KR101604633B1

KR101604633B1 - Medium composition for culturing lactic acid bacteria and producing method of powder of lactic acid bacteria using the same

Info

Publication number: KR101604633B1
Application number: KR1020150121738A
Authority: KR
Inventors: 정일선; 손민; 김현수
Original assignee: 주식회사 락토메이슨
Priority date: 2015-08-28
Filing date: 2015-08-28
Publication date: 2016-03-18

Abstract

Provided are: a medium composition of lactic acid bacteria comprising chicory dry powder, a chicory extract, devil′s tongue jelly (Gonjack) dry powder, Jerusalem artichoke powder, and a natural plant fiber selected from the group consisting of the combinations thereof; a method for manufacturing powder of lactic acid bacteria by using the same; powder of lactic acid bacteria manufactured by the manufacturing method; and uses of the powder of lactic acid bacteria. The medium composition of lactic acid bacteria of the present invention is capable of reducing chemical stress of lactic acid bacteria and increasing production yields, by culturing the lactic acid bacteria through a fed-batch culture method.

Description

TECHNICAL FIELD [0001] The present invention relates to a lactic acid bacterium culture medium, a lactic acid bacteria culture medium,

The present invention relates to a lactic acid bacterial culture composition comprising a natural plant fiber, a method for producing a lactic acid bacterial powder using the same, a lactic acid bacterial powder prepared by the method, and a use of the lactic acid bacterial powder.

It is essential for living microorganisms to function as probiotics, since microorganisms are ingested to survive in the host's field and to adhere to the host's field for survival to form microflora.

Generally, lactic acid bacteria are cultured purely through a fermentation process so as to be produced as probiotics, and the cells are separated from the culture broth, mixed with a cryoprotectant or the like, lyophilized, and prepared as a viable agent through pulverization. However, in order to maintain the physiological activity inherent to the probiotic agent, it is necessary to improve the acid resistance, resistance to bile acid, and improve the distribution stability of the lactic acid bacteriostatic agent, so that the lactic acid bacteria administered at the oral administration maintains rapid growth and high intestinal fixability in the intestines It is necessary. Therefore, manufacturers of lactic acid bacterium probiotics are striving not only to develop strains that are basically resistant to acid or bile acid, but also to improve the quality of probiotics by developing excellent freeze protection and stabilizer compositions (Korean Patent Laid-Open No. 10-2009-0014264).

Lactic acid bacteria are exposed to physical and chemical stress during incubation. Biochemical stress is caused by organic acids such as lactic acid and acetic acid accumulated during cultivation of lactic acid bacteria. These stresses can greatly affect the production yield during the cell collection period and the viability of the lactic acid bacteria present in the dried lactic acid bacteria, so it is very important to reduce this type of stress intensity during lactic acid probiotics manufacturing process.

The present invention provides a lactic acid bacterium culture composition comprising a natural plant fiber, a method for producing lactic acid bacterium powder using the same, a lactic acid bacterium powder prepared by the above method, and a use of the lactic acid bacterium powder.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

A first aspect of the invention provides a lactic acid bacterial culture composition comprising a natural plant fiber comprising a plant polysaccharide and a whey powder.

According to a second aspect of the present invention, there is provided a method for producing a lactic acid bacterium, comprising: inoculating a lactic acid bacterium strain into a lactic acid bacterium culture composition according to the first aspect of the present invention; Culturing the inoculated lactic acid bacteria strain; Preparing a lactic acid bacteria pellet by centrifuging the cultured lactic acid bacteria strain; And lyophilizing the lactic acid bacteria pellet, wherein the lactic acid bacterial pellet is lyophilized by adding a selected one selected from the group consisting of microalgae extract, lactic acid bacterium, tyndalized lactic acid bacteria, and combinations thereof. do.

A third aspect of the present invention provides a lactic acid bacterium powder prepared by the manufacturing method according to the second aspect of the present invention.

A fourth aspect of the invention includes a dietary supplement, comprising a lactic acid bacterium powder according to the third aspect of the present application.

A fifth aspect of the invention comprises a feed additive comprising a lactic acid bacterium powder according to the third aspect of the invention.

The lactic acid bacteria culture composition of the present application includes a natural plant fiber including a plant polysaccharide, for example, a dried chicory powder or a chicory extract. The inulin component, which is a plant polysaccharide contained in chicory or the inulin It is possible to protect the strain from the stresses that occur during the physical and chemical stresses and the bacterium recovery that occur during the cultivation of the lactic acid bacteria. In addition, by culturing the lactic acid bacteria through the fed-batch culture method, the chemical stress of the lactic acid bacteria can be reduced and the production yield can be increased as compared with the batch culture.

In the process for producing a lactic acid bacterium powder according to the present invention, by adding a microalgae extract and a lactic acid bacterium as a growth promoting agent for lactic acid bacteria, not only the growth of lactic acid bacteria is promoted, but also the natural plant fiber contained in the lactic acid bacteria culture composition acts as a freeze- Lactic acid bacteria can be protected.

Further, in addition to the use of the culture composition for lactic acid bacteria containing the natural plant fiber in the process of producing the lactic acid bacteria powder of the present invention, the production yield can be remarkably increased by adding a separate natural plant fiber in the lyophilization step.

