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1971

Radiation Induced Alterations in FlavorComponents in Penaeus Setiferus.Raja Jaffan IsmailLouisiana State University and Agricultural & Mechanical College

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Recommended CitationIsmail, Raja Jaffan, "Radiation Induced Alterations in Flavor Components in Penaeus Setiferus." (1971). LSU Historical Dissertationsand Theses. 1993.https://digitalcommons.lsu.edu/gradschool_disstheses/1993

71-29,37HISMAIL, Raj* Jaffan, 1938-RADIATION INDUCED ALTERATIONS IN FLAVOR COM­PONENTS IN PENEAUS SETIFERUS.Tha Louisiana Stats University and Agricultural and Mechanical Collage, Ph.D., 1971 Food Technology

University Microfilms, A XEROX Company , Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED

RADIATION INDUCED ALTERATIONS IN FLAVOR COMPONENTSIN PENEAUS SETIFERUS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of

Doctor of Philosophyin

The Department of Food Science

byRaja Jaffan Ismail

B.S., Cairo University, 1962 M.S., Louisiana State University, 1965

May, 1971

ACKNOWLEDGMENTS

The author is indebted to Dr. Arthur P. Novak, her major professor and Head of the Department of Food Science, whose interest and guidance have been a constant source of encouragement during the author's graduate tenure.

Special thanks are extended to Dr. M. R. Ramachandra Rao for hi3 valuable suggestions, and to Dr. Alworth D. Larson, Dr. William H. James, Dr. Robert M. Grodner, and Dr. Joseph A. Liuzzo foi serving as members of her advisory committee.

She also wishes to thank Dr. Ernest A. Epps, Jr.,Head of the Department of Feed and Fertilizer, and Miss Frances L, Bonner, for their constant encouragement and interest in the author's endeavors.

Above all she is grateful for the love, devotion and sacrifice of her husband, whose understanding and patience enabled her to complete this dissertation.

TABLE OP CONTENTSPufio

ACKNOWLEDGMENTS................. 1LIST OP T A B L E S ....................................... vLIST OF F I G U R E S ....................................... viA B S T R A C T .................................................vllINTRODUCTION .......................................... 1REVIEW OF L I T E R A T U R E ................................. 4

Mononucleotides and their derivatives ......... 4Amino acids and p e p t i d e s ...................... 6Lipids and free fatty acids .................... 9Carbohydrates and sugar phosphates ........... 12Carbonyl compounds ...................... . . . 14Volatile sulfur compounds ...................... 21Effects of radiation on food f l a v o r ........... 22

EXPERIMENTAL METHODS ................................. 30Procurement and preparation , ................. 30Irradiation source and location ............... 33Irradiation of s h r i m p .......................... 34-Extraction of carbonyls from s h r i m p ........... 35Equipment and procedure ........................ 37Regeneration of carbonyls ...................... 39Identification of free carbonyls in shrimp . . 39Extraction of lipids from shrimp ............. 40Saponification and esterification ............. 44Thin-layer chromatography ...................... 45Gas-liquid chromatography ...................... 45

RESULTS AND D I S C U S S I O N ............................... 47Total carbonyls................................. 47Gas chromatography analysis of volatile

carbonyls ........... . . . . . . . . . . . . 53Effects of irradiation and storage on

carbonyls..................................... 63Total lipid e x t r a c t ............................. 73Effect of irradiation on fatty acids ......... 73Effects of Iced storage and irradiation . . . . 76

SUMMARY AND C O N C L U S I O N ............................... 82BIBLIOGRAPHY .......................................... 85APPENDIX 97

VITA

iv

Pap;e . 101

LIST OP TABLESTable Page1. Operating parameters for gas chromatographic

analysis of carbonyls In shrimp ............... 4l2. Operating parameters for gas chromatographic

analysis of fatty acids In shrimp ............. 463. Recovery of added carbonyls (each in 1.0 ppm). . 554. Recovery of added carbonyls (each in 5.0 ppm). . 565. Recovery of added carbonyls (each in 10.0 ppm) . 576. Retention times of volatile carbonyls in shrimp

using two separate c o l u m n s ...................... 587. Effects of irradiation at two different dose

levels on volatile carbonyls in shrimp ......... 608. Changes in volatile carbonyl compounds during

ice storage on control non-lrradiated shrimp . . 649. Changes in volatile carbonyl compounds during

ice storage on irradiated shrimp at 0.15 Mrad . 6610. Changes in volatile carbonyl compounds during

ice storage on irradiated shrimp at 0.8 Mrad . . 6911. Sulfur and aromatic compounds from irradiated

m e a t s ............................................ 7212. Retention times of fatty acids found in shrimp

as compared with standards...................... 7413. Effect of irradiation at two different dose

levels on fatty acids in shrimp ............... 7714. Fatty acid analysis on lipid extracts obtained

from non-irradiated shrimp stored in ice . . . . 7815. Fatty acid analysis on lipid extracts obtained

from irradiated shrimp at 0.15 Mrad and storedin i c e ............................................ 79

16. Fatty acid analysis on lipid extracts obtained from Irradiated shrimp at 0.8 Mrad and storedin i c e ............................................ 80

LIST OP FIGURESFigure Page1. Distribution of hydrocarbon and carbonyl

compounds among irradiates and oxidizedbutter fat volatiles............................. 26

2. Plow sheet diagram showing steps involved In preparing the sample for carbonyl andfatty acid e x t r a c t i o n .......................... 32

3. Apparatus for carbonyls extraction .............. 384. Effects of irradiation on total carbonyl com­

pounds of ice-stored shrimp (measured as mg2-4 dinitrophenylhydrozon per 100 g of shrimp) . 48

5. Effects of irradiation on volatile carbonyls in shrimp irradiated at two different dosel e v e l s ............................................ 6l

6. Changes in volatile carbonyls in non-irradiatedshrimp iced-stored over a month ............... 65

7. Changes In volatile carbonyls In shrimp irradiated at 0.15 Mrad iced-stored over am o n t h ............................................ 87

8. Changes in volatile carbonyls in shrimp irradiated at 0.8 Mrad iced-stored over am o n t h ............................................ 70

9. Chromatogram of volatile carbonyls In non-irradiated shrimp................................. 98

10. Chromatogram of volatile carbonyls In shrimpIrradiated at 0.15 M r a d ........................ 99

11. Chromatogram of volatile carbonyls in shrimpirradiated at 0.8 M r a d ............................. 100

ABSTRACT

Flavor components in PeneauB setlferus and their alterations during gamma radiation and subsequent refrig­erated storage have been investigated.

The quality of iced-stored shrimp is influenced by lipid deterioration which is a result of the reactivity of its characteristic fatty acids. The fatty acid composition and the nature of volatile carbonyls was investigated in fresh, irradiated, fresh iced-stored, and irradiated iced- stored shrimp. Moreover, if the product is irradiated carbonyls are formed from the radiation induced oxidative chain action on fatty acids.

In the samples used in this study, the amount of total lipids extracted per 100 g shrimp ranged from 0.7 to 0.8 g. Fatty acids identified from lipid extracts by gas-liquid chromatography in both the non-irradiated and the irradiated samples were: myristic, myristoleic,pentadecanolc, palmitic, palmitoleic, stearic, oleic, llnolelc, and linolenic. Palmitic and oleic acids were predominant in every sample.

The predicted radiation-induced decrease in fatty acids was not observed in the fatty acids identified.

Trends shown in the results of this study appear to indicate that storage of both irradiated and non-irradiated shrimp had very little effects on the fatty acid composi­tion of the samples.

viii

No appreciable increase in carbonyls occurred at a gamma radiation dose of 0.15 Mrad. However, at a dose level of 0.8 Mrad, an increase in total carbonyls was Indi­cated by the higher quantity of 2, 4-dinitrophenylhydrazone. Acetylaldehyde, propionaldehyde, acetone, isobutyraldehyde, butyraldehyde, butanone, 3-methyl-2-butanone, diacetyl, hexanal, heptaldehyde, and £-heptanone were found to be present in fresh shrimp, and were identified by gas-liquid chromatography. Their presence in higher concentrations was noticed by the increase of the peak heights in chromato­grams made from shrimp samples irradiated at a dose of 0.8 Mrad. Increases were negligible when shrimp were irradi­ated at 0.15 Mrad. Plausible radiochemical mechanisms were suggested to account for the production of aldehydes and ketones at the higher radiation dose level.

Concentration of identified carbonyls In non-irradi­ated Iced-stored shrimp increased throughout 21 days storage, and then tended to fall off. However, hexanal and 2- butanone showed less Increase in the peak height than the other compounds investigated.

Samples irradiated at a dose of 0l15 Mrad showed stable carbonyl compounds during a storage period of 21 days, but resulted in a slight increase after 30 days.

The radiation-induced immediate increase in carbonyl concentrations in the iced-stored irradiated shrimp at 0.8 Mrad appeared to continue during the first seven days, and then tended to decrease.

INTRODUCTION

The development of new methods of food preservutLon such as radiation, mechanical freezing, cryogenic freezing with liquid nitrogen and freon as cryogens, canning, and chemical additives has contributed to the shelf life exten­sion of many food products. With fish and shellfish, the application of these methods of preservation was limited because these products lost their delicate flavor within a few hours. However, before the introduction and develop­ment of Irradiation as a method of food preservation, most chemical analyses were limited to tests for quality and freshness. Since irradiation, and subsequent refrigerated storage produced chemical changes in foods which altered their flavor, attention was necessarily directed to the chemical analysis of flavor components to determine and isolate the components responsible for these alterations, and to study the mechanisms involved in these changes.

The evaluation of food aromas and flavor has been an extremely difficult undertaking mainly for the following reasons:

1- The substances which contribute to flavor and aroma comprise many different classes of organic com­pounds .

2- The great variety of compounds within a given homologous series which arise naturally by bio­chemical processes, or by subsequent treatment and processing by man.

2

3- The low concentration of many of these compounds.4- The complex relationship of chemical composition

to olfaction.5- The highly specific molecular structure-flavor

relationship.However, the advent of gas chromatography has given

the research chemist a powerful tool for unlocking Nature's secrets of flavor composition. Tremendous progress has been made through the combined efforts of scientists with widely diversified Interests and experience. Yet, much investi­gative work remains to be done to understand the composi­tion and physical characteristics of food flavors, especially the delicate flavors of fish and shellfish. Gas chromatog­raphy has served to focus particular attention on compounds of relatively low boiling points (50° to 200°C). Compounding experience has shown, in most cases, that the relatively low boiling compounds modify flavors In important and demon­strable ways, but nonetheless fail to duplicate the total flavor. Low boiling alcohols, aldehydeB, ketones, and sulfur compounds are ubiquitous constituents of food vola- tiles although the ratios of their occurrence may vary widely, depending on the ratios of precursor substances present and other physical and chemical factors in the envi­ronment of the food.

Since the influence of lipid deterioration on the quality of chill-stored shellfish has been clearly docu­mented by several laboratories (reviewed by Ackraan, 1967)#

3

it has been indicated that the deterioration of shellfish lipid is due primarily to the reactivity of its character­istic fatty acids. The effects of Ionizing radiation on fatty acids are considered to be oxidative, and since a variety of radiolysis products such as hydrocarbons or carbonyls may be produced during or after radiation, the purpose of this study is to determine:

1- The nature and composition of flavor producing fatty acids and volatile carbonyls in fresh shrimp, and alterations produced during and after gamma radiation.

2- The effects of lced-storage of shrimp after irra­diation, and

3- The radiation Induced chemical and physical changes.

REVIEW OP LITERATURE

To increase consumer demand for fish and shellfish, it is essential to expand our knowledge of the physical and chemical reactions which cause the production of both acceptable and undesirable flavors and aromas.

Flavor is a term used to Indicate the over-all sensual response to food taken into the mouth. This comprises odor, taste and other mouth sensations. The term taste is applied In its narrow sense as a chemical tongue sensation producing one or more of the four sensations described as acid, bitter, sweet or salty.

Proteins, carbohydrates and lipids are the three main classes of food stuffs considered to be the Important sources of flavor components. Although the ribomononucleo- tides have been used as flavor additives or enhancers, their identification as flavorous compounds, occurring naturally in food stuffs, is equally important.

