JPS58869B2 - Method for producing microbial cell destruction products - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides 5 to 20 microbial cells (hereinafter referred to simply as microbial cells) of microbial cells frozen at a temperature of -100°C or lower.
%(weight.

An efficient and industrial method for producing a bacterial cell-destroying product, which is characterized by pulverizing a suspension with a concentration of (hereinafter the same) in a frozen state at a temperature below -40°C to destroy the bacterial cells. Regarding.

An object of the present invention is to provide an efficient and continuous method for producing a bacterial cell destroyer on an industrial scale.

Another object of the present invention is to separate intracellular proteins, enzymes, etc.
The objective is to provide a bacterial cell-destroying substance for use as a medicine.

Furthermore, another object of the present invention is to provide a bacterial cell destroyer that can be used as food or feed.

Conventionally, destroying bacterial cells and extracting cell walls, intracellular granules, intracellular enzymes, etc. has been carried out for a long time, and has been used in a wide range of fields such as microbiology, enzymology, biochemistry, medicine, pharmacy, and nutrition. Used in laboratory for research.

Industrial production of bacterial cell destroyers has conventionally been carried out mainly for enzyme production.

For example, for glucose oxidase, urecase, glucose isomerase, etc., bacterial cells can be destroyed using methods such as autolysis, repeated freezing and thawing to damage cell walls with ice crystals, sonication, French press, mortar, homogenizer, etc. Extraction is performed by simultaneously using extractants such as salt solutions, buffers, and surfactants.

These destructive treatments remain at an extremely mild stage because enzyme extraction causes loss of cell membrane permeability and the initial goal is leakage of intracellular components.

Autolysed products of yeast cells have been commercially available as seasonings for a long time, but yeast cells themselves are produced for food, medicine, and feed.

When these cells are destroyed, their nutritional value and digestibility are improved, but because their cell walls are so strong that they cannot be destroyed sufficiently, they cannot be produced by methods other than autolysis.

There is no known industrial method to destroy bacterial cells by freezing them, but on a laboratory scale, it is possible to freeze the bacterial cells at a temperature of -25 to -35°C and pressurize the frozen material to destroy the bacterial cells. Hugh
es press, x-press and their structure,
Devices similar in principle are commercially available.

In these devices, 5 to 25 ml of sample liquid is placed in a pressure-resistant cylindrical container, the container is cooled to -25 to -35°C, the sample is frozen, and then a piston is inserted into the cylindrical hole. By setting it in a press and applying sharp pressure, the sample is forced out (passed through) in a frozen state through the pores provided in the cylindrical body or the narrow grooves on the cylindrical joint surface.
This crushes the bacterial cells.

Regarding the mechanism of bacterial cell crushing action by these devices, Hu
Hughes surmised that most of the fracture effect was due to the anti-friction effect of ice crystals during the compression and refreezing of the frozen sample under high pressure (Hughes, D., E.
,: “The Disintegration of
Micro-organisms”, Method i
n microbi-010gy, vol, 5B, 1
~54 pages, 1971, Academic Pre
ss issue).

Furthermore, according to a study on X-press by Edebo, the crushing effect mainly involved is caused by changes in the morphology of ice crystals under high pressure (Edebo, L.: Acta Phatho
logica et Bio-1 logica 5can
dinavica, vol. 52, p. 300, 1961).

As described above, the conventional method for destroying frozen microbial cells has been extremely small-scale, in which microbial cells frozen at high temperatures (-25 to -35° C.) are destroyed by applying pressure.

Recently, a method has been published in which bacteria such as yeast are brought into contact with liquefied refrigerant gas to freeze them, and then the frozen bacteria are crushed under extremely low temperatures in an atmosphere of refrigerant gas (Japanese Patent Laid-Open No. 53-29983).
.

This method attempts to destroy the microbial cells by freezing the water present inside the microbial cells, but as described later, according to the tests conducted by the present inventors, freezing the water inside the microbial cells alone does not destroy the microbial cells. It was found that the rate was low.

