KR101605516B1 - Method for Increasing Viability, Storage Stability, Acid Tolerance or Oxgall Tolerance of Lactic Acid Bacteria - Google Patents

Abstract

The present invention provides a method of increasing viability, storage stability, acid resistance, and bile resistance of the lactic acid bacteria after freeze-drying the lactic acid bacteria by adding proline to the lactic acid bacteria while lactic acid bacteria are being cultivated or after lactic acid bacteria are cultivated. A method of the present invention not only increases stabilities for external environmental stresses such as freeze-drying stability, storage stability and others of lactic acid bacteria themselves, but also remarkably improves acid resistance and bile resistance as stability indices of intestinal canal environment during intaking of lactic acid bacteria by containing a protection agent such as proline in lactic acid bacteria in a high concentration. Conventional technologies have dropped economic feasibilities and are limited in industrial applications by using complicated methods of constructing physical barriers on outer parts of the lactic acid bacteria by performing multistep coating processes such as a double coating process, a triple coating process and a quadruple coating process on protein, polysaccharide, porous polymer, edible oil and fat, etc. On the other hand, the present invention is an economical method which increases viability of the lactic acid bacteria during passing of the lactic acid bacteria through the gastrointestinal tract, is capable of exhibiting inherent functionality of the lactic acid bacteria in an intestine, and is capable of overcoming a problem of cost rise according to existing multistep coating processes by greatly increasing resistance to external physiochemical stresses of the lactic acid bacteria themselves.

Description

[0001] The present invention relates to a method for increasing the survival rate, storage stability, acid resistance or biliary properties of a lactic acid bacterium,

The present invention relates to a method for increasing the survival rate, storage stability, acid resistance or biliary cholestasis of a lactic acid bacterium.

Lactic acid bacteria are bacteria that produce saccharides by using saccharides as an energy source. They are found in digestive tracts, oral cavity and vagina of humans and mammals. They are widely distributed in nature such as various fermented foods. Lactic acid bacteria is one of the microorganisms that humans have used extensively for a long time and is a microorganism that does not produce harmful substances in human or animal fields and has a pure function to prevent corruption in the intestines.

Currently, lactic acid bacteria are classified into 12 genera ( Lactobacillus, Carnobacterium , Atopobium , Lactococcus , Pediococcus , Tetragenococcus, Leuconostoc , Weisella , Oenococcus , Enterococcus , Streptococcus and Vagococcus ). Lactobacillus commonly used by us is bacterium Lactobacillus sp .), a strain of Lactococcus ( Lactococcus sp .), Streptococcus sp ., Leuconostoc sp . sp .), and Pedioccus spp. sp .) (BioWave, 2009, Vol.11, No. 7, pp. 1-20).

Probiotics, on the other hand, are foods that contain live microorganisms or live microorganisms that have a health effect on the host (FAO / WHO 2001), and have important functions to maintain and regulate human intestinal microorganisms at normal levels. Typical probiotics include lactic acid bacteria and Bifidobacterium , and yeast ( Saccharomyces cerevisiae and Saccharomyces boulardii ) and Aspergillus oryzae (BioWave, 2009, Vol. 11, No. 7, pp. 1-20).

Antibiotic activity, antibiotic-related diarrhea reduction, anticancer effect, anti-cancer effect, blood cholesterol lowering, Helicobacter pylori fungus inhibition, irritable colitis, Crohn's disease, ulcerative colitis relief, immune function control (International Dairy Journal, 2007, Vol. 17, pp. 12621277). Probiotics can be divided into the formulations of human drugs, lactose, lactic acid bacteria, probiotics as feed additives, and lactic acid bacteria, a kind of health food. Lactic acid bacteria food is made by cultivating live bacteria such as lactic acid bacterium, lactic acid bacterium and bifidus bacteria and mixing them with foodstuffs in powder, granules, tablets, capsules etc. for stable and easy ingestion. Lactic acid bacteria fermented food, lactic acid fermented milk, It says.

The process for producing the above-mentioned lactic acid bacteria food is largely classified into culturing of lactic acid bacteria, recovery of cells, freeze-drying, crushing, and commercialization. In this process, lactic acid bacteria are exposed to various physicochemical stresses. In other words, the cells are affected by the osmotic pressure due to concentration during the recovery of the cells. During the lyophilization process, the temperature and the osmotic pressure are influenced by the formation of ice crystals in the cytoplasm or the dehydration generated outside the cells due to the rapid temperature change At the same time. In addition, the stability of the lactic acid bacterium is lowered when exposed to high temperature and high pressure or by moisture in the air during pulverization and commercialization (International Dairy Journal, 2004, Vol. 14, pp. 835847; Journal of Applied Microbiology, 2005, Vol. 98, pp. 14101417), liquid foods such as fermented foods of lactic acid bacteria, fermented milk of lactic acid bacteria and drinks of lactic acid bacteria stored for a short period of time, It has been pointed out that even in the case of a product made in the form of a fatty acid, the fatty acid constituting the cell membrane is oxidized and the survival rate is reduced when exposed to oxygen (Comprehensive Reviews in Food Science and Food Safety, 2004, Vol. Curr Issues Intest Microbiol, 2004, Vol. 5, pp. 1-8; Letters in Applied Microbiology, 1996, Vol. 22, No. 1, pp. 3438).

