Research Reports No.10 Abstract（2004）

10-01

Influence of synthetic and natural food dyes on activities of CYP2A6, UGT1A6 and UGT2B7

Takaharu Mizutani
Department of Drug Metabolism and Disposition,
Graduate School of Pharmaceutical Sciences, Nagoya City University

Abstract

Synthetic or natural food dyes are typical xenobiotics, as are drugs and pollutants. After ingestion, a part of these dyes may be absorbed and metabolized by phase I and II drug-metabolizing enzymes, and excreted by transporters of phase III enzymes. However, there is little information about the metabolism of these dyes. We investigated whether these dyes are substrates for CYP2A6 and UDP-glucuronosyltransferase (UGT). We also studied the in vitro inhibition of drug-metabolizing enzymes by these dyes. The synthetic food dyes studied were Amaranth (Food Red No.2), Erythrosine B (Food Red No.3), Allura Red (Food Red No.40), New Coccine (Food Red No.102), Acid Red (Food Red No.106), Tartrazine (Food Yellow No.4), Sunset Yellow FCF (Food Yellow No.5), Brilliant Blue FCF (Food Blue No.1), and Indigo Carmine (Food Blue No.2). The natural additive dyes studied were extracts from purple sweet potato, purple corn, cochineal, monascus, grape skin, elderberry, beet red, gardenia, and curthamus. First, we confirmed that these dyes were not substrates for CYP2A6, UGT1A6 and UGT2B7. Then, we studied the inhibitory function of these dyes. In the synthetic additive dyes, only Indigo Carmine inhibited CYP2A6 in a non-competitive manner. Meanwhile, only Erythrosine B inhibited UGT1A6 (glucuronidation of p -nitrophenol) and UGT2B7 (glucuronidation of androsterone). In the natural additive dyes described above, only monascus inhibited UGT1A6 and UGT2B7. However, we could not find any inhibition of CYP2A6 by the natural dyes. Thus, we found that Indigocarmine out of the chemical pigments inhibited CYP2A6 reaction and Erythrosine B inhibited glucuronidation by UGT1A6 and UGT2B7. Monascus out of the natural additive dyes also inhibited UGT reaction.

10-02

Evaluation of Safety and Usefulness of Food Additives on Daily Intake
Level by Platelet Functions

Hiroyasu Yamazaki
Faculty of Pharmaceutical Sciences, Kobe-Gakuin University

Abstract

Immigration of chemical substances from plastic food packages to inpackaged food has been reported. Bisphenol A (BPA) is one of these chemicals (food contaminants) and is suspected as an endocrine disrupter.
On the other hand, many food additives are used in food, so that we usually intake food additives and food contaminants together. Therefore, it is important to know their interactive effects on our health in addition to effects by themselves.
In this study, to investigate the additional useful aspect of food additives, we carried out the effect of antioxidant, butyl hydroxy anisole (BHA), on cellular functions exerted by BPA. As a result, BHA itself had little effect on platelet functions, and abolished or reduced the stimulus or inhibitory action of BPA on platelet functions in vitro.
In addition, in spite of the stimuli induced aggregation of platelet prepared from rabbit given BPA of Tolerable Dally Intake (TDI) level for 5 days was accelerated, simultaneous intake of BHA of Acceptable Daily Intake (ADI) level ablished and reversed the effect of BPA. These results indicate the potential protective effect of BHA on platelet functions exerted by the food contaminant, BPA.

10-03

Studies on the molecular mechanism of plant sterol-mediated cholesterol lowering effects

Ryuichiro Sato
Department of Applied Biological Chemistry, The University of Tokyo

Abstract

Net cholesterol absorption in the small intestine is calculated by both the uptake and the efflux of cholesterol. Using differentiated Caco-2 cells, a novel system for evaluating cholesterol efflux from the cells was established. When the cells were treated with a synthetic LXR ligand, gene expression of several ABC transporters was enhanced, followed by increased efflux of cholesterol into both apical and basolateral sides. The addition of apo A-1 into the apical medium resulted in stimulated efflux of cholesterol, suggesting that ABCA1 is responsible for the cholesterol efflux on the basolateral surface through generation of HDL. On the other hand, it is postulated that a heterodimer of ABCG5 and G8 is involved in the cholesterol efflux from the apical side. It was found that bile acids stimulate the cholesterol efflux from the apical side.

