JPH0761273B2 - Phospholipid modification method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for modifying a phospholipid, which comprises reacting a phospholipid with a lipase to convert at least a part of the phospholipid into a lyso-type phospholipid.

(Prior Art) Lysophospholipid is a partially decomposed phospholipid obtained by hydrolyzing a part of a fatty acid bonded to a glycerin residue of the phospholipid. Such lysophospholipids have increased water solubility
It has been reported that excellent properties are imparted such that the emulsifiability of the O / W type becomes strong and the emulsifying power does not decrease even when ions such as calcium and magnesium coexist at high concentrations (J. Amer.Oil Chem.Soc. 53 , 425.1976), examples of the application of this are milk replacer (see above), mayonnaise thermal stability improvement (J. Sci. Food Agric. 32 , 451.198).
1), a bread-making improving agent (JP-A-60-30644) and the like are known.

There are two methods of hydrolysis for converting phospholipids into lyso-type phospholipids, which are scientific methods using acids, alkalis, etc. and methods using enzymes, but chemical methods have no specificity for the decomposition reaction, so lyso-type The yield of phospholipid is low, and the reaction requires high temperature, which causes the inconvenience that the decomposition products are colored,
Today it is barely neglected.

Phospholipase is known as an enzyme that decomposes phospholipids. The enzyme is further subdivided according to its action site. Of these, phospholipase A which selectively hydrolyzes fatty acids bound to the 1-position and 2-position of a glycerol residue in order to decompose phospholipids into lyso-type phospholipids, respectively. Either ₁ , or A ₂ , or those with phospholipase B activity that hydrolyzes both the 1 and 2 positions are required.

It has long been known that enzymes having phospholipase A or B activity are present in snake venom and bee venom, but other than this, pancreatic lipase, Rhizop dermer (Rhizop
The presence of the activity has been reported only in a small number of lipases of microbial origin such as us delemar), Rhizopus alhizus (R.arrhizus) and Mucor javanicus (Mucor javanicus). Currently, some lyso-type phospholipids, which are obtained by degrading phospholipids using enzymes, are also commercially available, but this uses porcine pancreatic phospholipase A ₂ , and modification of phospholipids using lipase of microbial origin. Is not yet done industrially.

(Problems to be Solved by the Invention) The present invention broadly searches industrially available lipases of microbial origin to decompose a phospholipid into a lyso-type phospholipid, which is a strong follilipase A or B.
It is intended to find a lipase agent having an activity, and thus to open a road to practical use for modification by modifying an phospholipid into a lyso-type phospholipid using an enzyme.

(Structure of the Invention) The present invention relates to a phospholipid, a microorganism belonging to the genus Aspergillus or the genus Pseudomonas, Rhizopus javanicus (Rh
izopus javanicus) Rhizopus niv
eus) and Mucor Mehhi produced lipase produced by the action of at least a part of the lyso-type phospholipid. The microorganisms belonging to the genus Alpergillus are Aspergillus niger or Aspergillus wenti, and the microorganisms belonging to the genus Pseudomonas are Pseudomonus fluorescence or Pseudomonus fragi. It is particularly advantageous because the lipase produced has a high phospholipase activity. Many of the enzymes produced by these microorganisms are commercially available and can be used in the present invention.

Phospholipase activities of lipase agents obtained from these microorganisms include those that preferentially hydrolyze the 1-position fatty acid of phospholipids and that that hydrolyze both 1- and 2-position fatty acids almost uniformly depending on their origin. Examples of the former are lipases produced by Aspergillus niger, Rhizopus japonicas and Niveus, and examples of the latter are Pseudomonas fluorescens and frage. To produce a large amount of lysophospholipids only,
It is advantageous to use the former type of lipase.

