JPH09316483A - Separation and production of oil and fat and protein from seed of oil and fat - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain high-quality oils and fats and protein from seeds of oils and fats friendly to an environment, simply, inexpensively and in good reproducibility, by adding and reacting seeds of oils and fats to with a specific fruit body (extracted substance) of a fungus and separating the reaction solution into the oils and fats and the protein. SOLUTION: (A) Ground or flattened seeds of oils and fats (e.g. soybean or colza) are reacted with (B) (B1 ) a fruit body of Basidiomycetes or Ascomycetes or (B2 ) an extract of a mycorrhizal part after the harvesting of the component B. with water or reacted with (D) a mixture of a plant tissue disintegrating enzyme derived from a microorganism in the presence of (C) an acidic protease inhibitor or reacted with (E) a fraction obtained by passing the component D through a column having immobilized the component C, and the reaction solution is separated. The reaction is preferably carried out in the presence of citric acid at 30-70 deg.C for 2-60 hours.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating and producing oils and fats and proteins from oil seeds. More specifically, the present invention relates to a method for producing high-quality fats and proteins by separating fats and proteins from oil seeds by a novel method based on an idea significantly different from the method for producing fats and oils by mechanical separation by pressing or solvent extraction. .

[0002]

2. Description of the Related Art Conventionally, as a method for producing fats and oils from oil seeds, a method of extracting using an organic solvent (extraction method), a squeezing method, or a method of producing a combination of these two methods has been used. It was Further, when a high oil content seed such as rapeseed is used as a raw material, a method of squeezing oil only by a pressing method is also performed, but this method has a low oil recovery rate. Further, in the case of low oil content seeds such as soybean, oil extraction is extremely difficult. Therefore, even if it is possible to industrialize the oil-and-fat manufacturing method by the pressing method, it is unavoidable that it will be economically disadvantageous.

On the other hand, the extraction method using an organic solvent such as hexane requires an indispensable extraction device because the organic solvent is used, and after the solvent extraction, the organic solvent remaining in the crude oil and protein is completely removed. Equipment for removal is required. Therefore, a large amount of initial investment is required for these devices and equipment. In addition, this method causes a loss of organic solvent during the extraction operation,
The lost solvent is likely to be released into the atmosphere. A recent report points out that the released solvent affects the destruction of the ozone layer and the like. Furthermore, by using an organic solvent, it is necessary to be strictly careful in terms of equipment and operation. This is because hexane, which is generally used as an organic solvent, has explosive properties, and in the past, there have been tragic damages due to explosion accidents.

Under these circumstances, in recent years, olives (Abstract (Basic): JP0505939) containing a high concentration of oil have been investigated.
Regarding the oil extraction method using 0) as a raw material, a method of performing oil extraction using an enzyme has been proposed and is in the stage of practical application. Similarly, a method of recovering fats and oils from grains by enzymatic treatment (Abstract (Basic): DD2902912), or a method of recovering fats and oils by treating crushed plant tissue with enzymes such as protease and cellulase (Abstract (Basic Basic): DE3
843027) and others have been proposed. However, although the enzymatic method can be easily applied to the recovery of oils and fats from mesocarp such as olives, it is not always possible to efficiently separate oils and proteins from oil seeds that store stored oils and fats in organelles called oil bodies. This is impossible and it is extremely difficult to produce high-quality fats and oils and proteins.

Incidentally, in the case of soybeans with low oil content oil seeds, a method of improving the oil / fat extraction ratio by spraying an enzyme solution on the crushed raw material to perform an enzymatic pretreatment and then performing hexane extraction is proposed. (Food Chemistry, Volume 46,
277, 1993; ibid. 54, 223, 1995). However, as long as extraction is performed with hexane, the same environmental problems as described above remain, so a drastic improvement measure cannot be achieved. For rapeseed with high oil content oil seeds, a method has been proposed in which the oil is separated by using a centrifuge after crushing it, heat-treating it, and then subjecting it to enzyme treatment (JAOCS. Vol. 69, (No. 3) Page 195,
1992). However, according to additional tests by the inventors, the oil and fat recovery rate is not sufficient. Furthermore, after enzymatic pretreatment,
Recently, a method of recovering an oil component by the X-Peller oil extraction method has been reported (JAOCS. 70, (No. 9) page 825, 1993). Using this method, the fat recovery rate by the conventional method is at most 72.
However, when the defatted meal is used as a feed, the utilization rate as a feed can be improved. However, this method does not recover high-concentration proteins.

