JPH0789863B2 - Cream for beverages containing peroxidase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cream for beverages such as coffee and tea containing peroxidase. The cream of the present invention reduces the cytotoxicity of the beverage by decomposing hydrogen peroxide generated in coffee and tea during drinking, and at the same time inactivates the mutagenicity of the beverage. With the goal.

(Prior Art) Elimination of mutagens in foods by peroxidase is disclosed in Japanese Patent Application Laid-Open No. 60-6294 related to the inventors of the present invention, and the present invention relates to improvement thereof.

(Technical problem to be solved by the invention) It goes without saying that safety of food and drink is a serious problem for human health. Here, the safety of foods and drinks basically means that the foods and drinks do not contain substances (pathogens, poisons) that adversely affect human health. Regarding microbial control of products produced by the food industry, each company has undergone strict sterilization and sterilization processes,
In addition, a base such as an oxygen scavenger that makes growth of microorganisms difficult has been developed, so that it is rarely a problem at present. However, there are many unsolved problems regarding poisons. That is, frill flamide (AF-
2), the use of food additives such as cyclamate (cyclo) and hydrogen peroxide whose carcinogenicity has been revealed is prohibited or should not remain, and has been excluded from food additives. Is a carcinogen originally contained in edible plants such as Aquilide A, and Trp
-For carcinogens such as P-1, MeIQ, which are generated endogenously during the cooking process of meat, their risks cannot be excluded by mere legal regulation.

Therefore, the development of a method for decomposing / removing these poisons has become the most important issue for the health of humans who actually ingest foods and drinks containing these substances.

The present inventors have examined the cytotoxicity and mutagenicity of coffee on human small intestinal cells, and as a result, immediately after making coffee (that is, in the case of instant coffee, immediately after dissolving coffee powder in water or hot water, regular coffee). In the case of coffee, immediately after extraction of roasted coffee beans), it was confirmed that hydrogen peroxide was generated by autoxidation of coffee components (Fig. 1).

According to FIG. 1, hydrogen peroxide was generated immediately after the coffee powder was dissolved in water, and the concentration thereof increased with time. In contrast, hydrogen peroxide was not detected in the coffee solution prepared under anaerobic conditions (100% N ₂ ). When the coffee produced under aerobic conditions was transferred to anaerobic conditions, the hydrogen peroxide concentration stopped increasing, but the concentration did not decrease.

Similar reports can be found in Nagao, M. et al., Banbury Report.
17: Coffee and Health, pp69-77, Cold Spring Harbor
Also done in Laboratory, 1984.

The concentration of hydrogen peroxide is instant coffee (15 mg powder / m
l, 5 minutes after dissolution) 2.4 ± 1.2 ppm, 1 with regular coffee
It was 0.6 ± 4.8 ppm. Therefore, as shown in the Examples below, the present inventors have found that enzymatic decomposition of hydrogen peroxide significantly reduces the above-mentioned cytotoxicity of coffee and almost eliminates mutagenicity. . Hydrogen peroxide is considered to be the main cause of amphoteric toxicity.

In addition, as a result of examining the presence and degree of hydrogen peroxide generation in beverages other than coffee, it was found that even though the concentration was lower than that of coffee, hydrogen peroxide was generated at around 1 ppm in black tea, and hydrogen peroxide was enzymatically generated. It was clarified that the degradation of tea into human small intestinal cells and its mutagenicity were also reduced by the decomposition into black tea.

The toxicity of hydrogen peroxide has already been clarified [Ito, A. et al., Gann, Vol. 73, 315-322, 1982;
Thacker, J., Mutation Res., Vol.33,147-1
56,1975], the production of hydrogen peroxide from foods and drinks including the heating step (including roasting and cooking) and the drying step is considered to have an unfavorable effect on mutagenicity and cytotoxicity.