On the other hand, unlike other industrial microorganisms, probiotics products are exposed to various stresses in the human body before reaching the post-intake stage because they use live bacteria. In the gastrointestinal tract, the exposure to acidic environment falls below pH 3, and in the small intestine, the survival rate may be greatly reduced due to the effects of secreted digestive enzymes and bile acids. In addition, even when it reaches the intestine, it competes with the microorganisms established in the intestinal tract and is inhibited by various harmful components and active oxygen (Immunology and Cell Biology, 2000, Vol. 78, pp. 8088; Am J Clin Nutr, 2001, Vol. 73, pp. 393S398S (suppl); Probiotic Bacteria and Enteric Infections, 2011, Chap.2, pp. 41-63).

Various methods for coating lactic acid bacteria have been developed in order to solve the above problems. Initially, encapsulation using an encapsulating agent and microencapsulation using gelatin, polysaccharide, gum, etc. have been performed. However, Has been pointed out. To solve this problem, a method of producing a double structure jelly in which a high concentration of a lactic acid bacterium is active or a method of double coating a protein with a polysaccharide (Korean Patent Application No. 10-2003-0020375, Korean Patent Application No. 10-2001-0010397) A triple coating method in which nanoparticles are added to a protein and polysaccharide coating has been introduced (Korean Patent Application No. 10-2008-0008267). However, the improved lactic acid bacteria coating technique also fails to completely coat the surface of the lactic acid bacteria, so that the heat resistance, acid resistance and bile resistance of the lactic acid bacteria are not sufficiently excellent. In order to overcome this problem, there has been proposed a method in which a triple coating is further coated with an additional edible oil (Korean Patent Application No. 10-2011-0093074), a method of coating a water-soluble polymer, hyaluronic acid, a coating agent having porous particles, Korean Patent Application No. 10-2011-0134486) have been competitively developed. However, the above-mentioned lactic acid bacteria coating technology has a disadvantage in that aseptic manipulation is difficult as a probiotic agent because the lactic acid bacteria cultured by a conventional method is recovered and then the multi-step process of mixing and stirring the coating composition is carried out. There is a problem of falling.

On the other hand, it is known that probiotic microorganisms can be exposed to various stresses such as osmotic pressure, pH, and oxygen from the time of cultivation, and thus exhibit various physiological responses to cope with changes in the external environment. According to culturing conditions, (Comparative Biochemistry and Physiology Part A, 2001, Vol. 130, pp. 437-460; Journal of Applied Microbiology, 2005, Vol. 99, pp. 13301339; Dairy Sci., 2005, Vol. 88, pp. 21-29, Biochemical Engineering Journal, 2010, Vol.52, pp. 65-70).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors of the present invention have made efforts to increase the stability of lactic acid bacteria in culture and storage. As a result, it has been found that when amino acids including proline are added during or after lactic acid bacteria culture, the survival rate and storage stability after lyophilization can be remarkably increased, Acidity and brittleness were greatly increased, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for increasing survival rate, storage stability, acid resistance and biliary cholesterol after lyophilization of lactic acid bacteria.

Another object of the present invention is to provide a lactic acid bacterium having increased survival rate, storage stability, acid resistance and biliary cholesteriness after lyophilization produced by the above method.

Other objects and advantages of the present invention will become more apparent from the following description and claims of the present invention.

According to one aspect of the present invention, there is provided a method for producing a lactic acid bacterium, comprising adding at least one amino acid selected from the group consisting of proline, aspartic acid, serine, threonine, glutamic acid, lysine and valine, A method for increasing the survival rate, storage stability, acid resistance, and bile resistance after lyophilization is provided.

The inventors of the present invention have made efforts to increase the stability of lactic acid bacteria in culture and storage. As a result, it has been found that when amino acids including proline are added during or after lactic acid bacteria culture, the survival rate and storage stability after lyophilization can be remarkably increased, Acidity, and biliary properties were significantly increased.

The term " survival rate " as used herein means the percentage of viable cell count after lyophilization divided by viable cell count before lyophilization.

The method of the present invention will be described in detail as follows:

According to the present invention, lactic acid bacteria are first cultured.

The lactic acid bacteria used in the present invention are not particularly limited and include, for example, Lactobacillus sp., Streptococcus sp., Lactococcus sp., Enterococcus sp. Pediococcus sp., Leuconostoc sp., Weissella sp., Or Bifidobacterium sp. Lactic acid bacteria.

According to another embodiment of the present invention, the lactic acid bacteria to be used in the present invention may be selected from the group consisting of L. plantarum , L. acidophilus , Bifidum bifidum , Bifidobacterium longum ( B. longum ) or leucono stokescensenteroid ( L. mesenteroides ).

