阅读说明：本技术 用于提高益生菌肠道存活率和粘附性的益生菌冻干物再激活组合物 (Probiotic freeze-dried substance reactivation composition for improving intestinal survival rate and adhesiveness of probiotics ) 是由 金镇学 金秀珍 金秀姃 池约瑟 于 2021-03-29 设计创作，主要内容包括：公开了一种Zeta-bio组合物,用于通过在益生菌的细胞表面上赋予负zeta电位来重新激活冻干益生菌,所述组合物包含选自由L-赖氨酸、L-鸟氨酸、L-酪氨酸和L-组氨酸组成的组中的至少一种作为激活剂。所述组合物通过赋予冻干益生菌负zeta电位来激活冻干益生菌,从而提高冻干益生菌的肠道存活率和粘附性,此外还表现出对由于冻干造成的细胞损伤的恢复作用。(Disclosed is a Zeta-bio composition for reactivating freeze-dried probiotics by imparting a negative Zeta potential on the cell surface of the probiotics, the composition comprising at least one selected from the group consisting of L-lysine, L-ornithine, L-tyrosine and L-histidine as an activator. The composition activates freeze-dried probiotics by imparting a negative zeta potential to the freeze-dried probiotics, thereby improving the intestinal viability and adhesion of the freeze-dried probiotics and further showing a recovery effect on cell damage caused by freeze-drying.)

1. A composition for reactivation of freeze-dried probiotics that imparts a negative zeta potential to the surface of probiotic cells to reactivate freeze-dried probiotics.

2. The composition of claim 1, wherein the composition comprises at least one selected from the group consisting of L-lysine, L-ornithine, L-tyrosine and L-histidine as an activator.

3. The composition of claim 2, wherein the composition comprises L-lysine as an activator.

4. A composition according to claim 2 or 3, wherein the activator is present in an amount such that it forms a final concentration of 0.01M to 0.15M when dissolved in a solvent.

5. The composition according to any one of claims 1-3, further comprising at least one carbohydrate selected from the group consisting of fructose, sucrose, sorbitol, glucose, maltose, trehalose, and fructo-oligosaccharides.

6. The composition according to claim 5, wherein the carbohydrate is present in an amount of 0.1 to 8 g.

7. The composition according to any one of claims 1 to 3,it has a concentration of 1 × 10⁸To 1X10¹²CFU/g of freeze-dried probiotic bacteria.

8. The composition of any one of claims 1-3, wherein the lyophilized probiotic is a lyophilized form of Lactobacillus, lactococcus, enterococcus, Bifidobacterium, Pediococcus, Streptococcus, or a combination thereof.

9. The composition of claim 8, wherein the lyophilized probiotic is a lyophilized form of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Streptococcus thermophilus, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus reuteri, Lactobacillus gasseri, enterococcus faecium, Clostridium butyricum, Lactobacillus rhamnosus, Streptococcus thermophilus, Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus salivarius, lactococcus lactis, enterococcus faecalis, Bifidobacterium bifidum, or a combination thereof.

10. The composition of any one of claims 1-3, wherein the composition is dissolved in a solvent prior to ingestion to reactivate the lyophilized probiotic.

11. A lyophilized probiotic product comprising the composition of any one of claims 1-3.

12. The lyophilized probiotic product of claim 11 wherein said product is dissolved in a solvent prior to ingestion to reactivate the lyophilized probiotic.

13. A method of reactivating freeze-dried probiotics comprising contacting the freeze-dried probiotics with an activator, wherein the activator comprises at least one selected from the group consisting of L-lysine, L-ornithine, L-tyrosine and L-histidine.

14. A method of screening a substance having a function of reactivating freeze-dried probiotics, the method comprising selecting a substance that confers a negative zeta potential on the cell surface of freeze-dried probiotics.

Technical Field

The present disclosure relates to a composition that rehydrates and activates lyophilized probiotics in a short period of time, and a method of activating lyophilized probiotics.

Background

It is well known that probiotics have a beneficial effect on the health of the host when ingested in appropriate amounts. Scientific evidence is accumulating that probiotics have beneficial effects on human health in various ways, including relief of immune disorders, inflammatory bowel disease, type 2 diabetes, arteriosclerosis, and the like. Although recommendations are biased towards high dosages of probiotic bacteria, neither the specific dosages nor the minimum number of viable bacteria required for the assumed probiotic strain is well defined.

Although strains with potential probiotic properties can be obtained "naturally" from fermented foods such as yoghurt, the distribution of freeze-dried probiotic powders packaged in sachets or capsules on the market is also rapidly expanding. Commercially available probiotics should be readily transportable, shelf stable concentrates to ensure their effect on their inherent functional properties at room temperature. Commercialization of non-dairy probiotic products requires precise optimization of the final processing steps, such as the freezing and drying processes.

