includes guar gum or a guar gum derivative, the above water-soluble sugar includes trehalose, and the above protein may include pea peptone or pea protein.

[0012] The above lactic acid bacteria may include at least one of the genus Lactobacillus sp., the genus Bifidobacterium sp., the genus Streptococcus sp., the genus Lactococcus sp., the genus Enterococcus sp., the genus Clostridium sp., and the genus Bacillus sp.

[0013] The above lactic acid bacteria may include at least one of Streptococcus thermophilus IDCC 2201, Lacticaseibacillus rhamnosus IDCC 3201, Lactiplantibacillus plantarum IDCC 3501, and Bifidobacterium animalis ssp. lactis IDCC 4301.

[0014] The above lactic acid bacteria may include Lactibacillus plantarum IDCC 3501.

[0015] The step of forming the above secondary coated lactic acid bacteria can induce the formation of a gel of the amidated lowmethoxyl pectin using only calcium ions naturally present in the lactic acid bacteria culture solution or slurry, without the addition of separate calcium chloride (CaCl2).

[0016] Another objective of the present invention is achieved by multi-coated lactic acid bacteria prepared by the above method. Effects of the invention

[0017] According to the present invention, a method for producing multi-coated lactic acid bacteria using amidated lowmethoxyl pectin and multi-coated lactic acid bacteria produced therefrom are provided. Brief explanation of the drawing

[0018] Figure 1 is a graph comparing freeze-drying survival rates (HA vs LMA pectin) in an experimental example of the present invention, and FIG. 2 is a graph comparing acid resistance (pH2) survival rates in an experimental example of the present invention, and Figure 3 is a graph comparing the survival rates of bile-resistant (bile 0.3%) specimens in an experimental example of the present invention, and FIG. 4 is a comparison graph of logarithmic reduction in storage stability at 4°C in an experimental example of the present invention, and Figure 5 is a comparison graph of logarithmic reduction in storage stability at 25°C in an experimental example of the present invention. Specific details for implementing the invention

[0019] The present invention will be described in more detail below with reference to the drawings.

[0020] The attached drawings are merely examples illustrated to further explain the technical concept of the present invention, and therefore the concept of the present invention is not limited to the attached drawings. Additionally, the sizes and spacing, etc., in the attached drawings may be exaggerated from reality to explain the relationships between the components.

[0021] The present invention relates to a multi-layer coating technology for improving the viability and storage stability of probiotic lactic acid bacteria, and in particular, to a method for producing probiotics with improved freeze-drying viability, acid resistance, bile resistance, and long-term storage stability by applying amidated low methoxyl pectin (LMP-amide) to a secondary coating layer.

[0022] Existing probiotic lactic acid bacteria preparations have a problem in that they easily die during the freeze-drying process due to cell membrane damage, ice crystal formation, and pH changes. To solve this problem, multi-coating technology has been proposed. Among these, there is a technology that uses hyaluronic acid (HA) as a secondary coating agent, but (1) HA is an expensive raw material and has poor formulation stability, and (2) Ca 2+ There is a problem with low structural strength because it fails to induce base gel formation.

[0023] HM (High Methoxy) Pectin forms a gel only at high sugar concentrations and low pH, while LM (Low Methoxyl) Pectin Ca 2+ Due to high dependency, there is a problem where the effectiveness is limited if there is a lack of Ca in the culture medium.

[0024] In the present invention, naturally occurring Ca in culture media and slurries 2+ A manufacturing method using LMA pectin capable of stable coating is provided.

[0025] The lactic acid bacteria of the present invention may include at least one of the genus Lactobacillus sp., the genus Bifidobacterium sp., the genus Streptococcus sp., the genus Lactococcus sp., the genus Enterococcus sp., the genus Clostridium sp., and the genus Bacillus sp., but are not limited thereto.

[0026] The lactic acid bacteria of the present invention may include at least one of Streptococcus thermophilus IDCC 2201, Lacticaseibacillus rhamnosus IDCC 3201, Lactiplantibacillus plantarum IDCC 3501, and Bifidobacterium animalis ssp. lactis IDCC 4301, but is not limited thereto.

[0027] The lactic acid bacteria of the present invention may be a species of Streptococcus thermophillus, and in particular may be Lactiplantibacillus plantarum IDCC 3501.