The phospholipid raw material for obtaining the lyso-type phospholipid by acting such an enzyme includes, for example, soybean lecithin, commercially available lecithin of animal and plant origin such as egg yolk lecithin, and a degumming step that is one of the steps for refining vegetable oil. The so-called extracted slag obtained can also be used. The reaction is optimal for water or enzymes
Dispersed in a concentration of phospholipids 1-50 wt% buffer PH, per phospholipids 1g to the dispersion, the method of Yamada et al [Japan Agricultural Chemistry Journal 36, 860.1962] lipase utilization measured by 10000 units Add an amount of lipase corresponding to, while shaking the reaction solution 20 ~ 60 ℃, more preferably 25 ~ 40 ℃
It is recommended to react for 3 to 24 hours.

The reaction liquid after a lapse of a predetermined time can be used as it is as kneading water for the dough when it is used for, for example, baking, but in general, the lipase is inactivated by heating if necessary, and further if necessary. Concentration by an appropriate concentration machine such as thin layer concentration, and further appropriate drying means such as spray drying and freeze drying may be performed to obtain a concentrated or dried product.

The present invention will be specifically described below with reference to examples.

(Example) Example 1 Phospholipid 1-palmitoyl 2-oleylphosphatidylcholine 10 mg and commercially available lipase agent 400 units of each microbial origin listed in Table 1 were mixed with 0.1 M phosphate buffer (PH7.0) 1 ml.
It was dispersed in the solution and allowed to react for 3 hours with stirring in a 37 ° C constant temperature bath. After a lapse of time, the reaction solution was freeze-dried, and 2 mg of stearic acid as an internal standard substance was added to the chloroform-methanol (2: 1) extract solution of this product, which was spotted on a silica gel thin layer plate, and chloroform: methanol: water (60:30 : 3) was used as a developing solvent to perform thin layer chromatography. After deployment,
The free fatty acid fraction and the lysophosphatidylcholine fraction were scraped off.For the former, after fatty acid was methyl esterified by the trifluoromethanol method, palmitic acid and oleic acid were quantified by gas chromatography. After decomposition, phosphorus was quantified to calculate the amount of lysophosphatidylcholine produced. The results are summarized in Table 1.

As seen in Table 1, the microorganisms belonging to the genus Aspergillus and the genus Pseudomonas according to the present invention, the lipase agents obtained from Rhizopus javanicus, Niveus, and Mucor Mihai all have phospholipase activity, and phospholipid 1- By acting on palmitoyl 2-oleylphosphatidylcholine, lysophosphatidylcholine (abbreviated as lysoPC in the table) which is a lysophospholipid was produced.

On the other hand, lipases produced by Candida cylindracea, Chromobacterium viscosum, Arthrobacter ureafaciens and the like had little or no phospholipase activity and were not practically applicable.

In addition, even in lipase agents having phospholipase activity, there is a considerable difference in the ability to produce lysophosphatidylcholine depending on the mechanism of action, and in order to produce a large amount of lyso-type phospholipids, especially the 1st fatty acid of phospholipids is preferentially It was suggested that it is preferable to use a lipase agent of the type that decomposes (in which the amount of palmitic acid (fatty acid at 1st position) is higher than that of oleic acid (fatty acid at 2nd position) in the column of free fatty acid composition in Table 1).

Example 2 Soybean phospholipid (Sternper PM manufactured by Sternchemy, Germany)
10% (W / W) buffer solution of 1000 g each of 1,000 units of each lipase of microbial origin listed in Table 2 was added to 50 g of the dispersion, and the mixture was reacted in a rotary shaker at 37 ° C. for 24 hours.

* Buffer 0.1M phosphate buffer (PH7.0) for Pseudomonas fluorescens, FRAGE, Rhizopus javanicus, and Niveus produced enzyme preparations, 0.1M acetate buffer (PH7.0) for Aspergillus niger produced enzyme preparations ( PH5.6) is used.