The freeze-drying (or lyophilization) process is considered to be a suitable method to ensure an extended shelf life for most probiotic products. However, it is well known that this process also puts stress on the live bacteria. The freeze-drying process is indeed a "challenge" to the viability of the probiotic strains. Thus, to maintain an effective dose, most probiotic products are manufactured with three to ten times higher numbers of bacteria than are noted on the product label.

During the freeze-drying process, probiotics are fatally damaged to a large extent, and when exposed to gastric acid and bile in the human body in such a damaged state, the viability of the probiotics is further reduced due to the fatality stress. Even probiotics that survive in the intestine have difficulty in fully performing their functional role unless they are able to effectively adhere to the intestine. Thus, key requirements to ensure the effectiveness of probiotics include their survival under gastrointestinal stress conditions and adhesion to the intestinal wall. The colon cell line can be used as an in vitro model for determining the adhesion potential of putative probiotic strains (non-patent document 1). The adhesion properties of bacterial cell membranes are determined by surface properties such as hydrophobicity, extracellular polymeric substances and charge. At the same time, the cell membrane plays a key role in maintaining cellular homeostasis and supporting intracellular functions. Upon interaction with the environment, bacteria are subjected to various physical forces that are transmitted to the cell by specific surface structures. Various functional acidic and basic groups (such as phospholipids, Lipopolysaccharides (LPS) on the surface of gram-negative bacterial membranes) and surface proteins, as well as hydrocarbon-like proteins (such as lipoteichoic acid and teichoic acid on the cell surface of gram-positive bacteria) determine the response of the strain to the environment (non-patent documents 2 and 3).

Protection of the cell membrane to maintain cell viability and adhesion functions is important, which is necessary for effective potential probiotics. To promote immune regulation and metabolic function, help strengthen the intestinal barrier, and competitively inhibit adhesion of pathogens, cells must survive gastric pressure and attach properly to intestinal cells. Most adhesion factors, such as lipoteichoic acid, S-layer proteins, mucus binding proteins, etc., are located around the cell surface of bacteria. Although the lyophilization process helps to maintain the shelf life of the bacteria by reducing water activity, it also destroys cell membranes, which may result in loss of its original function (non-patent document 4). Restoring the integrity of the cell membrane may help to reactivate its function by increasing cell viability and cell adhesion.

The electrostatic charge on the cell surface is believed to be a reflection of its functional groups. The surface charge of bacterial cells when in contact with a liquid can be measured in millivolt units as zeta or electrokinetic potential. Both the surface composition of the cell and the properties of the surrounding medium (such as conductivity/ionic strength and pH) determine the zeta potential of the cell.

Recently, the zeta potential around bacteria has become an important indicator of bacterial viability and overall efficacy, particularly in terms of physiology. However, there has been no report on the use of zeta potential for cell reactivation by rehydration of probiotic lyophilisates.

(non-patent document 1) Arellano K.K., Vazzezez J.H., Lim J.J., Ji Y.K., Kang H.H., Cho D.J.H.W.and Holzapfel W.H (2019) Safety evaluation and book-gene evaluation of Lactobacillus plant strain from parameter sources with special focus on enzymes from grid

(non-patent document 2) Dufrene, Y.F. and Persat, A. (2020) mechanomicromology: how bacteria sense the response to forces. Nat. Rev. Microbiol.18:227-

(non-patent document 3) Boonaert, C.J.P.and Rouxhet, P.G. (2000). Surface of lactic acid bacteria: relationship shifts between chemical composition and physical properties.appl.Environment.Microbiol.66: 2548-.

(non-patent document 4) Govender, M., Choonara, Y.E., Kumar, P., du Toit, L.C., van Vuuren, S., & Pillay, V. (2014). A review of the advance in biological delivery: general v.non-general for interaction flow administration.Aaps PharmScich, 15(1),29-43.

Disclosure of Invention

According to various embodiments thereof, the present disclosure provides a composition for probiotic reactivation and a method for freeze-dried probiotic reactivation, wherein freeze-dried probiotics are activated within a short time and provide improved survival and gut adhesion by imparting an appropriate negative zeta potential thereon, which results in maximization of probiotic efficacy.

According to other various embodiments thereof, the present disclosure provides a composition for activating lyophilized probiotics whereby the probiotic cells may recover from damage caused by the lyophilization pressure.

According to other various embodiments thereof, the present disclosure provides a screening method wherein a substance conferring a negative zeta potential on the cell surface of a lyophilized probiotic is selected and used as an activator to improve the survival rate and intestinal adhesion of the lyophilized probiotic.