[0028] Lactic acid bacteria can be formed with a quadruple coating, and depending on the coating stage, first-coated lactic acid bacteria, second-coated lactic acid bacteria, third-coated lactic acid bacteria, and fourth-coated lactic acid bacteria are formed.

[0029] Each coating is performed by stirring the filtered lactic acid bacteria solution with a coating agent. Stirring may be performed at room temperature, 10°C to 30°C, or 20°C to 30°C for 1 minute to 60 minutes, 2 minutes to 30 minutes, or 3 minutes to 10 minutes.

[0030] The coating material for each stage may be a combination of the following.

[0031] Primary coating: Guar gum or guar gum derivative

[0032] Secondary coating: Amidated lowmethoxyl pectin (Amidated LMP)

[0033] 3rd Coating: Trehalose

[0034] 4th coating: Pea protein or pea peptone

[0035] The primary coated lactic acid bacteria is obtained by mixing a water-soluble polymer with the lactic acid bacteria.

[0036] The water-soluble polymer may include at least one of carboxymethyl cellulose (CMC), hydroxyethylcellulose (HEC), xanthan gum (XG), guar gum (GG), polyvinylpyrrolidone (PVP), carbopol, sodium alginate, and propylene glycol alginate.

[0037] Water-soluble polymers are suitable for acting as a binder that blocks the ingress of external air during coating due to their excellent adhesion to the surface of lactic acid bacteria and has excellent binding affinity with hyaluronic acid, a secondary coating agent. Since most lactic acid bacteria are anaerobic, a base with excellent gas barrier properties that can be used for food and pharmaceutical purposes is desirable.

[0038] In the first coating lactic acid bacteria formation step, the concentration of the water-soluble polymer may be 0.01 to 0.2 wt%, 0.01 to 0.1 wt%, or 0.02 to 0.08 wt%.

[0039] Secondary coated lactic acid bacteria are obtained by mixing Low Methoxyl Amidated Pectin (LMA pectin) with primary coated lactic acid bacteria. LMA pectin is a natural polymer material with water activity regulating function that controls external moisture inflow. In the secondary coated lactic acid bacteria formation step, the concentration of LMA pectin may be 0.01 to 0.20 wt%, 0.02 to 0.30 wt%, or 0.05 to 0.30 wt%.

[0040] The second coating is achieved by inducing gel formation of the amidated lowmethoxyl pectin using only calcium ions naturally present in the lactic acid bacteria culture solution or slurry, without the addition of separate calcium chloride (CaCl2).

[0041] Third-coated lactic acid bacteria are obtained by mixing water-soluble sugars with second-coated lactic acid bacteria.

[0042] Water-soluble sugars form a coating film on the surface of the bacterial cells to block the inflow of external moisture and humid air, and when applied to high-concentration bacterial solutions, they increase the freeze-drying recovery rate and enable the securing of a high initial bacterial count during production.

[0043] Water-soluble sugars can be completely water-soluble. Water-soluble sugars are plant-derived, but corn-derived components are excluded. Water-soluble sugars function as freeze-drying protective agents.

[0044] Water-soluble sugars may include at least one of sucrose, trehalose, maltose, lactose, and fructo-oligosaccharide.

[0045] In the third coating lactic acid bacteria formation step, the concentration of the water-soluble sugar may be 2 to 20 wt%, 3 to 13 wt%, or 3 to 6 wt%.

[0046] Fourth-coated lactic acid bacteria are obtained by coating third-coated lactic acid bacteria with a protein substance that does not cause allergies.

[0047] Proteins bind to the surface of a tertiary-coated porous sugar coating agent, playing a role in inducing a higher moisture absorption prevention effect, providing excellent cell protection and excluding allergens.

[0048] The protein material includes at least one of pea peptone, rice protein, and yeast extract.

[0049] Pea peptone is isolated pea protein, and rice protein is isolated rice protein.

[0050] Pea peptone, rice protein, and yeast extract are products produced by treating peas, rice, and yeast, respectively, with enzymes such as proteases, cellulases, and amylases to hydrolyze them to an appropriate degree depending on the application, and then adding amino acids as needed to the dried powder; these products contain protein molecules of various sizes and amino acids and are effective as microbial growth agents or freeze-drying protective agents.

[0051] In the fourth coating lactic acid bacteria formation step, the protein concentration may be 0.1 to 2.0 wt%, 0.2 to 1.5 wt%, or 0.3 to 0.7 wt%.