According to a particular embodiment of the present invention, the lactic acid bacteria of the present invention are selected from the group consisting of Lactobacillus plantarum KCTC3108, Lactobacillus acidophilus KCTC3142, Bifidobacterium bifidum KCTC3202, Bifidobacterium longum KCTC3128 or Leuconostomercenteroid KCTC3100.

The cultivation of lactic acid bacteria in the present invention is carried out under conventionally known lactic acid bacteria culture medium and culture conditions known in the art.

According to the present invention, in order to increase the survival rate, storage stability, acid resistance and bile resistance after lyophilization of lactic acid bacteria, amino acid is added during cultivation of lactic acid bacteria or after cultivation of lactic acid bacteria.

According to one embodiment of the present invention, the amino acid is added at the time of preparing the culture medium for lactic acid bacteria, at 0 hours after the cultivation of the lactic acid bacteria or at the end of the culture, or before the lyophilization after the recovery of the lactic acid bacteria.

According to another embodiment of the present invention, an amino acid (for example, proline) is added at the time when the growth of the lactic acid bacteria reaches the stationary phase during the culture of the lactic acid bacterium.

According to one embodiment of the present invention, the amino acid added to increase survival rate, storage stability, acid resistance and biliary cholesterol after lyophilization of lactic acid bacteria is selected from the group consisting of proline, aspartic acid, serine, threonine, glutamic acid, lysine and valine One or more amino acids selected.

According to another embodiment of the present invention, the amino acid used in the present invention is proline.

According to one embodiment of the present invention, the added concentration of proline is 1 g / L to 50 g / L, 2 g / L to 20 g / L or 2 g / L to 10 g / L.

According to an embodiment of the present invention, there is further included a step of adding a cryoprotectant or a coating agent to the lactic acid bacteria having increased stability by addition of an amino acid to prepare a film-forming lactic acid bacterium.

The cryoprotectant used in the present invention is proline, trehalose or glycerin, and the coating agent is chitosan, malto-dextrin, indigestible dextrin, It is known that xanthan gum (XG), guar gum (GG), carboxymethyl cellulose (CMC), hydroxyethylcellulose (HEC), polyvinylpyrroridone (PVP) But are not limited to, carbopol, sodium alginate, propylene glycol alginate, alginate, polyethyleneglycol (PEG), triacetin, propylene glycol, acetyl Acetyl triethyl citrate or triethyl citrate.

According to the present invention, the lactic acid bacterium of the present invention is freeze-dried after completion of cultivation in accordance with addition of amino acid.

As verified in the following examples, the lyophilized Lactobacillus after the culture according to the method of the present invention exhibited remarkably increased survival rate, storage stability, acid resistance and bile resistance.

According to the present invention, the method of increasing the resistance of the cells themselves to extreme environments by adding amino acids during or after culturing is simpler and more economical than the method of constructing physical barriers with the conventional multi-stage coating, Very high. On the other hand, when the multistage coated lactobacillus product produced by the prior art is ingested, there is a high possibility that the lactic acid bacterial coating agent is not dissociated in the intestines and is discharged through the intestines. Even if dissociated, the survival rate is difficult to be secured in a rapidly changing intestinal environment . On the other hand, the lactic acid bacterium produced by the method of the present invention has a high survival rate in the process of rehydration when the lactic acid bacterium itself has a high resistance to the extreme environment.

The present invention relates to a process for the production of microorganisms capable of accumulating high concentrations of amino acids in microbial cells by adding amino acids during lactic acid bacterial cultivation, recovering the microbial cells, mixing them with a protective agent or a coating agent and lyophilizing the microbial cells so that the microbes are highly resistant to acid, Lactic acid bacteria can be produced.

According to another aspect of the present invention, there is provided a lactic acid bacterium having increased survival rate, storage stability, acid resistance, and bile resistance after lyophilization prepared by the above-described method.

The lactic acid bacteria produced by the above method have hydrogen peroxide resistance and hydrogen peroxide decomposing activity.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a method for increasing survival rate, storage stability, acid resistance, and bile resistance after lyophilization of lactic acid bacteria by adding proline during culturing or culturing of lactic acid bacteria and lactic acid bacteria produced by the method .

(Ii) The method of the present invention not only increases the stability against external environmental stresses such as freeze-drying stability and storage stability of the lactic acid bacteria itself by including a proline or the like such as proline in the lactic acid bacterium at a high concentration, Acid resistance, and bile resistance.

