JPH0638745A - Neutral phytase and its production - Google Patents

Abstract

PURPOSE:To provide the neutral phytase produced with Bacillus natto, useful for improving the qualities of feeds, beverages and foods and for preventing environmental pollution, and having action pHs in a specific pH range. CONSTITUTION:Bacillus.natto N-77 (FERN P-12751) is aerobically cultured in a medium containing a calcium salt and D-mannose at 37-40 deg.C to produce the objective phytase having action pHs within a pH range of 5-8.5.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a neutral phytase used for improving the quality of feed and food and drink and preventing environmental pollution, as well as a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, 18 to 88% of phosphoric acid contained in beans and cereals is storage type phytic acid (6-phosphate ester of myo-inositol) [Advance in.
Food Research (Adv. Food Res.) 2
8: 1 (1982)]. Therefore, even if livestock or humans ingest the feed or food or drink containing the storage-type phytic acid, the storage-type phytic acid is excreted in the feces as it is,
Not only is the waste of phosphoric acid, which is a constituent of the storage type phytic acid, not utilized, it causes problems such as environmental pollution due to phosphoric acid in phytic acid contained in the feces. Furthermore, the phytic acid has the disadvantage of inhibiting the activity of digestive enzymes when ingested by livestock and humans. Therefore, conventionally, for example, in feed, it is generally supplemented with a phosphoric acid content that is deficient as nutrients in the form of calcium phosphate,
As a result, it becomes an expensive compounded feed, which is not preferable. on the other hand,
In order to solve the above-mentioned problems, Poultry Sci. 47: 1842 (196)
8); Journal of Nutrition (J. Nu
tr. ) 101: 1289 (1971); Food magazine 32:
174 (1985); British Journal of Nutrition (Br. J. Nutr) 64: 5.
25 (1990); Nutrition and Food 21:24 (196
8); Agricultural and Biological
Chemistry (Agr. Biol. Chem) 32: 1
275 (1968); Acta Microb. Pol. 27:41.
(1978); Preparative Biochemistry (P
rep. Biochem. ) 17:63 (1987), the acid phytase derived from a microorganism is added to feeds or foods or their raw materials, and the phytic acid contained therein is decomposed to be used in livestock or humans. It has been reported that the available effective phosphoric acid is increased and the amount of excreted phytic acid, and thus the amount of phosphoric acid, is reduced to prevent environmental pollution.

[0003]

However, the previously reported microorganism-derived acid phytase has a significantly low activity in the weakly acidic to weakly alkaline range because its action pH is on the strongly acidic side. It is not suitable for degrading cereals and beans, which are the main ingredients of feed and food processing raw materials in the pH range, and therefore it is necessary to adjust the pH of such an object to be treated to a strong acidity. Not only is it troublesome, but under acidic conditions, it causes denaturation of the object to be treated, and the resulting decomposed product is sour that it is often not suitable for daily intake by livestock and humans. , Its use is limited to acidic drinks and acidic foods. Also, acid phytase is active in the stomach of livestock and humans,
In the neutral to weakly alkaline intestinal tract that controls the absorption of phosphoric acid, the activity is remarkably reduced, and phytic acid is hardly decomposed. Therefore, in place of such acid phytase, it is desired to use neutral phytase which is active in the range of weak acidity to weak alkalinity suitable for general feed, food and drink, and intestinal pH. In this respect, it is conventionally known that some plant-derived phytase has such neutral and active activity.
ysiol. 45: 4 (1970); Biokim. Eto Biophysica Actor (Biochim. Biop).
hys. Acta) 302: 259 (1973), but since it is extracted from plants, the yield is low and the cost is high, which is not suitable for practical use. Therefore, in view of the above conventional circumstances, in the processing of feed and food and drink, added without performing the pH adjustment work, the decomposition activity of the phytic acid contained is extremely high, enzymatically decomposed products easily without quality deterioration Further, it is desired that a neutral phytase which is obtained in human intestines and has a weak activity in the intestinal tract, and which acts in a weakly acidic to weakly alkaline manner, can be produced at a high yield and at a low cost.

[0004]

The present invention solves the above-mentioned conventional problems and provides a neutral phytase satisfying the above-mentioned needs, and has a pH of 5 to 8.5 produced by Bacillus natto.
Exists in a neutral phytase having a working pH in the range of. The present invention also provides a method for producing the neutral phytase, which comprises culturing in a medium containing a calcium salt.

