KR102314881B1 - Feed additive comprising fermented mealworm and feed composition using the same - Google Patents

Abstract

The present invention relates to a feed additive and/or a feed composition comprising a lactic acid bacteria fermented product of Mealworm Mealworm extract, antibacterial activity against pathogens, excellent microbial safety against sanitary indicator organisms, growth promotion of animals, and/or It has a heavy metal content that meets the criteria, overcoming the limitations of conventional insect feed.

Description

Feed additive comprising fermented mealworm and feed composition comprising the same {Feed additive comprising fermented mealworm and feed composition using the same}

The present invention relates to a feed additive and/or feed composition for animals using edible insects, and more particularly, to a feed additive and/or feed composition comprising a fermented product of Mealworm Mealworm extract.

Recently, as the production burden on aquaculture increases due to the high demand for fishmeal and the resulting increase in price, the link to the development of insect proteins for aquaculture and livestock is being made. The search for alternative and sustainable proteins requires an edible solution in the short term and the insect benefits as a feed option are being emphasized. In particular, insects are a natural food source for various fish and poultry, and chickens peck worms and larvae in top soil and straw, and maggots are used as baits in fishing.

The FAO's Anival feed resources information system (Feedipedia) provides sources, processing, and feeding cycles for insect feeds, including desert fern (Schistocerca gregaria), common housefly maggot (Musca domestica) and silkworm (Bombyx mori) used as livestock and fish feed. Guidelines, feeding cycle experiments and categories for nutritional properties are provided.

The main insect species used as feed include the common housefly larvae, silkworms, brown mealworms, grasshoppers, or termites. Common housefly larvae have been used as a substitute for fish meal in broiler production, but it has been reported that there is a risk of disease transmission and other bacterial propagation in livestock feed. In the case of termites, there are cases where they have been used as food for fish or birds, but breeding is not easy and there is a problem of safety due to the emission of methanol. In the case of silkworm, it was reported that there was no effect compared to general feed in terms of feed intake, weight gain, feed efficiency or protein efficiency.

Therefore, in order to use edible insects in general feed, it is necessary to secure safety through evaluation of the heavy metal content and toxicity of edible insects, to prepare for contamination and spoilage by microorganisms such as bacteria and viruses, and to store and distribute safely for a long period of time, storage methods and feed counting, growth , research on immunity enhancement is needed.

On the other hand, in the case of the fermentation method using microorganisms, a new efficacy different from the existing efficacy of the raw material may be given, and a number of harmful ingredients may be changed into safe ingredients. In addition, by reducing the molecular weight of nutrients by the action of degrading enzymes, absorption can be increased, irritation can be lowered, and allergic components can be reduced.

Recently, research to search for substances with antibacterial activity from natural materials and apply them to food and feed is being actively conducted. There are no examples of it applied to and entered into force. Produces natural antibacterial substances with bioconversion technology using lactic acid bacteria and applies them to edible insects to produce fermented products of edible insect extracts with improved antibacterial and antioxidant functions, and improved preservatives in storage, storage and distribution, and lactic acid bacteria There is still no known fermented product of edible insect extract, which improves absorption and functionality in the body by decomposing amino acids, peptides, fats, and carbohydrates with its own decomposing enzymes, and there is a high need for research on this.

The present invention provides a feed additive and/or feed composition comprising the lactic acid bacteria fermented product of Mealworm Mealworm extract, a feed composition comprising the feed additive, and uses thereof.

The present invention provides a feed additive comprising a lactic acid bacteria fermented product of Mealworm Mealworm extract or a feed composition comprising the feed additive.

The present invention provides a pharmaceutical composition for the prevention or treatment of livestock or farmed fish, comprising a lactic acid bacteria fermented product of brown mealworm extract.

Hereinafter, the present invention will be described in more detail.

The feed additive provided by the present invention includes a lactic acid bacteria fermented extract of Mealworm brown.

As used herein, "brown mealworm" means at least one selected from the group consisting of eggs, larvae, pupae, and adults of Mealworm brown ( Tenebrio molitor ), and in one example, Mealworm brown is a larva and / or pupae of Mealworm brown. and in another example, Mealworm Brown may be Mealworm larvae.

Brown mealworm larvae, also called mealworms, have been used as breeding food for various animals such as fish, birds, mammals, carnivorous plants, and/or arthropods. As one of the insects approved as edible insects by the Ministry of Food and Drug Safety of the Republic of Korea, brown mealworm larvae used for human consumption are also distributed under the name of 'Gosoae'.

Brown mealworm, which is a raw material of the feed additive provided by the present invention, may be used in powder form by lyophilization, and may be additionally subjected to washing and fasting steps before the freeze-drying step if necessary. The fasting step is a step for emptying the intestines of brown mealworm, and may be performed by stopping the feeding of brown mealworm or feeding glutinous rice flour within the target range, but is not limited thereto.

In one embodiment of the present invention, the brown mealworm larvae of the old age were fasted for 3 days, the intestines were washed with running water, and then the brown mealworm larvae were dried in hot air at 60° C. for 48 hours to be insecticidal. The hot air-dried brown mealworm larvae were prepared into powder using a food grinder and used for subsequent experiments.

The powdering process can be performed by appropriately using the grinding and resting time as necessary, and by having the resting time, it has the advantage of minimizing damage to the brown mealworm larvae sample due to the heat generated in the grinder. In one embodiment, the grinding cycle of grinding for 1 minute and resting for 3 minutes was repeated 5 times to prepare brown mealworm larvae powder (Tm).

The brown mealworm extract can be prepared by using a freeze-dried meal of brown mealworm, a dry meal of mealworm brown, and/or a freeze-dried product of mealworm brown, and, if necessary, a freeze-dried product of mealworm brown, a dry meal of brown mealworm, and/or a freeze-dried product of mealworm brown. It can be used in powder form.

The brown mealworm extract may be used by selecting an appropriate extraction solvent as needed, and the extraction solvent may be at least one selected from the group consisting of alcohol and water having 1 (C1) to 3 (C3) carbon atoms. For example, it may be water, ethanol (EtOH), or an aqueous ethanol solution. The extraction method for preparing the extract may use a conventional extraction method, for example, extraction with an extraction solvent, supercritical fluid extraction, ultrasonic extraction, hot water extraction, etc. may be used. Preferably, a hot water extraction method may be used. The solvent extraction time may be 4 to 6 hours, or 5 to 6 hours.

The extraction temperature during the solvent extraction may be 10 to 90 ℃, 20 to 80 ℃, 30 to 70 ℃ or 30 to 60 ℃. For example, when the solvent is water, the extraction temperature may be 40 to 80 ℃, 50 to 70 ℃, or 55 to 65 ℃, for example, 60 ℃, when the solvent is alcohol (EtOH), the extraction temperature is 10 to 50°C, 20-40°C, or 25-35°C, for example 30°C.

The lactic acid bacteria fermented product of the brown mealworm extract may be used as a raw material for fermentation as it is, or it may be used as a fermentation raw material through a post-treatment process step such as centrifugation or filtration. In order to reduce the quantitative loss in order to utilize the active substance of Mealworm of Brown, the whole extract of Mealworm of Brown may be used, or only the supernatant of Mealworm of Brown may be used.

The brown mealworm extract may be (i) an extract from which the brown mealworm powder has been partially removed by centrifugation and filtration, or (ii) an extract precipitate from which the brown mealworm powder has not been removed.

In the feed additive and/or feed composition comprising the lactic acid bacteria fermented product of Mealworm brown extract provided by the present invention, the lactic acid bacteria fermented product of the Mealworm brown extract is one or more types of lactic acid bacteria fermented product of the Mealworm brown extract. It can be produced through the step of fermentation by adding (inoculation) lactic acid bacteria.

