In the previous paper, we reported radio-sterilizing effect and change of microflora of unpolished rice harvested in Japan, and polished rice imported from Spain. They were irradiated in order to store a long period. In this investigation, Thai rice unfumigated with methylbromide was used as a sample.
(1) The main microflora of unirradiated Thai rice was mainly occupied by secondary contaminated microorganisms such as Bacillus megaterium, B.cereus, B.subtilis, Brevibacterium and Micrococus which were peculiar to long-time-stored polished rice, and also Actinomycetes was isolated at a considerable amount. Fluorescent Pseudomonas and Chromogenic Pseudomonas which are main microorganisms of new rice, were not isolated.
(2) When the total dose on Thai rice reached between 0.1 and 0.2 MR, a rapid decrease of the number of survival microorganisms, as can be seen in case of new rice reported in our previous paper, was not observed. Bacillus and Actinomycetes were main survivors at the dose of more than 0.2 MR, but Bacillus was more resistant to irradiation than Actinomycetes.
(3) Moulds found, mainly Aspergillus, were much less than Bacteria. Some Penicillium were also isolated. Aspergillus and Penicillium were sterilized at the dose between 0.3 MR and 0.4 MR.
(4) Polyethylene bag was better than craft paper bag for the purpose of longer storage of polished rice.

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Effectiveness of gamma irradiation as a protective means against post-harvest decay in ‘Donner’ strawberries was studied.
Very fresh strawberries packed in monolayers in polyvinylchloride boxes were wrapped with ordinary cellophane or water-proof cellophane, and irradiated immediately.
When strawberries were stored at 23°C, 10% infection due to mould fungi, such as Botrytis cinerea, Rhizopus and Penicillium, was found 3 days after 0 KR irradiation, 4 days after 100 KR irradiation, and 5 to 6 days after 200 KR irradiation. This means that their shelf life can be extended by 2 or 3 days by irradiation treatment. Cellophane has been found to be more favorable than waterproof one when it is used as wrap-film at room temperature.
Weight of irradiated berries decreased, independently of dose, linearly with storage time.
When strawberries were stored at 8°C, berries were found to be kept clean for 7 to 8 days without irradiation, for 11 to 12 days with 200 KR irradiation and for 14 to 16 days with 300 KR. In this case, water-proof cellophane is more effective.
Main pathogen, strain no. TO-1 of ‘Donner’ berries produced in Gumma Pref. was isolated and identified as Bot. cinerea. From its survival (vs. dose) curve and the survival curve of B. cinerea IAN 5128 (one of the collection of the Institute of Applied Microbiology, Univ. of Tokyo) as a standard rot, their lethal doses were determined as 970 KR and 540 KR, respectively. It has been found that Botrytis are more resistant to radiation than Aspergillus and Penicillium.

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In connection with food irradiation in the frozen state, radiation sensitivity of Micrococcus radiodurans, an extremely radioresistant vegetative bacterium, was determined under different conditions of irradiation. Stationary- phase cells suspended in 0.067M phosphate buffer were irradiated at liquid nitrogen temperature (-196°C) and also at room temperature. The induction dose and D₁₀ value were 6.12 Mrads and 0.53 Mrads at liquid nitrogen temperature and 0.87 Mrads and 0.11 Mrads at room temperature, respectively. The protection factors afforded by freezing were 7.0 for the induction dose and 4.8 for the D₁₀. In comparison with the protection factors obtained with other species of vegetative bacteria which have been previously reported (2.5-8.5), the factor of 4.8 for the D₁₀ is not unusual. However, the factor of 7.0 for the induction dose in M. radiodurans is comparatively large. It is noteworthy to mention that the induction dose of this radioresistant bacterium, about 6 Mrads, in frozen state irradiation is larger than the sterilization dose in radappertization, 4.5 Mrads, which corresponds to the 12D of Clostridium botulinum spore (type A and type B). Thereby, it would be worthwhile to calculate the numbers of hits in DNA per cell. If one could postulate that an average ion cluster contains about 2.3 primary ion pairs and subsequently the mean energy dissipated in one effective event is 78 eV (=34eV× 2.3). On this basis, the direct hits by effective primary ion clusters in DNA at a given dose, D, are calculated as follows: hits in DNA=8× 10¹¹× gmDNA (rads). The survivors of M. radiodurans were about 90% by 3 Mrads at liquid nitrogen temperature and by 0.3 Mrads at room temperature. Hits in DNA per cell are calculated as 1.2× 10⁵ for 3 Mrads and 1.2× 10⁴ for 0.3 Mrads. To elucidate the very high radioresistance of this bacterium, further investigations are needed. Those should include the study on the non-DNA damage caused by radiations as well as the repair mechanism for DNA damages. The D₁₀ values at the above two temperatures of five strains of E. coli, which were obtained directly or indirectly from strain B, were also determined. As the result, the D₁₀ ratio defined as the D₁₀ for liquid nitrogen temperature divided by the D₁₀ for room temperature was found higher with the more radioresistant strain. The possible interpretation was briefly discussed.