After the passage of time, freeze-drying was performed, and the chloroform-methanol extract was subjected to chloroform-methanol-water (65: 2
Two-dimensional thin layer chromatography was carried out using two developing solvents of 5: 4) and butanol-acetic acid-water (60:20:20). Phosphatidylcon (PC), Phosphatidylethanolamine (PE), Lysophosphatidylcholine (lyso
PC) and lysophosphatidylethanolamine (lyso
Each composition of PE) was scraped, and phosphorus was quantified after decomposition of perchloric acid to determine the composition ratio of each. The results are shown in Table 2.

As shown in Table 2, even when soybean phospholipid is used as the substrate, the lipases of microbial origin according to the present invention all produce lyso-type phospholipids, but (lyso PC + lyso PE) / (PC + PE + lyso PC + Aspergillus niger had the highest “riso conversion rate” calculated by Rhizo PE), and below was Rhizopus javanicus and Mucor.
The results of Example 1 above in which the use of a lipase that preferentially acts on the 1-position fatty acid in order to obtain a lyso-type phospholipid is desirable in the order of Mi-Hai, Rhizopus nibeus, Pseudomonas fluorescens and Fleis It was a proof.

Example 3 The soybean phospholipid of Example 2 was used as egg yolk phospholipid (eg, egg lecithin manufactured by Wako Pure Chemical Industries, for biochemistry), and the amount of lipase used was 30.
The same operation as in Example 2 was carried out except that the units were changed to 00 units, and the results shown in Table 3 were obtained. It was confirmed that the lipase of microbial origin according to the present invention also acts on egg yolk phospholipid to produce lyso-type phospholipid. The tendency of the reaction was almost the same as that of soybean phospholipid.

Example 4 20% of the same soybean phospholipid used in Example 2 (W /
W) To 50 g of the aqueous dispersion, 2000 units of Aspergillus niger-derived lipase agent (Amano Pharmaceutical Lipase A) was added, and the mixture was reacted in a rotary shaker at 37 ° C. for 24 hours.

The results of analysis of the reaction solution according to Example 2 are as shown in Table 4, and it was found that the decomposition reaction with lipase was normally performed even in the high concentration aqueous dispersion.

Example 5 The emulsifying power of the hydrolyzate obtained by the method of the present invention was tested as follows.

A freeze-dried product of the enzymatic degradation product obtained in Example 4 was used as a phospholipid according to the method of the present invention, and undecomposed soybean lecithin was used as a control, respectively, and 0.5 g of each sample and chloride of each concentration shown in Table 5 were used. Put 5 ml of calcium aqueous solution into a test tube and 4.5 m with a homogenizer "Hiscotron" manufactured by Nichine Medical Science Instruments Co., Ltd.
Using an mφ shaft, the dispersion was performed at 10.000 rpm for 2 minutes and the emulsified state was observed. .

As described above, it was shown that the enzyme-treated lecithin according to the present invention exhibits excellent emulsifying power even in a system with high ionic strength, which has almost no emulsifying power with untreated lecithin.

(Effect of the Invention) According to the present invention, a wide range of raw material phospholipids can be modified into lyso-type phospholipids by using commercially available lipase agents of microbial origin that are commercially available. The lyso-type phospholipid is a substance having an excellent emulsifying property particularly in an aqueous dispersion medium, and the present invention that makes it possible to provide this at a low cost makes a great contribution to the industrial side, particularly in the food industry. It is a thing.

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Claims (3)

[Claims] 1. A phospholipid-containing microorganism belonging to the genus Aspergillus or the genus Pseudomonas, Rhizopus javanicus,
A method for modifying a phospholipid, which comprises causing a lipase produced by one of Rhizopus nibeus and Mucor mihai to act, and at least a part thereof being a lyso-type phospholipid. 2. The method for modifying a phospholipid according to claim 1, wherein the microorganism belonging to the genus Aspergillus is Aspergillus niger or Aspergillus wenchi. 3. The method for modifying a phospholipid according to claim 1, wherein the microorganism belonging to the genus Pseudomonas is Pseudomonas fluorescens or Pseudomonas frage.