JP7619557B2 - Bacteria Tablets - Google Patents

Description

The present invention relates to a tablet that carries bacteria that purify the water in an aquarium.

In aquariums where aquarium creatures are kept, bacteria are used to decompose organic matter such as excrement and leftover food, which can cause deterioration of water quality. One method of supplying bacteria to an aquarium is to add powdered or liquid bacteria to the water, but this has various problems, such as the effort required to measure out the powder or liquid, the effort required to clean up if the powder or liquid falls outside the aquarium, and the discomfort it can cause when it gets on your hands.

As a method to solve this problem, a method has been proposed in which bacteria are supported on tablets and the tablets are directly introduced into water (Non-Patent Document 1).

Product name: "Natto bacteria tablets", manufactured by Ito Sui Co., Ltd., searched on August 7, 2020, Internet <URL: http://www.itosui.co.jp/series/505/>

However, when an aeration device that dissolves air into the water was operated in an aquarium containing such tablets, there was a problem in that bubbles formed on the water surface.

The object of the present invention is to provide a bacterial tablet that has minimal effect on foaming caused by an aeration device.

The bacterial tablet is formed into a tablet shape by mixing a bacterial carrier carrying Bacillus bacteria with an excipient whose main component is sodium sulfate.

By using sodium sulfate as the main excipient, it is possible to produce a bacterial tablet that is less affected by foaming caused by an aeration device. Foaming is a phenomenon that occurs when air is adsorbed to the air-liquid interface of a surface active substance or surfactant. This is because sodium sulfate is an inorganic substance and has less of an effect on foaming compared to components that combine organic substances such as polyethylene glycol with hydroxyl groups.

In addition, in the bacterial tablets, the Bacillus bacteria form spores.

The formation of highly durable spores improves the shelf life of bacterial tablets.

In addition, in the bacterial tablets, the Bacillus bacteria is Bacillus licheniformis.

By using Bacillus licheniformis, which has the best ability to decompose organic matter among the Bacillus bacteria, the organic matter decomposition effect of the bacterial tablet can be increased. This is because Bacillus licheniformis contains many types of enzymes, such as alkaline phosphatase, esterase, esterase lipase, β-galactosidase, and glucosidase.

The bacterial tablet may also contain magnesium sulfate, and the weight ratio of the magnesium sulfate in the entire bacterial tablet may be 10% or less.

By including magnesium sulfate and reducing the proportion of sodium sulfate, the amount of sodium can be reduced, and the increase in the salinity of the water can be prevented. In addition, by keeping the weight ratio of magnesium sulfate to 10% or less, the increase in water hardness can be prevented.

FIG. 1 shows the results of an aeration test on a bacterial tablet of a comparative example. FIG. 2 shows the results of an aeration test on the bacterial tablet of the embodiment.

<Bacteria tablets>
The bacterial tablet according to the present embodiment is formed into a tablet shape by mixing a bacteria support carrying Bacillus bacteria with an excipient mainly composed of sodium sulfate. This allows the bacterial tablet to be directly put into water, reducing the labor of measuring powder or liquid, the labor of cleaning when it falls outside the water tank, and the discomfort caused when it adheres to the hands. Here, the tablet shape refers to, for example, a cube, a rectangular parallelepiped, a sphere, a cylinder, a prism, a cone, a pyramid, an ellipsoid, a cylinder, or a tablet shape that is a combination of these. In particular, from the viewpoint of moldability and usability of the bacterial tablet, a tablet shape that is approximately triangular is preferable.

(Bacteria carrier)
The bacterial support according to this embodiment uses a support substrate and a spore liquid or spore powder of Bacillus bacteria. As the support substrate, an inorganic mineral salt is preferable from the viewpoints of moldability, storage stability, and water solubility. As the inorganic mineral salt, sodium sulfate or magnesium sulfate, which is easily soluble in water and has little effect on the liquid property (pH), is preferable. In particular, sodium sulfate, which has no effect on the total hardness of water, is preferable as the support substrate. For example, a liquid or powder containing Bacillus bacteria (in a non-spore-formed state) may be used as the bacterial support.

The Bacillus bacteria supported on the bacterial support are preferably Bacillus firmus, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus licheniformis, which form highly durable spores. Spore formation can improve the shelf life of the bacterial tablet (e.g., suppressing bacterial activity during storage). Bacillus licheniformis is particularly preferred, as it has many types of enzymes, such as alkaline phosphatase, esterase, esterase lipase, β-galactosidase, and glucosidase.

(Excipients)
The excipient according to this embodiment is sodium sulfate or a combination of sodium sulfate and magnesium sulfate, which is non-foaming, easily soluble in water, and has little effect on liquidity. In order to reduce the effect on the total hardness of water, it is preferable that the weight ratio of magnesium sulfate in the entire bacterial tablet is 10% or less. This makes it possible to suppress an increase in the total hardness of water. In addition, by including magnesium sulfate, the amount of sodium can be reduced, and an increase in the salinity concentration of water can be suppressed.