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The flesh of TMAO-rich fishes, such as cod, Alaska pollack, shark and squid were subjected to gamma-ir-radiation at dosages from 0.6 to 5.6 Mrad, to see how TMAO, a specific constituent in marine fish, would undergo of its breakdown and if it would result in causing undesirable effect on the quality of irradiated fish.
TMA, DMA, and FA were all identified in any sample of fish flesh irradiated, and a decomposition product of TMAO. An increase in the amount of ammonia was observed in the same samples after irradiation, but very little of ammonia was formed in TMAO solution. The difference in the amount of TMA produced by irradiation was not so apparent among the fishes by species.
But, the formation of DMA was considerably large in the quantity, particularly in the flesh of gadoid fish. And, a remarkable difference in DMA level was noted between two individuals of Alaska pollack. A significant finding was that the amount of DMA in one of the irradiated flesh of Alaska pollack was extremely larger than the amount produced from pure TMAO solution under the same irradiation condition.
The precursor of FA formed by irradiation of fish flesh would not be limited to TMAO, however, a comparatively large amount of “free” FA extracted from irradiated flesh of gadoid fishes was presumed to be derived from TMAO.

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In the previous paper, it was revealed that the amount of DMA produced during gamma-irradiation on the flesh of gadoid fishes was higher than of other fish, and, in particular case, even higher than the amount of DMA found in pure TMAO solution irradiated under the same condition.
The tissues of gadoid fishes, especially the viscera, have been known to contain an enzyme system which participatein the decomposition of TMAO to DMA. The enzymeaction was supposed to have a relation with the differenceabout the level of DMA formed by irradiation.
To make this problem clear, the effect of cod viscerawas investigated by adding five percent of it into codflesh and irradiated with 3.0 Mrad of gamma ray.
The results obtained are as follows:
1. The formation of DMA during irradiation on cod flesh revealed a marked acceleration by the addition of codviscera, especially by pyloric caeca of the fish. Amajor factor involved in the “acceleration” should be anenzyme, because the enhancement of DMA formation never happened when pyloric caeca had been heat-processed.
No acceleration was observed also in the instances, where the irradiation on flesh and pyloric caeca was conducted separately or either of these materials was irradiated alone. These findings may seem to support the authors'estimation described in the introductory part.The acceleration should be dependent on the active role of free radicals generated from radiation decompositionof water molecules, since the “acceleration” only occursunder irradiation treatment.
2. To the contrary of the effect of “acceleration” possessed by pyloric caeca, “inhibition” of DMA formation was demonstrated during irradiation of cod flesh, whenthe blood of horse mackerel or of carp was added to thesystem. Heat treatment did not much reduce the “inhibition” effect of the blood. And, a similar effect was also confirmed by haemoglobin preparation from fish. Some component (s), very stable under heating, was thought responsible for the “inhibition” effect in the blood.

