KR20200107384A - Novel Pediococcus pentosaceus FB145 with removing cadmium and use thereof - Google Patents

Abstract

The present invention relates to a Pediococcus pentosaceus microorganism among lactic acid bacteria, and more specifically, to a Pediococcus pentosaceus FB145 strain exhibiting excellent cadmium adsorption activity and an application of the strain.

Description

Pediococcus pentosaceus FB145 strain for removing cadmium and its application TECHNICAL FIELD [Novel Pediococcus pentosaceus FB145 with removing cadmium and use thereof]

The present invention relates to a Pediococcus pentosaceus- based microorganism among lactic acid bacteria, and more specifically, to a Pediococcus pentosaceus FB145 strain exhibiting excellent cadmium adsorption activity and an application of the strain.

Cadmium (Cd) is a harmful heavy metal in biological systems. Industrial emissions and extensive use of chemical fertilizers have serious consequences for heavy metal contamination in the environment. Cd can be absorbed from the soil by crops and aquatic organisms. Long-term exposure to Cd has serious detrimental effects such as lung disease and impaired calcium metabolism in humans. In addition, Cd is carcinogenic. The recent increase in Cd contamination in food and drinking water is one of the major concerns of the World Health Organization and the International Cancer Research for Cancer. There have been studies on the health effects of heavy metals contaminated with food or water on humans. However, no effective technique for removing Cd from the body has been proposed.

The decontamination of heavy metals is divided into non-biological (based on physical and chemical properties) and biological processes. In addition to the usual chelation therapy for cadmium poisoning, some studies have shown that the use of a microbial-based diet is more effective and safer for patients. During the past 20 years, several microorganisms (5-7) such as Saccharomyces cerevisiae, Pseudomonas, Rhodovulum and Bacillus have been reported to exhibit metal binding capacity. However, the use of these microorganisms as a therapeutic strategy for Cd toxicity involves important issues because of the protection of humans and the tolerance of human gut to extreme environments.

Lactic acid bacteria (LAB) are generally recognized as safe, and when ingested, they promote health benefits and are now considered probiotics, and are known to inhibit pathogens. Lactobacillus is a bacterium that decomposes carbohydrates and uses them to create lactic acid, and is a tragic anaerobic bacteria or anaerobic bacteria that proliferate well in places with low oxygen. Lactobacillus can be divided into five genera, Streptococcus, Lactobacillus, Leuconostoc, Bifidobacteria, and Pediococcus. The microorganisms of the genus Lactobacillus are lactic acid bacillus that undergo homogeneous or heterogeneous fermentation, and are commonly found in the fermentation process of dairy products or vegetables, and the microorganisms of the genus Lukonostak, diococcus, undergo heterogeneous fermentation and are mainly involved in fermentation of vegetables. In addition, microorganisms in the genus Bifidobacteria are organized anaerobic bacteria that cannot grow in the presence of oxygen, and produce L(+)-type lactic acid that can be usefully metabolized to infants by fermenting sugar. Lastly, the microorganisms in the genus Pediococcus are homozygous fermentation bacteria that have the form of bacterium, and are present in kimchi or pickled foods, and are involved in the fermentation of meat such as sausages. Among them, the most common lactic acid bacteria in the human intestine is Bifidobacteria, which is an anaerobic lactic acid bacterium, which is known to exist about 100 to 1,000 times more than the anaerobic lactic acid bacteria such as Lactobacillus and Streptococcus. Lactobacillus keeps the intestinal pH acidic, inhibits the reproduction of harmful bacteria such as E. coli and Clostridium sp., improves diarrhea and constipation, and plays a role in vitamin synthesis, anticancer activity, and lowering serum cholesterol. . In particular, lactic acid bacteria have a specific protein that can strongly bind to the intestinal mucosa and epithelial cells, which helps a lot in the intestinal action of preventing the growth of harmful bacteria. In addition, lactic acid bacteria are known to promote the proliferation of macrophages to enhance the recognition and sterilization of harmful bacteria in the intestine of macrophages, and to promote the secretion of immune-related substances to enhance immunity.

According to studies so far, many lactic acid bacteria are known to have resistance to heavy metals due to bioaccumulation and bioabsorption. For example, in Korean Patent Application No. 10-2001-0005325, Nucono, which can adsorb cadmium and lead. It refers to the lactic acid strain of Leuconostoc spp, and Korean Patent Application No. 10-2003-0066998 describes the removal of heavy metals using Enterococcus faecium, which has a high ability to adsorb heavy metals, among lactic acid bacteria. Registered Patent No. 10-0760987 describes Lactobacillus excidophilus PM1, which has the ability to remove heavy metals against mercury and arsenic in addition to cadmium and lead, but their detoxification properties and mechanisms have not been investigated so far.

