. FISHERIES AND MARINE SERVICE

Translation Series No. 3117 ,4,/ctJuip-tô

The effect of decomposed products of triméthylamine oxide on quality of frozen Alaska pollack fillet

by Toshio Tokunaga

,

Original title: Reito Sukerodara no Hinshitsu ni oyobosu Torimechiruamin okisaido Bunkaibutsu no Eikyo

From: Nihon Suisan Gakkai.7Shi(Bulletin Of the Japanese Society of.Scientific Fisheries), 40(2) : 167-174, 1974

Translated by the Translation Bureau(JO/PS) Multilingual Services Division

Department of the Secretary of State of Canada

Department of the Environment Fisheries and Marine Service'

Halifax Laboratory - Halifax, N.S.

1974

15 pages typescript

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The effect of decomposed products of trimethylamine oxideon quality of frozen Alaska pollack fillet

TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS)TITRE EN LANGUE ETRANGERE ( TRANSCRIRE EN CARACTÉRES ROMAINS)

Reito Sukerodara no Hinshitsu ni oyobosu Torimechiruaminokisaido Bunkaibutsu no Eikyo

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Bulletin of the Japanese Society of Scientific Fisheries

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The Effect of Decomposed Products of Trimethylamine Oxide on Quality of Frozen Alaska Pollack Fillet e l Bulletin of the Japanese Society of Scientific Fisheries, Vol. 40, No. 2, pp. 167-174, 1974

By TOKUNAGA, ToshiP

Received: October 2, 1973

LU

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Formaldehyde (FA) begins to form in the fish when

they are kept frozen. Its amount, however, varies from fish

to fishl) . It is well known2) that the major part

is. formed along with dimethylamine (DMA) as products of

enzymic degradation of trimethylamine oxide (TMAO). The

rate of TMAO degradation during frozen stora9e is rapid, es-

pecially in the gadoid fish family, and, as pointed out

before l) , it is possible that FA formation may be one of the

causes of a rapid reduction in the extractability of the

muscle protein. Castell et. al. 3) have also carried out a

frozen storage experiment on nine species of fish caught in

1. 1 -Achievements by Tokai Regional Fisheries Research Laboratory B - 586. Summary of this study was reported at the fall meeting of the Japanese Society of Scientific Fisheries

* (Kochi City) in October, 1c72. 2Tokai Reg. Fish. Res. Lab., Kachidoki, Chuo-ku, Tokyo.

the Atlantic Ocean, and reported that TMAO degradation

advanced in the gadoid family only. They further reported

that even in the gadoid family, there was a difference in

the rate of degradation depending on the species. And, in

general the fish with a rapid TMAO degradation tended to have

a marked protein insolubility.

In this paper we have tried to find out the effect

of storage temperature on the rate of TMAO degradation, and

to find out to what extent the degradation products are

related to the change in muscle protein extractability,

using Alaska pollack (Theragra charcogramma) as sample .

Using samples obtained during different seasons and at

various places, we also studied whether TMAO degradation

rates differ in individual fish or fish parts. Further, the

relationship between these differences and the extractability p.168

of muscle protein was examined.

The quality of frozen fish fillet can usually be

assessed by taking into account many factors such as firmness,

water retaining quality, flavour, colour or muscle protein

extractability, etc. However, in this paper we have

limited the study only to the changes in protein extracta-

bility.

Method of experiment

Sample fish: The origin and treatment of the Alaska pollack

samples that were used for this study, are described in

each of the following experiments.

3

Methodofariabrsis DMA-N and TMA-N were measured with regard to the

trichloride acetic acid solution extracted frail -11e sample, etlpioying

the copper-dithiocarbamate methodl) and the picrate method5) respectively.

FA was measured in the same extractives by theNashmethcd) .

As to actomyocin, 0.6M KC1 (adjusted to pH 7.2 by NaHCO3)

extractives of the samples were diluted in 10 times cold

distilled water, sedimented and the protein content in the

sedimènt was measured by the biuret method? ) .