1 is a graph showing the effect of inhibiting the growth rate of lactic acid bacteria (LD ₅₀ ) on the concentration of acetic acid and lactic acid according to one embodiment of the present invention.
FIG. 2A is a graph comparing the culture results of L. plutarium with the fed-batch culture and the batch culture in one embodiment of the present invention. FIG.
FIG. 2B is a graph comparing the culture results of B. longom with oil-fed culture and batch culture in one embodiment of the present invention. FIG.
FIG. 3 is a photograph showing the difference in the amount of EPS produced during the fermentation of L. plutarium and B. lactis according to whether or not the chicory extract is added, in one embodiment of the present invention.
FIG. 4 is a photograph showing the difference in the amount of centrifugally precipitated EPS of L. plutarium according to whether or not the chicory extract is added, in one embodiment of the present invention.
FIG. 5 is a photograph showing the difference in the amounts of EPS produced by drying of L. plutarium and B. lactis according to whether or not the chicory extract is added, in one embodiment of the present invention.
FIG. 6A is a graph showing the results of the lyophilized microbial cells obtained by culturing L. plutarium in the absence of a chicory extract and lyophilized without adding a lyophilized protective agent to obtain dried lactic acid bacteria powder, Which is a graph comparing the results obtained through the test.
FIG. 6B is a graph showing the stability of a live bacterium in a dry powder by performing lyophilization without adding a lyophilized protective agent after culturing B. longudum according to the addition of a chicory extract, The results are shown in Fig.
FIG. 7 is a graph showing the growth promoting effect of lactic acid bacteria by addition of microalgae extract and lactic acid bacteria lysate in one embodiment of the present invention.
8 is an SEM image of the lactic acid bacteria powder according to one embodiment of the present invention.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains. It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout this specification, when a part is referred to as being "connected" to another part, it is understood that it includes not only "directly connected" but also "electrically connected" do.

Throughout this specification, when a member is "on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

As used herein, the terms "about," " substantially, "and the like are used herein to refer to or approximate the numerical value of manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to prevent unauthorized exploitation by unauthorized intruders of the mentioned disclosure.

The term " step "or" step of ~ " as used throughout the specification does not imply "step for.

Throughout this specification, the term "combination (s) thereof " included in the expression of the machine form means a mixture or combination of one or more elements selected from the group consisting of the constituents described in the expression of the form of a marker, Quot; means at least one selected from the group consisting of the above-mentioned elements.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout the present specification, the term "Fed batch culture" means a culture method in which a nutrient medium is added to a fermentation tank over a plurality of stages during the culture, and the culture is not extracted from the fermentation tank until the completion of the culture.

Throughout this specification, the term "whey powder" refers to a powder obtained by dry-pulverizing whey, which is a byproduct produced when milk is processed into cheese. Specifically, the term " whey powder " The whey solution is dried and pulverized. In general, the protein content of the whey powder is about 11.0% to about 14.5%, the lactose concentration is about 65%, and the ash content is about 8.5%. Skim milk, on the other hand, is a product that removes fat from milk and powdered milk. It differs from whey powder in that it has a high protein content of 52% carbohydrates and 35% protein. In addition, whey protein is a raw material that improves protein content by adding protein to whey powder by more than 35%, and is different from whey powder.

Throughout the specification, the term " tyndallization "is a sterilization method developed by John Tyndall. The sterilization method of generalized microorganisms is sterilization at 121 ° C and 1.2 atm for 15 minutes. Most of these sterilization methods can kill microbial spores that are highly heat-resistant, but they can cause the destruction of heat-sensitive components. Thymarization is a sterilization method developed for sterilizing microorganisms at a lower temperature (for example, 80 to 100 DEG C), for example, a method in which a substance to be sterilized is first sterilized at a high temperature such as a boiling point, Microbial spores can be sterilized by allowing them to stand for a few days to allow them to germinate by heat shock applied first and then heat them again.

Hereinafter, embodiments of the present invention are described in detail, but the present invention is not limited thereto.

In one embodiment of the invention, the natural plant fiber may be selected from the group consisting of chicory dry powder, chicory extract, Gonjack dry powder, porcine potato dry powder, and combinations thereof.

The natural plant fiber comprises a plant polysaccharide. For example, the chicory dried powder or chicory extract includes inulin, the dried potato powder includes fructan, and the dried konjac powder includes glucomannan.

In one embodiment herein, the natural plant fiber may comprise, but is not limited to, at least about 60 weight percent vegetable polysaccharide. For example, the extract of chicory or chicory-like analogues may include, but is not limited to, about 60% by weight or more of inulin and / or fructan. The inulin and fructan can protect the lactic acid bacterial strain from the physical and chemical stresses occurring during the culture and from the stresses occurring during bacterial recovery. Inulin is a polysaccharide polymer that repeats a unit structure of three molecules of fructose and one molecule of glucose and can be used as a sugar as a carbohydrate in the culture of lactic acid bacteria and can be used as a structural protecting material for lactic acid bacteria such as EPS Exo-polysaccharides and can also be converted into inulin-like during the culture to stimulate the selective growth of intestinal microorganisms except for the growth of pathogenic microorganisms as pre-biotics . In addition, it is a protective substance that increases the resistance of the lactic acid bacteria to the external stress generated during the culturing process, and consequently increases the yield of viable cells and the stability of the viable cells in the final product, and then, As a cryoprotectant. Inulin is consumed in the form of short or long oligosaccharides by Bifidobacterium spp and other lactic acid bacterial species, and is used to synthesize EPS (Exo-polysaccharides), which act as a structural material of cells, and can be used as a protective substance against external stress of lactic acid bacteria species have. These EPSs are synthesized with EPS of various molecular weights depending on the kind of lactic acid bacteria. Especially, lactic acid bacteria producing EPS with high molecular weight can be used as industrial strains important for increasing viscosity in the production of concentrated yogurt. These EPSs synthesized by lactic acid bacteria are synthesized by using galactose, rhamnose, fructose and glucose as substrates and glucopyranosyl, galactopyranosyl, fructopyranosyl, rhamnopyranosyl, And the like. In case of lactic acid bacteria digesting the sugars in the medium with carbon source during metabolic process and accumulating them as structural materials or energy storage materials such as EPS, it can be controlled according to the energy level of lactic acid bacteria. That is, when there is sufficient energy in the cells, that is, when ATP is present, the lactic acid bacteria can produce a structural material such as EPS instead of producing energy by consuming the carbon source of the medium, and synthesize and store the energy storage form. Lactic acid bacteria synthesizes EPS by decomposing lactose in milk into glucose and galactose. Especially, when the carbon source is abnormally increased or the culture environment deteriorates compared to the nitrogen source in the medium, it is synthesized in a large amount. Streptococcus, Lactobacillus, and Bifidobacterium, which are used in the production of probiotic fermented milk, synthesize and accumulate various EPSs for survival in fermented milk, and they play an important role in the bile salts in the human intestines when they are taken, .