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Keeping quality of horse-mackerel was examined as to the effect of gamma-irradiation. Fresh fillets of horse-mackerel with skins were divided into two groups: one was cooked in steam for 15 minutes and sealed in a film package and the other was packed first and boiled in water at 85 to 90°C for 30 minutes. Film bags employed were cellophane laminated with polyethylene and the fillets were sealed in air or in vacuum.
The samples were irradiated at 5°C at dosages of 0.1 and 0.2 Mrad of gamma-ray from Co-60 source with about 1, 400 Ci, and stood at 30°C after the treatment. Organoleptic tests revealed that no discernible change in flavor or odor was noticed in the irradiated fillets. In addition to the sensory test, chemical and bacteriological examinations indicated that the shelf life of the group packed after heating and irradiated at 0.2 Mrad was as short as five days at 30°C, where the control was two days. No remarkable difference was noted between air-packed and vacuum-packed samples.
The fillets heated after packed in raw and irradiated showed 13 days of storage life in air-packed group and 5 days in vacuum-packed, while control run showed 2 days.
A rather longer period of storage seemed to be secured in the group irradiated at 0.2 Mrad level than that at 0.1 Mrad.

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L-Cysteine, L-cystine, thiazolidine-4-carboxylic acid, 2-(2-furyl)thiazolidine-4-carboxylic acid and 2-phenylthiazolidine-4-carboxylic acid in 0.0004 - 0.01 Maqueous solutions were irradiated at 20 - 25°C byexposure to γ-rays from Co-60 source for about 50 hrs.(5 Mrads) in sealed Pyrex glass tubes under nitrogen and oxygen atmospheres.
Some estimations of degradation products were carried out by gas chromatography, thin-layer chromatographyand by spectrophotometric methods.
As the results of H₂S and NH₃ determinations, a cnsiderable oxygen effect was observed. G values of H₂S were unexpectedly small (in case of thiazolidine-4-carboxylic acid at oxygen atmosphere G_H2S = 0.034), and a large proportion of sulfur fragments seems to be changed into disulfides and oxides of sulfur compounds.

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Radurization of Citrus fruit have been conducted many European countries and those results have suggested high feasibility of radiation treatment to the extension of their shelf life. According to theformer experimental, “unshiu” had higher radiosensitivity than European species and the acceptability was completely lost at 50 Krad of gamma-irradiation. However, shallow irradiation would be likely for such a radiosensitive citrus fruit and in fact, 50-150 Krad of irradiation by 0.5-1.0 mev cathode ray have extended the shelf life of “unshiu” without organoleptical deterioration.The radiation damage on the peel colour such as browning and fading have occured when the dosage was over 200 Krad.
For the purpose of examining the radiation effects on the carotenogenesis of orange peel, “unshiu” were irirradiated at the dosage of 1 Mrad and 5 Mrad by 1 mev cathode ray and were measured of their carotenoid pattern immediately after irradiation and after stored 2, 5, 8 days at room temperature. Carotenoid pattern was measured by thin layer chromatography and all carotenoids have been divided into eight fractions according to number of hydroxy and epoxide groups.
Immediately after the irradiation, 30-40% of total carotenoids destroyed and then gradually increased during the storage time and the rate of change in the pattern of 1 Mrad group was greater than that of 5 Mrad group. Eight days after the irradiation, carotenoid pattern irradiated 1 Mrad showed that polyol, diol-monoepoxide fractions have increased to 5, 3 and 2 times respectively against tothose of non-irradiated ones, on the other hand diol-diepoxide B (cis-violaxanthin) and monol-monoepoxide fractions have decreased to 1/4 and 1/3 respectively against to those of non-irradiated ones. Another three fractions, hydrocarbon, monol and diol-diepoxide A (violaxanthin), were about the sameas those of non-irradiated ones.
These results have shown that each carotenogenetic pass-way has different radiosensitivities and also they have suggested that the radiation treatment at suitable dosage would be useful for the studies on carotenogenesis of fruit.

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The volatile compounds from cooked potato irradiated with doses of about 10 K and 100 K rad weredetermined by gas chromatography and chemical derivatization immediately after irradiation.No significant differences were observed between the volatile compounds 10 K rad radiated potato and that from the control; compounds recognized were acetaldehyde, propyonaldehyde, acrorein, acetone, iso-butylaldehyde, butylaldehyde, valeraldehyde, hydrogen sulfide and methyl mercaptan.treatment with doses of 100 K rad yielded the increase of volatile compounds, especially acetaldehyde, propyonaldehyde, iso-butylaldehyde, butylaldehyde, and iso-valeraldehyde.