Results and observation • Effect of storage temperature on TMAO degradation in minced meat:

It has been clearly indicated in the previous

report 8) , as well as in a recent study conducted by Babbitt

et. al. 9) , that DMA formation in minced muscle was much more

rapid than in fillet , when the muscle was minced and kept

frozen. We used minced muscle here in the belief that the

effect of storage temperature on TMAO degradation would

appear more clearly in such a sample.

Ten very fresh Alaska pollacks,that were caught by

dragnet off the coast of Onahama in Fukushima Pref. in

February 1971, were used as samples. They were skinned,

deboned, and made into fillets, and then separated into

ordinary and dark meats. The ordinary and the dark muscles

from the ten fish were chopped separately i sealed in plastic

bags, stored at different temperatures of -5°C, -10 °C, -20°C

and -40°C, and used for the analyses according to needs. The

results obtained for ordinary meat are shown in Fia.1-a, ordinary meat are shown in Fig.1-a, and

4

for dark meat in Fig. 1-b. Depending on the storage

temperature, DMA and FA formations accompanying TMAO degra-

dation were observed even in the ordinary meat. It was most

marked in the sample stored at -100C. With the sample stored

at -50C, the formationsof DMA and FA was quicker than in the

one at -109C for the first two days, but after the third day

the increase was slow. The change at -20°C was very small (169

and at -40°C the formation of DMA and FA were hardly noted.

With the sample stored at -10°C, the degradation products

showed their maximum level after 20 days and stayed there.

DMA-N and FA in this case were 16 to 18mg/100g and 13 to _

16mg/100g respectively, and the detected level of FA was much

lower than the value estimated (34 tc 38mg/100g) from DMA-N.

TMAO degradation in dark meat occurred very rapidly

compared to that of ordinary meat. FA level in dark meat

stored at -1000 reached the maximum of about 30mg/100g as

early as aften124ys storage. The change in DMA-showed

a similar pattern, but even after a rapid formation it

tended to increase gradually as the storage period prolonged.

The change in dark meat stored at -5°C was small compared to

that at -100C. However, there was no great difference in

ordinary muscle.

It has been reported6)

thet etorege temperature

greatly influences the degradation rete of TMAO in frozen

Alaska pollack. The reetat obtained in that report ehowed

that within the range of .2 9Ç to 4eço the lower the

5

temperature the less the degradation of TMAO. However, the

experiment carried out with minced muscle showed the maximum in

formation of DMA and FA/à sample stored at -10°C and not at

-5°C. This difference was more noticeable in minced

ordinary meat. At -5°C the FA level increased rapidly at the

beginning of the storage and it rather decreased after that.

Could it be that the amino acids of FA and protein combined

relatively easily at this temperature, and thatas a result

a tendency to slow down appeared in the formation of

FA? However on the other hand, the change in DMA shows that

its formation was not substantial, and it is a fact that at

-5°C storage TMAO degradation was rapid at the very beginning

of the storage but that later its progress was comparatively

slow. The disparity between the results reported previously

and the results obtained with this experiment can be

explained by the different forms of samples (previous report

used meat in 3 cm square pieces). That is, it is presumed

that the enzymes contained in a living body are generally

stable so long as they exist in a perfect structure, but in

a destroyed structure the loss of enzymic activities is

easily promoted by denaturation or by air oxidation etc.

Especially under very adverse freezing conditions at around

-5°C the loss of this sort of activity is further accelerated,

and enzymic degradation of TMAO may almost stop at the latter

half of the storage period. In the case of the storage at

-20°C, an increase of DMA and FA was observed even though it

6

was very slight; but at -40°C there was no change. At this

temperature it seems that enzymic action of-TMAO degradation

almost completely stops.

Like in many other fish, the formationsof DMA and FA

was far greater in the dark muscle of Alaska pollack than

that in the ordinary muscle, clearly indicating that there is

a concentration of degradative enzymes of TMAO in this

structure. However, the changes in TNA-N content in the dark

muscle, as shown in.Table 1, are minimal compared to those in

DMA-N, and the maximum change was less than 1mg/100g. This

is in quite a contrast with the.fact that TMA formed in the

dark muscle of many other fish, such as mackerel, bonito,

tuna etc. during their storage at -6aC, was in excess of the

amount of DMA10). A reasonable interpretation would be that

the enzymes related to the reductive reaction of TMAO-=TN]A

in the dark muscle of Alaska pollack are largely absent.

