JPS5988086A - Production of protoplast of bacterium of lactobacillus - Google Patents

Abstract

PURPOSE:To obtain a protoplast of a bacterium belonging to the genus in a short time in a high yield, by treating end-N-acetylmuramidase with a bacterium belong to the genus Lactobacillus in the presence of a dimer. CONSTITUTION:In a method wherein a protoplast of a bacterium belonging to the genus Lactobacillus is produced by treating end-N-acetylmuramidase prepared by Sterptomyces globisporus with the bacterium in the presence of a substance for adjusting osmotic pressure, a dimer is used as the substance for adjusting osmotic pressure. Sucrose, lactose, maltose, trehalose, etc. may be cited as the substance for adjusting osmotic pressure used. Lactobacillus casei, L. acidophilus, L. bulgaricus, L. lactice, L. heleveticus, etc. may be used as the bacterium belonging to the genus Lactobacillus.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for preparing protoplasts of bacteria of the Lactobacillus range. More specifically, Streptomyces glopisvorus (Streptobacillus) is a bacteria in the Lactobacillus area.
Protoplasts of Lactobacillus bacteria are prepared by allowing endo-N-acetylmuramidase produced by Myces globisporus to act in the presence of trisaccharides such as sucrose and lactose. It is related to the method.

Traditionally, egg white lysozyme has been used to prepare bacterial protoplasts. However, since the cell walls of Lactobacillus bacteria are less sensitive to egg white lysozyme, protoplasts have not yet been successfully prepared using egg white lysozyme. Henderson
on) et al., -BEsu), hereinafter referred to as Lactobacillus auris,
(abbreviated as) ATCC7469 with 30% egg white lysozyme
By reacting with 'C for 16 hours, L. casei (
(J, Biol, Chem), 252.
No. 3760-3765.1977) repeated this experiment under the conditions described in the above report and observed the morphology of the bacteria using an optical microscope and an electron microscope.According to the results, almost no protoplast formation was observed. In addition, since egg white lysozyme treatment of bacteria of the genus Lactobacillus requires a long treatment time, such a long enzyme treatment significantly inhibits the physiological activity of the bacteria.

In view of the problems of low sensitivity to egg white lysozyme and long processing time in the preparation of protoplasts of bacteria of the genus Lactobacillus, the present inventors conducted intensive research and found that Streptomyces globisvorus (Streptomyces globisvorus)
End-N-acetylmuramidase (end-N-acetyl muramidase) isolated and purified from
The present inventors have completed the present invention by discovering that protoplasts can be obtained in a short period of time and at a high rate by allowing tylmuramidase to act on bacteria belonging to the genus Lactobacillus in the presence of trisaccharides.

To be more specific, (1) When treated with endo-N-acetylmuramidase alone, no formation of protoplasts is observed and only bacteriolytic action is observed; however, treatment with endo-N-acetylmuramidase alone Formation of protoplasts is achieved only in the presence of trisaccharides; in other words, trisaccharides effectively act as osmotic pressure regulators, preventing the destruction of bacterial cells due to osmotic pressure differences during the protoplast preparation process. The presence of these trisaccharides is an essential condition for protoplast formation because they are effective in inhibiting protoplast formation. The present invention was completed based on the findings that protoplasts prepared in the presence of the above-mentioned trisaccharides retain their physiological activity.

Endo-N-acetylmuramidase, which is one component of the present invention, is derived from Streptomyces globisvorus (Streptomyces glo-bisp).
orus), and in the experimental examples and examples described in detail later, it was collected from the culture supernatant of Streptomyces globisvorus (3).
, myces globisporus ) 1829
(manufactured by Dainippon Pharmaceutical Co., Ltd. and commercially available from Seikagaku Kogyo Co., Ltd.; hereinafter referred to as M-1 enzyme) is used.

Bacteria of the genus Lactobacillus, which is another component of the present invention, include all species, including L.
casei (L, casei), L, andophilus (
L. acidophilus ), L. bulgaricus (L. bulgaricus ), L. lactis (
L. 1actis), plantarum),
L, fermentum (L, fermentum)
buchner i), L, previs (L, bre
vis) etc.

