JPS6058276B2 - natural antioxidants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antioxidant that exhibits an excellent antioxidant effect on various products whose quality is significantly impaired by oxidation, such as foods, fragrances, pigments, cosmetics, etc.

Conventionally, antioxidants (nucleic oxidants) for the various products listed above
Chemically synthesized products such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA),
It is known to be extracted from natural substances such as natural tocopherol.

However, in view of the growing concern over the safety of food additives made from chemically synthesized drugs in recent years, the use of the above-mentioned BHT and BHA as antioxidants is gradually decreasing, and the use of naturally occurring antioxidants is gradually decreasing. Demand for antioxidants is increasing. As mentioned above, representative examples of naturally occurring nucleic oxidation components include:
Examples include tocopherol and its congeners, which are abundantly contained in vegetable oils such as soybean oil, cottonseed oil, rapeseed oil, and palm oil, but there are also oryzanol constituents such as unsaturated terpene alcohols contained in rice and wheat. , gossypol in cottonseed, sezamol in sesame oil, caffeic acid and its derivative polyphenols in coffee beans, and eugenol, ganfen, thymol, cineole, syogaol, cinnamaldehyde, etc. present in various spices. ing. These nucleic acid components mentioned above can generally be collected by extraction methods, but in addition to the low extraction yield, they also contain components unsuitable for use as antioxidants, so they are not practical except for tocopherols. It has not yet become a reality.

In addition to the natural antioxidant components mentioned above, various amino acids such as tryptophan, proline, and methionine, their homologues, peptides as hydrolysates of animal and plant proteins, and various other natural substances that exhibit nucleic oxidizing properties have been used. Melanoidins, which are reaction products of amino acids and reducing sugars, have been reported.

However, most of the various amino acids and peptides have a physical problem of being poorly soluble in fats and oils.
Alternatively, these alone do not have very strong antioxidant effects, but rather other antioxidants such as BHT and BHAL
It is applied in a so-called synergistic use for the purpose of synergistic effects with natural tocopherols, etc., or in a raw material use method in which it is used in large quantities in foods and the like.

In addition, reductones and melanoidins, which are reaction products of amino acids and reducing sugars, have very strong coloring, have an unpleasant odor when used, and are similar to protein hydrolysates in terms of properties and are sensitive to lipids. However, due to its poor solubility, in actual use, only a few methods are used, such as pre-existing amino acids and sugars, which are precursors of reductones and melanoidins, in foods and fats and oils, and then heating and adding them to foods. Can only be used in limited fields. As mentioned above, conventionally known natural antioxidant ingredients or antioxidants made from them have lower economic efficiency, antioxidant potency, and their uses and applications compared to chemical products such as BHT and ⑍. The current situation is that the results are not completely satisfactory.

In view of the above-mentioned current situation, the present inventors have studied the provision of an effective antioxidant of natural origin and have arrived at the present invention. The present invention will be explained in detail below.

The antioxidant according to the present invention can be applied to a substrate consisting of a vegetable oil, a residue from the extraction process thereof, a residue from the extraction and purification process of the above-mentioned oil, a mixture thereof, or a substance obtained by adding a carbohydrate substance to Aspergillus. It is characterized in that the active ingredient is a product obtained by reacting filamentous fungi belonging to the genus.

The term “vegetable oil feedstock” as used herein refers to soybeans, cottonseed, raw materials for vegetable oil, crude oil, edible vegetable oil, industrial vegetable oil, etc.
rapeseed, olive, sesame, sunflower, corn,
Refers to substances that are harmless from a food hygiene point of view, such as plant fruit and germs such as peanuts and coconuts, and sugars, and which have antioxidant effects, and are in accordance with the terminology defined by JAS. It is.

In addition, "extraction processing residue" means the so-called extraction residue after mainly extracting oil and fat components from the vegetable oil material, and defatted soybeans can be exemplified, and "゛extraction and purification processing of vegetable oil material""Residue" refers to the residue after refining the extract (extracted crude oil) from the above-mentioned vegetable oil substance, and refers to the residue after refining soybean crude oil (lecithin, other impurities in crude oil). In the present invention, these vegetable oil substances, their extraction treatment residues, or the extraction and purification treatment residues of these oil substances are usually impregnated with an appropriate amount of water; heating,
Products that have been subjected to processing such as swelling, coloring, and flavoring can also be applied.