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The experiment was conducted to study changes of volatiles in apples irradiated with gamma radiation, using the headspace vapor analysis of gas chromatography.There were detected three aldehydes, fivealcohols, fifteen esters and seven unknowns. Radiationat the dose of 1.25×10⁵r considerably inhibited theproduction of low boiling substances which increased inthe un-irradiated apples during storage of 10 days atroom temperature and of three months at 0°C.

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Effect of γ-irradiation on the non-enzymatic browning of foods was investigated with the model system of glucoseglycine (1:1) solution, and with white shoyu (soya source).
The browning, the increase in E_420mμ, of the glucoseglycine solution with heating after irradiation was enhanced by irradiation of the doses above 0.5 Mrad, and the effect was remarkable at the initial stage of browning reaction.
Irradiation of the mixture under anaerobic condition brought about the development of characteristic UV absorption spectrum, and its λ_max., 292 was not so altered by change in pH from acid to alkali as in the case of irradiated glucose solution.
The doses of 1 Mrad and over, especially in the presence of oxygen, weakly accelerated the browning of white shoyu.

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Many physical, chemical and biological effects caused by ionizing radiations on chemical constituents of biological materials, are brought about depending upon various states in which the constituents are contained.
On irradiating food materials for preservation, sterization and related objects, it is of important that the materials are under the most effective condition of the state for the purpose.
It deals with basic observation to find relationships between the irradiation effects and the irradiating conditions of amino acids, peptides, polyamino acids, proteins and enzymes.
1. The effects in purification steps of the enzyme
It has been well known that sulfhydryl enzymes are more radio-sensitive than non-sulfhydryl enzymes in solution. When any other compound is contained in the enzyme solution the coexisting compound may protect the enzyme from irradiation inactivation.
It has been found that crystalline urease in solution is more radiosensitive than the enzyme contained in beans.
2. The effects on crystalline amino acids and proteins in vacuo at low temperatu
At low temperature, it may be possible to detect less-stable radicals such as radical cations and anions by electron spin resonance spectroscopy.
It was found that when polycrystalline polyglycine and polyalanine were irradiated with γ-rays at 77°K, ionized radicals were detected by the ESR method.The ionized radicals changed into more stable radicals at room temperature by heat treatments.
Similar observations have been reported on cysteine, cystine, methionine, polyglycine and several proteins.
Generally speaking, it appears likely that the cations and anions may occur at initial processes of irradiation on food materials.
3. The effects on amino acids in solution
Oxidative deamination of amino acids and proteins in solution by γ-irradiation has been reported and many carbonyl compounds were detected by the 2, 4, dinitrophenyl hydrazone method. Radiosensitivity and protecting effects of amino acids were also reported from the results by the automatic amino acid analysis.
4. The effects on enzymes in solution
Radiolytic disappearance of amino acid components was observed on bacterial proteinase after exposure to γ-ray.
On α-amylase, it was found that before the radiolytic decomposition of amino acids residues conformational changes occur to cause inactivation of the enzyme.
5. The effects on enzymes in homogenates and cellular particles
The succinoxidase system and each component enzyme, i.e. succinic dehydrogenase and cytochrome oxidase, which are obtained from liver of mice and rats, and from beef heart, were irradiated in vitro with cobalt-60 γ-rays. Inactivation of the enzyme systems was observed to occur by relatively large does of γ-irradiation. Succinic dehydrogenase was found to be more radiosensitive than cytochrome oxidase in all preparations.
Effect of gamma irradiation on the enzyme systems localized in mitochondria and nuclei of rat liver was compared. The enzyme systems in the nuclear fraction were relatively more radio-sensitive to γ-ray inactivation than those in the mitochondrial fractio.

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The formation of reductone from sugars and ascorbicacid in solids and aqueous solutions by ⁶⁰Co gammaradiationwas examined by measuring both amounts of acireductoneand reductonate with dose (in range of total dose 1×10⁴-1×10⁸ R). Decomposition rate of these substances, color, changes of acidity, UV absorption spectra, paperchromatograms were also compared.
It was found that aci-reductone was produced from sugar in the dose above 10⁶ R in 0.1 M solution and 10⁷ R in solid respectively. The maximum yield of acireductonein 0.1 M sugar solutions were 3.9×10^-5, 1.7×10^-4 and 6.4×10^-3 M in 10⁶, 10⁷ and 10⁸ R respectively.The production of reductonate was also similar to aci-reductone formation but the yield of the former wasmore higher than the latter.
On the other hand, ascorbic acid was, in contrast to solid, rapidly decomposed by gamma-radiation in dilute solution (2×10^-3M), and oxidized to dehydroascorbicacid and thereafter to 2, 3-diketogulonic acid.No other reductone was observed from the radiolysis of ascorbic acid.
In view of the above facts it seems that in the dose range of food irradiation (less than 5×10⁶ R)ascorbic acid was damaged inevitably but the quantity of reductone produced from sugar was very little and quality of food would not be affected by the reductone.