For culturing these bacteria of the genus Lactobacillus, a conventional medium and conventional culture conditions may be used.

(4) Furthermore, the trisaccharide as an osmotic pressure adjusting substance, which is the remaining component of the present invention, includes sucrose (shakerose), milk sugar (lactose), maltose, trehalose, and the like.

Next, the present invention will be explained in more detail with reference to experimental examples. In addition, in Experimental Examples 1 to 5,
The test strain was L. casei ATC.
C7469 was used.

EXAMPLE This experiment concerns the basic conditions of M-1 enzyme treatment (pH conditions, type of buffer, temperature conditions, shaking conditions, bacterial suspension concentration, etc.).

In order to examine the enzymatic action of the M-1 enzyme on bacteria of the genus Lactobacillus, L. ca-aei was first cultured using a medium with the following composition under the following culture conditions. That is, in the medium 11, 12 g of nutrient prosthesis (manufactured by Tifco) yeast extract (manufactured by Difco)
5I Tween 80 Punishment to 2HPO<
31KHzPO
41.5g Glucose 0.49M g S
04.7H200,1g Mn SO4" 4H200,025, including j9, with the final voice adjusted to 6.8.

Among the above compositions, glucose, MgSO4・7H20
As for ゜MnSO4.4H60, these were added to the above-mentioned final concentration after autoclaving separately. Cultivation was performed at 37° C. under static conditions, and bacterial growth was measured by changes in absorbance at a wavelength of 650 nm.

For M-1 enzyme treatment, bacteria cultured overnight at 37°C were collected by centrifugation at 4,000Xp for 5 minutes.
2 with 201rLM potassium phosphate buffer (pH 7,0)
After washing twice, add 0.5 to 5.0 μ wet weight/
The collected bacteria were suspended so that the result was as follows.

The suspension was placed in a 20 m/capacity T-shaped tube and heated at 37°C.
After incubation for 5 minutes at room temperature, enzyme treatment was started by adding M-1 enzyme. Regarding the temperature conditions for enzyme treatment, enzyme activity was examined within the range of 25°C to 40°C.

Regarding shaking of the bacterial suspension, a mono-type shaker is used to
It was performed under mild shaking conditions of ~30 cycles/min. The degree of enzyme action was determined by the decrease in absorbance at 650 nm.

Furthermore, protoplasts were prepared under exactly the same treatment conditions as the enzyme treatment described above, except that the enzyme treatment was performed in the presence of a disaccharide. At that time, the degree of protoplast formation was determined by observing the formation of rounded cells under an optical microscope.

The experimental results are shown below.

M-1 enzyme action against bacteria of the genus Lactobacillus occurs at pH 6,
It was observed in the range of 0 to 7.5. It was also confirmed that the type of buffer is unrelated to the enzyme action.

Regarding the temperature conditions for enzyme treatment, strong enzyme activity was observed at high temperatures within the range of 25°C to 40°C. However, in order to maintain the physiological activity of the formed protoplasts, 37
A temperature around 30°C (30°C to 40°C) was suitable.

Regarding the shaking conditions for enzyme treatment, protoplasts were successfully formed under weak shaking conditions of 30 cycles/min or less, preferably 5 to 8 cycles/min.

Regarding the concentration of bacterial suspension for enzyme treatment, 0.5
〜5■ wet weight/d is preferable, especially 1~3■ wet weight/d
d was the most preferred.

EXAMPLE This experiment concerns the effect of cell growth phase on M-1 enzyme action.

FIG. 1(A) shows the growth curve of casei (L, Ca5ei) in the same medium as that for Experimental Example 1.

Figure 1 (B) shows the decrease in turbidity caused by the M-1 enzyme in cells in each growth phase. After collecting the bacteria in each growth phase and washing them, they were diluted with 20 mM phosphate buffer. .5■Wet weight/
Suspend to 1nl and add 1.5μ9/ml to each
The results show the results of treatment with the addition of IILI M-1 enzyme, and the lysis process was followed by a decrease in turbidity. The experimental method was the same as in Experimental Example 1.