On the other hand, the principle of freeze-grinding technology has been studied for more than 50 years, and in 1962 H0B, Wist
The first application to food was that of spices by Reich et al.

After that, it was widely used in the food industry (Food Industry Technology Information, Vol. 11, No. 5, pages 1-15, 1979).
It is used for grinding various foods or food ingredients.

The advantages of this method are: firstly, materials that are difficult to crush can be easily crushed at room temperature, such as rubber tires, aluminum or plastic containers, etc., and secondly, the heat generated during crushing at room temperature is prevented. Therefore, substances that are easily denatured by heat can be pulverized, and thirdly, liquid nitrogen used as a refrigerant fills the voids in the powder, which not only prevents oxidation but also reduces the risk of dust explosion.

The only disadvantage of freeze-grinding is that it is more expensive than grinding at room temperature, but this disadvantage is said to be offset in some cases by its use in high-value-added products and the aforementioned advantages. .

As for food products, we have already introduced beans such as peanuts and almonds that are difficult to crush at room temperature, crunchy foods, and meat, as well as various spices, fragrances, and fruit juices whose aromas or ingredients change as the temperature rises at room temperature. However, green tea, black tea, and coffee, which contain ingredients that are easily oxidized,
Seaweed etc. are processed.

In addition, products such as bone-in fish meat, shrimp, or shrimp, which are difficult to crush at room temperature because they are made up of parts that differ greatly in hardness and moisture content, are products with high added value that can absorb costs, such as Chinese herbal medicines, herbal medicine raw materials, and germs. , chlorella, etc. are also processed.

In addition, we are reusing materials that have traditionally been discarded or have lost their commercial value, such as bones of cows, pigs, fish, etc., soybeans, rice husks, etc.
New uses have been found for squid waste by freezing and crushing it.

Various types of freeze-grinding equipment are commercially available, and all of them are comprised of a raw material hopper, a cooling device, a crushing device, a device for supplying the cooled raw material to the crushing device, a classification device, and the like.

First, raw materials are put into a hopper and frozen in liquid nitrogen that has been supplied to the hopper in advance.

Next, it is sent to a crusher (mill) by a screw set feeder,
After being crushed and classified at a predetermined temperature, it is transported using vaporized nitrogen gas, collected by a cyclone, and taken out to the outside.

Impact mills such as hammer mills, ball mills, and pin mills are often used for pulverization, and depending on the sample, jet mills and attritors are also used.

A typical device is the Linrex Mill (trademark).

(manufactured by Seiyo Ironworks), Cryomill (trademark. Manufactured by Nippon Sanso and Turbo Kogyo), Complex Mill (trademark).

(manufactured by Makino Sangyo and Alpine), a complete atomizer mill (manufactured by Fuji Paudal and Toyo Sanso), etc.

As mentioned above, various cryo-pulverizers are commercially available.
In addition, any material of visible size that is edible is processed as the material to be crushed.

The present inventors conducted research on an efficient and continuous production method for bacterial cell destruction products on an industrial scale, and obtained 5 to 2 bacterial cells.
The unexpected fact that freezing a suspension in water or physiological saline at a concentration of 0% and pulverizing it in a frozen state results in a better bacterial cell destruction effect than conventionally known methods. discovered this and completed the present invention.

In the method of the present invention, a suspension of bacterial cells with a concentration of 5 to 20%,
-11 by ultra-low temperature liquefied gas such as liquid nitrogen or liquid helium
Freeze to a temperature below 00°C, and freeze the frozen material at -40°C in a frozen state.
This is a method for producing a destroyed microbial cell material, which is characterized by pulverizing and destroying cells at a temperature of 0.degree. C. or below.

Next, the method of the present invention will be explained in detail.

The bacterial cells used in the method of the present invention include, for example, lactic acid bacteria,
These are useful microorganisms such as bifidobacteria, yeasts, molds, and actinomycetes, and are well-known microorganisms that are widely used in the pharmaceutical, food, or feed industries.

These bacterial cells are cultured by conventional methods under culture conditions suitable for each bacterial cell.