[0005]

Since the neutral phytase of the present invention is produced by Bacillus natto, it can be mass-produced easily and inexpensively. In addition, since its action pH is in the range of 5 to 8.5, it matches the pH of cereals and beans that are the main components of most feeds and foods or drinks, or the pH of the intestinal tract of livestock and humans, which is in such a pH range. Therefore, it can be immediately added to the processing raw material, and when kept at its working temperature, the decomposition of phytic acid is favorably carried out, and a decomposition-treated product containing phosphoric acid or a phosphate salt liberated is obtained. When livestock or humans ingest the feed or food or drink to which the neutral phytase is added, the phytase is reactivated in the intestinal tract, and the phytate contained therein is satisfactorily decomposed to produce phosphate. It is released and these are effectively ingested from the intestine and used effectively. Also,
As a result, the amount of phytic acid excreted outside the body decreases,
Environmental pollution is prevented as much. Examples of the Bacillus natto of the present invention include Bacillus natto.
Atto) N-77 (No. 12751 from Microbiology Research Institute) is used.

The neutral phytase of the present invention can be produced reliably and satisfactorily by culturing the Bacillus natto in a medium containing a calcium salt.
Higher yields of neutral phytase are produced when cultured in medium containing mannose.

[0007]

EXAMPLES Next, examples of the present invention will be described in detail. The present invention is
As a result of culturing and searching each isolate as described in detail below, natase phytase shows remarkable activity in the pH range of 5 to 8.5 regardless of the strain (hereinafter referred to as neutral phytase). Were found to produce Thus, Bacillus natto is commercially available and can be obtained very easily, and its culture method is easily performed by any known method, so that it is possible to produce neutral phytase in an extremely easy and high yield. Brings the convenience of being able to produce dynamically. It was found that the medium can be satisfactorily cultured if it contains calcium salt, and that the production of neutral phytase can be increased particularly in the medium containing calcium salt and D-mannose. The medium may be a liquid medium, a semi-solid medium, a solid medium, various feeds such as beans and cereals,
It is also possible to use a natural semi-solid medium that uses a food material, for example, soybean meal, malt, or the like containing water. Also,
If a medium containing no glutamic acid is used, a non-viscous decomposition product is obtained, and subsequent handling such as recovery of neutral phytase is convenient. The culturing temperature of Bacillus natto may be a desired temperature at which neutral phytase is produced,
It is preferable to culture aerobically at 40 ° C.

As described above, Bacillus natto produces neutral phytase, but the strain may be of any kind. As a result of isolating a large number of strains of natto from commercially available natto in various places in Japan and conducting a search by the agar block method described in the below-mentioned Example, it was confirmed that they have a neutral phytase-producing ability. Below, as a typical example, Bacillus natto (Bacillus natt)
o) The mycological properties of N-77 strain (No. 12751 from Microbiology Research Institute) were examined and the results are shown in Table 1 below.

[0009]

[Table 1]

As is clear from Table 1, it was confirmed that the natto had the general mycological properties.

Since the neutral phytase of the present invention is an extracellular enzyme secreted by Bacillus natto, it can be separated from the bacterial cells, collected and purified by conventional means, if necessary. For example, a culture obtained by culturing Bacillus natto in a liquid medium is separated into cells by centrifugation or the like, and the culture supernatant is subjected to a salting out method (addition of ammonium sulfate or the like) or a solvent precipitation method (acetone, ethanol). Neutral phytase as a precipitate, or as a concentrate containing neutral phytase by an ultrafiltration method, and if necessary, the precipitate or the concentrate is subjected to gel filtration, ion It can be recovered as a purified product of neutral phytase by appropriately combining exchange chromatography, affinity chromatography and the like.

A culture containing a neutral phytase obtained by culturing Bacillus natto using the above various media can be used by pulverizing it if it is a solid substance, and if it is a liquid substance, it is usually a bacterium. After separation and removal, the pulverized product or liquid product is mixed with, for example, a desired amount of the compounded feed, and the mixture is stirred at the action temperature of neutral phytase to decompose phytic acid contained in the feed for a desired time, It is used to produce decomposition products containing free phosphoric acid. Further, as described above, what is made into a crude product or a purified product such as a concentrate of neutral phytase from the culture supernatant is appropriately added to feed or food / beverage processing raw material or food / beverage,
Alternatively, it is used by directly supplying it to livestock or humans in the form of powder, tablets, capsules or the like. Thus, since the neutral phytase of the present invention acts at pH 5 to 8.5, when it is added to a general feed or food / beverage raw material mainly composed of cereals and beans, which has a similar pH range, it immediately shows remarkable activity. Indicates. For example, if it is added to soy milk which is a non-acidic beverage as it is, it shows remarkable activity. As in the case of using conventional acid phytase, the acid of soy milk is adjusted to pH 2 to 3 suitable for the activity. The adjustment work can be omitted and the protein denaturation of soymilk due to strong acidity is eliminated, resulting in a delicious product. Further, when a decomposition product is obtained by decomposing phytic acid into a feed or a food and drink processing raw material, it causes an increase in phosphoric acid, so that it is not necessary to add a phosphoric acid component such as calcium phosphate as in the conventional case, Alternatively, the amount added can be reduced, which leads to improvement of feed and food and drink. Furthermore, when the neutral phytase is ingested by livestock and humans by the various means described above,
In the intestinal tract, for a relatively long period of time, it shows remarkable activity against phytic acid contained in feed and food and drink due to neutral phytase, and can effectively absorb and utilize the separated phosphate, while in feces excreted outside the body. The amount of phytic acid can be reduced, and thus the environmental pollution due to phosphoric acid can be prevented.