Mononucleotides and their derivativesThe nucleotide of primary Interest in contributing

flavor to fish and shellfiBh Is inoslne 5 1-monophosphate, because of Its structure (6-hydroxypurine ribonucleoside 5 1-monophosphate) as reported by Kuninaka al^ (1964), and its concentration which is high enough to contribute to the meaty flavor of fish. While the concentration of adenosine 5 1-triphosphate is higher in shrimp than In fish (Sonu, 1970; Ouardla and Dollar, 1970), its contribution to

5

flavor enhancing la questionable. Whereas Hashlmoto (lQti4) found it to be active in enhancing flavor, Kunlnaka et a l . (1964) found it lacked the structure which is associated with the flavoring action. However, adenylated deaminase appears to be lacking in invertebrate muscles, so that adenosine 5 1-monophosphate accumulates rather than inoaine 5 1-monophosphate, and fulfills a similar function. Hashlmoto (1964) suggested that there is strong natural enhancement of flavors between adenosine 5 1-monophosphate and glutamate, and related this combination to invertebrate flavors. Ob­viously, the environment imposed by the other compounds of a musculature has a critical effect on mononucleotide flavor and vice versa. Similarly, inosine 5 1-monophosphate occurs in fresh fish muscle and meat extracts in concentrations at which solutions are strongly flavorous in a "meaty" sense {Wood, 196I; Salto, 1961). However, it is apparent that such compounds are of greater significance through their inter-relations with other compounds. These can be purely physiological (Hashlmoto, 1964) or in flavor-precursoring reactions (Batzar at &1., 1962).

There is disagreement between workers on the pertinence of degradation products such as inosine and hypoxanthine. While Kazeniac (1961) considered Inosine to contribute bitterness to chicken, Jones (1961) found this compound to be flavorless at the concentrations and pH obtained in chill-stored fish muscle. Both workers agreed that hy­poxanthine solutions were bitter, and suggested that they

6

were of relevance to these meats; but Hashlmoto (1964) found that the purine was not a “positive taste substance" In "Uni" (sea urchin gonad), and Sato (i960) reported It to be taste­less. Spinelli (1966) appears to have clarified the point somewhat. In agreement with Jones (1961) and Kazeniac (1961), he found that solutions of the purine were bitter at con­centrations which occur in muscle post-mortem.

Amino acids and peptidesSeveral studies on the taste of amino acids in food,

and their production by extraction, fermentation, or by chemical synthesis, have resulted in mass production of various amino acids. The ta3te of cheese had been known, in the United States and Europe, to be characterized by amino acids formed during the ripening process. Today, amino acids are used in food processing not only to enhance the nutritive value of many processed foods such as cereals, but also to enhance or improve otherwise the taste and natural characteristics of many foodstuffs.

The tastes of individual amino acids were character­ized by Kirimura et al. (1969) as being sweet, salty, sour, bitter, or monosodium L-glutamate-like. Solms (1969) re­ported that the common, pure amino acids have the following taste properties near neutrality: no taste at all or abarely perceptible taste: D-alanine, D- and L-iBoleucine,D- and L-lysine, D- and L-arginine, D- and L-aspartic acid, D-glutamic acid, L-histidine, D- and L-proline, D- and L- serine, D- and L-threonine, D- and L-valine. Sweet taste

(in order of decreasing Bweetness): D-tryptophan, D-hlstidlne, D-phenylalanine, D-tyrosine, D-leucine, L- alanine, Bitter taste (in order of decreasing bitterness): L-tryptophan, L-phenylalanine, L-tyrosine, L-leuclne. Sulfurous taste; D- and L-cysteine, and D- and L-methionlne. L-glutamlc acid has a unique taste-potentiating property.

Klrimura et_ al. (1969) noted a pronounced mutual en­hancing of flavor in combination of the amino acid fraction with Inosine 5'-monophosphate. In a further study on frac­tionated extracts of abalone meat, Konosu and Hashlmoto (1964) demonstrated a similar key inter-relationship to meaty character between glutamic acid and adenosine 5 '- monophosphate. Hashlmoto (1964) obtained similar results with "Uni", the unripe gonad of the sea urchin. In the amino acid fraction In combination with mono-nucleotideB, he found glycine, valine, alanine, glutamic acid and par­ticularly, methionine to be Important.

Recent studies on the taste of various combinations of amino acids showed that the distribution of free amino acids differs from food to food, and that quantitative changes within each food may also be seasonal. Some of these amino acids per se are essential for the taste of some foods. Thus, glycine is very Important in contributing to the distinct sweet taste of lobster and crab (Amano and Bito, 1951), and histidine, the predominant amino acid in (Debrowski et al., 1969) contributes to the distinct meaty character. However, this is not always the case. The

8

Individual amino acids of gadoid muscles were found to occur below the flavor threshold, but they were readily detectable in a composite simulated extract (Jones, I96I). Similarly, Konosu at a_l. (i960) found that a simulated amino acid fraction (which included histidine) of an extract of Katsuobushi (dried bonito) was almost flavorless. The elim­ination of glycine resulted in an increase in bitterness and a decrease in sweetness. Taurine, which is a major constituent in most fish muscle and shrimp (Debrowski et al., i960), is variously reported as serumy, somewhat as­tringent, slightly bitter (Konosu and Hashlmoto, 1964), and tasteless.

Free amino acids undergo carbonyl-amino reactions in dehydrated, frozen, and canned fish. Consequently, they are precursors of other flavorous compounds.

It has been previously reported by Klrimura et al. (1969) that the taste intensities of peptides are generally weak compared to those of the corresponding free amino acids. The taste characteristice of peptides are complex but can be classified into three groups:Bitter taste: peptides such as L-leucine - L-leucine,L-arginlne - L-proline.Sour taste: peptides such as L-alanine - L-aspartic acid -

- L-glutamic acid - L-glutamic acid.Little or no taste: Glycine - glycine - glycine - glycine.

It is interesting to note that dipeptides composed of sweet amino acids, gly-glycine, gly-L-alanine, and gly-L-

9

p rolling ut'u almost tasteless. The disappearance of sweet - in’:i:i may bo a result of chain lengthening. The combination of an acidic and a basic amino acid, such as L-lyslne-L- glutamic acid and L-argininel-glutamic acid is also taste­less. This may be due to interactions between the amino groups and the carboxyl groups in the dipeptide molecule.

Many peptides have been isolated from plants and animals, but have not been tested for their taste properties, At least two peptides isolated from chives and onions were evaluated for taste, and they exhibited a characteristic biting taste sensation (Virtanen, 1965). An agreeable taste has been described for anserine, a major extractive of some muscles (Shewan and Jones, 1957), which is also highly sus­ceptible to reaction with sugars. Certain flavor-enhancing properties have been assigned to calcium pantothenate (Dixon, 1968), and to several copolypeptides containing glutamic acid, aspartic acid, lysine, and ornithine. For many years it has been claimed that glutathione is respon­sible for meaty taste (Bouthilet, 1951).

It is generally accepted that proteins exert no taste activity. However, they contribute to flavor indirectly, with tactile and similar effects. This Is especially true for protein-rich foods like milk, milk products, meat, fish and shellfish.

Lipids and free fatty acidsThe most widely recognized contribution of lipids to

flavor is as precursors. The compounds formed may be

10

readily volatile and have appreciable odors -- e.g., lower molecular weight aliphatic aldehydes, ketones, and fatty acids. Both the intact lipids, and their low volatile breakdown products, contribute to flavor largely through mouth stimulation (PorBS, 19&9).

Lipids of low volatility are tasteless in terms of sour, sweet, bitter, and salty, partly because they are insoluble in water. They modify the taste and flavor of other compounds in foods. They also influence the physical state of food, which affects the movement of compounds to the taste and odor receptors. Taste and odor perception might be expected to be more rapid from liquid foods than solid foods.

Free fatty acids are generally formed by hydrolysis of the lipid through action of autolysis and bacterial enzymes (Gardner, 1957)* and may contribute to desirable flavors such as those present in cheese, or undesirable flavors such as those which are found in fish and shellfish.

A compound responsible for a metallic flavor in many fatty foods, l-octen-3-one, (Stark, 19^7) may be responsible for a fifth taste which is attributed to the presence of inorganic salts of iron and copper. These metals often catalyze lipid breakdown, and may be responsible for lipid oxidation in the mouth, and the formation of l-octen-3-one.

i

Formic, acetic, and propionic acids taste acid and sour, but these characters decrease as the chain length Increases. The peppery taste of blue cheese has been attributed to

11

butyric, hexanoic, and octanoic acids (Day, 1967). Since the lower fatty acids are volatile, it is probable that most taste measurements are really over-all flavor measure­ments involving both olfaction and mouth feel. Fatty acids of low volatility (above C^Q ) do not taste acid or sour, nor do they have much flavor. Their flavor has been described as candlelike. Glycerol results from hydrolysis of lipids and has a sweet taste.

The increases in total lower fatty acids such as acetic acid, formic acid, propionic acid, n-butyric acid, and iso-valeric acid, and their concentration, were studied by several workers (Hillig et_ al., 1959; Hughes, I960; Miyahara, 1961) as an index of spoilage in fish and shell­fish. The thresholds of pure solutions of the above acids were studied by Patton (1964).

Succinic acid, a potent substance giving the "meaty" taste to shellfish, was reported by Hashlmoto (1964). He noted that the disodium salt can be used as a flavor addi­tive, but that its application is far more limited than monosodium glutamate, because an excess confers too much of a shellfish-like flavor to foods. Hillig at al . (1959) reported a fair degree of correlation between succinate concentration and an overall organoleptic evaluation in the flesh of tuna. Results with other species were insig­nificant .

Opinion varies as to the relevance of lactic acid as a flavor constituent of flesh food. Wood (1961) considered

12

that the compound contributes nothing to the flavor of meat extract, but Kazeniac (1961) reported that It was responsible for the "sour, astringent" character In chicken broths. It has been recognized for many years that high concentrations can occur in fish muscle (Sharp, 1935)*

Carbohydrates and Sugar PhosphatesAlthough flavorless, glycogen Is claimed to contri­

bute what Is known as body, harmony, and smoothness in taste to a simulated extract of abalone meat (Hashlmoto,1964). However, glycogen is substantially degraded in most fish muscle within a short period after death, If it has not disappeared beforehand during the death struggle in catching operations.

The sugars commonly occurring In fish muscle are glucose, ribose, and a trace of maltose (Tarr 1953* 1955*1958 A, B, 1964; Jones 1958 A, B; Burt 1961; Tarr and Leroux 1962; Hughes 1964 B ) . Glucose, In considerably greater quantities than that occurring In fin fish, has been re­ported to be present in canned crab, along with rhamnose, galactose, and other sugars (Nagasawa, 1958, i960). Jones (1961) suggested that much of the characteristic sweetness of fresh fish flesh results from the presence of glucose in high initial concentrations. A weak sweetness In "Uni" ex­tracts, deriving from glucose was reported by Hashlmoto (1964).

Very little work has been done on the flavors of sugar phosphates. While emphasizing the difficulties of simula­ting conditions in the foodstuff, Jones (1961) concluded,

13

from a study on solutions of these compounds at their maxi­mum concentration, that they contributed "sweetish salty" character to fresh or chill-stored fish. While the validity of certain findings has not yet been established, a body of data has become available in recent years on concentrations of glucose 1- and 6- phosphates, fructose 1- and 6- phos­phates, and ribose 1- and 5- phosphates in fish muscles (Jones and Burg, I960; Burt, 196I; Burt and Jones, 1961;Tarr and Leroux, 1962; Burt, 1964; Tarr, 1964; Jones and Burt, 1964).

The combined effect of free amino acids, and par­ticularly of hiBtidine, aspartic acid, glutamic acid, and proline, may be to enhance sweetness (Tarr, 1966). The importance of the interaction between amino nitrogen com­pounds and carbohydrates in the production of odors was reported by Herz and Shallenberger (i960), who studied reactions of these compounds at relatively high tempera­tures, and by Keeney and Day (1957)* who discussed the im­portance of the Strecker degradation of amino acids in flavor development in cheese.