Then, the culture solution itself containing the proliferated microbial cells, or a suspension of the microbial cells separated from the culture solution by a conventional method and suspended in water, physiological saline, etc. at a concentration of 5 to 20%, or A suspension of bacterial powder dried by a known method such as freeze-drying or spray-drying is suspended in water, physiological saline, etc. at a concentration of 5 to 20%.

The microbial cells contained in the suspension may be live microbial cells or dead microbial cells.

Alternatively, the bacterial cells in the suspension may be treated in advance by a known method, such as lysozyme treatment, so that the bacterial cells are easily destroyed.

The type of suspension to be used is appropriately determined depending on the intended use of the destroyed microbial cells.

For example, when separating intracellular enzymes, the cells are separated from the culture solution to avoid contamination with components in the culture solution, and the collected cells are washed and suspended in water before use.

When using bacterial cells isolated from a culture solution or dried bacteria, the bacterial cells are suspended in water, physiological saline, etc. at a ratio of 5 to 20%, preferably 5 to 10%.

The suspension is pre-frozen and crushed into pieces of about 1 cm3 size, or the suspension is directly poured into a hopper filled with ultra-low temperature liquefied gas to freeze the suspension.

As the cryogenic liquefied gas, liquid nitrogen, liquid helium, liquid argon, liquid oxygen or liquid air can be used, with liquid nitrogen being particularly preferred due to its low cost and protection against oxidation.

The suspension is frozen at a temperature below -100°C.

By freezing at a temperature below -100℃, it is possible to promote the weakening of bacterial cells in the suspension, to reduce the temperature rise during grinding of the frozen suspension, and to mill the frozen suspension. improve mobility to.

Fine pulverization can be carried out using commercially available equipment, such as the Linrex Mill (trade name).

Made by Seiyo Iron Works. XL-0 type).

The apparatus is pre-cooled to below -40°C using ultra-low temperature liquefied gas, and the frozen suspension is supplied and pulverized.

Since the temperature of the pulverizing part (mill) rises, it is necessary to prevent the temperature from rising by adjusting the amount of ultra-low temperature liquefied gas supplied.

If the temperature of the mill rises above 140° C., it is undesirable because the bacteria-destroyed substances will adhere to the mill and the movement of the bacteria-destroyed substances within the apparatus will be hindered.

The temperature inside the device during pulverization varies depending on the type of bacteria to be destroyed, the concentration of bacteria in the starting material, the degree of destruction of bacteria, and the purpose of use of the destroyed bacteria, but -40 to -120°C is the optimum temperature for destruction efficiency. It is desirable from the point of view.

The microorganism-destroyed material, which has been finely ground into snow-like particles, moves through the device together with ice crystals under the pressure of the ultra-low temperature liquefied gas that vaporizes inside the device.
It passes through a classifier and is taken out into a receiver.

The microbial cell destruction material taken out in this manner may be subjected to destruction treatment again if necessary.

The destroyed bacterial cells removed in this manner can also be immediately freeze-dried to form a powder.

Moreover, this microbial cell destruction product can also be used for the production of useful substances such as enzymes and vitamins.

Furthermore, this microbial cell destruction product can also be used as it is as a component for the production of food or feed.

Next, the method of the present invention will be further explained in detail by showing test examples.

Test 1 Destruction of Baker's Yeast Cells (1) Sample 1) Sample by the method of the present invention: Produced by the same method as Example 1.

2) Sample prepared by a known method: 100 g of a suspension of baker's yeast cells prepared by the same method as in Example 1 was heated using an experimental ultrasonic generator (manufactured by Kubota Seisakusho).

200M type) with an output of 9KHz and 200W.
The cells were treated for 60 minutes while being maintained at a temperature of 0.degree. C. to destroy the bacterial cells.

(2) Test method Observe the above two types of samples with a microscope, calculate the percentage of the number of destroyed bacteria to the total number of bacteria in one field of view, and measure the destruction rate of bacteria as the average value of 20 fields of view. did.

