/ CI-IIV 1,7", S

FISHERIES RESEARCH BOARD OF CANADA

Translation Séries No. 2528

Retardative mechanism of protein denaturation by addition of saccharides during cold storage of minced fish meat (Surimi)

I. Behaviour of electrolytes in sucrose solution

by Eiji Niwa, Bunji Mon, and Masato Miyake

Original title: Torui ni yoru surimi no toketsu hensei yokusei kiko I. Shoto yoeki chu ni okeru denkaishitsu no kyodo

From: Nihon Suisan Gakkai-Shi (Bulletin of •the Japanese Society of Scientific Fisheries ), 39(1) : 61-67, 1973

Translated by the Translation Bureau(JW) Foreign Languages Division

Department of the Secretary of State of Canada

Department of the Environment Fisheries Research Board of Canada

Halifax Laboratory Halifax, N. S.

1973

19 pages typescript

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ISSUE NO. NUMÉRO VOLUME

1 39 1973

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E. Niwa, B. Mori and M. Miyake.

TITLE IN ENGLISH - - TITRE ANGLAIS

Retardative Mechanism of Protein Denaturation by Addition of

Saccharides during Cold Storage of Minced Fish Meat (Surimi).

Behaviour of Electrolytes in Sucrose Solution. TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÈRES ROMAINS)

Torui ni yoru surimi no toketsu hensei yokusei kiko 1. Shoto yoeki chu ni okeru denkaishitsu no kyodo.

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Nichi sui shi (1) 61 - 67 (1973)

REFERENCE IN ENGLISH RÉFÉRENCE EN ANGLAIS

Bulletin of the Japanese Society of Scientific Figheries 39 (1) 61 — 67 (1973)

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PERSON REQUESTING DEMANDÉ PAR

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165280 Japanese JWC APR 2 4 1973

-eRetardative Mechanism of Protein Denaturation by Addition of Saccharides ' during Cold Storage of Minced Fish Meat (Surimi)—I..

Behavior of Electrolytes in Sucrose Éolution . .

Eiji NIWA, Bunji MORI, and the late MaSatO MIYAKE*

(Received 6 September 1972).

• , To clarify the retardative mechanism of 'protein denaturation by the addition of . saccharides during cold storage of minced fish meat (Surimi), the behavior of electro- -

lytes in sucrose solution was studied by the electrochemical method and the following results were obtained.

' - 1) The electrical conduction of electrolytes in sucrose solution is small in com- parison with that of electrolytes in water. This is caused by the decrease in the mig-ration of ions.

2) The degree of dissociation of electrolytes in sucorse solution is exactly equal - to that of electrolytas in water.

• 3) The relation between conductivity and viscosity of the solvent, as to the sucrose solution, follows WALDEles rule.

4) The denaturation of actomyosin.during storage under various circumstances is retarded also by the addition of polyethylene glycol (PEG).

* Faculty of Fisheries, Prefectural University of Mie, Tsu, Japan.

'SOS-200-10-91

UNEDITED TRAI",.LATION

rot informon only

TRADUCTION NON W.:NISEE

Iniemalion slr.ent 7530-21-029-5332

'2

Neutral organic solutes such as saccharides and P~l

polyhydric alcohols are known to retard the denaturation of

frozen surimi during storage . It has been suggestedl'~ that

these substances interfere with the interactions between the

protein molecules and the water molecules, or that the y

3interfere with the motion of the protein molecules , but the

detailed me!:~hanism is not yet clear* However salted surimi

is very much more easily denatured than is unsalted purimij

and the reasons for stability are of g)?eat interest .

From the chemical point of view, the denaturation of

surimi is a chemical reaction involving the partial 4eqompo~-

sition of a supersaturated solution of .actomyosin in a

solution of sodium chloride .

It has long been known that the speed of re4pt~qns

occurring in solutj:on may be greatly affectedby the physical

and chemical properties of the solvent and by the electrolytic

properties of widely used mixtures of -neutral organic solvents

with water* It is known that in some solvents-an increase in

the reaction may be accompanied by a reduction in ponductiv .ityt

in ion-pair formation, or in ionization . Yasui et a15 have

suggested that the effectiveness of neutral organic solutes

in restraining denatiiration is fundamentally due to reduction

in reaction rate produced by reduced polarization, but it is

probable that other effects also contribute to denaturation.