(Iii) The prior art uses a complicated method of building a physical barrier outside the lactic acid bacteria by applying a multi-step coating such as a double coating, a triple coating, or a quadruple coating with protein, polysaccharide, porous polymer, edible oil, The present invention significantly increases the resistance to external physicochemical stress of the lactic acid bacteria itself, thereby increasing the viability during passage through the gastrointestinal tract and allowing the lactic acid bacteria to exert their inherent functionality in the intestines. In addition, This is an economical way to overcome the problem of cost increase caused by

FIG. 1 shows the results of confirming osmotic pressure and intracellular proline concentration changes during the culture of Lactobacillus plantarum KCTC3108.
FIG. 2 is a graph showing the relationship between proline (a) and proline (b), proline (b) and proline (c) This is the chromatogram of my proline.
FIG. 3 is a result of quantifying intracellular proline concentration according to the concentration of proline.
FIG. 4 shows the results of quantifying intracellular proline concentration according to the time point of addition of proline.
5 is a schematic diagram of a prior art manufacturing method using a multistage coating.
6 is a schematic view of the manufacturing method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Amino acid addition after cell recovery

In order to cultivate lactic acid bacteria at a high concentration for use in the production of probiotics, medium and culture conditions optimized for each species were used. After the centrifugation, the cells were rapidly frozen in a freezer at -60 ° C, ≪ / RTI > and < RTI ID = 0.0 > 45 C < / RTI > The following experiment was conducted to investigate the effect of amino acid added after cell recovery on stability during freeze drying and storage. In the present invention, the culture medium of Lactobacillus plantarum KCTC3108 and Lactobacillus acidophilus KCTC3142 were concentrated and 20 amino acids were added to the culture medium to have a concentration of 5 g / L, respectively. The 20 amino acids were added to the concentrate by lyophilization, and then the survival rate, accelerated stability (40 ° C, 70% humidity), acid resistance and biliary cholesterol were observed upon freeze drying.

In order to evaluate the storage stability of the probiotics during long term circulation, the stability of the probiotics was evaluated under the conditions of 40 ℃ and 70% humidity for 4 weeks. After exposure to artificial gastric conditions, viable counts were analyzed. Specifically, artificial gastric juice conditions were artificial gastric juice conditions (2.0 g of sodium chloride, 24.0 ml / L of diluted hydrochloric acid, pH 1.2) in the food shattering test, and 10% concentration of probiotics powder was added, 100 reciprocating movements per minute were carried out and the exposure time was 2 hours considering the passage time. Experimental groups exposed to artificial gastric conditions were analyzed by the usual method of measuring viable counts after pH was adjusted to 7.0. Probiotics without amino acids were used as comparative test groups.

On the other hand, bile acid is produced in the liver, secreted into the small intestine through the bile duct, absorbed again by the ileum at the end of the small intestine, and circulates intestinally into the liver again. Because bile acid affects the small intestine, the survival rate of lactic acid bacteria exposed to bile acid was compared in vitro. Specifically, a medium in which bile acid was not added and a medium in which 0.5% of bile acid was filtered and aseptically added was used. 1 g of each lactic acid bacterium sample was inoculated into the prepared medium, reacted for 2 hours, And the number of viable cells was measured.

As shown in Table 1 and Table 2, the amino acid treatment of the concentrate after culturing the lactic acid bacteria was analyzed as follows.

The highest survival rate of Lactobacillus plantarum KCTC3108 after lyophilization of Lactobacillus plantarum KCTC3108 was in the group treated with proline and aspartic acid. The results of accelerated experiment showed that the survival rate of Lactobacillus plantarum KCTC3108 was highest in the proline, Respectively. In addition, the amino acid which showed excellent results in acid resistance and bile acid resistance was proline. Furthermore, it was confirmed that acid tolerance and bile resistance were also excellent when threonine and glutamic acid were used.

Lactobacillus acidophilus KCTC3142 ( Lactobacillus acidophilus KCTC3142) survived after lyophilization. Lecithin (lysine) and proline treatment were the best survival rates. The accelerated test (40 ℃, 70% Treated group showed excellent results. And acid tolerance and biliary properties showed excellent results in proline and glutamine treatment groups, respectively.

Effect of amino acid added after cell recovery (Lactobacillus plantarum KCTC 3108) amino acid Freeze-dried Accelerated test
(40 DEG C, 70% humidity) Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) One Not added 21 8.00E + 10 22 19 14 2 Alanine 23 2.40E + 10 10 11 9 3 Cysteine 13 9.70E + 09 8 13 8 4 Aspartic acid 28 4.20E + 10 18 19 11 5 Glutamic acid 10 1.60E + 10 16 30 21 6 Phenylalanine 19 4.20E + 10 23 27 14 7 Glycine 14 1.80E + 10 13 28 17 8 Histidine 16 5.00E + 10 21 24 12 9 Isoleucine 19 3.50E + 10 19 21 11 10 Lysine 22 3.40E + 10 16 27 9 11 Leucine 20 3.60E + 10 18 22 13 12 Methionine 10 4.30E + 10 13 18 17 13 Asparagine 15 1.70E + 10 17 19 11 14 Proline 32 1.00E + 11 30 34 20 15 Glutamine 11 1.10E + 10 10 7 18 16 Arginine 23 2.60E + 10 11 17 13 17 Serine 12 6.50E + 10 25 7 10 18 Threonine 9 2.10E + 10 23 32 19 19 Balin 13 8.30E + 09 6 10 11 20 Tryptophan 19 4.40E + 10 23 28 20 21 Tyrosine 18 3.40E + 10 19 7 10