10-04

Molecular authentication of Ephedra Herb based on the nucleotide
sequences of a chloroplast ChlB gene

Hajime Mizukami
Graduate School of Pharmaceutical Sciences, Nagoya City University

Abstract

DNA profiling (DNA-based polymorphic assay) has several merits over morphological and chemical methods for authenticating medicinal plants and crude drugs: (1) genotype, as opposed to phenotype, is directly analyzed and, therefore, the assay is not affected by environmental factors; (2) one or more sequences appropriate for solving the particular problem can be selected; (3) the required amount of sample is very small.
The chloroplast ChlB gene which encodes subunit B of light-independent protochlorophyllide reductase was amplified by PCR from various Ephedra plants, herbarium specimens and crude drugs, and sequenced.
Based on the alignment of the sequences, we could authenticate and distinguish Ephedra Herb originating from E. sinica, E. intermedia and E. equisetina. A rapid and convenient protocol for DNA authentication of Ephedra Herb was established.

10-05

Investigation of Metabolic Pathway of Flavonoid Oligosaccharide Food
Additive with Antioxidative Function

Nobutoshi Murakami
Graduate School of Pharmaceutical Sciences, Osaka University

Abstract

Metabolic pathway of flavonoid oligosaccharide food additive, which consists of a mixture of isoquercitrin (IQC) glucosides, by human salivary and pancreatic a-amylase was examined. Consequently, IQC oligoglucosides except for treatment of a tri-glucoside by pancreatic (a-amylase afforded a mono- and a di-glucoside as predominant metabolites. On the other hand, the IQC tri-glucoside was resistant to hydrolysis of pancreatic (a-amylase to be remained as a major product. In addition, IQC oligosaccharides linking more than three glucosyl residues were little observed in either case.

10-06

Evaluation of photo-mutagenicity and photo-cytotoxicity of food coloring agents

Arimoto-Kobayashi, S., Machida, M., Okamoto, K. and Yamaguchi, A.
Faculty of Pharmaceutical Sciences, Okayama University

Abstract

Pigments extracted from natural products are being used for food coloration. Photo-mutagenicity and photocytotoxicity of colorants were examined. Using photo-Ames test, colorants from lac, cochineal, carthamus (yellow), tamarind, monascus (red), grape skin, gardenia (yellow and blue) and beet (red) showed no mutagenic activity towards S. typhimurium TA98, TA100 and TA102 strains under the UVA irradiation. The solutions of colorants from lac, cochineal, carthamus and tamarind irradiated with UVA for 4-hr were mutagenic toward S. typhimurium TA98 without metabolic activation. In the dark these colorants except monasus pigments was not cytotoxic on the human derived lymphoblastoid WTK-1 cell by itself. If these colorants were added into the suspension of WTK- 1 cells in PBS in the combination with UVA, the cytotoxicity of purpurin and grape-skin colorants were observed. Significant decrease of survival of the cells was also observed after 24-hr incubation after UVA-irradiation with colorants from cochineal, carthamus, grape skin, gardenia (yellow and blue) and beet.

