AN ABSTRACT OF THE THESIS OF
Jeffry Dean Culbertson
for the degree of
Master of Science
in Food Science and Technology presented on "TTtnnPyU ID, IQ05?
Title;
ACID STABILIZATION OF AUTOLYZED FISH; STORAGE
AND NUTRITIONAL CHARACTERISTICS
Abstract approved;
rr-™-- „,,—.^^
Dr. David 1^. Crawford
The effects of ambient temperature storage on the chemical
and nutritional characteristics of phosphoric and sorbic acidstabilized fish autolysates were determined.
Variations in sample
composition due to autolysis, deboning, and acidification were also
evaluated.
Autolysates of English sole (Parophrys vetulus), true cod
(Gadus macrocephalus), Dover sole (Microstomus pacificus), and
orange rockfish (Sebastodes pinniger) carcass wastes, whole Pacific
hake (Merluccius productus) and dogfish shark (Squalis acanthias),
and a hake/tuna viscera (70/30 wt/wt) mixture were utilized in the
investigation.
Samples were acidified to ca. pH 3.25 with phosphoric
acid (85% w/v) and contained potassium sorbate at the 0. 2% (wt/wt)
level.
Screen separation of bone reduced the ash levels of samples,
with the exception of dogfish shark.
Loss of bone minerals, spe-
cifically calcium and phosphorus, was responsible.
Phosphoric acid
addition elevated ash and phosphorus contents to higher levels than
for raw materials.
Acid-stabilized autolysates were stable to microbial outgrowth
throughout 8 months of ambient temperature storage.
creases in pH levels were generally observed.
Slight in-
Sorbate levels de-
creased at an apparently exponential rate with respect to time
(r=-.9146, P > .005).
Hydrolytic rancidity did not proceed during storage.
Stability
towards microbial outgrowth, inactivation of endogenous lipases
during pasteurization, and maintenance of acidic environmental conditions enhanced fat stability.
Oxidative rancidity, as measured by
2-thiobarbituric acid (TBA) numbers, progressed during storage.
Carcass wastes showed higher rates and overall levels of oxidation.
Initial free fatty acid levels correlated in a linear manner with 0 and
4 month TBA numbers (r=:. 9548, P > . 005; r=. 9187, P >. 005, respectively).
The regression of TBA numbers at 4 months on zero time
values increased in a linear manner (r=. 9346, P > . 005).
Proteolysis during storage, as monitored by free amino groups,
was not detected.
Inactivation of native proteases during processing
and the microbial stability of samples were responsible.
Levels of
available e-amino lysine and tryptophan were stable throughout storage.
The protein quality of acidified hake, dogfish shark, orange
rockfish, and English sole autolysates, stored for 0, 4, and 8 months,
was evaluated using protein efficiency ratio (PER) determinations.
Protein quality was not affected by storage in samples of acidified
hake, English sole, and orange rockfish (P=.05).
Ratios for all
samples of hake and the 0 and 8 month samples of dogfish shark did
not vary significantly (P=.05) from the casein control.
English sole
and orange rockfish samples yielded PER values that were inferior
to casein and round fish samples (P=. 05).
The regression of feed
consumption and PER values on TBA numbers decreased in a linear
manner (r=-. 7999, P > . 005; r=-.8424, P > . 005, respectively).
Higher contents of nutritionally inferior visceral proteins and increased rates of oxidative rancidity in the carcass waste samples
probably were responsible for their reduced protein qualities.
Acid Stabilization of Autolyzed Fishs Storage
and Nutritiojial Characteristics
by
Jeffry Dean Culbertson
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
June 1978
APPROVEDs
Professor of Food Science and Teoeiiology
in charge of major
Head of Department of Food Solence and Technology
Dean of Graduate School
Date thesis is presented
n^c\^oV>
\ O ^ lOj^lfi
Typed by Opal Grossnicklaus for Jeffry Dean Culbertson
ACKNOWLEDGMENT
The author wishes to express sincere gratitude to Dr. David L.
Crawford for his many years of guidance and support.
Thanks are
also given to Duncan K. Law, Dr. Dennis T. Gordon, Dr. Jerry
Babbitt, and the staff of the Astoria Seafoods Laboratory for their
worthy advice, cooperation, and endless patience.
I am also indebted
to Dr. A. F. Anglemier and Dr. W. D. Davidson for their timely
contributions to a balanced education.
Appreciation is extended to Bioproducts, Inc. , Warrenton,
Oregon and to the National Oceanic and Atmospheric Administration
(maintained by the U. S. Department of Commerce) Institutional Sea
Grant 04-6-158-44094 and 04-7-158-44085 for partial support of
this investigation.
TABLE OF CONTENTS
INTRODUCTION
1
REVIEW OF LITERATURE
4
Industrial Fish Products
Meals, Oils and Solubles
Direct Animal Feeds
Non-edible products
Fish Silage
Production
Storage Characteristics
Fish Protein Concentrates
Processes
Nutritional Characteristics
Autolysis of Fish Proteins
MATERIALS AND METHODS
Preparation of Samples
Apparatus
Autolysis and Stabilization of Samples
Mineral Composition
Storage Characteristics
Stability towards Microbial Outgrowth
Lipid Stability
Protein Stability
Protein Quality Stability
RESULTS AND DISCUSSION
Sample Composition
Storage Characteristics
Stability towards Microbial Outgrowth
Lipid Stability
Protein Stability
Protein Quality Stability
4
4
6
7
7
8
9
10
11
12
13
16
16
17
17
18
18
19
19
21
22
23
23
28
28
37
43
49
SUMMARY
56
BIBLIOGRAPHY
59
LIST OF FIGURES
Figure
1.
2.
3.
Page
Loss of sorbic acid during storage of acidstabilized autolysates.
32
Log of % sorbic acid retained (based on initial
contents) versus storage.
34
Malonaldehyde levels (mg/kg) in stored acidstabilized autolysates.
41
LIST OF TABLES
Table
1.
Page
Proximate composition of raw, autolyzed and
deboned, and acid-stabilized fish samples.
24
Macromineral content of raw, autolyzed-deboned,
and acid-stabilized samples.
26
Micromineral composition of raw, autolyzeddeboned, and acid-stabilized samples.
27
4.
Effect of storage on pH.
29
5.
Sorbic acid levels (% wt/wt) of stored acidstabilized autolysates.
31
Regression of the log % sorbic acid retained on
storage time (mos).
35
Stability of acidified autolysates toward microbial
outgrowth (organisms/gm x 10 ).
36
Free fatty acid levels (% wt/wt in total lipid expressed
as oleic acid) in stored acid-stabilized autolysates.
38
Malonaldehyde levels (mg/kg) in stored acidstabilized avitoly sates.
40
Proteolysis (mg glycine equivalents/16 gm total N) in
stored acid-stabilized autolysates.
44
Available e-amino lysine content (gm/16 gm total N)
of stored acid-stabilized autolysates.
46
Tryptophan content (gm/16 gm total N) of stored
acid-stabilized autolysates.
48
Protein efficiency ratio (PER) of stored acidstabilized autolysates. Summary of analysis of
variance; factorial design.
50
2.
3.
6.
7.
8.
9.
10.
11.
12.
1 3.
Table
14.
Page
Protein efficiency ratio (PER) of stored acidstabilized autolysates. Summary of analysis of
variance. Randomized block design; ranking of
individual treatment means.
52
ACID STABILIZATION OF AUTOLYZED FISHs STORAGE
AND NUTRITIONAL CHARACTERISTICS
INTRODUCTION
The increasing population of the world has sharpened the demand
for high quality protein and made it imperative that none of our potential harvest from the ocean be wasted.
While there remains consider-
able disagreement about the extent to which the seas can provide
increasing amounts of food for man, there is general agreement that
the present catch can be substantially increased.
The 197 3 world
landings of 145 billion pounds of seafoods represented an approximate 20 percent harvest of the estimated resource (U. S. Fish and
Wildlife Ser. , 197 3).
Increasing the total catch is not the only method of providing
more fish protein.
Solid waste from fish processing plants consti-
tutes a significant portion of the highly nutritional catch from the
sea.
The overall loss of protein for fish processed for human
consumption is estimated to be approximately 50 percent (Finch,
1970).
The annual U. S. volume of solid fishery wastes thus gener-
ated is about 1.2 billion pounds (Soderquist et al. , 1970) and only
50 percent of this waste is recovered for animal feeds.
Based on an
estimated mean protein content of 14 percent, the remaining 600
million pounds of waste would represent approximately 84 million
pounds of protein.
The under utilization of fishery waste not only represents a
significant loss of high quality protein, but creates environmental
and economic problems.
Until recently, solid fishery wastes were
disposed of in one of the following manners? incineration, dumping
into waterways, or landfills (Kreag and Smith, 1970).
In these days
of increasing environmental concern, public pressure and more
stringent pollution control laws have caused more emphasis to be
placed upon developing methods for transferring seafoods processing
waste into saleable products (Crawford, 1976).
Marketable products
for which fishery waste could be utilized as a raw material include
meals, pelletized animal or fish foods, silage, fertilizers, fish
glues, oils and protein concentrates (Brody, 1965).
Upgrading or
recovering the high grade proteins from seafood wastes (or more
appropriately expressed "secondary raw materials") will, directly
or indirectly, assist in solving the world protein shortage and reduce
pollution problems.