In a study on the effects of heating on amino nitro­gen constituents and carbohydrates, it was reported that taurine, anserine - camosine, and alanine were the major constituents of unheated Bamples, and losses during heating were relatively large (Macy et al., 1964), Similarly,Jones (1959) noted that taurine was one of the major con­tributors to browning in fresh and muscle extracts.

14

Carbohydrates present were alBO affected by heating. As reported by Macy e£ al. (1964) glucose, fructose, ribose, and some unidentified carbohydrates were found in extracts from beef, lamb, and pork. Glucose was present in greatest quantities followed by fructose, ribose, and the unknown fraction. Ribose appeared to be the most labile to heating, and fructose the most stable. Tarr (1954) reported that In fish muscle, ribose liberated enzymically from ribonu­cleic acid was responsible for browning.

Particulate enzyme (I) of Pseudomonas fragl was shown to have a powerful ribose and glucose dehydrogenase activity which catalyzed formation of ribone ~ f ~ lactone from ri­bose, and glucone - y - lactone from glucose. "I" wa3 applied to lingood fish, which had been stored three days at 0°C to ensure post-mortem formation of ribose and glu­cose, After several days at 0°C, "I" was capable of remov­ing both ribose and glucose from fish muscles and thereby strongly retarded the browning induced by heating for one hour at 120°C (Von Tigerstaom and Tarr, 1966).

A recent study on reducing sugars in fresh fish showed that no change in their values took place after boiling, treatment, and storage at room temperature and in ice (De and Haque, 1967)-

Carbonyl compoundsAldehydes belonging to the three groups, n-

alkanalB, the C^_12 n-2-alkenals, and the C^_1£ n-2,4- alkadienals, are formed from oxidized unsaturated fatty

15

acids, particularly oleic, llnoleic, llnolenic, and arachidonlc (Berry, 1958; Day, 1961; and Hill, 1965).They are reported by Schultz et al. (1962) aB being mainly responsible for oxidized flavors described as tallowy, painty, oily, or cardboardy. In an earlier study, Fross et al. (i960) attributed the fishy flavor of milk fat to n-hexanol, n-heptanol and 2-hexenal. Thus methyl llnoleate forms three monohydroperoxides which decompose to 2,4- decadienal, 2-octenal, and n-hexanal.

During the last ten years, a number of other carbonyl compounds with intense and characteristic flavors 3uch as cis-4-heptenal and trans-2,cis-6-nonadienal have been iso­lated from oxidized lipid materials. Since the formation of these compounds from oleic, linoleic, llnolenic, or arachldonic acids can be explained only through complex pathways, it has been suggested that these flavor com­pounds resulted from the oxidation of other C^0 unsaturated fatty acids occurring in trace amounts. Fross et al. (1962) reported that 2,6-nonadlenal is the main compound which con­tributes to the pleasant flavor in cucumber, and this re­sult was confirmed in a recent study by Fleming et al. (1968).

Only a small number of ketones derived from oxidized lipid are important in flavor, dtark et aJ., (1967) repor­ted that l-penten-3-one has an oily flavor. Methyl ketones (2-alkanones) play a major role in the flavor of blue cheese. Methyl ketones may also be formed by the thermal decarboxy­lation of 3-ketoacids occurring In triglycerides. Day et al.

16

(i960) found that the following components, i.e., 2- nonanone, 2-heptanone, 2-pentanone, butanone, 2-octanone, and acetone are the major compounds In Cheddar cheese. Tn a recent study, Mattick (1969) Isolated and identified ethyl vinyl ketone as the volatile component contributing to the raw bean odor and flavor in soybean. He further speculated that the following reactions might offer a possible mechanism of Its formation by the oxidation of llnolenic acid:

. /CH3-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)-C -0-RLlnolenic acid

1 M /CH3-CH2-CH=CH -CH2-CH -CH-CH=CH -CH=CH - (CH2 ) -C -0-R

/CH3-CH2-CH=CH-CH2-CH-CH=CH-CH=CH-(CH2)-C -0-R

\ Lo o h CH3-CH2-CH=CH-CH2— ♦CH3-CH2-CH-CH=CH2

|@CH3 7CH2-CH-CH=CH2

Lo o hLH20CH3 -CH2-C -CH=»CH2

I0Ethyl vinyl ketone

Delta - and gamma-lactones play important roles in flavor, the former in dairy products such as butter, and the latter in fruits Buch as peaches and apricots. Such lactones may be formed from the corresponding triglyceride

17

by heat - e.g., 130°C for one hour In vacuum.Alkanols generally play a minor role In flavor. They

may be derived from carbohydrates, amino acids, or oxidized unsaturated acids, and in the last two cases, may be pro­duced by the formation and reduction of the corresponding aldehyde. Stark and Pross (1966) isolated C^_q n-alkan- 1-ols from oxidized butter, and discussed their formation from 0 ^ ^ Q unsaturated acid hydroperoxides.

Unsaturated aliphatic alcohols are much more potent flavor compounds. Hoffmann (1962) isolated l-octen-3-ol from oxidized methyl linoleate and soybean oil. This com­pound is an important flavor constituent in mushrooms.Another compound which contributes to the oily, grassy flavor in milk and meat is l-penten-3-ol.

A great number of Investigators have demonstrated the presence of a number of carbonyl and alcohol compounds in fresh raw or cooked fish and shellfish. However, early studies varied In specificity and usually had quality testing as an objective. Thus, Parber (1952) examined the steam volatile carbonyls of tuna by iodometric titration; Yu and Sinnhuber (1957) related Btorage time to carbonyl develop­ment by the 2-thlobarbituric acid test which they developed later to estimate malonaldehyde specifically (Sinnhuber and Yu, 1958)• A similar procedure was used by Schwartz and Watts (1957) to test the spoilage of cooked oyBters;Mendelson and Steinberg (1962) trapped the carbonyl com­pounds from spoiling fish as the 2,4-dinitrophenylhydrazones,

IB

and concluded that though some oarbonyls of relatively low molecular weight were formed during storage of haddock filets at 2°C, by far the largest quantities were those of higher molecular weight and were liberated at maximum con­centration after 8-11 days of storage. However, the quan­tity of carbonyls of relatively low molecular weight in­creased linearly with storage time.

It had been suspected for some time, Lundberg (1957) that the odor and flavor of oxidized fish oils was derived mainly from the presence of unsaturated carbonyl and di- carbonyl compounds; and it was reasonable to suppose that the carbonyl fraction would be of considerable significance in fish products generally. Amano and Yamada (1964) pre­sented a modern view of the formation of formaldehyde from the degradation of trimethylamine oxide, formaldehyde being one of the carbonyl compounds present in fish and shellfish.

A tentative identification of volatile distillates from fresh and frozen stored skinless haddock filets was attempted by Mangan (1959). The precipitate for 2,4- dinitrophenylhydrazones of the frozen fish yielded a greater amount than did fresh fish. Mangan et al. (1959) reported the presence of acetaldehyde, and probably dicarbonyl and *<-hydroxycarbonyl compounds in frozen haddock. Merritt and Mendelsohn (1962) found both acetaldehyde and methyl ethyl ketone in chill-stored haddock. Hughes (1961) iso­lated acetaldehyde, proplonaldehyde, acetoin, isobutyralde- hyde, and 2-methylbutyraldehyde as the main components of

19

the neutral volatile fraction of cooked herrings* wLth smaller quantities of longer-chain carbonyl-compounds. Groninger (1961), also found acetoin in chill-stored fish. Subsequently, Hughes (1963) confirmed the presence of these compounds and identified tentatively: undecanal* nonanal,Cg-> C^-* Cg-* anti C^-alkan-2-ones* n-and lsovaleral-dehyde, ethyl methyl ketone* hex-2-enal* hept-2-enal* 2- pentenal* and formaldehyde. He also isolated a group of fractions that were probably dicarbonyl and dienal in nature, and others that were branched-chain Cg-* and C^- aldehydes and branched-chain Cg-* C ,-* Cg-* C^-, and C-^- alkan-2-ones. These findings were similar in some respects to those of Yu et jhL. (1961) on isolates from oxidized salmon oil* and to the reports of Diemair and Schams (1962)* and Diemair (1964) on uncooked stored fish. Hughes (1963) reported also on changes occurring in uncooked herring.His results were in agreement with other workers (Mendelsohn and Steinberg* 1962; Ota, 1958) in that there is a general increase in the concentration of carbonyls during chill stor­age* but it is apparent that proportional increases in dif­ferent constituents can vary from species to species.Groninger (1961) failed to detect an increase in the acetal­dehyde concentration in whole fish when they had been in chill-storage for ten days.

Shigeki (1967) in a recent study, obtained volatile monocarbonyl in frozen halibut. Ethenal was the main carbonyl in the water extract, whereas the n-hexane extract contained

20

propanone, butanone, 2-pentanone and £-heptanone. Several alkanals including methanal, ethanal and n-nonal were de­tected. In a recent attempt to identify the flavor com­ponents in Brazilian fish and shellfish and their changes during decomposition, Novak et al. (1970) showed that a number of hydrocarbons as well as carbonyls and alcohols such as n-butanol, n-heptanol are among the components which contribute to flavor in freshly caught fish and shellfish.

Although Hughes (1963) noted early work on the thresh­olds of some carbonyl compounds, he concluded after an ex­tensive study that nothing was known regarding the influence of "these carbonyls" on the flavor of cooked fish. Never­theless, he did make the very pertinent observation that the fall in rancidity that occurs during post-canning storage is accompanied by a decrease in the concentration of carbonyls. Almost concurrently with this work, Guadagni et al. (1963) published further threshold data on some of the compounds reported upon by Hughes (1963).

Evidence relating to the flavors of individual car­bonyls to specific attributes of fish flavor has been reported by several workers (Diemair and Schams, 1962; Diemair, 1964; Stansby and J>llinck, 1964). On the basis of his work, Diemair (1964) suggested that the carbonyls of low molecular weight are associated with fresh flavor,The "tranig" odor components of fat-containing species come mainly from hexanal, heptanal, and hexenal, while the rancid

21

and "talgigen" components belong to the higher constituents of the saturated and unsaturated fatty acids (nonanal, hep- tadlenal, decanal). A strongly "metallic" component dis­cussed by Diemair (1964) had many of the properties of 1-octen-3-one (n-amyl vinyl ketone) previously reported by Pross (1963). Diemair and Schams (1962) isolated from stored fish a carbonyl component with a "mushroom" odor.From its properties this was thought to have a dihydroxy- propane skeleton.

Volatile Sulfur CompoundsAlmy (1925) and Hughes (1964 A) reported the presence

of hydrogen sulfide in fish and shellfish products. Di­methyl sulfide was identified in raw haddock by Mangan (1959). Diethyl sulfide and other sulfides in similar material were found in an advanced state of spoilage by Merritt and Mendelsohn (1962).

Bailey et al. (1956) found that one of the tests that signify the onset of spoilage of shrimp, as well as being a quality test, is the sulfhydryl group. Thus, the pre­sence of large amounts of hydrogen sulfide, methyl mereap- tan and organic sulfide is characteristic of "well aged" spoiled fish and shellfish. The above was agreed upon by Miyahara (1961) who identified methyl mercaptan as a con­stituent of spoiling tuna flesh.

Hydrogen sulfide is considered to contribute to cooked chicken flavor by accumulating in significant amounts in freshly cooked chicken meat.

22

Kramlich and Pearson (i960) using QLC were able to Identify methyl mercaptan and methyl sulfide from the vola­tile constituents of cooked ground beef. Similarly, Hughes (1964 B) waB able to identify such compounds from cooked fish flesh and shellfish.

Merritt et al . (1959) studied the effect of irradia­tion on beef using 2, 4, and 6 Mrad. Among the volatile components that were identified In irradiated beef were the following: methyl mercaptan, dimethyl sulfide, dimethyldisulfide, ethyl mercaptan, and lsobutyl mercaptan.

Effects of radiation on food flavorThe undesirable odor and flavor produced in food

products such as milk, meat, fish and shellfish by irra­diation has been a continuing problem in efforts to pre­serve food by irradiation. In search of the causes of flavor deterioration, some investigators attempted to isolate and identify the volatiles from irradiated meats (Batzer et al., 1955* 1957* Burks et al., 1959* Merritt et al.* 1959; Monty et al., 1961; Salvador et al.* 1962;Wick et al.* 1961), and they established the presence of several types of compounds including carbonyls, alcohols, thiols, thioalkanes, and esters.