The lactic acid bacteria are Lactobacillus plantarum (ATCC 10241, NCTC7220), Lactococcus lactis (Lactococcus lactics, ATCC 11454, NCIC 8586), Lactobacillus sakei (Lactobacillus sakei, ATCC 31063, ATCC 15521) Fermentum (Lactobacillus fermentum, ATCC14931, NCDO 1750), Lactobacillus rhamnosus (ATCC7469, KCTC3326), Lactobacillus brevis (Lactobacillus brevis, ATCC15521), Lactobacillus graminis, Lactobacillus graminis (Lactobacillus graminis, ATCC15521) Lactobacillus dextrinicus (ATCC 33087), Lactococcus garvieae (ATCC11454, LMG 8162), Enterococcus faecium (Enterococcus faecium, ATCC 19657alis), Enterococcus faecium (Enterococcus faecium, ATCC 19657alis), Enterococcus faeces , Leuconostoc citreum (ATCC 49370), Weissella hellenica (ATCC 51523), Weissella paramesenteroides (ATCC 33313), Pediococcus acidilactici (Pediococcus acidilactici) ATCC33314), Pediococcus pentosaceus (Pediococcus pentosaceus, ATCC 33316), Bifidobacterium animalis (Bifidobacterium animalis, ATCC 27672) and Bifidobacterium infantis (Bifidobacterium infantis, ATCC15697) selected from the group consisting of bacteria It may be one or more lactic acid bacteria, but is not limited thereto . The above bacteria can be purchased and used from the Bioresource Center of the Korea Research Institute of Bioscience and Biotechnology or the American Type Culture Collection (ATCC).

In one example, the lactic acid bacteria is Lactobacillus plantarum (Lactobacillus plantarum), Weissella paramesenteroides, and Pediococcus acidilactici (Pediococcus acidilactici) at least one selected from the group consisting of, two types It may be more than, or three types of lactic acid bacteria.

The Lactobacillus planterum strain may be a strain comprising a 16S rRNA gene having 99% or more, 99.5% or more, or 99.9% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3, for example, It may be a strain containing the 16S rRNA sequence consisting of the nucleotide sequence of SEQ ID NO: 3, and more preferably, the accession number KCCM12299P strain deposited with the Korea Microorganism Conservation Center (KCCM) on August 10, 2018.

The Weissella paramesenteroid strain may be a strain comprising a 16S rRNA gene base sequence having at least 99%, 99.5% or 99.9% sequence homology with the base sequence of SEQ ID NO: 4, and the base sequence of SEQ ID NO: 4 It may be a strain containing a 16S rRNA gene consisting of

The Pediococcus acidi lactisi strain may be a strain comprising a 16S rRNA gene having 99% or more, 99.5% or more, or 99.9% or more sequence identity with the nucleotide sequence of SEQ ID NO: 5, For example, it may be a strain comprising a 16S rRNA sequence consisting of the nucleotide sequence of SEQ ID NO: 5, and preferably, it may be a strain of accession number KCCM12472P deposited at the Korea Microorganism Conservation Center (KCCM) on March 29, 2019. have.

The lactic acid bacteria fermented product of Mealworm Mealworm extract of the present invention may have one or more of the properties listed below:

(1) antibacterial activity against one or more pathogens selected from the group consisting of Vibrio, Listeria, and Streptococcus;

(2) excellent microbial safety against sanitary indicator bacteria;

(3) promote animal growth; and

(4) The content of heavy metals that meet the standards of the Food Standards Code.

The lactic acid bacteria fermented product of Mealworm brown extract of the present invention, the feed additive and / or feed composition comprising the lactic acid bacteria fermented product of the Mealworm Mealworm extract of the present invention, Vibrio bacteria, Listeria bacteria, and one or more pathogens selected from the group consisting of Streptococcus. It may have antibacterial activity and/or growth inhibitory activity.

Vibrio bacteria are known as the causative agent of vibriosis, and the symptoms include darkening of body color, ulceration, and/or muscle loss. Streptococcus is a causative agent of streptococcosis, and the typical symptoms of infection include blackening of the body, protrusion of the eyeball, secretion of mucus from the gills or body surface, and/or intestinal bleeding. Listeria is known to cause problems when used as food by attaching to the body surface of fish.

In one embodiment, the test strain Vibrio central nine days rareom (Listonella anguillarum, ATCC19264), Streptococcus para Ube-less (Streptococcus parauberis, ATCC 35668), Streptococcus am in (Streptococcus iniae, ATCC 29177), and L. monocytogenes to Ness ( Listeria monocytogenes ) After culturing in LB agar medium, respectively, hot water extract (HeTm) of mealworm larvae on 8 mm paper, lactic acid bacteria fermented product of hot water extract of mealworm larvae (HeTm LpWpPa), and Lactobacillus planterum as a control (KCCM12299P), Weissella paramesenteroid (KCCM12301P), and Pediococcus acidi lactisi (KCCM12472P) were respectively injected to confirm whether the inhibition ring was formed. As a result, almost no inhibition ring was generated around the paper disc treated with the HeTm sample that had not undergone lactic acid fermentation, whereas in the case of the HeTm LpWpPa sample, the inhibition ring was generated at a level similar to the case of treatment with each strain of the control group. Therefore, it was confirmed that the brown mealworm extract obtained growth inhibitory or antibacterial effects on the test strains (Vibrio bacteria, Listeria bacteria and/or Streptococcus) at a level similar to that of each lactic acid bacteria during the fermentation process by three types of lactic acid bacteria.

Accordingly, the lactic acid bacteria fermented product of the Mealworm Mealworm extract provided by the present invention, the feed additive and/or feed composition comprising the lactic acid bacteria fermented product of the Mealworm Mealworm extract, and/or the feed composition comprising the feed additive is a fish disease, For example, it can be utilized for preventing, ameliorating or treating vibriosis, streptococcosis, and/or listeriosis. These characteristics suggest that safety problems such as concerns about disease transmission or bacterial propagation reported in conventional insect feeds can be overcome.

In one embodiment, the lactic acid bacteria fermented product of the Mealworm Mealworm extract may be used as a vaccine composition or vaccine additive for the prevention of vibriosis, streptococcosis, and/or listeriosis.

In one embodiment, the lactic acid bacteria fermented product of Mealworm Mealworm extract may be used as a feed additive and/or feed composition for improving vibriosis, streptococcosis, and/or listeriosis.

In one embodiment, the lactic acid bacteria fermented product of the Mealworm Mealworm extract may be used as a pharmaceutical composition for the prevention or treatment of vibriosis, streptococcosis, and/or listeriosis. The pharmaceutical composition may be administered to animals other than humans, for example, fish, reptiles, birds, mammals and/or arthropods. In one example, the animal may be a fish, for example, flounder, halibut, rockfish, rainbow trout, yellowtail, eel, and/or carp.

The pharmaceutical composition may be administered by an appropriate method if necessary, for example, oral administration, intravenous injection, intramuscular injection, intraperitoneal administration, intrathecal administration, subcutaneous administration, sublingual administration, buccal administration, rectal administration, vaginal administration It may be one or more administration methods selected from the group consisting of administration, nasal administration, and transdermal administration, but is not limited thereto.

In one embodiment, the pharmaceutical composition may be administered to fish by intraperitoneal administration, immersion administration, and/or mixing with feed.

The lactic acid bacteria fermented product of the Melancholic Melancholy Extract of the present invention, the feed additive and/or feed composition comprising the lactic acid bacteria fermented product of the Mealworm Mealworm extract of the present invention may have microbial safety against sanitary indicator bacteria. The microbial safety means that the sanitary indicator bacteria are not detected or are detected below the sanitary standard value in the field of medicine, food or feed.

The hygiene indicator bacteria may be Listeria (Environmental Listeria), yeast (yeast), mold (Fungi), Escherichia coli ( Escherichia coli ) and / or general bacteria. The general bacteria refer to all microorganisms that grow under aerobic conditions.

In one embodiment, whether or not to detect sanitary indicator bacteria for the lactic acid bacteria fermented product (HeTm LpWpPa) of the brown mealworm larva powder (Tm), the hot water extract (HeTm) of the mealworm larvae powder (HeTm) and the brown mealworm larvae extract that have not undergone the extraction and fermentation steps As a result of confirming using a commercially available kit, Listeria and general bacteria were detected in Tm, while no sanitary indicator bacteria were detected in both HeTm and HeTm LpWpPa, indicating that microbial stability was increased through extraction and fermentation. Confirmed.