The above configuration makes it possible to provide a bacterial tablet that has minimal effect on foaming caused by the aeration device.

<How to prepare bacterial tablets>
An example of a method for producing a spore liquid, a spore powder, a bacterial carrier, and a bacterial tablet is described below. Note that the methods for producing the spore liquid, the spore powder, the bacterial carrier, and the bacterial tablet are not limited, and bacterial tablets produced by methods and conditions other than those described below are also included in the present invention.

(Preparation of spore liquid)
(1) A strain of Bacillus bacteria is cultured on an agar medium at 30° C. for 18 to 48 hours.
(2) The strain cultured in (1) above is subcultured in a liquid medium and gradually scaled up at 30° C. for 18 to 24 hours.
(3) The culture solution scaled up in (2) above is cultured in a jar fermenter at 30° C. for 120 to 168 hours to form spores.
(4) After confirming that spores have been formed in the culture medium of (3) above, the spores are centrifuged and collected from the culture medium using a refrigerated centrifuge.
(5) The spores collected in (4) above are washed several times with saline and sterile water.
(6) The spores washed in (5) above are suspended in 1 L of sterile water, and a highly concentrated spore liquid can be obtained by confirming that the cell count of the Bacillus bacteria is 1.0 x 10 ⁹ cfu/mL or more.

(Preparation of bacterial carrier)
The spore liquid or spore powder obtained by drying the spore liquid obtained above is mixed with a support substrate in an arbitrary ratio, whereby the Bacillus bacteria are supported on the support substrate. Then, by confirming that the cell count of the Bacillus bacteria is 1.0 x ¹⁰⁸ cfu/g or more, a bacteria support can be obtained. Note that, for example, a bacteria support may be prepared by mixing a liquid or powder containing the Bacillus bacteria (in a non-spore-formed state) with a support substrate in an arbitrary ratio.

When the bacterial carrier is large (e.g., 1 mm or larger), it is preferable to crush the bacterial carrier with a mixer or crusher in order to improve the tableting properties described below. In order to incorporate a high concentration of Bacillus bacteria into the bacterial tablet, it is preferable to mix the spore liquid or spore powder with the carrier substrate in a weight ratio of 3:7 to 2:8.

Mixing of Bacterial Carrier with Excipients
The bacterial support and excipient obtained above are placed in a thick vinyl bag or powder mixer in a weight ratio of 0.1:9.9 to 2:8, and mixed. In order to blend a high concentration of Bacillus bacteria into the bacterial tablet and form a tablet with sufficient hardness, when using a bacterial support having magnesium sulfate as a support base, it is preferable to mix the bacterial support and excipient in a weight ratio of 1 (or less) to 9 (or more). When using a bacterial support having sodium sulfate as a support base, it is preferable to mix the bacterial support and excipient in a weight ratio of 2:8. In addition, when the bacterial support and excipient are mixed, if the powders of each are solidified, it is preferable to remove the solidified powders using a 1 mm mesh in order to improve the tableting property described later.

(Tableting)
The mixture of the bacteria carrier and excipient obtained above is tableted with a tablet press, and it is confirmed that the number of bacteria of the genus Bacillus is 1.0 x ¹⁰⁶ cfu/g or more. This makes it possible to obtain bacteria tablets (e.g., 0.5 g per tablet). The bacteria tablets are preferably stored in an aluminum bag, for example, in a tank of 10 L of water, so that one tablet is suitable, to prevent moisture.

It is preferable to measure 10 bacteria tablets at a time. If the bacteria tablets are weighed one by one, it is difficult to judge whether the measured value is within the margin of error. In order to provide a high concentration of Bacillus bacteria in the aquarium by adding one tablet, the number of Bacillus bacteria per 10 tablets is set to 5.0 x ¹⁰⁶ cfu or more.

<Excipient selection test>
Bacterial tablets containing Bacillus licheniformis with magnesium sulfate, sodium sulfate or wood chips as the bacterial carrier were prepared by the above process using the excipients shown in Table 1. The weight ratios shown in Table 1 indicate the weight ratios of the entire bacterial tablet.

Three bacteria tablets from the above selection example were placed in a beaker containing 100 mL of tap water to conduct a selection test for excipients. In the selection test, the tablet compression properties of the tablets, their water solubility and turbidity, pH, and total hardness were evaluated. The tablet compression properties were evaluated to see if they could be compressed without problems using a tablet press, and the water solubility was evaluated to see if the tablets dissolved in water after being placed in the water. In addition, from the perspective of minimizing the effects on ornamental fish, the presence or absence of turbidity in the water, whether the pH of the water did not fluctuate by 0.5 or more, and whether the total hardness was 100 mg/L or less were evaluated. Furthermore, the presence or absence of foaming when aeration was applied was evaluated. The evaluation results of the selection test are shown in Table 2 below. For reference, the evaluation results of a comparative example (manufactured by Ito Sui, product name "Natto Bacillus Tablets") are also shown.