In one embodiment of the invention, the natural plant fiber is included in an amount of from about 0.01 to about 10 parts by weight based on 100 parts by weight of the lactic acid bacteria culture composition, and the whey powder may be included in an amount of about 0.1 to about 10 parts by weight However, the present invention is not limited thereto.

In one embodiment of the present invention, the lactic acid bacteria culture composition may further comprise at least one member selected from the group consisting of casein peptone, caspase, peptone of milk origin, and combinations thereof. For example, But may not be limited to, about 0.001 part by weight to about 10 parts by weight based on 100 parts by weight of the culture medium composition.

In one embodiment of the present application, the lactic acid bacteria culture composition may include, without limitation, components used for culturing lactic acid bacteria in the art, for example, glucose, yeast extract, L-cysteine, Tween- But may not be limited thereto. The ingredients and the content of the lactic acid bacteria culture composition may be controlled depending on the kind of the lactic acid bacteria to be cultured, but may not be limited thereto.

The inoculated lactic acid bacteria strain can be cultured by a fed-batch culture method. The Fed batch culture method is a method in which the concentration of lactic acid and acetic acid accumulated during cultivation of lactic acid bacteria is maintained relatively low compared with batch cultures conventionally used for culture of lactic acid bacteria, It can reduce the chemical stress it receives. Lactic acid and acetic acid are produced during fermentation of lactic acid bacteria through metabolism of sugars and accumulate in the medium during fermentation. If the organic acid is accumulated above a certain concentration, the consumed saccharides are seriously adversely affected by the growth of the cells, which is consumed in making the organic acid rather than being used for the production of the cell product. In addition, the fed-batch culture method has an advantage in that the concentration of sugar added at the beginning is lower than that in the batch culture, while the production rate of the organic acid is kept low by controlling the sugar concentration added during the culture. As a result, fed-batch culture reduces the chemical stress (lactic acid concentration) during cultivation, thereby inducing physiological production of the product according to consumption of the sugar, thereby increasing the yield in the fermentation process. At the same time, Can contribute to increase production yield (recovery yield) based on important live bacteria in probiotics.

The lactic acid bacteria pellet means a substance precipitated when the cultured lactic acid bacteria strain is centrifuged, and the culture by-products and the like are removed, and a high content of the lactic acid bacteria strain is included.

In one embodiment of the invention, the lactic acid bacteria strain is selected from the group consisting of Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, For example, Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus spp., Lactobacillus spp. Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salvilus, Lactobacillus spp., Lactobacillus spp. Lactobacillus salivarius, Lactobacillus reuteri, But are not limited to, Leuconostoc mesenteroides, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium bifidum, lactis, Bifidobacterium infantis, and combinations thereof. The term " bifidobacterium infantis "

In one embodiment of the invention, the microalgae extract may serve as a microbial growth stimulator, but the present invention is not limited thereto. The microalgae extract may be in the form of dry microalgae powder or microalgae lumps, but may not be limited thereto. Exo-polysaccharides (EPS), a major component of the microalgae extract added as a protective agent, and FOS (Fructo-oligosaccharides) or GOS (Galacto-oligosaccharides), which are structural materials thereof, are synthesized during the cultivation of lactic acid bacteria. It is known to accumulate as a substance and protect the cells. Some also promote selective growth of intestinal microorganisms and inhibit the growth of other pathogenic microorganisms. Therefore, when these materials are used as a cryoprotectant, they can act as pre-biotics to help selective growth of intestinal lactic acid bacteria, thereby helping intestinal colonization of probiotics.

In one embodiment of the invention, the microalgae extract is present in an amount of from about 0.01% to about 10% by weight, such as from about 0.1% to about 10% by weight, from about 0.5% About 10 wt%, about 1 wt% to about 10 wt%, about 3 wt% to about 10 wt%, about 5 wt% to about 10 wt%, about 7 wt% to about 10 wt% From about 0.01% to about 5%, from about 0.01% to about 3%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, or from about 0.01% % To about 0.1 wt.%, Based on the total weight of the composition.

In one embodiment of the invention, the microalgae extract may be, but is not limited to, those derived from microalgae Spirulina ( Arthrospira platensis ). The microalgae extract may be obtained by hydrolyzing the microalgae spirulina concentrate cultured.

In one embodiment of the invention, the lactic acid bacterium is selected from the group consisting of Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, For example, Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus spp., Lactobacillus spp. Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salvilus, Lactobacillus spp., Lactobacillus spp. Lactobacillus salivarius, Lactobacillus reuteri, But are not limited to, Leuconostoc mesenteroides, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium bifidum, but are not limited to, lactic acid bacteria, lactis, Bifidobacterium infantis, and combinations thereof. The lactic acid bacterium can be produced by culturing a lactic acid bacterium, collecting it by centrifugation, disrupting the cells, sterilizing, lyophilizing and pulverizing the lactic acid bacteria. The lactic acid bacterium may act as a microbial growth stimulator, but the present invention is not limited thereto. The above-mentioned lactic acid bacteria lysate contains components such as EPS (Exo-polysaccharides) and FOS (Fructo-oligosaccharides) or GOS (Galacto-oligosaccharides) as its structural material, and can act as a growth promoter. When these substances are added as a cryoprotectant during the manufacturing process and introduced into the intestines of humans together with lactic acid bacteria, they act as pre-biotics for promoting the growth of intestinal lactic acid bacteria in the intestines of humans. At the same time, To inhibit the growth of lactic acid bacteria (intestinal colonization) can help.