Therefore, it was confirmed that by using the lactic acid bacteria fermented product of the brown mealworm extract in a feed additive or feed composition, it was possible to overcome the problems of the prior art in which there was a concern of bacterial propagation when conventional housefly larvae were used in feed.

The lactic acid bacteria fermented product of the Melancholy Mealtimes brown extract of the present invention, the feed additive and/or the feed composition comprising the lactic acid bacteria fermented product of the Mealworm brown extract may be an animal growth promoter. The animal may be an animal fed a feed or feed composition containing the feed additive.

The animal may be any animal except humans, and may be a fish, reptile, bird, mammal, and/or arthropod, but is not limited thereto. In one example, the animal may be a fish, for example, flounder, halibut, rockfish, rainbow trout, yellowtail, eel, and/or carp.

Growth promotion of animals fed with the feed additive provided by the present invention and/or the feed composition of the present invention can be expressed in the form of a growth rate or daily growth rate, and the growth rate is expressed by the following [Equation 1], The growth rate can be calculated by the method of [Equation 2] below.

[Equation 1]

[Equation 2]

The feed additive to which the lactic acid bacteria fermented product of brown mealworm extract is added and/or the animal fed the feed composition of the present invention provided by the present invention is a feed that does not contain the lactic acid bacteria fermented product of the brown mealworm extract (hereinafter 1.05 times to 3 times, 1.05 times to 2 times, 1.05 times to 1.5 times, 1.05 times to 1.2 times, 1.1 times to 3 times, 1.1 times to 2 times, 1.1 times than animals fed with "general feed") to 1.5 times, or 1.1 times to 1.2 times higher growth rate.

In one embodiment, as a result of measuring the growth rate by feeding each feed containing or not containing the lactic acid bacteria fermented product of the brown mealworm extract, respectively, in the control group, the general feed group, at the 3rd week after the start of feeding, 93.77% In the experimental group, the weight gain rate in the experimental group was 110.08% at the 3rd week after the start of feeding, which was higher in the experimental group. It was found to be 25%p higher than that of this control group.

The feed additive to which the lactic acid bacteria fermented product of brown mealworm extract is added and/or the animal fed the feed composition of the present invention provided by the present invention is a feed that does not contain the lactic acid bacteria fermented product of the brown mealworm extract (hereinafter It may have a daily growth rate of 1 to 3 times, 1 to 2 times, 1 to 1.5 times, or 1 to 1.2 times higher than that of animals fed with "general feed").

When the lactic acid bacteria fermented product of the brown mealworm extract of the present invention is included in a fish feed additive or a fish feed, it is possible to promote the growth of fry. In one embodiment, as a result of feeding the fry mixed with the lactic acid bacteria fermented product of the brown mealworm extract, the daily growth rate in the experimental group fed the mixed feed compared to the control group fed with a general feed that does not contain the fermented product This drug was 0.4%p higher, and the daily growth rate was similar to that of the control group from 3 to 6 weeks. Therefore, the lactic acid bacteria fermented product of the brown mealworm extract can be used as a growth promoter for fry in aquaculture of fish.

If the feed additive or feed composition comprising the lactic acid bacteria fermented product of the brown mealworm extract provided by the present invention is used for aquaculture of fish, for example, flounder, within 3 weeks or within 4 weeks after starting the breeding of seedlings, Or it may be paid within 5 weeks.

Therefore, when the feed additive and/or the feed composition of the present invention is fed to animals, it can contribute to the improvement of aquaculture and/or breeding productivity than when fed a general feed. Therefore, it was confirmed that, unlike silkworms used as feed in the prior art, it showed more excellent effects in terms of weight gain and feed efficiency.

The lactic acid bacterium fermented product of the Melancholic Melancholy Extract of the present invention, the feed additive and/or the feed composition comprising the lactic acid bacteria fermented product of the Mealworm Meal brown extract may have a heavy metal content that satisfies the standards of food standards. Therefore, it is excellent in biosafety as a food additive and/or a feed composition as well as food.

The heavy metals are chromium, cadmium, arsenic and/or mercury, and the heavy metal standard values of edible insects presented in the Food Code of the Ministry of Food and Drug Safety are cadmium 0.05 mg/kg, mercury 0.5 mg/kg, and arsenic 0.1 mg/kg.

The chromium content of the lactic acid bacteria fermented product of the brown mealworm extract may be 0.03 mg/kg or less, 0.02 mg/kg or less, 0.01 mg/kg or less, 0.001 mg/kg or less, or 0.0005 mg/kg or less.

The cadmium content of the lactic acid bacteria fermented product of the brown mealworm extract may be 0.05 mg/kg or less, 0.01 mg/kg or less, 0.008 mg/kg or less, 0.007 mg/kg or less, or 0.006 mg/kg or less.

The arsenic content of the lactic acid bacteria fermented product of the brown mealworm extract may be 0.1 mg/kg or less, 0.095 mg/kg or less, 0.09 mg/kg or less, or 0.085 mg/kg or less.

The mercury content of the lactic acid bacteria fermented product of the brown mealworm extract may be 0.5 mg/kg or less, 0.05 mg/kg or less, 0.005 mg/kg or less, 0.001 mg/kg or less, 0.0001 mg/kg or less, or 0.

In one embodiment, as a result of measuring the content of chromium, cadmium, arsenic, and mercury in the lactic acid bacteria fermented product of the brown mealworm larvae extract, chromium, cadmium, arsenic, and mercury are 0.0002 mg/kg, 0.0057 mg/kg, 0.083 mg, respectively. /kg, and 0 mg/kg, and all showed lower values compared to brown mealworm larvae powder (Tm), and it was confirmed that heavy metal components harmful to the living body were removed due to the lactic acid bacteria fermentation process.

Therefore, it was confirmed that the heavy metal safety of the lactic acid bacteria fermented extract of the brown mealworm larvae of the present invention was excellent.

In the feed additive comprising the lactic acid bacteria fermented product of Mealworm brown extract provided by the present invention, the content of the lactic acid bacteria fermented product of Mealworm brown extract is 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30 wt% or more, 40 wt% or more, 50 wt% or more, 60 wt% or more, 70 wt% or more, 80 wt% or more, 90 wt% or more, 95 wt% or more, or 99 wt% or more.

In the feed composition comprising the lactic acid bacterium fermented product of Mealworm brown extract provided by the present invention, the content of the lactic acid bacteria fermented product of Mealworm brown extract is 0.1 to 50% by weight, 0.1 to 40% by weight, 0.1 to 30% by weight, 0.1 to 20 wt%, 0.1-15 wt%, 1-50 wt%, 1-40 wt%, 1-30 wt%, 1-20 wt%, 1-15 wt%, 5-50 wt%, 5-40 wt% %, 5 to 30% by weight, 5 to 20% by weight, or 5 to 15% by weight.

In the feed composition containing the lactic acid bacteria fermented product of Mealworm brown extract provided by the present invention, the feed may be fed alone, or mixed with and/or alternately fed with a general feed that does not contain the lactic acid bacteria fermented product of Mealworm brown extract. can The amount of feed and the number of feedings can be appropriately adjusted and used by those skilled in the art according to the type of target animal, the number of individuals, the age and/or size of the animal. In one embodiment, when feeding the feed composition provided by the present invention to a fish tank containing 100 flounder, 30 g of feed was fed twice a day.

The present invention provides a feed additive or feed composition comprising a lactic acid bacteria fermented extract of Mealworm brown. The fermented lactic acid bacteria has antibacterial properties against sanitary indicator bacteria, so it has excellent storage safety, and improves the growth rate and / or daily growth rate of the breeding target animal compared to the commercial feed, so that it can properly crawl for the growth of the breeding target animal, heavy metal The content is lower than the content by the food standard or not detected, so it is excellent in safety to the living body.