For the purpose of the present disclosure, the present inventors have intensively and thoroughly studied the reactivation of a freeze-dried probiotic substance in a short time and found that a substance imparting a negative zeta potential to a probiotic bacterium can be used as an activator for activating the freeze-dried probiotic bacterium, improve the intestinal survival rate and adhesiveness of the freeze-dried probiotic bacterium, and recover the freeze-dried probiotic bacterium from cell damage caused by freeze-drying. Surprisingly, carbohydrates, amino acids and proteins with a cell membrane protecting function, in particular amino acids selected from the group consisting of L-lysine, L-ornithine, L-tyrosine and L-histidine, can act as very effective activators to reactivate various lyophilized probiotics by imparting a negative zeta potential on the cell surface of the lyophilized probiotics.

The compositions and methods according to one aspect of the present disclosure have the effect of reactivating and improving the functionality of freeze-dried probiotics by imparting an appropriate negative zeta potential on the cell surface of the freeze-dried probiotics.

With such effects, the compositions and methods of the present disclosure can improve the survival rate and intestinal adhesion of lyophilized probiotics and restore damage to cell membranes.

Furthermore, due to these effects, the compositions and methods of the present disclosure can achieve cost reduction and superior efficacy in the probiotic market where probiotic efficacy is guaranteed only by the input of large quantities of lyophilized probiotics.

Drawings

FIG. 1 is a zeta potential diagram of freeze-dried Lactobacillus plantarum HAC03 to which 9 amino acids were added, respectively.

FIG. 2 shows the results of measurements of the adherence to intestinal cells based on the bacterial count of Lactobacillus casei Lc-11(A), Bifidobacterium longum Bl-05(B), Lactobacillus plantarum Lp-115(C) and the 7-strain mixture (400B) (D), in the form of fresh cells, lyophilized probiotic, L-lysine activated lyophilized probiotic or proline mixed lyophilized probiotic; and the results of measurement of adhesion to intestinal cells based on the relative adhesion (%) of Lactobacillus casei Lc-11(E), Bifidobacterium longum Bl-05(F), Lactobacillus plantarum Lp-115(G) and 7-strain mixture (400B) (H) in the form of fresh cells, lyophilized probiotics, L-lysine-activated lyophilized probiotics or proline-mixed lyophilized probiotics, as compared to the number of adherent bacteria in the test group and the control group.

Fig. 3 shows the results of the determination of the adhesion to intestinal cells based on the bacterial count of lactobacillus plantarum Lp-115(a) and 7-strain mixture (400B) (C), in the form of fresh cells, lyophilized probiotics or lyophilized probiotics activated with the Zeta-bio composition of the present disclosure, and the results of the determination of the adhesion to intestinal cells based on the relative adhesion (%) of lactobacillus plantarum Lp-115(B) and 7-strain mixture (400B) (D) (ratio of the number of adherent bacteria in the test group to the number of adherent bacteria in the control group), in the form of fresh cells, lyophilized probiotics or lyophilized probiotics activated with the Zeta-bio composition of the present disclosure.

Detailed Description

Embodiments of the present disclosure are shown to describe the technical spirit of the present disclosure. The scope of the claims according to the present disclosure is not limited to the embodiments described below or the detailed description of these embodiments.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used herein is for the purpose of describing the disclosure more clearly and is not intended to limit the scope of the claims in accordance with the disclosure.

Unless otherwise stated in a phrase or sentence including such expressions, the expressions "including," "providing," "having," and the like, as used herein, are to be understood as open-ended terms that imply the possibility of including other embodiments.

Expressions such as "consisting of … … only" as used in the present disclosure should be understood as closed terms, which exclude the possibility of including other configurations than the corresponding configuration.

As used herein, the singular forms "a", "an" and "the" may include plural referents unless the context clearly dictates otherwise.

In one aspect of the present disclosure, the term "about" is used for the purpose of including errors in manufacturing of specific numerical values or slight numerical adjustments falling within the technical idea of the present disclosure. For example, the term "about" means within + -10% of the value it refers to, on one side + -5% and on the other side + -2%. In the field of this disclosure, approximations to the levels are suitable unless the value is specifically indicated to require a narrower range.

Next, a specific description will be given of a composition according to an aspect of the present disclosure.