Furthermore, the carbohydrate substances used in the present invention include starchy grains such as rice, wheat, corn, and processed products thereof, other starchy substances, and substances containing sugars such as glucose. These carbohydrate substances effectively participate in the growth of filamentous fungi of the genus Aspergillus, which will be described later. In the present invention, the above-mentioned vegetable oil substance or its extraction processing residue, the extraction and purification processing residue of the oil substance, or a mixture thereof uses a mixture of these substances and the carbohydrate as a substrate, and the substrate contains Aspergillus. The active ingredient is a product obtained by reacting filamentous fungi belonging to the genus, and the substrate used here has a content ratio (weight) of total carbohydrate content and total protein content of 0.5. ~5
．． 01 preferably in the range of 1.0 to 3.0. The filamentous fungi belonging to the genus Aspergillus that act on the substrate may be non-toxic ones that are commonly used in the brewing and fermentation industries, such as Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, etc., which are generally easily available. Examples include Aspergillus awamori. In the present invention, in order to cause the filamentous fungus belonging to the genus Aspergillus to act on the substrate, the filamentous fungus may be aseptically inoculated onto the substrate and cultured using known fermentation techniques.

Therefore, during this culture, the amount of carbohydrates in the substrate, the moisture content of the substrate, and the temperature should be adjusted and controlled as necessary to suit the growth and action of the filamentous fungi used. In addition, during the above culture, if bacteria such as Bacillus are present, the filamentous fungi used will be contaminated and the production of the desired effective antioxidant component in the substrate will be inhibited, so on an industrial scale, culture equipment that can be operated aseptically is used. Alternatively, it is actually necessary to maintain the growth of the filamentous fungus used under optimal conditions by carrying out the above-mentioned management strictly. In particular, when defatted soybean extract residue is used as a substrate component, the protein content tends to be high and the carbohydrate source necessary for the good growth of the filamentous fungi used is likely to be insufficient, resulting in contamination by harmful microorganisms. Therefore, it is important to adjust the amount of carbohydrates in the substrate. Although the culturing time described above varies depending on the filamentous fungus used, the desired antioxidant component can generally be produced in about 3 days.

In the present invention, the mechanism of production of antioxidant components by the action of Aspergillus filamentous fungi on the above-mentioned substrate and the contents of these components have not yet been elucidated, but as can be understood from the experimental results described later, Aspergillus filamentous fungi are used on the above-mentioned substrate. In view of the fact that the resulting culture product exhibits an antioxidant power several to several tens of times higher than that of the raw material substrate, it has conventionally been found that it exists as a natural product in edible oil substances or their extraction residues. In addition to the natural antioxidant components that have been reported, such as tocopherol in soybeans, gossypol in cottonseed, and terpene alcohols that are constituents of oryzanol in rice bran, the above-mentioned Precursors of natural antioxidant components or unknown components present in the above-mentioned edible oil substances or their extraction residues, so-called components that have potential antioxidant power due to their functions, may be affected in some way. As a result, it was actually expressed as an active ingredient with antioxidant effect, and the various antioxidant ingredients that were expressed, singly or in combination, resulted in the need for anti-oxidation, such as in fatty people. It is presumed that a substance with strong antioxidant power is produced by exerting an additive or additive effect on a certain product or material.

It should be noted that there are almost no antioxidant components present in the growing cells of the filamentous fungi of the Aspergillus genus used in the present invention or in the spores that adhere to them during their growth, and even if they exist, the amount is so small that they do not have any antioxidative components. No substantial oxidation effect can be expected.

Furthermore, it is presumed that the amount of antioxidant components accumulated in the substrate in the present invention does not increase proportionally with the growth of Aspergillus filamentous fungi, but is rather influenced by the type and composition of the substrate. Ru. Regarding the reason why filamentous fungi of the genus Aspergillus are particularly used in the present invention, according to the test results conducted by the present inventor, filamentous fungi other than the genus Aspergillus, such as Penicillium, Monascus, Neurospora, and Mucor, Filamentous fungi belonging to the genus Aspergillus and the genus Rhizopus also produce antioxidant components when acting on the substrate of the present invention, but as shown in the experimental results below, the antioxidant components produced by the action of filamentous fungi of the genus Aspergillus Its effective antioxidant potency is quite low, and therefore, from the viewpoint of practicality as an antioxidant, it is necessary to use filamentous fungi of the genus Aspergillus.

In addition, in view of the fact that the various filamentous fungi mentioned above act on the substrate of the present invention to produce antioxidant components, it is naturally thought that the enzymes of these filamentous fungi are involved in the production of the antioxidant components. However, as shown in the experimental results below, it has not been confirmed that the action of the enzyme alone exhibits an antioxidant potency comparable to that of filamentous fungi of the genus Aspergillus.