The significance of these characteristics in -

the dark muscle of this fish is not clear. Like many other fish,

. formation of TMA in the ordinary muscle during the frozen

storage of Alaska pollack was hardly observed.

Changes in TN^A.O degradation and muscle protein extractability:

Alaska pollack is one of the fish that are easily

denatured during frozen storage. It is well known that its

denaturation rate is greatly influenced by its temperature

during storage. As clearly seen from the results given in

the previous report 8) , and also the above-reported

7

results, the degradation rate of TMAO is greatly affected by

storage temperature, and FA formed at the same time has a

very strong denaturation action on protein. There is, (170

therefore, some relationship between TMAO degradative reaction

and muscle protein extractability.

Thus, while the fillets of Alaska pollack were stored

at various temperatures, their muscle protein extractability

and DMA content were compared and the changes were watched.

Among the degradative products of TMAO the one that acts on

protein is really FA. However, as noted from the test results

stated before., there was a variation in the value of FA.

Moreover, under certain conditions, FA that was produced

began to decrease. This makes a btudy of the proper rate of

degradation very difficult, even if FA_is measured. Therefore,

in this study the change in DMA-N, that is the other product

of TMAO degradation, was measured.

Alaska pollacks that were used for the experiment

were purchased at Tsukiji Central Market in March 1971. They

were fairly fresh. The dark muscle was completely removed, and

filleted for use as samples. All the fillets were frozen

overnight at -40°C, after which they were transferred to

storage under -5°C, -10°C and -20°C temperatures. Some,

however, were left at -40°C. Three fillets were taken out

from each temperature group as samples for any singleto

analysis. The results are shown in Fig. 2-a/d. Although DMA

formation in each of the samples stored at -SoC and'-10 0 C

8

showed marked individual differences, the graphic line

joining the average values revealed a definite increase, in

sharp contrast with the decrease in protein extractability.

In the experiment using minced ordinary muscle, the degra-

dation rate of TMAO was markedly greater at -10°C than that

at -5°C while there was hardly any difference in the case of

fillets at both temperatures. After 8 to 10 days DMA-N in

the samples reached a level of about 5mg/100g and more than

80% actomyocin becate insoluble. &t -20°C storage, an

irregular fluctuation was observed until after 20 days,

apparently due to individual differences, but, on the whole,

there was little decrease in protein extractability and in

formation of DMA. However, after that period the changes in

both became very apparent, and after 72 days DMA-N reached an

average of 8.5mg/100g. In the samples stored at -40°C, there

was no change either in DMA or protein extractability during

the whole period of the experiment. Although the DMA levels

in each of the three samples used in one analysis showed

wide fluctuations, the sample with the maximum level of DMA

had the lowest protein extractability. This clearly

suggested that TMAO degradaticn product (essentially FA) has

a strong effect on muscle protein.

Correlation between DMA level and Protein extractability: (172

In the previous experiment, using fillets, it was

observed that DMA contents differed greatly in individual

samples. In order to clarify the extent of individual

9

differences, therefore, DMA levels and muscle protein

extractability were measured in each sample using a number of

Alaska pollacks from the same school. Individual differences

in those samples as well as their correlation were then

studied.

Samples were grouped as Samples I and II--the 30

Alaska pollacks purchased at Tsukiji Central Market as Sample

I; and the 30 Alaska pollacks caught off East Kamchatka in

June 1971 as Sample II. Sample I was all in the form of

fillet , and stored at -10°C. After two weeks,only the

ordinary muscle was collected from the centre of the back, and

DMA content and protein extractability were measured. Sample

II was frozen on board, immediately after the catch, at

-30 00 and kept for two weeks at that temperature. At the

time of the experiment they were in a frozen state. They

were then sliced into twos by an electric saw along the

spine. One side was stored at -10°C, and the other at -20°C.

