JPH0299196A - Putrefaction prevention of organic material-containing aqueous liquid - Google Patents

Abstract

PURPOSE:To prevent putrefaction and degradation of aqueous liquid effectively by adding bacteriophage, which is active bacteriolysis against bacteria putrefying an aqueous liquid, to an organic material-contg. aqueous liquid such as an aqueous treatment agent. CONSTITUTION:A sample containing bacteriophage searched by checking whether bacteriolysis spot is or not is diluted with sterilized physiological salt solution. A certain amount of the dilute solution is taken, a mixture of a culture liquid of an isolated host bacteria and soft agar culture is put on agar culture and statically cultured. Bacteriophage is collected from bacteriolysis spots formed on the agar culture by a platinum wire, it is added to an isolated bacteria culture liquid, cultured, and a supernatant obtained by centrifugation of the culture liquid is filtered to give a bacteriophage-contg. solution. When a dilute solution of the bacteriophage-contg. solution is added to an organic material-contg. aqueous liquid, bacteriolysis of the putrefying bacteria in the aqueous liquid is carried out by the bacteriophage, thus propagation of the putrefying bacteria is efficiently suppressed and putrefaction of the aqueous liquid is effectively prevented.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a method for preventing spoilage of organic substance-containing aqueous liquids, such as water-soluble cutting and grinding fluids, paint mist treatment agents, and water-soluble cleaning agents.

Summary of the Invention This invention provides a method for preventing spoilage of aqueous liquids containing organic matter such as aqueous treatment agents, by adding Bacterio 7. It is designed to effectively prevent spoilage and deterioration of aqueous liquids.

Conventional technology In the past, preservatives such as triazine-based compounds and isothiazoline-based compounds have been used to prevent aqueous processing agents such as water-soluble cutting and grinding fluids, paint mist processing agents, and water-soluble cleaning agents from rotting and deteriorating due to bacteria. Generally, a method is adopted in which the additive is added to the processing agent at any time before and/or after its use.

However, adding large amounts of this type of preservative and disinfectant can cause skin roughness and inflammation of the mucous membranes of the eyes and nasal passages for workers, so it is important to use an amount sufficient to effectively prevent decomposition and deterioration. This is problematic from an occupational hygiene perspective. For this reason, the reality is that after spoilage has occurred, a preservative and fungicide is added as needed at a concentration of several hundred ppm. Furthermore, if the same type of preservative and fungicide is used continuously, bacteria resistant to the agent will appear, making it impossible to effectively prevent spoilage.

Furthermore, in the case of a water-soluble cutting and grinding fluid, aeration treatment may be performed. This method suppresses the growth of anaerobic bacteria, thereby suppressing the generation of hydrogen sulfide, etc., but the problem is that aerobic bacteria grow, making oil components such as lubricants and surfactants more susceptible to biodegradation. There is.

Problems to be Solved by the Invention This invention can effectively prevent rotting and deterioration of organic matter-containing aqueous liquids such as aqueous treatment agents without using chemicals such as preservatives and disinfectants that pose problems in terms of work hygiene. This was done to provide a method.

Means for Solving the Problems, That is, the present invention provides a method for preventing spoilage of an organic matter-containing aqueous liquid, which is characterized by adding to the organic matter-containing aqueous liquid a bacteriophage having bacteriolytic activity against bacteria involved in the spoilage of the aqueous liquid. Regarding.

The basic unit operations for implementing the present invention will be explained below.

The putrefactive bacteria contained in the putrefactive liquid of the aqueous liquid to be treated are removed by a conventional method.
Culture is spread on a suitable medium, and bacteria are isolated from the resulting colonies.

Isolated bacteria may be identified by conventional methods such as morphology, Durham staining, motility, growth under anaerobic conditions, and O-F test.

Many bacteria involved in the spoilage of ordinary organic matter-containing aqueous liquids have already been isolated and identified in the field;
This unit operation can be easily performed.

This type of bacteria is Escherichia coli (Escherichia
coli), Klebsiella pn
eumoniae), Paracoli spp.
bactrum sp, ), Proteu vulgaris
svulgaris), Pseudomonas aeruginosa
s aeruginosa), Pseudomonas oleoporans
s), Pseudomonas sp.
), Salmonella typhoid
sa), Staphylococcus aureus (SLaphylococcus
aureus), Desulfovibrio desulfuricans (Desulfovibri).

desulfuricans), sulfate-reducing bacteria species (Des
ulfovibrio sp, ), Bacillus subtilis
lus 5ubtillis) and Bacillus (Ba
cillus sp, ) and the like.