The present invention will be described in more detail with reference to specific examples. Example 1 Search for phytase-producing bacteria 2 mM sodium phytate (Aldrich), 10
mM Cacl ₂ · 2H ₂ was sterilized dissolved 1.5% agarose containing 0.1 M Tris-Hcl buffer pH 7.0 consisting of O, poured into a sterile petri dish, about 3
It was solidified to a thickness of mm. On the other hand, 10m sterilized and solidified
A large number of 8 mm diameter × 5 mm thick agar blocks were hollowed out from a HIA (heart infusion agar, Difco) plate containing M CaCl ₂ .2H ₂ O by a sterile cork borer and placed on the agarose plate. After inoculating each isolate on each agar block, cover the dish and
It was cultured at 37 ° C for 3 days. Next, after removing each agar block, in order to detect the phosphoric acid released in the agarose plate, a coloring solution ( ₄ % of 1.5% ammonium molybdate / 5.5% H ₂ SO ₄ was added, A mixture of 2.7% ferrous sulfate / H ₂ O (1 volume mixture) was poured in 2-3 ml to develop a color (formation of a molybdic acid reduced blue complex).
The neutral phytase-producing ability of each strain was judged from the size and shade of the blue colored circle. As a result, all the strains of Bacillus natto isolated from natto in various regions in Japan showed strong phytase-producing ability. In contrast, Bacillus subtilis (Bacill)
The phytase-producing ability was scarcely found in each of the isolates other than natto, such as the Bacillus cereus isolate, the Bacillus cereus isolate, and the Bacillus cereus isolate.

Method for measuring Bacillus natto phytase activity The phytase activity produced by Bacillus natto was measured as follows. That is, 2 mM sodium phytate (Ald
Rich Tris-HCl (pH) (pH)
7.0) Incubate 600 μl of buffer solution at 37 ° C., and add phytase sample 150 such as supernatant of culture solution of Natto
After adding μl and reacting at 37 ° C. for 15 to 30 minutes,
The reaction was stopped by adding 750 μl of 5% trichloroacetic acid. The same color developing solution 1.
After adding 5 ml and leaving it for 10 minutes at room temperature, colorimetric determination (O
D _{700 nm} ). The protein was measured by "BCA protein quantification reagent" (Funakoshi), and the phytase activity unit (U) was expressed as the amount of phosphate (μM) released by 1 mg of enzyme protein per minute.

Examples of phytase production by Bacillus natto N-77 strain are shown in Examples 2 to 4 below. Example 2 1000 ml Erlenmeyer containing 500 ml of HI (heart infusion, Difco) broth supplemented with 10 mM CaCl ₂ .2H ₂ O, 1% D-mannose, 1% glucose and 1.5% yeast extract. Inoculate the flask with spores of strain N-77 (OD
_660nm = 0.005, Shimadzu Spectronic
20A colorimeter and shake culture at 37 ° C (150 rp)
Then, the bacterial amount and the phytase activity in the culture supernatant were measured daily. The result is shown in FIG. As a result, culture 5
It was found to show maximal phytase production on day 7.

Example 3 To examine the effect of Ca ⁺⁺ addition on phytase production, HI broth containing 1% glucose without CaCl ₂ .2H ₂ O and 0.5% yeast extract was used, and CaCl ₂ · 2H ₂ O with the above composition
Was added in the same manner as in Example 2 except that the HI medium was added by changing the addition concentration of each of them in the range of 1.3 mM to 40 mM.
The −77 strain was cultured, and the bacterial amount and the phytase activity in the culture supernatant were measured daily. The measurement results of phytase activity on the 5th day of culture are shown in FIG. As is clear from the figure, addition of Ca ⁺⁺ is effective for phytase production, and
Phytase production increases with increasing Ca ⁺⁺ concentration,
Particularly, the concentration of CaCl ₂ · 2H ₂ O added is 1.3 mM to 1
In the 5 mM range, phytase production was especially increased,
It was found that the phytase-producing ability was good.

