Patent Grant
12005108
The present invention relates to a novel Lactococcus lactis subspecies lactis isolate WFLU-12, and a use thereof.
The world fish food aquaculture production in 2012 was recorded as 44.2 million tons of fish (66%), 15.2 million tons of mollusks (23%), 6.4 million tons of crustaceans (10%), and 0.9 million tons of other species (1%). The inland aquaculture production was 38.6 million tons, which accounts for 58% of the world total aquaculture production in 2012. The proportion of aquaculture in the total fish production is steadily on an increase around the world. In Asia, the fish production of aquaculture exceeded the fish production of the general fishing industry (capture) since 2008, and the fish production of aquaculture accounted for 54% of the total fish production in 2012. This is a remarkably high figure compared to 18% in Europe and less than 15% in other countries, and aquaculture accounts for a very high share in the food industry of Asia (FAO yearbook, Fishery and Aquaculture Statistics. 2012). For fish aquaculture, high quality feed is important, and feed in this regard is a concept including not only essential nutrients but also supplementary feed additives. The feed affects the health, growth and development of the fish body, and should be eco-friendly.
Feed additives are defined as substances added to the feed in trace amounts, affecting not only the nutrition and growth rate but also the health of the fish body. Most feed additives used to promote growth include hormones, antibiotics, ionospheres, and some salts. Probiotics use living microorganisms among these feed additives to improve the intestinal microbial balance in the host and promote the growth of the host. For aquaculture, probiotics have been evaluated in terms of pathogen suppression, water quality improvement, immune response activity of host, promotion of nutritional absorption through the production of additional digestive enzymes, etc. (Verschuere et al., 2002; Carnevali et al., 2006). For such reasons, the use of probiotics is drawing attention in fish aquaculture around the world. Representative probiotic strains commonly used include Lactobacillus acidophilus, L. bulgaricus, L. plantariu, Lactococcus lactis, Saccharomyces cerevisiae (FAO, 2004).
The most significant benefit of using probiotics as a feed additive is that probiotics may be used as a substitute for antibiotics. At the moment, fish aquaculture depends on the use of antibiotics and chemotherapeutics in order to control and prevent bacterial diseases. However, the use of antibiotics and chemotherapeutics may cause selective pressure, thereby creating antibiotic resistant bacteria in the aquatic environment, increasing antibiotic tolerance in fish pathogens, and subsequently delivering tolerance determinants which exist in bacteria and fish pathogens to the pathogens of land animals and humans. Therefore, it is imminent to develop a substance which can replace antibiotics and chemotherapeutics for fish aquaculture. In this aspect, probiotics are the most suitable substitute capable of eliminating the risk of creating antibiotic resistant bacteria. In many researches, the use of probiotics has been proved to be effective in decreasing disease infection and mortality due to the host immune activity and direct pathogen suppression, making it an eco-friendly aquaculture technology.
In Korea, the rapid expansion of the aquaculture industry has been severely impacted by bacterial pathogens. Specifically, the loss caused by Streptococcus iniae and S. parauberis, which are the major pathogens of streptococcal diseases in flounder, accounted for the greatest portion. A long-term use of antibiotics for controlling streptococcal diseases in flounder contributed to a higher rate of antibiotic tolerance in S. parauberis than in S. iniae (Park et al., 2009). Therefore, as an alternative, it may be considered to reduce the use of antibiotics and use proper probiotics to prevent the propagation of tolerance in S. parauberis.
In light of the above, as a result of conducting continuous research on a probiotic isolate which has not only an excellent antibacterial activity against pathogens and excellent effect in preventing infection, but also an excellent effect in promoting the growth of fish body, and a feed additive comprising the same, the inventors of the present invention completed the present invention.
The present invention aims to provide an isolate with an excellent antibacterial activity against pathogens which is used in a probiotic composition to enhance innate immune response of fish and promote the growth of fish body.
Also, the present invention aims to provide a probiotic composition, an antibacterial composition, and a feed additive and a feed for fish farming, comprising the isolate.
Also, the present invention aims to provide a method of promoting the growth of farmed fish and a method of preventing infection by pathogens of farmed fish, using the isolate.
An embodiment of the present invention provides a Lactococcus lactis subspecies lactis isolate WFLU-12 with the accession number KCTC13180BP.
The isolate may show resistance to a temperature of at least 4° C., preferably a temperature of 4-10° C., a pH of 2-10, and a bile acid.