Merritt et al. (1959) showed an increase of several volatile carbonyl-and-sulfur-contalning compounds with increased irradiation dosage. Owing to the lack of suitable separation techniques, most of the earlier investigators were able to Identify only a small number of the total

compounds produced. However, they failed to demonstrate any but a quantitative relationship between the presence of theae compounds and the irradiation dose. All of the compounds were found to be present in non-irradlated meat. Although their presence in greater amount in the irradiated product possibly contributed to the overall "off-odor" sen­sation, it could not be described as peculiar to irradia­tion "off-odor" — at least from the viewpoint of chemical composition,

Other workers studied the effects of irradiation on meat components such as fats and proteins (Chipault et al., 1966; Hedin et aJ., 1961; Merritt et al., 1966; Rhodes et al., 1964; Sribney at ad., 1955; Witting et al.., 1958; Merritt et al., 1967). Amines, sulfur compounds and some carbonyl compounds were presumed to originate from the meat proteins, while the lipid fraction is believed to produce carbonyl compounds and hydrocarbons. In some cases they were typical of the classic products of fat oxidation.

As early as 1949, Burton studied and speculated on the compounds produced by irradiation of oleic acid, and the mechanism of their formation. He concluded that irra­diated fatty acids lose C0£ to yield a hydrocarbon with one less carbon in the alkyl chain.

A similar mechanism may also be responsible for hydro­carbon formation in irradiated lipid. The alkyl free radi­cal obtained from fat could form hydrocarbons

24

R-CHg - COO- » R-CH3 + C02 (1)R-CH2 + XH----->R-CH3 - X (2)

Alkenes may be formed as follows:R-CH2-CH2 ;— » R-CH =* CH£ + H

The short chain hydrocarbons could result from chain scission reactions. The branched-chain compounds could be formed also as a result of irradiation, by combination of two alkyl free radicals.

It is only recently, with the advanced analytical techniques available, that it was possible to acquire suf­ficient analytical data about the composition of the vola­tile compounds produced in irradiated food, and to relate this information to the mechanisms Involved. Thus, Merritt et al. (1959) were the first to demonstrate hlgh-vacuum, lower-temperature distillation of the volatiles of irradi­ated beef. This procedure was followed by subsequent gas chromatographic separation and mass spectrometric analysis. The initial experiment was followed by extensive reporting on the refinements of the analysis (Merritt, 1959; Merritt and Walsh, 1963; Merritt <5t al., 1964; Merritt et al., 1965)- More recently, Merritt et al, (1966) irradiated beef, veal, mutton, lamb and pork meats, and identified the normal al- kanes from to C ^ , the alkenes from C£ to C ^ , and the C- q and alkynes. With the exception of undecyne, these compounds were found in all five samples In similar quanti­ties. No aldehydes higher than Cg were detected. The pre­sence of large amounts of hydrocarbons and the relative

25

lack of carbonyl compounds, even In the presence of air, show that the mechanism for irradiation production of vola­tile s Is different from the mechanism for autoxidation (Figure 1). The sensory response is likewise different. Whereas the oxidized fat is typically rancid, the irradiated fat has a characteristic fat irradiation odor without being rancid. The above workers concluded that the hydrocarbons originated from the meat lipids and that their formation resulted from random splitting at carbon-carbon bonds along the fatty acid chains. Illustrating their conclusion by glycerostearate, they showed that all of the n-alkanes from methane to heptadecane could be formed as a result of the scission of the bonds at all points of the chain, with re­combination or hydrogen termination of the resulting alkyl- free radicals.

CH,CH0CHoCHoCHoCHoCHoCHoCH0CHoCni0CHoCH0CH0CH0CH-CH-COOCH-COOCH-COOCH

Cn

Moreover, if secondary collisions, extracting a second electron, occur, a similar homologous series of alkenes is predicted and these also are detected In quantity.

Of even greater importance, however, Is the study of the radiation products induced in methyl oleate and methyl stearate. Merritt et al. (1966) showed that the principal

26

c3O6<a)>tH.PnjrHa>K

OxidizedN - Alkanes

i

IrradiatedN - Alkanals

> i i ■2 4 6 8 10 12 4 6 8

Carbon Number

Figure 1. Distribution of hydrocarbon and carbonyl compounds among irradiates and oxidized butter fat volatiles. From Merritt et al. (1966)

27

products were the alkaneB, alkenes, and a homologous series of methyl esters. The highest member of the alkane series found in irradiated methyl oleate Is n-nonane and the high­est methyl ester is methyl caprylate i.e., the Cg acid.The series of unsaturat-ii hydrocarbons goes up to or higher. These compounds can arise from methyl oleate by the mechanism described for tristearin, as follows:CHoCHoCHoCHoCHoCHoCH^CH^CH = CHCH^CH^CH^CH^CH CH CHCOOCHo2 2 2 2 2 2 i

+

CH3

n-Nonane

Methylacetate

Methylpropionate

Methylbutyrate

Methyl caprylate

Although no methyl esters have been detected among the radiation products, acetone was a major component.

In agreement with Merritt (1966), Champagne and Nawar (1969) reported that the hydrocarbons are the major radiolytic products in fats. They pointed out that the alkadienes and some of the longer chain alkanes and alkenes Identified in their study have never been reported previous­ly In meats or meat fats. The significant part of their study was that the major hydrocarbons possessed either one or two carbon atoms less than the major fatty acids present in the fat studied. Thus, their Interpretation was that radiolytic splitting of fatty acid chains is not random as suggested by Merritt et al. (1966), but follows a preferential

28

pattern resulting in uneven distribution of the hydrocar­bons formed.

Dubrovcic and Nawar (1969)# ih their study on irra­diated fish oils, agreed with Champagne and Nawar (1969). Their quantitative analysis of the volatiles from the irra­diated fish oils demonstrated that the major products of irradiation were the longer chain compounds which were considered by Dubrovcic and Nawar (1969) from the fatty acids near the carboxyl group.

This comprehensive literature review contained ab­stracts of previous investigations on compounds which con­tribute to the flavor and odor of fish and shellfish, and how they are modified under certain physical and chemical conditions.

To summarize the work on irradiated products, the formation of hydrocarbons, and to a lesser extent of carbonyl compounds in the absence of oxygen, is charac­teristic of irradiated off-odors and flavors in meats, meat fats, fish and fish oils, milk and butter fats. An increased production of volatiles obtained during radiation sterilization (4-6 Mrad) was obtained by all of the inves­tigators, and their role in flavor production is now estab­lished. However, at sterilization doses, the radiation changes were so numerous that it was impossible to ascertain their interrelationships.

Although most of the studies on flavor contributing compounds were made with radiation sterilized foods, it is

29

urgent to collect Information on the modifications obtained during radiation pasteurization. Most of the United States Atomic Energy Commission's programs were limited to radia­tion pasteurization of foods (.05-.80 Mrad) using Cobalt 60 as the ionizing radiation source. Under these circum­stances research on post irradiation storage problems were necessary, because many reactions were initiated during radiation, and continued during subsequent storage periods.In contrast, virtually no changes occurred during storage when products were sterilized by radiation.

Since radiation pasteurization is likely to receive federal approval through the responsible regulatory agencies, it was necessary to organize a research project to study the nature and composition of flavor producing fatty acids and volatile carbonyls in shrimp.

Shrimp is a seafood of considerable economic impor­tance, and can be preserved successfully by radiation pas­teurization.

Therefore, this investigation was designed to study the products of various reactions occurring during low-dose radiation pasteurization of shrimp, and subsequent refrig­erated storage to determine what compounds contribute to flavor before and after radiation, and whether or not, and to what degree, these reactions can be used as an index of decomposition for Gulf shrimp.

Results of this project will become part of the U.S. A.E.C. petition to the F.D.A. for requesting approval to pasteurize fresh shrimp by gamma radiation.

EXPERIMENTAL METHODS

In studying such compounds s b carbonyls and fatty acids, and their interrelationships , it is necessary to procure samples that are a3 biologically free from con­tamination, bacterial and otherwise, as are readily obtain­able. Even when ideal circumstances exist, shrimp are so susceptible to microbial activity that it is Impossible to ascertain with any degree of assurance that the results obtained reflect only those chemical and physical processes normally Inherent to the tissue under examination.

When first removed from the water, shrimp carry an indigenous microbial flora which cannot be completely re­moved without extensive tissue destruction and the conse­quent production of components not normally present. While it is not technically feasible to obtain a biologlcally- pure condition with shrimp, this state can be approximated by exercising caution in obtaining, handling, and preparing the sample for study. It is to the effectiveness of obser­ving these precautions which are directly related to the quality of the product at the time of irradiation that Novak et al. (1966) attributed the successful application of gamma irradiation in the preservation of oysters and other shellfish.

I . Procurement and PreparationFresh shrimp (Peneaus Betlferus) of known history

used in this study were procured from a commercial shellfish

31

packing plant In New Orleans, Louisiana.The shrimp were caught from boats in butterfly nets

in the vicinity of New Orleans, and immediately following removal from the water, they were separated from trash fish, washed with water and packed in crushed ice. The shrimp were de-iced upon arrival of the boat at the plant, washed in running tap water for two minutes, and allowed to drain for a period of five minutes. After being washed and drained, the shrimp were packed in a layer of ice and transported to the Louisiana State University at Baton Rouge. The elapsed time from catching until delivery at the laboratory was limited to a maximum of 15 hours. Upon arrival at the Department of Food Science on the Baton Rouge campus, they were rewashed with running cold water to remove surface contamination. After being drained, the shrimp were deheaded and peeled. All the above processing pro­cedures were performed in the cold room at 40°F to mini­mize the deterioration of shrimp which would increase at room temperature.

Figure (2) is a flow diagram showing the steps in which the shrimp were processed.

Weighed amounts of the headed and peeled shrimp were placed in Scotchpak 20A5 pouches and heat sealed. These packages were chilled for 30 minutes prior to irradiation to preserve the fresh quality of shrimp during irradiation.

32

\ Shrimp racKing| Boat Plant

I De-icingV • _____________

IfeaCTna1*) ood aa.en<®TDe-icing

T/Ashing .anaI draining

“■WagftiS j kd.3dto.ceIrradiation

Food Sciencep Z Z I Z -lice Storage |

Figure 2. Flow sheet diagram showing steps involved In preparing the sample for carbonyl and fatty acid extraction.

33

II. Irradiation source and locationPreliminary studies with gamma irradiation were per­

formed upon receipt of the shrimp to determine the approxi­mate levels of irradiation which should be used on the pro­duct .

All samples were irradiated at various Mrad levels in an 11,000-curie Cobalt-60 irradiator located in the Nuclear Science Center at the Louisiana State University.It was determined by Fricke Dosimetry (1963) that the aver­age dose rate in the center of the diving bell was .0012 Mrad per minute. The dose rate is measured in the range of 0.15 to 0.8 Mrads by oxidation of ferrous ammonium sul­fate and determination of the ferric ion spectrophoto- metrically. In this procedure a specimen of dosimetric solution is placed in the radiation field for a carefully measured length of time. After removal from the radiation field, the optical density of the specimen is read in a spectrophotometer on the same day. A portion of the unir­radiated ferrous solution is used as a blank in the spec­trophotometer. From the optical density of the irradiated sample, the radiation dose Is read in Mrads directly from a calibration curve.

The measurement of optical densities with the spec­trophotometer is performed at constant temperature, since the extinction coefficient is + 0.7?6 per degree C. Alter­natively, a correction for temperature may be made according to the following equation:

34

Dose (corrected) - doae measured at Tg1 + 0.007 (T2 - T 1

where:T 1 = temperature at which the calibration curve

was prepared.T2 = temperature at which the unknown sample is read.

Ill. Irradiation of shrimpThe weighed and packaged samples of shrimp were

divided Into three groups:a. those that served as the non-irradiated control.b. those Irradiated at a dose of 0.15 Mrad. Although

a high reduction in microorganisms wa3 noted at this dose, and the shelf life of shrimp was exten­ded up to 25 days without Impairing the accepta­bility of the product, it Is necessary to study the effects of radiation on the fatty acids and carbonyls.

c. those irradiated at a dose of 0.8 Mrad. This dose was selected specifically to observe fatty acids and carbonyl changes in shrimp as related to varying levels of Ionizing radiation.