Example 4 Next, in order to investigate the effect of adding D-mannose on the production of phytase, 10 mM CaCl ₂ .2H ₂ O and 1 were added.
In the same manner as in Example 2, N-broth was added in the same manner as in Example 2 except that the concentrations of D-mannose added to HI broth containing 0.5% glucose and 0.5% yeast extract were changed in the range of 0.5 to 4%. The -77 strain was cultured. The measurement results of phytase activity on day 5 of culture are shown in FIG. As is clear from the figure, it was found that the addition of D-mannose further increased the phytase production, resulting in an increased phytase productivity.

That is, according to the above comparison test, the medium contains C
by incorporating both a a ⁺⁺ and D- mannose, compared with media containing non or Ca ⁺⁺ alone containing Ca ^++, it is possible to perform the cultivation of Bacillus natto particularly well, phytase production is significantly increased I found out that

Example 5 Purification of Bacillus natto phytase Supernatant of culture of N-77 strain cultured in Example 2 above 20
Ultrafiltration from 0 ml (YM3 membrane, Amicon), Se
phadex G-100 (Pharmacia) chromatography and DEAE-Sepharose C
The phytase was purified to a single protein using L-6B (Pharmacia) chromatography. Table 2 shows the measurement results of each activity at each stage until the purification.

[0020]

[Table 2]

When the properties of the purified phytase obtained in Example 5 were examined, it was found that it had the properties listed below. (1) Molecular weight 36,000 to 38,000 As a result of measurement by gel filtration and SDS-polyacrylamide gel electrophoresis, the molecular weight of phytase was 36, as shown in FIGS. 4 (a) and 4 (b). 000-38,000
Met. (2) Isoelectric point 6.25 (by isoelectric focusing method) (3) Working pH and pH stability The present enzyme contains 1 mM CaCl ₂ .2H ₂ O.
1 M maleic acid-Tris-NaOH buffer, 0.1
MTris-HCl buffer and 0.1 M glycine-N
The relationship with the relative activity due to the change in pH when the aOH buffer was treated for 15 minutes at 37 ° C. was examined. The result was as shown in FIG. In the figure, A is a 0.1 M maleic acid-Tris-NaOH buffer solution, B is a 0.1 M Tris-HCl buffer solution, and C is a 0.1 M glycine-NaOH buffer solution. As is clear from this, the action pH of this enzyme is 5
~ 8.5, the optimum pH is about 6-8. In addition, the present enzyme was further added to each of the above three types of buffer solutions, 0.1 M glycine-HC.
1 buffer and 0.1 M formic acid-NaOH buffer,
The relationship with the residual activity due to the change in pH when treated at 25 ° C. for 20 hours was examined. The result was as shown in FIG. In the drawing, D is 0.1 M glycine-HCl
Buffer E shows respective relationship curves when 0.1 M formic acid-NaOH buffer was used. As you can see,
The enzyme has a pH of 5 to 11 and is stable. (4) Action temperature and temperature stability This enzyme was added to 0.1M Tris-Hcl-1mM CaC.
The relative activity and the residual activity were measured after changing the treatment temperature for 15 minutes in a buffer solution containing 1 · ₂ · 2H ₂ O and having a pH of 7.0 and holding each temperature for 15 minutes. The result is shown in Fig. 7.
Shown in. As is clear from the figure, the action temperature increases the activity as the temperature rises, the optimum temperature is 50 to 60 ° C or around, and the temperature stability is stable up to around 50 ° C. The residual activity was 40% even at 60 ° C. (5) Michaelis constant (Km) and activation energy (ΔE) Km = 500 μM (37 ° C.) and ΔE = 9.87 Kca
It was calculated as 1. (6) Substrate specificity 0.02 U / ml of the present enzyme was added to each of the above pH 7.0 buffer solutions containing 2 mM of each substrate shown in Table 3, and 37
After reacting at 20 ° C. for 20 minutes, the relative decomposition activity was measured.
As a result, as shown in Table 3, the phytic acid showed a decomposing activity, but the substrate for phosphatase, sodium p-nitrophenylphosphate, showed no activity.

[0022]

[Table 3]

As is clear from the above, the phytase produced by Bacillus natto is stable in the weakly acidic to weakly alkaline range of pH 5 to 8.5, and exhibits remarkable activity against phytic acid in such a range. It is found to be sex phytase.