However, it is effective to use these enzymes in combination with filamentous fungi belonging to the genus Aspergillus, and it is particularly effective to use enzymes derived from the filamentous fungi as described above. Next, the results of an experiment on the antioxidant effect of the antioxidant according to the present invention are shown in the table below.

Experiment 1: Regarding the production of antioxidant components by various microorganisms on vegetable oil substances as substrate components.

Whole soybeans were soaked overnight and then steamed at 120°C under a pressure of 1 kg/al. This was used as an edible oil material, and Bacillus subtilis was added to it as a bacterium.
ubtilis) and Aspergillus as a filamentous fungus
AspergillLlsOryzae and RhizOpusOryzae
Inoculate each of
The antioxidant potency of the antioxidant components produced in the steamed whole beans after pure culture for 3 days was investigated as follows.

Each cultured product of the above whole soybeans was heated to 180% using commercially available soybean oil.
℃ for 3 minutes, and as a control, the above-mentioned steamed whole soybeans that were oiled and soaked in the same way without culture treatment were stored in a dark place at 63℃, and the oil and fat content of each sample during storage was In order to observe changes over time, each sample was extracted with n-hexane over time, and the peroxide value (POV) of the extract was measured according to the following method. Measuring method for peroxide value (P.O.V): 10% chloroform in a 250mt Erlenmeyer flask with a stopper.
ml and pass clean, dry CO2 into it to replace the solvent and the air in the vessel.

Next, about 0.1 to 2 ml of sample is added to the flask while passing CO2.
．． 0y was weighed correctly, and gently shaken to dissolve the sample. To this solution, 15 ml of glacial acetic acid and 1 ml of saturated potassium iodide were added. After turning off the carbon dioxide gas, the sample was shaken for 1 minute, and then left in a dark place for 5 minutes. After 5 minutes, add 75ml of water, stir vigorously, and use starch solution as an indicator to measure N/10.
Titrate with 0 sodium thiosulfate standard solution, and the end point is when the blue color due to starch disappears.

In addition, a blank test will be conducted in parallel with the main test. A...
Amount of N/100-sodium thiosulfate standard solution used in this test (Mt) B...Amount of N/100-sodium thiosulfate standard solution used in blank test (ml) F... N
Factor C of /100 sodium thiosulfate standard solution
・・・・・・Amount of sample collected (y) The results are calculated as P. O. The relationship between V and storage days is shown in Figure 1.

As can be seen in Figure 1, the P of the filamentous fungus treated with the filamentous fungi increased over time compared to the control. O. The tendency of increase in V was low, and the above tendency was almost the same as that of the control when bacteria were applied. It has particularly good antioxidant potency.

Experiment 2 Regarding the production effects of antioxidant components of various filamentous fungi on substrates.

After sprinkling 120% water on defatted soybeans,
A mixture of 7 parts by weight of roasted and cracked wheat as a carbohydrate substance was added and mixed with 7 parts by weight of steamed rice at 0'C under a pressure of 1k9/c for 30 minutes, and a mixture was used as a substrate. , Aspergillus as a filamentous fungus! S. oryzae, Aspergillus awamori, Rhizopus oryzae, Rhizopus deremois, Penicillium lockfordii, Mucor jiabanicus, and Neurospora kratusa were inoculated and cultured at 30°C for 3 days.

These culture products were each dried at 80° C. under ventilation and then powdered using a mixer.

Each powder obtained as described above was mixed with white soybean oil by 1%.
．． After adding 50% of the total amount, the samples were dispersed by stirring slowly at 55°C for 30 minutes, and 40y of each sample was placed in a 100ml beaker and stored in a dark place at 55°C. The changes in oil and fat of each sample over time were determined using the same method as in Experiment 1. ON. was measured and investigated.

The results are shown in Table 1. (Note) The numerical values in the table indicate the peroxide value (P.O.V.) in the fats and oils of each sample.

As seen in the above table, it is understood that the use of filamentous fungi of the genus Aspergillus is advantageous in expressing antioxidant components in substrates.

Experiment 3 Regarding the effects of Aspergillus filamentous fungi on the production of antioxidant components on various vegetable oil substances.

Using Aspergillus oryzae as the filamentous fungus, the following
The effects of filamentous fungi on the production of antioxidant components on the various vegetable oil substances shown in the table were investigated as follows.