Also, the isolate may have an antibacterial activity against at least one selected from the group consisting of a Gram-negative bacterium of Vibrio anguillarum, V. ichthyoenteri, Aeromonas salmonicida, Edwardsiella tarda, Photobacterium damselae or a Gram-positive bacterium of Streptococcus iniae, S. parauberis.
Another embodiment of the present invention provides a probiotic composition comprising the isolate, a culture thereof, a lysate thereof or an extract thereof as an active ingredient.
The probiotic composition may present at least one effect selected from enhancing innate immune response of fish, increasing body weight, increasing body length, enhancing body circulating metabolite, maintaining homeostasis of metabolite, increasing sulfur-containing amino acid level in body, increasing taurine level in intestine, enhancing citrulline level in body, and enhancing vitamin level in intestine.
Another embodiment of the present invention provides an antibacterial composition comprising the isolate, a culture thereof, a lysate thereof or an extract thereof as an active ingredient.
Another embodiment of the present invention provides a feed additive for fish farming, comprising the probiotic composition or the antibacterial composition as an active ingredient.
Another embodiment of the present invention provides a feed for fish farming, comprising the feed additive.
Another embodiment of the present invention provides a method of promoting the growth of farmed fish, comprising feeding the feed to the fish being farmed.
Another embodiment of the present invention provides a method of preventing infection by pathogens of farmed fish, comprising spraying the antibacterial composition to a fish farm.
The novel Lactococcus lactis subspecies lactis isolate WFLU-12 of the present invention has excellent storage stability and antibacterial activity against pathogens, and the probiotic composition comprising the same presents effects of enhancing innate immune response of fish and promoting the growth of fish body. Therefore, it would be possible to improve the profits of the aquaculture industry by using a probiotic composition, an antibacterial composition, and a feed additive and a feed for fish farming, comprising the isolate.
An embodiment of the present invention provides a Lactococcus lactis subspecies lactis isolate WFLU-12 with the accession number KCTC13180BP. The Lactococcus lactis subspecies lactis isolate WFLU-12 of the present invention was isolated from a gastrointestinal tract of a flounder. The 16S rRNA gene sequence analysis showed a homology of 99.6%, and was identified as a Lactococcus lactis subspecies lactis type strain (RDP SEMATCH program). The API CH50 test results showed a homology of 99.9% to same species.
Comparative Analysis of the Entire Genomes Between Isolate WFLU-12 and Isolates from Different Orientations
As a result of analyzing the synteny of the isolate WFLU-12 of the present invention and the isolates from different orientations, the isolate WFLU-12 of the present invention formed a distinctive synteny compared to the isolates from different orientations (
Also, the inventors of the present invention found that the Lactococcus lactis subspecies lactis isolate WFLU-12 of the present invention with its good functional genes is distinguished from other isolates, has excellent resistance to a low temperature, various pH environments and bile acids and excellent storage stability, and has an excellent antibacterial activity against various fish pathogens, and completed the present invention.
Specifically, the isolate WFLU-12 of the present invention grows well at a temperature between 4° C. and room temperature and under pH conditions of 2-10, and in particular has good resistance to bile acids. Also, the isolate of the present invention may have an antibacterial activity against at least one selected from the group consisting of a Gram-negative bacterium of Vibrio anguillarum, V. ichthyoenteri, Aeromonas salmonicida, Edwardsiella tarda, Photobacterium damselae or a Gram-positive bacterium of Streptococcus iniae, S. parauberis.
The present inventors deposited the isolate WFLU-12 of the present invention at the Biological Resources Center of the Korean Collection for Type Cultures (KCTC), which is an international depositary institution under the Budapest Treaty, on Jan. 3, 2017, and received accession number KCTC13180BP.
According to another embodiment of the present invention, the present invention provides a probiotic composition comprising the isolate, a culture thereof, a lysate thereof or an extract thereof as an active ingredient.
The term “culture” in the present invention means the entire medium including cultured strains obtained by culturing the isolate for a certain period of time in a medium capable of supplying nutrients so that the isolate WFLU-12 of the present invention can grow and survive in vitro, metabolites thereof, and extra nutrients, etc., and also includes culture solutions in which strains are removed after culturing the strains.
The term “probiotic” in the present invention means living microorganisms, i.e., biomicrobial species beneficial to the health of intestinal flora, i.e., the health of the host. In general, probiotics are consumed as part of fermented food such as yogurt, etc. or as dietary supplements. Microorganisms known as probiotics include lactic acid bacteria (LAB), bifidobacteria, some yeasts and bacillus, etc.