The group at -10°C was analysed after two and eight weeks,

and the other group, at -20 °C, was analysed after eight and to

nineteen weeks. The results are shown in Fig. 3-aie. Sample

I, stored at -10°C for two weeks, had DMA-N from 1.74

(minimum) to 7.28mg/100g (maximum), actomyocin 41.8 to 0.8mg/g

muscle, showing fairly wide distribution. However, a high

correlation of r= -0.7712 was observed between them. Sample

II, stored under similar conditions, also showed an even

distribution of DMA-N from 0.84 to 4.25mg/10%,and actomyocin

1 0

from 58.8 to 5.4mg/g muscle. This also showed a high

correlation of rug -0.8304. On the other hand, when the

absolute amount of DMA-N formed in group samples I and II was

compared, all except one sample in the former had more than

2mg/100g--the maximum being 7.2mg/100g. In the latter group,

five samples had less than 2mg/100g--the maximum being only

4.3mg/100g. On the average DMA content in sample I was fairly

high compared with sample II, showing that it is probable that

different schools have different DMA producing ability. When

sample II was stored at -10°C for the longer perioud of eight

weeks, DMA-N showed a large increase of 2.4 (minimum) to

11.3mg/100g (maximum)--averaging 6.7mg/100g. Actomyocin

decreased markedly, and in many samples it became less than

5mg/g muscle. When DMA-N exceeded roughly 7mg/100g, protein

extracted as actomyocin did no longer seem to exist. The

results of samples II, stored at -20°C for eight weeks and

nineteen weeks, also showed strong correlations (r= -0.7367

and r= -0.8354 respectively) between DMA-N content and

protein extractability.

rossibilitY of quality judgement of frozen Alaska pollack and other gadoid fish fillets by DMA-N contents

When the previously stated results obtained by storing at

Alaska pollack fillets/temperatures from -5°C to -40°C for to

different periods (Fig. 2-a/d), are arranged as in Fig. 4,

they clearly show that regardless à storage conditions, the

extractable protein decreaseà as DMA increases. In these

11

samples, whe'n DMA-N reached levels around 2.0 to 2.5mg/100g,

there was a decrease in extractive protein equivalent to about

50% of that in fresh fish; and when it exceeds, that much more

insolubility is observed. On the other hand, from the results

obtained by studying individual differences in DMA content and

protein extractability in samples I and II, a similar tendency

is observed. For instance, the majority of fillets in sample

II that were kept at -20 °C for eight weeks (Fig. 3-d), showed

great protein extractability when DMA-N was less than 2mg/100g.

From these results it seems possible to derive the muscle

protein extractability of frozen Alaska pollacks by measuring

DMA-N content. Also, since this extractability is one of

the important quality, indices, thore is a great possibility

of utilizing DMA value as a standard for judging the quality (173

of Alaska pollacks and other gadoid fish fillets. However,

in the samples (Fig. 3-e) stored at -20 00 for 19 weeks,

there were many fillets where protein extractability became

very low even though DMA-N was less than 2mg/100g--

indicating that when they are stored for a long period

there is another factor affecting the denaturation process.

Differences in changes in DMA formation and protein extractability in different parts of fish:

Considering the results described before, if DMA

formation is different in different parts of the same fish,

then there should be a difference in the changes in protein

extractability in different parts. So far many studies

12

have been carried out on denaturation due to freezing of

gadoid fish, but none has examined the differences in

denaturation rates in different parts of a fish. We have

conducted the following experiment in order to clarify the

points mentioned above. Fresh Alaska pollacks, caught off the

coast of Abashiri in Hokkaido in June 1971, were used as

samples. They were frozen on the spot at -30°C, and

transported by a freezer truck, at around -20 °C, to the

laboratory. This took three days. These 11 fish were

wrapped whole in polyethylene bags and stored at -10°C. They

were analysed-after two weeks and two months. Muscle used

for the measurement was collected from three places: the

back, the stomach and near the tall. The results are shown

in Fig. 5-a and b. With the samples stored at -10 °C for

two weeks DMA-N was as follows: in dorsal muscle DMA-N was

1.24 to 3.17mg/100g, averaging 1.78mg/100g. Its distribution

width as well as average value was comparatively low. On

the other hand DMA-N in the belly muscle fluctuated widely,

showing 2.84 to 10.74mg/100g. Its average value was the

highest. DMA-N in caudal muscle was in between those two.