Effect of Amino Acid Types Added After Cell Recovery (Lactobacillus ashophilus KCTC 3142) Amino acid source Freeze-dried Accelerated test
(40 DEG C, 70% humidity) Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) One Not added 22 6.10E + 10 11 24 23 2 Alanine 10 5.00E + 10 9 18 13 3 Cysteine 12 6.00E + 10 10 5 10 4 Aspartic acid 21 9.20E + 10 16 22 17 5 Glutamic acid 15 7.10E + 10 12 14 20 6 Phenylalanine 14 8.50E + 10 15 16 21 7 Glycine 21 7.80E + 10 13 22 18 8 Histidine 15 5.00E + 10 9 20 22 9 Isoleucine 18 6.20E + 10 12 5 4 10 Lysine 26 7.00E + 10 14 14 10 11 Leucine 20 4.20E + 10 7 22 15 12 Methionine 10 5.11E + 10 12 11 10 13 Asparagine 21 8.90E + 10 13 12 15 14 Proline 29 1.14E + 11 24 30 28 15 Glutamine 23 7.20E + 10 12 28 27 16 Arginine 9 5.45E + 10 10 12 11 17 Serine 18 6.60E + 10 11 6 5 18 Threonine 19 9.40E + 10 16 22 20 19 Balin 14 1.06E + 11 18 15 17 20 Tryptophan 10 9.00E + 10 15 20 13 21 Tyrosine 16 8.60E + 10 12 15 18

Example 2: Addition by concentration after cell recovery

Since 20 amino acids were added to the concentrate after the cultivation of the lactic acid bacteria, the proline was the most effective amino acid in the freeze-drying survival rate, the acceleration stability, the acid resistance and the bile resistance. Therefore, the lactobacillus plantarum KCTC 3108 culture was concentrated After lyophilization, proline was added to the concentrate in the concentration range of 0-50 g / L. After 4 weeks of accelerating test (40 ℃, 70% humidity) and acid tolerance and biliary tests, the concentration of proline was increased from 5 g / L to 10 g / L. Survival rates were all improved and acid resistance was also improved (Table 3).

Effect of concentration of proline added after concentration of culture medium Proline (g / L) Freeze-dried Accelerated test Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) 0 21 8.00E + 10 22 19 14 5 32 1.00E + 11 30 34 20 10 45 2.40E + 11 48 41 20 25 37 1.86E + 11 40 38 22 50 33 1.70E + 11 38 37 20

Example 3: Effect of proline addition on strains after concentration of culture medium

In order to confirm that the effect of adding proline to the concentrate after the cultivation of Lactobacillus plantarum KCTC 3108 in Example 2 was applied to other lactic acid bacteria, five kinds of lactic acid bacteria including Lactobacillus plantarum KCTC 3108 were cultured and recovered, 10 g / L, and lyophilization was carried out according to the method of Example 1. As a result, the survival rate, accelerated stability and acid resistance after freeze - drying were improved in all strains used in the experiment. In the case of Lactobacillus plantarum KCTC 3108 strain, the freeze - drying survival rate and survival rate after accelerated test were the highest when 10 g / L of proline was added and the acid resistance was greatly increased.

Effect of proline added after concentration of culture solution by strain Proline
Whether to add Strain Freeze-dried Accelerated test Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) Not added L. plantarum KCTC3108 21 8.00E + 10 22 19 14 L. acidophilus KCTC3142 22 6.10E + 10 11 24 23 B. bifidum KCTC3202 20 8.00E + 10 10 10 15 B. longum KCTC3128 25 8.70E + 10 15 20 22 L. mesenteroides KCTC3100 30 1.00E + 11 31 32 20 10 g / L
adding L. plantarum KCTC3108 45 2.40E + 11 48 41 20 L. acidophilus KCTC3142 32 2.50E + 11 34 38 31 B. bifidum KCTC3202 34 1.00E + 11 15 17 15 B. longum KCTC3128 38 2.10E + 11 31 24 25 L. mesenteroides KCTC3100 42 2.00E + 11 58 35 23

Example 4: Proline concentration according to osmotic pressure of lactic acid bacteria cells

In the present invention, Lactobacillus plan tarum KCTC 3108 (Lactobacillus plantarum KCTC3108), Lactobacillus ash FIG filler's KCTC 3142 (Lactobacillus acidophilus KCTC3142), Bifidobacterium bipyridinium bonus KCTC3202 (Bifidobacterium bifidum KCTC3202), Bifidobacterium ronggeom KCTC3128 (Bifidobacterium longum KCTC3128), Leuconostoc mesenteroides KCTC3100 ( Leuconostoc mesenteroides KCTC3100) and osmotic pressure and intracellular proline concentration. The changes in osmotic pressure and intracellular proline concentration during the culture of Lactobacillus plantarum KCTC3108 are shown in Fig.

