KR101823073B1 - New microorganism SJP-SNU KCTC12756BP and use thereof - Google Patents

Abstract

The present invention relates to a novel yeast strain, and more particularly, to a novel SJP-SNU yeast strain, a probiotic composition comprising the SJP-SNU yeast strain, and food or feed comprising the same. The SJP-SNU strain of the present invention is excellent in proliferation and long-term fixability at the temperature of 35 to 40C, which is similar to the temperature of a human body, and does not show pathogenicity. The SJP-SNU strain of the present invention has the characteristics, and thus can be widely used as a probiotic composition, a food or food additive, a feed or a feed additive.

Description

New strain SJP-SNU KCTC12756BP and its use {New microorganism SJP-SNU KCTC12756BP and use thereof}

The present invention relates to a novel SJP-SNU yeast strain, and more particularly to a novel SJP-SNU strain having excellent long-term fixability, heat resistance, alcohol decomposition efficacy, odor reduction efficacy and anti-influenza efficacy and no pathogenicity, Probiotic compositions, food compositions and feed compositions.

Yeast is a single cell microorganism composed of eukaryotic cells. It is capable of producing ethanol and carbon dioxide by fermenting sugars, and is generally used as a generic term for fungi that proliferate mainly by germination. Yeast can be widely distributed from the surface of fruits, soil, air, seawater or the epidermis of plants to flowers and animals and can be easily separated from the natural world. In addition, yeast has been used for human beings very usefully for alcohol production and baking.

Recently, it has been reported that yeast itself is used as a cheap protein and vitamin nutrient source, especially in maintaining intestinal microorganism balance, inhibiting the growth of pathogenic bacteria, and improving immunity. As a result, yeast has been used as a feed additive to improve the digestibility of feed nutrients and livestock productivity as probiotics or supplementary feed probiotics.

Yeast is used in various industrial fields including food field, and it plays an important role in maintaining intestinal health by inhibiting the development of harmful bacteria in the intestines while living in the intestines of human and animals. In addition, in the livestock sector, yeast is added to the feed of livestock to promote the secretion of digestive enzymes necessary for the growth of livestock, to stabilize intestinal bacterial counts, to prevent metabolic diseases, to improve livestock productivity, It is widely used as a probiotic agent.

Recently, glucan, a major component of yeast cell wall, has been reported as a substance that enhances the immune function of an animal and has been shown to have an effect of increasing the disease resistance of an animal when it is fed to an animal. Therefore, yeast has been attracting attention as a substitute substance to solve the residual and tolerance problem in livestock caused by antibiotics used for improving productivity of livestock. Therefore, it is necessary to develop new yeast strains which are excellent in the fixation property and the safety to the living body, and can be effectively used for the probiotic composition, food or feed.

A problem to be solved by the present invention is to provide an SJP-SNU strain excellent in the fixation property, the heat resistance, the alcohol decomposing effect, the odor reducing effect and the anti-influenza effect and having no pathogenicity.

Another object of the present invention is to provide a probiotic composition, food or feed containing the strain as an active ingredient.

The inventors of the present invention have made extensive efforts to discover new yeast strains which can be used in probiotic compositions, foods or feeds, and as a result, they have found a novel yeast strain of SJP-SNU of the present invention. The inventors of the present invention selected the yeast colonies showing high proliferation by cultivating the microorganisms present in the composted soil at 60 ° C. on a medium containing antibiotics and culturing at 37 ° C. Thereafter, it was confirmed that the colony yeast was superior to S. cerevisiae and S. boulardii in proliferation, long-term fixability and heat resistance, and had no odor reducing effect, ethanol degradation efficacy and anti-influenza efficacy, and was not pathogenic. The genome of the yeast was analyzed to confirm that it was a new yeast strain and named as SJP-SNU strain, thereby completing the present invention.

The present invention relates to an SJP-SNU strain which is a new yeast strain. The SJP-SNU strain of the present invention is excellent in long-term fixation property, heat resistance, alcohol decomposing effect, odor reducing effect and anti-influenza effect and is not pathogenic, and can be usefully used in probiotics composition, food or feed.