The results of this experiment revealed that bacteria cultured for a long time were more sensitive to DoM-1 enzyme treatment. However, in order to obtain protoplasts with high physiological activity, long-term culture is not preferable, and from the late logarithmic growth phase to the stationary phase (Fig.
A) It is desirable to use cells with a culture period of 3 to 6.

EXAMPLE This experiment concerns the effect of enzyme amount on M-1 enzyme action.

FIG. 2 shows the tendency of change in turbidity when M-1 enzyme was applied at varying concentrations to a cell suspension of 3.0 μ wet weight/Tnl. The experimental method was the same as in Experimental Example 1.

By adding 0.38 to 9.0 μfl/ml of M-1 enzyme, a decrease in turbidity was observed in proportion to the amount of enzyme added.

However, when preparing protoplasts, if the amount of enzyme is increased and the decrease in turbidity is increased, the yield of protogelast will be poor.On the other hand, if the amount of enzyme is decreased to suppress the decrease in turbidity, There is a tendency for it to take a long time to form.

Therefore, for the preparation of protoplasts, the cell suspension concentration should be adjusted to 3 wet weight/-I! Moderate i is 0.75~7
It is desirable to add 65 μg/IILI of M-1 enzyme and perform the treatment for 5 to 30 minutes.

EXAMPLE This experiment concerns the effect of various osmolytes on the formation of protogellasts.

Even if M-1 enzyme is added to the cell suspension, no protoplasts are formed, and only bacteriolysis is observed. Therefore, for the formation of protoplasts, it is necessary to add some kind of osmotic pressure regulator. found it to be effective. Therefore, in order to search for osmotic pressure regulating substances suitable for the preparation of protoplasts, we selected NaCl, choline chloride, glycerin, glucose, sucrose, and two types of polyethylene glycols with different molecular weights, and each osmotic pressure regulating substance had an effect on the M-1 enzyme. We investigated the effect on

The experimental method was the same as in Experimental Example 1, except that various osmotic pressure regulators were present, and the cell suspension concentration was adjusted to 2.
51v/ml was selected.

The experimental results are shown in FIG.

As is clear from the figure, the addition of two types of electrolytes (NaCl and choline chloride) was found to inhibit enzyme activity. Moreover, the addition of glycose, glycerin, and two types of polyethylene glycols was accompanied by only the same degree of turbidity reduction as the addition of M-1 enzyme alone.

Among the above-mentioned osmotic regulators, 3-4% protoplast formation was observed only when 0.9 M glucose was added. On the other hand, when 0.9M sucrose was added (9 in Figure 3), M-1 enzyme was added (1.5μg/
At the same time, a rapid decrease in turbidity was observed (ml), and it was confirmed that 95% or more of the cells had become protoplasts 5 to 10 minutes after the enzyme treatment.

From these facts, it has been found that sucrose not only has the effect of effectively suppressing cell destruction due to osmotic pressure difference during the protoplast formation process, but also has the effect of enhancing the cell wall-dissolving action of the M-1 enzyme.

The inhibitory effect on cell destruction caused by the osmotic pressure difference during the protoplast formation process was similarly observed for trisaccharides other than sucrose, such as lactose, trehalose, and maltose.

However, on the other hand, when the enzyme treatment lasted for a long time, the formed protoplasts were destroyed, and a decrease in the turbidity of the cell suspension was observed.

In addition, during the above experiment, determination of protoplast formation was performed by observing with a phase contrast microscope.

EXAMPLE This experiment concerns the enhancement of the lytic action of M-1 enzyme by various sugars.

When M-1 enzyme is activated in the presence of sucrose, L.

It was found that the lytic effect on the cell wall of L. casei (L, Ca5ei) was enhanced.In order to investigate whether such an enhancing effect on the M-1 enzyme action can also be expected for other saccharides, M-1 by various saccharides The presence or absence of an enhancing effect on enzyme action was investigated.

The experimental method was the same as in Experimental Example 1, except that the enzyme treatment was performed in the presence of various sugars.

The experimental results are shown in FIG.