Therefore, enzymatically decomposing this is considered to be sufficiently significant to protect the health of humans who ingest these foods and drinks on a daily basis. Incidentally, the Ministry of Health and Welfare has announced that there should be zero residual hydrogen peroxide in food (19).
80). In addition, among the coffee and tea creams currently on the market, 10 kinds of powdery or liquid creams were examined, and none of them decomposed hydrogen peroxide, and the cytotoxicity and sudden Mutagenicity did not change at all by addition of each cream (Figs. 2 and 3).
Figure).

Figure 2 shows that none of the commercial creams (3; 7 others examined) had any effect on coffee mutagenicity.

FIG. 3 shows that the commercial cream (creaming powder) had no effect on the cytotoxicity of coffee on human small intestinal cells. Although four types (4 brands) of cream were examined, all of them showed the same results as the white circles in FIG. 3, so the three types (3 brands) were omitted. Also, in the drawing, the white circles and the black circles are shifted, but the points are essentially the same.

The present invention is based on the new finding that hydrogen peroxide is generated in coffee, tea, etc. during drinking, which constitutes the cause of cytotoxicity and mutagenicity of both beverages. Peroxidase (donor: H ₂ O ₂ -oxidoreductase, EC1.11.7), which is an enzyme that decomposes hydrogen peroxide, is added to the mixture, and by using the cream, hydrogen peroxide in coffee and tea can be easily removed by a simple method. It is intended to completely decompose and eliminate the toxicity of hydrogen peroxide (carcinogenicity, mutagenicity, clastogenicity, cytotoxicity).

(Structure of Invention) The present invention is a beverage cream containing peroxidase. As the peroxidase used in the present invention,
Animal-derived substances such as lactoperoxidase and glutathione peroxidase, plant-derived substances such as wheat germ and horseradish peroxidase, and microorganism-derived substances that have been confirmed to be safe can be used. Among these peroxidases, plant-based peroxidases are particularly suitable for the use of the present invention because of their heat resistance, safety and stability. In particular, since the plant peroxidase used in the present invention is finally ingested by humans, it can be isolated and purified by a safe purification method from plants that have been used for a long time (for example, horseradish, wheat germ, rice germ). It is desirable to use the one that has been used. However,
If there are no problems in practical use such as dispersibility, solubility, and heat coagulation, even a crude product is sufficient.

The peroxidase may be handled easily by using an excipient in the operation when blending it into the cream. As the excipient, those which are safe, have no problems in palatability and have low hygroscopicity and reactivity are preferable. For example, lactose is suitable.

The type of cream to be added with peroxidase is not particularly limited. It may be either an instant powder cream obtained by pulverizing the cream or a liquid cream, and whether milk-derived cream or milk fat is replaced with vegetable fat does not cause a large difference in the peroxidase activity to be incorporated or its stability.

When adding peroxidase to the cream, if the cream is a powder, mix powdered peroxidase preparations that have been added with an excipient powder, or spray peroxidase liquid with the excipient by spray granulation or fluidized bed formation. It can be blended by the granule method, or when the cream is liquid, crudely purified or purified peroxidase may be dissolved in the cream without using an excipient. The blending ratio varies depending on the heat resistance of the peroxidase used and the temperature of the beverage during the beverage, but the peroxidase activity is 0.5 to 60 units / g cream for powder cream, and 0.4 to 50 units for liquid cream. Blend so that it becomes / ml (both units are purprogarin units).

Hereinafter, the present invention will be described in detail with reference to Examples. First, the test methods and types of peroxidases used are as follows.

I) Measurement of hydrogen peroxide concentration The test solution was placed in an oxygen measurement reaction tank [Type BOM-11, Ishikawa Seisakusho Co., Ltd., Tokyo], and after being shielded from the outside air, catalase (manufactured by beef liver, Pharmacia P -L Biochemica
ls Inc.) 1,500 units / 10 μl was added, and the amount of oxygen generated by the decomposition of hydrogen peroxide was measured by the dissolved oxygen electrode [Type DG-5,
Ishikawa Seisakusho Co., Ltd.]. The hydrogen peroxide concentration was calculated from the generated dissolved oxygen concentration.