In addition, the probiotic composition of the present invention may present at least one effect selected from enhancing innate immune response of fish, increasing body weight, increasing body length, enhancing body circulating metabolite, maintaining homeostasis of metabolite, increasing sulfur-containing amino acid level in body, increasing taurine level in intestine, enhancing citrulline level in body, and enhancing vitamin level in intestine.
According to another embodiment, the present invention provides an antibacterial composition comprising the isolate, a culture thereof, a lysate thereof or an extract thereof as an active ingredient. Since the isolate WFLU-12 has an antibacterial activity, particularly an antibacterial activity against fish bacterial pathogens, the isolate WFLU-12 or a culture thereof may be used as an antibacterial composition, preferably as an antibacterial composition against fish bacterial pathogens.
According to another embodiment, the present invention provides a feed additive for fish farming, comprising the antibacterial composition or the probiotic composition as an active ingredient. In addition to the above active ingredient, the feed additive of the present invention may further comprise an additive such as a known carrier or stabilizer, etc. that is pharmaceutically or sitologically acceptable, or is acceptable as feed. If necessary, the feed additive may comprise various nutrients such as vitamins, amino acids, minerals, etc., antioxidants, antibiotics, antibacterial agents, and other additives. At this time, the feed additive may be in a suitable form such as powder, granule, pellet, suspension, etc.
When the antibacterial composition of the present invention is included in the feed as a feed additive, the composition may be added as it is or mixed with other feed ingredients, and properly used according to a known method. The amount of active ingredients mixed may be properly determined according to use. Since the composition of the present invention is derived from a strain, eco-friendly, and has no particular problem in terms of stability, there is no particular limitation on the amount thereof.
The fish targeted in the present invention preferably includes marine fish such as sea bream, flounder, rockfish, red sea bream, croaker, mullet, sea bass, etc., and land fish such as eel, sweetfish, masu salmon, trout, mandarin fish, etc., and more preferably may be fish such as flounder, turbot, etc., whose taurine production is suppressed, but is not particularly limited thereto.
The feed additive of the present invention is not limited to the above mentioned fish, and can be used for all farmed fish. Preferably, the feed additive may be used for farmed fish which are likely to be infected with a Gram-negative bacterium of Vibrio anguillarum, V. ichthyoenteri, Aeromonas salmonicida, Edwardsiella tarda, Photobacterium damselae or a Gram-positive bacterium of Streptococcus iniae, S. parauberis.
Also, the feed additive of the present invention may be prepared by a method comprising culturing a Lactococcus lactis subspecies lactis isolate WFLU-12 in a solid medium; and centrifuging the cultured isolate to harvest a culture. Preferably, the step of culturing may be carried out in an MRS liquid medium at 28° C. for at least 24 hours, preferably 46-50 hours, most preferably 48 hours. Also, the step of harvesting the culture may be carried out by centrifuging the cultured isolate at 3,000 g for 5-30 minutes, preferably 15 minutes. The method for preparing the feed additive may further comprise re-suspending the culture in physiological saline. A feed comprising the feed additive may be prepared by spraying a suspension suspending the culture in physiological saline onto the feed at a concentration of 109 CFU/g to uniformly apply the suspension, and naturally drying the feed.
As described above, since the feed additive is manufactured by a simple method of culturing the isolate and harvesting the culture, the feed additive may be very simply manufactured and may be mass-cultured in a short time. Also, when the feed additive is stored at a temperature of at least 4° C., preferably 4-10° C. after being added to the feed, the number of bacteria in the feed of the isolate of the present invention may be maintained for a certain period of time, and thus it may be stored for a long period of time after being added to the feed. Accordingly, the feed additive may be very useful when practically used as a feed additive in fish farms.
According to another embodiment, the present invention provides a feed for fish farming comprising the feed additive. The form of the feed of the present invention is not particularly limited, and any feed such as powder feed, solid feed, wet pellet feed, dry pellet feed, extruder pellet (EP) feed, raw feed, etc., may be used.
According to another embodiment, the present invention provides a method of promoting the growth of farmed fish comprising feeding the feed to the fish being farmed. At this time, preferably, the feed is supplied in the same amount and feed interval as ordinary feed.