One aspect of the present disclosure relates to a composition that can reactivate lyophilized probiotics to impart an appropriate negative zeta potential on the cell surface of the probiotics. As an activator of freeze-dried probiotics, the compositions of the present disclosure can improve intestinal viability and adhesion by reactivating freeze-dried probiotics and imparting them with an appropriate negative zeta potential instead of simply rehydrating the probiotic lyophilizate. The zeta potential can be used as an indicator of bacterial viability, and changes in zeta potential reflect membrane damage and changes in permeability. The depolarizing zeta potential explains the intact cell membrane. Thus, the inventors first introduced the zeta potential concept into the reactivation of freeze-dried probiotics and found that changing to a negative zeta potential results in cell reactivation, recovery from injury and improved viability.

In one aspect of the present disclosure, the composition for probiotic reactivation according to the present disclosure may include at least one selected from the group consisting of L-lysine, L-ornithine, L-tyrosine, and L-histidine as an activator, preferably L-lysine and/or L-tyrosine, more preferably L-lysine.

In the present disclosure, L-lysine may be L-lysine hydrochloride.

In one aspect of the disclosure, when the activator is dissolved in the solvent, the composition of the disclosure may comprise the activator at a concentration of about 0.01M to about 0.15M, about 0.01M to about 0.1M, about 0.02M to about 0.07M, about 0.02M to about 0.05M, or about 0.03M to about 0.05M. When used at concentrations below the lower limit, the activator may not be able to sufficiently activate the lyophilized probiotic. At concentrations above the upper limit, there are problems in that the probiotics may die, it is difficult to obtain high concentration products, and the taste of the products comprising the composition of the present disclosure deteriorates.

In one aspect of the present disclosure, the composition for probiotic reactivation according to the present disclosure may further comprise at least one carbohydrate selected from the group consisting of fructose, sucrose, sorbitol, glucose, maltose, trehalose, and fructo-oligosaccharides, preferably fructo-oligosaccharides.

In the present disclosure, at least one carbohydrate selected from the group consisting of fructose, sucrose, sorbitol, glucose, maltose, trehalose, and fructo-oligosaccharide has not only a cell damage preventing function for the lyophilization of probiotics, but also can prevent the lyophilized probiotics from suffering cell membrane damage caused by osmotic pressure upon rehydration and increase the intestinal viability of the probiotics. Preferred in the present disclosure are carbohydrates that have no effect on the function of the activator to impart a negative zeta potential on the cell surface of the lyophilized probiotic.

In the present disclosure, fructooligosaccharide is a functional oligosaccharide with sweetness and physical properties similar to sucrose, thus improving the mouthfeel of products containing the composition of the present disclosure. As a prebiotic, fructooligosaccharides can extend shelf life, are stable to heat and acids, and have significant resistance to acids, proteases, and bile acids when passed through the gastrointestinal tract. Due to the resistance to gastric pressure, fructooligosaccharides may improve the viability of the probiotic. In addition, fructooligosaccharides may provide beneficial health effects in preventing weight gain and intestinal disorders by producing short chain fatty acids. Fructooligosaccharides also regulate the microbiota to a healthy state by increasing the proportion of beneficial bacteria such as bifidobacteria and reducing harmful bacteria in the human gut. In particular, fructooligosaccharides may enhance the resistance of probiotics under unbalanced osmotic conditions when used in combination with amino acid activators.

In one aspect of the disclosure, the compositions of the present disclosure may comprise carbohydrate in an amount of from about 0.1g to about 8g, from about 0.2g to about 6g, from about 0.3g to about 5g, from about 0.5g to about 4g, or from about 1g to about 4 g. When the content of the carbohydrate is below the lower limit, it is difficult to expect a synergistic effect on the recovery of damaged cell membranes after rehydration of the probiotic lyophilizate, and to sufficiently improve the intestinal survival rate and adhesion to intestinal cells.

In one aspect of the disclosure, the compositions of the disclosure may comprise a concentration of about 1x10⁸To about 1X10¹²CFU/g of freeze-dried probiotic bacteria.

In one aspect of the disclosure, the lyophilized probiotic that may be reactivated with the composition may be lactobacillus, lactococcus, enterococcus, bifidobacterium, pediococcus, streptococcus, or combinations thereof. Specifically, the lyophilized probiotic of the present disclosure may be lactobacillus plantarum, lactobacillus acidophilus, lactobacillus casei, streptococcus thermophilus, bifidobacterium animalis, bifidobacterium longum, bifidobacterium breve, bifidobacterium lactis, lactobacillus reuteri, lactobacillus gasseri, enterococcus faecium, clostridium butyricum, lactobacillus rhamnosus, streptococcus thermophilus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus helveticus, lactobacillus fermentum, lactobacillus paracasei, lactobacillus salivarius, lactococcus lactis, enterococcus faecalis, bifidobacterium bifidum, or a combination thereof.