II) Mutagenicity test Mutagenicity is the Salmonella mutagenicity test (Salmonella m
utagenicity test) (Ames, BNet al., Mutation Res.,
Vol.31,347-364,1975) and Chinese hamster lung fibroblasts (CHL cells) using diphtheria toxin resistance as an index (Nakasato, F., Mutation Res., Vol.141,109-112,1).
984).

(1) Salmonella mutagenicity test Preincubation method (Sugimura, T. and M. Naga
o,: FJ de Serres and A. Hollaender (co-edited), Chemica
l Mutagens, Principles and Methods for Their detect
ion, Vol.6, Plenum, New York, pp41-66, 1980). The strain used is Salmonella teifimulium ( Salmon
ella typhimuritum ) TA100 strain (hereinafter abbreviated as “S.TA100 strain”) was used.

(I) Preparation of sample i) Coffee: Instant coffee (powder) is dissolved in distilled water. In the case of regular coffee, roasted coffee beans are extracted with 250 ml of hot distilled water per 20 g of the coffee, and the extract is passed through coffee filter paper (Carita No. 12). The solution is freeze-dried, the dry weight is measured, and then the solution is dissolved in distilled water.

B) Cream: In the case of instant powder cream, dissolve in distilled water to a prescribed concentration. When peroxidase was added to the cream, a predetermined amount was added using lactose as an excipient by a spray granulation method. In the case of liquid creaming, a predetermined amount of peroxidase was added and dissolved.

C) Black tea: 2.5 g of commercially available black tea is extracted with 200 ml of boiling distilled water, the leaves are removed after extraction, the extract is dried under reduced pressure at 40 ° C with an evaporator, and the residue is converted to dimethylsulfoxide (DMSO). Dissolve.

(Ii) Measurement of mutagenicity Each of the samples obtained by the above a) to c) was used alone, or after mixing [a] and b), b) and c)], 100 μl was added to each, and 500 μl of this was added. Add 100 mM sodium phosphate buffer (pH 7.4) and 100 μl of S.TA100 strain culture solution. This mixture was shaken at 37 ° C for 20 minutes and then dissolved
Mix in 2 ml of soft agar and spread on a 0.1% glucose agar plate. For the soft agar, 0.1 μmole / 2 ml soft agar / plate L necessary for dividing the bacteria several times on the plate was used.
-Add histidine. After standing at 37 ° C for 48 hours, count the number of colonies on the plate as revertant strains.

(2) Mutation test using the appearance of diphtheria toxin-resistant cells as an index Tested by the method of Nakayasu et al. [Nakayasu, M. et al.,
Carcinogenesis, Vol.3,917-922,1982; Sakamoto and Terada,
Toxicology Forum, Vol.8 (2), 101-104, 198
Five〕.

48 hours before the start of the mutagenicity test, CHL cells are released from the flask using 0.25% trypsin, and 1 × 10 ⁵ cells are transferred to a 5.5 cm ² tube (A / C, Nunc).

After culturing the subject with CHL cells at 37 ° C for 3 hours, the culture solution was removed, and the cells were released by trypsin treatment and the cells were VAMEM containing 10% fetal bovine serum [MEM containing vitamins and amino acids.
Wash 3 times with medium (Eagle's minimum essential medium)]. For measurement of cytotoxicity, CHL cells cultured with a subject and CHL cells cultured in a culture medium containing no subject were treated with 60 m
Sprinkle 200 to 400 seeds on a m-chalet and incubate for 7 to 8 days.
The cells are fixed with methanol and stained with Giemsa. Number of cells
Count more than 50 colonies as colonies. Cytotoxicity is expressed as cell viability, which is the ratio of the colony formation rate of subject-treated cells to the colony formation rate of untreated cells.