According to another embodiment, the present invention provides a method of preventing infection by pathogens of farmed fish comprising spraying the antibacterial composition to a fish farm. Since the antibacterial composition comprising the Lactococcus lactis subspecies lactis isolate WFLU-12 of the present invention, a culture thereof, a lysate thereof or an extract thereof as an active ingredient shows an antibacterial activity against fish bacterial pathogens, infection by the pathogens of farmed fish may be prevented by spraying the antibacterial composition to a fish farm so as to inhibit the activity of the bacterial pathogens. The infection with pathogens preferably such as a Gram-negative bacterium of Vibrio anguillarum, V. ichthyoenteri, Aeromonas salmonicida, Edwardsiella tarda, Photobacterium damselae or a Gram-positive bacterium of Streptococcus iniae, S. parauberis may be prevented by spraying the antibacterial composition of the present invention to the fish farm, but the pathogens are not limited thereto.
The Lactococcus lactis subspecies lactis isolate WFLU-12 of the present invention and isolates not derived from same marine-derived species were tested for their resistance to low temperature, various pH environments and bile acids, and the results are shown in Table 1 below.
Lc. lactis WFLU-12
Lc. lactis KCTC 3899 (Earth
Lc. lactis KCTC 3769 (Milk)
Lc. lactis KCTC 3768 (Plant)
Lc. lactis KGCM 40699 (Milk)
As described above, the isolate WFLU-12 grows well at a low temperature and in various pH environments, and in particular has good resistance to bile acids. Also, the fact that the isolate WFLU-12 survives at 10° C. indicates that the isolate may be preserved at a low temperature.
In addition, when the feed to which the isolate WFLU-12 of the present invention is added was stored at 4° C., the isolate WFLU-12 survived up to about 8 months, which indicates that the isolate is very capable of long-term survival (
In addition, no clinical symptoms were observed when the isolate WFLU-12 was injected into a fish by intramuscular or intraperitoneal injection. Also, no adverse effects were observed when the isolate was fed to a flounder at a very high concentration (˜109 CFU/g feed) for 8 weeks. In fact, the rate of intake of the fish was very good during the 8 weeks. Therefore, it may be confirmed that the isolate WFLU-12 is a very safe substance which does not adversely affect the fish body.
The antibacterial activity against fish pathogens was tested using the cross-streak method, and the results are shown in Table 2 and
Aeromonas
Edwardsiella
Photobacterium
Streptococcus
Streptococcus
Vibrio
Vibrio
salmoicida
tarda
damselae
iniae
parauberis
anguillarum
ichthyoenteri
a Inhibition zone(mm): ++++ = 16-20 mm; +++ = 11-15 mm; ++ = 6-10 mm; + = 1-5 mm; − = no inhibition
Also, in Table 2, isolates of same species from different orientations have very weak or no inhibitory effect against fish pathogens, but the isolate WFLU-12 of the present invention showed a broad and strong antibacterial activity against all Gram-negative pathogenic bacteria (Vibrio anguillarum, V. ichthyoenteri, Aeromonas salmonicida, Edwardsiella tarda, Photobacterium damselae) and Gram-positive pathogenic bacteria (Streptococcus iniae, S. parauberis).
In addition, the substance (supernatant) obtained from the culture medium of the isolate of the present invention was heat-treated to 65° C. and 100° C., and the results are shown in Table 3 and
A. samonicida
E. tarda
S. iniae
S. parauberis
V. anguilarium
V. ichthyoenteri
V. harvey
Preparation of Feed
A feed comprising the Lactococcus lactis subspecies lactis isolate WFLU-12 feed additive used in the present invention was prepared through the following steps.
In a solid medium, Lactococcus lactis subspecies lactis isolate WFLU-12 was cultured in MRS liquid medium at 28° C. for 48 hours. Then, a culture was harvested by centrifugation at 3,000 g for 15 minutes, resuspended in physiological saline, and adjusted to a concentration of 109 CFU/g feed weight, and the suspension was applied by being sprayed onto the feed. Then, the feed was dried naturally.
The following test was carried out having a flounder fed with a general compound feed as a control group and a flounder fed with a feed to which WFLU-12 probiotic was added as an experimental group (probiotic).
After orally infected with Streptococcus parauberis, the numbers of bacteria in the intestinal and renal tissues were compared (
The beneficial effect of the isolate WFLU-12 in flounder is proven by the natural infection rate of bacteria. As shown in Table 4 below, in a pilot-scale prey test, the infection rate of the experiment group (33%=10/30) was significantly lower than that of the control group (60%=18/30) (Fisher's exact test, p<0.05). Mixed infection occurred more frequently in the control group than in the experimental group (experimental group: 6.7%=2/30; control group: 26.7%=8/30). In particular, as for Streptococcal infection disease by S. parauberis, 60% was infected in the control group at week 2 (6/10), whereas infection by this bacterium was not detected in the experimental group for the 8 weeks. This result shows that the isolate WFLU-12 of the present invention may provide protection against bacterial pathogens in the intestinal tract.