Phospholipids are present in the crude crude oil recovered by the conventional oil extraction method. Therefore, in order to produce high-quality fats and oils, it is necessary to remove them in the refining process (degumming process). In order to remove these phospholipids, it is necessary to incorporate a plurality of steps such as addition of a chemical agent such as phosphoric acid and centrifugation after the heat treatment, and the direct cost for that purpose also increases. In addition, secondary costs such as waste liquid processing costs are added. Furthermore, neutral oil is also involved in the removal of these phospholipids, resulting in loss of oil and fat recovery.

In view of the above background and actual conditions, as an alternative method to the organic solvent extraction method, in order to recover oils and fats and proteins present in soybean seed cotyledon tissue in good yield,
A method for disintegrating oils and fats by disintegrating the fibers, proteins, etc. that compose these tissues using an enzyme complex containing cellulase, hemicellulase and protease activity has also been studied and reported (Proceeding of AOCS Annual Meet
ing, May, 1995). As a result of additional tests by the inventors of this method, it was possible to extract fats and proteins,
Only the emulsion mainly composed of both was recovered. Moreover, once an emulsion is formed, oil and fat from this emulsion,
Separation and fractionation of partially decomposed products such as proteins and peptides was even more difficult.

[0008]

SUMMARY OF THE INVENTION The present invention overcomes the problems and drawbacks of the conventional methods as described above, is environmentally friendly,
The purpose of the present invention is to provide a high-quality oil / fat and protein with good reproducibility and at low cost by separating the oil / fat and protein by an easy operation. As one of the measures for avoiding the above-mentioned "emulsion formation", the present inventors depress the soybean (protein) to high strength by heating and compressing the raw soybean by "extrusion" and the like,
A plant tissue-disintegrating enzyme (eg, Novo Co., trade name: Viscozyme, Pectinex, SP311 etc.) was allowed to act to successfully suppress the emulsification and separate the oil layer, and a patent application has been filed (Japanese Patent Laid-Open Publication No. HEI-7). -179881). However, under the conditions of this method, the protein is strongly denatured, and therefore, its use in collecting and utilizing it is limited. Therefore, the present inventors have made it a next task to develop a method for separating and recovering fats and oils and proteins without strongly pre-treating the raw materials with heat and suppressing emulsion formation as much as possible. The present inventors have been conducting various studies on the cause of such emulsion formation, but the protein partially decomposed by the protease mixed in the enzyme source to be used to produce peptides, which was emulsified from oil and fat and protein. It is presumed that it accelerates the formation of things.

[0009]

[Means for Solving the Problems] As described above, a plant tissue-disintegrating enzyme of microbial origin having both pectinase, cellulase and hemicellulase activities is used to separate and recover fats and proteins from soybean raw materials without intensive heat treatment. If an attempt is made, an emulsified layer will be produced and it will be difficult to separate the two. The present inventors have found that when this enzyme reaction is carried out at pH 3 to 5 in the presence of an acidic protease inhibitor, the production of emulsion is suppressed and the oil layer can be separated. That is, when a mold-derived plant tissue-disintegrating enzyme (protease activity is mixed together in many cases) as seen in conventional publicly known documents, most proteases are added by addition of an acidic protease inhibitor or an inhibitor-immobilized column. It has been found that avoiding emulsion formation is extremely difficult unless a procedure to remove activity is performed. In addition, this knowledge, genetically breeding microorganisms having the ability to produce plant tissue-disintegrating enzymes, and breeding of strains that do not have the ability to produce enzymes or proteases that do not meet the purpose, or have weak productivity, have the need for development. It is a strong suggestion.