It is also known that denaturation may be supptessed by the

3

addition of amino-acids which increase the polarization of

the solution: 6, so that there is some doubt whether the

suppression of denaturation by saccharides and by amino-acids

#can have similar explanations. In the work reported here

the denaturation of surimi during frozen storage is treated

as a chemical reaction. The mechanism by which neutral

organic solutes such as saccharides and polyhydric alcohols p62

prevent.denaturation was related to an investigation of the

behaviour of electrolytes in saccharide solutions.

Experimental methods.

Measurement of relative conductivity.

A Toa-Denpa conductivity meter type CM-2A was used.

This is an AC bridge with accuracy t 4/, sensitivity ± 0.3%

and electrodes immersed in the solution. About 25m1 of the

solution to be measured was placed in a50m1 beaker and

measured after it had been left for 30 minutes in a temperature--

controlled bath at 25 ± 0.010C. The water used for conductivity

measurement was distilled water which had been further puHfied

with an ion exchange resin.

Visc.osity measurement.

Viscosity was measured in an Ostwald viscosity meter,

with capillary length 12cm and a flow time for water of

25 ± 0.2 seconds. The flow time of the solution under test

# Summaries of papers given to the General Meeting of theJapanese Society of Scientific Fisheries, p227 (1972. 4).

wae measured after it had been left at constant temperature

. for 10 minutes. The viscosity relative to water was calculated ccroi

from the densityflow time, relative to water, for each sample.

Reagents.

The sucrose and all electrolytes used for the

measurement of conductivity were of special quality. The

acetic acid used in measuring the degree of dissociation was

also of special quality, and was further purified by

distillation with a 30cm high column.

Results.

The conductivity of small-molecule electrolytes in

sucrose solution.

The concentration C of a strong electrolyte in

solution in water, the equivalent electrical conductivity•

and the equivalent conductivity at . infinite dilution are

connected by the Kohlrausch relation

in which k is a constant.

In order to study the behaviour of electrolytes in

•a solution of sucrose, the relative electrical conductivity

of a number of 1 - 1 electrolytes was measured at various

concentrations in a solution of sucrose. The equivalent

conductivity for each was obtained and plotted,graphically

according to equation 1. The equivalent conductivity at

(1)

5

0.1 0.2 0.3

130

120

e m

o

U

00

a

a 90

I . 0.0

jConeentration(Equiv. per Liter)

Fig. 1. Relation between concentration of sodium chloade and the equiva-lent conductivity in aqueous solution of sucrose

infinite dilution was then obtained by extrapolation.

Figure 1 shows the results obtained with sodium chloride.

Sucrose solutionswith concentrations up to 10% (w/v) were

used to determine k. Other electrolytes gave exactly the same

type of results, and the equivalent conductivity at infinite

dilution of these electrolytes in a 10% sucrose solution is

shown in the second column of Table 1. For comparison the

experimental values of the conductivity at infinite dilution

in aqueous solution are shown in the third column.

Since the conductivity is proportional to the product

of the dissociation of the electrolyte and the sum - of the

mobillties of each of its ions, it may be supposed that the

decrease of conductivity in sucrose solution is in some way

Table 1. Equivalent conductivity at infinite dilution of various électrolytes (;1') in âqueoussolution of 10i° sucrose at 25°C and the relation to viscosity of the soh•ent (71r).

Electrolyte

HCl

HBr

LiOH

: LiCI

LiBr

LiOAc

NaOH

NaCI

NaBr,

NaOÀc

KOH

KClKBr•

KOAc

d° in sucrose soln ' d° in. water(cm^ equiv.-1 ohm-') (cm" equiv.'1 oh-m-1)

351.0 426.0

349.8 - _'- _432:0•

74.0

99-2,

92.1

62.6

85.0

105.2 .

101.271:6 ..: ï.;..

108:0

118.0-

118.0 -

88.0

c

234.0

121.0

117.:0

80.0

247.0

126.0

129.5

91.1

271.0

149.0.