In one aspect of the invention, the composition may be reactivated in vitro by dissolution in a solvent prior to ingestion, or in vivo by drinking a solvent prior to or after ingestion. In the present disclosure, when the lyophilized probiotic is reactivated, the probiotic may have a negative zeta potential on its cell surface.

In one aspect of the disclosure, the lyophilized probiotic may be in the form of a lyophilized powder.

In one aspect of the present disclosure, the composition may further comprise an ingredient, such as an amino acid (e.g., L-glutamic acid, L-serine, L-threonine, L-tryptophan, L-phenylalanine, etc.), betaine, taurine, riboflavin, thiamine, etc., all of which can improve the survival rate of the lyophilized probiotic bacteria by restoring cell membranes. The additional ingredient may be present in the composition in an amount such that the additional ingredient has a final concentration of from 0.01M to 0.15M when the composition is dissolved in a solvent.

One aspect of the present disclosure relates to a method of reactivation of lyophilized probiotics, wherein a composition of the present disclosure is dissolved in a solvent to impart a negative zeta potential to the cell surface of the probiotics prior to ingestion, whereby the lyophilized probiotics are reactivated. In one embodiment, the lyophilized probiotic may be reactivated by ingestion of a composition of the present disclosure either before or after ingestion of the solvent. Any method may be used as long as it allows rehydration of both the composition of the present disclosure and the probiotic lyophilizate, without limitation thereto.

One aspect of the present disclosure relates to a method of reactivating freeze-dried probiotics, the method comprising contacting the freeze-dried probiotics with an activator comprising at least one selected from the group consisting of L-lysine, L-ornithine, L-tyrosine and L-histidine.

In one aspect of the present disclosure, the solvent may be any drinkable solvent, is not particularly limited, and may preferably be water.

In a method of freeze-dried probiotic reactivation according to the present disclosure, a composition of the present disclosure is dissolved in a solvent to activate probiotics in about 30 minutes, about 10 minutes, about 5 minutes, about 1 minute, about 30 seconds, or a few seconds. Whether the lyophilized probiotic bacteria are activated within a short period of time may be determined by adding an edible dye to the composition of the present disclosure that changes color as a function of pH.

In one aspect of the disclosure, the compositions of the disclosure may be mixed with a solvent in an amount of 0.1 to 20 times the weight of the composition, especially 0.5 to 15 times or 1 to 10 times the weight of the composition. For example, the solvent may be used in an amount of 1 to 200ml, 5 to 150ml or 10 to 100ml per 10g of the composition.

One aspect of the present disclosure relates to probiotic products comprising the compositions of the present disclosure. In one aspect of the present disclosure, a probiotic product comprising a composition of the present disclosure may be provided in a separate package of lyophilized probiotic bacteria and other ingredients or in an integrated package of lyophilized probiotic bacteria and other ingredients. The probiotic product comprising the composition of the present disclosure may be provided in the form of a sachet or capsule. In one aspect of the present disclosure, the composition of the present disclosure may be dissolved in a solvent to reactivate the lyophilized probiotic prior to ingestion, or may be ingested to reactivate the lyophilized probiotic prior to or after ingestion of the solvent, as appropriate.

One aspect of the present disclosure relates to a method of screening for a substance capable of reactivating freeze-dried probiotic bacteria, the method comprising selecting a substance that confers a negative zeta potential on the cell surface of the freeze-dried probiotic bacteria. Substances that confer a negative zeta potential on the cell surface of the lyophilized probiotic may reactivate the lyophilized probiotic, help increase survival and adhesion to intestinal cells, and restore damaged cell membranes.

Experimental example 1 screening of ingredients capable of reactivating lyophilized probiotics by imparting a negative Zeta potential to the cell surface of the lyophilized probiotics

In this experimental example, Lactobacillus plantarum HAC03 (accession No.: KCTC13242BP, Korea institute of bioscience and Biotechnology) (hereinafter, referred to as "HAC 03") described in Korean patent application No. 10-2017-0051574 was used as the probiotic bacterial strain.

At 1x10⁹CFU/g concentration lyophilized HAC03 strain powder was transferred to a 50mL tube and mixed with each of nine single amino acid components (L-tyrosine, L-ornithine, malic acid, L-lysine, L-histidine, L-aspartic acid, L-ascorbic acid, L-arginine and proline) to form a final concentration of 0.1M. To the mixture of lyophilized HAC03 and the single component was added 1mL DW and rehydrated for 1 minute. Then, 2mL of distilled water pH 2.5 was added to each sample, the pH was readjusted using 0.1N HCl, and 800 μ L of the calibration sample was loaded into a DTS1080 cuvette. After 2 minutes of equilibration, electrophoretic mobility was measured by a Zetaizer Nano ZEN 3600(Malvern Panalytica, UK) and the data were converted to zeta potential values using the Smoluchowski equation. The converted zeta potential values are shown in FIG. 1.