, For the past several years various research projects at the
Oregon State University Seafoods Laboratory, Astoria, Oregon have
been undertaken to find avenues of utilization for various fishery
wastes, as well as for "trash" or undesirable species of fish.
Fish
with this unappropriate designation include hake, spiny dogfish,
dogfish shark (mammal), and starry flounder.
Although the
abundance of "trash" fish, particularly the Pacific hake (Merluccius
productus ), was once ignored and often irritating, it is now recognized that such fish could make a substantial contribution to the
Pacific fishery, particularly as a raw material for protein concentrates (Finch, 1970).
The production of fish protein concentrates (FPCs), based upon
the hydrolytic or autolytic digestion of fishery wastes or "trash"
fish, has been under investigation for several years.
Normally,
preservation of such concentrates depends upon the energy intensive
removal of water to produce a dry powder.
The objectives of this
investigation were to (1) evaluate the stabilization of autolyzed fish
and fishery wastes using phosphoric and sorbic acids for storage at
ambient temperatures and (2) to determine the chemical and nutritional effects of such treatment.
LITERATURE REVIEW
Industrial Fish Products
At the present time the bulk of industrial fish products is produced from species too small, boney, or oily for direct human consumption (Bucove and Pigott, 1976).
The term "industrial fish" has
been used to define fish which are processed directly into feedstuffs
for the production of livestock such as chicken, beef, and pork.
This
classification includes species such as herring, menhaden, anchovie,
and sprat (Windsor and Thomas, 1974).
Slightly over 50 percent of
the 4. 8 billion pounds of fish caught in the U. S. in 197 3 was converted
directly into industrial products (U. S. Fish, and Wildlife Ser. , 197 3).
Meals, Oils and Solubles
Fish meal, which has won wide acceptance in the feed trade
because of its proved nutritional value, is obtained through the "fish
reduction process" which consists essentially of grinding, cooking,
pressing and drying of whole fish or fishery waste (Brody, 1965).
Depending on the raw material and the desired end-products, two
general types of reduction, wet and dry, can be used.
The wet reduction process utilizes live steam under pressure to
soften the flesh and bones and to release the oil.
Pressing the cooked
material expels the liquid portion, known as the "press liquor," and
5
produces a "presscake" which is subsequently dried and ground.
The
"press liquor" is processed by continuous centrifugation to obtain fish
body oils and "solubles." In the past the "solubles, " containing
water soluble vitamins and a variety of proteins, were discarded
(Brody, 1965).
However, current technology involves either concen-
tration to yield a product with approximately 50 percent solids which
can be marketed separately, or complete dehydration and restoration
to the presscake meal (Karrick, 196 3).
As the name infers, dry reduction utilizes dry heat to cook and
dehydrate raw fish material which is subsequently ground to produce
a meal.
The process has several advantages over the wet reduction
process; namely, the water soluble materials are retained in the
meal and batch processing allows for changes in operating conditions
when a variety of raw materials is processed (Brody, 1965).
How-
ever, the high fish oil content of meals produced in this manner has
limited their use in animal feeds.
The protein content of fish meals varies from 55 to 70 percent
by weight.
Herring meals have the highest protein content, while fish
scrap meals exhibit the lowest values (Nilson, 1950).
Fish meal
proteins contain all of the essential amino acids and are particularly
nutritionally valuable because of their relatively high content of lysine
(McBride et al. , I960).
Supplementation of animal feeds, whose pro-
tein content is based primarily on cereal sources and are deficient
in this amino acid, represents the largest current use of fish meals
(Finch, 1970).
Direct Animal Feeds
Industrial fish and fishery wastes have wide application in the
broad field of animal feeding.
They are utilized extensively in pet
food formulations, particularly for cat food, in rations for fur bearing
animals, and for the preparation of pelletized rations for various
species of fish (Brody, 1965).
European technology also includes
the production of fish silage which is used extensively in swine production (Tatterson and Windsor, 1974).
In 1973 over 116 million pounds of pet foods was produced in
the U.S. (Fishery Statistics, 1973).
Comparing this value with the
1958 production of 3 million pounds (Jones, I960) indicates the
dynamic nature of the pet food industry.
Although the lower protein
content and high mineral content of most fishery wastes limits its
scope of utilization in certain pet food formulations, machine separation of skin, flesh, and bone could yield highly nutritional proteinaceous raw materials for such products (Crawford jit ah (1972).
Crawford ^t al. (1972) found the protein quality, as measured by
protein efficiency ratio (PER) evaluations, of machine separated
flesh from fillet carcass wastes to be superior or equal to casein.
The use of fishery wastes in the preparation of rations for
domestically reared fish for human consumption or governmental
propagation efforts represents a means of recycling protein resources.
Fillet carcass waste, generated in large quantities during the processing of groundfish for human consumption, was found to be a satisfactory replacement for round fish (such as turbot) in the formulation of
Oregon moist pellets (Crawford, 1976).
Non-edible Products
A wide variety of non-edible products can be produced from
industrial fish and fishery wastes.
These include liquid fish glues,
gelatin, leathers, and fertilizers (Brody, 1965).
Numerous proces-
sing techniques and patents have been developed for the manufacture
of non-edible products, however, with the increased food demands of
today, greater economic emphasis has been placed on the utilization
of fishery wastes as protein sources for consumption by fish, livestock, and man (Tannenbaum et al. , 1974).
Fish Silage
Fish silage is commonly defined in the literature as a liquid
product made from whole or parts of fish to which no other material
has been added other than an acid.
Liquification of the fish mass
is carried out by enzymes already present in the fish.
True ensilage
differs from this in that the breakdown is accomplished by the action
8
of lactic acid bacteria (McBride et ah , I960).
Liquification of fish
silage usually occurs over a period of several weeks; the rate of
autolysis being dependent on the ambient temperature, pH, and the
relative amount of visceral organs present (Mackie, 1974).
Ensilage
processes have been most extensively studied in the Scandinavian
countries where liquified fish products are utilized primarily as
protein supplements for swine (Tatterson and Windsor, 1974).
Several advantages of having animal ration supplements in liquid,
rather than solid form, are apparent.
transported in bulk.
Liquid products can be readily
Secondly, liquid protein supplements can act
as binders in otherwise dry rations.
A third advantage is that a
liquid product could theoretically be produced at a lower cost since
the energy costs of drying and grinding could be saved.
The trans-
portation of bulk water is one obvious disadvantage for liquid products.
Production
The principle involved in the manufacture of fish silages is
based upon the rapid action of endogenous enzymes spread throughout
the fish mass by grinding and mixing and accelerated by favorable
conditions of acidity.
Acid conditions also inhibit bacterial out-
growth during the process.
been used.
Various acids or mixtures of acids have
The first successful process, developed in Finland in the
1920's, utilized acidification to pH 2. 5 with a mixture of sulfuric and
hydrochloric acids (Tatterson and Windsor, 1974).
Work done by
Hanson and Lovern (1951) indicated that acidification to pH 4 with
formic acid produced an acceptable product which was superior to
those produced by mineral acids because neutralization prior to feeding was not required.
(1974).
This was confirmed by Tatterson and Windsor
McBrideetal. (I960), however, found that liquification
occurred more rapidly in silages acidified to pH 3 with mineral acids
and that neutralization was not necessary if the supplement did not
exceed 20 percent of the diet.
Storage Characteristics
The effects of storage on freshly prepared silage are primarily
reflected in changes in the protein and lipid fractions.
Studies by
various workers show an increase in soluble nitrogen levels, indicating that a rapid breakdown of proteins to low molecular weight peptides and free anaino acids occurs.
Tatterson and Windsor (1974)
found that the rate of change decreased greatly after approximately
one month of storage when 75 to 85 percent of the total nitrogen content
was soluble.
McBrideetal. (I960) and Mackie (1974) indicated that
the rate of protein hydrolysis was dependent upon the pH, ambient
temperature and relative amounts of visceral organs present.
The
level of free fatty acids, as dictated primarily by the action of endogenous Upases, rises rapidly during the first month of storage, reaching
10
levels as high as 20 percent of the total lipid present (Tatterson and
Windsor, 1974).
Fish Protein Concentrates
Fish protein concentrates (FPCs), often containing over 90
percent protein, have been proposed for use in various parts of the
world as an important source of inexpensive complete protein for
both man and animals (Finch, 1970).
There is no singular FPC,
rather a family of products each with its own characteristic balance
of physical, nutritive, and sensory properties and cost which makes
it applicable to one particular area of usage.
Processes for the pro-
duction of FPCs consist primarily of stages of separation and concentration.
They have in common the concentration of protein; the
stabilization of the product against the growth of microorganisms,
mostly by reduction of the water level; and its stabilization against
chemical change, especially lipid oxidation, by removal of the lipid
fraction (Tannenbaum et al. , 1974).
In the United States, the produc-
tion of FPC for human consumption is limited by the Food and Drug
Administration to the species of fish (hake and hake-like fish, menhaden, and herring of the genus Clupea) and the solvent extraction
process (extraction with isopropyl alcohol or with ethylene dichloride
followed by isopropyl alcohol) used and requires the product to meet
certain chemical and nutritional specifications (Kreag and Smith, 1973),
11
FPC Processes
The processes involved in the production of FPC can be categorized into the following general classifications;
1) solvent extraction,
2) non-solvent extraction, and 3) biological (Finch, 1970).
The
solvent extraction processes utilize lipid solvents to extract nonpolar compounds, such as oils, and may or may not remove water
from the ground, deboned raw material.