154-0

114.4

K .at W-a.LDEN's rule*

-959.8

S 6.9

128.6

120,£>

82.0

111.3

1S7.8

132.5

93.7

141.4 •

154.4

154.5

115.5

* K was given by K=d° in sucrose solnxi7,. (1.310 at 10% sucrose soln)

Table 2. Application of ,KoxLxAUSCH's.law (law of independent migration of ion)to various electrolytes in aqueous solution of 10°C sucrose. The figure in

parenthesis shows value in water.

.. . • rl°oH-li'ct !i°cl1A.°8r '. ..:.

'

11°.cl1àpAaIon

_ . (cmR equiv.-1 ohm-' .

,

(cm2 equiv.-a ohm YI (=2 tqniv.-1 ohm-'

H 1.2 ( 6.0):° . - ( - )

Li -24.2 (113.0) 6.1 ( -4.0) . 35.6 ( 41.0)

Na -20.2 (121.0) 4.0 ( -3.5) 33.6 ( 34.9)-

K -10.0 (122.0) 0.0 ( -5.0) ' - 20.0 ( 35.0)

°H-!i°LI/i 1i°LI-!i°Na • 1^°^a-Ii°SIon

(cm2 equiv.'i ohm-') (cm2 equiv.-1 ohm-') (cm2 equiv.-1 ohm-')

OH - ( - ) -11.0 (-13.0) • -23.0 (-24.0)

C1 252.8 (305.0) -7.0 (- 5.0) -12.8 (-23.0)

Br • 257.7 (315.0) -9.1 (-12.5) -16.8 (-25.0)

^-OAc - ( - ) - -9.0 (-11.0) . -16.4 (-23.3)

7

due to a decrease in the mobility of the ions. Since

Figure 1 shows that equation 1 is satisfied, it was

thought necessary to confirm that the decrease in conductivity

in sucrose solution with concentrations up to 10% is

principally due to decrease in ionic mobility.

The rule for independence of the mobilitï of ions in

a sucrose solution.

The ions of an electrolyte in solution in water

conduct electricity independently, and the conductivity of

the electrolyte is made up of the total of the conductivities

of each ion (the mobility of totally dissociated substances).

In order to investigate whether this law is preserved in a

10% sucrose solution, we investigated whether or not the

limiting equivalent conductivity of each ion is independent

of other ions which may be simultaneously present. Thus

it was found that when for example sodium and potassium p63

compounds were used, the difference between the conductivities

of the sodium and potassium compounds with the same anion

did not depend on the anion used.

* When the saine anion X is present in the sodium compound NaX

and the potassium compound KX and the conductivities of each

are A° Kx and JC . NaX' then if A° XX= e K /)X and

,eNaX = eNa A° X, one would expect that /10 KX NaX K Na should be independent of X. = e

r

8

By extracting-from Table 1 the equivalent conductivities

at infinite diLution and taking the difference between -those

with a common ion, the results shown in Figure 2 are obtained .

The values are fairly consistent, so that the independence of

the mobility of ions is a 10% solution of sucrose can b e

taken to be confirmed .

To Qbtain absolute values of the mobilities or p64

equivalent conductivities of each ion measurements of th e

amount transported should be made . However it is possible to

obtain relative values from the columns of Table 2 . The

conductivity of cations in a 10% solution of sucrose are in

the order H+ ~~ K+ > Na+

:,, Li + , which is the same as in

aqueous solution, but the order of the anions is Br- A: Cl>a

OH_ > OAC-, and the mobilityof the hydroxyl ion has been

greatly reduced from its value in aqueous solution .

.The relation between conductivi scosity

the confirmation of Walden's law .

- . On the premise that the radius of the cloud around

the ion is fixed, and that according to Walden's law the

change of conductivity in various solvents is caused by the

change of the viscosity I of the solvent itself, we can

obtain from Stork's law the relatio n

A-9=K (2 )

in which K is a constant .

f

9

In order to investigate the reason for the decrease

of mobility of ions in a 10% sucrose solution and the relation

of this decrease to the increase of viscosity, the viscosity

of each solution was measured relative to the viscosity of

water at the same temperature. Table 3 shows the results for

sodium chloride, and the results for the other electrolytes

in a 10% solution of sucrose are given in the fourth column

of Table 1.

