JP7395344B2 - Bacterial count inhibitor for Frischella bacteria - Google Patents

Description

The present invention provides an agent for suppressing the number of Frischella bacteria and a feed for suppressing the number of bacteria, which contains 1-kestose as an active ingredient, an agent for suppressing DNA damage in honeybees, and a feed for suppressing the number of bacteria, and a food for suppressing DNA damage in honey bees using the same. Regarding breeding methods.

Bees such as honeybees and bumblebees play an extremely important role in pollinating crops such as vegetables and fruits. Bees also produce honey, beeswax, propolis, and royal jelly that are useful for human life. For these reasons, honey bees have traditionally been bred by humans and used as pollinators for agricultural crops and in the production of honey and the like.

On the other hand, Frischella Perrara (hereinafter abbreviated as Perrara), a bacterium belonging to the genus Frischella, is one of the main bacterial species that inhabit the intestines of honey bees (Non-Patent Document 1). It has been reported that Perara induces megalocytosis (enlargement of the cytoplasm and cell nucleus without mitosis) in human-derived cultured cells, resulting in DNA damage (Non-Patent Document 2). Furthermore, it has been reported that there is a negative correlation between the growth rate of honeybees and the amount of bacteria in the intestines of Perala (Non-Patent Document 3). Based on these facts, it can be said that Perara is an intestinal bacterium that has harmful effects on the host.

Powell J.E., et al., Appl Environ Microbiol, Volume 80, Issue 23, Pages 7378-7387, 2014. Engel P, et al., Appl Environ Microbiol, Volume 81, Issue 4, Pages 1502-1512, 2015 Maes P. W., et al, Moleclar ecology, Volume 25, Issue 21, Pages 5439-5450, 2016.

Therefore, the present inventors believe that if the number of Frischella bacteria in the intestine can be suppressed, the harmful effects of Frischella bacteria on the host (e.g., induction of megalocytosis, DNA damage, growth inhibition, etc.) We thought that this could contribute to maintaining, improving, or promoting the host's health condition by suppressing the

In particular, when the host is a beneficial insect such as a bee, by maintaining, improving or promoting its health condition, it is possible to improve the productivity of beneficial insect products such as honey and agricultural products through pollination and pest control. I thought it could be done.

The present invention has been made to solve the above problems, and provides an agent and feed for suppressing the number of bacteria of the genus Frischella, an agent and feed for suppressing DNA damage, and a method for breeding honey bees using the same. The purpose is to provide.

As a result of intensive research, the present inventors discovered that 1-kestose can suppress the number of bacteria of the genus Frischella. Therefore, based on this knowledge, the following inventions were completed.

(1) The agent for suppressing the number of Frischella bacteria according to the present invention contains 1-kestose as an active ingredient.

(2) The agent for suppressing the number of bacteria of the genus Frischella according to the present invention may be used to suppress the number of bacteria of the genus Frischella in bees.

(3) The inhibitor of DNA damage in honeybees according to the present invention contains 1-kestose as an active ingredient.

(4) The feed for suppressing the number of Frischella bacteria according to the present invention contains 1-kestose as an active ingredient.

(5) The feed for suppressing DNA damage in bees according to the present invention contains 1-kestose as an active ingredient.

(6) The method for breeding honeybees according to the present invention includes the step of causing the bees to ingest the agent for suppressing the number of Frischella bacteria or the feed for suppressing the number of bacteria, or the agent for suppressing DNA damage in honeybees or the feed for suppressing the number of bacteria according to the present invention. has.

According to the present invention, the number of bacteria of the genus Frischella in the living body or intestine of a host can be effectively suppressed. Further, according to the present invention, by suppressing the number of Frischella bacteria, harmful effects of the bacteria on the host (e.g., induction of megalocytosis, DNA damage, growth inhibition, etc.) are suppressed, It can contribute to the maintenance, improvement, or promotion of the host's health condition. In particular, when the host is a beneficial insect such as a bee, by maintaining, improving or promoting its health condition, it is possible to improve the productivity of beneficial insect products such as honey and agricultural products through pollination and pest control. can.