To measure the mutation rate, after culturing the cells with the subject, transfer 1 × 10 ^{5 to} 3 × 10 ⁵ cells to a 25 cm ² flask and add 10 ml of 10% fetal bovine serum to the culture medium. Culture for 7 to 8 days (corresponding to the expression period). During this time, the cells are once stripped with trypsin and reseeded in another flask to keep the cells in the growth phase at all times. After the onset period, 2.5
× 10 ⁵ cells are transferred to a 90 mm dish and 10 ml of VAMEM containing 10% fetal bovine serum is added. After 3-4 hours, diphtheria toxin is added at a concentration of 0.1 Lf / ml. Simultaneously after the expression period, transfer 200-400 cells to a 60 mm dish and add 5 ml of 1
The cells are cultured in VAMEM containing 0% fetal bovine serum for 7 to 8 days, and the colony formation rate is measured. The mutation rate is expressed as the mutation rate per 2.5 × 10 ⁵ viable cells by dividing the number of colonies formed in the presence of diphtheria toxin by the number of colonies in the absence of toxin.

The mutagenic potency in this system correlates better with the carcinogenic potency of the subject than the mutagenic potency against Salmonella.

(3) Cytotoxicity test against human small intestinal cells Human small intestinal epithelial cells (Intestine 407) were seeded on a 6-well dish (bottom area 9.6 cm ² × 6) by 9.2 × 10 ³ cells / 2 ml each, and 10
MEM medium containing% fetal bovine serum (Eagle's minimal essent
Incubate for 3 days in a CO ₂ gas incubator. After 3 days, the MEM is removed, the subject is dissolved in MEM or PBS, 2 ml each is added to the dish, and left in a CO ₂ gas incubator for 3 hours, and then the MEM or PBS containing the subject is removed. Further ME
Replace M and incubate for 3 days. After culturing, the cells are washed with PBS, fixed with methanol and stained with Giemsa. Cytotoxicity (degree of cytotoxicity) is evaluated in 5 levels.

III) Peroxidase The following peroxidases were tested.

(1) Peroxidase [derived from horseradish; 120 units ^* / m
g: Toyobo Co., Ltd., Osaka) (2) Peroxidase [derived from horseradish; 180 units ^* / m
g: Amano Pharmaceutical Co., Ltd., Nagoya] * All are purpurogallin units Example (1) Hydrogen peroxide decomposition effect in coffee and tea containing cream containing peroxidase II) (i) b) and c) To the hydrogen peroxide generated in the prepared regular coffee and tea for 20 minutes, peroxidase blended cream (from horseradish, 6 purpurogallin unit / g powder cream) was added instantly when 3g of coffee or tea of 150ml was added. Was disassembled (Fig. 4). The amount of cream added was 20 mg per ml for both regular coffee and black tea.

(2) Decomposition of hydrogen peroxide and mutagenic inactivation Dissolve instant coffee in distilled water, and after 10 minutes, add peroxidase (horseradish, 10 units / ml coffee) to decompose hydrogen peroxide. did. At this time, the mutagenicity against S.TA100 was also inactivated (Fig. 5). According to FIG. 5, the mutagenicity of coffee has a close correlation with the amount of hydrogen peroxide generated in coffee, but peroxidase can extinguish hydrogen peroxide and mutagenicity extremely effectively. Is recognized. Moreover, it was also found that the action of this enzyme is reversible, because both of them return to their original levels when the peroxidase is removed by ultrafiltration. However, with peroxidase for a long time (at least 1 hour)
Upon treatment or addition of an electron acceptor serving as a substrate for peroxidase to completely react and consume the electron donor in coffee, the mutagen derived from hydrogen peroxide in coffee disappears irreversibly.

FIGS. 6A and 6B show as data the method of irreversibly inactivating the mutagenicity of coffee by long-term treatment with peroxidase (POD). PO
Coffee S. Tefimurium TA1 without D
If the mutagenicity for strain 00 is 100%, POD (0.125 ~
1.25 units / ml coffee solution) and the longer the reaction time, the more mutagenicity is irreversibly lost.
About 90% of the mutagenicity is irreversibly inactivated by reacting D (the above concentration) for 2 hours with shaking at 37 ° C.