1 is a graph showing the result of measuring the number of lactic acid bacteria after fermentation culture by co-inoculating 3 types of lactic acid bacteria into 4 types of medium for the production of a fermented product of brown mealworm larvae.
2 is a result of performing a test for detecting harmful microorganisms in the powder of Mealworm larvae of Brown Meal, the hot water extract of the powder, and the fermented product of the hot water extract.
Figure 3 shows Lactobacillus planterum, Weissella paramesenteroid, Pediococcus acidi lactisi sample (control) and lactic acid bacteria fermented product (HeTm LpWpPa) of Mealworm larvae extract, Brown Mealworm larvae hot water extract (HeTm) each harmful It is the experimental result confirming the formation of the inhibitory ring in the case of treatment with the strain.
4 is a graph showing the results of analysis of changes in average body length and average body weight over time after feeding a feed added with a lactic acid bacteria fermented product of general feed or brown mealworm larvae extract to stone fluke.
Figure 5 is a graph comparing the growth rate over time after feeding the fermented lactic acid bacteria of a normal feed or brown mealworm larvae extract to stone fluke.
Figure 6 is a graph comparing the daily growth rate over time after feeding the fermented lactic acid bacteria of general feed or brown mealworm larvae extract to stone fluke.

Example 1. Preparation of extract of Mealworm of Brown

1-1: Preparation of Mealworm Powder

Brown mealworm larvae were raised for 90 days and fasted for 3 days to clean the intestines of the brown mealworm larvae. Next, the brown mealworm larvae were washed in running water and then dried with hot air at 60° C. for 48 hours to be insecticide-treated.

The dried brown mealworm larvae were prepared into powder using a food grinder. Specifically, in order to prevent heat generation due to the use of a food grinder, the grinding cycle of grinding for 1 minute and then resting for 3 minutes was repeated 5 times to prepare brown mealworm larvae powder (Tm).

1-2: Preparation of hot water extract of brown mealworm

A hot water extract was prepared using the brown mealworm larva powder (Tm) prepared in Example 1-1. After mixing 100 g of brown mealworm larvae powder (Tm) and 900 ml of water in a ratio of 1 : 9, ultrasonic waves were irradiated at 750 watt, 10 kHz at 10 kHz intervals for 5 minutes and mixed (Sonics Vibra-Cell, USA). Next, the mixture was incubated with shaking in hot water at 60° C. for 4 to 6 hours, and the cultured extract was centrifuged (3,000 rpm, 10 minutes) and vacuum filtered using a 0.45 um corning bottle-top (Sigma, USA). (vacuum filter) to prepare about 800 to 900ml (final yield 80 to 90%) of brown mealworm hot water extract (Hotwater Extract of Tenebrio molitor , HeTm).

The liquid hot water extract was stored at -70°C and then freeze-dried, or freeze-dried immediately after preparation and powdered, and then used for subsequent experiments.

Example 2. Isolation and identification of lactic acid bacteria for production of fermented products

2-1. Isolation of strains

For the production of fermented products using effective strains for perfumery and food, Lactobacillus plantarum (Lp), Weissella paramesenteroides (Wp) and Pediococcus acidilactici ( Pediococcus acidilactici , Pa) was isolated and identified from deep sea water and fermented sawdust, respectively, and used.

Lactobacillus plantarum ( L. plantarum ) was isolated from deep sea water. The deep sea water was transported in a refrigerated state by taking seawater from the surface at a depth of 200 m from the surface in Taeha, Jeodong and Hyeonpo, Ulleungdo from November to December 2015. The deep sea water was smeared on MRS agar medium supplemented with 0.1% (v/v) BCP and cultured at 37° C. for 1 day. Thereafter, the selection of yellow colonies formed by the reaction with BCP was repeated to isolate a single species.

Weissella paramesenteroid and Pediococcus esidiraci strains were isolated and identified from fermented sawdust, a food for edible insects. The fermented sawdust for feeding the edible insects was purchased from Yeongcheon Insect Industry (Korea). Fermented sawdust stored at 25° C. was added to PBS at a concentration of 1% (w/v) and mixed by vortexing. The fermented sawdust mixture was smeared on MRS (Difco, USA) agar medium supplemented with 0.1% (v/v) BCP (Bromocresol purple, Sigma, USA) and cultured at 37°C for 1 day. The BCP is acidified by the lactic acid of lactic acid bacteria to perform a function to form yellow colonies. Only the yellow colonies formed by the reaction with BCP were selected, spread on MRS agar medium, and the process of reselection was repeated to isolate a single species of Weissella paramesenteroid and Pediococcus esidilactici.

The three isolated strains were cultured in MRS liquid medium, mixed with 50% (v/v) glycerol so that the final glycerol concentration was 25%, and then stored at -70°C.

2-2. Identification of strains

The strain isolated in Example 2-1 was identified using 16S rRNA sequencing analysis. To identify three strains, first, genomic DNA of each strain was isolated using a mini genomic DNA extraction kit (Promega).

Using the isolated genomic DNA as a template, the following 27F and 1492R primer sets and AccPower PCR premix kit (Bioneer, Korea) were used to amplify the 16S rRNA gene regions of the three strains using TaKaRa PCR Thermal Cycler (Japan).

27F Primer (SEQ ID NO: 1): 5'-AGAGTTTGATCCTGGCTCAG -3'

1492R Primer (SEQ ID NO: 2): 5'-GGTTACCTTGTTACGACTT-3'

The PCR product was purified by PCR clean-up Gel extraction (Macherey Nagel, USA) and the gene sequence was analyzed with an ABI3730 DNA analyzer (Applied Bioxyxtems, USA). The 16S rRNA sequence of the Lactobacillus planterum strain isolated in the present invention is shown in SEQ ID NO: 3, and the 16S rRNA sequence of the Weissella paramesenteroid strain and Pediococcus esidiraticsi is shown in SEQ ID NOs: 4 and 5.

The 16S rRNA sequence similarity analysis was performed using the 16S rRNA sequence information of the isolated Lactobacillus planterum, Weissella paramesenteroid and Pediococcus esidiraci strains, and the results are shown in Table 1 below. As shown in Table 1 below, the lactic acid bacteria isolated from deep sea water showed 99% sequence similarity with Lactobacillus planterum as a result of 16S rRNA analysis, and Weissella paramesentroid and Pediococcus escidilatic isolated from fermented sawdust. Shi also showed a sequence similarity of 99%, and was named Lactobacillus planterum, Weissella paramesenteroid, and Pediococcus esidilatixi, respectively.

number lactic acid bacteria isolate strain Proximal 16S rRNA 16S rRNA sequence similarity One deep sea water Lactobacillus plantarum DSW#2-4
(16S rRNA of SEQ ID NO: 3) Lactobacillus plantarum MK311261.1
(SEQ ID NO: 6) 99% 2 Fermented sawdust Weissella paramesenteroides D20
(16S rRNA of SEQ ID NO: 4) Weissella paramesenteroides LC094432.1
(SEQ ID NO: 7) 99% 3 Fermented sawdust Pediococcus acidilactici D17
(16S rRNA of SEQ ID NO: 5) Pediococcus acidilactici LC311071.1
(SEQ ID NO: 8) 99%

The isolated Lactobacillus planterum strain was deposited with the Korea Center for Microorganisms (KCCM) on August 10, 2018, and the accession number is KCCM12299P. The Weissella paramesenteroid strain was deposited with the Korea Microorganism Conservation Center (KCCM) on August 10, 2018, and was given an accession number KCCM12301P. The Pediococcus acidi lactisi strain was deposited with the Korea Microorganism Conservation Center (KCCM) on March 29, 2019, and received KCCM12472P as an accession number.

Example 3. Confirmation of optimal conditions for the preparation of fermented mealworm extract

In preparing the fermented product of the brown mealworm larvae extract, an experiment was performed to determine whether the addition of a carbon source is necessary as an optimal condition to effectively cause lactic acid bacteria fermentation.