In one embodiment herein, the lactic acid bacterium is present in an amount of from about 0.01% to about 10% by weight, for example, from about 0.1% to about 10% by weight, from about 0.5% About 10 wt%, about 1 wt% to about 10 wt%, about 3 wt% to about 10 wt%, about 5 wt% to about 10 wt%, about 7 wt% to about 10 wt% From about 0.01% to about 5%, from about 0.01% to about 3%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, or from about 0.01% To about 0.1% by weight, based on the total weight of the composition.

In one embodiment of the invention, the tinctured Lactobacillus plantus is selected from the group consisting of Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus fermentum ), Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus reuteri, Lukono Leuconostoc mesenteroides, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis (Bifidobacterium lactis), Bifidobacterium bifidum, Bifidobacterium lactis ), Bifidobacterium infantis, and combinations thereof. , Followed by drying, followed by tilization of the selected lactic acid bacteria. The dried tinctured lactic acid bacteria are cultured by lyophilizing the lactic acid bacteria, centrifuging the cells, separating the cells from the culture broth, disinfecting the cells by the tincturing process, and lyophilizing the dried product. The EPS , FOS, and GOS as important components. When added as a protective agent, it is possible to add a single strain or a combination of two or more species.

In one embodiment herein, the tinctured lactic acid bacterial product is present in an amount from about 0.01% to about 10% by weight, for example, from about 0.1% to about 10% by weight, from about 0.5% About 10 wt% to about 10 wt%, about 1 wt% to about 10 wt%, about 3 wt% to about 10 wt%, about 5 wt% to about 10 wt%, about 7 wt% From about 0.01 wt.% To about 0.5 wt.%, Or from about 0.01 wt.% To about 5 wt.%, From about 0.01 wt.% To about 3 wt. May be added in an amount of 0.01% by weight to about 0.1% by weight, but the present invention is not limited thereto.

In one embodiment of the present invention, the method for preparing the lactic acid bacteria powder may further include adding the amino acid mixture to the lactic acid bacteria pellet, but the present invention is not limited thereto. The amino acid mixture may include, but is not limited to, a mixture of amino acids selected from the group consisting of arginine, glutamic acid, penicillamine, and combinations thereof.

The use of amino acids or combinations of amino acids can protect microbial cells during the freezing process. Several amino acids, including lysine, stabilize liposomes during the freezing process of liposomes, which have cell membrane structure and abortive structure of microorganisms. These properties are the properties of amino acids (both hydrophilic and lipophilic) . In addition, proline may play an important role as a cryoprotectant in freezing and re-dissolving plant cells cultured in liquid nitrogen. In the lyophilization of catalase enzymes important for the survival of non-aerobic microorganisms, amino acids such as alanine, glycine, lysine, or serine are important for stabilizing protein enzymes It plays a role. Amino acids play an important role in the stabilization of cell membrane and protein enzyme structure, which can be most damaging to microorganisms during freeze-drying process. The arginine, glutamine, or phenylalanine used in the present application serves as an auxiliary cryoprotectant in addition to the cryoprotective effect on the chicory extract and whey powder contained in the lactic acid bacterium culture composition according to the present invention, so that the mixture of the recovered lactic acid bacterium pellet and the cryoprotectant May be added during the process.

In one embodiment of the invention, the amino acid mixture is present in an amount from about 0.01% to about 10% by weight, for example, from about 0.1% to about 10%, from about 0.5% About 10 wt%, about 1 wt% to about 10 wt%, about 3 wt% to about 10 wt%, about 5 wt% to about 10 wt%, about 7 wt% to about 10 wt% From about 0.01% to about 5%, from about 0.01% to about 3%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, or from about 0.01% To about 0.1% by weight, based on the total weight of the composition.

In one embodiment of the present invention, the method for producing the lactic acid bacteria powder may further include adding the natural plant fiber to the lactic acid bacteria pellet, but may not be limited thereto. In addition to the use of the culture composition for culture of lactic acid bacteria containing the natural plant fiber, the production yield can be remarkably increased by adding the natural plant fiber even in the freeze-drying step. Adding the step of adding the chicory dry powder or the chicory extract to the lactic acid bacteria pellet may result in an increase in the production yield of about 2 times.

In one embodiment of the invention, the natural plant fiber may comprise selected from the group consisting of chicory dry powder, chicory extract, Gonjack dry powder, porcine potato dry powder, and combinations thereof. The natural plant fiber may include vegetable polysaccharides, for example, but may not be limited to, at least about 60 weight percent vegetable polysaccharides. For example, the chicory dried powder or chicory extract includes inulin, the dried potato powder includes fructan, and the dried konjac powder includes glucomannan.

In one embodiment herein, the natural plant fiber is present in an amount from about 0.1% to about 5.0% by weight, for example, from about 0.1% to about 4.0% by weight, from about 0.1% About 0.1 wt% to about 2.0 wt%, about 0.1 wt% to about 1.0 wt%, about 0.5 wt% to about 5.0 wt%, about 1.0 wt% to about 5.0 wt%, about 2.0 wt% To about 5.0 wt%, about 3.0 wt% to about 5.0 wt%, about 4.0 wt% to about 5.0 wt%, about 1.0 wt% to about 3.0 wt%, and about 2.0 wt% to about 3.0 wt% But may not be limited thereto.

With respect to the third to fifth aspects of the present application, all of the descriptions of the first and second aspects of the present application can be applied.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are given for the purpose of helping understanding of the present invention, but the present invention is not limited to the following Examples.

[ Example ]

[Comparative Example]

The composition of the culture medium used in the conventional batch culture method, which is mainly used for culture of lactic acid bacteria, is as follows.