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The Strawberries obtained from a market, were washed with detergent and rinsed with sterilized water several times to eliminate the microbial contamination from natural source. The fruits were inoculated or not inoculated with a portion of Botrytis cinerea spore suspension, and were irradiated 200 krad at room temperature (20 °C) for 1, 8 and 24 hours, at the dose rate of 200, 25 and 8.3 krad/hr, respectively, and were stored at refrigeration temperature (2 °C). From the results obtained, it was shown that the fruits which were irradiated for 1 hour, sound and not contaminated, could be stored for more than 4 weeks (Table I). On the contrary, the fruits which were irradiated for 8 or 24 hours (Fig. 1), contaminated (Fig. 2) or wounded (Fig. 3), were infected early in the refrigeration.
A possible application of room temperature irradiation for storing strawberries was discussed.

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For determining the feasibility of using irradiation in the preparation of tobacco, a preliminary study on irradiation of tobacco was attempted at our laboratory. The results obtained are as follows:
1) Tobacco leaves in the drying process acquired good aroma of fermented or aged tobacco by irradiation at a dose of 1 × 10⁶ rads of Co-60 γ-rays, whereas control unirradiated leaves had green-note smell of green leaves.
2) Tobacco flea beetles and tobacco moths and their larvae were killed in a dose less than 1.7×10⁵ rads of γ-rays.
3) “ Peace” (one of the representative brand of cigarette in Japan) irradiated in a dose of l×10⁶ rads of γ-rays was prefered after 3-4 weeks to unirradiated cigarette, many of panel members appealed that irradiated cigarette became milder than the control.
4) Bacterial plate count on the irradiated cigarette after one month storage in a show window case was only a few per piece, whereas that of the control of unirradiated cigarette was of the order of 2×10⁴ per piece.
5) Reduction in concentration of nicotine in aqueous solution was observed by exposure of various doses of γ-irradiation, but nicotine content of irradiated cigarette was not significantly different from the control unirradiated cigarette. For the explanation of the difference between the results in vitro and in vivo, tobacco leaf was stained with Dragendorfs reagent and many red spots (nicotine) were found to localize which led to the assumption that nicotine in a leaf might be contained not diffusely but concentratedly.
No significant difference in nicotine contents of main stream of cigarette smoke between the cigarette irradiated in a dose of 1×10⁶ rads and unirradiated cigarette was observed.
6) There was no significant difference in crude tar content from cigarette smoke between the cigarette irradiated in a dose of 1×10⁶ rads and unirradiated cigarette.
7) No significant difference was observed in maximum temperature of smoking between the irradiated and unirradiated cigarette.
8) Toxicity test of the water in which the main stream smoke of the irradiated cigarette was dissolved by using Japanese gold fishes did not show the difference from the unirradiated.

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Garlic bulbes were irradiated by Co-60 and stored one to four months at room temperature. The irradiations were carried out at the doses from 0.1 to 6.4 Krads. The earlier experiments before August were successfully carried out. But the later experiment failed as the result of the onset of sprouting mechanisms. The minimum dose of irradiation to prevent sprouting of garlic was 3-4 Krads. The overall quantities and the physico-chemical properties of alliin-lyase obtained from irradiated garlic were unchanged compared with the enzyme from unirradiated one. But the enzyme obtained from irradiated garlic was less soluble than that of unirradiated one.