The SJP-SNU strain of the present invention has high proliferative (temperature resistance) at 35 to 40 占 폚, preferably 35 to 37 占 폚, and may have heat resistance at 65 占 폚. The SJP-SNU strain exhibits very high proliferation at 35 to 40 ° C., preferably at 37 ° C., unlike a conventional yeast strain cultured at 25 to 30 ° C., and thus has excellent advantages as probiotics. In addition, the SJP-SNU strain of the present invention does not show pathogenicity in fetuses or 4-week-old chickens, and has excellent advantages in being used as probiotics because of its excellent long-term fixability.

The SJP-SNU strain of the present invention may be a yeast strain deposited with the Korean Society for Biotechnology Research, Biotechnology Center, Daejeon, on February 5, 2015 and deposited with the deposit number KCTC12756BP.

The present invention also provides a probiotic (probiotic) composition comprising the SJP-SNU strain. The composition may include at least one selected from the group consisting of a strain of SJP-SNU, a culture of the strain, a concentrated solution of the culture solution, and a dried product of the culture solution. The SJP-SNU strain of the present invention can stably grow at a temperature (35 to 40 ° C) similar to a living body temperature, has excellent long-term fixability and is not pathogenic, and thus can be effectively used as an effective ingredient of a probiotic composition.

In the present invention, the culture means that a specific microorganism is cultured in a culture medium or a culture solution, and the culture may contain the specific microorganism. The culture is not limited in its formulation, and may be, for example, liquid or solid. Preferably, the culture medium of the present invention may mean that the culture medium is inoculated with a specific microorganism in a liquid medium. In the present invention, the concentrate of the culture liquid may mean that the culture liquid is concentrated, and the dried material of the culture liquid may mean that moisture is removed from the culture liquid.

In the present invention, probiotics can be a probiotics in the form of a single or multiple strains which are provided to humans or animals to provide beneficial effects by ameliorating intestinal flora of a human or animal host. In general, probiotics have the effect of treating and ameliorating all the symptoms caused by abnormal fermentation of intestinal bacterial flora, and they function to prevent harmful microorganisms from settlement by being densely settled and fixed on the walls of the digestive tract in the host's intestines. The live bacterial active agent may mean a probiotic agent that improves intestinal microflora of a human or livestock to give health-beneficial effects. In order for the microorganism to be used as a live-active agent, a certain number of bacteria must survive the process of passing through the stomach. Therefore, the microorganism should be able to maintain its activity for a certain period of time after ingestion because of its excellent heat resistance and intestinal viability. The SJP-SNU strain of the present invention satisfies the requirement of the live microorganism activator as described above, and thus can be used as an excellent probiotic composition.

Further, in the present invention, the probiotic composition has a dressing action and can therefore be used as a composition for dressing. Formal application may mean the treatment or amelioration of various symptoms caused by abnormal fermentation of intestinal flora of a person or an animal. Such symptoms may include, for example, diarrhea (for example, infectious diarrhea or intestinal diarrhea caused by harmful microorganisms), gastroenteritis, inflammatory bowel disease, neurodegenerative syndrome or small intestine microorganisms and growth conditions, It is not.

The probiotic compositions of the present invention can be prepared and administered in a variety of formulations and methods according to methods known in the art.

The present invention also provides a food or food additive comprising the probiotic composition. The present invention also provides a feed or feed additive comprising the probiotic composition. The probiotic composition comprising the SJP-SNU strain can be effectively delivered into the intestines of a human or animal through the food or feed.

In the present invention, the term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing. Typically, the food may be, but is not limited to, a health functional food, beverage, food additive or beverage additive. For example, the food may be selected from the group consisting of various foods, beverages, gums, tea, vitamin complex, functional foods, special nutrition foods (such as crude oil or infant formula), health supplements, confectionery Food, and the like. The food can be prepared according to a conventional manufacturing method known in the art.