Various solvents, including
isopropyl alcohol, ethylene dichloride, acetone, and hexane, have
been utilized (Tannenbaum et al« , 1974).
The extracted material is
then dehydrated yielding a product with 80 to 95 percent protein
(Yanezetal. , 1967).
Problems stemming from the use of solvents in FPC production, namely residual solvent toxicity and loss of protein functionality
due to denaturation, have precipitated research into FPC production
using non-solvent techniques.
Various patents have been developed
which rely on the use of alkali to dissolve the protein in the raw material.
Freed oil is separated by centrifugation and protein is recovered
by subsequent acid precipitation (Tannenbaum, 1970).
Surface active
agents have also been proposed as alternatives to the use of solvents,
but the procedures currently suffer from technological problems
(Connell, 1969).
Three biological approaches have been studied in the manufacture
12
of FPC.
These include the use of microorganisms and the use of
endogenous or exogenous enzymes (Finch, 1970).
Regardless of the
approach, the main objective is to hydrolyze or solubilize the proteins
present in the raw material so that oil separation is facilitated and
a maximum of protein functionality is retained.
Burkholder jst al.
(1965) reported the discovery of two microorganisms which were
able to hydrolyze fishery wastes and produce acceptable products.
The use of Aspergillus species followed by yeast to ferment fish has
been investigated by Jeffreys and Krill (1965).
Nutritional Characteristics
Numerous references to studies on the nutritional quality of
FPCs prepared from different species and various processes have
been reported.
With few exceptions, FPCs have been shown to be
high in protein with a quality at least equivalent to casein when
evaluated by laboratory animals (Finch, 1970).
The amino acid
pattern of an "average" FPC compares favorably with those of egg
and milk; methionine, isoleucine and total aromatic amino acids levels
are somewhat lower and lysine levels higher (FAO, 1965).
Methionine
has been shown to be the first limiting amino acid in FPC (Miller,
1956, Yanez^tal. , 1967, and Stillings et al. , 1969).
A variety of studies have amply demonstrated that FPC supplementation at levels from 3 to 8 percent can effectively enhance the
13
quality and quantity of protein in foods that contain proteins of
vegetable origin (Finch, 1970).
The addition of 3. 5 percent FPC
to either rice, wheat, or corn increased protein efficiency ratio by
35, 100, and 300 percent, respectively (Sidwell _et al. , 1970).
Power (1964) compared the nutritive value of FPC prepared
from whole fish, fillets, headed and eviscerated fish, and processing
scrap.
The use of either headed and eviscerated fish or fish scrap
had little effect on the protein quality of FPC as compared to that
made from whole fish.
Tarkey jst al. (197 3) and Pigott and Chu
(1969) have produced FPC from fishery wastes that are nutritionally
equivalent to casein.
Autolysis of Fish Proteins
The preparation of liquified fish products based on autolytic
activity has been extensively studied in Japan, where a variety of
products for human consumption are produced, and in various
Scandinavian countries which utilize fish silages as animal feeds.
Ensilage processes depend primarily upon the addition of acids to
modify the environmental pH to enhance the activity of various endogenous proteolytic enzymes and to prevent microbial growth.
Wide
variations in the time required for autolysis or liquification have been
reported (Tatterson and McBride, 1974).
Autolytic activity of various
species of fish has been found to be affected by several parameters
14
including season of the year and dietary intake (Kaiwai and Ikeda,
1973).
Freeman and Hoogland (1956) found that the quantities of digestive enzymes present in cod and haddock viscera were sufficient for
autolysis at native pH in a 48 hour period.
McBride jet al. (1961)
reported the complete autolytic digestion of whole Pacific herring in
72 hours.
The entire mixture was acidified to pH 2 with hydrochloric
acid and maintained at 37 C.
Liquification of ground whole herring,
acidified to pH 4. 0 with formic acid, was accomplished in 48 hours
by Tatterson and McBride (1974).
Sullivan (1975) reported that extracts of whole tuna viscera
yielded protease activity optimums at pH 1.5, 3. 1-3. 5, and 9-5,
with an approximate 30 fold increase in activity from pH 6. 0 to 3. 5.
Work by Timmerman (1977) indicated that the optimum pH for hydrolysis of whole hake was approximately pH 3. 5-3. 6 at 55 C.
A pH 3. 0
optimum at 30 C was obtained in similar work by Koury (1971).
The
optimum pH for the liquification of whole hake based upon viscometric properties was found to be between 4. 3 and 5. 1 which varied
considerably from that for overall proteolytic activity (pH 3. 6-3. 7)
(Timmerman, 1977).
It was theorized that since the connective tissue
of the fish was degraded releasing bone, protease systems specific
for connective tissues with higher pH optimums may have been responsible for the enhanced liquification.
15
The optimum temperature for the broad mix of proteases contained in whole hake has been shown to be near 60 C (Timnnerman,
1977 and Koury, 1971).
Proteases contained in the pyloric caeca of
albacore tuna showed activity near 60 C, but were rapidly inactivated
at higher temperatures (Sullivan, 1976).
16
MATERIALS AND METHODS
Preparation of Samples
Samples of Dover sole (Microstomus pacificus), true cod
(Gadus macrocephalus), English sole (Parophrys vetulus), and
orange rockfish (Sebastodes pinniger) carcass wastes were obtained
through Astoria Seafoods, Inc. , Astoria, Oregon.
Round Pacific hake
(Merluccius productus) and dogfish shark (Squalis acanthias) samples
were obtained from Pacific Shrimp, Inc. , Warrenton, Oregon.
Twenty-four hours prior to digestion, samples were passed
through a dual-cut meat chopper equipped with a 1/2" plate, packed
into 1" x 4" x 24" stainless steel trays, and frozen at -34.4 C
(-30 F).
Immediately prior to processing, the frozen blocks were
removed from the trays and passed twice through a 1/4" plate of the
chopper.
The resulting minced material was then held at 2 C
until digestion.
A sample of albacore tuna (Thunnus alalunga) viscera was
obtained soon after evisceration of thawed frozen fish from Bumble
Bee Seafoods, Inc. , Astoria, Oregon.
The viscera sample was
immediately frozen (-34.4 C), cut into slabs, vacuum sealed in
moisture-vapor proof film, and held at -17.7 C (0 F).
The frozen
viscera was passed twice through the chopper equipped with a 1/4"
plate prior to its use.
17
Apparatus
Samples were digested using a reaction unit developed by
Duncan K. Law of the Oregon State University Seafoods Laboratory,
Astoria, Oregon.
The digestion unit consists primarily of an open
sample vessel, equipped with a stirring device, a pump, and a simple
shell-in-tube heat exchanger.
Hot water served as the exchange
medium during digestion, while live steam was introduced into the
heat exchange system to achieve pasteurization temperatures.
Reac-
tion mixture temperatures were controlled by thermostatically activating the water flow through the heat exchange system.
Autolysis and Stabilization of Samples
Processing waste from Dover and English sole, orange rockfish
and true cod and round dogfish shark and hake and a hake/tuna viscera
(70/30 wt/wt) mixture were included in the investigation.
Approxi-
mately 125 kg of minced sample was utilized in each digestion run.
Samples were heated to 55 C in the digestion unit and held at this
temperature for a period of 1 hr. to effect liquification.
Bone par-
ticles were subsequently removed by passing the liquified material
through a 30 mesh screen.
The sample was then heated to 83. 7 C
o
(180 F) for five min to inactivate native enzyme systems and effect
pasteurization.
Using cold water as the exchange medium, the
18
sample was cooled to ca. 25 C, pumped into 50 gal plastic drums,
and held at 2 C overnight.
quantities of H PO
The cooled material was stabilized with
(85% wt/wt) required to achieve a pH of ca. 3. 25
and 0.2% potassium sorbate (wt/wt).
The samples were then stored
at ambient non-heated room temperature.
Mineral Composition
The mineral composition of the initial raw material and their
resulting deboned and acid stabilized autolysates was determined.
Approximately 500 gm of each sample was dried in vacuo at 65 C and
passed through a 1 mm sieve of a high speed mill (with dry ice when
necessary).
Mineral composition was determined by emission spec-
troscopy (WARF Institute, Madison, Wisconsin).
The moisture con-
tent of raw and autolyzed samples was determined by AOAC (1970)
procedures so that mineral composition could be expressed on a wet
weight basis.
Storage Characteristics
Various chemical and biological assays were performed at 0, 2,
4, 6, and 8 months of storage to determine the stability of the acidified autolysates towards microbial outgrowth and to assess deteriorative changes in their protein and lipid fractions.
At appropriate times
samples were placed in styrofoam containers, frozen at -34. 4 C
19
(-30 F), vacuum sealed in moisture-vapor proof film and stored at
-17.70C (0OF) for later analysis.
Stability toward Microbial Out-growth
Changes in acidic conditions and microbial numbers were determined during storage to evaluate the degree of autolysate stability.
The pH of each sample was determined at 0, 1, 2, 3, and 4 days and
at 2, 4, 6, and 8 months of storage.
Sorbic acid levels were moni-
tored using a spectrophotometric method developed by Wilamowski
(1974).
The method relied on the ether extraction of the protonated
form of the acid, followed by a quantification based on absorption at
250 nm.
A sample of the potassium sorbate used to stabilize the
autolysates, dried under vacuum at 65 C for 4 hr, was used in the
preparation of a standard curve.