In addition, a report from the European Food Safety Authority found the highest concentrations of Cd in fish and shellfish and related products. Therefore, it is a functional fermented seafood food that removes heavy metals by adhering heavy metals such as lead, mercury, and cadmium, which are fatal to the human body among beneficial lactic acid bacteria that are easy to ingest and do not harm the human body. Technology that can be provided needs to be developed.

Domestic Patent Registration No. 10-0407663

The present inventors have completed the present invention by developing a new strain showing excellent cadmium adsorption activity as a result of a number of studies.

Accordingly, an object of the present invention is to provide a Pediococcus pentosaceus FB145 strain capable of removing cadmium from the body because it exhibits excellent cadmium adsorption activity as well as viability in the intestine.

Another object of the present invention is to provide a variety of application products including one or more of Pediococcus pentosaceus FB145 strains and cultures thereof exhibiting excellent cadmium adsorption activity, health functional foods, fermented seafood, feed additives, etc. Is to do.

Objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention described above, the present invention provides a pediococcus pentosaceus FB145 strain (KACC 81089BP) having a cadmium adsorption ability.

In a preferred embodiment, the cadmium adsorption ability is exhibited by a strong chemical bond with cadmium by amine groups, carboxyl groups and ketone groups formed on the surface of the strain.

In a preferred embodiment, the strain is isolated from fermented seafood.

In a preferred embodiment, the strain removes more than 50% of Cd within 12 hours under 100 mg/mL Cd stress.

In a preferred embodiment, the strain has a maximum absorption capacity of Cd of 50 mg/g or more.

In addition, the present invention is one of the cultures of the Pediococcus pentosaceus FB145 strain (KACC 81089BP) and the Pediococcus pentosaceus FB145 strain (KACC 81089BP) of any one described above It provides a pharmaceutical composition for the prevention and treatment of cadmium poisoning comprising the above as an active ingredient.

In addition, the present invention is one of the cultures of the Pediococcus pentosaceus FB145 strain (KACC 81089BP) and the Pediococcus pentosaceus FB145 strain (KACC 81089BP) of any one described above Provides health functional foods for the prevention and treatment of cadmium poisoning containing the above as an active ingredient.

In a preferred embodiment, the food is at least one selected from the group consisting of yogurt, kimchi, fermented seafood, cheese, probiotics, confectionery, and gum.

In addition, the present invention is one of the cultures of the Pediococcus pentosaceus FB145 strain (KACC 81089BP) and the Pediococcus pentosaceus FB145 strain (KACC 81089BP) of any one described above Provides a feed additive composition containing the above as an active ingredient.

In a preferred embodiment, the strain prevents or treats cadmium poisoning in animals by adsorbing cadmium in the body of the animal.

In addition, the present invention is one of the cultures of the Pediococcus pentosaceus FB145 strain (KACC 81089BP) and the Pediococcus pentosaceus FB145 strain (KACC 81089BP) of any one described above It provides a method for removing cadmium using the above.

In addition, the present invention comprises the steps of culturing the Pediococcus pentosaceus (P ediococcus pentosaceus) FB145 strain (KACC 81089BP) of any one of the above; Separating the strain from the culture medium; It provides a method for producing probiotics comprising; lyophilizing the isolated strain.

Pediococcus pentosaceus FB145 strain of the present invention described above not only exhibits excellent cadmium adsorption activity, but also can survive in the intestine, so that cadmium can be removed from the body.

In addition, according to the present invention, Pediococcus pentosaceus ( Pediococcus pentosaceus ) FB145 strain exhibiting excellent cadmium adsorption activity and various application products including one or more of its culture medium, fermented seafood, and feed additives are provided. can do.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Figure 1a is a result graph showing the effect of cadmium ions on the growth of the investigated lactic acid bacteria (LAB) isolated from fermented food, Figure 1b is analyzed based on the 16S rRNA gene sequence of the Pediococcus pentosaceus FB145 strain of the present invention It shows the phylogenetic diagram.
Figure 2a shows Cd removal and cell density (average SD) during growth of Pediococcus pentosaceus FB145 in MRS medium, and Figure 2b shows Cd removal and cell density (average SD) during growth of Pediococcus pentosaceus FB181. I did it.
3 is a graph showing the Cd bioabsorption rate of Pediococcus pentosaceus FB145 and FB181.
4 is an electron micrograph of Pediococcus pentosaceus FB145 and FB181, where A and C are pictures of the surfaces before Cd bioabsorption of P. pentosaceus FB145 and FB181, respectively, and B and D are surfaces of P. pentosaceus FB145 and FB181, respectively, after Cd bioabsorption. It's a picture.
Figure 5a shows the FTIR spectrum of Pediococcus pentosaceus FB145 before (a) and after (b) bioabsorption of Cd, and Figure 5b shows the FTIR spectrum of Pediococcus pentosaceus FB181 before (a) and after (b) bioabsorption of Cd.

The terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate the existence of features, numbers, steps, actions, elements, parts, or a combination thereof described in the description of the invention, but one or more other It is to be understood that it does not preclude the presence or addition of features, numbers, steps, actions, components, parts, or combinations thereof.

Terms such as first and second may be used to describe various elements, but the elements should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another component. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may be referred to as a first component.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present invention. Does not.

In interpreting the constituent elements, it is interpreted as including an error range even if there is no explicit description. In particular, when the terms "about", "substantially" and the like of degree are used, it can be interpreted as being used in or close to that value when manufacturing and material tolerances specific to the stated meaning are presented. .

In the case of a description of a temporal relationship, for example,'after','following','after','before', etc. It includes cases that are not continuous unless' is used.

Statistical analysis in the present invention was performed with the SPSS 20.0 software program (IBM Corp., Armonk, NY). A tonsil analysis was performed to compare Cd removal by the strain and Duncan's post-hoc test. Differences with p <0.05 are statistically significant.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The same reference numerals used to describe the present invention throughout the specification denote the same elements.

Technical features of the present invention are not only exhibiting excellent cadmium adsorption activity, but also in the intestinal viability Pediococcus pentosaceus FB145 strain and its use.

That is, in general, many lactic acid bacteria have heavy metal resistance due to bioaccumulation and bioabsorption, but their detoxification properties and mechanisms have not been investigated until now, but in the present invention, Pediococcus pentosaceus FB145 strain is excellent. This is because the mechanism of cadmium adsorption activity was investigated and revealed.

Since most of the bacteria are not degraded or metabolized to fix cells, the binding of Cd to cell wall components or surface factors is rather lactic acid bacteria (LAB, including Pediococcus pentosaceus ) FB145 strain for preventing toxicity. ) Can be the main mechanism. This is because LAB cell membranes are rich in peptidoglycans and exopolysaccharides (EPS), EPS reacts with metal ions to promote the bioadsorption process, and LAB's EPS is negative in polysaccharides such as protein, phosphate and carboxyl groups of hydroxyl groups. This is because the proportion of positively charged groups is higher, and the amount of these functional groups as a whole is predicted to increase the bonding surface to the sequestration of charged metals such as Cd. According to this mechanism, as described below, in the present invention, it can be seen that the Pediococcus pentosaceus FB145 strain exhibits excellent Cd adsorption ability, and the application of the Pediococcus pentosaceus FB145 strain of the present invention is It may be a remedy to prevent Cd toxicity, reduce absorption in the intestines of animals and humans, and remove cadmium from water.

Accordingly, the present invention provides a Pediococcus pentosaceus FB145 strain (KACC 81089BP) having cadmium adsorption capacity.

Here, Pediococcus pentosaceus FB145 strain is a biological resource unique to Korea as a fungus isolated from fermented seafood such as salted fish. In particular, Pediococcus pentosaceus FB145 strain has excellent cadmium adsorption ability, and it can remove more than 50% of Cd within 12 hours under 100 mg/mL Cd stress, and the maximum absorption capacity of Cd is expected to be 50 mg/g or more. do. In addition, Pediococcus pentosaceus FB145 strain shows a survival rate of 80% or more for 4-5 hours in a low pH environment (pH 2-3), and this characteristic is that Pediococcus pentosaceus FB145 strain is It shows that it can survive in the stressful environment of the digestive system including, and can pass through it and attach to the mucous membrane cells in the intestine to exhibit bioactivity.

Pediococcus pentosaceus of the present invention Using the characteristics of the FB145 strain (KACC 81089BP), it is possible to detoxify humans and animals from Cd toxicity by preventing or removing Cd poisoning from humans and animals through excretion.Therefore, various applications such as biological treatments, health functional foods and feed additives It is possible.

As a result, the pharmaceutical composition for the prevention and treatment of cadmium poisoning of the present invention is Pediococcus pentosaceus (P ediococcus pentosaceus ) FB145 strain (KACC 81089BP) and the Pediococcus pentosaceus ( Pediococcus pentosaceus ) FB145 strain (KACC 81089BP) may be included as an active ingredient. The pharmaceutical composition of the present invention may have various formulations, and as an embodiment, granules (GRANULES), limonades (LEMONADES), powders (POWDERS), syrups (SYRUPS), liquids (LIQUIDS AND SOLUTIONS), X agents (EXTRACTS) ), ELIXIRS, FLUIDEXTRACTS, Suspensions (SUSPESIONS), DECOCTIONS, INFUSIONS, Tablets, Spirits (SPIRITS), Capsules, Troches ( TROCHES), pills (PILLS), and may be any one selected from soft or hard gelatin capsules. This is because the pharmaceutical composition prepared in the above formulation not only can deliver the contained ingredients well into the human body, but also is easy to take. In addition, the dosage of the pharmaceutical composition for preventing or treating cadmium poisoning of the present invention is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease. In one embodiment, the pharmaceutical composition for preventing or treating cadmium poisoning of the present invention can be administered at least once at 0.00001 to 100 mg/kg (body weight) per day based on the active ingredient. However, the above dosage is only an example for illustration, and may be changed by a doctor's prescription according to the state of the user.