Parts of the two types of samples were centrifuged at 28.0 OOG for 40 minutes, the supernatant was collected, and the soluble solid content and soluble protein content in 10 ml of the supernatant were measured by the following method.

Then, calculate the percentage of soluble solid content and soluble protein content in 10 ml of centrifuged supernatant relative to the total solid content and protein content in 10 ml of sample before centrifugation,
The transfer rate of each component to the supernatant liquid was measured.

The total solid content of the sample before centrifugation and the soluble solid content of the supernatant were determined by the drying method at 100°C ± 0.5°C, and the protein content of the sample before centrifugation and the soluble protein content of the supernatant were , respectively, were measured by the Kaersol method.

(3) Results The test results are shown in Table 1.

As is clear from Table 1, although the destruction rate of microbial cells by microscopy for both samples was the same, the transfer rate of soluble solids and soluble proteins to the supernatant was lower in the sample obtained by the method of the present invention. About 1.33 times and about 1 times that by the conventional method, respectively.
．． It showed a high value of 68 times.

This result proves that bacterial cells are more completely destroyed by the method of the present invention than by the conventional method.

That is, according to the method of the present invention, since the sample is frozen at an ultra-low temperature using liquid nitrogen, it easily causes freezing inside the bacteria, weakening the bacteria, and causing ice crystals outside the bacteria that coexist in the frozen state. By playing the role of anti-wear agent,
Bacterial cells are easily crushed by slight pressure or impact.

Test 2 Extraction of enzyme from disrupted lactic acid bacteria cells (1) Sample ■) Sample according to the method of the present invention: Produced by the same method as in Example 2.

2) Sample prepared by conventional method: prepared by the same method as in Example 2, or prepared using 150 g of a suspension of P. helveticus cells.
5orval-Ribi refrigera-te
d cell fractionator (Ivan
20,000 by 0RF-1 type manufactured by 5orva1)
The microbial cells were destroyed by maintaining the temperature at 8 to 1.5° C. under a pressure of psi.

(2) Test method The above two types of samples were centrifuged at 28,000 g for 40 minutes, the supernatant was collected, and the amount of β-galactosidase (a type of intracellular enzyme) in the supernatant was measured using the Dahlgvist method (Analytica
Biochemistry, Vol. 7, pp. 18-25, 1964).

The amount of enzyme required to hydrolyze 1 micromole of lactose per minute at 30°C was expressed as one unit.

(3) Results The test results are shown in Table 2.

As is clear from Table 2, the bacterial cell destruction product produced by the method of the present invention contains approximately the same amount of β-galact as the conventional method, which is said to have good destruction efficiency.
osidase, the bacterial cells are more effectively destroyed and intracellular enzymes are taken out of the bacterial cells.

Test 3 Destruction of bacterial cells at various bacterial cell concentrations (1) Sample Commercially available fresh baker's yeast (moisture content: 33%). This baker's yeast was suspended in purified water and the bacterial cell concentration was adjusted to 3°5.20, 30.
, and 6 types of samples adjusted to 50%, each weighing 15 kg, were prepared.

Each sample was treated in the same manner as in Example 1 to destroy bacterial cells.

(2) Test method For each sample subjected to bacterial cell destruction treatment, the bacterial cell destruction rate, soluble solid content transfer rate, and soluble protein transfer rate were measured by the same method as in Test 1, and the state of bacterial cell destruction was tested.

(3) Results The results are shown in Table 3.

As is clear from Table 3, the bacterial cell concentration of the suspension is 5 to 20.
%, especially in the range of 5% and 10%, the soluble solid content transfer rate, soluble protein transfer rate, and destruction rate are high, and it is recognized that the bacterial cells are destroyed.

Comparing the measured value of the bacterial cell concentration at 5%, where the most bacterial cells are destroyed, with that of the control, the former has a soluble solid content transfer rate of 2.45 times, a soluble protein transfer rate of 2.83 times, The destruction rate is 3.17 times higher.

Based on this test result, in the present invention, the bacterial cell concentration of the suspension was
It has been found necessary to adjust it to ~20%, preferably 5-10%.