Further, in order to develop a new enzyme source, the present inventors conducted a comparative test using the above-mentioned mold-derived commercial plant tissue-disintegrating enzyme and an edible mushroom extract obtained by artificial cultivation as the enzyme source. As a result, in the experimental section to which a commercially available plant tissue-disintegrating enzyme (trade name: Viscozyme, Pectinex, SP311) was added, the reaction solution was centrifuged after the enzymatic reaction, and the aqueous layer was found to be a protein (protein partially decomposed product). The transparency of the aqueous layer was low, and it was cloudy, probably because the emulsion was easily formed from the oil. On the other hand, it was found that when the mushroom extract was used, the transparency of the water layer was high, the oil layer floated and floated, and the amount of the emulsified layer was obviously small. Furthermore, we have found that Basidiomycetes or Ascomycetes
(Ascomycetes) After harvesting the fruiting bodies of the fruit body, the mycorrhizal part was immersed in water and added as an enzyme source to react to minimize the amount of emulsion formation and to contain the oil layer part and protein. It was confirmed that the water layer portion to be separated could be separated and fractionated, various studies were repeated, and the present invention was completed based on these findings.

According to the first aspect of the present invention, the fruit seeds of basidiomycetes or ascomycetes or the fruit bodies of the mycorrhizal portion after harvesting the oil seeds subjected to the crushing treatment and / or the pressing treatment are collected. A method for separating and producing fats and oils and proteins from oil seeds, which comprises separating the reaction liquid after adding and reacting the extract liquid. Further, according to the invention of claim 2, (1) a plant tissue-disintegrating enzyme mixture of microbial origin is allowed to act on oil seeds which have been subjected to crushing treatment and / or pressing treatment in the presence of an acidic protease inhibitor. After or
(2) After the mixture of the plant tissue-disintegrating enzyme mixture of microbial origin and the fraction obtained through an acidic protease inhibitor-immobilized column are allowed to act, the reaction solution is separated to separate fats and proteins from oil seeds, It is a manufacturing method.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, oil seeds include soybean, which is a low oil content oil seed, and rapeseed, which is a high oil content oil seed. In the method of the present invention, this oil seed is first subjected to a crushing treatment and / or a pressing treatment. If necessary, further pulverization treatment is performed. When the oil seed is a low oil content oil seed, it is usually crushed, then pressed, and then optionally pulverized.
In addition, the crushing process (cracking) performed by the method of the present invention
Is the same as the crushing process performed in the current oil extraction equipment. On the other hand, when the oil seeds are oil seeds with a high oil content such as rapeseed, crushing or crushing treatment tends to cause loss of fats and oils. Therefore, when oil-rich oil seeds are used in the present invention, a roller having a thickness of 0.1 to 0.1 is usually used.
A tissue obtained by subjecting it to a compression treatment of crushing to 1.0 mm and destroying the tissue is used. In addition, from the raw material that has been subjected to the pressing process, after the oil is extracted by the pressing method in the first stage and the oils and fats are recovered,
In the second step, it is also possible to adopt a method of recovering the remaining oils and fats by subjecting the pressed meal to the enzyme treatment of the present invention described below.

As an enzyme source which acts on the oil seeds,
In the present invention, fruiting bodies of basidiomycetes or ascomycetes, water extracts from mycorrhizal portions of the fruiting bodies after harvesting, or a mixture of plant tissue-disintegrating enzymes of microbial origin is used. Enzyme sources derived from basidiomycetes or ascomycetes, so-called mushrooms, are shiitake mushrooms (Lentinus edodes) and hiratake (Pleurotus ostreatu).
s), Maitake (Grifola frondosa), Hatshimeji (Lyop
hyllum decastes) and fungal fruiting bodies, or the mycorrhizal portion after harvesting the fruiting bodies, is ground and mixed with an approximately equal amount of water, and then solid-liquid separation operations such as centrifugation and filtration are performed. The recovered extract may be used. In addition, the extraction efficiency can be improved by leaving it at low temperature (0 to 10 ° C.) for 1 to 2 days after grinding. Further, the cost can be reduced by using the water extract from the mycorrhizal portion after harvesting the fruiting bodies as the enzyme source. Incidentally, depending on the production site such as basidiomycete and artificial cultivation conditions, protease activity may be observed in the harvested fruiting bodies or mycorrhizal roots called "Ashi" after harvesting the fruiting bodies. The protease activity may be inhibited or removed by the same treatment as in the case of the microorganism-derived enzyme described below.