Furthermore, 1-kestose, which is the active ingredient of the present invention, is a type of oligosaccharide that is also contained in vegetables and grains such as onions, garlic, barley, and rye, and is a substance that has been eaten since ancient times. , No toxicity was observed in any of the mutagenicity tests, acute toxicity tests, subchronic toxicity tests, and chronic toxicity tests, so safety is extremely high (Food and Development, Vol. 49, No. 12, 9 pages, 2014). Therefore, according to the present invention, it is possible to suppress the number of Frischella bacteria in a living body or intestine, or to suppress DNA damage in honey bees, without worrying about safety or side effects.

In addition, 1-kestose is highly water-soluble and has good sweetness similar to sugar, so it can be easily ingested on a daily basis as it is or as an additive or sweetener, and it can also be used in various feeds. It can be easily incorporated into foods, medicines, etc. Therefore, according to the present invention, there is provided an agent for suppressing the number of bacteria of the genus Frischella that is highly safe and can be easily ingested on a daily basis as it is or by easily blending it into various feeds, foods, medicines, etc. Feed for suppressing the number of bacteria, as well as an inhibitor and feed for suppressing DNA damage in honeybees can be obtained.

The photo on the left shows a bee on the 3rd day after emergence (within 7 days after emergence), and the photo on the right shows a honeybee on the 11th day after emergence (more than 7 days after emergence). The white arrow in the later photo points to the hair on the chest. 1- Copy of Frischella perarra rpoE in the intestines of bees fed a sugar solution containing kestose (test group) and bees fed commercial beekeeping sugar (control group) It is a bar graph showing percentages of numbers.

The present invention will be explained in detail below. The present invention provides an agent for suppressing the number of bacteria of the genus Frischella, a feed for suppressing the number of bacteria of the genus Frischella, an agent for suppressing DNA damage in bees, and a feed for suppressing DNA damage in bees (hereinafter, all or any of these). (This may be referred to as "the agent/feed of the present invention.")

In the present invention, "bacteria belonging to the genus Frischella" refers to bacteria belonging to the genus Frischella. Examples of such bacteria include Frischella Perrara.

The agent/feed of the present invention contains 1-kestose as an active ingredient. 1-kestose is a trisaccharide oligosaccharide consisting of one molecule of glucose and two molecules of fructose. 1-kestose can be produced by carrying out an enzymatic reaction using an enzyme as disclosed in JP-A-58-201980 using sucrose as a substrate. Specifically, β-fructofuranosidase is first added to a sucrose solution, and the solution is allowed to stand at 37° C. to 50° C. for about 20 hours to carry out an enzyme reaction, thereby obtaining an enzyme reaction solution. Since this enzyme reaction solution is a sugar solution containing a considerable amount of 1-kestose, it can be used as it is as the agent/feed according to the present invention.

On the other hand, when purifying 1-kestose, 1-kestose and other sugars (glucose, fructose, Sucrose, oligosaccharides of 4 or more sugars) are separated and purified to obtain a highly pure 1-kestose solution. Subsequently, this high-purity 1-kestose solution is concentrated and then crystallized by a crystallization method as disclosed in Japanese Patent Publication No. 6-70075, whereby 1-kestose can be obtained as a crystal.

Furthermore, since 1-kestose is contained in commercially available fructooligosaccharide, it may be used as it is or after separating and purifying 1-kestose from fructooligosaccharide by the method described above. In addition, in the present invention, the "purity" of a specific oligosaccharide refers to the mass % of the oligosaccharide when the total amount of sugar is 100%.

Examples of the form of the agent for suppressing the number of Frischella bacteria or the agent for suppressing DNA damage in honeybees of the present invention include pharmaceuticals, feed additives, and supplements. The form of this drug is not particularly limited, and may be, for example, a solid form such as a powder or lump, a paste form, or a liquid form. In addition, other components may be added to this agent as long as they do not impair the characteristics of the present invention. Examples of such additives include excipients, stabilizers, sugars other than 1-kestose, and attractants depending on the animal species.

The form of the feed for suppressing the number of Frischella bacteria or the feed for suppressing DNA damage in bees of the present invention is also not particularly limited, and may be in any form, for example, solid form such as powder or lumps, paste form, or liquid form. It may be. In addition, other ingredients (for example, ingredients contained in normal feed) may be added to the feed as long as the characteristics of the present invention are not impaired. Specifically, feed for bees includes sugars other than 1-kestose, vitamins, minerals, amino acids, excipients, stabilizers, soybean powder, casein, brewer's yeast, commercially available pollen substitutes, pollen, and bees. Examples include attractants (Cyrus chinensis and its components, Nasanoff's gland pheromones, honey, etc.).