10-07

Evaluation of safety and physiological function of polyphenol utilizing
gene engineering and cell technology

Satoshi Nagaoka
Department of Agriculture, Gifu University

Abstract

Soy isoflavones, are analogous to estrogen on chemical structures, have estrogenic action, and their favorable influences are noticed on various sorts of cancers, osteoporosis and others. It has known that the level of apolipoprotein A-I (ApoA-I) which is the negative factor atherosclerosis is influenced by various hormones such as estrogen and it's hoped that soy isoflavones have same activity. We showed previously that genistein, one of soy isofravones, increased ApoA-I levels because ApoA-I mRNA levels was increased mediated by the estrogen receptor (ER) a in HepG2 cells for the first time 1). However, some studies about the mechanism of action have shown little or nothing. Therefore, the aim of this study was to elucidate whether genistein which is soy isoflavone effect on ApoA-I gene transcriptional activity and its mechanism. By using HepG2 cell transfected with ApoA-I-CAT (chloramphanicol acetyl transferase) mutations, we showed that presence of -256 to -190 region of ApoA-I promoter is related to 50 mM genistein-induced increase of its gene transcriptional activity. Furthermore, in this condition, we found that addition of genistein increase HNF-4 a mRNA level and decrease ARP-1 mRNA level, which are transcriptional factors that bind siteA in -256 to -190 region of ApoA-I promoter. Our bandshift result indicate that ERa binds to site A region. These results suggest that HNF-4 a and ARP-1 in addition to ERa may relate to the increase in ApoA-I transactivation induced by genistein.

10-08

Elucidation of oxidation mechanism of gallic esters and the related compounds

Jun Kawabata
Laboratory of Food Biochemistry, Graduate school of Agriculture, Hokkaido University

Abstract

Phenolic acids and their esters are widely distributed in plants and regarded as antioxidants. Among them, ester derivatives of gallic acid, a typical phenolic acid, are used as food additives. However, mechanism of oxidation reactions of such phenolic acids and esters is complex and not yet elucidated. In this research, we have prepared a series of phenolic esters, methyl, ethyl, propyl, isopropyl and butyl gallares, and 2,3,4-trihydroxybenzoates and determined their radical scavenging activities using 2,2-diphenyl-1-picrylhydrazyl radical. The radical scavenging rate of gallic esters was fast and there was no significant difference in reactivity of free acid and each ester in both protic and aprotic solvents, methanol and acetonitrile, respectively. On the other hand, radical scavenging equivalence of 2,3,4-trihydroxybenzoic esters was higher than the free acid in both solvents. It was also found that the alkyl chain length of the esters of both acids did not affect their antiradical efficiencies. In addition, a new type of dimeric product was identified in the reaction mixture of methyl 2,3,4-trihydroxybenzoate and o -chioranil. Its lactonic structure was determined by the spectroscopic evidence.

10-09

Identification of anaphylaxis due to cochineal extract

Yuko Yamakawa
Department of Dermatology, Yokohama City University Medical Center

Abstract

Cochineal extract (carmic acid) is a natural red dye extracted from the dried bodies of the female insect
Dactylopius coccus var. Costa, which lives as a parasite on some species of cacti, particularly Nopalea
cochenillifera. Carmic acid has been implicated as an etiologic agent of occupational asthma, extrinsic allergic aleveolitis, and carmine allergy. Three female patients presented with a history of anaphylaxis after drinking alcohol or juice containing cochineal extract. Skin prick tests of all patients with campari was ++ or +++, and that with cochineal extract was ++. However, prick test of purified carmic acid was - or +. IgE of the two patients for campari or cochineal extract was positive, but IgE for purified carmic acid was negative. These results suggest that the allergen responsible for her anaphylaxis might be cochineal extract, especially insect-derived proteins, possibly complexed with cochineal extrat. SDS-PAGE of cochineal extract and immunoblotting for IgE antibody with sera from all patients were performed. The protein bands (39-45 kDa) were recognized by all three patients. These results suggest that allergens which induced anaphylaxis of cochineal extrat are insect-derived 39-45 kDa proteins.

10-10

Changes of intestinal flora by chemicals: PCR-DGGE-based approach

Norio Itoh
Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University

Abstract

Host-intestinal flora interaction alters absorption, metabolism, and toxicity of chemicals. Major part of
intestinal flora is composed of bacteria which are uncultureable or difficult to culture. Analytical methods to detect uncultureable bacteria by 16S rDNA-based RFLP, FISH and DGGE were developed and already applied in the field of environmental microbiology. We tried to apply PCR-DGGE to analyze the change of intestinal flora, and newly developed a statistic test to evaluate PCR-DGGE images. Various preservatives were evaluated by the newly developed methods. Risk assessment was performed under our experimental conditions.

