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Nutritive Utilisation of Canned Sardine Proteinin Olive Oil The Influence of the DifferentElaboration Process Stages and Storage for6 Months and 5 Years

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J Sci Food Agric 1996,72,135-140
Nutritive Utilisation of Canned Sardine Protein
in Olive Oil: The Influence of the Different
Elaboration Process Stages and Storage for
6 Months and 5 Years
Baltasar Ruiz-Roso Calvo de Mora, * Mercedes Perez Alvarez-Quiiiones,
Isabel Cuesta de la Plaza, Manuela Garcia-Cuevas, Gregorio Varela Mosquera
Department of Nutrition, Faculty of Pharmacy, Complutense University, Avda Complutense s/n,
Madrid 28040, Spain
(Received 10 October 1995; revised version received 13 February 1996; accepted 10 May 1996)
Abstract: The true digestibility coefficient (TD), the metabolic utilisation (BV)
and the net protein utilisation (NPU) of canned sardines from the same production lot have been studied in the different process stages of conservation and
maturing (canned): raw (RS), precooked (PS), just canned (CS) and with 6
months (6MS) and 5 years (5YS) of storage. We find that during the process there
are no significant quantitative fish protein losses. Qualitatively, the PS show the
lowest digestibility values (TD, 0.90 & 0.02) and metabolic utility (BV,
0.55 & 0.08). The highest digestibility is showed for the CS (TD, 0.94 0.01) and
the highest BV for the 6MS (0.65 & 0.07). All these values are high if they are
compared with their casein controls (TD, 0.95, BV, 0.71), the 6MS have the
highest NPU (0.61) and although this value decreases in the 5 years of storage
(NPU, 0.56), the differences are not significant.
Key words: sardine, protein, canned, maturation.
(Bender 1984). Regarding fish protein, in the scientific
literature there is insufficient information about nutritional modifications in the canning process and in the
resulting maturation of the stored product. It appears
that the protein quality can be affected during the
process (Garcia Arias 1989). There is no reason to think
that the total protein content of the canned food was
reduced during the storage. However, it is obvious that
if the time and temperature of the storage is increased
the protein bioavailability is diminished (Kramer 1986).
The same fact has been confirmed in canned tuna with
respect to the time of storage (Garcia Arias 1989). So,
the influence of the canning process and the storage
time on the protein nutritive quality of sardines has
been studied.
Spain, together with Portugal, has the highest fish consumption per person (76 g day-') in Europe (INE
1991). The canning of sardines is a very popular preservation process for this food. The surplus of this food is
stabilised and an important market value is reached
(Varona 1986). However, in this process the fish is subjected to treatments that may modify its composition
and the nutritive value. The changes must be taken into
account to determine the nutritive value of the fish
when it is consumed, that is to say its real nutritive
value (Moreiras-Varela 1966; Ruiz-Roso 1983; Varela
1985; Moreiras-Varela and Ruiz-Roso 1986; Krzynowck 1987; Varela et a1 1990).
The preparation of canned fish is a complex process
that consists of different phases in which nutrient composition can be modified. Several nutrients, aminoacids,
minerals, vitamins etc, can pass into the coating oil
The nutritive value of raw sardine protein and the influence of the canning process on it were studied in order
to analyse the specific or additive effect of precooking
* To whom correspondence should be addressed.
J Sci Food Agric 0022-5142/96/$09.00
0 1996 SCI. Printed in Great Britain
(baking and dehydration), coating oil addition, sterilisation and maturation time over 5 years of storage.
Therefore, isocaloric diets adapted to the rats requirements during the growth period were prepared; the sole
source of protein was the different kinds of sardines:
raw and the different canning and maturation phases.
Wistar rats in their growth period were used.
B Ruiz-Roso et a1
food and the coating oil, all the samples were immediately frozen to -40°C once the sampling had been
performed. After draining the coating liquids, the fishes
were dried at 60°C in vacuum and stored at -40°C in
nitrogen atmosphere.
Sardina pilchardus species sardines weighing roughly
93 g and with an average length of 22 cm were
employed in our assays. They were caught in Sada
(Spain) during the month of July 1988, and refrigerated
in the fishing ship at 3-4°C for approximately 24 h.