The health functional food for the prevention and treatment of cadmium poisoning of the present invention may be any known food, but as an embodiment, any selected from the group consisting of yogurt, kimchi, fermented seafood, cheese, probiotics, confectionery, and gum It could be one. In particular, fermented seafood includes all foods fermented by salting foods derived from the sea, including various salted fish.

In addition, according to the method for removing cadmium of the present invention, the culture medium of Pediococcus pentosaceus FB145 strain (KACC 81089BP) and the Pediococcus pentosaceus FB145 strain (KACC 81089BP) One or more of them can be used to effectively remove cadmium from cadmium-contaminated objects.

Pediococcus pentosaceus (P ediococcus pentosaceus ) FB145 strain (KACC 81089BP) of the present invention may be formulated in various forms for convenience such as transportation or storage. For example, it may be lyophilized with a cryoprotectant and used in the form of a powder, or may be mixed with a storage carrier, adsorbed, dried, and solidified. The cryoprotectant and the preservation carrier are not particularly limited as long as they are commonly used in the art. For example, glycerol, skim milk, honey, etc. may be used as the cryoprotectant, and diatomaceous earth, activated carbon, and skim steel as the preservation carrier Etc. can be used.

Example. Isolation and identification of Pediococcus pentosaceus FB145 strain

1. Isolation of strain

20 Cd-resistant LABs were separated from Yeosu fermented seafood and stored at -20°C. For each LAB, 1 g of the sample was thoroughly mixed with 9 mL of sterile saline, and then 200 μL of the supernatant was transferred to an incubator and stirred with 20 mL of MRS broth (Difco, USA) at 37° C. for 2 to 4 days. After the lapse of the period, the culture medium was plated on MRS agar medium containing 5 μg Cd2 + / mL. The cadmium adsorption capacity of each isolated strain was determined by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) recommended in Clinical and Laboratory Standards Institute methods M07-A9 and M26-A). The bacterial concentration of the strain was monitored in 96-well plates using a microplate reader (BioTek Instruments, Inc., Winooski, USA) at OD600. The results are shown in Fig. 1A. As shown in Figure 1a, each strain tested showed a wide range of resistance among (5-200 μg/mL), especially two strains (FB145 and FB145) that showed resistance to high concentrations of Cd ions (> 200 μg/mL). FB181) was isolated.

2. Identification of strains through 16s-rRNA sequencing

Genomic DNA was extracted using the AccuPrep Genomic DNA extraction kit (Bioneer Co., Daejeon, Korea) for the two isolated strains. To amplify the 16S rDNA high resistance strain, 27F and 1492R primers against bacteria were used, and PCR amplification of the sequence was confirmed with a 1% agarose gel. The purified PCR products were sequenced by Bioneer Corporation (Daejeon, Korea).

The 16S rRNA gene sequence of the isolated two strains was analyzed by Nucleotide Blast having a sequence known from the National Center for Biotechnology Information GenBank database. As a result of BLAST analysis, the sequences of the two isolated strains (FB145 and FB181) showed high similarity (98%) to closely related Pediococcus pentosaceus A5 (KX262845). Phylogenetic analysis between LABs was constructed from evolutionary distances by neighboring junction methods, and the results are shown in FIG. 1B.

As shown in FIGS. 1A and 1B, two strains showing high concentration resistance to cadmium ions were identified as Pediococcus pentosaceus FB145 and FB181 strains. Of the two strains, in particular, Pediococcus pentosaceus (P ediococcus pentosaceus ) FB145 strain was deposited with the National Academy of Agricultural Sciences Agricultural Microbiology Bank on February 27, 2019, and received KACC 81089BP accession number.

Experimental Example 1. Pediococcus pentosaceus (P ediococcus pentosaceus ) Confirmation of cadmium removal characteristics of FB145 strain

Pediococcus pentosaceus (P ediococcus pentosaceus ) FB145 and FB181 strains were pre-cultured twice for 18 hours at 100 xg and 37°C in MRS medium for further analysis. Thereafter, the cadmium removal characteristics of the FB145 and FB181 strains were tested as follows. FB145 and FB181 strains were cultured for 36 hours at 37°C in MRS medium containing 20, 40, 60, 80 or 100 mg/L of Cd ²⁺ . The growth status of these strains was periodically evaluated by absorption with a microplate reader at 600 nm. The concentration of Cd ²⁺ was measured with a NexION 300X ICP-MS spectrometer (Perkin Elmer, USA). The results are shown in FIGS. 2A and 2B. 2A and 2B, "■" = Cd 0 mg/L; "●" = Cd 20 mg/L; “▲”= Cd 40 mg/L; “◆” = Cd 60 mg/L; "□" = means Cd 80 mg/L.