The irradiation process consisted of placing the samples into a water-tight canister which was then lowered to the bottom of an 18-foot water well, where It remained in close proximity to the cobalt-60 source until the desired level of gamma radiation had been administered to the samples.

35

Since the dosage distribution was found to vary with the distance between the position of the samples and the source, the same positions were used with all the samples to be Irradiated. The irradiated samples were brought back to the laboratories of the Department of Food Science where they were packed in ice and stored in a cold room throughout the investigation. The samples prepared for carbonyl analysis were stored for a period of 30 day3. Analyses were run at 0, 7, 14, 21 and 30 days. Those pre­pared for fatty acid analysis were stored for a period of 24 days. Analyses were conducted at 0, 12 and 24 days.

IV. Extraction of Carbonyls from ShrimpVolatile carbonyls may occur naturally in shrimp

tissues as reported by Novak et al, (1970), or may be formed by autolysis, microbial action, pyrolysis or radi- olysls action.

The volatile fraction is defined arbitrarily as the materials Isolated in whole or in part by the specific method of separation employed, whether it be steam dis­tillation, vacuum distillation or aeration. In general, compounds comprising this fraction contain one, or rarely two, functional groups and not more than 10 carbon atoms. The volatile fraction can be chromatographed directly, or It may be separated first into acidic, basic, and neutral subfractions by auxiliary chemical and physical methods. The neutral components can be separated further as sulfur compounds, carbonyls, etc., by use of the appropriate

36

precipitating or complexing reagent. Aldehydes and ketones are part of the neutral fraction of the extract or distil­late, in as much as they cannot be isolated from alcohols, ethers, or sulfur-containing compounds by adjustment of pH. However, they can be separated from other neutrals by making use of the well-known capacity of the carbonyl groups for condensing with reagents containing labile hydro­gen, such as hydroxylamine, or 2-4-dinitrophenylhydrazine. Precipitation through reaction with this latter compound and regeneration of the free carbonyls by levulinic acid, and extraction of the free carbonyls with methyl phenyl ether form the basis of the principal method for analysis of these compounds in shrimp by gas liquid chromatography.

A slight modification of the method used by Pippen et al. (1958, and Pippen e_t al. 1957) to strip the carbonyl compounds free from cooked chicken was adopted for this study, and is described below.

1. Rinsing the equipment prior to its use with nanograde petroleum ether was necessary to elim­inate the extraneous peaks which were found in the reagent blank in the initial study.

2. a 600 g Bample of shrimp was found to be sufficient for the extraction.

3. Aeration over a period of 24 hours was found to be important to obtain a higher recovery, instead of the 20 hours suggested by Pippen et al, (1958).

37

V. Equipment and ProcedureThe equipment used for the Isolation of carbonyl

compounds by aeration is illustrated in figure 3- Trap A, containing 400 ml of a saturated solution of 2,4-dinitro- phenylhydrazine in 2N sulfuric acid, was used to remove carbonyls from the air that sweeps the volatiles from the sample. Trap B contains glass wool. C is a 5-liter flask containing the sample, D. Traps E and F collect the car­bonyls and contain 400 ml each of a 0.2^ solution of 2,4- dlnlt rophenylhydrazine solution in 2 N hydrochloric acid.

The 600 g of shrimp was mixed with an equal weight of distilled water and homogenized in a Waring Blendor. The mixture was transferred to a five-liter flask quantita­tively with distilled water so that the total amount of distilled water used was one-liter. The flask was closed, and air was drawn through the system by reducing the pres­sure sufficiently at trap F to cause approximately two bubbles per second to pass through the liquid in traps E and F. With water circulating through the condenser, suf­ficient heat was applied to flask C to maintain a gentle reflux throughout the run. Aeration was continued for a period of 24 hours. At the end of the run, hydrazones in traps E and F were recovered by filtration, washed with 2 N hydrochloric acid and then with water, and dried under vacuum at 35°C.

Figure 3- Apparatus for carbonyls extraction

39

V I . Regeneration of CarbonylsRequirements necessary In a successful reagent for

regeneration of carbonyls from 2,4-dinitrophenylhydrazones would be carbonyl groups, acidity, and water. Pyruvic acid as a source of carbonyl and acid groups was eliminated, because of the unstable ill-defined character of this com­pound. Levulinlc acid CH^-COfCHgJg-COOCgH^ was proven by Keeney (1957) to be useful for the regeneration of micro quantities of carbonyl compounds.

In conducting the experiments related to this study, the dried 2,4-dinitrophenylhydrozone were quantitatively transferred to a 50 ml distilling flask. Ten milliliters of a mixture of nine volumes of levulinlc acid to one vol­ume of 2N hydrochloric acid were added. The flask was fitted with a side arm tube attached to a condenser. The distilling flask was heated to 100°C In an oil bath for five minutes, then 10 ml of distilled water were added to the flask, and the temperature of the bath was rapidly raised to l60°C. About 2 ml of aqueous distillate was collected in the tube which was immersed In an ice bath.The carbonyls were extracted with a small amount (30-50jf 1) of redistilled methyl phenyl ether (anisole) and injected In (l-10y|l) quantities into a gas chromatograph.

VTI. Identification of free carbonyls in shrimpThe methods commonly uBed for fractionation and

identification of the micro quantities of carbonyl com­pounds, adsorption, partition, and paper chromatography,

40

have their limitations especially when good recovery is required. However, the gas liquid chromatography was suc­cessfully used in identifying micro quantity components responsible for food aroma.

Numerous chromatographic techniques were performed as preliminary investigations, before the final selection of procedures were made for this research. These proce­dures were selected to be able to determine the minute quantity of carbonyls.

The identification of free volatile carbonyls was conducted in a Microtek GC 2000-R gas chromatograph equipped with dual columns and flame-ionlzation detectors. The columns employed were:

1- 25{£ carbowax 20M coated on 80-100 mesh acid- washed cromosorb W described in table I.

2- a 1/8 in. by 8 ft. coiled helix stainless steel column packed with 80-100 mesh Porapak Q, porous polymer beads developed by the Dow Chemical Com­pany, Freeport, Texas.

The operating conditions as noted in table 2 were employed for both columns with the exception that the latter column was operated isothermally at 175°C.

VIII. Extraction of 11-Pldg from shrimpIn the course of method selection for lipid extraction

two important criteria were considered:1- Efficiency of the method of extraction and puri­

fication .

4i

TABLE 1

OPERATING PARAMETERS FOR GAS CHROMATOGRAPHIC ANALYSIS OF CARBONYLS IN SHRIMP

Column

Operating temperature detector

Inletcolumn

Program rate Carrier gas Carrier flow rate

Air flow rateHydrogen flow rateDetectorChart speedRecorderAttenuationSample size

1/8 in, by 8 ft. coiled, helix stainless steel, packed with 25/6 carbowax 20M coated on 80 to 100 me3h acid-washed cromosorb W.

225°C200°Cmaintained at 50°C for five minutes after sample injec­tion. Programmed from 50°C to 175°C.5°C per minuteNitrogen50 ml per minute (measured at the column inlet)283 ml per minute60 ml per minuteDual hydrogen flame1/2 in. per minute-0.2- to 1.0-mv HoneywellAs notedA b noted

Up

2- The method should Involve a mild treatment due to the unsaturated nature of lipids in fish and shellfish.

Several methods were performed. However, their applicability was not entirely suitable to the present study. The methods of Dambergs (1956) and Polch et_ al. (1951) were time consuming, and since the former method entailed heating and evaporation, it was considered un­suitable for lipid composition studies. The method of Folch et al^(l957) had the disadvantage of employing a large and inconvenient volume of solvents. The method used by Dyer and Morton (1956) extracted only a fraction of the total lipids.

The method used in this study was recommended by Bligh and Dyer (1959) whereby an optimum lipid extraction resulted when the tissue was homogenized with a mixture of chloroform and methanol, which when mixed with the water in the tissue, would yield a monophasic solution. The resulting homogenate could then be diluted with water and/ or chloroform to produce a biphasic system, the chloroform layer of which should contain the lipids and the methanol- water layer the non-lipidB. Hence, a purified lipid ex­tract is obtained when the chloroform layer is Isolated.It is imperative, however, that the volumes of chloroform, methanol, and water before and after dilution, be kept in the proportions 1 : 2 : 0.8 and 2 : 2 : 1.8, respectively. The procedure: Two 100 g portions of the sample were

43

homogenized In a Waring Blendor for 2 minutes with a mix­ture of 100 ml chloroform and 200 ml methanol. To the mixture was then added 100 ml chloroform, and after blending for 30 seconds, 100 ml distilled water was added and blen­ding was continued for another 30 seconds. The homogenate was filtered through Whatman No. 1 filter paper on a Coors No. 3 B u c h n e r funnel with slight suction. Filtration was quite rapid, and when the residue became dry, pressure was applied with the bottom of a beaker to ensure maximum recovery of solvent. The filtrate was transferred to a 500 ml graduated cylinder, and after allowing a few minutes for complete separation and clarification, the volume of the chloroform layer (at least 150 ml) was recorded and the alcoholic layer removed by aspiration. A small volume of the chloroform layer was alsu removed to ensure complete removal of the top layer. The chloroform layer contained the purified lipid.Determination of lipid content: A portion of the lipidextract containing 200 mg lipid was evaporated to dryness in a tared flask and the weight of the lipid residue was determined.

Evaporation, facilitated by a stream of nitrogen, was carried out in water bath at 40 - 50°C and the residue was dried over phosphoric anhydride in a vacuum desiccator. The dry weight of the residue was determined and subtracted from the Initial weight. The lipid content of the sample was calculated as follows;

44

Ttotal ltrrtd = 14?ld In aliquot x volume cf chloroform layer-upia = volume or aliquotthe weight of lipid In the aliquot In this study was 200 mg.

IX. Saponification and EsterlflcatlonForty ml of redistilled hexane was added immediately

to the dry extract obtained from the other portion of the extracted sample. A 10 ml aliquot was reduced in volume to 1 ml at 30°C using purified nitrogen. This was quantita­tively transferred with a small portion of hexane to a 50 ml volumetric flask. With the aid of purified nitrogen, the hexane was evaporated to almost dryness and the sample was ready for saponification and esterlfication, following the procedure recommended by Metcalf el; al . (1966): 4 mlof 0.5 N methanolic potassium hydroxide was added to the mixture which was heated on steam bath for about five minutes. Five milliliters of boron-trifluoride-methanol reagent (BFg methanol, Supelco, Inc., Bellefonte, Pa. 16823) was added to the flask and the mixture was boiled for two minutes. After cooling the mixture, 2 ml of nanograde hexane were added to the flask in order to transfer the methyl esters to this solvent for gas-liquid chromatography. Sufficient quantities of a saturated sodium chloride solu­tion were added to the flask to float the fatty acid methyl esters up into the narrow neck of the flask where they were withdrawn by means of a syringe.

X . Thin-layer ChromatographyThin-layer chromatography waB used to check the com­

pleteness of the conversion. Fifty grams of silica gel-G were mixed with 60 ml of distilled water for 15 seconds, and spread at 0.50 mm thickness over well-cleaned glass plates (20 cm2) with the aid of an adjustable applicator.The plates were air-dried for 30 minutes and activated in a drying oven at 120°C for 30 minutes, where they were placed in a special desiccator until ready to be used. Samples to be tested were spotted on an imaginary vertical line drawn on the plate alongside reference fatty acid and fatty acid methyl ester standards. The plates were then chromatographed in the developing tanks by adding 50 ml of a mixture of petroleum ether, diethyl ether, acetic acid 90:10:1 (v/V/V) to the trough in the tank. When the solvent front reached 1 in. from the top, the plate was removed from the tank and air-dried for 15 minutes at room tempera­ture .

Identification of the spots was accomplished by spraying evenly 0.2 percent solution of 2 ’, 7 1-dichloro- fluorescein in 95 percent ethanol, and observing the plate under an ultra-violet light source.