The effects of the method of the present invention are as follows.

(1) Bacterial cells can be efficiently and easily destroyed.

(2) Since heat generation during pulverization can be prevented by the latent heat of vaporization of the ultra-low temperature liquefied gas, denatured bacterial cell constituent substances, such as physiologically active substances such as enzymes, can be stably recovered.

(3) By using liquid nitrogen or liquid helium as the ultra-low temperature liquefied gas, the vaporized gas covers the destroyed bacterial cells and prevents the treated bacterial cells from being oxidized.

(4) It is possible to continuously destroy large amounts of bacterial cells.

(5) The production of yeast feed with high feed efficiency or the effective extraction of enzymes inside microorganisms, which were previously thought to be extremely difficult to achieve both technically and economically, becomes possible at low running costs.

Example 1 20 kg of purified water was added to 10 kg of commercially available fresh baker's yeast (water content approximately 33%) to prepare 30 kg of a suspension containing approximately 10% yeast cells.

20 kg of this suspension was mixed with Linrex Mill (trademark).

Made by Seiyo Iron Works.

XL-o type) as follows.

The inside of the apparatus was previously cooled to -100°C with liquid nitrogen.

Next, the suspension was dropped into a hopper filled with liquid nitrogen and frozen into pellets with a particle size of about 5 to 10 mm.

This frozen material was continuously supplied to the apparatus at a rate of 0.5 Yu per minute to finely pulverize the bacterial cells.

The apparatus was equipped with a hammer mill, and the peripheral speed of the rotor was 109 m/s.

The apparatus was operated continuously for about 40 minutes, and 19.5 ky of snow-like frozen fine powder of baker's yeast cells was obtained.

When the obtained bacterial cell destruction product was examined under a microscope in the same manner as in Test 1, approximately 90% of the bacterial cells were destroyed.

Example 2 Lactic acid bacteria, helveticus (ATCC8018)
A medium 6 consisting of yeast extract, peptone, lactose, and salts
001, cultured at 37°C for 18 hours, centrifuged to collect the bacterial cells, washed with purified water, and then added with physiological saline to obtain approximately 10 kg of bacterial cell suspension of approximately 12%. Ta.

Freeze 8 kg of this suspension in a freezer at -40°C, crush the frozen material into pieces of about 1 cm3, and freeze 0.3 kg for 1 minute.
The following Example 1 was supplied to the same equipment as in Example 1 at a ratio of
The bacterial cells were destroyed in the same manner as above to obtain 7.8 kg of crushed lactic acid bacteria cells in the form of snow-like frozen fine powder.

Example 3 Bifidobacterium breve (ATCC15700
), yeast extract, kashiton, which are all commercially available.
It was inoculated into a sterilized liquid medium 6001 consisting of meat extract, cysteine, and salts, and cultured at 37° C. for 16 hours while aerating carbon dioxide gas at a rate of 0.1 vvm.

Thereafter, the cells were collected by centrifugation, washed with purified water, and purified water was added to obtain about 12 kg of a suspension with a cell concentration of about 9%.

Immediately freeze 10 kg of this suspension in a freezer at -40°C, crush the frozen material into pieces of approximately 1Cm2, and freeze for 1 minute.
．． 3 kg was supplied to the same apparatus as in Example 1, and the bacterial cells were destroyed in the same manner as in Example 1 to obtain 9.5 kg of broken bifidobacterial cells in the form of snow-like frozen fine powder. .

Claims (1)

[Scope of Claims] 1. A suspension prepared by adding water or physiological saline to microbial cells to adjust the cell concentration to 5 to 20 parts (weight) is heated to a temperature of -100°C or less using ultra-low temperature liquefied gas. 1. A method for producing a microorganism-destroyed product, which comprises freezing and pulverizing the frozen product in a frozen state at a temperature of -40° C. or lower to destroy the microorganisms. 2. The method for producing a microbial cell destroyer according to claim 1, wherein the ultra-low temperature liquefied gas is liquid nitrogen, liquid helium, liquid argon, liquid oxygen or liquid air.