Total aerobic plate counts were
determined according to AOAC (1970) procedures.
Serial dilutions
2
3
4
5
6
9
of IslO , IslO , IslO , IslO , 1;10 and 1; 10 were used and plates
exhibiting between 30 and 300 colonies after 2 days of incubation at
o
37 C were counted.
Lipid Stability
The level of hydrolytic and oxidative rancidity that developed
during storage was estimated by the determination of free fatty acid
and malonaldehyde (2-thiobarbituric acid numbers) contents,
20
respectively.
A modified Folch (1957) procedure was used to extract lipid
from which sample free fatty acid levels were determined.
A 5-10
gm sample was. homogenized for 2 min in a Virtis blender with 20
volumes of CHCl ;CH OH (2/1 v/v).
Solids were separated by
vacuum filtration through a Buchner funnel fitted with Whatman No. 1
filter paper.
The filtrate was quantitatively transferred to a 500 ml
glass stoppered graduated mixing cylinder.
(w/v) CaCl
A volume of 0. 05%
solution equivalent to 20%-of the filtrate volume was
added and mixed by gentle inversion.
The phases were allowed to
separate overnight at 0 C and the interfacial volume of the CHCl
layer was recorded.
The aqueous phase was removed and a 100 ml
aliquot of the CHCl phase was placed in a pre-weighed 250 ml rour^d
bottomed flask.
The sample was dried under nitrogen on a rotary
evaporator and the flask weighed to determine the total lipid weight.
Free fatty acids were determined by direct titration according to
procedures outlined by Smith et al. (1957) and expressed as % free
oleic acid based on total lipid weight.
A procedure described by Sinnhuber and Yu' (1977) was used
for the determination of 2-thiobarbituric acid (TBA) numbers.
The
procedure is based upon the reaction of malonaldehyde (released
during the oxidative deterioration of polyenoic fatty acids) in the
intact sample lipid fraction with 2-thiobarbiturate yielding a pigment
21
which is spectrophotometrically quantified at 5 35 nm.
TBA numbers
represent the milligrams of malonaldehyde present per kilogram of
sample.
Protein Stability
The degree of protein hydrolysis occurring during storage was
evaluated by determining the formation of free amino nitrogen.
Free
amino nitrogen was determined using the ninhydrin analysis carried
out according to Moore and Stein (1954) and reported as mg glycine/16
gm total sample nitrogen.
Total nitrogen content was determined
using the semi-micro Kjeldahl procedure (AOAC, 1970).
The levels of available lysine were estimated using the spectrophotometric method developed by Carpenter (I960).
The analysis is
based upon the formation of dinitrobenzene derivatives of the constituent amino acids.
Lysine derivatives were separated on the basis of
their water solubility and quantified by their absorbtion at 435 nm.
Values were expressed as gm lysine/16 gm total sample nitrogen.
A standard curve was prepared using free e-amino lysine hydrochloride, obtained from Nutritional Biochemicals Corp. , Cleveland,
Ohio.
The tryptophan content of samples was estimated using a method
outlined by Spies (1950).
The procedure involved the reaction of
p-dimethylaminobenzaldehyde with tryptophan residues, under
22
strongly acidic conditions, to produce a chromophore which absorbs
at 590 nm after exposure to light.
Tryptophan hydrochloride obtained
from Nutritional Biochemicals Corp. , Cleveland, Ohio was used to
prepare a standard curve.
Results were expressed as gm tryptophan/
16 gm total sample nitrogen.
Protein Quality Stability
Acid stabilized autolysates of English sole, dogfish shark, hake,
and orange rockfish, stored for 0, 4, and 8 months at ambient temperatures were selected for nutritional evaluation.
At appropriate
storage times, approximately 7 kg samples were frozen into blocks
at -34.4 C (-30 F), placed in large plastic lined storage bags, and
o
o
held at -17. 7 C (0 F) prior to analysis.
Autolysates were mixed approximately 2s 1 (wt/wt) with corn
starch, drum dried, and passed through a 0. 02 in. sieve of a high
speed hammermill for incorporation into rations.
The addition of
corn starch facilitated drum drying and assured thorough milling.
The protein efficiency ratio (PER) of samples was determined by
AOAC procedures using a ANRC reference casein (Humko Sheffield
Chemicals, Lyndhurst, N. J.) control and ten male rats per assay
group.
Data were analyzed by analysis of variance and the differ-
ences in means determined by Duncan's multiple range test.
23
RESULTS AND DISCUSSION
The utilization of fishery wastes and "trash" fish as sources of
high quality protein represents a significant means by which rising
world food demands may be satisfied.
This investigation was designed
to assess the effects of phosphoric and sorbic acid stabilization on
autolyzed fish materiaL
Stabilization by this method would allow
preservation without costly refrigeration or dehydration.
Parame-
ters evaluated included the effect of processing and acidification on
sample mineral and proximate composition, and the effects of ambient
temperature storage of acid-stabilized autolysates with regards to
microbial outgrowth, lipid and protein stability, and nutritional characteristics.
Sample Composition
The proximate compositions of raw, autolyzed and deboned,
and acid-stabilized fish samples are shown in Table 1.
The removal
of bone fragments following autolysis resulted in decreased ash levels
for all samples except dogfish shark.
This observation would be ex-
pected since members of the shark family rely on collagen, rather
than bone, for supportive structures.
Acidification of samples with
phosphoric acid (85% w/v) to pH 3. 25 resulted in the elevation of ash
levels to values higher than for the raw materials.
Although moisture
(0
en
J3
I
T)
■H
O
<i
a
o
XI
<u
a
•v
0
u
o
to
o
6
o
u
o
u
ft
(ti
XI
(U
u
ft
a
•H
(U
4->
O
u
ft
(ti
03
XJ
<
3
(U
O
U
NO
ON
oo m rj
m o on
so "^ en en in Tt1
ON oo m
■^ [^- oo
NO
vD in oo »-( o ^f c^C^ oo ■—i en oo c")
NO
(\j —< ON roo o r- M
m in in in Tj( m in
ON
NO
ON
ON NO
ON
^
<M
NO
en (M CM oo ON o oo
c^
O
M
N£>
r^
■^ m eo -^ (\j TJI rj*
^H [NON NO
(\J
t-H
oo en en o en oo ^
en 00 in (M M O ON
nH
oo r~ oo '^ oo ^H en
.-H
Tjt vO (M NO rt< T}< O
•st* en so oo •* oo -^
O
in r(M Tf
r^ ^ (M
in r^
oo <\j
ON
ON^Hvoeor-enm
I-H
ND ON
oNenf-eoooenm
oo o oo ■<* m t- ON
r~<N^-(MNO(\jin
p-H O •—< ^ ^H fNJ ^H
eo o NO oo r~- ON ON
(\J
^H
(\J
r- m ^ oo oo (\j -^
o rj en t— oo -H oo
oo en (\j Tf en r-H
-X) t- en in ON NO eO
r- ON m ON oo <\j ■-<
NO
^H ON 00 O 00 (M NO
O ^H ON O C^ <M O
ri p p a w H a
NO NO 00 NO ON ON en
so NO vo r^ NO NO r^
o >
r-n ON m oo m oo rr- ND r^ r^ r^ r^ 1^-
Ui in
rt Q Q Sg w H x
2 a a aG ffi tr! ffi
o en o •<# oo r~
r- r- oo r^ r^ r^
ON
NO
h w
U >
(2j Q Q K W H ffi
pci pd pi tf pci crj a5
M
24
(U
a
(ti
XI (ti
to
XI
xj
to
•H
>+H
CuO
O
o
m
P u
« p
•^ p
o ..
^1 TJ
<u o
M
ti 0)
(ti 3
M u
0 +>
II
II
ft U
rt H
•«» ••
<u
-H
o
m
T3
(1)
• r-l
4-1
Tt XI
W
u CO
(ti
on
II ti
< W
n
»o.
G
O ^^^
X3 +>
-(->
<u *
Tt
>N
OJ
(ti
u
T) &
0>
N o
>, en
XT ■--.
o o
r)-l
XI
X)
II
(0
a • ow
• •. >
£
(ti (ti
(ti
J*
jj
II -u
|H
cd
a
ii
<D XI
^H
a
6
(ti
w
25
and protein levels decreased upon acidification of autolyzed samples,
close inspection reveals that this observation is probably due to a
replacement or dilution effect caused by the addition of phosphoric
acid.
The levels of macrominerals (P, K, Ca, Mg, and Na) for
samples of raw, hydrolyzed-deboned, and acidified fish materials
are listed in Table 2.
Autolysis and deboning resulted in decreased
levels of phosphorus and calcium as cpmpared to starting raw materials for all samples except dogfish shark.
Magnesium levels also
decreased slightly for all samples at this stage of processing.
Acidification of autolysates with phosphoric acid resulted in
increased levels of phosphorus; all samples except orange rockfish
exhibited levels decidedly higher than those for raw fish materials.
Acid stabilization decreased levels of calcium, magnesium, and
sodium in relation to those for autolyzed-deboned samples reflecting
the dilution of the ash contents by added phosphoric acid.
The precipi-
tation of insoluble calcium and magnesium phosphate complexes may
have complicated sampling techniques and might have been a factor
in this observation.
Potassium levels, overall, did not substantially
decrease after acidification.
The addition of 0.2% (wt/wt) potassium
sorbate at the time of acidification probably contributed to the finding.