The composition of the present culture medium LM-PRM-1 for culturing L. prostata room is 0.1-10.0 wt% glucose, 0.1-10.0 wt% soy peptone, 0.1-10.0 wt% yeast extract, 0.0001-5.0 wt% wt% L- cysteine, 0.01 to 5.0 wt.% Tween-80, 0.001 ~ 5.0 _{weight% K 2 HPO 4, 0.01 ~} 5.0 _{wt% MgSO 4 · 7H 2 O,} 0.0001 ~ 5.0 weight% MnSO ₄ · 4H ₂ O, and 0.01 to 5.0% by weight potassium citrate was used.

The culture medium LM-BLM-1 for the cultivation of B. longagi is 0.1-10.0 wt% glucose, 0.1-10.0 wt% lactose, 0.1-10.0 wt% soybean peptone, 0.1-10.0 wt% yeast extract, 0.0001-5.0 wt% 0.001-5.0 wt% MSG (monosodium glutamate), 0.0001-5.0 wt% ascorbic acid, 0.0001-5.0 wt% Tween-80, 0.001-5.0 wt% K ₂ HPO ₄ , 0.001-5.0 wt% 0.0001-5.0 wt% MgSO ₄揃 7H ₂ O, 0.0001-5.0 wt% MnSO ₄揃 4H ₂ O, 0.0001-5.0 wt% ferric sulfate, 0.0001-5.0 wt% calcium chloride, and 0.001-5.0 wt% A medium containing potassium citrate was used.

The cultures were incubated for 14 h and 11 h, respectively, with slight agitation at 37 ° C in all cases, and L. plutarum was incubated at pH 5.0-6.0, B. long black pH 5.0-6.0 Ammonia was used. During the incubation the pH was automatically controlled.

After culturing, each of the lactic acid bacteria was separated from the culture solution by centrifugation at 8,000 to 10,000 rpm, and the separated bacterial strains were mixed with each protective agent added for the purpose of the invention. Cells mixed with the protector were lyophilized.

The viable cell count of live lactic acid bacteria present in the fermentation broth, recovered cell pellet, and freeze - dried powder contained in this manufacturing process was analyzed and the stability of the lactic acid bacteria in the prepared dry powder was analyzed by decreasing the viable cell count of the dry powder at 40 ℃ .

[Example 1]

Experiments to inhibit the growth of lactic acid bacteria by lactic acid and acetic acid were carried out on a lactic acid bacteria vial medium having the following composition. L. prosta room was cultured in LM-PRVM medium containing glucose (2.0%, w / v), soy peptone (1.0%, w / v), yeast extract (1.0%, w / v) w / v), Tween-80 (0.1%, w / v), KH 2 PO 4 (0.1%, w / v), MgSO 4 · 7H 2 O (0.01%, w / v), and MnSO ₄ · 4H ₂ O in a vial containing culture medium having a composition of (0.005%, w / v) , B. Long black glucose (1.0%, w / v) as LM-BLVM medium, lactose (2.0%, w / v) , soybean (0.05%, w / v), Tween-80 (0.05%, w / v) (0.005%, w / v), KH ₂ PO ₄ (0.1%, w / v), MgSO ₄ .7H ₂ O (0.01%, w / v) and MnSO ₄ .4H ₂ O Lt; RTI ID = 0.0 > 37 C. < / RTI > The initial growth rate of each lactic acid bacterium was measured before 6 hours after the start of the culture, and the case of adding lactic acid and acetic acid to each concentration was compared with the case where no organic acid was added to the initial medium (Table 1 and Fig. 1 ). Were shown degree receives inhibited by the concentration of lactic acid or acetic acid present in the culture to LD _50, where the LD ₅₀ represents the concentration (mM) of each organic acid to reduce the growth rate (μ) of the cell (X) by 50% (Table 1).

[Table 1]

In FIG. 1, μ / μ _o represents growth rate inhibition. It can be seen that the initial growth rate (μ ₀ ) in the case where no organic acid is added . This means that the larger the inhibition of growth rate, the lower the value. As can be seen in FIG. 1, L. plutarium (blue curve) was found to be more resistant to organic acids than B. longum (red curve), and B. reacted more sensitively to acetic acid than black lactic acid It looked.

From the above experiment, it was confirmed that the growth of lactic acid bacteria was inhibited by lactic acid and acetic acid.

[Example 2]

The effect of fed - batch fermentation on the fermentation yield of lactic acid bacteria was investigated. Unlike the culture medium for the batch culture of the comparative example, the sugar and nitrogen source concentrations of the fed-batch culture medium used in this example were lower than those used in the batch culture and were added during the culture if necessary.