First, four types of media were prepared. Each of the four mediums is a medium in which 80 mL of brown mealworm hot water extract (HeTm) 1 L medium prepared in Example 2, 1 L of HeTm and 2% (w/v) dextrose (D-glucose, Sigma, USA) is mixed , MRS medium, which is a commonly used lactic acid bacteria medium, and an aqueous solution medium in which the HeTm lyophilized powder (HeTm Powder) is dissolved at 10% (w/v). Three kinds of lactic acid bacteria L. plantarum ( KCCM12299P ), W. paramesenteroides (KCCM12301P), and Pediococcus acidilactici (KCCM12472P) were inoculated in the above four media at a ^{concentration of 1x10 8} cfu/ml by 1 mL each, and co-cultured at 30° C. for 24 hours. did. After 24 hours, LAB Counts (log cfu/ml) were measured to confirm the optimal fermented culture conditions, and the measurement results are shown in FIG. 1 and Table 2 below.

culture conditions LAB Counts by incubation time
(log cfu/ml) culture medium strain 24 h incubation hot water extract
(HeTm) L. plantarum KCCM12299P
W. paramesenteroides KCCM12301P
P.acidilactici KCCM12472P 1.08E+9 HeTm with dextrose
(HeTm Dex) L. plantarum KCCM12299P
W. paramesenteroides KCCM12301P
P.acidilactici KCCM12472P 1.18E+9 MRS L. plantarum KCCM12299P
W. paramesenteroides KCCM12301P
P.acidilactici KCCM12472P 6.0E+8 HeTm lyophilisate solution
(HeTm Powder Solution) L. plantarum KCCM12299P
W. paramesenteroides KCCM12301P
P.acidilactici KCCM12472P 1.30E+9

The LAB Counts were obtained by diluting the culture to 1% (v/v) in a solid medium prepared by adding agarose (BD Difco, USA) to each liquid medium, spreading 1 ml on the LAB plate, and then at 30°C for 24 hours This was performed by measuring the number of colonies formed by culturing.

As can be seen in FIG. 1 , when L. plantarum, W. paramesenteroides and P.acidilactici were inoculated in MRS medium, LAB Counts was measured as ^{1.08x10 9 /} ml (1.08E+09) after culturing for 24 hours. . L. plantarum, W. paramesenteroides and P.acidilactici were co-inoculated in HeTm medium without dextrose and the number of lactic acid bacteria in the culture cultured for ^{24 hours was 1.18x10 9 /ml (1.18E+09) for lactobacillus culture.} Compared to the widely used MRS medium, LAB Counts was higher.

On the other hand, L. plantarum, W. paramesenteroides and P.acidilactici were co-inoculated into HeTm with dextrose and the number of lactic acid bacteria in the culture cultured for 24 hours was 6.0x10 ^{8 /} ml (6.0E+8). The number of lactic acid bacteria was rather reduced when compared to MRS medium or HeTm medium without dextrose.

On the other hand, L. plantarum , W. paramesenteroides and P.acidilactici were co-inoculated in a 10% (w/v) aqueous solution of HeTm and the LAB Counts in the culture cultured for 24 hours was 1.30x10 ⁹ /ml ( 1.30E+09), which was higher than that of commercially available MRS medium for culturing lactic acid bacteria, and showed a higher number of viable lactic acid bacteria than HeTm without lyophilization.

Therefore, it was confirmed that the medium to which dextrose was not added significantly increased the growth of lactic acid bacteria compared to the case in which dextrose was added. This is a different result from the previously applied lactobacillus culture experiment in the extract of white-spotted flower radish extract, in which the number of viable lactic acid bacteria was higher in the medium to which dextrose was added.

Therefore, in the subsequent experiment, without adding dextrose to the hot water extract of Mealworm larvae, 3 kinds of lactic acid bacteria were inoculated and cultured for 24 hours at 30° C. to prepare a fermented product (HeTm LpWpPa) of Mealworm larvae.

Example 4. Microbial stability analysis of fermented mealworm extract

In order to confirm the presence or absence of harmful microorganisms in the fermented product of the brown mealworm larva extract or the brown mealworm larva extract of the present invention, an experiment was performed to determine whether sanitary indicator bacteria were present.

As a sample for confirming the presence or absence of sanitary indicator bacteria, (i) 1% (w / v) aqueous solution of brown mealworm larvae powder (Tm) prepared in Example 1, (ii) Brown mealworm prepared in Example 2 Lactobacillus planterum, Weissella paramesenteroid, and Pediococcus in 1% (w/v) aqueous solution of lyophilisate powder of larvae hot water extract (HeTm) and (iii) 10% (w/v) aqueous solution of the lyophilisate. Three of a 1% (w/v) aqueous solution of a freeze-dried powder of a fermented product (HeTm LpWpPa) fermented under the conditions established in Example 3 (without dextrose, 30° C., 24 hours incubation) by inoculating acid lactisi branches were used.

1mL of the above three samples was inoculated into ^{petrifilm TM} (3M, USA) for measurement of Listeria, general bacteria, yeast and mold, Escherichia coli , E.coli , and inoculated at 37°C. After culturing for 24 hours, it was confirmed whether Listeria, general bacteria, yeast and mold, and E. coli were detected, and the results are shown in FIG. 2 . The general bacteria refer to all microorganisms that grow under aerobic conditions.

As shown in Figure 2, Listeria and general bacteria were detected in the brown mealworm larva powder (Tm) solution, but Listeria in the brown mealworm larvae hot water extract powder (HeTm) solution and the extract culture powder (HeTm LpWpPa) of the brown mealworm larvae (HeTm LpWpPa), Normal bacteria, yeast and mold, and Escherichia coli were not detected, confirming that the microbial stability was increased compared to the raw material, brown mealworm larvae powder.

Example 5. Confirmation of antibacterial effect of hot water extract or fermented product of brown mealworm

Vibrio bacteria ( Listonella anguillarum; Viobrio anguillarum ), Streptococcus parauberis and Streptococcus iniae ) and Listeria monocytogenes An experiment was performed for

Of test organisms to determine the antibacterial effect Vibrio central nine days rareom (Listonella anguillarum, ATCC19264), Streptococcus para Ube-less (Streptococcus parauberis, ATCC 35668), Streptococcus am in (Streptococcus iniae, ATCC 29177), and L. monocytogenes to Ness ( Listeria monocytogenes ) Inoculated in LB medium and pre-cultured for 24 hours at 37 ℃. Then, 20 mL of 1% (w/v) LB agar was dispensed into a Petri dish by 20 mL and coagulated to prepare a plate medium. After application, it was dried for 30 minutes. A paper disk (8 mm, Toyo Roshi Kaicha Ltd, Japan) was placed on the dried medium.

As a sample used to confirm the antibacterial effect, a hot water extract (HeTm) of brown mealworm larvae according to Example 1 and a lactic acid bacteria fermented product (HeTm LpWpPa) of a hot water extract according to Example 3 were used, and the antibacterial effect of each lactic acid bacteria was used. To confirm each, Lactobacillus planterum (KCCM12299P), Weissella paramesenteroid (KCCM12301P), and Pediococcus acidi lactisi (KCCM12472P) as a control were prepared by culturing each in MRS medium.

30uL of each of the five samples was injected into the prepared paper disk, and the diameter of the inhibition clear zone around the paper disk was measured by incubation at 37° C. for 24 hours. 3 shows a photograph of the result of the inhibition ring formation test.

As shown in FIG. 3, in the case of the control L. plantarum, W. paramesenteroides KCCM12301P, and Pediococcus acidilactici KCCM12472P strains, the test strain could not grow around the paper disc due to the antibacterial effect of each strain itself, so a circular inhibitory ring was confirmed. When the HeTm sample was treated, almost no circular inhibition ring was generated around the paper disk. On the other hand, in the case of the lactic acid bacteria fermented product (HeTm LpWpPa) of the present invention, a similar level of inhibition as in the case of treatment with each strain of the control group was generated. Therefore, it was confirmed that the brown mealworm extract obtained the antibacterial effect against the test strains (Vibrio bacteria, Listeria bacteria and Streptococcus) at a level similar to each lactic acid bacteria while going through the fermentation process by three types of lactic acid bacteria.

Vibrio bacteria, streptococci and Listeria bacteria are known as fish pathogens that cause diseases such as vibriosis, streptococcus, and listeriosis in fish. By going through the fermentation step, it was confirmed that it had an excellent antibacterial effect against fish pathogens. Accordingly, it was confirmed that the fermented product of the extract of brown mealworm of the present invention can be used as a substance for preventing, improving, or treating fish diseases.

Example 6. Preparation of fish feed

An experiment was performed to confirm the effect of the lactic acid bacteria fermented product of the brown mealworm extract provided by the present invention on the growth of fish.