Table 3 lists the micromineral (Al, Fe, Sr, B, Cu, Zn, Mn,
and Cr) composition of the raw fish materials, autolyzed-deboned,
(0
—I
m
ID
n)
J3
I
O
a)
T3
C
<4
a
o
A
(U
T)
I
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<U
N
n5
-p
o
o
—I
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u
<D
O
u
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(VJ
(U
H
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M
u
a
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z
so
flj
U
W
^
T3
O
u
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f—i
sD in
i—i
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r—(
(M
p—H
•
i—1
r-H
vO
i-H
■*
i-H
CM
oo r~
1—1
i-H
CO
sO
o
CM
i—i
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o
^
in
d d d d d
co m ^
r- ■* oo oo o
CM
i—i
d o d odd
cr- m i—i
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o o
00 ^H sO sO m
^H <\J 00 o vO
r-H r-H O CVJ r—t
f!
O
NO
o oo
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i—i
i—i
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•
00 oo
00 OO
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CM
I—H
d d
in
r- o rO 00 o
00
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CM
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CM
i-H
OO
r-H
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CM
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CM
CM
CO
CM
o
CM
a^
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d
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oo o
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00
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o
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d d
CM
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m r~
00
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d odd d d d d d d odd d d d d d
,-H
CM
o
r- r-
CM
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MOO sD in
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T^
r—(
o o
00 oo
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tCM
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in o co r- ■—i in o CO r^- a- rg t- -o o m CM <M •* oo
o co og o oo i—< o OO CM o "tf -i o (M
CM o
o o o o o o o o o o o o o o O O o o o o
d d d odd d
<M
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oo oo sO o M m o o
m C\] cr> f\] oo CM i—i sO
<\j vO CM ro t- in CM o
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T—1
00
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r—t
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CM
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i-H
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d d d
^
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vO oo m oo CO in oo
oo co oo
CM o
d d d odd
h h h U U u
> > >
X X X Q Q Q 0 Q P ffi pci PJ H H H W H W
oi X < Pi X < oi X < rt x <; PS X < Pi X <
d d d d d
*!
d d odd d d d d d d odd d d d d d d
CO
oo m
00 o (M M
00 (M CO oo
d odd
00
o
m oo rr~ (M o
d
^h
oo
ro
o
O
vD
.—(
Tf
O
ffi ffi ffi
rt ffi <
o
OO
o
c^
«J
M
D
U
M
'si
a
0)
n
26
CO
xl
(U
<->
bo o
lH
O
0
-a
•rH
^! VI
U
co
w
0
n
n
CO
o
CO
X!
ao
•rH
—H
n
Q n g a
W
n
X! T)
M
UJ
^H
m
o
> w fc u W
ffi Q BS H
T)
D
a
O
XI
<u
tJ
T)
<V
N T)
>s (U
o ^
fll
n
>
u
Q
U
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(t)
II
CCi
M XI
II
P
II
II
Pi X < ffi
6
27Table 3.
Micromineral composition of raw, autolyzed-deboned, and
acid-stabilized samples.
Micromineral (ppm wet weight)
Al
Fe
Sr
B
Cu
Zn
Mn
Cr
RH
22.2
23.4
34.2
1.4
1. 3
11.6
2.4
1. 3
HH
13.6
26.9
12.9
1.0
4.8
19-9
1. 3
0.9
AH
77.4
11. 5
7. 1
1.8
2.9
8. 1
0. 3
1.0
RHV
11.8
38.9
16. 1
1.0
2.5
19.1
1.4
0.9
HHV
10.4
39.2
4.6
0.7
4.0
18.6
0.8
0.6
AHV
64.6
27.8
2.5
2.0
1.2
14.4
0. 3
0.9
RD
26.0
39.9
37.2
1.6
1. 3
18.8
2.6
1.6
HD
15.4
55. 3
10.1
1. 1
3. 1
14.2
1.0
0.6
AD
154.2
19.5
3. 3
3. 1
2.4
12. 3
1.0
1.6
RDS
19- 6
21.0
21.0
1.4
1.5
8.6
1.0
1.5
HDS
28,9
81.7
10. 1
1. 1
1.8
8.7
1.2
0.9
ADS
142.8
5.7
5.7
2.5
1.0
6.6
0.2
1.6
RRF
27. 6
18.6
55.6
2. 1
1.5
9-6
0.6
2.7
HRF
12.8
36.7
28.0
1.0
2.5
4.9
0.2
1. 1
ARF
70.2
5.9
2. 5
1.6
1.6
4.8
0.3
1.5
RTC
24.6
29.0
61.8
1.7
1. 5
■ 12. 1
1.7
1.8
HTC
13.4
36.7
20.9
0.8
2.7
10.9
0.8
0.6
ATC
58.4
16.6
12.8
5.2
3.8
15. 3
1.4
3.4
RE
107.0
193. 1
87.6
3.6
2. 1
18. 3.
5.9
3.9
HE
85.6
163.0
24.8
1.9
2.7
14. 6
3.6
1.9
AE
104.6
30.7
7.4
2.4
1.0
8.5
2.0
1.9
Code
1
■
Samples R = rav/, H = hydrolyzed/deboned; A = acidified; H = hake;
D = Dover sole; HV = hake/tuna viscera (70/30 wt/wt); DS = dogfish
shark; RF = orange rockfish; TC = true cod; E = English sole.
28
and acidified (zero time) autolysate samples.
Manganese levels
decreased due to deboning processes for all samples except dogfish
shark.
The removal of bone minerals resulted in increased levels
of iron and copper in all samples.
Acidification subsequently lowered
levels of these minerals by either dilution with phosphorus or through
the formation of insoluble complexes which may have adversely
affected sampling procedures.
Storage Characteristics
Microbial Outgrowth
The influence of ambient temperature storage on the maintenance
of acidic environmental conditions was investigated.
The influence of
storage time on the pH of acid-stabilized fish autolysates is shown by
values listed in Table 4.
Carcass waste (English and Dover sole,
orange rockfish and true cod) and round dogfish shark required the
addition of ca. twice the amount of phsophoric acid (85% w/v) to
achieve a pH of ca. 3. 25 compared to round hake samples.
The in-
creased acid requirement of carcass waste was probably related to
neutralization by a higher bone-fragment and shell content of the
carcass waste over round hake.
The higher bone content of carcass
waste would increase the amount of small bone fragments that would
not be removed by screening.
Carcass wastes also possessed a higher
amount of digestive tract contents per unit weight than round hake
Table 4.
Code
Effect of storage on pH.
% H3P04
(85% w/v)
Storage time (days)
0
1
2
Storage time (mos
3
2
4
6
AH
4.5
3.22
3. 33
3.44
3.36
3. 38
3. 37
3.41
3. 31
3.44
AHV
4.5
3.24
3. 36
3. 38
3. 37
3.40
3.47
3.41
3.53
3.49
AD
8.5
3. 19
3.28
3. 30
3. 30
3. 34
3. 52
3.47
3.41
3.50
ATC
8.5
3.21
3.20
3.21
3.21
3.21
3.19
3.20
3.24
3.19
AE
8.7
3. 17
3.25
3.28
3.25
3.26
3. 34
3.21
3. 31
3.29
ARE
9.0
3. 18
3.20
3.24
3.23
3.24
3. 33
3. 37
3.40
3. 38
ADS
7.8
3.21
3. 10
3.09
3. 10
3. 11
3.07
3.09
3. 13
3.16
1
Samples
A
H
D
TC
=
=
=
=
acidified
hake
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
30
adding proportionately more bone and shell derived from partially
digested food.
The urea content of the dogfish shark sample probably
increased its requirement for acid over round hake.
The pH of all samples, with the exception of true cod and dogfish shark, rose slightly (5-10% of initial values amounting to 0. 120. 31 pH units) over the 8 months storage period.
Increases in pH
were most marked during the initial 4 days of storage, with only
slight fluctuations evident during the remainder of the storage period
(Table 4).
No increase in the pH of true cod or dogfish shark samples
was observed.
Tatterson and Windsor (1974), McBride et al. (I960),
and Hanson and Lovern (1951), found that pH levels for fish silages
prepared with a variety of mineral acids remained constant during
storage at ambient temperature for periods up to one year.
Changes in pH during the initial 4 days of storage reflected the
neutralization of acid by dissolution of bone salts or the delayed establishment of a pH equilibration due to the large sample size (ca. 50
gal).
The sorbate content of all samples decreased during ambient
temperature storage; the majority of change was apparent during the
initial 2 months of storage (Table 5; Figure 1).
The largest decrease
(77%) was observed for the Dover sole sample, while the smallest loss
(39%) was shown by the hake sample.
Pooled retention values (based
on % initial sorbate retained) for samples prepared from carcass
Table 5.
Sorbic acid levels (% wt/wt) of stored acid-stabilized autolysates.