[0052] The multi-coated lactic acid bacteria produced by the above method secured a high number of bacteria in the freeze-dried product of a high-concentration lactic acid bacteria solution and increased stability by effectively preventing moisture absorption. In addition, it does not cause allergies and has excellent acid and bile resistance.

[0053] Using amidated lowmethoxyl pectin (Amidated LMP) as a secondary coating has the following effects.

[0054] (1) Increase in freeze-dried survival rate by +11 to 15%p compared to HA

[0055] (2) Survival rate increased by +13 to 20%p under GI simulation conditions (pH 2, bile 0.3%)

[0056] (3) 4℃ / 25℃ storage stability 0.12 to 0.3 log decrease range

[0057] (4) Clear protective effect expressed only in amide LMP compared to HMP / LMP

[0058] The present invention will be explained in detail through the following experimental examples.

[0059] Coating experiment

[0060] The filtered high-concentration lactic acid bacteria concentrate was brought to room temperature (25℃), stirring was started, guar gum (0.03 wt%) was added, and stirring was performed for 5 minutes to complete the first coating. The lactic acid bacteria concentration in the high-concentration lactic acid bacteria concentrate was 200 to 300 billion CFU / mL.

[0061] Three types of lactic acid bacteria were used: Lacticase-Bacillus rhamnosus IDCC 3201, Lactiplantibacillus plantarum IDCC 3501, and Bifidobacterium lactis IDCC 4301.

[0062] 3 types of primary coated lactic acid bacteria were subjected to a secondary coating of 0.15 wt% LMA pectin to obtain secondary coated lactic acid bacteria. The mixing time between the primary coated lactic acid bacteria and pectin was 5 minutes.

[0063] A third coating was performed on the second coated lactic acid bacteria using 4.25 wt% trehalose to obtain third coated lactic acid bacteria.

[0064] The mixing time for the third coating was also 5 minutes.

[0065] Quaternary coated lactic acid bacteria are obtained by coating tertiary coated lactic acid bacteria with a protein substance that does not cause allergies. In the quaternary coated lactic acid bacteria formation stage, the concentration of the quaternary was 0.5 wt%.

[0066] The 4th coated lactic acid bacteria were frozen at -45℃ for 3 hours, freeze-dried to powder, and then the number of viable bacteria was measured.

[0067] The bacterial count of the concentrated mixture before initial freezing is as follows.

[0068] Lacticaseibacillus rhamnosus : 2.728± 0.33×10 11 CFU / mL

[0069] Lactiplantibacillus plantarum : 2.965± 0.54×10 11 CFU / mL

[0070] Bifidobacterium animalis ssp. Lactis : 2.446± 0.28×10 11 CFU / mL

[0071] The number of bacteria in the solid content of the concentrated mixture is as follows.

[0072] Lacticaseibacillus rhamnosus : 10.56×10 11 CFU / g

[0073] Lactiplantibacillus plantarum : 14.8×10 11 CFU / g

[0074] Bifidobacterium animalis ssp. lactis : 9.78×10 11 CFU / g

[0075] Replication: n=3 (mean ± SD)

[0076] The common conditions are as follows.

[0077] Bacteria count in powder after freeze-drying: 1.0×10 11 CFU / g

[0078] The strains are as follows, and

[0079] Lacticaseibacillus rhamnosus

[0080] Lactiplantibacillus plantarum

[0081] Bifidobacterium animalis ssp. lactis

[0082] The number of replicate experiments was n=3 (mean ± SD).

[0083] Analysis Results - Freeze-dried Survival Rate

[0084] Table 1 shows the freeze-drying survival rate (%) and bacterial count (mean ± SD).

[0085] As shown in Table 1 and Figure 1, LMA pectin exhibits a survival rate approximately 7.7 to 28.2% higher than HA in all strains. In particular, L. plantarum In the case of the IDCC3501 strain, the freeze-drying survival rate increased by approximately 12% compared to conventional hyaluronic acid when LMA pectin was applied.

[0086]

[0087]

[0088] Analysis Results - Acid Resistance (pH 2) and Bile Resistance (0.3% bile)

[0089] Table 2 shows pH 2 acid resistance (mean ± SD).