The composition of the medium used for fed-batch culture in this example is as follows. L. Pleuralarum is a LM-PRM-2 culture medium containing 0.1 to 10.0 wt% glucose, 1.0 to 10.0 wt% of soybean peptone, 0.1 to 10.0 wt% of whey powder, 0.1 to 10.0 wt% of yeast extract, 0.002 to 0.1 wt% L- cysteine, 0.1 to 10.0 wt% Tween-80, 0.1 ~ 10.0 % by weight of _{K 2 HPO 4, 0.1 ~ 10.0} % by weight of _{MgSO 4 · 7H 2 O, 0.005} ~ 1.0 wt% MnSO ₄ · 4H ₂ O, and 0.1 to 0.1 to 10.0% by weight of glucose, 0.1 to 10.0% by weight of soybean peptone, 0.1 to 10.0% by weight of yeast extract, 0.002 to 1.0% by weight of an initial medium containing 1.0% by weight of potassium citrate and a feed medium LM-PRFM- % L-cysteine, 0.1-5.0 wt.% K ₂ HPO ₄ , 0.1-5.0 wt.% MgSO ₄ .7H ₂ O, and 0.005-5.0 wt.% MnSO ₄ .4H ₂ O. B. Long black primary culture medium LM-BLM-2 was prepared by mixing 0.1-10.0 wt% glucose, 0.1-10.0 wt% lactose, 1.0-10.0 wt% soybean peptone, 0.1-10.0 wt% whey powder, 0.1-10.0 wt% Yeast extract, 0.01-1.0 wt% L-cysteine, 0.001-5.0 wt% MSG, 0.001-10.0 wt% ascorbic acid, 0.001-10.0 wt% Tween-80, 0.1-10.0 wt% K ₂ HPO ₄ , 0.1-10.0 0.001 to 10.0% by weight of ferric sulfate, 0.001 to 10.0% by weight of calcium chloride, and 0.01 to 10.0% by weight of sodium chloride, 0.01 to 10.0% by weight MgSO ₄ .7H ₂ O, 0.0001 to 10.0% by weight MnSO ₄ .4H ₂ O, 0.01 to 10.0% by weight of lactose, 0.01 to 10.0% by weight of soybean peptone, 0.01 to 10.0% by weight of yeast extract, 0.001% by weight of potassium citrate, and the following feed medium LM-BLFM- To 5.0 wt% L-cysteine, 0.001 to 5.0 wt% MSG, 0.0001 to 5.0 wt% ascorbic acid, and 0.01 to 5.0 wt% K ₂ HPO ₄ were used.

The culture was incubated at 37 ° C and ammonia was used to maintain the culture pH at pH 5.0 to 6.0 for L. plutarum and to maintain the culture pH at pH 5.0 to 6.0 for B longorm, It was automatically controlled.

L. Planta room and B. long black were cultured in this culture for a total of 14 hours and 11 hours, respectively. The culture was started at a total culture volume of 45% at the initial stage of culture, and 30% of the total culture volume was added first at 4 hours after the culture, and 25% of the total culture volume was added at the second time and cultured until the final time . As can be seen from Example 1, lactic acid bacteria consume sugar during the culture to synthesize lactic acid and acetic acid to reduce the pH of the culture and accumulate in the culture medium to cause a feedback inhibition. As a result, an important disadvantage of culturing lactic acid bacteria is that the production of the cells is limited by the accumulated organic acids during the culture, thus limiting the productivity of the cells. In this regard, we have overcome these limitations by applying the fed - batch culture method and obtained higher yield than the conventional batch culture method.

In the conventional batch culture method, lactic acid or acetic acid accumulated at a high concentration inhibits the growth of the cells, whereas when the organic acid is accumulated, the organic acid is appropriately diluted with a new medium and the new nutrient is added. The production of the cells was relatively high as compared with the batch culture. As a result, in the culture of L. plutarum, in the case of batch culture, a live cell density of 1,1E + 10 cfu / mL and 460 mM of lactic acid were produced while consuming 207 mM of glucose, whereas in case of batch culture, 287 mM glucose , A live cell density of 8.7E + 09 cfu / mL and 740 mM lactic acid were produced (Fig. 2A). That is, in case of fed-batch culture, the carbon balance between the substrate and lactic acid was 1.107, and in the case of batch culture was 1.288, the same 1 g / L sugar and amino acid, which is another carbon source of the medium, was used to produce more live cells.

In the culture of B. longom, lactic acid was produced at a concentration of 303 mM and acetic acid at 477 mM using 188 mM of glucose and 58 mM of lactose. In the case of fed-batch culture, 48.6 mM of glucose and 16.6 mM of lactic acid were used, mM and acetic acid of 141.5 mM (Fig. 2B). The carbon balance of the sugar and organic acids, which were substrates, was 1.42 for the fed-batch culture and 1.29 for the batch culture. B. In the case of batch culture using long gum, the lactic acid and acetic acid concentration in the medium increased to 4.55E + 09 cfu / mL in the culture medium, and 6.1E + 09 cfu / mL. These results suggest that the proportion of dead cells increased with increasing lactic acid and acetic acid concentration in the case of batch culture.

The higher the concentrations of organic acids accumulated in the fed - batch and batch - type cultures, the higher the stresses received by the lactobacilli during the recovery process and the less the resistance to stress. In the case of batch fermentation using a high sugar concentration, when the organic acid is accumulated at a high concentration during fermentation and the sugar concentration is initially set relatively low and added during fermentation, the degree of accumulation of organic acids during fermentation is relatively low, The degree of chemical stress to which the microbial cells are subjected may be low and the decrease in the resistance is likely to result in an increase in the lyophilization yield when the viable cell count is used in the recovery process of both L. plutarium and B. longom (Table 2 And Table 3).

[Table 2]

[Table 3]

As can be seen from the above experiment, the yield of lactic acid bacteria including fermentation and lyophilization yield was reduced to 12.66% in L. plutarum, B And increased to 48.24% for Longjack (Table 4).

[Table 4]

[Example 3]

The effect of the addition of chicory extract on the fermentation of lactic acid bacteria was investigated. In this example, in order to maximize the effect of producing lactic acid bacterium during the cultivation, cultivation was carried out as in Example 2, except that 2.0% by weight whey powder with 1.0% by weight of chicory extract was used in the initial culture medium, respectively, Was not further added through the injection medium. In the comparative example, the cells were cultured in a medium to which no chicory extract was added.

No inhibition or promotion of growth of lactic acid bacteria was observed in the concentration range in which chicory extract and whey powder were used, nor did it affect the consumption rate of glucose or the production of organic acid.

However, the addition of chicory extract significantly improved the lyophilization yield of the live bacteria in the recovery process of lactic acid bacteria. In the case of adding chicory extract and whey powder to the fermentation, the production yield of L. plantaum was increased from 42.36% to 58.16% in the case of L. platarium, and from 6.92% To 11.19% (Table 5 and Table 6).