In this experiment, the zeta potential was measured at a pH of 2.5, taking into account the highly acidic gastric environment, which is one of the first obstacles to significantly reduce the viability of the bacteria. As shown in fig. 1, after HAC03 was lyophilized, the zeta potential of bacterial cells significantly depolarized compared to freshly cultured cells. When bacteria are mixed with each of the nine amino acid components, L-tyrosine, L-ornithine, L-lysine and L-histidine produce a negative zeta potential, as in freshly cultured HAC 03. However, the remaining five components, L-arginine, L-aspartic acid, malic acid, L-ascorbic acid and proline, do not change the depolarizing zeta potential to a negative value, but exhibit a positive zeta potential.

Experimental example 2 increasing the intestinal viability of lyophilized probiotic bacteria by imparting a negative Zeta potential to the cell surface

The HAC03 strain and the eight components used in Experimental example 1 were evaluated for probiotic activity (acid resistance and bile resistance) using an in vitro simulated gastroduodenal channel (SSDP) according to Ji et al (Food control 31(2):467-473,2013) with some modifications. The HAC03 strain was mixed with each single ingredient and 1mL of distilled water for 1 minute. These ingredients were formulated to a final concentration of 0.1M when dissolved in 1mL of distilled water. Thereafter, 9mL of distilled water adjusted to pH 2.5 was added to each mixture. If due to the nature of some of the individual componentsIf the pH rises due to the substance, the pH is readjusted back to 2.5 (in order not to affect the survival of the probiotic). After adjusting the pH to 2.5, the tubes were incubated at 37 ℃ for 1 hour to apply a low pH gastric pressure regime. Then, the strain was combined in 4mL bile salt (10% oxgall) and 17mL pH 6.0 to synthesize duodenal juice (6.4g/L NaHCO)₃0.239g/L KCl and 1.28g/L NaCl) for 2 hours, mimicking small intestinal channels. During gastrointestinal testing, samples were taken immediately, 1 hour, and 3 hours (t ═ 0, 1, and 2) after incubation. Probiotic survival after gastric and biliary stress was calculated for each sample by counting viable colonies. The results of the measurements are summarized in table 1 below.

TABLE 1

As can be seen from the data of table 1, when cultured with four components (L-ornithine, L-lysine, L-tyrosine, and L-histidine) imparting a negative zeta potential, the acid resistance and bile resistance of the lyophilized probiotics were increased compared to fresh bacteria, thereby exhibiting improved intestinal survival rates. In contrast, the four components (arginine, ascorbic acid, aspartic acid and malic acid) which do not convert the zeta potential to a negative value, resulted in a much lower intestinal survival rate of the probiotic lyophilizate than distilled water (survival rate 3.40%). These data indicate that substances that confer a negative zeta potential on the cell surface of freeze-dried probiotic have a significant positive impact on the intestinal survival rate of freeze-dried probiotic.

Experimental example 3 intestinal adhesion assay capable of reactivating a component of a lyophilized probiotic

Of the four components conferring negative zeta potential and increasing intestinal survival rate in the freeze-dried probiotic, L-lysine, which showed the highest survival rate in experimental example 2, was used to reactivate the freeze-dried probiotic to assess the effect on cell adhesion to the intestine. This experimental example investigated whether the reactivation effect of freeze-dried probiotic was applicable to various strains. In this regard, lyophilized Bifidobacterium longum Bl-05 (Dupont; hereinafter simply referred to as "Bl-05"), Lactobacillus plantarum Lp-115 (Dupont; hereinafter simply referred to as "Lp-115"), Lactobacillus casei Lc-11 (Dupont; hereinafter simply referred to as "Lc-11"), and 7-strain mixture (400B) (Dupont; hereinafter simply referred to as "MIX") were reactivated with L-lysine, and then their ability to adhere to human enterocyte-like Caco-2/TC-7 cell lines was determined. The 7-strain mixture refers to a mixture of 7 strains listed in table 2 below. In this experimental example, proline was kept at a positive zeta potential on the cell surface of lyophilized HAC03 and used as a control.