(2) Search for bacteriophage Search for bacteriophage that lyses isolated bacteria is conveniently carried out by an indirect method.

The culture solution of the isolated bacteria is added to an appropriate liquid medium (e.g., an aqueous medium containing meat extract, peptone, sodium chloride, etc.) and cultured with shaking, and then this culture solution is thought to contain bacteriophages. The septic liquid of the aqueous liquid to be treated is added and cultured with shaking. This culture solution was centrifuged,
The supernatant liquid is subjected to filtration using a membrane filter or the like.

The search for bacteriophage in the filtrate is carried out by dropping the filtrate onto a double-layered agar medium inoculated with the isolated bacteria, and after static culture, checking for the presence or absence of a lytic bed formed in the area where the filtrate is dropped.

(3) Bacteriophage isolation A sample in which the presence of bacteriophage was confirmed was diluted 10 to 10' times using sterile physiological saline, etc., a certain amount of the diluted solution was placed on an agar medium, and the isolated host A mixture of bacterial culture solution and soft agar is layered on the agar medium and cultured stationary. Bacteriophages are collected from the lysis bed formed on the agar medium using a platinum wire, added to the culture solution of the isolated bacteria, and then cultured. This culture solution was centrifuged,
A bacteriophage-containing solution is obtained by filtering the supernatant using a membrane filter or the like.

The containing liquid is appropriately diluted with sterile physiological saline and the like before use.

(4) Bacteriophage lytic action and spoilage prevention effect When the bacteriophage-containing solution prepared as described above is added to the aqueous solution to be treated containing organic matter, the bacteriophage is a bacterium that is involved in the spoilage of the aqueous solution. A gene is injected into the cells of the bacteriophage, causing replication of the gene and synthesis of constituent proteins, and replication of the bacteriophage. The replicated bacteriophage is released outside the cell by lysing the bacterium. Therefore, once a bacteriophage is added to an aqueous liquid to be treated, the growth of spoilage bacteria in the aqueous liquid can be effectively inhibited without adding it thereafter, and spoilage of the aqueous liquid can be effectively prevented. .

It is effective to use multiple types of bacteriophage in combination depending on the type of spoilage bacteria in the aqueous liquid to be treated.

Furthermore, since bacteriophages are stable against commonly used preservatives and fungicides, they may be used in combination with these.

The timing of adding bacteriophages is particularly limited.Bacteriophages generally exhibit the highest lytic effect when spoilage bacteria are in the growth phase, so they should be added to the aqueous liquid to be treated in advance, or at the beginning of use. It is preferable to add it when replacing the liquid used.

Hereinafter, the present invention will be explained by examples.

Example 1 (1) Isolation and Identification of Bacteria Pseudomonas and Bacillus spp. A rotting fungus was isolated.

Identification of these bacteria was performed using conventional methods such as morphology, Durham staining,
Motility, growth under anaerobic conditions, O-F test, etc. were examined.

(2) Exploration of bacteriophage Concentrated liquid medium (meat extract 10g, peptone 20g 1m) - To! J Rum 10g 5IFt [Water IQ; pH 7,
0) l OQmQ and spoilage bacteria preculture solution [The bacteria were grown in a liquid medium (meat extract 5g, peptone 10g, sodium chloride 5g.

After overnight shaking culture (120 times/min, amplitude 20 mm) in purified water lQ; pH 7,0), 10.67 m12 of the culture solution was
The cells were placed in a 00ml shaking flask and subjected to shaking culture at 30°C for 4 hours (120 times/min, amplitude 70mm).

To this culture solution, add 100 m of putrefaction fluid that is thought to contain bacteriophage (aqueous putrefaction solution containing surfactants, rust preventives, solubilizers, dyes, oils, polymers, etc. as organic substances), Shaking culture was carried out overnight under the same conditions.

This culture solution was centrifuged for 10 minutes at 5°C (lO
The supernatant was subjected to filtration using a membrane filter (0.45 μ'm) and then collected in a sterile test tube.