X I . Gas-liquid ChromatographyFatty acid analysis was conducted in a Microtek GC

2000-R gas chromatograph equipped with dual columns and flame-ionization detectors. The operational conditions employed for the analysiB is shown in Table 2.

4o

TABLE 2

OPERATING PARAMETERS FOR GAS CHROMATOGRAPHIC ANALYSIS OF FATTY ACIDS IN SHRIMP

Column

operating temperature detector

inletcolumn

Carrier gas Carrier flow rate Air flow rate Hydrogen flow rate Detector Chart speed Recorder Attenuation Sample size

1/4 in. X 6 ft. coiled helix stainless steel, packed with 17# ethyleneglycol adipate coated on 80 - 100 mesh chromoport XXX.

235°C230°C180°Cnitrogen50 ml per minute283 ml per minute60 ml per minutedual hydrogen flame1 inch per minute-0.2- to 1.0-mv Honeywellas notedas noted

RESULTS AND DISCUSSION

Results reported In this Investigation represent qualitative and quantitative changes which occur in fresh shrimp of known history, when they are caught and handled by procedures employed for commercial production. Altera­tions during and after ionizing radiation did not result In the formation of known deleterious components.

Total CarbonylsThe total carbonyl derivatives of the non-irradiated

shrimp and those Irradiated at 0.15 and 0.8 Mrad were determined immediately before and after irradiation, in order to study the effect of irradiation at these two dose rates on the carbonyls in shrimp. Figure 4 shows that al­though the total carbonyl derivative values measured as mg of 2,4-dinitrophenylhydrazone were slightly higher in the samples Irradiated at 0.15 Mrad, than the non-irradiated samples at the zero time, this increase In total weight was not considered significant.

It was noted that irradiation at the 0.8 Mrad level caused an increase in the total weight of carbonyl deriva­tives. This should be expected, because the intense energy of radiation in the presence of oxygen has been found to produce aldehydes and ketones from various organic compounds (Chipault jet al. 1966; Rhodes et al. 1964).

It is widely accepted that OH radicals, hydrated elec­trons (egq ), H radlealB, Hg and HgOg are primary species

mg 2,

4-di

nitr

ophe

nylh

ydra

zone

48

A Non-irradiated

25 % Irradiated at 0.15 Mrad

B Irradiated at 0.8 Mrad

20

15

10

5

014 21

Storage time in days

Figure 4. Effects of irradiation on total carbonyl compounds of ice-stored shrimp (measured as mg 2-4 dinitrophenylhydrazcne per 100 g of shrimp)

ly 9

arising In the radiolysis of water. At the neutral pHregion, OH and e- mainly ariBe, while H is primarily lessaqimportant, G(0H}=2.25, G(e- )-2.30, and G(H)=0.55j where Gaqis defined as a number of molecules produced by absorption of 100 e^ irradiation energy (Fujimaki and Morita, 1968).

Burton (1969)* in studying the effect of high energy radiation on water, explained the formation of the above species as follows:

H20 ► H20+ + e‘

this being the important first elementary process. The next steps usually assumed were:

H£0+ + e" (H20) — v 2H + 20H

H20+ + H20# > H20+ + °HHgO + e" > H + OH"

followed by a variety of processes involving hydrogen atom and hydroxyl radical.

Generally, in food irradiation, water-soluble food constituents are affected directly by the attack; of radia­tion energy and indirectly by the attacks of the active species from water, such as OH, eg^ and H, If a solution is dilute, the direct attack of irradiation to solutes in it is negligible, and reactions occurring in the system are simplified. When a certain food is irradiated, each of these active specleB, OH, e”^ and H, is expected to be shared to each constituent in the food approximately in

50

proportion to the product of the rate constant for* the active species, and the concentration of the constituent.It appears that when shrimp were irradiated at 0.15 Mrad the energy absorbed was not enough to Induce the formation of such active species as those mentioned above, hence there was no Increase In total carbonyl compounds. However, at doses of 0.8 Mrad the energy absorbed by shrimp Induced the formation of these active species which in turn reacted with shrimp constituents such as amino acids and fatty acids to bring about an increase in total carbonyl compounds.

The amount of total carbonyl derivatives for shrimp samples stored over a period of 30 days were studied at 7/ 14, 21 and 30 days to find the effect of storage in Ice alone. As shown in figure 4 there was an increase In the total carbonyl derivatives with the Increase of the storage period. It was noted that the total carbonyl deriv­atives reached their maximum sometime between the l4th and the 21st day. The exact storage day at which the maximum concentration was reached would vary according to many fac­tors. However, at the end of the storage period a decrease in the total carbonyl derivatives waB noted.

In general, it was found that the amount of total carbonyl derivatives increases with time until spoilage occurs, and then the trend is reversed. This finding was reported earlier by Settoon (1967) who postulated the fol­lowing processes which may account for these decreases:

(a) Extensive proteolyBis may create a more favorable

51

diffusion gradient for the loss of volatlles from the system.(b) Release of compounds capable of condensing with carbonyls may occur in the latter stages of decom­position. Basicity of the system would result ina condition catalytic for the occurrence of the condensations.(c) Aldehydes may be oxidized to acids.(d) Carbonyls may Berve as precursors in microbial processes.(e) Increase in pH may create an environment ur>fa- forable to microbial activity responsible for car­bonyl production.(f) Carbonyl precursors in the tissue may be depleted. Carbonyl generation from the precursors may be viewed

as occurring through processes such as hydrolysis, in which aldehydes are liberated from plasmalogens or acetal phos­pholipids. Such hydrolytic reactions could readily be cata­lyzed by endogenous tissue enzymes or by exogenous microbial activity. It is equally feasible to consider the carbonyls as originating from microbial metabolism leading to the for­mation of these materials as end products or as intermedi­ates for other pathways.

The pattern In which the amount of total carbonyl derivatives change during iced storage over a period of one month for shrimp irradiated at 0.8 Mrad is shown in figure 4. There were Increases in the amount of total

52

carbonyl derivatives between 0 and 7 days, and a charac­teristic Irradiation odor emanated from the sample. Al­though the exact day at which the Increase in the amount of total carbonyl derivatives reached its maximum is not known, this Increase was followed by a decrease as the storage time Increased, and the irradiation odor decreased as well.

The Immediate Increase In the amount of total carbonyl derivatives of shrimp after irradiation at a dose of 0.8 Mrad and not at 0.15 Mrad was anticipated. Novak et al. (1966) had already established that shrimp exposed to doses in excess of 0.2 Mrad suffer a loss in quality as revealed by organoleptic tests.

The decrease in the amount of total carbonyl compounds during storage is due to the fact that carbonyls are very active compounds, and in the presence of oxygen and the free radicals formed by irradiation, oxidation and hydrol­ysis of such compounds take place during storage.

The following three correlated findings might suggest that the characteristic irradiation off-flavor results from the increase In total carbonyl compounds in irradiated food:

1- The decrease In the amount of total carbonyl derivatives during Iced storage as shown above,

2- The diminution of the irradiation odor during iced storage as reported by Sinnhuber (1961),

3- The decrease In irradiation off-flavor of ice-stored shrimp (Scholz et al., 1962).

53

Figure 4 showB that low dose gamma irradiation did not change the amount of total carbonyl derivatives during iced storage over a period of 21 days* Samples tested at the termination of a 30-day period showed a slight increase in total carbonyl derivatives.

These results were in harmony with those reported by Novak and Lluzzo (1967). Shrimp irradiated at a dose of 0,2 Mrad and iced stored for 25 days were tested by 40 expert panelists at the National Fisheries Institute meeting. These panelists judged that the 25-day old irradiated shrimp were less than ten days old. Over 90 per cent of them guessed the samples to be between five and eight days old. All these findings led the investigators to believe that irradiation at a dose of 0.15 Mrad prevents microbial activities that cause an increase in the total carbonyls in the stored, non-irradiated samples, and does not induce a characteristic irradiation off-flavor.

Gas Chromatography Analysis of Volatile CarbonylsThe profile in which each individual carbonyl con­

tributed to the irradiation induced changes and to the changes that took place during storage over a period of 30 days, was followed by gas liquid chromatography.

Although the method used in the present study for the extraction and isolation of the volatile carbonyls was applied efficiently to cooked chicken by PIppen et al.(1958) it was felt necessary to establish and determine the efficiency of this method when applied to Bhrimp.

54

The results in tables 3* 4, and 5 show that the first three compounds suffer greater losses due to their vola- tl.llty. However, the recovery was considered to be excel­lent In view of the number of steps Involved. It should be noted that the recovery of added carbonyls to shrimp itself would be slightly higher due to the presence of other compounds which will decrease the losses.

The carbonyl compounds were identified by comparing their retention times with those of known redistilled stan­dards on two different kinds of columns: 25 per cent Carbo-wax 20 M coated on 80-100 mesh acld-waBhed Chromosorb W., and Porapack Q 80-100 mesh.

Table 6 lists the identified compounds chromatographed Individually under similar conditions to the shrimp samples. The retention time of each compound is given for both columns used. Changes in volatile carbonyl compounds in this study were followed only with the 25 per cent Carbowax 2QM column.

The monocarbonyl compounds which were identified in shrimp are the following: acetaldehyde, propionaldehyde, acetone, isobutyraldehyde, butyraldehyde, butanone, 3- methyl-2-butanone, diacetyl, hexanal, 2-heptanone, and heptaldehyde. With the exception of 2-heptanone, all the carbonyls identified in the shrimp in the present study were reported by Novak e£ al, (1970) to be present with other compounds In freshly caught Brazilian fish and shellfish.

TABLE 3

RECOVERY OF ADDED CARBONYLS (EACH IN 1.0 ppm)

Carbonylsadded

Recovered carbonyls a/

(in ppm)Percent

recovery

Acetaldehyde 0.71 71Propionaldehyde 0.73 73Acetone 0.73 78Butanone O .85 85Hexanal 0.92 92

a/ Average of three runs

TABLE 4

RECOVERY OF ADDED CARBONYLS (EACH IN 5.0 ppm)

Carbonylsadded

Recovered carbonyls a/

(in ppm)Percent

recovery

Acetaldehyde 4.00 80Proplonaldehyde 4.05 81Acetone 4.35 87Butanone 4.55 91Hexanal 4.70 94

a/ Average of three runs

TABLE 5

RECOVERY OF ADDED CARBONYLS (EACH IN 10.0 ppm)

Carlo onyls added

Recovered carbonyls a/

(in ppm)Percent

recovery

Acetaldehyde 8.50 85Propionaldehyde 8.80 88Acetone 9.20 92Butanone 9.54 95.4Hexanal 9.68 96.8

a/ Average of three runs

58

TABLE 6

RETENTION TIMES OF VOLATILE CARBONYLS IN SHRIMP USING TWO SEPARATE COLUMNS

PeakNumber

CarbonylCompounds

Time In

25# Carbowax 20M

minutes

Porapak Q 80-100

1 Acetaldehyde 0.30 0.482 Propionaldehyde 1.06 1.423 Acetone 1.20 1.544 Ia obutyraldehyde 1.24 4.005 Butyraldehyde 1.58 3.186 But an one 2.12 3.307 3-Me thy1-2-butanone 2.48 5.488 Dlacetyl 2.58 3.249 Methyl Butyrate 3.24 8.00

10 4-Methyl-2-Pentanone 3-30 11.1211 Hexanal 6.00 14.2412 2-Heptanone 14.24 17.2413 Heptaldehyde 14.30 17.3614 Ani sole(solvent) 19.36 21.18

59

All the compounds Bhown in table 7 have been found to be present in non-irradiated shrimp, and their presence in greater amounts in the irradiated shrimp at a dose of 0.8 Mrad was estimated by the increase in peak heights.Thus it is clear that the presence of volatile carbonyl compounds in irradiated samples, from the viewpoint of chemical composition, is not peculiar to irradiated shrimp. This finding was reported earlier by Merritt (1959* 1965).

The increase in the volatile carbonyl concentrations may contribute to the overall off-odor sensation in the irradiated samples at doses greater than 0.2 Mrad. In determining whether a particular compound is of flavor significance, the concentration of the compound in the food products must be determined, and the flavor threshold- value of the compound should be evaluated. Therefore, it seems that the monocarbonyls in fresh non-irradiated shrimp are present below their threshold value, and when shrimp is irradiated the concentration of the carbonyls increases beyond their threshold value, allowing the formation of a characteristic irradiation off-flavor.