As the microorganism-derived plant tissue-disintegrating enzyme complex, enzyme complexes having activities such as pectinase, cellulase, hemicellulase and protease are commercially available, and these can be used. However, in order to avoid emulsion formation, it is necessary to inhibit all or most of the protease activity, or remove the protease activity in advance, for example, by coexisting with a commercially available acidic protease inhibitor. In order to remove the protease activity in the enzyme complex, it is effective to pass the enzyme solution through a column on which an acidic protease inhibitor is immobilized, and this operation causes most of the protease activity (usually about 85%) to be removed. Are removed. Here, the present inventors have already reported on a method for preparing an acidic protease inhibitor column in Enzyme Engineering, Volume 3, pp. 331-336 (1978). That is, the carboxyl terminus of a pepsin inhibitor (SPI) produced by actinomycetes is treated with p-aminobenzyl-, p-aminoethyl- or DEAE.
-Immobilized on cellulose (eg SPI: 14.7 mg /
p-aminobenzyl-cellulose 1 g), pH 4.5
When the enzyme solution is passed through, the mixed acid protease is adsorbed on and removed from the column, and the enzyme solution containing other enzymes as it is can be recovered.

According to the method of the present invention, the above-mentioned enzyme source is allowed to act on the raw oil seeds, and after the reaction, a separation treatment such as solid-liquid separation is carried out to separately produce the target oil and fat and protein. be able to. Moreover, according to this method, the fats and oils and the protein can be separated and collected while the amount of the emulsion produced is extremely small. Explaining a preferred embodiment of the method of the present invention, 3 to 1 is applied to the oil and fat seed that has been subjected to the crushing treatment and / or the pressing treatment.
Add 0 times the amount of water, pH 3.0-6.0, preferably pH 4.0 in the presence of 0.01-1.0 M citrate buffer.
The reaction is carried out at ˜5.0 at 30 to 70 ° C., preferably 40 to 60 ° C. for 2 to 60 hours, preferably 4 to 40 hours. When the enzymatic reaction is long, it is desirable to carry out the reaction at a temperature of about 50 ° C or lower, but to finish the reaction in a short time, about 50
Reactions between ℃ and about 60 ℃ are desirable. By doing so, spoilage during the reaction and deterioration of the quality of the recovered product can be prevented. Note that if the reaction solution is strongly stirred during the reaction, an emulsion is likely to be generated, so it is desirable to stir gently so that the reaction solution is uniformly stirred.

After the completion of the enzymatic reaction, in order to separate and collect the oil and fat and the protein, the reaction solution is first subjected to a solid-liquid separation operation such as centrifugation to divide into a solid layer for precipitation and an aqueous layer / oil layer portion. After that, once the water layer / oil layer portion was recovered in a NaHCO ₃ or Na ₂ CO ₃ solution having a pH of about 10 to dissolve the insoluble protein, centrifugation was performed again at a low speed to obtain an oil layer and a protein-containing water layer. Can be easily separated. If fat and protein remain in the precipitated solid layer even by such an operation and the recovery is somewhat insufficient, by adding the citrate buffer solution and the enzyme solution again to the solid part, and then repeating the same reaction. The recovery rate of fats and oils and proteins can be improved.

By the above-mentioned method, it is possible to separate the uppermost oil layer portion, the intermediate water layer (water-soluble protein, water-soluble polysaccharide or oligosaccharide), and the lower solid portion (insoluble protein, insoluble polysaccharide). Here, the protein portion contained in the aqueous layer can be isolated as an isolated protein by isoelectric precipitation, or by separating the saccharide and protein from each other by membrane separation and concentration, and pulverizing with a dryer such as a spray dryer.

On the other hand, the oil layer portion floating on the uppermost layer is continuously subjected to solid-liquid separation operation such as centrifugation as necessary to recover a pale yellow transparent oil having a water content of 0.5% or less. be able to. The crude crude oil thus obtained has a low phosphoric acid content (phospholipid) to the extent that conventional degumming operations are not required, so the degumming and decoloring steps of the conventional method can be omitted, and labor, cost, Environmental pollution etc. can be greatly reduced.