Storage time (mos)
Code
0
2
4
6
8
AH
0.211(100)2
0. 128(61)
0.072(34)
0.023(11)
0.014(7)
AHV
0.230(100)
0.076(33)
0.028(12)
0.023(10)
0.007(3)
AD
0. 197(100)
0.045(23)
0.037(19)
0.026(13)
0.009(5)
ATC
0.201(100)
0.068(34)
0.028(14)
0.017(8)
0.012(6)
AE
0.211(100)
0.086(41)
0.072(34)
0.059(28)
0.030(14)
ARF
0.207(100)
0.056(27)
0.033(16)
0.017(8)
0.017(8)
ADS
0.214(100)
0.066(31)
0.056(26)
0.043(20)
0.019(9)
0.210(100)
0.075(36)
0.046(22)
0.029(14)
0.015(7)
Group
Average
Sample:
A =
H =
D =
TC: =
acidified
hake
Dover sole
true cod
'( ) = % of zero time level
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
100
.a
2
50-
storage (mos)
0=hake
|_J = Dover sole
} = hake/tuna viscera B = true cod
( 70/30 wt/wt)
Figure 1.
/\ = English sole
\/ = dogfish shark
^^ = orange rockfish
Loss of sorbic acid during storage of acid-stabilized autolysates
33
waste (English and Dover sole, orange rockfish and true cod) and
round fish (hake, hake/tuna viscera (70/30 wt/wt), and dogfish shark)
were 28% and 45%, respectively, for the first 2 months of storage.
The log of pooled sorbic acid retention values for all samples
decreased in a linear manner with respect to time (r:=-. 9146,
P> . 005) strongly supporting an exponential decay in stored acidstabilized autolysates (Figure 2).
Statistical inspection of data for
individual samples revealed the rate of decay (as indicated by the
slope) was highest for the hake/tuna viscera (70/30 wt/wt) sample
followed by true cod, hake, Dover sole, orange rockfish, dogfish
shark, and English sole (Table 6).
The correlation of the regression
of the log of retention values with respect to time were all significant
at at least the P> .025 level.
The rate of sorbate decay in acid-
stabilized autolysates did not correlate with the microbiological load
(Table 7) of the samples.
Heat treatments provided during the autolysis and pasteurization
stages of sample preparation greatly reduced the microbial load of
the raw materials.
Although sorbic acid levels decreased greatly,
acid-stabilized autolysates were stable toward microbial outgrowth
during the storage period (Table 7).
This indicates that either preser-
vation was effected solely by the addition of phosphoric acid or mold
inhibition was possibly due to a sorbic acid metabolite.
Marth et al.
(1966) studied the metabolism of sorbic acid by Penecillium species
2.0«
i.sc
O
6^
CO
3
0.5Equation: y = -0. 1379) x + 1. 90
Correlation • oefficient = -. 9155
-1
4
"
""
Storage time (mos)
Figure 2.
Log
of % sorbic acid retained (based on initial content) versus storage.
35
Table 6.
Regression of the log % sorbic acid retained on storage
time (mos).
Code
AH
98872
-.1527
AHV
98202
-.1782
AD
94633
-.1425
ATC
97482
-.15 36
AE
95063
-.09 37
ARF
93003
-.1310
ADS
95423
-.1141
pooled;
91562
-. 1379
Sample: A
H
D
TC
2
P > .005
3
P > .025
m
r
=
=
=
=
acidified
hake
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
Table 7.
Stability of acidified autolysates towards microbial outgrowth (organisms/gm x 10 ),
Storage time (mos)
2
0
4
6
8
45
5.0
6.5
4.8
5. 1
4.6
126
TFC2
3.2
4.6
3.8
3.6
60
3.0
7.8
5.4
12. 1
15.2
35,000
30.0
26.0
34.0
17.2
21.4
AE
560
52.0
12.6
17.0
34.0
18.8
ARF
250
46.0
54.0
36.0
31.0
21. 3
ADS
81
5.5
8.8
6.7
10.6
7. 3
Raw
Code
AH
AHV
AD
ATC
1
Samples
A
H
D
TC
=
=
=
=
acidified
hake
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
'Too few to count
a-
37
of molds and proposed the theory that sorbic acid metabolites may
interfere with dehydrogenases associated with (3-oxidation.
The
metabolism of sorbic acid by molds to an inhibitory compound which
was not detectable may explain both the exponential decay rate previously noted and the stability towards mold outgrowth of the samples.
It should be noted that although the acid-stabilized autolysates
were stable to microbial outgrowth in the 8 month study, several
samples exhibited rapid spoilage shortly after termination of the
experiment.
Although the exact cause of this occurrence was not
determined, it presumably was due to sorbate depletion.
Long term
storage of acid-stabilized autolysates, therefore, would require the
periodic addition of potassium sorbate to maintain microbial stability.
Lipid Stability
The levels of free fatty acids, as an indication of hydrolytic
rancidity, remained relatively constant throughout storage (Table 8).
Since the autolysates were found to be stable toward microbial growth,
this tends to indicate that endogenous lipases were inactivated by
processing and non-enzymatic base-catalyzed hydrolysis was inhibited
by maintenance of acidic environmental conditions.
A wide range of zero time individual sample levels from 3. 4%
for dogfish shark to 22. 30% for hake/tuna viscera (70/30 wt/wt), was
observed.
Initial free fatty acid levels may have been influenced by
Table 8.
Free fatty acid levels (% wt/wt in total lipid expressed as oleic acid) in stored acid-stabilized
autolysates.
Storage time (mos)
1
0
2
4
7. 65 ± 0. 34
5. 85 ±. 0.71
6.28 ± 0. 53
7.91 ± 1.01
6.41 ± 0.42
AHV
22. 30 ± 1.46
19-82 ± 0.90
24. 91 ± 0. 73
23. 36 ± 1. 86
21.47 ± 1. 24
AD
11. 80 ± 0.83
11.93 ± 0.77
11.23 ± 0.66
8.48 ± 0.45
10.47 ± 0.67
ATC
19. 37 ± 1.27
23. 66 ± 1. 31
23.53 ± 0.74
20. 14 ± 0.96
22.76 ± 0.88
AE
7. 10 ± 0. 18
8.90 ± 0. 31
6.86 ± 0. 87
9. 34 ± 0.24
7.44 ± 0. 33
ARF
6.23 ± 1. 17
8.47 ± 0. 69
7.73 ± 1. 11
8. 31 ± 0. 73
8. 08 ± 0. 64
ADS
3. 14 ± 0. 62
2.09 ± 0. 56
6. 19 ± 0.69
4.42 ± 0.84
6. 11 ± 0.93
Code
AH
1
Samples
A
H
D
TC
=
=
=
=
acidified
hake
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
8
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
'n = 2
u>
oo
39
several factors including the handling of samples prior to acquisition,
the composition of samples with regards to endogenous lipase content,
and non-enzymatic base-catalyzed hydrolysis prior to and during
processing.
Carcass and visceral wastes from the fish processing industry
are frequently allowed to remain at ambient temperatures prior to
disposal. During this time the action of endogenous lipases and microorganisms may proceed unchecked.
hydrolysis may occur.
In addition, simple non-enzymatic
The high microbial load of the true cod sample
(Table 7) indicates the existence of such a situation and may explain
the high initial free fatty acid level of this sample.
The lipases of fish are largely confined to organ tissue (Brody,
1965).
Carcass waste samples and hake with added tuna viscera,
therefore, possessed higher levels of endogenous lipases than round
fish samples.
This would presumably result in the production of
higher initial levels of free fatty acids during processing.
A compari-
son of the zero time free fatty acid levels for the hake and hake/tuna
viscera (70/30 wt/wt) samples, 7. 65 and 22. 30% respectively, illustrates this point.
Oxidative rancidity, as measured by 2-thiobarbituric acid
numbers, developed in all samples during storage (Table 9).
Values
for all samples except hake and dogfish shark rose rather rapidly
during the. first 2 months of storage (Figure 3).
The largest increase
Table 9-
Malonaldehyde levels (mg/kg) in stored acid-stabilized autoly sates.
1
1
Storage time ( mos)
4
Code
0
2
AH
2. 31 ± 0. 342
2.96 ± 0.41
AHV
6.96 ± 0.51
AD
6
8
4.81 ± 0. 36
3.86 ± 0.27
5.46 ± 0.73
10.41 ± 1. 11
16.72 ± 1.03
12.42 ± 0.84
11.87 ± 1.99
4.92 ± 0. 67
12. 13 ± 1.46
11.73 ± 0. 38
14.89 ± 1.48
16. 16 ± 1.57
ATC
5.54 ± 0.81
9.43 ± 0.78
12.46 ± 1. 16
8. 19 ± 0.84
6.42 ± 1.89
AE
5.21 ± 0. 36
8.97 ± 0.63
14. 69 ± 1.42
13. 32 ± 1.19
13.92 ± 1.86
ARF
6.85 ± 0.99
20.77 ± 1.61
16.40 ± 1.93
17. 16 ± 1.84
15.98 ± 1.27
ADS
3.54 ± 0.23
3.48 ± 0.40
8.29 ± 1. 10
7. 64 ± 0.66
10.43 ± 0.73
Sample;
A
H
D
TC
=
=
=
=
acidified
hake
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
'n = 3
o
41
storage time (mos)
O = hake
/\ = English sole
^
J^ = orange rockfish
= hake/tuna viscera/ 70/'30 wt/wt)
ll = Dover sole
^f = dogfish shark
H = true cod
Figure 3.
Malonaldehyde levels (mg/kg) in stored acid-stabilized autolysates.
42
was shown for the orange rockfish sample; its 2-thiobarbituric acid
(TBA) number tripled in 2 months.
Tatterson and Windsor (1974)
reported that although the acid-stabilized silage they prepared was
stable to hydrolytic rancidity, oxidative rancidity, as measured by
iodine value, proceeded unchecked throughout ambient temperature
storage.