It appears from table 7 that acetaldehyde and proplo- naldehyde are major constituents of volatile carbonyls that occur in fresh shrimp. Only traces of hexanal and butanone were detected. The Increase in their concentration after Irradiation at a dose of 0.8 Mrad was not significant.

The Increase In peak heights of the following com­pounds: butyraldehyde, dlacetyl, 3-methyl-2-butanone,

TABLE 7

EFFECTS OF IRRADIATION AT TWO DIFFERENT DOSE LEVELS ON VOLATILE CARBONYLS IN SHRIMP

PeakNumber

CarbonylCompounds

NonIrradiated

Irradiated at 0.15 Mrad

Irradiated at 0.8 Mrad

1 Acetaldehyde +++ +++ +++

2 Propionaldehyde +-H-+ ++++ +-H-4 h

Acetone3,4 Isobutyraldehyde ++ ++ ++

5 Butyraldehyde + ++ +++

6 Butanone Trace Trace +

3-Me thy 1 - 2 - but an one7,8 DIacetyl -H- +++ +++++

9 Hexanal Trace Trace Trace2-Heptanone

10,11 Heptaldehyde ++ +++ ■ HHF

+ Represents the smallest relative peak heightTrace: Represents a slight hump

Relative Peak Height

• i0: i1

pp. t •pct(T>P.

l ■

>"3PIapi !OcxP'o

Pu

pp.1 •pr . ta&pi 11o03►:pa

A c e t o n eIsobutyraldehyde

Butyraldehyde

-utanone

hoxunal

Acetaldehyde

Propionaldehyde

3 -Methy1-2-butanone D l . a c e b y l

2-HeptanoneHeptaldehyde

T'J

(1?

2-heptanone, and heptaldehyde In shrimp irradiated at a dose of 0.8 Mrad was apparent when compared with the peak; height of the non-irradiated sample. The presence of oxy­gen and the formation of the free radicals from water hydrolysis which in turn react with the shrimp constituents account for the increase in the concentration of aldehydes and ketones.

In general, alkanone seems to be formed from unsatu­rated fatty acids as shown below:

R - C H - C H - C H - R '

l °R - C H - C H = C H - R 1

I0I

R -C H ~CH=CH - R ' t

0 0 HI

R - C - C H = C H - R '+ H 0II 2 0

In the formation of alkanal, the following is postu­lated:

RCHg - COO * R C H g + C 0 g

RCHp + CO

J -RCHO + H g

and the alkanal produced possessed one carbon atom less than the major fatty acid. Merritt (1965) reported the presence of COg as well as Hg in irradiated meat samples.

(1

There were no signifleant changes In the concentra­tion of the volatile carbonyls of samples irradiated at a dose of 0.15 Mrad when compared with the fresh non-irradi­ated samples.

The profile for non-irradiated shrimp, in which the identified individual volatile carbonyl compounds change during Iced storage, is shown in table 8 . They followed the same pattern in which the total carbonyl derivatives change during iced storage. With the exception of hexanal which did not show a significant change, all the volatiles showed a higher concentration at 21 days. The exact day In which the carbonyls reached their maximum is not known.A significant Increase was noticed after 14 days of storage, and continued to increase until 21 days, followed by a slight decrease.

These increases In the concentrations during storage are partially due to autolysis and partially to the enzy­matic system of the microflora. Shewan and Ehrenberg (1962) reported that most compositional changes that take place during 8-12 days storage period are associated with the development of particular groups of bacteria, principally Pseudomonads. Settoon (1967) postulated several mechanisms for the formation of general aldehydes, ketones, acetylalde- hyde, diacetyl, acetone and 3-Methyl-2-butanone.

Effects of irradiation and storage on carbonylsAs shown in table 9 » the variability in peak height

of the individual volatile carbonyl compounds did not show

TABLE S

CHANGES IN VOLATILE CARBONYL COMPOUNDS DURING ICESTORAGE ON CONTROL NON-IRRADIATED SHRIMP

Carbonyl 0.0 Day 7 Days 14 Days 21 Days 30 Days

Acetaldehyde +++ +++ ++-H- 1 t H-l t ■ m 4-1Fropionaldehyde ++++ -HH-+ ++-H-+ ++++++ i i i i iTTTTT

AcetoneIsobutyraldehyde ++ ++ +-H- ++++ +++Butyraldehyde + + -H~H- -H-H- +++2-Butanone Trace Trace Trace + Trace3-Methyl-2-butan oneDiacetyl -H- ++ H+l 1 -HH-H-+ ++++Hexanal Trace Trace Trace + +2-HeptanoneHeptaldehyde ++ +++ -H-++ fr+41 f + +++

+ Represents the smallest relative peak heightTrace: Represents a slight hump

Relative Peak Height

11 II II 11 |l II l| II II II IS / S ' s y s y y . y y y ' y yy y r

AcetaldehydeII

....... II ii ii il lly y y yy y y y y ,

Propionaldehyde

HEfcr1"AcetoneIsobutyraldehyde

Butyraldehyde

Butanorie

I ll II II II II 11 II *' y y y y y y y y y y

3-Methyl-2-butanoneDi acetyl

Hexanal

B J A K H F2-IteptanoneHeptaldehyde

TABLE 9

CHANGES IN VOLATILE CARBONYL COMPOUNDS DURINGICE STORAGE ON IRRADIATED SHRIMP AT 0.15 MRAD

Carbonyls Detected 0.0 Days 7 Days 14 Days 21 Days 30 Days

Acetaldehyde -H-+ ■f-f+ +++ +++ +++ a/Propionaldehyde + I'H ++++ ++++ ++++ ++-H* a/

AcetoneIsobutyraldehyde ++ ++ ++ ++ ++ a/Butyraldehyde ++ + + + + y

2-Butanone Trace Trace Trace Trace Trace3-Methyl-2-butanoreDiacetyl +++ -H- ++ ++ +++

Hexanal Trace Trace Trace Trace Trace2-HeptanoneHeptaldehyde +++ ++ ++ ++ +++

+ Represents the smallest relative peak height a/ Slightly less than the number of +Trace: Represents a slight hump

Relative Peak Height

p-P

III-JO:PM

-r-p.pM

I'ro&P

2*ujo&pw

Acetaldehyde

Proplonaldehyde

AcetoneTsobutyraldehyde

Hutyraldehyde

hutanone

Hexanal

3-Methyl-2- butanone Diacetyl

2-HeptanoneHeptaldehyde

68

any particular pattern in shrimp Irradiated at a dose of O .15 Mrad, in relationship to iced storage time over a 21 day period. The slight increase in the peak height which was detected at 30 days was not significant. It is highly probably that these increases represent the onset of spoil­age, but this must await further study on irradiated iced stored shrimp beyond a 30 day period.

Both the total carbonyl derivatives and the scans of those carbonyls followed the same pattern during the post irradiation Iced storage (0.15 Mrad). The undetectable changes in the irradiated sample at 0.15 Mrad, and during iced storage, are in agreement with the results of the organoleptic tests conducted cn the stored sample.

It appears that Bhrimp subjected to a dose of 0.15 Mrad showed a noticeable decrease in bacterial load. The primary factor In Increased storageability of the irradi­ated shrimp would be due to the selective action in reducing the pseudomonads, thus preventing a further increase in the formation of the volatile carbonyls during Iced Btorage.On the other hand, this dosage level would appear to par­tially inactivate tissue enzyme systems catalyzing pro­teolysis .

The carbonyl chromatographies of irradiated samples at a dose of 0.8 Mrad during iced storage followed more or less the pattern of the total carbonyl compounds, with increases in relative peak heights between 0.0 - 7 days, and slight decreases throughout the remaining period of

TABLE 10CHANGES IN VOLATILE CARBONYL COMPOUNDS DURINGICE STORAGE ON IRRADIATED SHRIMP AT 0.8 MRAD

Carbonyl 0.0 Day 7 Days 14 Days 21 Days 30 Days

AcetaldehydePropionaldehydeAcetoneIsobutyraldehydeButyraldehydeButanone3wMethyl-2-butanoneDlacetylHexanal2-Heptanone HeptaldehydeUnknownUnknown

+ + -H -

I H I t

I f f++ Trace

-H-HH-

TraceTrace

++-HH-

++++++*

+*-HH-*+*

■H-H-f * Trace

■t t H-+

Trace Trace

+++++*+-H-H-*

Trace

Trace

++++Trace

+ -H -+

++++-i 1iff

Trace

Trace

-H-H-

Trace

H i t*

++++*Miff*

Trace

Trace

+ Represents the smallest relative peak height* Slightly less than the number of +Trace: Represents a slight hump

Relative Peak Height

ouop-pUi

hii *■iwP■ 3oap •

o ;■0 pp n1 L n

m a>(t UiII o►3 1 *- \ a> n aap 0 0

< t—1in CD P l: ft

i 1*P p p30 a n p

w n; yV- ■ 04--P. -1

F rjirn i "n

r/j"11j* ■ ^

IIro . ..

\ "3P

P P .

o Pit-O&Pv t

W O

ri H ii it n n i Acetaldehyde

I II II I Propionaldehyde

AcetoneTsobutyraldehyde

butyraldebyde

’utanone

II II II I) IIII M l

i I exana 1

I II l| II II II I/ / / y y

■II 3-Me thy 1-2 -butanone

Dlacetyl

^Huwi 2-HeptanoneIleptaldehyde

0 ).

71

the study. The chromatographs also indicated the presence of a couple of components in shrimp irradiated at 0.8 Mrad but in such minute quantities as to preclude their Identi­fication .

At present, data obtained from this study insinuate that the role of carbonyl compounds in irradiation off- odors seems to be uncertain. Chemical evidence shows them to be present in both spoiled and irradiated shrimp at the same concentration (iced stored for 14 days non-irradiated shrimp, and those irradiated at 0.8 Mrad, as compared in tables 8 and 10). The irradiation odor of these samples is very definitely and unmistakably characteristic.

Spoiled shrimp is also reported by Settoon (1967) to have a high concentration of trimethylamine nitrogen 15 mg/100 g of shrimp, ammonia nitrogen 30 mg/100 g of shrimp,and very high total bacterial plate counts.

On the other hand, Merritt et al. (1966) working with four kinds of meats: veal, lamb, mutton, and pork foundthat the irradiation odor for all meats studied is the same, and the chemical composition of the volatiles is practicallythe same for all. He also reported the presence of sulfuracid aromatic compounds in irradiated shrimp. Table 11 summarizes those compounds.

Concurrently, he postulated the formation of many of these compounds from direct bond cleavage of amino acid moieties. Benzene and toluene were formed from phenylala­nine and phenol and p-cresol from tyrosine. Some of the

TABLE 11

SULFUR AND AROMATIC COMPOUNDS FROM IRRADIATED MEATS

SULFUR COMPOUNDS AROMATIC COMPOUNDS

Sulfur dioxide BenzeneCarbonyl sulfide TolueneDimethyl sulfide EthylbenzeneDimethyl disulfide PropylbenzeneMethyl ethyl sulfideDiethyl disulfideEthyl butyl sulfide

From Merritt et al. (1966)

73

sulfides, disulfides, and mercaptans can be formed directly from cystine or methionine.

Since the precursors of the above compounds are present in shrimp, the presence in irradiated shrimp of most of these compounds might have been expected. Results reported in this project do not indicate the synthesis of any of these compounds in quantities which would be suf­ficient to be considered a health hazard.

Total lipid extractOf all the samples used in this study, the amount of

total lipids extracted per 100 g shrimp ranged from 0.7 to 0.8 g.

Effect of irradiation on fatty acidsFatty acid methyl esters were identified by comparing

their chromatographic data with standards as shown in table 12. The relative amounts of each ester present were deter­mined .

1- The area of each peak:A = t . ht - retention time (distance from the leading

edge of the solvent front to the mid point of the peak v'idth at £ itB height),

h - peak height (distance from the baseline to the maximum point of the curve).