10-11

Evaluation of natural compounds binding to human and/or food proteins
by surface plasmon resonance-based detection

Koichi Inoue
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University

Abstract

An analysis is developed and used to model the binding and dissociation kinetics between natural com-
pounds in solution and estrogen receptor (ER) by surface plasmon resonance (SPR). The 17 b- estradiol-17-PeNH is immobilized on a sensor chip of SPR biosensor. The interaction between an estrogen receptor alpha (ER) and an analytes is reacted in a test tube. Then, this solution is monitored to binding to free-ER and 17 b-estradiol-17-PeNH immobilized in real time. In the presence of natural compounds such as genistein with estrogenic activity, this interaction is enhanced and the kinetic parameters are altered. This analytical method is validated to the assay in terms of its specificity, dose dependency, optimal reaction conditions and reproducibility. Therefore, the present method could be useful for a routine determination and evaluation of natural compounds biding to proteins.

10-12

Pigment production by intensifying the enzyme genes of anthocyanin biosynthesis

Takashi Yamakawa
Department of Global Agricultural Sciences, The University of Tokyo

Abstract

An anthocyanin synthase (ANS) gene which coded one of the enzyme on biosynthetic pathway of anthocyanin pigments from Ipomoea nil, was introduced into sweet potato (Ayamurasaki) using Agrobacterium rhizogenes. Hairy roots were induced and their anthocyanin contents were investigated on selected transgenic hairy root lines. A variation of anthocyanin content was observed among selected hairy root lines. An expression of introduced ANS protein was not observed by Western hybridisation but transcription of the gene was confirmed by RT-PCR employing the different primer sequences between original and introduced ANS genes.

10-13

Studies for analysis and Control of the Oxidative Change of Natural Food Colorants

Toshiya Masuda
Faculty of Integrated Arts and Sciences, University of Tokushima

Abstract

Antioxidant activity of laccaic acid A, a main pigment of lac color, was affected by pH conditions. Wide　range of pH effect to antioxidant activity of laccaic acid was examined in this study. Although laccaic acid A never showed antioxidant activity in methanolic homogeneous system, sodium salt derivatives of laccaic acid A was found to have very potent antioxidant activity in the homogeneous system in this study.
Synthesis of non-polar derivatives having antioxidant activity of laccaic acid A, which can be used for the analysis of antioxidant mechanism of laccaic acid A, was failed. However, NMR measurement of an antioxidation product of disodium salt of lacccaic acid A was successful. The obtained data indicated that the D ring of laccaic acid A concerned to the antioxidation reaction.
The antioxidant activity of laccaic acid A under pH 6.3-tween20 micelle system was found to be stronger than under pH 7.4, however, the reaction rate of laccaic acid A under pH 7.4 was higher than under pH 6.3.
This phenomenon is very interesting and gives some useful insight for using laccaic acid both for food pigment and food antioxidant.

10-14

Availability of Anthocyanin Pigments in the Skin of Vitis Coignetiae
Grapes as a Coloring Agent and Healthy Food

Goro Okamoto
Faculty of Agriculture, Okayama University

Abstract

Vitis coignetiae grape berry skin contains high levels of anthocyanin pigments as well as several phenolic compounds. Skin extracts obtained from V. coignetiae grapes showed higher tolerance against light-decomposition of anthocyanin comparison with those from Cabernet Sauvignon (V. vinifera), Campbell Early (V. labruscana), and Pione (V. vinifera x V. labruscana). This study aimed to clarify the mechanism of anthocyanin-light-stability in V. coignetiae skin extracts. Fractionations of red and colorless phenolic compounds were carried out using an ODS column chromatography. Finally, in V. coignetiae skin extracts, a colorless phenolic substance, "B-compound", with a maximum light absorption peak at 254 nm, was found to be an active anti-light-decomposition substance. The compound was not detected in Cabernet Sauvignon skin extracts. ESI-LC-MC analyses of the "B-compound" revealed that the compound is a kind of glucoside composed of aglycon with a molecular weight of 194 and two sugar molecules. This compound can be easily extracted quantitatively from the skin of V. coignetiae and could be useful as a food additive to stabilize anthocyanin pigments.