The food may comprise a pharmaceutically acceptable food-aid additive and may additionally comprise suitable carriers, excipients or diluents conventionally used in the manufacture of a functional food.

In the present invention, the food contains 0.0001 to 99% by weight, preferably 0.001 to 90% by weight, more preferably 0.1 to 80% by weight, most preferably 1 to 50% by weight of the total food weight of the probiotic composition But it is not limited thereto and can be appropriately adjusted depending on the kind or use of the food.

In the present invention, the feed additive may be added to feed, thereby improving the productivity of livestock by inducing resistance to harmful microorganisms in the intestines of an animal that feeds the feed.

In the present invention, the feed contains 0.0001 to 99% by weight, preferably 0.001 to 90% by weight, more preferably 0.1 to 80% by weight, most preferably 1 to 50% by weight of the total feed weight of the probiotic composition But is not limited thereto.

The animal species to which the feed or feed additive of the present invention can be applied may be at least one kind selected from the group consisting of cattle such as cattle, pig, sheep, goat, dog or cat, pet, But is not limited to.

The novel SJP-SNU yeast strain of the present invention is excellent in the fixation property, the heat resistance, the alcohol decomposing effect, the odor reducing effect, the anti-influenza effect and does not show pathogenicity.

The SJP-SNU strain of the present invention has the above-described characteristics, and thus can be widely used as a probiotic composition, food or food additive, feed or feed additive, and the like.

FIG. 1 shows the results of comparing the intestinal fixation of SJP-SNU yeast strains and S. cerevisiae (KCTC 50757).
Fig. 2 shows the results of comparing the proliferative activity of the SJP-SNU yeast strain and S. boulardii at 37 占 폚.
FIG. 3 shows the results of comparing heat resistance of SJP-SNU yeast strain with S. cerevisiae (KCTC 50757) and S. boulardii .
Fig. 4 shows the results of ethanol degradation of the SJP-SNU yeast strain. In the case of SJP-SNU yeast strain, ethanol was decomposed at 37 ° C to produce acid, and the color of phenol red as pH indicator changed from red to yellow.
Figure 5 shows the hydrogen sulfide-reducing activity of the SJP-SNU yeast strain.
FIG. 6 shows the results of the non-pathogenicity of the SJP-SNU yeast strain. As a result, A showed no cytopathic effect upon inoculation of SJP-SNU new yeast into fetal fibroblast. B was obtained by inoculating the SJP-SNU yeast strain into the umbilical cord at 10-day-old SPF developmental flora, and there was no mortality compared with the control group inoculated with the phosphate buffer solution, and no lesion was observed The results showed no pathogenicity.
FIG. 7 shows the results of identifying SJP-SNU yeast strains through genome analysis.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  One: SJP - SNU  Isolation of new yeast

9 ml of antibiotics-PBS (gentamycin, kanamycin, ampicillin, tetracyclin and streptomycin-50 ug / ml) were added to 1 g of composted soil collected at various sites. The composted soil was then incubated at 37 ° C for 2 hours and heat-treated at 60 ° C for 10 minutes. Then, 10-dilution with antibiotic-PBS was performed, 100 ul aliquots were applied to YMA and cultured at 37 째 C for 18 hours. The largest colonies of the cultured microorganisms were cloned and stained with methylene blue to isolate the yeast, which was named SJP-SNU.

Example  2: Comparison of the fixing property

SJP-SNU, S. cerevisiae (KCTC 50757) and PBS (control, Con) were inoculated once per day with 500ul each of 10 ⁷ cfu / ml of 4 weeks old (120913) SPF (specific pathogen free) Orally for 3 days. On the 4th day, 3 appendices were collected and weighed. After 10-fold dilution with antibiotics-PBS (gentamycin, kanamycin, ampicillin, tetracycline and streptomycin-50 ug / ml), 10 μl of dilution was applied to each well of 100 μl of YMA and incubated at 37 ° C. for 18 hours to measure the number of colonies . As a result, the control group and the S. cerevisiae treated group showed no separation of yeast (0 colony forming unit (cfu) / g), whereas SJP-SNU separated 3.8 x 10 ⁵ cfu / g per cecomorphogram, (Fig. 1).