First, as the test fish, the fish that reached a weight of 5 to 6 g were used by acclimatizing stone fluke produced in seedlings at the Fisheries Research Institute in Gyeongsangbuk-do to a compound feed (superiority). Using 3 tanks of 100 animals per tank as a control group and 3 tanks of 100 animals per tank as an experimental group, 300 animals were placed in the control group and 300 animals in the experimental group, respectively. During the experiment, clean seawater was constantly supplied to the breeding tank, and the excess was taken out through the drain.

For the feed used in the experiment, a commercially available powdered feed (superior feed) used mainly in fish farms was used as a general feed. The freeze-dried powder of the fermented product (HeTm LpWpPa) was mixed with 10% (w/w) and fed to the experimental group water tank. Feed was fed to 100 animals twice a day, 30 g each. The molded control or experimental feed was stored in a refrigerator and supplied to the test subject flounder. In the experiment, standard body length (from mouth to anterior caudal fin) and body weight were measured 3 and 6 weeks after the start of feed feeding, and 2-phenoxyethanol (2-phenoxyethanol, Sigma, USA) 0.05% (v /v) Anesthetized flounder in aqueous solution. The length and weight of each group were individually measured, one for every rockfish, and the average value of each group was calculated.

Weight gain (WG) and daily growth rate (SGR) were calculated by the methods of Equations 1 and 2 below to measure the growth rate of flounder according to the supply of general feed and experimental feed, and each control and The initial average height and the number of underweight individuals were investigated for rockfish in the experimental group.

[Equation 1]

[Equation 2]

In Equations 1 and 2, the initial fish weight means the average weight of the corresponding rockfish group immediately before the feed feeding experiment (day 0), and the final fish weight is measured at the 3rd or 6th week after starting the feed feeding experiment. It means the average weight of the corresponding stone flounder group.

Measured values of the average length and weight of 300 rockfish according to the type of feed and the corresponding result graphs are shown in Tables 3 and 4 below, respectively.

Metrics Average height (cm) average body weight (g) fry age Day 0 3 weeks 6 weeks Day 0 3 weeks 6 weeks control
(General feed feed) 6.145 ±0.773 7.2 ±1.038 8.263 ±1.198 6.098 ±2.389 11.817 ±5.548 19.714 ±8.796 experimental group
(Experimental feed feeding) 6.092 ±0.704 7.258 ±0.833 8.423 ±1.010 5.815 ±1.977 12.217 ±4.588 20.281 ±7.123

The average length of 300 control group of flounder fry immediately before feed feeding (day 0) was 6.145 ± 0.773 cm, and the standard length of 300 experimental group was 6.092 ± 0.704 cm, indicating that the experimental group was slightly smaller than the control group. After 3 weeks from the start of each feed, the average height of the control (general feed) fry was 7.2±1.038 cm, and that of the experimental group was 7.258±0.833 cm, which was measured similarly between the two groups. After 6 weeks, the control group (general feed) was 8.263±1.198 cm, and the experimental feed was 8.423±1.010 cm. 4).

In terms of body weight, at the start of the experiment (day 0), the weight of the control group of 300 flounder fry was 6.098±2.389 g, and the standard weight of the 300 rats of the experimental group was 5.815±1.977 g, which was slightly lower in the experimental group. Three weeks after the start of each feed, the weight of the control group (normal feed) was 11.817±5.548 g, and the weight of the experimental group was 12.217±4.588 g. appeared to be heavier on average. After 6 weeks of feeding, the average body weight of the control group was 19.714±8.796 g and that of the experimental group was 20.281±7.123 g. was heavier (Table 3, Figure 4).

The average weight of the fry at the start of the rockfish experiment (day 0) was 6.098±2.389 g for the control group and 5.815±1.977 g for the experimental group. , subjects with underweight at 3 weeks and 6 weeks were investigated (Table 3). Specifically, when the measured weight was less than 6.0 g or when the body length was less than 6.0 cm, it was judged as underweight. Table 4 below shows the number of underweight individuals and their ratio over time.

Feeding period Day 0 3 weeks Week 6 number of underweight individuals
(number of animals) control 129 27 17 experimental group 137 9 3 Percentage of underweight individuals (%) control 43 9 5.7 experimental group 45.7 3 One

Just before feeding (Day 0), the number of underweight individuals in the control tank was 129, and the number of underweight individuals in the experimental group was 137. After 3 weeks, 27 underweight in the control tank and 9 in the experimental group. The number of underweight individuals was 17 in the control group and 3 underweight individuals in the experimental group when 6 weeks had elapsed from the start of the experiment.

When the number of underweight individuals was calculated as a percentage (%) with respect to the total number of experimental animals, it was found to be 43%, 9%, and 5.7% at the 0th day, 3rd week, and 6th week of the control group, respectively. , in the experimental group, the number of underweight individuals decreased from 45.5% on day 0 to 3% and 1% at 3 and 6 weeks, respectively.

Therefore, it was confirmed that when the lactic acid bacteria fermented product of the brown mealworm larvae extract was added to the feed, the growth of stonefish fry was evenly leveled.

As for the weight gain rate, the control group, which was fed general feed, showed a weight gain of 93.77% at the 3rd week after the start of feeding, and in the experimental group, it was 110.08% at the 3rd week after the start of feeding. Even after that, the weight gain rate in the control group was 223.26%, and the weight gain rate in the experimental group was 248.76%, which was 25%p higher than that in the control group (FIG. 5).

The daily growth rate (%/day) was 3.14%/day for the control group and 3.52%/day for the experimental group from day 0 to week 3, and the experimental group grew about 0.4%p more per day than the control group, and from week 3 to week 6 The daily growth rate was 2.42%/day for the control group and 2.43%/day for the experimental group (FIG. 6). The average daily growth rate for 6 weeks (weeks 0 to 6) during the experiment was 2.79%/day in the control group and 2.98%/day in the experimental group, and the difference in the daily growth rate between the two groups was about 0.2%p.

Example 7. Confirmation of heavy metal content in the fermented product of Mealworm larvae extract

In order to confirm the effect of reducing the concentration of heavy metals in the final product according to the fermentation of lactic acid bacteria, an experiment was conducted to confirm the effect of reducing heavy metals in the freeze-dried powder of the lactic acid bacteria fermented product (HeTm LpWpPa) of Mealworm larvae powder (Tm) and Brown mealworm larvae extract did.

Tm powder and HeTm LpWpPa freeze-dried powder were each dissolved in water to prepare a 1% (w/v) solution, and then each was injected into an inductively coupled plasma emission spectrometer, luminescence intensity for two elements, arsenic and cadmium. was measured and the concentration of the element of interest was quantified by substituting it into the regression equation of the calibration curve. When the concentration of the element of interest in the sample solution was quantified in the inductively coupled plasma, the concentration of the element of interest in the sample was calculated according to Equation 3 below. In Equation 3, the final amount means the total amount of the sample solution used for the spectroscopic test.

[Equation 3]

Arsenic (As) and cadmium (Cd) were dissolved in water and prepared as a 1% (w/w) solution according to the Food Standards Standards Test Method, treated with nitric acid and inductively coupled plasma emission spectrometry (Inductiviely Coupled Plasma Atomic Emission Spectrometry) , ICP-MS, KHSI-A-158) was used for component analysis (Korea Polymer Testing Research Institute, KOPTRI). When the concentration of the element of interest in the sample solution was quantified in the inductively coupled plasma, the concentration of the element of interest in the sample was calculated according to Equation 4 below.

[Equation 4]

For mercury, each sample was analyzed with a Mercyry analyzer Hydra-C (KHSI-A-060). When the concentration of the element of interest in the sample solution was quantified in the analyzer, the concentration of the element of interest in the sample was calculated according to Equation 5 below.

[Equation 5]

Concentration of the element of interest in the sample (ng/g) = Detected amount (ng) / Sampled amount (g)

In the case of chromium, a certain amount of the sample is placed in a microwave (Microwave MARS CEM, KHSI-A-063), decomposed by treatment with nitric acid, etc., transferred to a flask, and distilled water is added to prepare a solution. Spectrometry, ICP-MS, agilent 7700, KHSI-A-059) was used for component analysis (Korea Polymer Testing Research Institute, KOPTRI).