10-15

Structure and application of highly water adsorbent polysaccharide
on the surface of seeds of Artemisia sphaerocephala Krasch (沙蒿)

Masahiro Tada
Laboratory of Bio-organic Chemistry, Tokyo University of Agriculture and Technology

Abstract

Desert wormwood, Artemisia sphaerocephala Krasch(沙蒿) is distributed over dry desert of yellow soil in northwest China, Ningxia and Neimenggu Zizhigu. The surface of seeds is coated with highly water adsorbent polysaccharide. When it is rain on the desert, the polysaccharide on the surface of the seeds becomes a gum by adsorbing water to spread over the yellow soil and the seeds can germinate. The seeds of A. sphaerocephala Krasch are used for additives of noodles to improve the quality in the northwest China. We found efficient method to separate the polysaccharide from the seeds and found that the polysaccharide contains water-soluble substances in it on the seeds surface. The isolated polysaccharide by the known procedures is partially crystallized and difficult to adsorb water again. The water adsorbing polysaccharide could be prepared by adding some hydrophilic substance before drying it. The polysaccharide consists of glucose, mannose (in a ratio of 3:1) and small amount of unknown carbohydrates. These informations give new possibility for application of the seeds of A. sphaerocephala Krasch as foods additives.

10-16

Exploitation of antibiotic browning-inhibitors from marine algae

Tadahiko Kajiwara
Department of biological chemistry, Faculty of Agriculture, Yamaguchi University

Abstract

Since ancient days, antimicrobial properties of seaweeds have been recognized. However, antimicrobial activities of volatile compounds in seaweeds have not been explored so far. Here, essential oils from seaweeds including green, brown and red algae such as Laminaria japonica, Kjellmaniella crassifolia, Gracilaria verrucosa and Ulva pertusa were prepared by using SDE (simultaneous distillation and extraction) apparatus. Volatile compounds in the essential oils were identified as aldehydes, ketones, carboxylic acids, alcohols and hydrocarbons by comparison of GC-retention times and MS data with those of authentic specimens. Flavor compounds such as (3Z)-hexenal, (2E)-hexenal and (2E)-nonenal in some essential oils showed strong antimicrobial activities against Escherichia coli TG-1, and Erwinia carotovora. Inhibition of browning can be achieved at either one step of the two, namely, oxidation reaction by tyrosinase or subsequent nonenzymatic polymerization. Tyrosinase activity was measured with monitoring absorbance at 475 nm originated from dopachrome formed from L-DOPA. Many kinds of aliphatic carboxylic acids, aldehydes and alcohols were used as inhibitors for PPO activity. The results obtained indicated that the a, b-unsaturated carbonyl compounds strongly inhibit tyrosinase activity. When seaweeds are damaged or macerated, the a, b-unsaturated aldehydes such as (2E)-hexenal and (2E)-nonenal are biosynthesized via the corresponding (3Z)-unsaturated aldehydes from linolenic acid and arachidonic acid. The formed flavor compounds and seaweed juices could be valuable as safe antimicrobial browning-inhibitory agents of edible seaweed origin for prevention of vegetable browning and poisoning with E. coli O157.