As can be seen from Figs. 2A and 2B showing the Cd removal efficiency of the P. pentosaceus FB145 and FB181 strains, the cadmium removal efficiency was significantly different according to the test strain. That is, under 100mg/mL Cd stress, FB145 strain removed more than 50% of Cd within 12 hours, whereas FB181 showed a relatively low removal rate of 25%. More than 90% of Cds were removed in both strains within 42 hours. These results showed that a Cd stress concentration of 0 to 50 mg/L had no effect on the growth of the tested strains, namely P. pentosaceus FB145 and FB181 strains. In addition, although the viability of P. pentosaceus FB145 and FB181 strains are slightly inferior to other concentrations after cultivation under high Cd stress (100 mg/L), since the strains P. pentosaceus FB145 and FB181 can maintain viability and grow in high Cd contaminated solutions, the toxicity of heavy metals, especially cadmium As probiotics that can play two important roles in preventing and reducing absorption into livestock and human body as a long-term treatment, it will be applicable in various fields such as the food industry.

Experimental Example 2

In order to describe the adsorption kinetics for Cd removal by P. pentosaceus FB145 strain, the amount of bioadsorbed Cd during treatment was estimated by applying the absorption kinetics as follows, and the results are shown in FIG. 3. In Fig. 3, "●" = FB145, "■" = FB181.

P. pentosaceus FB145 and FB181 strains were inoculated with 1% of the entire culture medium of LAB in 1 L MRS medium fermentation, respectively, and inoculated at 37°C for 24 hours. Thereafter, each strain was harvested by centrifugation at 1230 x g for 20 minutes and washed twice with phosphate buffer (pH 7.2). The weight of the strain in the suspension was freeze-dried (FD8508, Ilshin, Republic of Korea) for 18 hours at -50°C and 0.024 mBar and measured. The dried strain (0.1 g) was mixed with 100 mL PBS containing 10 mg Cd / L to determine the Cd removal capacity.

Thereafter, a Cd- strain suspension was obtained after 360 minutes, and then centrifuged at 1230 g for 20 minutes to remove bacterial cells (used for subsequent analysis). A 1 mL sample of the supernatant was used for CD analysis. The adsorption capacity of the strain for Cd (qe) was calculated using the following formula:

qe = (Ci-Ce) V / M (1)

Where Ci and Ce are the initial and final concentrations of metal ions in the solution (mg/L), V is the volume of the PBS solution (L), and M is the amount of strain used in the reaction mixture (g). The pseudo quadratic model shows that the proportion of adsorption sites is proportional to the square of the number of empty sites. The linear equation of this model is as follows.

t / qt = (1 / k2 qe2) + (1 / qe) t (2)

Here, qe and QT are the amounts (µg/mg) of metal ions adsorbed to the biosorbent at equilibrium and time (t), respectively, and k2 is the pseudo-secondary biosorbent rate constant of magnesium (Mg/µg).

The equilibrium adsorption isotherm experiment was performed by varying the Cd concentration from 1 to 100 mg/L for 12 hours under the same conditions as described above. Langmuir isotherms are often used for adsorption of metal cations. The linear equation is expressed as

qe = qmaxKLCe /(1 + KLCe)

Where Ce is the metal concentration in the equilibrium solution (mg/L), qmax is the maximum adsorption capacity (μg/mg), and KL is the Langmuir constant (L/mg). The Freundlich isotherm used for the relationship between the concentration of heavy metals and the surface area of the adsorbent in the liquid was expressed as follows.

qe = KFCe 1/n

Here, KF is the Freundlich constant (L/mg) and 1/n is the adsorption strength.

The experimental data of the two strains in the present invention fit both the pseudo-second order kinetics model with high correlation coefficient. The parameter values were calculated and shown in Table 1.

Strain Pseudo-second-order kinetics Langmuir Freundlich q _e k ₂ R ² q _max K _L R ² K _F 1/n R ² FB145 52.8 0.0027 93.20 52.65 0.138 95.14 8.46 1.78 99.74 FB181 50.3 0.0028 99.63 50.35 0.142 97.75 8.28 1.82 99.89

As can be seen from Fig. 3, in which a linear plot of the kinetic model is shown, cadmium adsorption occurred more effectively in P. pentosaceus FB145 than in FB181. In addition, in terms of the adsorption rate, it can be seen that the maximum removal of Cd occurred after 60 minutes.

As shown in Table 1, the bioabsorption doses calculated for P. pentosaceus FB145 and FB181 were 52.8 mg/g and 50.3 mg/g, respectively, and are close to the values determined by the experiment. In addition, the correlation coefficient (R ² ) for Cd bioadsorption was determined to be 0.9320 for FB145 and 0.9963 for FB181.