Table 5 shows the results of analysis of four heavy metals obtained according to the above method.

item Tm HeTm LpWpPa One chrome
(Chromium,ug/100g) 0.0226 0.0002 2 Cadmium (mg/kg) 0.0064 0.0057 3 Arsenic (mg/kg) 0.094 0.083 4 Mercury (mg/kg) 0.0008 0

In Table 5, in the Tm sample, chromium, cadmium, arsenic, and mercury were measured to be 0.0226 mg/kg, 0.0064 mg/kg, 0.094 mg/kg, and 0.0008 mg/kg, respectively, and the edible content presented in the Food and Drug Administration of the Ministry of Food and Drug Safety. All of the values were lower than the insect heavy metal standard values of 0.05 mg/kg cadmium, 0.5 mg/kg mercury, and 0.1 mg/kg arsenic. In the HeTm LpWpPa sample, chromium, cadmium, arsenic, and mercury were 0.0002 mg/kg, 0.0057 mg/kg, 0.083 mg/kg, and 0 mg/kg, respectively. decreased, and no mercury was detected.

Therefore, it was confirmed that the lactic acid bacteria fermented product of the brown mealworm larvae extract of the present invention has an effect of removing heavy metal components harmful to the living body due to the lactic acid bacteria fermentation process.

Korea Microorganism Conservation Center KCCM12299P 20180810 Korea Microorganism Conservation Center KCCM12301P 20190810 Korea Microorganism Conservation Center KCCM12472P 20190329

<110> Marine Industry Research institute for East sea rim (MIRE) <120> Feed additive comprising fermented mealworm and feed composition using the same <130> DPP20193763KR <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F_primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R_primer <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 1472 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum KCCM12299P 16SrRNA <400> 3 gaagcagtgg cgggtgctat acatgcagtc gaacgaactc tggtattgat tggtgcttgc 60 atcatgattt acatttgagt gagtggcgaa ctggtgagta acacgtggga aacctgccca 120 gaagcggggg ataacacctg gaaacagatg ctaataccgc ataacaactt ggaccgcatg 180 gtccgagctt gaaagatggc ttcggctatc acttttggat ggtcccgcgg cgtattagct 240 agatggtggg gtaacggctc accatggcaa tgatacgtag ccgacctgag agggtaatcg 300 gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360 cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaaaggt ttcggctcgt 420 aaaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt 480 taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta cgtggcaagc 540 gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa 600 gccttcggct caaccgaaga agtgcatcgg aaactgggaa acttgagtgc agaagaggac 660 agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa 720 ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag tatgggtagc aaacaggatt 780 agataccctg gtagtccata ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc 840 ttcagtgctg cagctaacgc attaagcatt ccgcctgggg agtacggccg caaggctgaa 900 actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct 960 acgcgaagaa ccttaccagg tcttgacata ctatgcaaat ctaagagatt agacgttccc 1020 ttcggggaca tggatacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caacccttat tatcagttgc cagcattaag ttgggcactc 1140 tggtgagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200 cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagttgc gaactcgcga 1260 gagtaagcta atctcttaaa gccattctca gttcggattg taggctgcaa ctcgcctaca 1320 tgaagtcgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc 1380 ttgtacacac cgcccgtcac accatgagag tttgtaacac ccaaagtcgg tggggtaacc 1440 ttttaggaac cagccgctaa gtgacagaat gg 1472 <210> 4 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> Weissella paramesenteroides KCCM12301P 16SrRNA <400> 4 cgctggcggc gtgcctaata catgcaagtc gaacgctttg tctttaattg atatgacgag 60 cttgctctga tgtgatttta tctgacaaag agtggcgaac gggtgagtaa cacgtgggta 120 acctacctct tagcagggga taacatttgg aaacaagtgc taataccgta taataccaac 180 aaccgcatgg ttgttggttg aaagatggtt ctgctatcac taagagatgg acccgcggtg 240 cattagctag ttggtaaggt aacggcttac caaggcaatg atgcatagcc gagttgagag 300 actgatcggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatgggcgc aagcctgatg gagcaacgcc gcgtgtgtga tgaagggttt 420 cggctcgtaa aacactgtta taagagaaga acggcactga gagtaactgt tcagtgtgtg 480 acggtatctt accagaaagg aacggctaaa tacgtgccag cagccgcggt aatacgtatg 540 ttccaagcgt tatccggatt tattgggcgt aaagcgagcg cagacggtta tttaagtctg 600 aagtgaaagc cctcagctca actgaggaat ggctttggaa actggatgac ttgagtgcag 660 tagaggaaag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctttctg gactgtaact gacgttgagg ctcgaaagtg tgggtagcaa 780 acaggattag ataccctggt agtccacacc gtaaacgatg agtgctagat gttcgagggt 840 ttccgccctt gagtgtcgca gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc ttgctaatcc tagaaatagg 1020 acgttccctt cggggacaag gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttatta ttagttgcca gcattcagtt 1140 gggcactcta gtgagactgc cggtgacaac cggaggaggg ggggggaatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcatataca acgagtcgcc 1260 aacccgcgag ggtgcgctaa tctcttaaag tatgtctcaa tttcggaatt gtaggctgca 1320 actcgcctac atgaagtcgg aatcgct 1347 <210> 5 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> Pediococcus acidilactici KCCM12472P 16SrRNA <400> 5 cgctggcggc gtgcctaata catgcaagtc gaacgaactt ccgttaattg attatgacgt 60 gcttgcactg aatgagattt taacacgaag tgagtggcgg acgggtgagt aacacgtggg 120 taacctgccc agaagcaggg gataacacct ggaaacagat gctaataccg tataacagag 180 aaaaccgcct ggttttcttt taaaagatgg ctctgctatc acttctggat ggacccgcgg 240 cgcattagct agttggtgag gtaacggctc accaaggcga tgatgcgtag ccgacctgag 300 agggtaatcg gccacattgg gactgagaca cggcccagac tcctacggga ggcagcagta 360 gggaatcttc cacaatggac gcaagtctga tggagcaacg ccgcgtgagt gaagaagggt 420 ttcggctcgt aaagctctgt tgttaaagaa gaacgtgggt gagagtaact gttcacccag 480 tgacggtatt taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540 ggtggcaagc gttatccgga tttattgggc gtaaagcgag cgcaggcggt cttttaagtc 600 taatgtgaaa gccttcggct caaccgaaga agtgcattgg aaactgggag acttgagtgc 660 agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac 720 cagtggcgaa ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag catgggtagc 780 gaacaggatt agataccctg gtagtccatg ccgtaaacga tgattactaa gtgttggagg 840 gtttccgccc ttcagtgctg cagctaacgc attaagtaat ccgcctgggg agtacgaccg 900 caaggttgaa actcaaaaga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960 attcgaagct acgcgaagaa ccttaccagg tcttgacatc ttctgccaac ctaagagatt 1020 aggcgttccc ttcggggaca gaatgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1080 gagatgttgg gttaagtccc gcaacgagcg caacccttat tactagttgc cagcattcag 1140 ttgggcactc tagtgagact gccggtgaca aaccggagga aggtggggga cgacgtcaaa 1200 tcatcatgcc ccttatgacc tgggctacac acgtgctaca aatggatggt acaacgagtc 1260 gcgaaaccgc gaggtttagc taatctctta aaaccattct cagttcggga ctgtaggctg 1320 caactcgcct acacgaaagt cggaatcgct 1350 <210> 6 <211> 1470 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum MK311261.1 16SrRNA <400> 6 cgcagggcgg gtgctataca tgcagtcgaa cgaactctgg tattgattgg tgcttgcatc 60 atgatttaca tttgagtgag tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa 120 gcgggggata acacctggaa acagatgcta ataccgcata acaacttgga ccgcatggtc 180 cgagtttgaa agatggcttc ggctatcact tttggatggt cccgcggcgt attagctaga 240 tggtggggta acggctcacc atggcaatga tacgtagccg acctgagagg gtaatcggcc 300 acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac 360 aatggacgaa agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa 420 actctgttgt taaagaagaa catatctgag agtaactgtt caggtattga cggtatttaa 480 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggattt attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc 600 ttcggctcaa ccgaagaagt gcatcggaaa ctgggaaact tgagtgcaga agaggacagt 660 ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc 720 ggctgtctgg tctgtaactg acgctgaggc tcgaaagtat gggtagcaaa caggattaga 780 taccctggta gtccataccg taaacgatga atgctaagtg ttggagggtt tccgcccttc 840 agtgctgcag ctaacgcatt aagcattccg cctggggagt acggccgcaa ggctgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg 960 cgaagaacct taccaggtct tgacatacta tgcaaatcta agagattaga cgttcccttc 1020 ggggacatgg atacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttattat cagttgccag cattaagttg ggcactctgg 1140 tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt 1200 atgacctggg ctacacacgt gctacaatgg atggtacaac gagttgcgaa ctcgcgagag 1260 taagctaatc tcttaaagcc attctcagtt cggattgtag gctgcaactc gcctacatga 1320 agtcggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc atgagagttt gtaacaccca aagtcggtgg ggtaaccttt 1440 taggaaccag ccgcctaagt gacagaatgg 1470 <210> 7 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> Weissella paramesenteroides LC094432.1 16SrRNA <400> 7 cgctggcggc gtgcctaata catgcaagtc gaacgctttg tctttaattg atatgacgag 60 cttgctctga tgtgatttta tctgacaaag agtggcgaac gggtgagtaa cacgtgggta 120 acctacctct tagcagggga taacatttgg aaacaagtgc taataccgta taataccaac 180 aaccgcatgg ttgttggttg aaagatggtt ctgctatcac taagagatgg acccgcggtg 240 cattagctag ttggtaaggt aacggcttac caaggcaatg atgcatagcc gagttgagag 300 actgatcggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatgggcgc aagcctgatg gagcaacgcc gcgtgtgtga tgaagggttt 420 cggctcgtaa aacactgtta taagagaaga acggcactga gagtaactgt tcagtgtgtg 480 acggtatctt accagaaagg aacggctaaa tacgtgccag cagccgcggt aatacgtatg 540 ttccaagcgt tatccggatt tattgggcgt aaagcgagcg cagacggtta tttaagtctg 600 aagtgaaagc cctcagctca actgaggaat ggctttggaa actggatgac ttgagtgcag 660 tagaggaaag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctttctg gactgtaact gacgttgagg ctcgaaagtg tgggtagcaa 780 acaggattag ataccctggt agtccacacc gtaaacgatg agtgctagat gttcgagggt 840 ttccgccctt gagtgtcgca gctaacgcat taagcactcc gcctggggag tacgaccgca 900 aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat 960 tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc ttgctaatcc tagaaatagg 1020 acgttccctt cggggacaag gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1080 gatgttgggt taagtcccgc aacgagcgca acccttatta ttagttgcca gcattcagtt 1140 gggcactcta gtgagactgc cggtgacaac cggaggaggg ggggggaatg acgtcaaatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcatataca acgagtcgcc 1260 aacccgcgag ggtgcgctaa tctcttaaag tatgtctcaa tttcggaatt gtaggctgca 1320 actcgcctac atgaagtcgg aatcgct 1347 <210> 8 <211> 1516 <212> DNA <213> Artificial Sequence <220> <223> Pediococcus acidilactici LC311071.1 16SrRNA <400> 8 gctcaggatg aacgctggcg gcgtgcctaa tacatgcaag tcgaacgaac ttccgttaat 60 tgattatgac gtgcttgcac tgaatgagat tttaacacga agtgagtggc ggacgggtga 120 gtaacacgtg ggtaacctgc ccagaagcag gggataacac ctggaaacag atgctaatac 180 cgtataacag agaaaaccgc ctggttttct tttaaaagat ggctctgcta tcacttctgg 240 atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc gatgatgcgt 300 agccgacctg agagggtaat cggccacatt gggactgaga cacggcccag actcctacgg 360 gaggcagcag tagggaatct tccacaatgg acgcaagtct gatggagcaa cgccgcgtga 420 gtgaagaagg gtttcggctc gtaaagctct gttgttaaag aagaacgtgg gtgagagtaa 480 ctgttcaccc agtgacggta tttaaccaga aagccacggc taactacgtg ccagcagccg 540 cggtaatacg taggtggcaa gcgttatccg gatttattgg gcgtaaagcg agcgcaggcg 600 gtcttttaag tctaatgtga aagccttcgg ctcaaccgaa gaagtgcatt ggaaactggg 660 agacttgagt gcagaagagg acagtggaac tccatgtgta gcggtgaaat gcgtagatat 720 atggaagaac accagtggcg aaggcggctg tctggtctgt aactgacgct gaggctcgaa 780 agcatgggta gcgaacagga tagataccc tggtagtcca tgccgtaaac gatgattact 840 aagtgttgga gggtttccgc ccttcagtgc tgcagctaac gcattaagta atccgcctgg 900 gggatacgac cgcaaggttg aaactcaaaa gaattgacgg gggcccgcac aagcggtgga 960 gcatgtggtt taattcgaag ctacgcgaag aaccttacca ggtcttgaca tcttctgcca 1020 acctaagaga ttaggcgttc ccttcgggga cagaatgaca ggtggtgcat ggttgtcgtc 1080 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt attactagtt 1140 gccagcattc agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg 1200 acgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt 1260 acaacgagtc gcgaaaccgc gaggtttagc taatctctta aaaccattct cagttcggac 1320 tgtaggctgc aactcgccta cacgaagtcg gaatcgctag taatcgcgga tcagcatgcc 1380 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatgag agtttgtaac 1440 acccaaagcc ggtggggtaa ccttttagga gctagccgtc taaggtggga cagatgatta 1500 gggtgaagtc gtaaca 1516