10-17

Establishment of agricultural plants producing high bioactive compounds

Koichiro Shimomura
Faculty of Life Sciences, Toyo University

Abstract

For callus induction of red cabbage (Brassica oleracea L. var. capitata DC), petiole segments of seedling in vitro were cultured on LS solid medium supplemented with 20 mM IBA and 1 mM BA. Induded callus was subcultured on the same medium, and adventitious roots and buds were obtained. Callus of purple corn (Zea mays L.) was obtained when cultured on MS solid medium supplemented with 1 mg/L 2,4-D using sheaths of axenic plantlets. Callus grew well on N6 solid medium supplemented with 1 mg/L 2,4-D and 25 mM proline, and a few shoots regenerated from a part of callus were observed. We established adventitious root cultures using roots formed at shoots since red pigments were accumulated at aerial roots of purple corn cultivated in a field. The adventitious roots grew in MS liquid medium supplemented with 1 mg/L NAA or 2 mg/L IBA in the dark or light, especially vigorous growth was observed under the light condition. The adventitious roots cultured in MS liquid medium (1 mg/L NAA, in the light) showed more than 6-fold higher anthocyanin content (ca. 4 mg/100 mL flask) compared with the adventitious roots cultured in the dark. The main anthocyanidin in seeds, roots of plant cultivated in the field and the adventitious roots cultured in the light was cyanidin. In contrast the main anthocyanidin in the adventitious roots cultured in the dark was peonidin. Interestingly the highest content of anthocyanin in adventitious roots cultured in MS liquid medium (2 mg/L IBA, in the light) was almost the same level as that in seeds cultivated in the field (average 0.8 % as dry weight).

10-18

Studies on the elicitation of sweetness of lysozyme

Tetsuya Masuda and Naofumi Kitabatake
pision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University

Abstract

Lysozyme is one of the most thoroughly characterized enzymes, and one of the sweet-tasting proteins.
However, a detailed explanation for the sweetness of the hen egg white lysozyme (HEL) is as yet unknown.
At first the relationship between thermal denaturation and loss of sweetness of lysozyme were investigated. The results showed that sweetness of lysozyme was not remarkably changed by heating at 70 oC for 25 hr or 80 oC for 8 hr. Second, to clarify the role of lysine residues for elicitation of sweetness of lysozyme, the effect of the side chain of lysine residues was investigated by chemical modification method. These results indicated that positive charges of side chain of lysine residues influenced the sweetness of lysozyme.
Finally, high yields of expression of lysozyme was attempted by methylotrophic yeast Pichia pastoris.
Expression of HEL was carried out by fermenter. Approximately 400 mg L^-1 of recombinant HEL was obtained. The purified recombinant lysozyme elicited sweet taste as efficient as does the lysozyme from egg white, and showed the full lyric activity against Micrococcus luteus. High yields of expression of lysozyme from this study would provide not only a large amount of sample for sensory analysis, but also help our understanding the mechanism of interaction of sweet proteins with their receptors.

10-19

Antibacterial Activities of Extracts and Constituents in the Complex of
Tea catechins and Soybean Protein

Abstract

Extracts and constituents in the complex of tea catechins and soybean protein, a new food material, were screened for their antibacterial activity against 2 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and 2 Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes). The minimum inhibitory concentrations (MICs) of extracts and constituents in the complex of tea catechins and soybean protein were determined by the microdilution method ranged from 0.49 to 16,000 mg/mL and 0.03 to 1,000 mg/mL, respectively. The 2 Gram-positive bacteria tested were only inhibited by the complex extracts, (-)-epigallocatechin 3-O-gallate (EGCG), and (-)-epicatechin 3-O-gallate (ECG). The pyrogallol moiety on the B ring of the flavonoid structure showed the stronger activity rather than catechol moiety against Staphylococcus aureus and Listeria monocytogenes.

10-20

Study on the bioavailability of biotin, a new food additive, using a biotin-deficient mice

Abstract

The aim of this study is to clarify the bioavailability of biotin by scientifically elucidating the digestion and absorption of biotin in the intestinal tract. In this year, we determined the biotin and 3-hydroxyisovaleric acid (3-HIA), which is related with biotin-dependent carboxylase, in biotin-deficient mice. 3-HIA was detected in urine of biotin-deficient mice fed a biotin-deficient diet for 4 weeks. 3-HIA could be assayed with high accuracy by the present HPLC method. It is suggested that 3-HIA may be useful to detect the biotin deficiency in the early stage of biotin deficiency.