In the presence of four types of monosaccharides (xylose, galactose, fructose, inositol), almost no change in turbidity was observed even after the addition of M-1 enzyme.

Even in the presence of the trisaccharide raffinose, almost no enhancement effect on the M-1 enzyme action was observed.

On the other hand, in the presence of four types of trisaccharides, namely maltose, trehalose J sugar (sucrose), and milk sugar (lactose), an enhancing effect on M-1 enzyme activity was observed, and in particular, sucrose and In the presence of lactose,
The degree of enhancing effect was extremely strong.

From these facts, trisaccharides are not only effective as osmotic pressure regulators to suppress the destructive effect of protoplasts due to osmotic pressure differences, but also have the effect of enhancing the cell wall lytic effect of M-1 enzyme against bacteria of the genus Lactobacillus. It turned out that it has.

Note that there are differences in the enhancing effect on the enzyme action described above depending on the type of bacteria of the genus Lactobacillus.

For example, L, helveticus (L, helvet 1
cus ), L. salivarius (L. sal 1va
rius), L. plantarum (TJ, plant
arum), L. fermentum (L.

A similar enhancing effect on the M-1 enzyme action has been observed for many other Lactobacillus genus bacteria, including L.

acidophilus), L, bulgaricus (L,
bulgari-cus), no enhancing effect on the M-1 enzyme action was observed. (See Table 1 in Experimental Example 6 below.) The experiment in this example concerns the difference in M-1 enzyme action depending on the type of bacteria.

The sensitivity of various Lactobacillus bacteria to the M-1 enzyme and the state of protoplast formation were observed according to Experimental Examples 1 and 4.

Regarding the sensitivity of cells to the M-1 enzyme, the degree of turbidity reduction in the presence of 0.85 M sucrose in the buffer solution and in the case of no sucrose addition, and regarding the protoplast formation rate, Table 1 summarizes the results of microscopic observation using a phase contrast microscope in the presence of .85M sucrose.

As is clear from Table 1, L. acidophilus (L.
acidophi Ius), L. helveticus (L.

helveticus), L. salivarius (L.
sal 1varius), L. plantarum (L.
plantarum), L, casei (L, casei)
i), L, fermentum (L, fermentu)
m), L, bulgaricus (L, bulgaricu)
A high rate of protoplast formation was confirmed for almost all bacteria of the genus Lactobacillus, including Lactobacillus s.

Table 1 Protogellast formation in various Lactobacillus bacteria 1) Sue indicates the absence of sucrose,
u c indicates the presence of sucrose.

Note 2) Shi, Ju, Ushi, and Kan are based on the observer's sensitive evaluation of the strength of the cell wall lytic effect of the M-1 enzyme on various lactic acid bacteria (the degree to which the turbidity decreases as a result of an 8-minute reaction). As shown, there is an increasing tendency from left to right.

Regarding L. acidophilus and L. bulgaricus, no influence of the presence or absence of sucrose on the sensitivity to M-1 enzyme was observed.

Example 1 As a test strain, L. casei (L, Ca5ei)
Using ATCC7469, add nutrient broth (manufactured by Difco) to the medium 121! , yeast extract (
Tifco￥rJn 5Ji', Tween 801 m1
%! HPO4311, KH2PO41,5&, glucose-0°411%Mg5O, 7Ht0.0.19,
MnSO4-4HtOo, containing 025 g, final)
The above test bacteria were cultured in a medium whose p)I was adjusted to 6.8.

Among the above medium composition, glucose, MgSO4・7Ht
Regarding O1MrL804''4HtO, each was sterilized under high pressure and added to the above final concentration.

Cultivation was performed at 37° C. under static conditions.

- Bacteria cultured in the apparatus overnight were collected by centrifugation at 4.0OOX for 95 minutes, washed twice with 20TrLM potassium phosphate buffer (pH 7.0), and then added to the above buffer at 2.5
The cells were suspended in a greenhouse/Wtl.

Enzyme treatment was started by adding 10 mA' of the bacterial suspension to a 20-volume T-shaped tube, pre-incubating at 37°C for 5 minutes, and then adding 3 μI/d of M-1 enzyme. did.