The change in protein extractability decreased in inverse

proportion of DMA content of dorsal muscle, caudal muscle

and abdominal muscle in that order. Increase in DMA was

noticed in all parts of the samples stored at -10 °C for two

months. However, it was, likewise, the greatest in the abdominal

muscle. At this point the decrease in protein extractability

13

in all parts was marked, and the remnants of actomyocin were

hardly observed. '

These results show that DMA formation as well as the

rate of protein denaturation are different in different

parts, even though they are all from the ordinary muscle of

the same fish. The cause of these differences may have some

connection with the following: As this study, the report by

Castell et. al. 3) and the previous reportn) clearly indicate

DMA formation activity is very strong in dark muscle, •

therefore the effect of the dark muscle enzymes 2) on

tue ordinary muscle and of the visceral enzymes on abdominarmuscle

may be great. -Considering that the samples used here were frozen whole, with the storage temperature at -10°C

which is relatively high and uroduces slower freezing, it

is presumed that TMAO degradative product that formed rapidly

in dark muscle and viscera had a chance to penetrate

gradually into ordinary muscle. In general, the ratio of

dark muscle to ordinary muscle is greater around the tail of

the fish, and since there is only a thin membrane separating the

.abdallimal. muscle and viscera it can be assumed that the effect

of the enzyme appears to be very great. For these reasons,

if the object is the ordinary muscle only, changes in DMA

formation and protein insolubility don't seem to be great in

different parts of the fish. However, we shall study this

later. It has already been reportedll) that the removal of

dark muscle is effective in quality stabilization of frozen

Alaska pollacks, and further when tnaating fish it seems

14

necessary also to avoid contamination of ordinary muscle by

enzymes in viscera or TMAO degradative products.

Summary(174

The study was done on the effect of storage tempera-

tures on TMAO degradation and the effect that TMAO

degradative products have on muscle protein extractability in

Alaska pollack, samples. The following results were obtained:

1. Ordinary muscle and dark muscle were minced and stored

at -5°C to -40°C. The production of DMA was observed as

being maximum at -10°C. TMAO degradation in dark meat was

fairly rapid and after 12 days at-10°C DMA-N reached 30mg/100g,

but during this period TMA did not increase much.

2. When fillets were frozenat -5°C.. to -40°C, protein

extractability decreased with DMA-formatiori; but at -40°'C,

DMA amount as well as protein extractability did not change

at all.

3. There was a large fluctuation in DMA formation in each

fillet and its correlation with protein extractability was

very high--r- -0.7712 to -0.8354.

4. When DMA-N exceeded 2.0 to 2.5mg/100g, decrease in muscle

protein extractability became striking. This relationship

makes it possible to make a quality

judgement of Alaska pollack by measuring the DMA level.

5. When fish was frozen whole, and the changes in DMA level

and protein extractability were compared in different parts,

formation of DMA was in the belly muscle, caudal muscle and

1 5

dorsal muscle--in that order. And the decrease in protein

extractability was in inverse ratio.

Thanks are due to Mr. Okada and Mr K. Miwa at this

institute for their guidance and advice. Also we thank Mr.

T. Hagiwara of Taiyo Fishery and Mr. N. Yanagiuchi of the

Fisheries experimental station in Fukushima Pref. as well as

Mr. M. Furuta of All Japan Frozen Fish Association for their

help in aquiring samplesi

Bibliography

1) Tokunaga, Toshio: Hokusuiken Hokoku (Hokkaido Fisheries Research Laboratory Report.), No. 29, 108-122, 1964.

2) Amano, K. and Yamada, K.: Bulletin of the Japanese Society of Scientific Fisheries, 29, 695-701, 1963.

3) Castell, C. H.: J. Fish. Res. Bd. Canada, 28, 1-5, 1971.