On the other hand, the above results were also confirmed in CHL cells.
That is, the mutagenicity of instant coffee against CHL cells was determined by adding the cream containing peroxidase (5
(Unit / ml coffee) significantly reduced. (7th
Figure). The effect of peroxidase was also confirmed in mammals.

(3) Reduction of cytotoxicity of coffee and black tea with cream containing peroxidase.

A commercially available cream (creaming powder) was spray-granulated with peroxidase (manufactured by Toyobo Co., Ltd.) to produce a compounding cream. This cream is PBS (phosphate saline)
The addition of 0.015 units / ml of citrate significantly reduced the cytotoxicity of instant coffee and tea on human small intestinal epithelial cells (Figs. 8 and 9). No effect was observed with the cream containing no peroxidase (Figs. 8 and 9). As for cream, other 3 kinds of commercially available creaming powder and 2 kinds of liquid cream were added to coffee or black tea and the effect was examined, but the same result as coffee without cream was shown, and no effect was observed on cytotoxicity. It was

(4) Heat resistance of cream containing peroxidase.

The heat resistance of this cream was examined. Enzyme activity sufficient to show the above-mentioned effect was lost by boiling for 10 minutes, but the conditions assuming normal drinking forms of coffee and tea,
That is, when treated at 80 ° C. for 10 minutes, the cream containing 6 units of enzyme per gram exhibited remarkable effects of decomposing hydrogen peroxide, cytotoxicity and mutagenicity. If the amount of enzyme is increased 10 times, sufficient enzyme activity will remain even if treated at 90 ° C for 5 minutes, and it will be sufficient if the boiling water is allowed to cool for a short time (usually about 1 to 2 minutes). There is practicality.
In addition, deterioration of the cream such as deterioration and deterioration of solubility and coagulation did not occur due to the addition of peroxidase. At the same time, regarding the effect of this cream on the palatability of coffee and tea, the addition of peroxidase did not impair the taste and aroma, and there was no practical problem in this regard.

Preparation Example (Cream containing Peroxidase) A cream containing 6 units of peroxidase per 1 g of the synthetic cream was produced according to a conventional method for producing a synthetic cream.
That is, in the raw material oil and fat consisting of rice bran oil and hardened coconut oil
Water, corn syrup, soy protein, sodium sterol lactate, polysorbate, sodium diphosphate, potassium diphosphate, pyrophosphate and peroxidase were mixed and whipped. Then homogenize at 65 ° C,
Perform batch sterilization at 80 ° C for 10 minutes, then cool rapidly,
Aging was performed at 5 ° C. for 10 hours to obtain a peroxidase-containing cream. Peroxidase also acted as an emulsifier and stabilized the emulsification.

[Brief description of drawings]

FIG. 1 is a graph showing hydrogen peroxide generation from a coffee solution, FIG. 2 is a graph showing that commercial cream does not inactivate the mutagenicity of coffee, and FIG. 3 is a graph showing that commercial cream does not. Fig. 4 is a graph showing that coffee does not inactivate the cytotoxicity to human small intestinal cells, Fig. 4 is a graph showing the hydrogen peroxide decomposing effect of peroxidase-containing cream in coffee and tea, and Fig. 5 is coffee. FIG. 6A is a graph showing the results of investigating the relationship between the amount of hydrogen peroxide generated in cells and mutagenicity by inactivating these with peroxidase. FIG. FIG. 6 is a process chart showing a method for examining activation, FIG. 6B is a graph showing irreversible inactivation of coffee mutagenicity, and FIG. 7 is a peroxidase-containing cream. Fig. 8 is a graph showing inactivation of mutagenicity of instant coffee by Fig. 8, Fig. 8 is a graph showing reduction of coffee cytotoxicity by cream containing peroxidase, and Fig. 9 is cytotoxicity of black tea by cream containing peroxidase. It is a graph which shows reduction of.

Claims (1)

[Claims] 1. A beverage cream comprising a peroxidase.