The values of K for sodium chloride were almost

independent of the concentration of sucrose, and apart from

substances which are either acids or bases the values in the

third and fourth columns of Table 1 are almost identical..

According to these.results the decrease in the conductivities

in sucrose solution of strong electrolytes other than acids

or bases can be taken to be principally due to the increase

in the viscosity of the solution.

Table 3. Relation between eqûivalent conductivity at infinite dilution of sodiumchloride d°xaoi in aqueous solution of sucrose at'25°C and relative viscosity

of the solvent

Concn of Sucrose i0.)

A°:adci(cmz equiv.-l ohm-)

Relative Viscosity ?jr d°xaci x rr =K

0 126.8 1.000 126.8

2 123.0 1.032 126.9

4 ' 120.2 1.090 131.0

6 117.0 1.102 128.9

8 112.5 1.183 133.0

10 105.2 1.310 137.8

1 0

The dissociation of weak acids in a,solution_of_SUcreàe.

1 Strong electrolytes are completely dissoôiated in

the sucrose solution, and it is supposed that only the

mobility is decreased. It was desirable that the condition

of dissociation of weak electrolytes should be investigated.

First, the relative conductivities of various

concentratiOns of acetic acid in water or in 10% sucrose

solution were measured at 2500 , and the extrapolated

equivalent conductivities shown in the second column of

Table 4 were obtained. Next, on the assumption that the law

of independent ionic mobilities would be satisfied in thé

10% sucrose solution, the equivalent conductivities A éllOAc • for each concentration were determined by meanS of équatiOn 3.

AeHOAc = A HX AMOAc ?( MX

where

AHX is the equivalent conductivity of the

• acid HX for X = Cl, Br.

A m0A.

and /MX

is the equivalent conductivity of the

M salt of acetic acid for M = Na, K, Li,

is the equivalent conductivity of the

compound MX.

11

The values of A A HX4 MOAc' and A mx

1.0 ( 1.0) 1.5 ( 1.6) 2.4 ( 2.4) _ 4.5 ( 4.6) 7.1 ( 6.7)

13.5 (12.6)

237 (291) 298 (369) 302 (381) 317 (396) 323 (413) 328 (421)

2.21 ( 2.82) 4.59 ( 5.78) 7.27 ( 9.14)

•-14.4 (18.2) . '22.8 (27.8 )

44.3 (53.1 )

0.400 0.100

'

• 0.040

0.010 0.004

;. 0.001

were read from graphs produced in the saine way as Figure 1. p65

The values obtained are shown in the third column of Table 4.

The degree of dissociationke- HOAc // eHOAc of the

acetic acid was calculated from these results, and as can be

seen is almost exactly the same in a 10% solution of sucrose

as in water'. According to this the reduction of conductivity

in the sucrose solution is not due to a decrease in

dissociation, and it must therefore be considered to be due

to a decrease in mobility.

Table 4. Degree of dissociation of acetic acid in aqueous solution of 10% sucrose

• . at 25°C. The figure in parenthesis shows value in water.

ro„"cn of HoAo Equivalent Conductivity Equivalent Conductivity Degree of Dissociation at Complete Dissociation

of HOAa enoAc a =100 X AllOAcideHOlc A e etIOAc (Equiv. per Liter) (cm2 equiv. -1 ohm-1) (cm' equiv.-1 ohm-1) (%)

The denaturation of actomyosin in highviscosity solutions.

The relations so far established show that the

viscosity of sucrose solutions has a great influence on the

behaviour of electrolytes. As we have already stated,

denaturation is to be considered as a chemical reaction.

Increasing the viscosity of the solvent will reStrain the

movement of the molecules, and may be expected to reduce the

12

opportunities for reaction. For this reason alone a

restraining influence on denaturation may be thought to

be likely.