To predict the mechanism of Cd adsorption by P. pentosaceus FB145 and FB181, Langmuir and Freundlich's modeling was performed by changing the initial Cd concentration to 10 ~ 200 mg / L to analyze the experimental results. The parameters of the Langmuir and Freundlich models are shown in Table 1 above. The predicted maximal bioabsorbable doses (qmax) by P. pentosaceus FB145 and FB181 were 52.65 and 50.35 mg/g, respectively. The highest R ² result showed that the biouptake of Cd by P. pentosaceus FB148 was higher than that of FB181. Although the Langmuir model fits well with the experimental data for Cd, it was desirable to invent a model fit with the Freundlich model based on an R ² value of 0.99. The 1/n values are 1.78 and 1.82, indicating that there is a chemical interaction between Cd and the cell surface of the P. pentosaceus FB145 and FB181 strains. In general, adsorption parameters are used to predict the maximum adsorption capacity of the adsorbent.

The experimental results of the present invention described above showed that there is no significance in the Cd adsorption capacity by P. pentosaceus FB145 and FB181 (P>0.05). However, its capacity increases as the initial concentration of Cd increases, and the adsorption equilibrium is reached when the concentration of the reactant is about 50 mg/L. This phenomenon indicates that P. pentosaceus FB145 and FB181 strains contain numerous adsorbates on the cell surface. The Freundlich model was the earliest known relationship explaining non-ideal and reversible adsorption. In the Freundlich model, 1/n is an index of non-linearity representing the strength between the adsorbent and Cd. A value of 1/n greater than 1 indicates cooperative adsorption. FB145 had a high bioabsorption constant KF of 8.45 compared to FB181 (8.28). Frederick (Freundlich) correlation coefficient (R ²⁾ it can be seen that the Cd is coupled P. pentosaceus FB145 and FB181 of the present invention according to well-Freundlich and Langmuir isotherm as 0.9987 at 0.9974, FB181 from P. pentosaceus FB145 achieved. As a result, it can be seen that P. pentosaceus FB145 and FB181 strains have a high Cd biouptake level.

Experimental Example 3

P. pentosaceus FB145 and FB181 strains were analyzed by energy dispersive spectroscopy (SEM-EDS) using a scanning electron microscope as follows, and a photograph of the results is shown in FIG. 4.

P. pentosaceus FB145 and FB181 strains were washed twice with saline buffer (0.85% NaCl) and freeze-dried, respectively. The lyophilized strains were coated with gold and the field emission SEM (Gemini 500; Carl Zeiss SMT, Germany) equipped with EDS (X-MaxN 80 EDS, Oxford Instruments, UK) was used to determine the selected five morphology and elemental compositions. area. Elemental composition was confirmed through SEM images using the EDAX Genesis software program.

SEM-EDS analysis was performed to compare the difference in surface morphology of P. pentosaceus FB145 strains before and after Cd in vivo absorption. As shown in FIG. 4, there was no significant change in morphology when comparing the cell surface morphology (A) of the strain P. pentosaceus FB145 before cadmium absorption and the cell surface morphology (C) of the strain after cadmium absorption. P. pentosaceus FB0 181 strain also showed no significant changes in the cell surface morphology of the strain before cadmium absorption (B) and the cell surface morphology of the strain after cadmium absorption (D).

According to these SEM analysis results, there was no significant change in the physiological structures of P. pentosaceus FB145 and FB181 having high Cd bioabsorbing capacity under 100 mg/mL Cd stress.

Experimental Example 4

Infrared spectra of cells obtained by lyophilizing each of P. pentosaceus FB145 and FB181 strains before and after Cd bioadsorption were obtained as follows. Samples were mixed with KBr, pressed into slices and then scanned with an FTIR infrared spectrometer (Jabco 430, Japan). The spectrum was set with a resolution of 4 cm ^-1 in the range of 4,000 to 450 cm ^-1 . The change in the vibration frequency of the functional groups on the cell surface was analyzed with PeakFit v4.12 software, and the results are shown in FIGS. 5A and 5B.

As shown in FIGS. 5A and 5B, the functional groups involved in the bioadsorption process can be known from the FTIR spectrum. In general, the spectra of P. pentosaceus FB145 and FB181 after Cd bioadsorption showed slight changes compared to the control group before Cd bioadsorption. P. pentosaceus FB145 and FB181 strains showed a difference at the peak of 3,417 cm ^-1 (NH stretch). A clear difference was also seen in 1,631cm ^-1 (C = O stretch). A slight difference in the spectrum of the treated samples was observed over a wide range of 1,034 to 1,424 cm ^-1 , indicating the presence of the -CH and O-OCH3 groups. In addition, in P. pentosaceus FB181, the shift of the detected peak indicated that the -C-CO-C band of ketones could participate in Cd bioabsorption. Gram-positive bacteria provide a broad functional group. Through these results, the present invention revealed a broad spectrum functional group on the surface of P. pentosaceus FB145 and FB181 strains capable of interacting with Cd through strong chemical bonding in the region in which the amine group, carboxyl group and ketone group were formed.

