United States Patent 0 "ice

1
FISH PROTEIN ISOLATE

3,798,126
Patented Mar. 19, 1974

2
times and the wash liquor may be recovered for addition, after desalting, to ?sh meal. After washing, the protein is

3,798,126

Rupert Josef Gasser, Kempttal, Grafstal, and Lienhard Bodo Huster, Winterthur, Switzerland, assignors to
Societe dAssistance Technique pour Produits Nestle

redissolved in alkali, preferably under conditions similar

to those applied in the ?rst treatment.

S.A., Lausanne, Switzerland No Drawing. Filed Sept. 10, 1971, Ser. No. 179,586 Claims priority, application Great Britain, Sept. 23, 1970,
Int. Cl. C12b 1/00

45,311/70

Before extraction of the lipids, the dissolved protein may be subjected to mild hydrolysis with a proteolytic enzyme, desirably at a pH of 7 to 10. The pH will pref erably have been adjusted to 7 to 8 after the alkali treat ment, and the enzyme used should be active at alkaline
10

pH. Papain is the preferred enzyme, with which hydrolysis

may be effected at a temperature of 60 to 80 C. during

Us. or. 195-s

19 Claims

5 to 30 minutes. The hydrolysis temperature will normally

ABSTRACT OF THE DISCLOSURE enzyme used. Protein is isolated from ?sh material (e.g. ?sh meal, 15 After hydrolysis the enzyme is inactivated by heating raw ?sh, ?sh offal) by solubilization with alkali and the and the solution cooled to ambient temperature. There lipids present in the ?sh are removed by liquid-liquid after, insoluble matter is removed (e.g. by centrifugation)
extraction with one or more solvents.

be adjusted having regard to the requirements of the

This invention is concerned with the production of a

and the lipids present extracted with a solvent. This opera tion may be carried out in continuous manner, using a 20 centrifugal extractor or any suitable extraction column,
with a water-immiscible solvent or a solvent system com

prising at least one such solvent. The term Water-im The object of the invention is to provide a process for miscible is used in its usual technological sense, and preparing a high-grade protein from ?sh and ?sh residues, the protein being in the form of a soluble white powder 25 hence includes solvents which are miscible with water to a slight degree as well as solvents which are water which is bland in odor and ?avor. immiscible. Care should be taken to avoid formation of According to the invention, protein is isolated from an emulsion and it is preferred to use a solvent system ?sh material (including fresh ?sh, ?sh meal and ?sh offal) comprising as primary solvent a water-miscible polar sol by a process which comprises treating the ?sh with alkali
in an aqueous medium at a temperature of 90 to 120 C. for not more than about ?ve minutes, the alkali concen

?sh protein isolate.

vent for the lipids, such as ethanol or isopropanol,

tration in the medium being between 0.02 and 0.5 N

whereby a substantial portion of the protein present in the ?sh is solubilized, adjusting the pH value of the medium
to a value of about 7 to 10, removing insoluble matter

together with a secondary solvent, less polar than the primary, for example methyl isobutyl ketone or hexane. Preferred solvent combinations are isopropanol/methyl

isobutyl ketone, ethanol/methyl isobutyl ketone and

isopropanol/hexane. The ratio of primary to secondary
solvent may be between 1:1 and 1:2.5 on a volume basis.

from the medium, removing the lipids present by liquid

liquid extraction with a solvent for the lipids and recov

ering the protein from the solution. The term lipids as used herein designates not only fats but also lipoproteins, phospholipids and like sub
stances.

The procedure which has been outlined comprises a number of operations each of which contributes to the

The proportion of protein solution to total solvent will generally be 60 to 80 parts solution (containing 3.5 to 10.5% by weight dissolved protein) to 20 to 40 parts of solvent. The solvent and extracted lipids may be recovered by conventional techniques, and solvent residues are pref erably removed from the protein solution by recti?cation
or steam stripping.