Saccharomyces boulardii , which is well- rounded at 37 ° C compared to S. cerevisiae and commercially available, was compared at 37 ° C with SJP-SNU. As a result, SJP-SNU showed very active growth in 18 hours, while S. boulardii did not colonize in culture for 18 hours. Furthermore, the colony of S. boulardii was smaller than that of SJP-SNU after 32 hours of incubation. Thus, it was confirmed that SJP-SNU was superior to S. boulardii at 37 ° C. (FIG. 2).

Example  3: Heat resistance comparison

S. cerevisiae (KCTC 50757) and S. boulardii were cultured in YM broth at 30 ° C for 18 hours and SJP-SNU at 37 ° C for 18 hours. Then, each of them was heat-treated at 55, 60 and 65 ° C for 10 minutes, spread on YMA and cultured at 37 ° C, and then viability was confirmed. As a result, all survived up to 60 ° C., but only SJP-SNU survived at 65 ° C. (FIG. 3).

Example  4: SJP - SNU  Anti-influenza virus effect of strain culture

SJP-SNU was cultured for 24 hours, and the culture solution (pH 4.59) was filtered. A / chicken / Korea / 01310/2001 (H9N2) (National Veterinary Research and Quarantine Service, Bird Disease Division, Registered Patent No. 10-0790801) The avian influenza virus culture was diluted 1/20 with an organic substance (5% BCS in PBS) And then mixed with the SJP-SNU culture solution at a ratio of 1: 1. They were reacted at 4 ° C for 30 minutes, diluted stepwise, and inoculated with Egg-bit and compared with Virus Control. As a result, there was a significant difference (P <0.05) in the decrease of virus titer (TCID ₅₀ /0.1 ml) of 10 times or more in SJP-SNU parent strain culture.

Dilution factor SJP treated group Virus control 10 ^-4 3/4 ^a 5/5 10 ^-5 1/4 5/5 10 ^-6 0/5 2/5 10 ^-7 1/5 10 ^-8 0/5 TCID _{50 /} 0.1ml 10 ^4.5 10 ^6.1

a HA positive / inoculated well

Example  5: Comparison of ethanol degradability

SJP-SNU 10 ⁶ cfu / 100ul was inoculated in 5%, 10% and 15% EtOH (in PBS + phenol red 0.1%) and cultured at 37 ° C for 16 hours. When SJP-SNU was inoculated in 5% EtOH, the color of Phenol Red was significantly changed to yellow compared to the control group inoculated with SJP-SNU. In addition, when SJP-SNU was inoculated in 10% EtOH, it turned yellow to be distinguishable from the control. However, there was no difference in 15% EtOH (FIG. 4A).

To compare the ethanol (EtOH) resolution of S. exiguus (KCTC7169) standard strain and SJP-SNU using 5% EtOH (in PBS), 10 ⁶ cfu / 100ul of each strain was inoculated, Lt; / RTI &gt; As a result, there was no change in S. exiguus (KCTC7169) standard compared to the control group in which the strain was not inoculated, but SJP-SNU showed distinct yellow color. Therefore, it was confirmed that the ability of SJP-SNU to decompose ethanol into acetic acid at 37 ° C was excellent (FIG. 4B).

Example  6: Comparison of hydrogen sulfide resolution

Hydrogen sulfide (H ₂ S), which is produced when organic matter is decomposed by microorganisms in an anaerobic state, is known as a colorless toxic gas. In order to evaluate the hydrogen sulfide-reducing effect of the SJP-SNU strain, Salmonella enteritidis (SE) producing the hydrogen sulfide was measured. For this, SE 4.3 × ^{10 9} cfu / ml cultured in LB broth was inoculated after diluting 10 ^-3 to 10 ^-10 in TSI slope medium and cultured at 37 ° C. for 18 hours. As a result, it was confirmed that the entire medium was blackened by hydrogen sulfide produced by SE when 10 ^-6 dilution (FIG. 5A).