[Table 5]

[Table 6]

As can be seen from the above results, it was found that the addition of a chicory extract containing 60% or more of inulin to the culture medium induces the cells in the culture to produce and accumulate EPS (Exo-polysaccharides) (Inulin-like), and they acted as a natural protecting agent, thereby increasing the stability of the prepared lactic acid germs. In addition, it was confirmed that the production yield was increased by 37.3% in B. Lantium and increased by 61.7% in B. longomg according to the addition of chicory extract (Table 7).

[Table 7]

As can be seen from the experiments conducted in the above examples, various lactic acid bacteria including B. longogens have an EPS-like structure outside the cell when the growth environment deteriorates (for example, the C / N ratio or environmental stress increases) They accumulate materials to protect themselves or accumulate nutrients. As in the present example, when fermented with fermentation by adding chicory extract and whey powder to fed-batch culture, the resistance to chemical stress of cultured lactic acid bacteria was increased by EPS-induced production by the added substances.

[Example 4]

When 1.0% by weight of chicory extract and 2.0% by weight of whey powder were added to fermented medium fermentation medium (LM-PRM-2, LM-BLM-2) as in Example 2 and 2.0% After fermentation by adding only powder, the microbial cells were recovered to prepare lactic acid bacterium pellets. Then, when the chicory extract was mixed with the lactic acid bacterium pellet, the difference in production yield was confirmed by lyophilization.

When fermented with the addition of chicory extract and whey powder as the representative probiotic lactic acid bacteria of L. plantaia, L. luteri and B. long black used in the experiment of this embodiment, the process yield was lower than that of fermented whey powder alone , Which was increased from 50.7% to 54.6% in the case of planta room, and in the case of addition of chicory extract in freeze drying process, the addition of chicory extract and whey powder compared with 65.7% %. In addition, the addition of chicory extract and whey powder led to the production of EPS during fermentation, and the additional yield of chicory extract containing inulin was increased by more than 49% to 97.9% (Table 8).

Similar results were obtained with other lactic acid bacteria tested. According to the experimental results, the addition of chicory extract to the fermentation process increased not only the fermentation yield but also the production yield of the lactic acid bacteria. Especially, L. luteri was about 50 %, And the production yield of B. long black chicory extract was significantly increased by 29.5% when the chicory extract was added in the fermentation process, compared to the yield of 1.4% without the fermentation process (Table 8). In addition, as can be seen in Table 8, it was confirmed that the addition of the chicory extract in the freeze-drying process after fermentation significantly increases the production efficiency. In particular, the addition of chicory extract to fermentation and lyophilization showed higher production yield than fermentation with fermentation of whey powder only during fermentation and addition of chicory extract during freeze-drying after fermentation, (Fig . 8 and Table 9) . The results are shown in Table 8 and Table 9, respectively . The results are shown in Table 8 and Table 9, respectively . Table 8 < tb & gt ; < tb & gt ; < tb & gt ; < tb & gt ;

[Table 8]

[Table 9]

Table 9 shows the increase in production yield depending on addition of chicory extract in fermentation and freeze-drying process. When fermentation was performed by adding chicory extract and whey powder, only whey powder was added, and then chicory extract was added after fermentation The yield of viable cells was increased in the final stage. Table 9 shows that when the chicory extract and the whey powder were added and the chicory extract was further added again in the freeze-drying process after fermentation, the production yield of the best lactic acid germ cells was obtained as a synergistic effect during the experiment.

[Example 5]

After the experiment of Example 4, in order to confirm that the EPSs were induced by lactic acid bacteria by the added 2.0 wt% whey powder and 1.0 wt% chicory extract, the amount of EPS produced after the addition fermentation was measured Respectively. After culturing the lactic acid bacterium according to Paulraj Kanmani [Bioresource Technology, 2011, 102 (7), 4827-4833], EPS produced in the culture broth was extracted and isolated.

In experiment A, chicory extract and whey powder were not added. B was added only whey powder. C was added with chicory extract and whey powder. After fermentation, 100 mL of fermentation broth was taken, EPS was extracted as well. The strains used in this experiment were B. lactis (LT) and P. flutarium (PR).

The procedure for isolating EPS from lactic acid fermentation broth is as follows. After 100 mL of each fermentation broth was taken and stirred at 100 ° C for 15 minutes, the EPSs were hydrothermally extracted from the cultured cells. The fermentation broth was centrifuged (10,000 rpm) after hydrothermal extraction, and the remaining water - insoluble materials in the culture broth were completely filtered off using a membrane filter of 0.2 mm pore size. The supernatant thus obtained was cooled in a cold refrigerator for 3 hours. To the thus prepared fermentation broth, 2-fold volume of cold ethanol (0 ° C) was added to precipitate EPS overnight. The next day, the EPSs were separated from the solution by centrifugation at 8,000 rpm.

As a result, the amount of EPS produced in each case is shown in Table 10 below.

[Table 10]

Table 10 shows the change in the amount of EPS produced when whey powder and chicory extract were added to the fermentation medium. As a result, it was confirmed that EPS was induced in a larger amount in case of all the lactic acid bacteria used.

FIG. 3 shows that the EPS produced during the fermentation of L. pleurantarium and B. lactis was treated with cold ethanol so that the EPS precipitated in the refrigerator overnight after the fermentation showed a quantitative difference according to each case FIG. 4 is a photograph showing the EPS precipitated in the centrifuge bottle after centrifuging it, and FIG. 5 is a photograph showing each EPS produced by drying the EPS. As can be seen from FIGS. 3 to 5, (C) when the extract of chicory and whey powder were added, although the EPS was filtered through a membrane of 0.2 mm pore to remove water-insoluble materials, Of EPS was produced by lactic acid bacteria during the culture and ethanol precipitated during the purification process.