The solvent-free protein solution is preferably concen desirable properties of the ?nal product. Of particular signi?cance is the initial treatment with alkali, which 45 trated to about 10 to 20% solids and acidi?ed to pH 4.5 to 5.5 (e.g. with hydrochloric acid) to precipitate an un causes solubilization of the protein. This treatment is pref desirable, dark colored protein fraction having a ?shy erably effected with intense mechanical disintegration at
taste. The precipitated fraction may be removed by cen
sufficient concentration for solubilization, but for a very

a relatively high temperature in the presence of alkali in

trifugation whereas the puri?ed protein is decolourized

and desalted prior to drying. Active charcoal is preferably used for decolorization, and desalting may be effected by ion-exchange or ultral?ltration. Before drying the protein
solution may be further concentrated, to about 30 to 50%

short period of time to avoid damage to the protein. After the ?rst alkali treatment and prior to separation of insolu
bles the pH is adjusted to a value of about 7 to 10. This

may be effected by adding an acid (sulphuric, hydro chloric) or by injecting gaseous sulphur dioxide. After

by weight solids, for example by reverse osmosis or other techniques'which avoid heat damage of the protein. The 55 pH adjustment substantially all the protein remains in dry product is a white powder, soluble in water, and solution, so that the insoluble non-protein matter such as having a bland taste. The protein content lies between 95 bones, scales and the like may easily be removed, prefer and 100% by weight. ably by centrifugation. The invention is illustrated by the following examples If the ?sh starting material is a ?sh meal (which may be in which the parts and percentages are by weight. partly defatted), it is preferably suspended in water to provide a slurry containing about 5 to 15% by weight of EXAMPLE 1

An aqueous slurry containing 10% of non-extracted grade ?shmeal is heated to 70 C. and su?icient sodium solids level is outside the limits indicated. .After removal of insolubles, the protein may be sepa 65 hydroxide solution is added to provide an alkali concen tration of 0.3 N. The alkaline slurry is rapidly heated to rated from the solution, for example by precipitation at 100 to 120 C. in a continuous stream by steam injection. pH 4.5 to 6.0. Precipitated protein, with which most of The ?ow is adjusted to give a holding time of about 30 the lipids are associated, may be separated by centrifuga
tion, and it is usually desirable to wash it with water or
seconds prior to cooling to '65 C.

dry matter. Fresh ?sh or ?sh offal has a relatively high water content, so that water need only be added if the

After cooling the pH is adjusted to about 8 by injection sodium chloride solution. Generally, the quantity of wash 70 of gaseous sulphur dioxide and the slurry is further cooled liquor will be between about 2 and 6 times the weight of

protein. The washing operation may be repeated several

to ambient temperature.

3,798,126

Insoluble matter present in the slurry, such as ?sh bones, is removed by centrifugation and a clear protein solution containing about 11% solids is recovered. This solution is acidi?ed to pH 5.2 by addition of sulphur dioxide and the

resulting precipitate is separated by centrifugation.

The mother liquor is desalted and dried for incorpora tion in feed grade ?sh meal. The crude protein precipitate is washed three times with water (or 10% sodium chloride solution), at a rate of 4 parts of wash liquor for each part

case preferably at a product temperature not exceeding 80 C. A white, water-soluble powder, having a bland taste and containing 95% protein is thus obtained. It is suitable for incorporation in various foods and beverages, and it may

also be shaped by extrusion or spinning.

EXAMPLE 3

of protein solution containing about 7% solids. The mother liquor is desalted and dried for incorpora The solvent stream leaving the extractor, containing fats, 25 tion in feed grade ?sh meal. The crude protein precipitate phospholipids, lipoproteins and like substances is proc is washed three times with 10% sodium chloride solution, essed for recovery of solvent and lipid fraction whereas at a rate of 4 parts of wash liquor for each part of
the protein solution is stream stripped to remove solvent residues and concentrated to 15% solids. The pH of the concentrated solution is adjusted to 5.0 with hydrochloric