Next, the action of degrading phytic acid when the natto-producing neutral phytase of the present invention is added to the object to be treated will be described. Example of Action Soymilk as a material to be treated, and crushed soybean meal, rapeseed meal, and defatted rice bran were added to water at 15% (w / v) to prepare respective suspensions, and neutral phytase prepared in advance was prepared. The purified powder was added to each of the above-mentioned objects to be treated at 1.1 U / ml, and
After reacting at 6 ° C. for 6 hours, free phosphoric acid was measured. On the other hand, after the purified powder of the neutral phytase was not added to each of the above-mentioned objects to be treated and kept at 50 ° C. for 6 hours, the amount of free phosphoric acid was quantified, and the free phytase reaction to each of the above-mentioned objects was released. The amount of phosphoric acid was calculated. The results are shown in Table 4 below.

[0025]

[Table 4]

As is clear from Table 4, phytic acid contained in the substance to be treated is effectively decomposed by this enzyme.

As is clear from the above, the neutral phytase produced by culturing Bacillus natto according to the present invention contains phytic acid contained in various livestock feeds, various human drink ingredients, food processing ingredients, and foods and drinks. Since it can be decomposed and liberate phosphoric acid, it can be decomposed after addition of the neutral phytase, or when it is immediately fed to livestock or humans without the decomposition treatment, it was previously decomposed in the intestinal tract. Phosphoric acid is immediately absorbed and utilized, and neutral phytase is remarkably activated in the intestine to decompose phytic acid in the intestinal tract, and its free phosphoric acid is absorbed and utilized, and as a result, phytic acid contained in feed, drink, or food is absorbed. The amount of phytic acid excreted outside the body is reduced, and it helps prevent pollution of the environment by phosphoric acid.

In this case, the use form of the neutral phytase is
It is used as a purified enzyme powder, tablets, capsules, etc., to be fed to livestock or humans alone, and when added to feed or food and drink, a liquid or solid culture in which Bacillus natto is cultivated Alternatively, a liquid containing a neutral phytase from which cells are removed may be supplied as a feed or a food or drink as a concentrate or a concentrate, and then dried and added in a powder form in a predetermined amount. It is possible to add it to the feed or beverage or the raw material for food processing to which it is added before feeding, hold it at about 50 to 60 ° C for a predetermined time, and perform a decomposition reaction by neutral phytase before use. be able to.

[0029]

As described above, the neutral phytase of the present invention is
By culturing Bacillus subtilis natto that can be easily and inexpensively obtained and is easy to culture, it can be mass-produced easily and inexpensively, and the neutral phytase has a pH of 5 which is suitable for most feeds and foods and drinks. Since it has a working pH in the range of ~ 8.5, it is possible to easily perform the decomposition of the contained phytic acid easily without the work of adjusting the acidity of the object to be treated necessary when using acid phytase. Can obtain feed or food and drink with improved quality, decompose phytic acid in the intestines of livestock and humans, and effectively utilize free phosphoric acid, while reducing the amount of excreted phytic acid. It is possible to reduce the amount of phosphoric acid, such as calcium phosphate, which has been conventionally mixed in feed, while reducing environmental pollution.

[Brief description of drawings]

FIG. 1 is a graph showing the daily changes in the amount of neutral phytase and the amount of bacteria produced by the culture of Bacillus natto.

FIG. 2 is a graph showing the relationship between the added concentration of Ca ^{++ in} the medium and the production of neutral phytase.

FIG. 3 is a graph showing the relationship between the concentration of D-mannose added in the medium and the production of neutral phytase.

FIG. 4 (a) is a graph showing the molecular weight of purified neutral phytase measured by gel filtration.

FIG. 4 (b) is a graph showing the molecular weight of purified neutral phytase measured by SDS-polyacrylamide gel electrophoresis.

FIG. 5 is a graph showing the relative activity of purified neutral phytase at each pH.

FIG. 6 is a graph showing the residual activity of purified neutral phytase after treatment at each pH.

FIG. 7 is a graph showing the relative activity of purified neutral phytase at each reaction temperature and the residual activity after treatment at each temperature.

Claims (4)

[Claims] 1. A pH of 5 to 8.5 produced by Bacillus natto.
Phytase with a working pH in the range of. 2. A method for producing neutral phytase, which comprises culturing Bacillus natto in a medium containing a calcium salt. 3. The method for producing neutral phytase according to claim 2, which comprises culturing in a medium containing calcium salt and D-mannose. 4. Bacillus natto
natto) N-77 (Ministry of Industrial Science, Microbiology No. 12751)
The method for producing the neutral phytase according to claim 2 or 3.