TABLE 2

Lyophilisates of the three strains and the 7-strain mixture were adjusted to 2X10⁸CFU/g, and mixed with 0.03M L-lysine or 0.03M proline. The pellet was washed 3 times with 1x PBS and resuspended in 10mL MEM cell culture medium supplemented with 20% FBS, 2mM glutamine, and 1% non-essential amino acids. Bacterial adhesion assays were performed according to the method of Botes et al (arch. microbiol,190(2008), pp.573-584) with some modifications. To assess bacterial cell adhesion, 5% CO at 37 deg.C₂And 95% air with 2mL of each bacterial suspension to 1X10⁵CFU/Caco-2/TC-7 monolayers were treated for 1.5 hours. Cells were washed 3 times with cold PBS to remove unattached bacteria and lysed by adding 40ng trypsin (Promega) for 15 minutes at 37 ℃. To measure the number of bacteria attached to Caco-2/TC-7 cells, samples were serially diluted, cultured on MRS agar plates for 48 hours (37 ℃) and then counted to measure the relative adherence ratio (the ratio of the count of attached bacteria in the test group to the count of attached bacteria in the control). The results are depicted in fig. 2. All experiments were performed in triplicate.

As can be seen in FIG. 2, the lyophilized Forms (FD) of Lc-11 (FIGS. 2A and 2E), Bl-05 (FIGS. 2B and 2F) and Lp-115 (FIGS. 2C and 2G) are significantly less adherent to the Caco-2/TC-7 cell line compared to fresh cells (fresh). . Reactivation with L-lysine significantly increased bacterial cell adhesion compared to the lyophilized form. The results were also repeated in the 7-strain mixture (400B) (FIGS. 2D and 2H). However, the intestinal adherence of the lyophilized form mixed with proline conferring a positive zeta potential on the cell surface of the lyophilized probiotic (control) is significantly lower than that of the lyophilized form itself (Lc-11, Bl-05, MIX) and those reactivated with L-lysine (Lp-115). In other words, when the freeze-dried probiotic bacteria are reactivated with a substance capable of imparting a negative zeta potential on their cell surface (e.g. L-lysine), the adhesion to the intestinal tract is increased, whereas substances imparting a positive zeta potential do not show an improved adhesion effect. In summary, the data indicate that negative zeta potential on the cell surface correlates with improved intestinal adhesion.

Experimental example 4 other ingredients that can be used with lyophilized probiotic activators

A test was performed to examine the components that can be used with an activator component capable of imparting a negative zeta potential on the cell surface of the freeze-dried probiotic. The HAC03 lyophilizate was mixed with each of the 11 carbohydrates listed in table 3 below, and the intestinal survival rate was determined in the same manner as in example 1. The results are summarized in table 3 below.

TABLE 3

As can be seen from table 3, seven components of fructose, sucrose, sorbitol, glucose, maltose, trehalose and fructo-oligosaccharide resulted in survival rates similar to or higher than that of fresh cells, and they were observed to have higher bile resistance than fresh cells. Thus, these ingredients can be used with an activator to reactivate probiotic lyophilisates and improve their intestinal viability.

Experimental example 5 survival assay after reactivation of lyophilized Probiotics with a composition of the invention

Experiments were performed to examine the effect of the compositions of the present disclosure on the reactivation of lyophilized probiotics. First, the composition of the present disclosure (hereinafter abbreviated as "Zeta-bio composition") was prepared to include L-lysine, Fructooligosaccharide (FOS), and microorganisms (probiotics) according to the compositions listed in table 4 below. Fructooligosaccharides were measured to have a zeta potential close to 0mV, and therefore did not affect the zeta potential of the lyophilized probiotic of the present disclosure. Lyophilized Lp-115 and MIX were used as lyophilized probiotics.

TABLE 4

The Zeta-bio compositions in table 4 were tested for probiotic survival by mimicking the gastroduodenal pathway (SSDP) in vitro, as in experimental example 2. The Zeta-bio compositions in Table 4 were each mixed with 1mL of distilled water at 25 ℃ for 1 minute. In this regard, the Zeta-bio composition is prepared to include L-lysine at a concentration of 0.01M, 0.02M, 0.03M, 0.04M, or 0.05M (when dissolved in 1mL of distilled water). Then, 9mL of distilled water adjusted to pH 2.5 was added to each mixture, readjusting back to pH 2.5 when the pH increased due to the nature of certain ingredients (to exclude any effect on probiotic survival). The tube was incubated at 37 ℃ for 1 hour to apply low pH gastric pressure thereto. Subsequently, the tubes were exposed to 4mL of bile salts (10% oxgall) and 17mL of synthetic bile at pH 6.0 (6.4g/L NaHCO)₃0.239g/L KCl and 1.28g/L NaCl) for 2 hours to mimic small intestinal channels. During the gastrointestinal tract assay, samples were taken immediately, 1 hour, and 3 hours (t ═ 0, 1, and 2) after exposure. Probiotic viability was calculated for each sample after exposure to gastric and biliary pressure by counting viable colonies. The results of the measurements are summarized in table 5 below.