The agent/feed of the present invention can be used by being orally ingested by animals. Since Frischella bacteria mainly live in the intestines, the active ingredient 1-kestose is added to enteral nutrition and this is administered by enteral nutrition via a tube inserted into the digestive tract. May be used in any method.

The agent/feed of the present invention can be suitably used for bees, especially among animals. That is, the present invention also provides a method for breeding honeybees using the agent/feed of the present invention. This breeding method includes the step of causing bees to ingest the agent/feed of the present invention.

Here, the term "bee" refers to insects belonging to the order Hymen optera and superfamily Apoidea, which have the habit of visiting flowers and collecting nectar and pollen. Specific examples of bees include honey bees (bees belonging to the genus Apis), bumblebees (bees belonging to the genus Bombus), carpenter bees (bees belonging to the subfamily Xylocopinae), and carpenter bees (bees belonging to the subfamily Xylocopinae). Examples include bees belonging to the family Meliponini (bees belonging to the genus Meliponini), and bees belonging to the genus Osmia.

The agent/feed of the present invention can be ingested by bees in the same manner as conventional bee feed. That is, the agent/feed of the present invention may be placed as it is or added to other feed or water, placed in a suitable container such as a feeder or waterer, and placed in or near the hive. When using the agent/feed of the present invention or feed containing the same in liquid form, use containers with shallow bottoms and avoid containers with smooth surfaces such as glass or resin to prevent bees from drowning. Wooden is better. It is also preferable to put something that can serve as a foothold, such as disposable chopsticks, twigs, or rope, into the container.

The intake amount (dose) of 1-kestose, which is the active ingredient of the present invention, can be appropriately set depending on the species, age, season, etc. of the animal. For example, for honey bees, according to the examples described below, the dose may be 0.1 to 10 g/1000 bees, 0.5 to 8 g/1000 bees, or 1 to 5 g/1000 bees per day. Can be done.

In the present invention, "suppressing the number of Frischella bacteria" refers to suppressing an increase in the number of cells of the bacteria in any cell or tissue/organ of a living body.

For example, the number of Frischella bacteria in the intestines is thought to be correlated with the number of bacteria in the intestinal contents or feces (hereinafter referred to as "intestinal contents, etc."). By measuring the number of bacteria in the intestines, it can be confirmed whether the number of Frischella bacteria has been suppressed in the intestines. Specifically, for example, intestinal contents, etc. before and after ingesting the agent/feed of the present invention, or intestinal contents, etc. from an individual (group) that has ingested it and from an individual (group) that has not ingested it. The number of copies of rpoE (a gene encoding the sigma E factor of RNA polymerase) is measured by performing real-time PCR using primers specific to Frischella bacteria as a sample. Since there is a correlation between the copy number of the rpoE gene of the bacterium and the number of bacteria, the rpoE copy number can be used as an index of the number of bacteria. Therefore, as a result of measuring the rpoE copy number of the bacteria, if the rpoE copy number in the sample after ingestion is smaller than before ingestion, or if the rpoE copy number in the sample from the ingested individual (group) is an individual that has not ingested it. If it is smaller than (group), it can be determined that the number of Frischella bacteria has been suppressed by the agent/feed of the present invention.

Primers specific to rpoE of Frischella bacteria can be designed based on known base sequences. For example, it can be designed based on the partial sequences of Perara rpoE (SEQ ID NO: 4, SEQ ID NO: 7) shown in Examples described later. In addition, the whole genome sequence of the type strain of Frischella perrara (Frischella perrara PEB0191 (DSM 104328, ATCC BAA 2450)) is available from GenBank accession number QGTI01000001.1. The sequence (SEQ ID NO: 1) is also disclosed, and based on this, primers specific to the rpoE gene of Frischella bacteria can be designed.

As mentioned above, it has been reported that DNA damage occurred in human-derived cultured cells infected with Perara (Non-Patent Document 2). Therefore, if the number of Frischella bacteria in the intestines of bees can be suppressed, it is thought that DNA damage that causes tumor formation, inflammation, etc. in the intestines of bees can be suppressed. Therefore, the agent/feed of the present invention can be used to suppress DNA damage in honey bees.