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This experiment was undertaken to determine the of sugars from wheat bran, incubated with cellulasepreparation subsequent to irradiation of β-ray produced by 1 Mev Van de Graaf, and also to determineeffect of irradiation on the nitrogenous components bran.
Wheat bran, irradiated or non-irradiated, was incubated with cellulase in 0.1 M acetate buffer (pH 4.6)at 40°C, and .then the formation of sugars was measured by colorimetry. Total and reducing sugar values obtained by Anthrone method and Somoghi-Nelsonmethod were respectively increased with the irradiation dose. Application of irradiation together with incubation with cellulase to the bran was brought about much increase of reducing sugar, as shownin Fig. 6. Extract of the bran, irradiated and incubated with cellulase, was concentrated to small volume under reduced pressure, and then subjected to paper chromatography by using n-butyl alcohol:pyridine:water (6:4:3) as a developing agent. Nine spots (A, B, C, ....., I) corresponding to xylose, an unknown sugar, glucose, galactose, mannose, cellobiose and two unknown sugars could be detected by0.3% o-amino diphenyl spray. The last two unknown sugars could be found only in the sample incubatedwith cellulase subsequent to irradiation, and might belong to oligosaccharides. The chromatographic bands corresponding to their locations of the sugars were cut off from the paper, and-eluted with water to measure the sugar content by the same colorimetry as the above. It was shown in Table l that the amount of glucose predominated among the sugars formed.
Wheat bran was treated successively with water, 99% ethyl alcohol and 0.2% NaOH to separate water soluble, 99% ethyl alcohol soluble, 0.2% NaOH soluble fractions and the residue. Nitrogen content of each fraction wasdetermined by Kjeldahl method and Conway's microdiffusion method (Table 2). Total nitrogen of wheat bran was somewhat decreased by irradiation. On the other hand nitrogen content of water soluble and 0.2% NaOH soluble fractions were slightly increased.

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Many reports have been published about the inferior quality of the oils extracted from rice bran which has been stored for long period than that from new rice bran owing to the rise in the acid value of the oil contained in rice bran during the storage.
The work on the exposure of rice bran to gamma irradiation before the oil extraction was attempted to know if the treatment would be effective to control the rise of the acid value. Acid value of 35 was obtained for the rice oil extracted from rice bran that had been exposed to 7×10⁶ rad of gamma irradiation from a Co⁶⁰ source at our laboratory, whereas the value of the rice oil from unirradiated bran was 60.
There was no significant difference between two groups of the oils as to the growth rate of mice given oral administration during three months.

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An use of gamma irradiation was studied as a meansremoval of chemical contaminants from water. Phenol and dodecyl benzene sulfonate were added as pollutants to raw water and sedimentation effect by coagulation was examined after irradiation of this sample water under the doses of 1× 10⁴rad or 1× 10⁵rad of gamma ray fromCo⁶⁰source.
Appreciable reduction in the concentration of these pollutants was observed in the irradiated water, when compared with the water treated only by sedimentationwith coagulation. Administration of the treated water to mice ad libitum as drinking water revealed no deleterious effect on their growth after one month's feeding. Toxicity test was also performed by using Japanese gold fish.
A Co⁶⁰sourde with 1.1× 10⁷Ci will be required when polluted water is irradiated at the rate of one metric ton per hour under a dose of 1×10⁴rad.Further studies of the effects of irradiation are under way on such water pollutants as organic pesticides anddyes and also on COD and BOD values, etc.

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For the purpose of simple and convenient monitoring radiation in irradiation processing of foods, we have developed polyvinyl chloride films dyed with the mixture of aniline blue and butter yellow dissolved in ethylene bromide. These were slightly modified from the color films which had been developed by us for the use of color radiography intended to amplify discernibility of the image in nondestructive testing.
Indicator film dyed only by yellow dye such as butter yellow, diethylyellow or meta-methyl red showed a narrow range of the color change on irradiation, but, this newly developed one indicated a stepwise conversion of its color by irradiation from blue-green to red and finally to colorless through green, yellow and orange indue course.
By varying the dye concentration and thickness of film, film indicator possessing capacity to detect different dose range can be prepared, for example, from 0.5×10⁴ to 5×10⁴ rad, from 2×10⁵ to 6×10⁵ rad, or from 1×10⁶ to 6×10⁶ rad, respectively.
Such blue dye as methylene blue which had been known in the field of radiation chemistry was found not suitable for the indicator owing to its low sensitivityto radiation. Quinoline blue or indophenol was observedfeasible for the use as well as aniline blue.

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