[0019]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. Example 1 500 containing 25 g of raw soybean obtained by pressing and crushing a soybean crushed product
100 ml of water and 100 ml of 0.5 M citrate buffer (pH 4.5) were added to a ml Meyer flask, and heated in boiling water for 5 minutes. Then, a commercially available plant tissue-disintegrating enzyme (trade name: Viscozyme, Novo Co.) solution 300 μm as an enzyme source
1 and 1 ml of a solution of an acidic protease inhibitor (SPI, 5 mg / methanol 1 ml) were added, and the mixture was reacted at 40 ° C. for 24 hours on a rotary shaker (120 revolutions). After completion of the reaction, centrifuge the reaction solution (9000 rpm, 30 minutes)
Then, the oil layer and the slightly existing emulsion layer portion were
Transfer to a 50 ml Falcon tube containing 15 ml of HCO ₃ solution and spin again (3000 rpm, 20 minutes).
A small amount of emulsion was also formed by addition of 3., but 3.
An oil layer of 5 ml (oil and fat recovery rate of 65%) floated, and 45% of protein was recovered in the water layer. In the control group to which the acidic protease inhibitor (SPI) was not added, the emulsified layer was mostly used and the oil layer was not separated.

Example 2 500 containing 25 g of raw soybean obtained by pressing and crushing a soybean crushed product
100 ml of water and 100 ml of 0.5 M citrate buffer (pH 4.5) were added to a ml Meyer flask, and heated in boiling water for 5 minutes. Next, as an enzyme source, 3 ml of an extract obtained by grinding and extracting 5 g of various commercially available edible mushroom bodies shown in the table below with 5 ml of water was added, and the mixture was heated to 50 ° C. with a rotary shaker (120 revolutions). And reacted for 4 hours. After completion of the reaction, centrifuge the reaction solution (9000 rpm, 30 minutes)
Then, the oil layer and the slightly existing emulsion layer portion were
Transfer to a 50 ml Falcon tube containing 15 ml of HCO ₃ solution and spin again (3000 rpm, 20 minutes).
When was added, an oil layer floated to the top layer. The recovery rate of the oil layer is shown in Table 1.

[0021]

[Table 1] Table 1 Enzyme extract solution Oil layer recovery rate (%) ─────────────────── Shimeji 15 Maitake 45 Shiitake 24 Nameko 17 No enzyme added 0

Example 3 Maitake extract enzyme solution (4 ml of enzyme solution obtained by extraction under the same conditions as in Example 2) was added as an enzyme source, and the reaction was carried out at various temperatures according to the method of Example 2. The optimum temperature was examined. The results are shown in Table 2. However, the reaction time was 4 hours.

[0023]

[Table 2] Table 2 Reaction temperature Oil layer recovery rate (%) ─────────────────── 25 ° C 5 30 10 10 40 45 45 50 65 60 60 40 70 70 12 75 5

Example 4 As the enzyme source, the Maitake extract prepared by the same method as in Example 2 was used, and the enzyme reaction was carried out for a predetermined time in the same manner as in Example 2 except that the reaction temperature was 50 ° C. The reaction time and the oil and fat recovery rate were investigated. The results are shown in Table 3.

[0025]

[Table 3] Table 3 Reaction time Oil recovery (%) (hours) AB ─────────────────────────── 2 23 32 4 40 65 8 45 67 67 16 60 69 69 24 58 67 36 50 60 * A: 2 ml of Maitake extract added per 25 g / 200 ml of soybean raw material * B: 4 ml addition of Maitake extract per 25 g / 200 ml of soybean raw material