Whether or not DNA damage has been suppressed can be confirmed by known DNA damage detection methods (comet test, UDS test, Rec assay, umu test, DNA adduct detection, γ-H2AX focus, GreenScreenHC, etc.). For example, the phosphorylated H2AX The amount of (γ-H2AX) is measured. γ-H2AX is a sensitive marker for DNA damage because it is generated rapidly when DNA double strands are cleaved, and kits that can easily detect γ-H2AX using a secondary antibody method are commercially available. As a result of measurement, if the amount of γ-H2AX in the sample after ingestion is smaller than before ingestion, or if the amount of γ-H2AX in the sample from the ingested individual (group) is smaller than that of the individual (group) that did not ingest it. It can be concluded that DNA damage was suppressed by the agent/feed of the present invention.

Hereinafter, the present invention will be explained based on each example. Note that the technical scope of the present invention is not limited to the features shown by these examples. In this example, "%" means mass percentage (mass %) unless otherwise specified. Moreover, the solid content concentration represents the content of soluble solid content contained in the sample. The solid content concentration is measured with a sugar refractometer and expressed as a Brix value in the unit of "°Bx".

<Example 1> Production of 1-kestose Sucrose was used as a substrate according to the method described in Japanese Patent Publication No. 59-53834 (pages 2 to 3) and Japanese Patent Application Publication No. 2010-273580 (paragraph [0096]). An enzymatic reaction using fructosyltransferase was performed to produce 1-kestose. Specifically, first, Aspergillus niger strain ACE-2-1 (deposit number: FERM P-5886) was incubated with an enzyme production medium (5% sucrose, 0.7% malt extract, 1% polypeptone, 0.5% carboxylic acid). After inoculating the cells into methylcellulose (0.3% NaCl) and culturing them at 28°C for 3 days, the cells were disrupted by ultrasonication to prepare a crude enzyme solution. The crude enzyme solution was added to a 45% sucrose aqueous solution (pH 7.5) at a rate of 2.5 units per 1 g of sucrose, and the mixture was reacted at 40° C. for 24 hours to obtain an enzyme reaction solution. The enzyme reaction solution was heated at 100° C. for 10 minutes to stop the enzyme reaction, and then filtered to collect the filtrate. The filtrate was decolorized using activated carbon in a conventional manner and further desalted using an ion exchange resin to obtain a sugar solution containing 1-kestose (test sample).

<Example 2> Sugar Composition Commercially available beekeeping sugar (solid product: Japan Beekeeping Association) was prepared and used as a comparison sample (control sample). The test sample and the control sample were subjected to high performance liquid chromatography (HPLC) under the following conditions to confirm the sugar composition (types of monosaccharides and oligosaccharides and their content ratios). The content ratio of each sugar was calculated as a percentage as the ratio of the area of each peak to the sum of the areas of all detected peaks. That is, the value calculated thereby indicates the mass % (purity) of each sugar when the total amount of sugar contained in the sample is taken as 100%. The results are shown in Table 1. In Table 1, "-" indicates that it is below the detection limit (0.1% or less).
《HPLC conditions》
Column: Shodex SUGAR KS-802 HQ (8.0mm ID x 300mm) 2 columns Eluent: High purity water Flow rate: 1.0mL/min Column temperature: 50℃
Injection volume: 200μL
Detection: Differential refractive index detector Shodex RI

As shown in Table 1, both the test and control samples contained relatively large amounts of sucrose and small amounts of glucose and fructose. On the other hand, the test sample contained about 20% of 1-kestose, whereas the 1-kestose content of the control sample was below the detection limit. This result revealed that the test sample contained 1-kestose as a unique component.

<Example 3> Effect of suppressing the number of bacteria (1) Breeding of honey bees Honey bees were reared in a plastic greenhouse to exclude external factors as much as possible. The solid content concentration of the test sample was 70°Bx. The control sample was set at 50°Bx, which is a commonly used concentration (since the control sample contains a large amount of sucrose, setting it at 70°Bx would result in high viscosity, easy precipitation of crystals, and poor operability).

Eight plastic greenhouses and eight beehives (numbers 1 to 8) were prepared. One bee hive was installed for each greenhouse, and approximately 8,000 bees/box were placed in each empty hive frame. Half of the beehives (bee hives Nos. 1 to 4) were used as a test group and fed with a test sample, and the remaining half (bee hives No. 5 to 8) were used as a control group and fed with a control sample. Feeding was carried out every two weeks by placing 3000 mL of sample into the beehive feeder. In order to eliminate the effects of confusion on bees due to environmental changes, the first 4 weeks were used as an environmental adaptation period, and the first day after 4 weeks was designated as "day 0", and the bees were kept for 8 weeks. On day 0, it was confirmed that there were no major differences in either the environment inside the greenhouse or the conditions inside the beehive (number of bees, amount of activity, etc.).