Now since polyhydric alcohols such as ethylene glycol.

and glycerin can be included in the same class of neutral

organic solutes as sucrose for the purposes of hindering

denaturation, it is supposed that the mutual interaction of

their hydroxyl radicals with the proteins is importantl. We

have not so far supposed that the viscosity of the solvent

would have an effect on the equilibrium to be reached.

Polymers of ethylene glycol (polyethlene glycol, abbreviated

as PEG) were added to solutions of actomyosin and their

-effectiveness in hindering denaturation was investigat.ed...

In deciding the concentration to be used it is

helpful that it should be reasonably similar to the concen-

tration of sucrose in surimi (10ô is about 0.3M). Since a

wide range of PEG molecular weights was to be investigated

there was the possibility that with the increase of molecular

weight the structure of the solvent would be increasingly

disrupted, and for 0.1M in the reaction solution comparison

of substances of the same molecular weight also limit the

sucrose to 10%.

A Weber-Edsall solution with 1.98 mgN/ml of p66

actomyosin was prepared from the flat-fish Kareius bicoloratus

by means of Arai's method?. 5ml of a 0.3M solution of each

molecular weight of PEG were added to 10m1 of the actomyosin

Molecular Weight of PEG . Residual Amount of Actomyosin (%)

2 Weeks After 4 Weeks After

Control 60 (Ethylene Glycol)

• 150 (Triethylene Glycol) 200

400

• 600 1000

2000

11.8

21.7

34.6

37.3

48.6

12.8

6.0

2.1

11.2

14.4

16.5

16.5

23.2

6.5

4.9

0.0

1 3

solution, and stored in a sealed test-tube at -20 °C. The

quantity of actomyosin remaining in the solution was

periodically determined8 . The results are shown in Table 5.

Table 5. Effect of molecular weight of PEG added to actomyosin (1.98mgN/m/ WEBER-EDSALL soln) during storage at —20°C on the denaturation.

The effectiveness in hindering denaturation increases as

the molecular weight increases to 400, but at 600 there is a

sharp decline and at 1000 or more the action is harmful.

In order to investigate the potency of PEG in

conditions of storage other than frozen storage, solutions of

actomyosin to which had been added the PEG 400 which had been

found to be the most potent were stored in a variety of

conditions and the effectiveness was measured.

5m1 of a 30% (w/v) solution of PEG was added to 10m1

of a Weber-Edsall solution of actomyosin containing 1.93 mgN/ml.

The proportion of actomyosin remaining was measured periodically

after storage at 38°C, 18 °C, 3 °C and -20 °C. The results, which

-14

'are given -in Figure 2 t showed that PEG had the power t o

-tebtrain denaturati6h in all §t6j~a~6 bohditions .

Omin ., - . . b L -i r--;x--

ft~ 2-. Effeft of PEG 400 ~dded (10 -jo)6h ili~e~ dieina~uiilti6ix of actoffiyosih

PEG iaddedi~oi iLdded

A ~0% (W/v) bblutibP- bf polyVih:Vlpyrodone of

hibibb,ulb± weight 24j~oo (abbi~eViated as PVP) was also used .

5hil bf this solution 'was add6d.-: to i0ml bf the Weber,gosall

bblution of act6myobin containing le~3mgN/ml and th e

proportion of Actomyosin remaining aftei~ 1 week was measured,

but no effect could be found .

15

Discussion.

Because the enauiry was made ffom the viewpoint of

elucidating the condition of the ions in the sucrose

solution, the particular properties of the polymers were

completely neglected. This may be thought to be quite

unjustifiable, but nevertheless the following interesting

observations can be made.

(i) The dissociation of electrolytes dissolved in

a 10% solution of sucrose is almost exactly the same

as when dissolved in water.

(ii) Viscosity causes the mobility of ions to be

less in the sucrose solution than in pure water.

(iii) When the viscosity is high, PEG which has a

small number of hydroxyl radicals in comparison to

its molecular weight can also hinder the denaturation

of actomyosin.

(iv) When comparisons are made at a fixed mol •

concentration of added PEG, an extremely great

dependence on the molecular weight of its ability

to hinder denaturation is found.

(v) No effect could be found for PVP although its

viscosity was high and its molecular weight was

extremely high.