Statistical evaluation of data for hydrolytic and oxidative rancidity revealed that free fatty acid levels at zero time correlated in a
linear manner with 0 and 4 month TBA values (r = . 9548, P > . 005;
r=.9187, P>.005, respectively).
Conditions which lead to the ap-
pearance of free fatty acids in the zero time samples may have been
conducive to the oxidation of lipids, e. g. , the poor handling of raw
materials with regards to conditions of time and temperature prior
to sample procurement.
A second possible explanation may be that
the free fatty acids present at zero time may have been more susceptible to oxidation than intact triglycerides.
Zero time TBA values correlated (r=. 9346, P > . 005) in a
linear manner with 4 month values.
Since lipid oxidation proceeds
through hydroperoxide formation and free radical propagation, the
presence of oxidized lipid at zero time would explain the continued
increase to 4 month values.
The TBA numbers for samples of orange rockfish, hake/tuna
viscera (70/30 wt/wt), and true cod rose and subsequently decreased
43
during storage (Figure 3).
Variations may have been due to sampling
techniques or a portion of the malonaldehyde originally released
through oxidation may have reacted with protein present in the sample
to form a linkage which inhibited detection.
Protein stability
The protein fraction of the acid-stabilized autolyzed fish
samples was stable toward acid catalyzed proteolysis during storage.
The levels of free amino groups (as measured by ninhydrin)
did not appreciably vary with time (Table 10).
The stability of the
samples towards mierobial outgrowth and the inactivation of endogenous proteases during pasteurization (180 F/83. 7 C for 5 min) are
most likely responsible for this observation.
Sullivan (1976) reported
the complete inactivation of proteases isolated from the pyloric cacea
of albacore tuna fish (Thunnus alalunga) by heating to 70 C for 10 min.
Work by Katsuma et al. (1974) supported these findings.
The levels of free amino groups indicate that the degree of
proteolysis that occurred during processing was the greatest for the
hake/tuna viscera (70/30 wt/wt) sample, followed by hake, the carcass wastes (English and Dover sole, orange rockfish and true cod),
and dogfish shark.
The apparently higher degree of proteolysis for
whole hake, as compared to the carcass wastes whose content of
viscera was higher, is interesting.
The feeding rate of fish greatly
Table 10.
Proteolysis (mg glycine equivalents/16 gm total N) in stored acid-stabilized autolysates.
Storage time (mos)
Code
2
4
6
8
8.94 ± 1.04
9. 36 ± 1. 36
9.69 ± 0.89
9.42 ± 1. 11
11. 67 ± 1.46
12.21 ± 1.06
11.99 ± 1.40
11.44 ± 0.78
11.81 ± 1. 17
AD
6.45 ± 0.89
6.74 ± 0.96
6. 32 ± 0.74
7. 01 ± 1.03
6.49 ± 0.94
ATC
7.02 ± 0.70
8. 13 ± 1.24
7.97 ± 1. 13
7.46 ± 1.45
7.55 ± 1.20
AE
6. 31 ± 0.86
6. 30 ± 0.97
6.02 ± 0.73
6.41 ± 1. 14
6.68 ± 1.23
ARF
7.26 ± 0.71
6.59 ± 0.90
7.03 ± 1. 33
6.78 ± 1.26
6.92 ± 1. 34
ADS
3.69 ± 0.81
3.41 ± 0.96
3.05 ± 1. 18
3. 17 ± 0.64
3. 36 ± 0.59
AH
AHV
1
Sample;
0
9.51 ± 1.212
A
H
D
TC
=
=
=
=
acidified
hake
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
n = 2
4^
45
affects the proteolytic activity of the gastrointestinal tract (Kaiwai
and Ikeda, 197 3).
Although the carcass wastes from groundfish con-
tained a higher proportion of viscera, it cannot be directly assumed
that the total proteolytic activity per unit weight of material was
greater than for round fish due to possible differences in feeding
states prior to harvest.
McBride ^t al. (I960) showed that liquifica-
tion rates for silages prepared from whole versus gutted herring
were essentially equal.
Members of the elasmobranchii, namely sharks, rays, and
skates, are known to contain rather high levels of urea in their
tissues.
Between 42 and 55% of the osmotically active blood solutes,
amounting to approximately 2-2. 5% (wet weight) of total tissues, are
in the form of urea (Smith, 19 36).
The nitrogen content of urea would
be determined in the Kjeldahl protein determination, but the ninhydrin
assay would fail to detect its presence.
Since the value for free amino
groups was based on total Kjeldahl nitrogen, the presence of urea
would theoretically result in low values for dogfish shark.
The levels of available e-amino lysine in acid-stabilized hydrolysates were not affected by storage (Table 11).
The available lysine
content of carcass waste samples was lower than that of whole hake.
The addition of 30% tuna viscera to the sample of whole hake reduced
its lysine content.
The absolute levels of available e-amino lysine
in the acid-stabilized autolysates of carcass waste samples are
Table I].
Available e-amino lysine content (gm/16 gm total N) of stored acid-stabilized autolysates.
1
Code
6
8
AH
6.42 ± 0.312
6.89 ± 0.46
6.91 ± 0. 15
6. 64 ± 0.61
6. 51 ± 0. 34
AHV
5.89 ± 0. 35
6.02 ± 0. 58
6. 36 ± 0.23
5.74 ± 0. 13
6. 17 ± 0.40
AD
5. 31 ± 0-61
5. 12 ± 0.22
5.69 ± 0. 37
5. 34 ± 0.46
5. 41 ± 0.71
ATC
5.56 ± 0. 14
5. 36 ± 0.66
5.89 ± 0.43
5.26 ± 0.77
5. 40 ± 0.58
AE
6.08 ± 0. 34
5.74 ± 0.49
6.70 ± 0.21
5.93 ± 0. 39
5. 59 ± 0.61
ARE
5.77 ± 0. 54
5.46 ± 0. 39
5.62 ± 0.46
5.99 ± 0.20
5. 36 ± 0.58
ADS
4.63 ± 0.28
4.81 ± 0.83
4.47 ± 0. 35
4. 55 ± 0.40
4. 42 ± 0.81
Sample:
n
0
Storage time (mos;!
4
2
A
H
D
TC
=
=
=
=
acidified
hake
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
orange rockfish
dogfish shark
47
somewhat lower than those reported by Crawford (1976) for pooled
raw samples of carcass wastes of the same species.
Autolysis may have converted some free e-amino lysine to
lysine with both a and e groups free reducing the amount of monoderivative available for determination by the procedure used.
It is
also possible that some lysine destruction or interaction with oxidized
lipid breakdown products occurred during processing.
The values for
dogfish shark are lower because of the nitrogen contribution of urea
to total sample nitrogen.
Tryptophan is an acid labile, essential amino acid.
Possible
destruction during prolonged storage in an acid environment would
decrease protein quality.
Tryptophan levels were stable in acid-
stabilized autolysates stored up to 8 months at ambient temperature
(Table 12).
Carcass waste samples (English and Dover sole, orange
rockfish and true cod) had higher overall levels of tryptophan than
hake, hake/tuna viscera (70/30 wt/wt) and dogfish shark.
The
tryptophan levels for acid-stabilized carcass waste samples were
approximately 30% higher than those for raw carcass wastes of the
same species as reported by Crawford (1976).
The method of analysis
used in this investigation (Spies, 1950) has been criticized previously
for yielding high values (Spies, 1967).
However, for this investiga-
tion the detection of overall changes in levels, rather than absolute
content, was the objective.
Table 12.
Code
Tryptophan content (gm/16 gm total N) of stored acid-stabilized autolysates.
1
2
Storage time (mos)
4
AH
1.78 ± 0.212
2.04 ± 0. 37
1.69 ± 0.09
1.90 ± 0.46
2. 17 ± 0. 18
AHV
2.21 ± 0. 36
2.46 ± 0.42
2. 05 ± 0. 17
2. 19 ± 0.20
2. 31 ± 0. 36
AD
2.89 ± 0. 11
3.07 ± 0.23
2.64 ± 0.47
3. 11 ± 0. 38
2. 69 ± 0.21
ATC
2. 63 ± 0.09
3. 18 ± 0.22
3.02 ± 0. 31
2.70 ± 0. 17
3. 37 ± 0.44
AE
3. 14 ± 0. 33
3.46 ± 0.26
2.99 ± 0. 16
3.27 ± 0.25
3. 35 ± 0. 32
ARF
3.06 ± 0. 13
2.88 ± 0.41
2.96 ± 0. 37
3.41 ± 0.40
3. 29 ± 0. 19
ADS
0.94 ± 0. 16
1.22 ± 0. 34
0.76 ± 0. 16
0.85 ± 0. 11
1. 04 ± 0.24
Sample:
A
H
D
TC
=
=
=
=
acidified
hak e
Dover sole
true cod
HV
E
RF
DS
=
=
=
=
hake/tuna viscera (70/30 wt/wt)
English sole
or■ange rockfiLSh
dogfish shark
'n = 2
oo
49
Protein Quality Stability
The protein efficiency ratio of samples of acidified hake,
English sole, orange rockfish, and dogfish shark autolysates stored
for 0, 4, and 8 months at ambient temperatures was determined.
Isonitrogenous diets, including a ANRC casein control, were utilized.
Factorial analysis of data, excluding that for casein, showed
that sample source and length of storage both significantly affected
protein efficiency ratios (PERs) (Table 13).