10-21

Enzyme-catalyzed Direct and Regioselective Acylation of Flavonoid
Glucosides for Stabilization and Functionarization of Plant Pigments

Abstract

Regioselective acylation of flavonoid glucosides was achieved by lipase-catalyzed transesterification in dry organic solvent. The participation of the acyl group in flavonoid glucoside molecules resulted in increasing of the physiological function (light-resistibility and radical-scavenging activity) of the acylated flavonoid glucosides.

10-22

Development of food additives with in situ gelling properties of
ionic responsible intelligent polysaccharides

Shozo Miyazaki
Health Science University of Hokkaido, Faculty of Pharmaceutical Sciences

Abstract

The purpose of this study was to develop liquid food additives with in situ gelling properties. The food
additives were dilute solutions of gellan gum and sodium alginate both containing complexed calcium ions that form gels when these ions are released in the acidic environment of the stomach. The liquid food additives of gellan gum (1.0 %w/v) and sodium alginate (1.5 %w/v) resulted in the formation of gels in the acidic environment (pH<3.0) of the gastric-acidity controlled rabbits. In situ gelations in the stomach of the high-acidity (pH 1.01) rabbits were similar to those of low-acidity (pH3.17) rabbits. These results suggest that gastric acidity has no effect on the in situ gelations of the liquid food additives in the gastric pH range investigated.

10-23

Fundamental studies for safety evaluation of insoluble minerals that are
existing food additives

Abstract

As molten lava cools, it tums into glass-like rock such as perlite, pitchstone and obsidian. Using these as a base, insoluble minerals (perlite, activated acid clay, diatomaceous earth and zeolite, etc.) are made into existing food additives that are used as processing agents in manufacturing food products (filtering aids or settling aids). Being that the basic material is rock, there is a danger that these food products, depending on the raw ore, may contain a large amount of bioactive substance such as heavy metal that is detrimental to the human body. However, in the list of existing food additives only the scientific name and the processing method is recorded of the original plant and animal, but not the form, characteristics or specifications. Furthermore, although perlite, talc, kaolin and diatomaceous earth are listed in Japan's specifications and standards of food additives, there are no standards set for any harmful substances other than for heavy metals, lead and arsenic. By the Food Sanitations Low, these processing agents may not be found remaining in the final food product. However, there is the danger that should any of these insoluble minerals containing harmful bioactive substances be found remaining in the food or should harmful substances leaching out from these agents enter into
the food products, it will cause enormous health damage.
Therefore, it would be an extremely significant step to create basic references for the purpose of executing safety evaluations by measuring the harmful materials contained in the insoluble minerals or their eluates. At the same time in one single swoop, it will resolve the many problems surrounding the food hygiene issues including that of the above mentioned enormous health damage.
In 2003, material testing and elution testing laws for insoluble minerals were deliberated.

10-24

Fundamental Safety Assessment of the Registered Existing Food Additives
(Natural Source Food Additives)

Abstract

In 1995, the 489 items of existing food additives (natural source food additives) were registered by the
Ministry of Health, Labor and Welfare. These additives were assessed the safety by existing research data and classified in following three categories.
1) The additives verified the safety by the several qualified scientific data: 214 items.
2) The additives recognized the verification would not be urgently executed from the nature of the source, process etc: 150 items.
3) The additives recognized the safety verification should be urgently executed, since the scientific results have not been sufficiently corrected: 125 items.
The fundamental safety assessment in this report was focused to the items classified to second category; the additives recognized the verification would not be urgently executed, since the original natural sources of the additives should be verified the safety properties as food materials and the components should not be contained any harmful substances even these additives were classified as less necessity of the safety assessment.
The procedure of assessment was carried on the following properties of the additives.
1) The identification of the original natural sources (animal and plant) of the additives.
2) The survey of eating experience as food of the natural sources.
3) The investigation of the components included in the natural sources and the scientific safety data obtained from RTECS.
4) The survey on the commercial enzyme products.