SJP-SNU (8.8x10 ⁹ cfu / ml) and S. boulardii (2x10 ⁸ cfu / ml) stock solution and stock solution were cultured in YM broth. Diluted by them respectively, Jean 10 were inoculated on a medium surface and each dilution mixed with 450ul of yeast with the SE 10 ^-6 dilution bacteria 50ul. As a result of culturing at 37 ° C. for 18 hours, it was observed that the darkness of the medium due to hydrogen sulfide production tended to be inhibited as the number of yeast was increased in both SJP-SNU and S. boulardii (FIG. 5B).

Example  7: Pathogenicity assessment

SJP-SNU was inoculated in YM broth and incubated at 37 ° C for 24 hours. The culture was diluted 100 times and cultured. After 5 days of inoculation with fetal fibroblasts, no cytopathic effect was observed (Fig. 6A). SJP-SNU (10 ⁷ CFU / ml) was inoculated into the SPF seedlings at a concentration of 0.1 ml / ml and incubated for 60 hours. The SJP-SNU-inoculated fetus survived for 60 hours, and the fetus did not show any pathogenicity to the fetus because it did not differ from the control group. (Fig. 6B).

In order to evaluate the pathogenicity of SJP-SNU by immunosuppression, four groups of three 4-week-old SPF test systems were set up, and 10 ⁷ cfu / ml of SJP-SNU, S. cerevisiae (KCTC 50757) 500 μl per day for 3 days. Thereafter, the feeding was stopped (negative supply) for 4 days to inhibit the immune system (protein-energy malnutrition model), and the blood of the test system was collected on the fifth day. Cotton swabs were taken from kidneys and liver and plated on YM agar medium (antibiotics) to determine the presence or absence of yeast. As a result, yeast was not isolated at all and it was confirmed that SJP-SNU was not invasive to the internal organs.

Number of bacteria Bacterial isolation blood kidney Soy sauce SJP-SNU 10 ⁷ cfu / ml voice voice voice S. cerevisiae 10 ⁷ cfu / ml voice voice voice PBS 0 cfu / ml voice voice voice

SJP-SNU was injected subcutaneously at 10 ⁹ cfu / ml / water in the 3-week-old SPF group and clinical symptoms were observed for 14 days. Three days after the inoculation, eosinophilic lesions were observed. As a result, no clinical symptoms compared to the control group, no autopsy results, and no SJP-SNU pathogenicity.

Example  8: Identification of strains using genome analysis

The genome of the SJP-SNU new yeast strain of the present invention was extracted using DNeasy Blood & Tissue Kit (QIAGEN), and the nucleotide sequence was determined by the NGS technique. Genomic analysis such as nucleotide sequence assembly of SJP-SNU new yeast, ORF (open reading frame) prediction, BLAST search and yeast identification (Krona tool) was performed by Intron Biotechnology. As a result, the genome of the SJP-SNU new yeast strain was Pichia 74.40 % with kudriavzevii, 13.05% with Candida glycerinogenes , and Debaryomyces hansenii and 10.72% (Table 3).

From these results, it was confirmed that the new yeast strain SJP-SNU was a new yeast that evolved through heterospecific hybridizations with various yeast strains in order to adapt to the surrounding environment (FIG. 7).

Korea Biotechnology Research Institute KCTC12756BP 20150205

Claims (4)

It has a total genome sequence similarity of 74.40% with Pichia kudriavzevii , 13.05% with Candida glycerinogenes , and 10.72% with Debaryomyces hansenii SJP-SNU strain for probiotics (KCTC12756BP). A probiotic composition comprising the strain of claim 1. A food comprising the probiotic composition of claim 2. A feed comprising the probiotic composition of claim 2.

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