Claims (13)

Lactobacillus planterum of accession number KCCM12299P, accession number KCCM12301P Weissella paramegentoid and accession number KCCM12472P Pediococcus acidi lactisi All three types of lactic acid bacteria were inoculated and fermented into the brown mealworm extract and fermented lactic acid bacteria,
A feed additive having antibacterial activity against one or more pathogens selected from the group consisting of Vibrio, Listeria and Streptococcus. The feed additive according to claim 1, wherein the brown mealworm extract is a solvent extract obtained by extracting at least one selected from the group consisting of alcohol and water having 1 (C1) to 3 (C3) carbon atoms as an extraction solvent. The feed additive according to claim 1, wherein the Melancholy Mealtimes brown extract is an extract of Mealworm larvae. According to claim 1, wherein the brown mealworm extract is extracted by hot water extraction, feed additive. delete The feed additive according to claim 1, wherein the lactic acid bacteria fermented product content of the brown mealworm extract is 1% by weight or more. The feed additive according to claim 1, wherein the lactic acid bacteria fermented product of the Mealworm Mealworm extract is an animal growth promoter. The feed additive according to claim 1, wherein the feed is a fish feed. The feed additive according to claim 8, wherein the fish is at least one selected from the group consisting of flounder, halibut, rockfish, rainbow trout, yellowtail, eel, and carp. According to claim 1, wherein the animal fed the feed with the feed additive added to the fish growth rate calculated by the method of [Equation 1] below than the animal fed the feed that does not contain the lactic acid bacteria fermented product of the brown mealworm extract. Feed additive having this 1.05 times to 3 times higher growth rate:
[Equation 1]

11. The method of claim 10, wherein the animal fed the feed additive is added to the fish growth rate calculated by the method of [Equation 1] than the animal fed a feed that does not contain the lactic acid bacteria fermented product of the brown mealworm extract. A feed additive having a growth rate that is 1.1 times to 1.5 times higher. Lactobacillus planterum of accession number KCCM12299P, accession number KCCM12301P Weissella paramegentoid and accession number KCCM12472P Pediococcus acidi lactisi containing all three types of lactic acid bacteria inoculated and fermented in the brown mealworm extract, A composition for preventing or treating a fish disease, wherein the fish disease is vibriosis, listeriosis or streptococcosis. The composition for preventing or treating fish diseases according to claim 12, wherein the composition is administered by being mixed with feed.

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