Example 5 1 L of water and 1 L of a 1M citrate buffer solution (pH 4.5) were added to 250 g of raw soybeans obtained by pressing and crushing a soybean crushed product, and the mixture was heated at 80 ° C.
For 10 minutes. Next, add 2 Shizuoka Maitake extract
0 ml (corresponding to 20 g of Maitake mushroom) was added, and the enzyme reaction was carried out at 50 ° C. for 4 hours while gently stirring (130 rotations). After the reaction was completed, the reaction solution was centrifuged (9000 rpm, 30 minutes) to collect the oil layer and the slightly floating oil layer, which was then transferred to 150 ml of 0.5 M NaHCO ₃ solution and 3000 rpm for 20 minutes. Centrifuge,
The amount of oil and fat that floated was collected and used as the first amount of recovered oil and fat. On the other hand, water was added to the oil layer, the separated precipitate layer and the aqueous fraction to make the total volume 2000 ml, and 20 ml of the new maitake extract (the same as above) was added to this, and then the enzyme reaction was performed again under the same conditions. It was The oil layer was separated by the same operation as the first operation, and the recovered oil was used as the second amount of recovered oil and fat. Further, the remaining precipitation layer and the aqueous layer fraction were used to carry out a third reaction in the same manner to recover oils and fats (3rd step).
The amount of fat and oil recovered the second time) As a result, the amount of recovered oil and fat (crude crude oil) was 18 g for the first time, 11 g for the second time and 7 g for the third time (total amount of oil and fat was 36 g), and the total recovery rate was 72%. Met. The total nitrogen recovery rate in the water layer was 64% with respect to the raw material. The crude crude oil thus obtained has an acid value (AV value) of 1.0 and an inorganic phosphorus content of 4.22 pp.
m.

Example 6 25 g of compressed rapeseed crushed to 12 mesh or less
100 ml of water was added to the starting material of Example 1 and treated at 100 ° C. for 10 minutes, and then 1M citrate buffer (100 ml, pH 4.5) and Maitake extract 4 were added.
After adding ml, the enzyme reaction was carried out while stirring at 50 ° C. for 6 hours. After completion of the reaction, the first, second and third fats and oils were collected according to the method of Example 5. As a result, the oil and fat recovery rate in each operation was 8.1% and 49%.
And 22.9%, and the total recovery rate from the raw fats and oils was 80%.

[0028]

INDUSTRIAL APPLICABILITY In the method of the present invention, since solvent extraction is not carried out in the production of fats and oils, a large-scale apparatus is not required, and an aqueous and environmentally friendly production process of fats and oils can be provided regardless of the scale. The method for producing oils and fats and proteins from the oil seed raw material according to the present invention has the following features. (1) When the fruiting body of basidiomycete or ascomycete or the extract of the mycorrhizal portion after harvesting the fruiting body is used as an enzyme source, the enzyme source is derived from so-called edible mushrooms,
It can be used as a highly safe enzyme source for food and can reduce or avoid emulsion formation. (2) When a microorganism-derived plant tissue-disintegrating enzyme is used as the enzyme source, the formation of an emulsion layer can be reduced or avoided by removing most of the protease mixed in the commercially available enzyme complex. (3) Since the degumming step can be omitted, the amount of acid and alkali used can be reduced. (4) It is possible to provide an eco-friendly and energy-saving oil / fat manufacturing method. (5) In addition to fats and oils, an intermediate liquid layer portion (water-soluble protein, water-soluble polysaccharide) can be separated and collected from fats and oils by the isoelectric precipitation method or membrane treatment. In addition, the recovered protein portion has a small thermal history, and thus it is expected to have a wide range of uses as a source of undenatured or moderately denatured proteins.

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Claims (4)

[Claims] 1. Reaction is carried out by adding a fruiting body of basidiomycetes or ascomycetes or a water extract from the mycorrhizal portion of the fruiting body to oil seeds subjected to crushing and / or pressing. A method for separating and producing fats and oils and proteins from oil seeds, which comprises separating the reaction liquid after the reaction. 2. An oil seed that has been subjected to a crushing treatment and / or a pressing treatment is treated with a mixture of (1) a plant tissue-disintegrating enzyme of microbial origin in the presence of an acidic protease inhibitor,
Or (2) Separation of fats and proteins from oil seeds characterized in that the reaction mixture is fractionated after the fraction obtained by passing a plant tissue-disintegrating enzyme mixture of microbial origin through an acidic protease inhibitor-immobilized column is reacted. , The method of manufacturing. 3. The method according to claim 1, wherein the oil seed is soybean or rapeseed. 4. The reaction is carried out at 30 to 70 ° C. in the presence of citric acid.
The method according to claim 1 or 2, which is carried out for 2 to 60 hours.