For various vegetable oil substances, the final moisture concentration in the substrate is approximately 4
After sprinkling water to a concentration of 5% and heating at 120°C for 30 minutes, Aspergillus oryzae was aseptically inoculated and pure cultured in a flask for 3 days at 30°C using a conventional method. Samples were prepared by extracting with 95% ethanol and adding each of the obtained extracts to purified lard (no antioxidant added) at a solid content of 0.5% by volume.

10 years of this sample was stored in a 100mt beaker and stored in a dark place at 1000C, and the change in each sample over time (rotten odor of lard) during storage was sensually identified, leading to the development of a rotten odor. The antioxidant efficacy of each of the above extracts was measured using time as an index. In addition, as each control, those that were not subjected to the above culture treatment were similarly differentiated. The results are shown in Table 2. (Note) ± The odor of lard that develops during storage is almost the same or slightly weaker than the control.

+ The odor of lard that develops during storage is slightly weaker than the control.

10. The odor of lard that develops during storage is relatively weak compared to the control.

110 The odor of lard that develops during storage is considerably weaker than that of the control.

+++10 The odor of lard that develops during storage is very weak compared to the control.

＋＋＋＋＋ I can hardly smell the rotten smell of lard.
stomach.

From the above results, it is clear that the antioxidant power of various oily substances is increased by allowing Aspergillus oryzae to act on them.

Experiment 4 Regarding the effects of carbohydrates (carbohydrate substances) on substrates.

This experiment was conducted to investigate the effect of the presence of carbohydrates in the substrate on the production of antioxidant components by filamentous fungi of the genus Aspergillus.

Using commercially available washed white sesame seeds as a vegetable oil substance, the white sesame seeds were ground in a mixer, sprinkled with 6% water, and then steamed for 3 times at 120°C under a pressure of 1k9/Clt. To this, roasted and cracked wheat was added as a carbohydrate material in various proportions as shown in Table 3 below, mixed, and then water was added to this to adjust the final moisture content to about 43%. "Substrate".

Each of the substrates obtained as described above was inoculated with Aspergillus oryzae, which had been pure cultured in a bran/bread crumb medium, and cultured in an open state at 28°C. The effects of contamination) and antioxidant efficacy were investigated.

Note that the antioxidant efficacy was measured according to the procedure described in Experiment-1. As shown in Table 3, when carbohydrates are present in the substrate, the carbohydrate/protein weight ratio is preferably between 1.0 and 3.
It can be understood that when present in the range of 0, a substance with excellent antioxidant efficacy is produced by the action of filamentous fungi of the genus Aspergillus.

Experiment 5 This experiment compared the antioxidant component production effects of Aspergillus filamentous fungi and commercially available enzyme preparations on vegetable oil substances.

Defatted soybeans were used as the vegetable oil feedstock, and the mixture was sprinkled with -1% by weight of water and then steamed at 120°C for 3 minutes under a pressure of 1k9/Cjl to obtain reaction substrates. The samples were obtained by reacting filamentous fungi of the genus Aspergillus and commercially available enzymes.

(b) Effects of filamentous fungi of the genus Aspergillus. The above reaction substrate was inoculated with Aspergillus oryzae, and cultured in a pure flask at 30°C for 3 days using a conventional method, water was added to the culture weight, and the mixture was sterilized by heating at 100°C, followed by extraction. Then, the p solution was collected by passing it through a filter paper, and the obtained p-liquid was freeze-dried to obtain a pale yellow powder.

(b) Effect of commercially available enzymes Biobrase T-S (Nagase Sangyo KK), a commercially available enzyme,
Add 0.5% by weight of each of Protin PC-10F' (Daiwa Kasei KK) and Denabcin powder (Nagase Sangyo KK) and let them react at 55°C for 24 hours, then heat each enzyme-treated solution obtained at 100°C for 10 minutes. After deactivating the enzyme, the extract was collected by filtration with filter paper, and the obtained extract was freeze-dried to obtain a pale yellow powder.

Next, the antioxidant power of each sample prepared as described above against linoleic acid was measured as follows.

10 ml of a 0.02 M solution of linoleic acid dissolved in 25% alcohol.1 ml of an 8% and 20% dilution of each of the above samples to each of the mixtures with 9 mt of 0.2 M phosphate buffer.
P.1 of linoleic acid was added to each 50 ml Mayer, sealed with a rubber stopper and stored in a dark place at 37°C. O. V was measured over time and 500r1m(7)0. D. The number of days required for the value to reach 0.300 (induction period) was used as an index of antioxidant power.