4) Dyer, W. J. and Mounsey, Y. A.: Bulletin of the Jap. Soc. of Sci. Fish., 6, 359-367, 1945.

5) Hashimoto, Y. and Okaichi, T.: Bulletin of the Jap. Soc. of Sci. Fish., 23, 269-272, 1957.

6) Nash, T.: Biochem. J.„55, 416-421, 1953.

7) Umemoto, S.: Bul. of the Jap. Soc. of Soi. Fish., 32, 427- 435, 1966.

8) Tokunaga, T.: Hokusuiken Hokoku (Hokkaido Fisheries Res. Lab. Report), No. 30, 90 -97, 1965.

9) Babbitt, J. K., Crawford, D. L., and Law, D. K.: J. Agric. Food. Chev., 20, 1052-1054, 1972.

10) Tokunaga, T.: Bul. of the Jap. Soc. of Sci. Fish., 36, 510-515, 1970.

11) Castell, C. H.: J. Am. Oil Chemist's Soc., 48, 645 -649, 1971.

(1-1,) emeed dark muscle.

so

I 7/ «e r fi. --g â 1 • ■ I

Time in storage 26 days 47 days

DMA-N TMA-N DMA-N TMA-N

29.94 0.-60

36.00 0.61

12.04 0.27

Storage temperature

—5°C —10°C —20°C

0.49 0.52 0.28

28.15 30.98 9.41

( 1 6 8

1,

10

30 Dore in enrage

Fig. 1. Formation of DMA and FA in minced ordinary (1-a) and dark (1-b)znascle of Alaska • pollack during frozen storage at —5°C (0), —10°C (0), —20°C (0), and —40°C (4)), respectively.

10

• Table .1, Formation of DMA and TMA in dark muscle (mg/ 100 g). (16 9

.."

i 8

• ea

ico

so 1

40 Dail in storage

40 60

"Qt4

1

( 17 0

Fig. 2. Change in DMA—N and protein extractability in Alaska pollack fillets during frozen • storage at —5°C (2—a), .-10°C (2-b), —20°C (2—c) and —40°C (2—d).

• (171 L• 0,3

C5-s) 7.-0,7712

5.

34

3

• E 2

• • t

5. 8 Z

i; E 6

e

• • •

•

40 50 60

4 4

-a

2 . 2

10 20 30

• • 13-61

r=-0.8304

•

•

• 10' 20" 30' 40 50 60'

Actou,s,sin (cme4muscle)

4 •

10 20 30

•Actocnyosiu (mglg muscle _

4

-a 8 A

2

• i3-d) • -Om?

•

• • • 41

• ,

•• • •

•• • • • a •

(3-e) t'..-02354

•

• • • ,••• 10

•* • • • •

a

•

2 1 40 50 so 100

Actomyosia (meil muscle)

3

. Z

2

20 40

Actomyosin (roes muscle)

Fig. 3. Relation between DMA—N contents and protein extractabilities of frozen Alaska pollack fillets. (3—a): The fish purchased at Tokyo Central Fish Market were filleted and stored at

• —10°C for 2 weeks. (3-1, ,..e): The fish caught off Kamchatka were stored at —10°C for 2 weeks (3—b), at

—10°C for 8 weeks (3—c), at —20°C for 8 weeks (3—d), and at —20°C for 19 weeks (3—e), respectively.

12

3

Cb

•O

G e C

% Oà e O m m

OmmO•

e iP

mm• •••

W •

• • •

20 40 60 &)

Actnmyosia (mgfg muscle)

Fig. 4. Relation between levels of DMA-N andactomyosin in frozen fillets stored under variousconditions. This figure is arranged from the data 'given in Fig. 2-a- d.

(172

(173

M

r-••:DNLI

p: Actom)rosin q

i _._.._-^s^e ' Y__• _• }J^

• -^ . W...^â29A0o--,:_^-.9.0^

10

I.

.Docsrnl Eeuy ' Caudal

muscle muscle muscle -

Fig. 5. Formation of DMA and insolubilization_of.__.._. -

protein at different parts of body during storage

at -10°C for 2 weeks (5-a) and 2 months (5-b),

iespectively.