S. parauberis
P. damselae
V. harveyi
V. ichthyoenteri
Vibrio spp.
(A) Increase in Body Length and Body Weight
The growing trend, i.e., body weight change (A) and body length change (B) of the control group and the experimental group (109 CFU/g) were observed for 8 weeks (˜80 g/fish, n=100), and the results are shown in
There was no significant difference in body weight increase and body length increase in both groups until week 2. However, at week 4, the average body weight of the control group being 147.40±22.47 g, and the average body weight of the experimental group being 164.15±24.73 g, there was a significant difference in average body weight (p<0.05), and at week 8, the average body weight of the control group being 192.91±29.31 g, and the average body weight of the experimental group being 217.88±33.36 g, there was a statistically significant difference in average body weight (p<0.01) (
(B) High Feed Efficiency and Specific Growth Rate
As a result of observing the feed conversion rate and specific growth rate for 8 weeks, there was a significant difference between the control group and the experimental group (p<0.01). The results are shown in
(C) Confirming Metabolite (Nutrient) in Intestinal Tract of Fish
The metabolites of the control group and the experimental group were compared assuming that the difference in fish growth between the control group and the experimental group is related to the fact that different patterns of gut microbiomes would be associated with beneficial metabolites of intestinal substances, total proteins, enzyme activity promotion, etc.
As a result of analyzing the metabolites,
(D) Maintaining Homeostasis of Metabolites Circulating in Fish Body
In order to confirm the effect of maintaining homeostasis of metabolites circulating in fish body, a graph of box plots showing the relative standard deviation (RSD %) of all metabolites detected in the intestinal fluid and serum was prepared and presented in
The difference in relative standard deviation (RSD) values of the metabolites indicate the difference in individual metabolites, and the metabolites in serum are affected by the metabolites produced by intestinal bacteria (Matsumoto et al., 2013; Front Syst Neurosci. 2013 Apr. 23; 7:9). The RSD values of intestinal substances were similar in both groups in terms of variability of metabolites (p=0.348), but the RSD values of serum were the most stable in the experimental group (p<0.001). That is, serum in the experimental group generally showed good homeostasis, whereas some fish in the control group showed relative unevenness (metabolites synthesized in host tissues). Just like the addition of probiotic to feed could gradually enhance the digestion capacity of the flounder over a period of weeks, the high homeostasis could be attributed to the appropriate nutritional status required by the fish. The isolate WFLU-12 of the present invention is considered to help maintain a strong homeostasis of metabolites circulating throughout the body.
The degree of increase in sulfur-containing amino acid level in the fish body was measured in the control group and the experimental group, and the results are shown in
Like other amino acids, sulfur-containing amino acids affect protein metabolism. They are components of tissue protein, and lack of these amino acids decreases protein synthesis. It has been reported that supplementing methionine has an effect on the muscle growth of chickens, and as a result of adding methionine to methionine-deficient feed (with other balanced amino acids added), protein synthesis and attachment increased in skeletal muscles.
As a result of comparing N-acetyl-methionine, which is a precursor of sulfur-containing amino acids in fish, in the control group and the experimental group, the present invention shows that N-acetyl-methionine increased 1.7 times in the intestine of the experimental group. Other sulfur-containing amino acid derivatives such as methionine sulfoxide (p<0.05), cysteine (1.6-fold increase), cysteine sulfonic acid (3.1-fold increase), cysteic acid (p<0.05) were expressed significantly higher in the experimental group than in the control group. This increase in sulfur-containing amino acid level leads to a significant increase in cystathionin (p<0.05) in circulating metabolites in which probiotics are involved in the synthesis of sulfur-containing amino acids, and to an increase of protein synthesis in fish organs.
In addition to the above, cystathionin increased in fish tissues (muscles) fed with cystine-added feed, and the growth also enhanced as compared with the control group (Park et al., 2002; Fisheries science Vol. 68 (2002) No. 4 P 824-829). Therefore, sulfur-containing amino acid precursors, especially cystathionin, act as an important marker for fish growth. They also play an important role in taurine biosynthesis, and in particular since cysteic acid produces taurine successively after enhancing the secretion of taurocholic acid (bile acid), cysteic acid may be used as the sole source of taurine formation.