In the experiments of this example, whey powder and chicory extract were added to the fermentation medium and increased production of EPS by cultured lactic acid bacteria during fermentation by these substances was confirmed. The EPS thus produced plays a role in protecting the cells from the stresses that the cells undergo during the culturing and subsequent processing.

[Example 6]

This study analyzed the effect of adding chicory extract to the fermentation broth by adding chicory extract to fermented broth. A 1.0% by weight chicory extract and a 2.0% by weight casein peptone were added to each of the culture media of L. planta (FIG. 6A) and B. longom (FIG. 6B) to ferment (" 6A and 6B), followed by centrifugation to obtain respective lactic acid bacterium pellets. Then, the lactic acid bacteria pellet was added with a cryoprotective agent And lyophilized without any treatment to obtain a lactic acid bacterial dry powder. In order to confirm the viability of each of the lactic acid bacteria obtained in this way, the stability was evaluated at 40 ° C. for 4 weeks.

In a severe stability test, the tendency of the viable cell count to decrease while periodically inspecting the stability of the live bacteria in the raw material of lactic acid bacteria was measured while keeping the seeds well sealed to prevent moisture from penetrating and storing at 40 ° C for 4 weeks.

The safety of the lactic acid bacteria obtained after lyophilization was evaluated twice in the L. plantata and B. longomi. The number of lactic acid bacteria present in the raw material obtained after 4 weeks of the harshness test was 11.95% in the case of L. plutarum obtained by adding only whey powder and fermented, whereas the content of chicory extract and whey powder was added The fermented broth showed a 2.18 fold increase in the number of yeast extracts (26.1%) (Fig. 6a) and a slight but slightly increased tendency in the B. longtrim leaf (Fig. 6b).

[Example 7]

The microalgae extracts containing EPS and the lysed lactic acid bacteria lysate were added for use as a cryoprotectant and prebiotics in the mixing process of recovered lactic acid bacterium cells and the preservative after incubation. In this experiment, microalgae extract was added to vial cultures to measure growth promoting effect to determine whether microalgae extract could serve as a prebiotics by helping four kinds of lactic acid bacteria grow.

Experiments have demonstrated the effects of microalgae extracts and lactic acid bacterial lysates as growth promoters in the growth of L. Pleantarum, B. longgom, L. luteri, and L. kaijie. Glucose was added to the MRS (Difco, UK) medium as the medium for the experiment in an amount of 1.0 wt% each. Microalgae extracts and lactic acid bacteria lysates extracted from microalgae spirulina were prepared in the laboratory and added at 0.5 wt% concentration. Lactic acid bacteria growth experiments were carried out in vials as non-standard. Microalgae extracts and lactic acid bacterium extracts added as a cryoprotectant have a mitogenic effect on the growth of lactic acid bacteria, promote nutritional growth, inhibit the growth of other intestinal bacteria, and selectively inhibit the growth of lactic acid bacteria and intestinal colonization ) As a prebiotic.

As can be seen from FIG. 7, it was confirmed that the microbial extract and the lactic acid bacterium-added sample were able to stimulate the growth of the four kinds of lactic acid bacteria used in the experiment, as compared with the comparative example.

As can be seen from the above results, the microalgae extracts and the lactic acid bacterial debris that were developed by the inventors stimulated the growth of four representative probiotic lactic acid bacteria. The mitogenic effect of these microalgae extracts and lactic acid bacterium isolates showed their full potential as prebiotics and they were added to the production of probiotics and used as freeze protectants and prebiotics to help intestinal activity of lactic acid bacteria Suggesting that it can be utilized.

[Example 8]

An SEM image of the dried powder of L. plutarium and B. longum obtained by lyophilizing the recovered lactic acid bacteria pellet by centrifugation (10,000 rpm) in a culture medium with or without added chicory extract is shown in FIG. As can be seen from Fig. 8, the materials formed during fermentation during the lyophilization process are concentrated and crystallized during lyophilization and are present on the surface of the cells, which is in contrast to the case where the fermentation medium is not supplemented with the chicory extract .

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention .

Claims

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Inoculating a lactic acid bacterium strain into a lactic acid bacterial culture composition comprising chicory dry powder or chicory extract and whey powder;
Culturing the inoculated lactic acid bacteria strain;
Preparing a lactic acid bacteria pellet by centrifuging the cultured lactic acid bacteria strain; And
Adding a chicory dry powder or a chicory extract to the lactic acid bacteria pellet, followed by lyophilization.

The method according to claim 6,
Wherein the inoculated lactic acid bacteria strain is cultured by a fed-batch culture method.

The method according to claim 6,
The lactic acid bacteria strain inoculated into the lactic acid bacteria culture composition may be selected from the group consisting of Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp. Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius (Lactobacillus spp.), Lactobacillus spp. Lactobacillus salivarius, Lactobacillus reuteri, But are not limited to, Leuconostoc mesenteroides, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium bifidum, lactis, Bifidobacterium infantis, and combinations thereof. < RTI ID = 0.0 > 21. < / RTI >

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The method according to claim 6,
Wherein the method further comprises the step of adding to the lactic acid bacteria pellet an amino acid mixture comprising an amino acid selected from the group consisting of arginine, glutamic acid, penicillamine, and combinations thereof.

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The method according to claim 6,
Wherein the chicory dry powder or chicory extract comprises inulin.

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13. The method of claim 12,
Wherein the amino acid mixture is added in an amount of 0.01 wt% to 10 wt% with respect to the weight of the lactic acid bacteria pellet.

A lactic acid bacterium powder produced by the method according to any one of claims 6 to 8, 12, 15 and 18.

A food supplement comprising the lactic acid bacterium powder according to claim 19.

A feed additive comprising the lactic acid bacterium powder according to claim 19.

The method according to claim 6,
Wherein the lactic acid bacteria culture medium composition further comprises at least one selected from the group consisting of casein peptone, caspase, peptone of milk origin, and combinations thereof.