hydroxide solution is added to provide an alkali concen dissolved by adding alkali to a concentration of 0.2 N. The tration of 0.3 N. The alkaline slurry is rapidly heated to solution is heated rapidly to 100 C., held for 30 seconds, 100 C. in a continuous stream by steam injection. The and cooled to 60 C. The pH of the solution is adjusted ?ow is adjusted to give a holding time of about 30 seconds to 7.5, 2% of papain, on protein basis, added and the 15 prior to cooling to 65 C. temperature is maintained at 60 C. for 10 minutes. The After cooling the pH is adjusted to about 8 by addition enzyme is then inactivated by heating for 3 minutes at of N-hydrochloric acid and the slurry is further cooled about 100 C. and the solution is cooled to ambient tem- I to ambient temperature. perature. Insoluble matter present in the slurry, such as ?sh bones, Insoluble matter is removed by centrifugation and the is removed by centrifugation. and a clear protein solution protein solution, after mixing with 17% of isopropanol, containing about 11% solids is recovered. This solution is defatted in a centrifugal extractor to which methyl iso is acidi?ed to pH 5.2 by addition of N-hydrochloric acid butyl ketone is supplied at a rate of 1 part per 4 parts and the resulting precipitate is separated by centrifugation.

of protein. After washing the protein is suspended in water and

An aqueous slurry containing 10% of partially defatted

10 grade ?shmeal is heated to 70 C. and suf?cient potassium

protein.

After washing the protein is suspended in water and

dissolved by adding alkali to a concentration of 0.1 N. The solution is heated rapidly to 100 C., held for 30 seconds and cooled to 60 C. The pH of the solution is

acid, which causes the precipitation of a small amount of an undesirable protein fraction which is removed by cen

trifugation. The clear solution from the centrifuge is de colorized with active charcoal and desalted by ion-ex change treatment using a strongly basic and a strongly acidic resin. After concentration (reverse osmosis) to 30% solids the protein solution is spray-dried at a product tem perature not exceeding 80C.

adjusted to 7.5, 2% of papain, on protein basis, added and

the temperature is maintained at 60 C. for 10 minutes.

The enzyme is then inactivated by heating for 3 minutes

"at about 100 C. and the solution is cooled to ambient

temperature.
Insoluble matter formed during the enzyme treatment is

The resulting dry powder is substantially completely

soluble in water and has a bland taste. The protein con

removed by centrifugation and the protein solution, after

mixing with 17% of isopropanol, is defatted in a centrif

chloric acid and the insoluble matter, together with a 55

ing about 7% solids. The solvent stream leaving the extractor, containing fats, 45 phospholipids, lipoproteins and like substances is processed An aqueous slurry containing 25% ?sh ?lleting waste for recovery of solvent and lipid fraction whereas the is prepared and its pH is adjusted to 9 by addition of 30% protein solution is steam stripped to remove solvent resi sodium hydroxide solution. The slurry is vigorously stirred dues and concentrated to 15% solids. The pH of the for one hour and thereafter heated to 100 C. by steam concentrated solution is adjusted to 5.0 with hydrochloric injection after adjustment of the alkali concentration to acid, which causes the precipitation of a small amount of 50 0.04 N by addition of 30% NaOH. an undesirable protein fraction which is removed by cen The heating is carried out in a continuous system, the trifugation. The clear solution from the centrifuge is de ?ow rate being adjusted to give a holding time of about colorized with active charcoal and desalted by ultra?ltra 2 minutes at 100 C. prior to cooling to 60 C. After tion. After concentration (reverse osmosis) to 30% solids cooling the pH is adjusted to 7.5 by addition of N-hydro the protein solution is freeze-dried.

tent is 99% and the product may be incorporated in vari ous foods and beverages. It may also be shaped, by extru sion or spinning. EXAMPLE 2

ugal extractor to which methyl isobutyl ketone is supplied

at a rate of 1 part per 4 parts of protein solution contain

fatty phase, removed by centrifugation. A clear solution containing 4% of protein is thus obtained.
1% of papain, on protein basis, is added to the solution,
the temperature is maintained for 30 minutes at 65 C. and then the enzyme is inactivated by heating at about 100 C. for 3 minutes. After cooling to ambient tempera~ ture, insoluble matter precipitated during the enzyme treat

The resulting dry powder is substantially completely

soluble in Water and has a bland taste. The protein content

is 99% and the product may be incorporated in various foods and beverages. It may also be shaped, by extrusion
or spinning.