Weight gain was simi-
larly affected, while feed consumption was affected significantly only
by sample source.
The interaction of sample source and storage
time was significant for PER values, feed consumption, and weight
gains (P > . 01; P > . 05; P > . 005, respectively).
Acidified autolysates of hake and dogfish shark yielded feed
consumptions and weight gains superior to orange rockfish and
English sole.
fish shark.
The PER value for hake was superior to that for dog-
Acidified orange rockfish and English sole autolysates
yielded PER values which were inferior to dogfish shark and hake.
Storage of samples resulted in significantly (P > .05) inferior
weight gains and PER values.
Protein quality reduction occurred
during the first 4 months of storage.
equal at 4 and 8 months of storage.
affected by storage time.
PER and weight gain were
Feed consumptibn was not
Table 13.
Protein efficiency ratio (PER) of stored acid-stabilized autolysates.
of variance; factorial design.
Summary of analysis
F-- value
Sample
Storage
3
12.472
38. 933
Feed (gm)
9.221
6. 691
4
4.09
Gain (gm)
72. 373
10. 582
PER
36. 13
Sample x Storage
6.71
Factor Mean Ranking'
Sample
Storage
PER
AH > ADS > ARF > AE
0 > 8 > _4
Feed (gm)
ADS > AH > ARF > AE
0 > 4 > 8
Gain (gm)
AH > ADS > ARF > AE
0 > 4 > 8
Significant at P > .05
2
Significant at P > .01
3
Significant at P > . 005
4
NS at P > .05
Mean values with same underline did not vary significantly (P=. 05) from each other
Sample: A = acidified
H = hake
E = English sole
DS = dogfish shark
RF = orange rockfish
o
51
Analysis of variance including data for the casein control
showed that feed consumption, weight gain, and PER values varied
significantly at levels of P > . 05, P > . 025, and P > . 005, respectively (Table 14).
Although factorial analysis showed PER values affected by
storage time, inspection of individual sample means (Table 14) indicated that ratios for 0, 4, and 8 month samples of acidified hake,
English sole, and orange rockfish did not vary significantly ( P = . 05).
The dogfish shark sample showed a significant PER decrease during
the first 4 months of storage, but yielded a value at 8 months equal
to the zero time sample.
Weight gains for samples of hake and English sole autolysates
did not vary significantly with respect to storage time.
Storage of
orange rockfish and dogfish shark autolysates yielded significant
decreases in weight gains at 4 months.
A further reduction (though
not significant with the number of replicates involved) in weight gain
was observed at 8 months for orange rockfish, but the weight gain
for dogfish shark was equal to that for the zero time sample.
Though not revealed by factorial analysis, consumption of
acidified orange rockfish was significantly (P = . 05) reduced by
storage.
Feed consumption for other samples did not vary with
storage time.
The relatively high degree of oxidative rancidity that
developed in the orange rockfish sample during storage, which was
Table 14.
Protein efficiency ratio (PER) of stored acid-stabilized autoly sates. Summary of analysis
of variance. Randomized block design; ranking of individual treatment means.
Code
Storage time
(mos)
Feed
consumption
AH
0
363. 0
AH
4
363. 1
AH
8
349. 6
AE
0
317.4
AE
AE
ARF
0
4
ARF
8
ADS
0
ADS
4
Casein
Sample;
8
ARF
ADS
2
4
8
_
ab
ab
abc
cd
298. 8C
284.7C
385. i'
bed
325. 1
cd
310.7
369.2C
ab
364.4
ab
358. 2
372. 62
Weight
gain3
PER
% of ANRC
casein
100. 9
.ab
95. 8'
abc
93. 2
ef
67.4
3.05
103.7
f
60. 6
60. 3f
de
77. 6
ef
71.5
63. 5i
ab
99. 1
cd
83. 6
be
88. 8
103. 8'
ab
2. 91"
2.93
ab
def
2. 39*
2.23
2. 34
2. 54
2.41
f
def
cde
def
.ab
2.97'
2. 72
2.94
99.8
81. 3
75. 9
79- 6
86.4
81.9
77.6
2.28
2. 56
98.9
cde
be
ab
101.0
87. 1
92.4
100.0
A = acidified; H = hake; E = English sole; RF = orange rockfish; DS = dogfish shark;
3
4
P > . 05; P > . 025; P > . 005; Mean values with same exponent letter did not vary significantly
(P= . 05) from each other.
53
reflected by a three-fold increase in the 2-thiobarbituric acid number
after 2 months of storage (Figure 3), appeared to have played a role
in mediating feed consumption.
The regression of feed consumption
for all samples on TBA values decreased in a linear manner
(r=-.7999; P > .005).
Feed consumption for samples of acidified hake and dogfish
shark autolysates and the zero time sample of orange rockfish was
not significantly (P=. 05) different than that for casein.
The lower
feed consumption of acidified English sole and the 4 and 8 month
samples of orange rockfish may have been responsible for their
decidedly lower weight gains.
Protein efficiency ratios for all samples of acidified hake and
the 0 and 8 month samples of stabilized dogfish shark did not vary
significantly (P=.05) from the ANRC casein control (Table 14).
PER
determinations for round dogfish shark were complicated by the
presence of urea, resulting in lower PER values than what would be
obtained if based solely upon protein nitrogen.
The protein quality of acid stabilized English sole and orange
rockfish was inferior to casein and round fish samples.
Crawford
(1976) evaluated the protein quality of several carcass wastes as
compared to round turbot.
Carcass wastes yielded PER values that
were 80.9 to 95.7% of round turbot ratios.
Olley et al. (1968) found
the quality of visceral proteins of herring and whitefish, as
54
determined by net protein utilization studies, to be 72. 5 and 76. 9%,
respectively, of whole herring.
Low levels of histidine and possibly
lysine and production of toxic factors, including histamine, prior
to processing were theorized to be responsible.
The higher content
of nutritionally inferior visceral protein in the carcass waste samples
was most probably involved in their reduced PER values.
The more rapid rate and overall higher levels of oxidative
rancidity in the carcass wastes (Table 9) may also have played a role
in reducing their protein quality.
The regression of PER values on
TBA numbers decreased in a linear manner (r=-.8424; P >.005).
Autooxidation of lipids yields a variety of products, including free
radicals, hydroperoxides, and carbonyls, which may react with
proteins to form indigestible crosslinks or block essential amino
acids, resulting in a decrease in protein quality (Fennema, 1976).
Roubal (1970), working with solid state model systems utilizing
isolated rockfish myofibrillar protein, showed that the amino acids
most sensitive to free radical attack were* in decreasing order,
cystine, tyrosine, methionine, alanine, and lysine.
Shin et al.
(1971) found the pH optimum for crosslinking of ribonuclease with
malonaldehyde to be between pH 4 and 5.
Although the lysine levels
of acid-stabilized carcass waste samples appeared to remain constant,
it may have been possible that lipid oxidation products reacted with
55
proteins, reducing their nutritional quality, in a manner that was
not detected.
56
SUMMARY
The effects of ambient temperature storage on the chemical and
nutritional characteristics of phosphoric and sorbic acid stabilized
autolysates, prepared from fishery processing wastes and undesirable species, were investigated.
The study yielded the following resultsi
(1) The deboning stage of processing reduced sample ash contents,
with the exception of dogfish shark; this was reflected by decreased levels of bone minerals, particularly calcium and
phosphorus.
Acidification with phosphoric acid increased
sample ash and phosphorus contents to levels decidedly higher
than for starting raw materials.
(2) Acidified autolysates were stable to microbial outgrowth during
8 months of ambient temperature storage despite slight increases in pH levels and an apparently logarithmic loss of
sorbic acid with respect to time (r=. 91458, P >. 005).
(3) Hydrolytic rancidity, as monitored by free fatty acid levels,
did not progress during storage.
Inactjvation of endogenous
lipases during pasteurization, maintenance of acidic environmental conditions
inhibiting base catalyzed hydrolysis, and
stability toward microbial outgrowth contributed to fat stability.
(4) Oxidative rancidity proceeded in all samples during storage.
57
Initial free fatty acid contents correlated with 0 and 4 month
levels of malonaldehyde, as determined by 2-thiobarbituric acid
(TBA) numbers (r=:. 9548, P >. 005; r= . 9187, P> .005, respectively).
The regression of 4 month TBA values on zero
time values increased in a positive manner (r^. 9346, P > . 005).
(5) Proteolysis, as monitored by the appearance of free amino
groups, did not occur during storage of acid-stabilized autolysates; inactivation of endogenous proteases during pasteurization
and the stability of samples toward microbial outgrowth were
responsible.
Levels of available e-amino lysine and tryptophan
remained constant in stored acid-stabilized autolysates.
(6) Protein quality, as measured by protein efficiency ratios (PER),
was not affected by storage in samples of acidified hake, English
sole, and orange rockfish.
Ratios for all samples of hake and
the 0 and 8 month samples of dogfish shark did not vary significantly (P=. 05) from the casein control.
English sole and orange
rockfish samples yielded PER values which were inferior to
casein and roundfish samples (P=. 05).
The regression of feed
consumption and PER values on TBA numbers decreased in a
linear manner (r=-. 7999, P >• 005; r=-.8424, P >. 005, respectively).
Higher contents of nutritionally inferior visceral pro-
tein and increased levels of oxidative rancidity in the carcass
58
waste samples were probably responsible for their reduced
protein qualities.
59
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