The degree of increase in taurine and bile acid in fish intestines was measured in the control group and the experimental group, and the results are shown in
Taurine (2-amino ethanesulfonic acid) is a sulfur-containing amino acid that participates in a wide variety of physiological processes, in particular in the production of bile acid complexes, osmotic pressure regulation, calcium homeostasis, skeletal muscle, nerves and retinal function in vertebrate animals. In vivo synthesis of taurine in fish varies widely depending on species, and in particular it is well known that the synthesis of taurine in flounder and turbot is limited. Taurine is conjugated with bile acids to produce taurocholic acid, which accounts for 95% or more of the total bile acid complex in flounder. Therefore, taurine is a very essential nutrient that affects the growth of young flounders.
The addition of sulfur-containing amino acids to the feed did not promote the biosynthesis of taurine in the flounder, but it appears that the addition of the isolate WFLU-12 of the present research to the feed controls the expression of the related amino acids in the intestines of the flounder. The isolate of the present invention contributes to enhancing the level of intermediate amino acids such as cysteic acid (CA), cysteine sulfinic acid (CSA), etc., in the intestines of the fish, thereby increasing the level of taurine (
The degree of increase in citrulline in the intestine and serum was measured in the control group and the experimental group, and the results are shown in
Fish requires a high level of arginine in the feed because arginine is rich in protein (as a peptide bound to amino acids) and tissue fluid (phosphoarginine), which mainly stores ATP. Ureogenic teleost may convert citrulline to arginine in the liver by arginosuccinate synthase and lyase (Mommsen et al., 2001). However, it is unknown whether there is a net synthesis of citrulline or arginine in the liver of aquatic animals.
The level of citrulline increased in the intestines and circulating blood (serum) of the experimental group in the present invention (
The degree of biosynthesis of vitamin B1 (Thiamine), B2 (FAD_divalent), B3 (Nicotinamide), and C (Ascorbic acid) was measured in the control group and the experimental group, and the results are shown in
Vitamins are organic compounds which are essential nutrients for fish growth and health. Some vitamins are not synthesized in the fish body, and must be ingested through a food source. It has been confirmed that fish fed by adding the isolate WFLU-12 of the present invention showed an increase in the level of vitamin B and vitamin C (ascorbic acid) (
This application is a continuation of U.S. patent application Ser. No. 16/652,373, filed on 30 Mar. 2020 (now abandoned), which is a national phase application of PCT Application No. PCT/KR2017/001615, filed on 14 Feb. 2017, which claims priority to Korean Patent Application No. 10-2017-0010635, filed on 23 Jan. 2017. The entire disclosure of the applications identified in this paragraph is incorporated herein by reference.
| Number | Date | Country |
|---|---|---|
| 2006-265181 | Oct 2006 | JP |
| 10-2009-0035960 | Apr 2009 | KR |
| 10-2014-0090854 | Jul 2014 | KR |
| Entry |
|---|
| Thanh Luan Nguyen et al., “Improved growth rate and disease resistance in olive flounder, Paralichthys olivaceus, by probiotic Lactococcus lactis WFLU12 isolated from wild marine fish”, Aquculture, www.elsevier.com/locate/aquaculture, Jan. 9, 2017, V. 471, p. 113-120, Republic of Korea. |
| Won-Seok Heo et al., “Effects of dietary probiotic, Lactococcus lactis subsp. lactis 12, supplementation on the growth and immune response of oliver flounder (Paralichthys olivaceus)”, Aquaculture, www.elsevier.com/locate/aqua-online, Nov. 16, 2012, V. 376-379, p. 20-24, Republic of Korea. |
| “Lactococcus lactis subsp. lactis strain: WFLU12,” NCBr, BioProject accession No. PRJNA338902, Aug. 15, 2016. |
| International Preliminary Report on Patentability from corresponding PCT Application No. PCT/KR2017/001615, dated Jul. 23, 2019. |
| International Search Report from corresponding PCT Application No. PCT/KR2017/001615, dated Oct. 24, 2017. |
| Non-Final Office Action dated May 11, 2021 in U.S. Appl. No. 16/652,373. |
| Number | Date | Country | |
|---|---|---|---|
| 20230235276 A1 | Jul 2023 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16652373 | US | |
| Child | 17643942 | US |