Ions CJB145 CJB145+Cd CJB181 CJB181+Cd C 52.69 ± 52.69 53.9 ± 53.90 54.36 ± 54.36 56.22 ± 56.22 N 5.55 ± 5.55 4.26 ± 4.26 5.12 ± 5.12 5.35 ± 5.35 O 34.62 ± 34.62 34.65 ± 34.65 33.74 ± 33.74 32.36 ± 32.36 Na 0.36 ± 0.36 0.31 ± 0.31 0.31 ± 0.31 0.29 ± 0.29 Mg 0.8 ± 0.80 0.03 ± 0.03 0.59 ± 0.59 0.06 ± 0.06 Al 1.26 ± 1.26 1.28 ± 1.28 1.3 ± 1.30 0.98 ± 0.98 Si 0.05 ± 0.05 0.06 ± 0.06 0.06 ± 0.06 0.08 ± 0.08 P 3.45 ± 3.45 2.85 ± 2.85 3.56 ± 3.56 2.75 ± 2.75 CD - 0.83 ± 0.83 - 0.79 ± 0.79 Si 0.43 ± 0.43 0.26 ± 0.26 0.39 ± 0.39 - Cl 0.12 ± 0.12 1.12 ± 1.12 0.12 ± 0.12 0.92 ± 0.92 K - - - - Ca 0.67 ± 0.67 0.45 ± 0.45 0.45 ± 0.45 0.23 ± 0.23 Values are mean of molecular weight percentage ± SD of six measuring areas.

In addition, as shown in Table 2, the presence of Cd after bioadsorption on the surfaces of FB145 and FB181 was confirmed by the EDS pattern. The Cd content ions adsorbed on the cell surface of the two strains were the same. These results strongly support the high ability of P. pentosaceus FB145 and FB181 strains to adsorb metal ions. In particular, it can be said that the interaction of Cd with negatively charged components (polysaccharides, membrane proteins and peptidoglycans) including the main elements of O, S, P, and N is responsible for the biological absorption of Cd.

Experimental Example 5

The ability of P. pentosaceus FB145 and FB181 strains for GIT tolerance and Cd bioaccessibility was determined as follows according to the method described by Kumar, N., Kumar, V. and shown in Table 3. P. pentosaceus FB145 and FB181 strains were activated twice before Cd biosorbent analysis, respectively. Cd-strain suspension was prepared as described above, and 10 mg Cd/L in PBS solution was used as a control. For oral stress, 4 mL of each Cd-suspension was mixed with 6 mL of immersion (containing 0.3 mg/mL-amylase in PBS buffer, pH 6.5) in a 50 mL Falcon tube. The tube was covered with nitrogen and placed in a water bath shaking for 10 minutes at 37°C. For the stomach, 5 mL of gastrointestinal solution (including 4.5 mg/mL pepsin in PBS buffer) was added and the pH was adjusted to 3 with HCl (1 N). The volume was normalized to 40 mL with PBS buffer and placed in a shaking water bath at 37° C. for 30 minutes. After the incubation period, the gastrointestinal tract pH was adjusted to 2 and the stomach was repeated. As intestinal stress, the pH of the reaction mixture of pancreatin (10 mg/mL), lipase (2 mg/mL) and bile (30 mg/mL) was adjusted to 7.2. The volume was normalized to 40 mL and placed in a shaking water bath at 37° C. for 120 minutes. After each step of the digestion process, the concentration of isolates was evaluated by counting the MRS agar plate. The survival rate was calculated by comparing the final concentration and the control group. The biological accessibility (%) of Cd was measured and expressed by normalization to the initial Cd concentration.

Considering the strains of P. pentosaceus FB145 and FB181 of the present invention as probiotics organisms, they must be able to survive in the stressful environment of the digestive system including the gastrointestinal tract and intestine, which means that probiotics pass through the gastrointestinal tract and adhere to the mucous membrane cells in the intestine. Because it represents. In general, the digestion process takes 4-5 hours at low acidic pH (2-3).