The theoretical and practical advantages of this method have been described by Burlett and Iverson (1966)

TABLE 12

RETENTION TIMES OF FATTY ACIDS FOUND IN SHRIMP AS COMPARED WITH STANDARDS

Peak Carbon Fatty Standards UnknownNumber Length Acids (min.) (min.}

1 cl4 Myristic 3.0 3.02 ii—i

O Myrlstoleic 4.1 4.13 C15 Pentadecanolc 4.5 4.54 cl6 Palmitic 6.3 6.35 cl6- Palmitoleic 7.0 7.06 C17 Heptadecanoic 9*5 9.57 C17- Heptadecanoleic 11.5 11.58 cl8 Stearic 12.4 12.49 Cl8 Oleic 13.5 13.5

10 C18=. Linoleic 16.7 16.711 Ill00rH

O Linolenic 21.6 21.6

75

and Meffered et al. (1968).R - h.t.f R - relative area h - peak height t « retention time f = attenuation factor

2- The percentage of each ester was determined:56 composition = R (lQO)

"5"where S 1b the sum of all relative areas.

The gas chromatography results have been summarized in tables 13, 14, 15 and 16. These tables include results related to the effect of irradiation on fatty acids and to the effect of storing the shrimp samples in ice over 24 days. The sequence of listing these fatty acids is based on structural similarities rather than on their retention times.

On the basis of Nawar's predictions (1968), the effect of irradiation at a dose of at least 0.8 Mrad should be evidenced by quantitative decreases in fatty acid con­centrations. Although the scope of the present investiga­tion precluded the employment of internal standards, some of the results in table 13 are in general agreement with this hypothesis.

From table 13 it appeared that palmitic as well as oleic acids are predominant compounds of fresh non-irradl- ated shrimp. Neither heptadecanoic nor heptadecanolelc

76

acidB were detected in fresh non-irradiated samples or in the irradiated ones at either dose.

A large increase of the amounts of palmitic acid obtained from the irradiated samples at 0.8 Mrad might fce expected, due to their relative chemical inertness, except for the fact that phospholipids contain about twice as much palmitic as non-phospholipids, Adison et al. (1968). Phos­pholipids are highly reactive in vitro.

Effects of iced storage and irradiationThe effect of iced storage of fresh non-irradiated

shrimp and those irradiated at 0.15 and 0.8 Mrad, are shown in tables 13 through 16. The interpretation of the results is based on the effect of irradiation per se, plus the po­tential for microbial or autolytlc enzymatic activity in these samples during the iced storage periods. The general trend shown by these results seems to be that storage has very little effect on the fatty acid composition of the shrimp. It is assumed that lipid hydrolysis did occur in these samples for it is well established by Olley and Duncan (1965) that the lipids of non-irradiated fish muscles are deesterified lri situ during chilled storage.

Whether irradiation has an effect on the rate of lipid hydrolysis or on the disorganization of the tissue, a pro­cess which accelerates the hydrolysis of lipids, has not been studied extensively.

77

ta ; - 13

EFFECT OF IRRADIATION AT TWO DIFFERENT DOSE LEVELS ON FATTY ACIDS IN SHRIMP

CarbonLength

FattyAcids

NonIrradiated

Irradiated at 0.15

Irradiated at 0.8

Percentage

Cl4 Myristic 0.42 0.68 0.94C15 Pentadecanolc 0.21 0.31 0.32

C16 Palmitic 15.80 9.38 16.11

c17 Heptadecanoic — — - -

0 0

CO Stearic 3.93 3.63 3.11

1-=

t

r-iO Myristoleic Trace Trace Trace a/

11—t

0 Palmitoleic 1.14 1.25 1.33

c17 Heptadecanoleic — —

0

t—*

00 1 Oleic 9.10 9.44 10.44

C18= Llnoleic Trace 0.56 1.00

C18= Linolenlc 0.29 0.36 0.34

a/ Less than 0.2

78

TABLE 14

FATTY ACID ANALYSIS ON LIPID EXTRACTS OBTAINED FROM NON-IRRADIATED SHRIMP STORED IN ICE

CarbonLength Fatty Acid f)ay

Percentage 12 Days 24 Days

cl4 Myristic 0.42 0.73 0.88

C15 Pentadecanoic 0.21 0.20 0.35

C16 Palmitic 15.80 13.91 15.00

C17 Heptadecanoic —

cl8 Stearic 3.93 2.93 3.14

C U - Myristolelc Trace Trace Trace a.

°16- Palmitoleic 1.14 1.62 1.62

°17- Heptadecanoleic — — --

0 t-J 00 1 Oleic 9.10 7.42 7.56IICOH

o Linoleic Trace Trace Trace

Cl8* Linolenic 0.29 0.34 0.31

a/ Less than 0.2

79

TABLE 15

FATTY ACID ANALYSIS ON LIFID EXTRACTS OBTAINED FROM IRRADIATED SHRIMP AT 0.15 MRAD AND STORED IN ICE

CarbonLength Fatty Acid 0 Day

Percentage 12 Days 24 Days

pHO Myristic 0.68 0.42 O .89

C15 Pentadecanoic 0.31 0.28 O .58

C16 Palmitic 9.38 13.89 12.80

c17 Heptadecanoic — -- —

Cl8 Stearic 3.63 4.88 3.41

cl4- Myristoleic Trace Trace Trace a.

cl6- Palmitoleic 1.25 1.14 1.43

0 H1 1 Heptadecanoleic -- —

Cl8- Oleic 9.44 10.68 9.05

ci8« Linoleic 0.56 0.79 0.98

Cl8* Linolenic O .36 0.31 0.34

a/ Leas than 0.2

80

TABLE 16

FATTY ACID ANALYSIS ON LIPID EXTRACTS OBTAINED FROM IRRADIATED SHRIMP AT 0.8 MRAD AND STORED IN ICE

CarbonLength Fatty Acid 0 f)ay

Percentage 12 Days Days

Cl4 Myristic 0.94 1.31 1.29

C15 Pentadecanolc 0.32 0.28 0.54

cl6 Palmitic 15.11 16.64 15.79

C17 Heptadecanoic — --

o i-* 00

Stearic 3.11 2.96 2.98

cl4- Myristoleic Trace Tracd Trace a,

C!6- Palmitoleic 1.33 1.52 1.63

c17- Heptadecanoleic — — —

i00 1—1u Oleic 10.44 10.83 11.09

Cl8- LInoleic 1.00 i.o4 0.98

C18- Linolenic 0.34 0.52 O .58

a/ Less than 0.2

8l

Although the current work has provided greater in­sight into the mechaniamB of irradiation induced flavor component alterations in shrimp, it is desirable that further investigations should be conducted to expand upon these results.

SUMMARY AND CONCLUSION

Freshly caught shrimp of known history (Peneaus setlferus) were used to determine the effects of low dose ionizing radiation, and subsequent iced storage of tuose products on their fatty acid and carbonyl composition.

Shrimp irradiated at 0.15 and 0.8 Mrad were compared to non-irradiated controls. Products tested for their fatty acid composition were Iced stored and analyzed after 0, 12, and 24 days. To identify the carbonyls, the samples were iced-stored and analyzed after 0, 7j 14, 21 and 30 days.

Total carbonyls were determined as mg of 2,4-dinitro- phenylhydrazone. Gas chromatography was used to tenta­tively identify individual fatty acids and carbonyls.

Results revealed that an irradiation dose of 0.8 Mrad Increased the amount of 2,4 dinitrophenylhydrazone, reflecting the increase In total carbonyls. No significant Increase was obtained with 0.15 Mrad. Carbonyls tentatively Identified to be present In fresh 3hrimp were: acetylalde-hyde, propionaldehyde, acetone, isobutyraldehyde, butyralde- hyde, butanone, 3-methyl-2-butanone, dlacetyl, hexanal, heptaldehyde, and £-heptanone. Their presence In greater concentration was noticed by the Increase in the peak heights in shrimp irradiated at a dose of 0.8 Mrad.

Concentration of identified carbonyls in non-irradiated iced-stored shrimp Increased throughout 21 days storage,

83

and then tended to fall off. However, hexanal and butanone showed less Increase In the peak height than the others.

Shrimp irradiated at a doae 0.15 Mrad contained stable carbonyl compounds during an iced storage period of 21 days, but there was a slight increase after 30 days.

The radiation-induced immediate increase in carbonyl concentrations of iced-stored shrimp irradiated at 0.8 Mrad appeared to continue during the first seven days, then tended to diminish.

Analyses of all samples used in this study showed the amount of total lipids extracted per 100 g shrimp ranged from 0.7 to 0.8 g. Myristlc, myristoleic, pentadecanoic, palmitic, palmitoleic, stearic, oleic, linoleic, and lino- lenic fatty acids were tentatively identified from lipid extracts in both the non-irradiated and the irradiated samples. Palmitic and oleic acids appeared to be predomi­nant in all of the samples.

The predicted radiation-induced decrease in fattyacids was not detected for all of the fatty acids identified.

Analytical results indicate that iced storage of both irradiated and non-irradiated shrimp had minor effects on the fatty acid composition of the samples.

The invariability in fetty acid composition of irra­diated, non-irradiated, and iced-stored shrimp requires further study on the fatty acid composition of lipid classes such as sterols, phospholipids, triglycerides, and others, and on the effects of ionizing radiation on each of these classes.

84

A slight modification in the eaterlfication method must be considered to prevent the loss of volatile fatty acids.

Low dose gamma irradiation at 0.15 Mrad proved to be a superior method for the extension of the shelf life and the preservation of the chemical, physical and organoleptic qualities of iced-Btored shrimp over a period of 25 days. This dose maintained the good quality by stabilizing the chemical composition of shrimp.

Doses in excess of 0,15 Mrad would increase the shelf life even further, but the undesirable radiation-induced chemical changes in the product would create consumer acceptability problems.

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Oh

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a p p e n d i x

Figure 9.

Chromatogram of

volatile carbonyls

in non-irradiated

shrimp.

Peak Height

Acetaldehyde P ropi onaldehyde

AcetoneIsobutyraldehyde

Butyraldehyde

Butanone3-Methyl-S-Butanone Diacetyl

Hexanal

2-HeptanoneHeptaldehyde

Solvent

96

Figure 10.

Chromatogram of

volatile carbonyls

in shrimp

irradiated at

0.15 Mrad.

Peak Height

3 Acetaldehyde 3 Propionaldehyde

AcetoneIsobutyraldehyde

Butyraldehyde

Butanone3 -Methy1-2-Butanone Diacetyl

Hexanal

2-Heptanone Heptaldehyde

] Solvent

66

Figure 11.

Chromatogram of

volatile carbonyls

in shrimp

irradiated at

0.8 Mrad.

Peak Height

AcetaldehydePropionaldehyde

AcetoneIs obutyraldehyde

Butyraldehyde

Butanone3-Methyl-2-Butanone Diacetyl

Hexanal

H*3(C S-HeptanoneHeptaldehyde

J Solvent

001

VITA

The author was b o m In Damascus, Syria on June 6, 1938* She was graduated from A1 Wataniya High School in 1958, and In June, 1961 obtained her B.S. in Agriculture, with a minor in Chemistry, from Cairo University. The author joined the Ministry of Agriculture in Damascus as Chief of the Chemistry Section, where she worked as a research counterpart to an F.A.O. Nutrition and Quality Control expert.

She entered the Graduate School of the Louisiana State University in September of 1963* and was awarded her M.S. In Food Science In August, 1985* Mrs. Ismail Joined Tracor Inc., Baton Rouge, as a research chemist, and in September, 19^7 re-entered Graduate School, where she has been full-time instructor in the Feed and Ferti­lizer Laboratory at Louisiana State University since 1968,

She was married to Nahed M. Ismail of Beirut, Lebanon in 1963, and they have been blessed with two children,Rana and Marc.

The author Is now a candidate for the degree of Doctor of Philosophy In the Department of Food Science at Louisiana State University.

EXAMINATION AND THESIS REPORT

Candidate:

Major Field:

Title of Thesis:

Raja Jaffan Ismail

Food S cien ce

"R adiation Induced A lte r a t io n s In F lavor Components In Peneaus s e t i f e r u s .”

Approved:

jl, 3 YUnfaAMajor Professor and Chairman

Dean of the Graduate School

EXAMINING COMMITTEE:

»flP A

O - s.±

Date of Examination:

A p ril 1 , 1971