As a control sample, defatted soybeans were steamed in the same manner as described above, 10@wt. A diluted solution was used. The results are shown in Table 4. As shown in Table 4 above, the antioxidant components obtained by the action of Aspergillus oryzae have significantly superior antioxidant power compared to those obtained by the action of enzymes, and the antioxidant properties obtained by the action of Aspergillus oryzae alone are significantly superior to those obtained by the action of enzymes. It is understood that the antioxidant efficacy of the antioxidant components used in the study is low.

The antioxidant according to the present invention can be applied in the form of a solid, powder, paste, emulsified, liquid, or the like of a product obtained by causing Aspergillus filamentous fungi to act on the substrate.

That is, the antioxidant according to the present invention is prepared by drying a powder of a culture obtained by treating a substrate with a filamentous fungus of the genus Aspergillus, adding an appropriate amount of an appropriate solvent to the culture, digesting or extracting the culture, and then separating it. The obtained solution may be purified, concentrated, and dried, or may be added with excipients, and depending on the form of use, the above-mentioned culture may be used as it is or diluted to an appropriate concentration. .

Furthermore, the antioxidant according to the present invention may be used in combination with an enzyme agent derived from a filamentous fungus, preferably a filamentous fungus of the genus Aspergillus.

As mentioned above, the antioxidant according to the present invention is an easily available vegetable oil substance or its extraction treatment residue, or an extraction and purification treatment residue of the oil substance, and if necessary, a carbohydrate substance is added thereto. It is easily obtained by using the substrate as a substrate and allowing easily available filamentous fungi of the genus Aspergillus to act on this substrate using conventional fermentation techniques.In addition, its effective antioxidant components are also derived from natural products. Because it is a food additive, it is safe and has the advantage of being an excellent antioxidant.

The present invention will be specifically described below by way of examples.

Example 1 5 parts by weight of coarsely crushed sunflower seeds were steamed at 120°C under a pressure of 1k9/d for 3 hours, followed by 25 parts by weight of rice bran and flour that was roasted at 180'C for 3 minutes and then crushed. Crushed food 2
The final moisture content was adjusted to 43% by adding 5 parts by weight and used as a substrate.

This was inoculated with Aspergillus Aspergillus (commercially available Aspergillus, product name Three Diamond, obtained from Higuchi Matsunosuke Shoten Hi).
The cells were cultured at a constant temperature of 0° C. for 3 days. This culture was dried in a dryer for 8 hours.
After drying with air at 0° C. for 5 hours to reduce the moisture content of the culture to a few percent or less, the mixture was pulverized with a mixer to prepare an antioxidant in powder form. The antioxidant obtained in this way is 1.0% and 2.013.0% (W/W), respectively, of genuine lard (no antioxidant added) that has been heated and dissolved.
The oil-soluble antioxidant component in the antioxidant is eluted into the lard by stirring gently for 3 minutes at 55℃, and the powder residue precipitated at the bottom is removed to contain the transparent antioxidant component. I got lard.

In addition, as a control material, lard obtained by processing in the same manner as above except that Aspergillus Aspergillus was inoculated and cultured on the raw material substrate was used. Next, instant ramen noodles were prepared by a conventional method using each lard obtained as described above, and each of the instant ramen noodles was heated in a dark place at 80°C under ventilation for 2 hours.
After being stored for a day, the lard of each instant ramen was extracted using n-hexane, and its P. ON was measured.

The results are shown in Table 5. As shown in Table 5, the antioxidant effect of the instant noodles prepared using lard to which the antioxidant of the present invention has been applied is clear compared to the control.

Example 2 Defatted soybeans were sprinkled with 12% water and the temperature was 1k9/C at 120℃! 3 parts of wheat flour that has been steamed under pressure under 1 liter of pressure;
The weighing portion was mixed and the water content of the mixture was adjusted to about 43% and used as a substrate.

Aspergillus on this substrate Awamori IFO-4033
(Ferment Research Institute) was inoculated and cultured at 30°C for 3 days).

Claims (1)

[Scope of Claims] 1. Aspergillus on a substrate consisting of a vegetable oil substance having an antioxidant effect, a residue from its extraction process, a residue from an extraction and purification process of the oil substance, a mixture thereof, or a carbohydrate substance added thereto. An antioxidant whose active ingredient is a product obtained by the action of filamentous fungi belonging to the genus. 2. The antioxidant according to claim 1, wherein the weight ratio of carbohydrate/protein contained in the substrate is in the range of 1.0 to 3.0:1.