It was confirmed that the osmotic pressure was increased by the various organic acids produced by the lactic acid bacteria and the concentration of intracellular proline was increased with the lapse of incubation time.

Example 5: Analysis of intracellular amino acid concentration after addition of proline during culture

After adding 5 g / L of proline in the culture of lactic acid bacteria, experiments were conducted to confirm the change of the concentration of proline in the intracellular amino acids. The culture was centrifuged, washed with MES buffer, and the washed cells were disrupted using an ultrasonic generator (Vibra-Cell-Sonics & Materials, Inc.). Amino acid concentrations in the disrupted cells were analyzed using a high performance liquid chromatography-vaporization light scattering detector (HPLC-ELSD) system.

A solution (0.1% aqueous formic acid) and a solution B (acetonitrile) were used as the mobile phase, using Xbridge amide column (Xbridge amide 3.5 μm, 250 × 4.6 mm And the flow rate was 1.5 mL / min. Gradient conditions were as follows: Initial 20% solution A, 80% solution B, 0-5 min: 25% solution A, 75% solution B, 5-13 min: 30% solution A, 70% -18 minutes: 20% of solution A and 80% of solution B were used, and the column temperature was 35 ° C. The detector was 380-ELSD (Agilent Technology). The nitrogen gas injection rate of the nebulizer was 2 L / min and the drift tube temperature was 90 ° C.

FIG. 2 is a chromatogram showing intracellular proline when proline is added to proline at a concentration of 5 g / L when proline is not added to a proline standard product by high-performance liquid chromatography-vaporization light scattering detector (HPLC-ELSD) FIG. 3 is a result of quantifying intracellular proline concentration according to the concentration of proline.

Example 6: Effect of concentration of proline added during culture

Considering that intracellular proline concentration is increased in the late stage of culture in Example 4 and that the stability of the lactic acid bacterium is improved in the subsequent stage when proline is added after concentrating the culture from Example 1-3, To induce intracellular proline accumulation at a high concentration, followed by lyophilization and accelerated stability.

For this purpose, proline was added at 0 g / L, 2.5 g / L and 5 g / L (1 g / L) at 8 hours after the culture corresponding to the log phase of the growth phase of the Lactobacillus plantarum KCTC3108 , 7.5 g / L, and 10 g / L, respectively. After 18 hours of incubation, each culture was concentrated by centrifugation, lyophilized, and then subjected to an accelerated test (40 ° C, 70% humidity) for 4 weeks and an acid-fast bacilli test (Table 5).

As a result, the freeze - dried survival rate increased as the concentration of proline increased, which was the highest at 5 g / L, and then showed a similar level. The 4 - week accelerated stability was the best when added at 5 g / L and the accelerated stability was lowered at the higher concentration. In addition, as the concentration of proline increased during the culture, the effect on acid resistance tended to increase, but the biliary properties were slightly increased.

Effect of concentration of proline addition During cultivation
Proline addition (g / L) Freeze-dried Accelerated test Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) 0 21 8.00E + 10 22 19 14 2.5 48 2.00E + 11 24 34 20 5 55 3.81E + 11 57 43 22 7.5 53 3.67E + 11 50 45 21 10 53 3.50E + 11 49 45 21

Example 7: Influence of proline addition at the time of culture

The addition of 5 g / L of proline to the medium during the fermentation period in Example 6 was effective in improving freeze-drying and accelerated stability. Therefore, the effect of proline addition during the culture period was examined based on the results. Specifically, the effect of adding proline during culturing was investigated using Lactobacillus plantarum KCTC 3108 ( Lactobacillus plantarum KCTC 3108). 5 g / L of proline was added at the initial stage of culture or 5 g / L of proline was added at the time of reaching the log phase or stationary phase in the growth stage of the cells. After 18 hours of incubation, (Fig. 4). ≪ tb >< TABLE >

In addition, each culture was centrifuged and then lyophilized according to a conventional method. After 4 weeks of accelerated testing (40 ° C, 70% humidity), acid resistance and biliary cholesterol were tested (Table 6) . When the growth of the bacteria reached the stopping point, proline was added, and after 2 hours, the culture solution was recovered and lyophilized, and the freeze-drying survival rate, the survival rate after the accelerated test, and the acidity and bile resistance were improved there was.