Generally, honey bees within seven days of emergence have long and large amounts of hair on their chests, as shown on the left side of Figure 1. On the other hand, bees that are more than 7 days old after emergence have shorter chest hair and less hair, as shown on the right side of Figure 1. Based on this, on day 0 of the test period, those with long chest hair and a large amount of hair were judged to be young bees within 7 days of emergence, and a felt-tip pen was placed on the backs of 300 young bees per beehive. Marked with (marking). Thereafter, six marked bees were collected every two weeks.

(2) Evaluation of bacterial count in perara The collected bee intestines were pulled out and subjected to phenol extraction using the DNA extraction kit "ISOPLANT II" (Nippon Gene) to extract DNA (total honeybee intestine DNA).

Subsequently, the following primers (SEQ ID NO: 2, 3) were designed based on the sequence of Perara rpoE gene (Perara rpoE) (SEQ ID NO: 1). Then, PCR was performed using honey bee intestinal total DNA as a template and a PCR enzyme (KOD FX Neo; Toyobo Co., Ltd.) to amplify Perara rpoE fragment (SEQ ID NO: 4). After this PCR product was purified, it was inserted into a vector using In-Fusion HD Cloning Kit (Takara Bio Inc.) according to the attached instruction manual, and this was used as a standard sample plasmid. The concentration of the standard sample plasmid was measured and a predetermined dilution series was prepared. In SEQ ID NOs: 2 and 3, lowercase letters indicate sequences homologous to the vector side, which were necessary for inserting the PCR product into the vector.
《Primer for standard sample preparation》
Forward; 5'-acggccagtgaattcGCCTTTAATTTATTAGT-3' (SEQ ID NO: 2)
Reverse; 5'-gattacgccaagcttAAAATACGGGAACGCAC-3' (SEQ ID NO: 3)

On the other hand, the following primers (SEQ ID NOs: 5 and 6) were designed based on the sequence of Perara rpoE. These primers, real-time PCR reagent "SYBR GREEN Master Mix" (Applied Biosystems), and real-time PCR device "ABI PRISM7700 sequence detection system (Applied Biosystems) Real-time PCR was performed using The copy number of rpoE was estimated. The perara rpoE fragment amplified by this real-time PCR is shown in SEQ ID NO: 7. The standard curve is the result of real-time PCR performed under the same conditions using a standard sample plasmid at a predetermined concentration as a template. The obtained copy numbers were averaged for all individuals in each group, and the percentage was calculated with the value on day 0 in each group as 100%.The results are shown in FIG.
《Primers for real-time PCR》
Forward; 5'-AGCTTATCGGTCTTTGGGTTC-3' (SEQ ID NO: 5)
Reverse; 5'-ATCATAGCTCTCTGCCTCCAC-3' (SEQ ID NO: 6)

As shown in FIG. 2, in the test group, the copy number percentage was less than 100% at any of the 2nd, 4th, 6th, and 8th weeks. In other words, it is presumed that in the test group, the number of Perara bacteria in the intestines decreased as the bees grew. In contrast, in the control group, the copy number percentage exceeded 100% at all of the 2nd, 4th, 6th, and 8th weeks. In other words, it is presumed that in the control group, the number of Perara bacteria in the intestines increased as the bees grew.

These results revealed that 1-kestose, a component unique to the test sample, was able to suppress the number of Frischella bacteria in the intestines of honey bees.

Claims (6)

A bacterial count inhibitor for Frischella bacteria that contains 1-kestose as an active ingredient. The agent according to claim 1, which is used to suppress the number of Frischella bacteria in honey bees. An inhibitor of DNA damage in honey bees, containing 1-kestose as an active ingredient. 1-A feed for suppressing the number of bacteria of the genus Frischella, which contains kestose as an active ingredient. A feed for suppressing DNA damage in honeybees, which contains 1-kestose as an active ingredient. A method for breeding honey bees, comprising the step of causing the honey bees to ingest the agent according to any one of claims 1 to 3 or the feed according to claim 4 or claim 5.