This filtrate was mixed with an agar plate double layer medium [3 mQ of a soft agar medium prepared by adding 5 g of agar to a liquid medium and dissolving it by heating, and 5 m12 of the above preculture solution Q of putrefaction bacteria, and then adding 15 g of agar to a liquid medium. The bacteriophages were detected by the presence or absence of lytic plaques after the mixture was poured onto an ordinary agar medium supplemented with 100% agar and then dropped onto the solidified medium and left to stand overnight at 30°C.

(3) Isolation of bacteriophage The sample in which the presence of bacteriophage was confirmed was diluted up to 106 times with sterile physiological saline, and 1 ml of the diluted solution (+
0.5 m of spoilage bacteria preculture (2 and 3 m of soft agar medium)
After mixing with Q and coagulating it on an ordinary agar medium,
Static culture was performed overnight at 0°C.

A sample collected by puncturing the resulting plaque with a platinum wire was suspended in 5 m+2 of sterile physiological saline, and this was again diluted to 105 times using the same method to generate bacteriophage plaques.

(4) Preparation of bacteriophage-containing liquid liquid medium 10m
Add 1 ml (1 liter) of host bacterial preculture to f2 and incubate at 30°C for 4 hours.
After a period of shaking culture (120 times/min, amplitude 20 mm), the bacteriophage plaques were collected using a platinum wire and suspended.

After culturing this suspension overnight under the same shaking conditions,
Centrifugation treatment (LOOOORPM) was performed for 10 minutes at °C, and the supernatant was filtered through a membrane filter (0°45 μm).
A bacteriophage-containing solution was prepared by subjecting the solution to a filtration process using . The containing liquid is collected in a sterile test tube,
Stored at 4°C.

The number of bacteriophages corresponding to Pseudomonas and Bacillus spoilage bacteria is 8-9X, respectively.
I O'pfu/mα and 3.5 X I O'pf
u/mQ (To measure the number of bacteriophages, the bacteriophage-containing solution was diluted with sterile physiological saline as described above, and this was placed on an agar plate double layer medium.
(performed by counting plaques formed by overnight incubation at 30°C).

(5) Bacteriophage lytic activity test 500 m (2
In a shaking flask, 150 ml of liquid medium, 1 mQ8 preculture of spoilage bacteria, and 1 ml of a solution containing bacteriophage for lysing spoilage bacteria were placed, and cultured with shaking at 30°C (120 times/min, amplitude 70 mm). A sample was taken at a specific time, and the absorbance at 650 nm was measured. in this case,
For comparison, the absorbance of a sample to which no bacteriophage-containing solution was added was also measured over time.

The viable counts of Pseudomonas and Bacillus spoilage bacteria at the start of the test were each 1.4 x I O'cf.
u/m+2 and l −8X l O'cfu/ mQ
It's here.

11J determination results are shown in Figure 1 I. In Figure a, a and a'' respectively indicate the case where a bacteriophage-containing solution corresponding to the putrefactive bacteria of the genus Pseudomonas was added and the case where the containing solution was not added, and b and b. ′ indicates the case where a bacteriophage-containing liquid corresponding to a spoilage bacterium belonging to the genus Bacillus was added and the case where the liquid containing the bacteriophage was not added, respectively.

(6) Anti-corrosion test by adding bacteriophage Organic substances include surfactants, rust preventives, solubilizers, dyes,
Aqueous liquid (1/N O
After sterilizing 00mQ for adding a concentrated liquid medium, 1m+2 of the preculture solution of the two types of spoilage bacteria and a bacteriophage-containing solution for lysing each putrefaction bacteria were added. Even when this aqueous solution was stored at 30° C. for IO days, almost no putrid odor was detected.

On the other hand, when these bacteriophage-containing solutions were not added, a putrid odor was detected after the aqueous solution was stored at 30°C for 1 day, and a strong putrid odor was detected after 3 days of storage.

Example 2 Biodegradability test Machine oil 5.02449 in a shake flask (500 mQ)
．． 100 ml of liquid medium with a concentration of 1/10. Example 1 (
Similarly to 6), 1 ml of the preculture solution of the two types of spoilage bacteria and the bacteriophage-containing solution (prepared in Example I) for lysing each bacteria were added, and the mixture was incubated at 30°0,120 times/min for 10 minutes. A machine oil spoilage test was conducted by shaking culture for days. As a control test, a sample to which no bacteriophage-containing solution was added was also conducted in the same manner.