16

One mechanism for denaturation that has been widely

9 #suggested is coagulation or the formation of conglomerat:es .

On the analogy of a mechanism for the "setting" of salted

surimi conglomerates could be formed by hydrophobic bonding,

10 *and hydrogen bonding could also occur Another explanation

is that protein denaturation is induced by the concentrated

salts produced by the "salting out" process during freezingl.

Only the results (ii) and (iii) have a bearing on these

theories. Treating the inauguration of the denaturation

r.eaction by means of the molecular collision theory of a

perfect gas, and considering the mutual reactions between the

protein molecules or between the protein molecules and the

salt ions in their neighbourhood, it appears that an increase

of viscosity will hinder or slow down the approach of one

molecule to another,-and this can be expected to result in

retarding denaturation..

However it would be dangerous to suppose that this

process alone will account for the retardation of denaturation.

There are many points which can in no way be so explained.

For example, according to (iv) the molecular wei?;ht, to

which the viscosity is of course proportional, has a very

large influence on the restraining of denaturation,.but PVP

which has a large viscosity had no influence. Also it is

# Papers of the symposium at the general meeting, of theJapanese Society of Scientific'Fisheries. (1971, 10).,

17

known that the influence of monocarboxylic acids and of

glycols of thc same series decreases as the molecular weight

increases. Not only viscosity but also many other physico-

chemical properties such as solubility or hydrophobic nature

are intimately related to the molecular weight, particularly

in compounds of the same series, and it is difficult to

discriminate.a priori between them.

In connection with the phenomenon mentioned in (iv)

we may remember that there is often a progressive change in

the pharmacological activity of members of the same alkyl

family, so that properties opposing the retardation of

denaturation may appear as the molecular weight increases.11

To sum up, substances such as PEG Lpoo which have a

large molecular weight but relatively few hydroxyl radicals

have been found to be unique among the substances hitherto

used in their effectiveness in retarding denaturation, and,

using them as a model of neutral organic retardants, we

intend to investigate their physical-chemical properties

and the mutual action between them and actmyosin.

* Summaries of papers at the general meeting of the Japanese Society of Scientific Fisheries. (1971, 10, p.104).

18

References.

S. Matsumoto.

Shokuhin hozo pp329 -- 355. Asakura shoten Tokyo 1966.

The Storage of Food. pp 329 - 355.

(Asakura, Tokyo, 1966).

2, W. Shimizu.

Suisan neri seihin pp 151 - 153 Korin shuppan.

(Tokyo).

Fish Pastes. pp 151 - 153. Korin Publications.

(Tokyo).

3. M. Okada.

Reito surimi no seizo ho.

Toku ko showa 41 - 15499.

A method of manufacture of frozen surimi.

Patent Gazette, 1966 -- 15499.

4. K. Iwai, K. Koichi, S. Umemato.

Hon shi 3? 626 - 633 (1966.)

Bull. Jap. Soc. Sci. Fish. 3? 626 -- 633 (1966.)

5, T. Yasui, J. Morita and K. Takahashi.

J. Biochem. 60 303 - 316 (1966)

6. S. Noguchi and J. J. Matsumoto.

Bu_ll.Jap. Soc. Sci. Fish. ]6 1078 - 1087 (1970)

19

7 , K. Aral, H. Takahashi and T. Saito.

Hon shi ..5b. 232 - 236 (1970).

Bull. Jap. Soc. Sci. Fish. 3 232 - 236 (1970).

8. E. Niwa, M. Okada and T. Kondo.

Mitsue ken dal sui kiyo 8 139 - 143 (1970).

Bulletin of the faculty of fisheries of the

Mitsue Prefectural University. 8 139 - 143 (1970).

9. J. J. Connell.

Nature. 183 664 - 665 (1959).

10. E. Niwa, M. Miyake.

Hon shi 31 877 - 883 (1971).

12 884 - 890 (1971).

Bull. Jap. Soc. Sci. Fish.

.1Z 877 - 883 (1971). -

.52 884 - 890 (1971).

11. G. M. Dyson.

MAY'S Chemistry of Synthetic Drugs, pp 1 - 18,

Longman's, London (1959).