[0090] Table 2

[0091]

[0092] As shown in Table 2 and Figure 2, L. rhamnosus , L. plantarum , B. animalis ssp. lactis When LMA pectin was applied, acid resistance increased by 4%, 1%, and 1%, respectively, compared to conventional hyaluronic acid.

[0093] Table 3 shows the biliability of 0.3% bile (mean ± SD).

[0094] Table 3

[0095]

[0096] As shown in Table 3 and Figure 3, L. rhamnosus , L. plantarum , B. animalis ssp. lactis When LMA pectin was applied, bile tolerance increased by 2%, 2%, and 4% compared to hyaluronic acid, respectively.

[0097] Analysis Results - Storage Stability (4℃ and 25℃, 30 days)

[0098] Table 4 shows the 30-day storage stability (mean ± SD) at 4℃.

[0099]

[0100]

[0101] As shown in Table 4 and Figure 4, L. rhamnosus , L. plantarum , B. animalis ssp. lactis When LMA pectin was applied, the 30-day storage stability at 4°C increased by 13.9%, 20.6%, and 13.7% compared to hyaluronic acid, respectively. Furthermore, compared to non-amidated LMP, it increased by 18.2%, 8.9%, and 15.1%, respectively. Through this, the application of LMA pectin, compared to hyaluronic acid L. plantarum It can be seen that the strain is particularly excellent.

[0102] Table 5 shows the 30-day storage stability (mean ± SD) at 25°C.

[0103] Table 5

[0104]

[0105] As shown in Table 5 and Figure 5, L. rhamnosus , L. plantarum , B. animalis ssp. lactis When LMA pectin was applied, the 30-day storage stability at 25°C increased by 9.1%, 12.1%, and 12.4% compared to hyaluronic acid, respectively. Furthermore, compared to non-amidated LMP, it increased by 0.9%, 5.4%, and 6.1%, respectively. Through this, the application of LMA pectin, compared to hyaluronic acid L. plantarum and B. animalis ssp. lactis It can be seen that the strain is particularly excellent.

Claims

Claim 1 A method for manufacturing multi-coated lactic acid bacteria using amidated lowmethoxyl pectin comprises: a step of forming a first-coated lactic acid bacterium by mixing a water-soluble polymer with the lactic acid bacteria; a step of forming a second-coated lactic acid bacterium by mixing amidated lowmethoxyl pectin with the first-coated lactic acid bacterium; a step of forming a third-coated lactic acid bacterium by mixing a water-soluble sugar with the second-coated lactic acid bacterium; and a step of forming a fourth-coated lactic acid bacterium by coating the third-coated lactic acid bacterium with a protein substance that does not cause allergies, wherein the lactic acid bacteria include Lactiplantibacillus plantarum IDCC 3501, and the step of forming the second-coated lactic acid bacterium induces gel formation of the amidated lowmethoxyl pectin using only calcium ions naturally present in the lactic acid bacteria culture medium or slurry without the addition of separate calcium chloride (CaCl2). Claim 2 A method for manufacturing, wherein the water-soluble polymer comprises at least one of carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC), xanthan gum (XG), guar gum (GG), polyvinylpyrrolidone (PVP), carbopol, sodium alginate, and propylene glycol alginate. Claim 3 A method of preparation according to claim 2, wherein the water-soluble sugar comprises at least one of sucrose, trehalose, maltose, lactose, and fructo-oligosaccharide. Claim 4 In paragraph 3, the protein material comprises at least one of pea peptone, rice protein, and yeast extract. Claim 5 A method for manufacturing according to claim 4, wherein the concentration of the water-soluble polymer in the first coating lactic acid bacteria formation step is 0.02 to 0.08 wt%, the concentration of the amidated lowmethoxyl pectin in the second coating lactic acid bacteria formation step is 0.05 to 0.3 wt%, the concentration of the water-soluble sugar in the third coating lactic acid bacteria formation step is 3 to 13 wt%, and the concentration of the protein in the fourth coating lactic acid bacteria formation step is 0.2 to 1.5 wt%. Claim 6 A method of preparation according to claim 5, wherein the water-soluble polymer comprises guar gum or a guar gum derivative, the water-soluble sugar comprises trehalose, and the protein comprises pea peptone or pea protein. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 In a multi-coated lactic acid bacterium, the multi-coated lactic acid bacterium prepared according to any one of claims 1 to 6.

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