Such enzymatic treatment was carried out in the presence of 1.0 M sucrose and
The mixture was incubated for 30 minutes under gentle shaking at cycles/minute.

When the protoplast formation rate was determined by the same method as in Experimental Example 4, the value was 95%.

Example 2 As a test strain, L. helveticus (L, he+-v
eticus) was used to collect bacteria cultured in the same medium and under the same conditions as in Example 1.
v wet weight/ml, and the bacterial suspension was diluted with 1 Mg/ml under gentle shaking conditions of 8 cycles/min for 15 minutes at 37°C in the presence of 1.0 M lactose. M- of d
Protoplasts were prepared by treatment with 1 enzyme.

The protogellast formation rate was determined by microscopic observation similar to that in Experimental Example 4, and the value was 95%.

Example 3 (19) As a test strain, L. casei
When ATCC7469 was used and treated in the same manner as in Example 2, a protoplast formation rate of 90% was confirmed.

[Brief explanation of drawings]

Figure 1 (A) is a turbidity curve representing the growth stages of L. casei, and Figure 1 (B) is a turbidity curve representing the sensitivity of cells to the M-1 enzyme at each growth stage. . Figure 2 shows the turbidity curve showing the influence of the amount of M-1 enzyme on the lytic action of L. casei;
The figure shows the turbidity curve showing the influence of various osmotic pressure regulators on the bacteriolytic action of the M-1 enzyme, and Figure 4 shows the turbidity curve showing the influence of various saccharides as osmotic pressure regulating substances on the action of the M-1 enzyme. It is a degree curve. In FIG. 1, each curve specified by each number in FIG. 1 (8) corresponds to each growth stage ( 20) concerning bacteria. In Figure 2, 1...No enzyme added, 2...0,3
8μI/- addition, 3...0.75μg/P addition, 4.
...1.5 μm1/It added, 5...3.0 μI/G added, 6...4.54/d added, 7...6.0 μl
/ml added, 8...7, 5μE/ml added, 9...
9.0μm1/ml addition In Figure 3, 1...20
mm! No enzyme added to J acid buffer, 2...20
mm! 1.5μg/dM-1 enzyme added to J phosphate buffer, 3-9...20mM The following various substances were added to phosphate buffer, and enzyme was added, i.e. 3...0
．． 45 M NaCl, 4-0.45 M Choline Chloride,
5...0.9M glycerin, 6...0.9M glucose, 7...0.5M polyethylene glycol (
Average molecular weight 300), 8...0.5M polyethylene glycol (average molecular weight 1000), 9...0.9M sucrose In Figure 4, 1... Control (no sugars and M-1 enzyme added), 2 ...Control (no sugar added, 1.5μ9/d
M-1 enzyme addition), 3... xylose addition, 4...
Galactose addition, 5...Fructose addition, 6...
・Inositol addition, 7... Raffinose addition, 8.
...Addition of maltose, 9...Addition of trehalose, 10
...Lactose addition, 11...Sucrose addition. (The amount of each addition is 0.5 M) Patent applicant: Yakult Honsha Co., Ltd. (23) Figure 1 (A) @ Tomo (h') Figure 1 (B) Bleaching time (min) Figure 2 Tomo Maki (h') n) Fig. 3 Interlayer (mjn) Fig. 4 Crossing M (min) Continuation of page 1 0 Inventor Yasuhiro Kanemasa 2-5-1 Shikata-cho, Okayama-shi Oka Ryo University School of Medicine Applicant Dainippon Pharmaceutical Co., Ltd. 3-25 Doshomachi, Higashi-ku, Osaka City

Claims (2)

[Claims] (1) Streptomyces globisvorus (St
-reptomyces globisporus)
by allowing endo-N-acetylmuramidase produced by to act on bacteria in the Lactobacillus range in the presence of an osmotic pressure regulator,
The method for preparing protoplasts of bacteria in the Lactobacillus range, wherein the osmotic pressure regulating substance is a trisaccharide. (2) The method for preparing protoplasts of Lactobacillus bacteria according to claim 1, wherein the trisaccharide as the osmotic pressure regulating substance is sucrose or lactose.