Influence of addition of proline during culture During cultivation
Proline addition (5 g / L) Freeze-dried Accelerated test Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) Not added 21 8.00E + 10 22 19 14 0 hr addition 30 1.20E + 11 35 31 18 8 hr addition 55 3.81E + 11 57 43 22 16 hr addition 78 5.18E + 11 80 52 22

Example 8 Effect of Proline Addition on Cultures by Strain

It was confirmed that the addition of proline during the cultivation of Lactobacillus plantarum KCTC3108 in Example 7 increased the freeze-drying survival rate, the survival rate after the accelerated test, and the acid resistance, so that experiments were performed to confirm whether the same effect was observed in other lactic acid bacteria Respectively. Five different strains including Lactobacillus plantarum KCTC3108 were cultivated, and 5 g / L of proline was added at the time of arrival of the larvae. After 2 hours, the culture broth was recovered, centrifuged and frozen according to a conventional method Drying was carried out. As a result, the freeze-drying and accelerated stability and acid resistance of all the strains were improved, and the biliary properties of some strains were improved (Table 7).

Effect of proline addition during culture Proline
Whether to add Strain Freeze-dried Accelerated test Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) Not added L. plantarum KCTC3108 21 8.00E + 10 22 19 14 L. acidophilus KCTC3142 22 6.10E + 10 11 24 23 B. bifidum KCTC3202 20 8.00E + 10 10 10 15 B. longum KCTC3128 25 8.70E + 10 15 20 22 L. mesenteroides KCTC3100 30 1.00E + 11 31 32 20 5 g / L
adding L. plantarum KCTC3108 78 5.18E + 11 80 52 22 L. acidophilus KCTC3142 62 4.24E + 11 75 62 32 B. bifidum KCTC3202 65 3.30E + 11 62 48 17 B. longum KCTC3128 68 3.70E + 11 68 53 30 L. mesenteroides KCTC3100 72 6.10E + 11 70 55 48

Example 9: Hydrogen peroxide resistance after lyophilization with addition of proline during culture

Experiments were carried out to confirm the oxygen tolerance of the products obtained by lyophilization using the cells recovered from the proline-free culture medium and the 5 g / L-containing culture medium as in Example 8, respectively. Hydrogen peroxide tolerance was compared indirectly to confirm resistance to oxygen. After lyophilization, each sample was suspended in 0.05 M phosphate buffer (pH 6.8) containing 0 ppm, 5,000 ppm, 10,000 ppm, 15,000 ppm and 20,000 ppm of hydrogen peroxide, and reacted at 37 ° C for 1 minute. 1 ml of the reaction solution was suspended in a buffer containing 2 mg / ml catalase, diluted 10-fold, and then plated on MRS medium. The survival rate (%) was determined as 100% for the number of bacteria added to the phosphate buffer before the reaction, and the results are shown in Table 8. In general, the survival rate decreased with increasing hydrogen peroxide concentration. In addition, the product after lyophilization with proline added significantly increased the resistance to hydrogen peroxide compared with the case without proline added during the culture.

Survival rate (%) when exposed to hydrogen peroxide During cultivation
Proline addition   Strain  Concentration of hydrogen peroxide treatment (ppm) 0 5,000 10,000 15,000 20,000 0 g / L L. plantarum KCTC3108 98 72 50 34 16 L. acidophilus KCTC3142 99 74 56 30 18 B. bifidum KCTC3202 92 58 37 12 0 B. longum KCTC3128 96 54 41 9 0 L. mesenteroides KCTC3100 97 66 52 38 24 5 g / L L. plantarum KCTC3108 98 87 85 79 62 L. acidophilus KCTC3142 98 89 83 80 76 B. bifidum KCTC3202 96 85 78 72 66 B. longum KCTC3128 99 86 80 71 64 L. mesenteroides KCTC3100 98 84 79 70 65

Example  10: Hydrogen peroxide decomposition activity after lyophilization with addition of proline during incubation

The hydrogen peroxide decomposition ability of the products obtained by lyophilization was compared using the cells recovered from the proline-free culture medium and the 5 g / L-containing culture medium as in Example 9, respectively. A solution containing 2% of water-saturated phenol solution, 0.04 mg / ml of 4-aminoantipyrine, 0.04 Unit / ml of peroxidase, and 300 nmol of hydrogen peroxide in 0.4 mM phosphate buffer (pH 6.9) The lyophilized lactic acid bacterium was added to the reaction solution, reacted at 37 ° C for 1 hour, and analyzed by a spectrophotometer at 505 nm to measure the concentration of residual hydrogen peroxide.

As a result of the experiment, the decomposition activity except for the residual concentration at the initial 300 nmol was significantly increased when the proline was added to the lyophilized product after lyophilization.

Comparison of hydrogen peroxide decomposition activity of lactic acid bacteria   Strain Hydrogen peroxide decomposition activity
(nmol of H ₂ O ₂ / mg of cell per hr) No proline added during culture Addition of proline (5 g / L) L. plantarum KCTC3108 57 126 L. acidophilus KCTC3142 105 172 B. bifidum KCTC3202 26 94 B. longum KCTC3128 48 133 L. mesenteroides KCTC3100 65 122

Example 11: Addition of cryoprotectant or coating agent

By accumulating intracellular proline at a high concentration using a culture process in which proline is added during the culture, it was possible to make the strain itself resistant to stress environments including freeze drying. The addition of proline to the intracellular proline increased the storage stability and acid resistance of the cell itself to the extreme environment. However, the effect of intracellular proline on the biliary properties was not significant.