FB145 FB181 LGG GIT
tolerance Initial 9.1 ± 0.04 9.1 ± 0.04 9.0 ± 0.02 Oral stress
(10 min) 8.5 ± 0.04 8.9 ± 0.02 8.4 ± 0.02 Gastric stress,
pH 3 (30 min) 8.4 ± 0.01 8.9 ± 0.02 8.4 ± 0.02 Gastric stress,
pH 2 (30 min) 7.7 ± 0.02 7.9 ± 0.04 7.3 ± 0.04 Intestinal stress
(120 min) 7.4 ± 0.01 7.6 ± 0.01 7.3 ± 0.01 Survival rate (%) 81.7 ± 0.31 ^b 83.4 ± 0.27 ^a 80.9 ± 0.21 ^b Cd bioaccessibility (%) of 44.7 ± 0.24 ^ab 46.8 ± 0.14 ^a 43.2 ± 0.24 ^b Values are mean ± SD of three independent experiments performed in duplicate with different alphabets are statistically significant (P <0.05).

As shown in Table 3, both strains showed a survival rate of 80% or more in a probiotic characteristic in vitro model for GIT resistance, which was similar to the commercial probiotic strain L. rhamnosus GG ATCC 53103 (LGG). It is estimated that the long-term adaptation period of P. pentosaceus FB145 and FB181 strains to the low pH environment of traditional fermented seafood can confer high GIT tolerance due to natural selection.

In addition, in the Cd bioaccessibility in vitro model, P. pentosaceus FB145 and FB181 strains significantly decreased from 44.7% to 46.8%, respectively, compared with the control group within 3 hours. These experimental results once again confirm the ability of lactic acid bacteria to absorb cadmium in a wide range of 24.71 to 41.62%.

The present invention proved for the first time the reduction of Cd through bioabsorption of P. pentosaceus FB145 and FB181 isolated from fermented seafood in a Cd bio-accessible in vitro model through the above-described experiment.

From the above-described experimental results, the P.pentosaceus FB145 strain as well as the FB181 strain of the present invention showed stable complex formation during bioadsorption and all test strains showing the ability to bind to Cd, as well as high binding ability and Cd bioaccessibility. It can be seen that there was a decrease. As a result, it is shown that the strains of P. pentosaceus FB145 and FB181 of the present invention can be potent probiotics or biotherapeutic agents capable of resisting and detoxifying Cd.

Therefore, the P. pentosaceus FB145 strain of the present invention can prevent or treat Cd poisoning of humans and animals by removing Cd from the human body or animals through excretion, so it includes biological treatments such as pharmaceutical compositions, health functional foods, as well as feed additives. Thus, a wide range of applications will be possible.

Although the present invention has been illustrated and described with a preferred embodiment as described above, it is not limited to the above-described embodiment, and within the scope of the spirit of the present invention, those of ordinary skill in the art to which the present invention belongs. Various changes and modifications will be possible.

National Academy of Agricultural Sciences Agricultural Microbiology Bank KACC81089BP 20190227

Claims (12)

Pediococcus pentosaceus FB145 strain (KACC 81089BP) with cadmium adsorption ability.
The method of claim 1,
The cadmium adsorption ability is Pediococcus pentosaceus ( Pediococcus pentosaceus ) FB145 strain (KACC 81089BP), characterized in that the amine group, carboxyl group and ketone groups formed on the surface of the strain appear by strong chemical bonding with cadmium.
The method of claim 1,
The strain is Pediococcus pentosaceus FB145 strain (KACC 81089BP), characterized in that isolated from fermented seafood.
The method of claim 1,
The strain is Pediococcus pentosaceus ( Pediococcus pentosaceus ) FB145 strain (KACC 81089BP), characterized in that it removes more than 50% of Cd within 12 hours under 100 mg / mL Cd stress.
The method of claim 1,
The strain is Pediococcus pentosaceus ( Pediococcus pentosaceus ) FB145 strain (KACC 81089BP), characterized in that the maximum absorption capacity of Cd is 50mg / g or more.
Claim items 1 to the culture medium of any one of Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) and the Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) of claim 5, wherein A pharmaceutical composition for the prevention and treatment of cadmium poisoning, comprising one or more as an active ingredient.
Claim items 1 to the culture medium of any one of Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) and the Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) of claim 5, wherein Health functional food for the prevention and treatment of cadmium poisoning containing one or more as an active ingredient.
The method of claim 7,
The food is a health functional food, characterized in that selected from the group consisting of yogurt, kimchi, fermented seafood, cheese, probiotics, confectionery, and gum.
Claim items 1 to the culture medium of any one of Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) and the Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) of claim 5, wherein Feed additive composition containing one or more as an active ingredient.
The method of claim 9,
The strain is a feed additive composition, characterized in that to prevent or treat cadmium poisoning of animals by adsorbing cadmium in the body of the animal.
Claim items 1 to the culture medium of any one of Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) and the Phedi OKO kusu pen soil three mouse (Pediococcus pentosaceus) FB145 strain (KACC 81089BP) of claim 5, wherein Cadmium removal method using more than one.
The step of culturing the Pediococcus pentosaceus (P ediococcus pentosaceus) FB145 strain (KACC 81089BP) of any one of claims 1 to 5;
Separating the strain from the culture medium;
Freeze-drying the isolated strain; Probiotics production method comprising a.

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