TABLE 5

As can be seen in table 5, the viability of the lyophilized form of Lp-115 was significantly reduced (0.02%) after SSDP compared to the fresh cell control (fresh). Lyophilized Lp-115 showed similar or higher survival rates as fresh cells under gastric and biliary pressure when reactivated with Zeta-bio compositions containing L-lysine at concentrations of 0.03M or higher. Activation with the Zeta-bio composition with L-lysine at a concentration of 0.03M or higher also ensured high survival of the lyophilized 7-strain mixture (400B) under gastric and bile pressure. Thus, a probiotic lyophilizate activated with a Zeta-bio composition with a concentration of L-lysine of 0.03M or higher shows a higher survival rate after passage through the gastrointestinal tract than the lyophilized probiotic alone.

Experimental example 6 determination of intestinal adhesion after reactivation of lyophilized Probiotics with a composition according to the invention

Experiments were performed to examine the effect of the Zeta-bio compositions of the present disclosure on freeze-dried probiotic intestinal adhesion. The Zeta-bio compositions in Table 3 were observed to increase intestinal adhesion in lyophilized LP-115 and MIX, when measured by the same assay for the ability to adhere to human intestinal cell-like Caco-2/TC-7 cell lines as in Experimental example 3. The results are shown in FIG. 3.

As can be seen in FIG. 3, the lyophilized Form (FD) of Lp-115 (FIGS. 3A and 3B) is significantly lower than fresh cells (fresh) in terms of adhesion of bacterial cells to the Caco-2/TC-7 cell line. However, reactivation with the Zeta-bio composition (LP-3 to LP-5) significantly restored bacterial cell adhesion compared to the lyophilized form. Lp-115 showed higher intestinal adhesion at all tested L-lysine concentrations compared to the lyophilized form. Also, the 7-strain mixture (400B) exhibited significantly high adhesion when reactivated with MIX-3 and MIX-4 compositions containing L-lysine at concentrations of 0.03M and 0.04M, respectively. Thus, the composition containing L-lysine according to the present disclosure may reactivate the freeze-dried probiotic and increase the survival rate and intestinal adhesion of the freeze-dried probiotic.

Experimental example 7 survival rate of lyophilized probiotic bacteria reactivated with the composition according to the present invention was determined according to L-lysine concentration

Experiments were performed to examine the effect of Zeta-bio compositions with L-lysine at concentrations of 0.1M or higher and at concentrations of 0.01M to 0.05M (as shown in table 2) on the survival of freeze-dried probiotics reactivated therewith. The Zeta-bio compositions used in this experimental example were the same as those in Table 2 in terms of formulation and content, but different in L-lysine concentration and were prepared to contain L-lysine at a concentration of 0.1M, 0.15M, 0.3M, 0.15M or 3M, respectively, when dissolved in 1mL of distilled water for MIX-6, MIX-7, MIX-8, MIX-9 or MIX-10.

The probiotic survival rate (acid and bile resistance) of the Zeta-bio compositions was determined by simulating the gastroduodenal pathway (SSDP) in vitro as shown in experimental example 5. The measurement results are summarized in table 6 below.

TABLE 6

As can be seen from Table 6, the compositions MIX-3, MIX-6 and MIX-7 containing L-lysine at concentrations of 0.03M, 0.1M and 0.15M, respectively, exhibited significant survival rates of 57% or more for gastric acid and 11% or more for bile acid. Thus, it can be understood from the data of experimental example 5 and this experimental example that the lyophilized probiotic bacteria treated with the Zeta-bio composition having 0.01 to 0.15M activator (e.g. lysine imparting a negative Zeta potential on its cell surface) exhibited higher survival rates through the gastrointestinal tract than the lyophilized probiotic bacteria per se.

Rehydration of probiotic lyophilizates with the compositions of the present disclosure imparts a negative zeta potential to reactivate cells, thereby improving survival therein. The compositions of the present disclosure are believed to play a role in promoting the recovery of damaged cells by providing nutrients and essential cellular components to the damaged cells. Furthermore, an increase in cell viability and a change in zeta potential may increase the rate of adhesion of the probiotic to the intestinal cells. Thus, even after exposure to gastric and biliary stress, reactivation of the lyophilized probiotic product with the Zeta-bio composition of the present disclosure may improve the viability of the probiotic and may restore its beneficial effects due to increased adhesion of bacteria to intestinal cells.

Although the technical spirit of the present disclosure has been described by way of examples described in some embodiments and shown in the drawings, it should be noted that various substitutions, modifications and changes may be made without departing from the scope of the present disclosure as can be understood by those skilled in the art to which the present disclosure pertains. Further, it is noted that such alternatives, modifications, and variations are intended to fall within the scope of the appended claims.