To this end, proline was added during the culture, and then the freeze-drying or lyophilization was carried out by adding a commonly known cryoprotectant or coating agent to the concentrated culture (Table 10).

The cryoprotectants used in further experiments were proline, trehalose, glycerin and coatings were chitosan, malto-dextrin, indigestible dextrin, Starch, xanthan gum, XG, guar gum, carboxymethyl cellulose (CMC), hydroxyethylcellulose (HEC), cellulose, polyvinylsulfone Polyvinylpyrrolidone (PVP), carbopol, sodium alginate, propylene glycol alginate, alginate, polyethyleneglycol (PEG), triacetin Propylene glycol, acetyltriethyl citrate, and triethyl citrate. The term " anionic surfactant "

In the case of addition of proline, trehalose, malto-dextrin, indigestible dextrin or cellulose, and addition of a cryoprotectant and a coating agent, Table 10 shows the results of the acid resistance and the bile resistance test of the control group. For each of the five strains used, the result of the addition of the cryoprotectant or coating composition was slightly different, but the overall acidity tended to improve. In addition, it was confirmed that the bile resistance was significantly increased by treatment with a cryoprotectant or a coating agent.

Effect of cryoprotectant or coating agent Strain Cryoprotectants and coatings freezing
dry 4 weeks Acceleration stability Acid resistance
(Artificial gastric juice pH 2.5) My bile
(0.5% oxgall) Survival rate (%) CFU / g Survival rate (%) Survival rate (%) Survival rate (%) L. plantarum KCTC3108 Not added 78 5.18E + 11 80 52 22 Proline
(proline) 80 5.82E + 11 81 69 35 Trihalose
(trehalose) 82 5.66E + 11 82 64 41 Maltodextrin
(malto-dextrin) 83 6.17E + 11 84 72 67 Indigestible maltodextrin
(indigestible-dextrin) 81 6.03E + 11 83 67 43 cellulose
(cellulose) 80 5.70E + 11 81 62 40 L. acidophilus KCTC3142 Not added 62 4.24E + 11 75 62 32 Proline
(proline) 65 5.70E + 11 78 70 45 Trihalose
(trehalose) 71 5.85E + 11 80 69 44 Maltodextrin
(malto-dextrin) 75 5.92E + 11 85 79 61 Indigestible maltodextrin
(indigestible-dextrin) 72 5.62E + 11 83 75 57 cellulose
(cellulose) 69 5.80E + 11 81 68 48 B. bifidum KCTC3202 Not added 65 3.30E + 11 62 48 17 Proline
(proline) 70 4.70E + 11 68 54 40 Trihalose
(trehalose) 68 4.85E + 11 66 53 39 Maltodextrin
(malto-dextrin) 78 4.92E + 11 71 63 56 Indigestible maltodextrin
(indigestible-dextrin) 72 4.62E + 11 63 59 52 cellulose
(cellulose) 65 4.80E + 11 62 52 43 B. longum KCTC3128 Not added 68 3.70E + 11 68 53 30 Proline
(proline) 69 3.95E + 11 67 60 46 Trihalose
(trehalose) 68 3.87E + 11 66 62 44 Maltodextrin
(malto-dextrin) 69 4.10E + 11 76 72 58 Indigestible maltodextrin
(indigestible-dextrin) 70 4.17E + 11 71 70 54 cellulose
(cellulose) 68 3.90E + 11 70 55 50

L. mesenteroides KCTC3100 Not added 72 6.10E + 11 70 55 48 Proline
(proline) 84 7.08E + 11 85 68 60 Trihalose
(trehalose) 81 7.40E + 11 85 68 68 Maltodextrin
(malto-dextrin) 85 8.00E + 11 91 74 70 Indigestible maltodextrin
(indigestible-dextrin) 78 7.48E + 11 82 65 64 cellulose
(cellulose) 77 7.10E + 11 87 70 62

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

A method for increasing survival rate, storage stability, acid resistance and biliary cholesterol after lyophilization of a lactic acid bacterium comprising the following steps:
(a) culturing lactic acid bacteria by adding proline in the culture, wherein the lactic acid bacteria are selected from the group consisting of Lactobacillus sp., Bifidobacterium sp., and Leuconostoc sp. A lactic acid bacterium selected from the group consisting of;
(b) concentrating the culture liquid of the lactic acid bacteria; And
(c) adding a cryoprotectant or coating to the concentrated culture, wherein the cryoprotectant is proline or trehalose and the coating agent is malto-dextrin, indigestible maltodextrin, dextrin or cellulose.
delete The method of claim 1, wherein the lactic acid bacterium is selected from the group consisting of L. plantarum , L. acidophilus , B. bifidum , Bifidobacterium longum ( B longum ) or L. mesenteroides .
delete The method according to claim 1, wherein the added concentration of proline is 1 g / L to 50 g / L. delete delete delete delete delete

Images