From Table 2, it was confirmed that sodium sulfate (Selection Examples 19, 20, 22) or a combination of sodium sulfate and magnesium sulfate (weight ratio 10% or less) (Selection Examples 18, 21) that are good in all respects of tablet compression, water solubility, total water hardness, pH, foaming, and the presence or absence of turbidity are suitable as excipients for the bacterial tablet of the present invention. In addition, it was confirmed that when the weight ratio of magnesium sulfate in the entire bacterial tablet exceeds 10% (Selection Examples 6, 7), the total water hardness exceeds 100 mg/L, and therefore is not suitable for the bacterial tablet of the present invention.

<Aeration test>
As an example, a bacterial tablet containing Bacillus licheniformis (bacterial count: 1.0×10 ⁶ cfu/g) with sodium sulfate as the excipient and magnesium sulfate as the bacterial carrier was prepared by the above process. Then, an aeration test was carried out for the above comparative examples and examples.

The bacterial tablet of the comparative example or the example was placed in a beaker containing 100 mL of tap water, and the bacterial tablet was allowed to completely dissolve. An air stone (small round stone made by GEX) attached to an aeration device (e-AIR1000 made by GEX) was then placed in the beaker, and the aeration device was operated to check for bubbling of the water. In addition, the bubbling of the water in a blank state, where no bacterial tablet was placed, was also checked. The experimental results of the comparative example are shown in Figure 1, and the experimental results of the example are shown in Figure 2.

Figure 1(a) shows the state in which the comparative bacterial tablet was added and the aeration device was operated, and Figure 1(b) shows the state in which the aeration device was operated as a blank. As shown in Figures 1(a) and 1(b), unlike the blank beaker, the beaker into which the comparative bacterial tablet was added showed bubbling (countless small bubbles) caused by the aeration device. The comparative bacterial tablet dissolved in water in about 30 minutes.

Figure 2(a) shows the state in which the bacterial tablet of the embodiment was added and the aeration device was operated, and Figure 2(b) shows the state in which the aeration device was operated as a blank. As shown in Figures 2(a) and 2(b), the beaker into which the bacterial tablet of the embodiment was added did not show any foaming caused by the aeration device, as did the blank beaker. This confirmed that the bacterial tablet of the embodiment had little effect on foaming caused by the aeration device. The bacterial tablet of the embodiment dissolved in water in about 60 minutes.

<Salt concentration test>
A salinity test was carried out using the bacterial tablets of the above example.

1 kg of soil (Gex Pure Soil Black) and fermented carbon dioxide (Gex Fermented _CO2 Starter Set) were placed in an aquarium filled with 3.5 L of tap water, and aquatic plants (Glossostigma, Rotala, Pinnatifida, Myriophyllum) were planted. Then, 20 tablets (about 10 g) of the bacterial tablets of the embodiment were added, and a blank was used in which no bacterial tablets were added. The salinity was measured and the condition of the aquatic plants was confirmed after 15 days. The experimental results (salt concentration) are shown in Table 3 below.

As shown in Table 3 above, 15 days after the start of the experiment, the salinity of the water in the tank into which the bacterial tablets of the embodiment were added rose by 0.3%. This confirmed that the salinity level that would affect freshwater fish was below 1.0%, and therefore the bacterial tablets of the embodiment did not affect freshwater fish. It was also confirmed that there was no change in the condition of the aquatic plants before and after the experiment.

<Water quality testing>
Water quality tests were carried out using the bacteria tablets of the above examples.

50 ml of sterile water and three carp pellets (Hartz Pond Pellets) were placed in a sterilized Erlenmeyer flask. Then, an Erlenmeyer flask containing one bacterial tablet of the above embodiment and a blank Erlenmeyer flask containing no bacterial tablet were each shaken for one month with a shaker (speed: 100 min ^-1 ). The water in each Erlenmeyer flask was then evaporated in a dryer, and the weight of the residue was measured and compared based on the rate of weight loss. The experimental results are shown in Table 4 below.

As shown in Table 4 above, when the bacteria tablets of the embodiment were added, the weight loss rate increased by an additional 7% compared to the blank state. This confirmed that adding the bacteria tablets of the embodiment accelerated the decomposition of carp feed and improved water quality.

Claims (4)

A bacterial tablet formed into a tablet shape by mixing a bacterial support in which Bacillus bacteria are supported on an inorganic mineral salt support base material with an excipient made of an inorganic mineral salt containing sodium sulfate. The bacterial tablet according to claim 1, wherein the Bacillus bacteria form spores. The bacterial tablet according to claim 1 or 2, wherein the Bacillus bacterium is Bacillus licheniformis. The bacterial tablet according to any one of claims 1 to 3, which contains magnesium sulfate and the weight ratio of the magnesium sulfate in the entire bacterial tablet is 10% or less.