We claim: 1. A process for isolating protein from ?sh material

ment is removed by centrifugation and the protein solution concentrated to 10% dry matter by reverse osmosis. The concentrated solution is mixed with 17% of iso 65
propanol, and the lipids are extracted in a centrifugal ex tractor to which methyl isobutyl ketone is fed at a rate of

which comprises: (a) solubilizing a substantial portion of the protein of

the ?sh material by slurrying said material for not
more than about 5 minutes in an aqueous medium at a

1 part per 4 parts of protein/isopropanol solution. The exiting solvent stream is treated for recovery of solvent 70 and lipids whereas the protein solution is recti?ed for re moval of solvent traces.

temperature of from 90 to 120 C., said medium having an alkali concentration of between 0.02 and

0.5 N; (b) adjusting the pH medium to a value of about 7

to 10;

The protein solution is then treated with active char

coal and desalted and concentrated to 30% solids by ultra ?ltration. It may be spray- or freeze-dried, in the former 75

(c) removing insoluble matter from the aqueous

medium;
(d) removing lipids present in the aqueous medium through liquid-liquid extraction with a solvent for said

5
(e) recovering protein from the aqueous medium.

3,798,126

lipids, said solvent comprising an essentially water immiscible liquid; and

2. A process according to claim 1 wherein the slurry

13. A process according to claim 1 in which 20 to 40 parts of solvent per 60 to 80 parts of aqueous medium are

employed in the extraction of step (d).

14. A process according to claim 9 in which 20 to 40 parts of solvent per 60 to 80 parts of the aqueous medium

of step (a) comprises 5 to 15% by weight of dry matter

of -?sh material.

3. A process according to claim 1 in which the aqueous medium produced by step (c) is adjusted to a pH of about

4.5 to 6.0 in order to precipitate protein which is ?rst washed and then redissolved in an aqueous alkali medium 10 remaining aqueous protein-containing medium. 16. A process according to claim 15 in which, prior to prior to the extraction of step (d) . acidi?cation, the extracted aqueous medium of step (d) 4. A process according to claim 3 in which the pre is concentrated to about 10 to 20% by weight of protein cipitated protein is redissolved in an aqueous alkali me solids. dium having a concentration of 0.02 to 0.5 N. 17. A process according to claim 16 in which, following 5. A process according to claim 3 in which the pre 15 removal of the precipitate, the aqueous medium is con cipitated protein is redissolved in an aqueous alkali medium centrated to yield a protein isolate. at a temperature of 90 to 120 C. for not more than about 18. A process according to claim 1, in which the aque 5 minutes.

are employed in the extraction of step (d). 15. A process according to claim 1 in which the pH of the extracted aqueous medium of step (d) is acidi?ed to 4.5 to 5.5 and the resulting precipitate removed from the

ous medium of step (d) is at ambient temperature prior 6. A process according to claim 1 in which, prior to step (d), the solubilized protein in the aqueous medium 20 to liquid-liquid extraction. 19. A process according to claim 8, in which the aque is subjected to mild hydrolysis through treatment with a
proteolytic enzyme at a pH of 7 to 10 and a temperature of 60 to 80 C. 7. A process according to claim 6 in which the enzyme

ous medium is cooled to ambient temperature after treat

ment with an enzyme.

is papain.
8. A process according to claim 6 in which, after treat
ment with an enzyme, insoluble material formed during said treatment is removed. 9. A process according to claim 1 in which the solvent

25
2,416,196 3,561,973 3,649,294

References Cited UNITED STATES PATENTS

2/1947 2/1971 3/1972 Mortenson ______ __ 260-412.8 Rutman ____________ .._ 99-18 Thijssen __________ __ 260112

of step (d) comprises both a water-miscible solvent and 30 an essentially water-immiscible solvent. 10. A process according to claim 9 in which the water

2,686,126 2,602,031

8/1954 7/1952

Lovern et al _______ .... 99-l8 X Ugelstad _______ __ 260112 R

miscible solvent comprises isopropanol.

11. A process according to claim 9 in which the essen

2,875,061

2/ 1959

Vogel et al. _______ __ 99-18 X

tially water-immiscible solvent comprises methyl isobutyl

ketone. 12. A process according to claim 9, in which the water miscible and essentially water-immiscible solvents are in a
ratio of between 1:1 and 1:25 on a volume basis.

35

A. LOUIS MONACELL, Primary Examiner

R. B. PENLAND, Assistant Examiner
US. Cl. X.R.

426-364; 260112 R