For extraction of machine oil, 200 ml of n-hexane was used, and 50 g of activated anhydrous sodium sulfate was added to the extract as a dehydrating agent. This extract was distilled under reduced pressure at room temperature, and after removing the solvent, it was precisely weighed to measure the weight of the machine oil after the putrefaction test.

To measure the acid value, a certain amount of the sample from which the solvent had been removed was dissolved in ethanol benzene (1,1) and titrated with 1/l ON potassium hydroxide solution using phenolphthalein as an indicator.

Gas chromatograph (Shimadzu model)
The operating conditions for C+C-C-4C are as follows.

Detector and inlet temperature: 320°C, column temperature: 30
0°C Length of 1 column 21 ml Inner diameter of 1 column: 3 mm,
Nitrogen flow rate: 40ml, 'min, filler: 5ilico
ne GE 5E-3060-860-8Olo%, detector: FID.

The results are shown in Table 1, Table 2, and Figure 2. As is clear from Table 1, the addition of the bacteriophage-containing liquid was found to be more than 10 times more effective in reducing the amount of machine oil. This is considered to be because the bacteria were lysed by the addition of the bacteriophage-containing solution, so that almost no biodegradation occurred.

Also, looking at the acid value in Table 2, no change was observed in the acid value of the samples to which the bacteriophage-containing solution was added. However, in the case where the bacteriophage-containing solution was not added, the acid value increased from 0.05 to 0.11. This is thought to be because alkanes and the like in the machine oil undergo β-oxidation and become carboxylic acids and the like due to decay.

FIG. 2 shows the results of a gas chromatograph analysis of machine oil extracted before and after a 10-day spoilage test without adding a bacteriophage-containing liquid to the machine oil. After adding the bacteriophage-containing solution to machine oil and culturing it with shaking for 10 days, it showed exactly the same chromatogram as the original machine oil.

Effects of the Invention According to the present invention, bacteriophage existing in nature can be used to prevent the deterioration of organic matter-containing aqueous liquids such as aqueous treatment agents, instead of using agents such as preservatives and disinfectants that pose problems in terms of work hygiene. It can be effectively prevented.

The bacteriophage used in the present invention selectively and specifically attacks and kills only specific bacteria, and is harmless to humans and animals, and does not cause secondary pollution.

Therefore, in addition to aqueous treatment agents (e.g., water-soluble cutting and grinding fluids, paint mist treatment agents, water-soluble cleaning agents, etc.), the present invention also applies to organic substance-containing aqueous liquids used in various fields, such as surfactants, paints, etc. It can be applied to prevent corrosion of antirust liquids, cooling water, adhesives, pastes, latex, dispersants, inks, fire extinguishers, polymers, etc.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of a bacteriophage lytic activity test. a and a' respectively indicate the case where a bacteriophage-containing solution corresponding to the spoilage bacteria of the genus Pseudomonas is added and the case where the solution containing the solution is not added, and b and b' respectively indicate the case where the solution containing the bacteriophage corresponding to the spoilage bacteria of the genus Bacillus is added. The case where the liquid is added and the case where the containing liquid is not added are shown. FIG. 2 is a gas chromatogram of the machine oil component (n-hexane extract) in the water-soluble cutting and grinding fluid before and after the rot test. C is a chromatogram of machine oil components obtained by n-hexane extraction from a water-soluble cutting and grinding fluid containing a bacteriophage-containing solution, and d is a chromatogram of machine oil components obtained by n-hexane extraction from a water-soluble cutting and grinding fluid without a bacteriophage-containing solution. The obtained chromatogram of the machine oil component and e shows the chromatogram of the machine oil component. Figure 2 Patent applicant Juzo Iizuka, Director of the Agency of Industrial Science and Technology, and two other representatives
One patent attorney: Aoyama Aoyama,
5 (now)

Claims (1)

[Scope of Claims] 1. A method for preventing spoilage of an organic matter-containing aqueous liquid, which comprises adding to the organic matter-containing aqueous liquid a bacteriophage having bacteriolytic activity against bacteria involved in the spoilage of the aqueous liquid. 2. The method for preventing spoilage according to claim 1, wherein the organic substance-containing aqueous liquid is an aqueous treatment agent. 3. Organic matter-containing aqueous liquid containing bacteriophage having bacteriolytic activity against bacteria involved in spoilage. 4. The organic matter-containing aqueous liquid according to claim 3, wherein the organic matter-containing aqueous liquid is an aqueous treatment agent.