Then the fish was canned in the canning factory Bernardo Alfageme SA (Vigo, Spain) with the same
machinery and under the same conditions as usually
adopted. The treatment began with the insertion of the
sardines (nearly 120 kg) into a saturated NaCl solution
for 6 h. After this, the upper third, fin and viscera were
removed by hand, up to 50% of the initial sample
weight was lost. The remaining pieces were then canned
into 105 x 60 x 20 mm cans three per can; 20 cans
were randomly chosen and these are referred to as raw
sardines (RS).Then the fish cans were steam cooked at
110°C for 30 min in a continuous system type Flash
cooker, followed by a drying and degreasing phase with
a hot air flow at 110°C for 15 min. Shortly after the
precooking process a second sampling was carried out;
ie 20 randomly chosen cans (precooked sardines, PS).
After precooking, 33 g of refined olive oil (Aceites
Toledo Refineria Andaluza, SA) was added to each can
by means of an automatic injection system. Fatty acid
composition of the olive oil was as follows (g per 1 kg of
fat): C16 : 0, 118; C16 : 1, 5; C18 : 0, 29; C18 : 1, 773;
C18 : 2, 64; C18 : 3, 5 and C20 :1, 6. Immediately after
this the cans were closed air-tight by a continuous cycle
machine without overflow. Once closed they were submitted to an sterilisation procedure for 45 min at 115°C
and a pressure of 1.72 kPa gauge in a steam vertical
autoclave. Next the cooling and washing of the cans
was achieved through a shower system. Again, 20 randomly chosen cans were sampled (just canned sardines,
When this canning process finished and before the
entering the market, the product is stored at room temperature for 6 months. In these 6 months the maturation preservation process takes place. After 6 months
another sampling was done, 20 randomly chosen cans
referred to as: 6 months maturation sardines (6MS).
Since the maturation process continues up the moment
of consumption, we took another sample of 20 cans
sample 5 years after storing them (5YS).
In an attempt to stop any further modification of fish
composition or interchange of substances between the
Chemical analysis
The following determinations were carried out in all
samples of sardines: moisture, by weight loss at 105°C
to constant weight; total protein was determined by the
Kjeldahl procedure (kjeltec autoanalyst Auto 1030,
Tecator, Sweden) using a 6.25 conversion factor (AOAC
1975); total lipid was extracted from dried samples with
petroleum ether 40-60°C in a Soxtec System 1040
Tecator extraction Unit, with the ether extract gravimetrically evaluated; and ash by incineration in a
mume furnace at 450-500°C to constant weight (AOAC
Rat bioassays
Isocaloric and semi-synthetic diets were prepared with
the dried fishes as the sole source of protein prepared
from the different sardine samples. Assay 1: raw (RS)
and different canning (PS, CS); assay 11: 6 months
maturation (6MS); assay 111: 5 years maturation (5YS).
Likewise, a control diet with casein and DL-methionhe
(20 g kg-' total protein) and a 'white diet' without
protein were used to determine the endogenous elimination of nitrogen from the animals. Moisture, total
protein, fat and ash were determined in the diets.
The composition of the diet per 1 kg was: protein,
casein (Casein alkaliloslich. E Merck, Darmstadt,
Germany) DL-methionine (E Merck) or sardine,
120 g; fat (olive oil Carbonell SA) 98 g; microcrystalline
cellulose, 50 g; mineral mixture (Salt Mix AIN-76,
Dyets Inc, PA, USA) 33.43 g (composition in mg kg-'
of diet): Ca, 5.2; P, 4; K, 3.6; Na, 1.02; C1, 1.56; S, 337;
Mg, 507; Fe, 35; Cu, 6; Mn, 54; Zn, 30; Cr, 2; I, 0.2; Se,
0.1; vitamin mixture (Vitamin mix AIN-76, Dyets Inc)
1.168 g (composition per kg of diet): thiamine HCl,
6 mg; riboflavin, 6 mg; piridoxine HC1, 7 mg; niacin,
30 mg; calcium pantothenate, 16 mg; folic acid, 2 mg;
biotin, 0.2 mg; cyanocobalamin (BIZ), 10 pg; menadione sodium bisulphite 0.8 mg, vit A, 4000 IU; vit E,
50 IU; vit D3, 1000 IU; equal parts wheat starch and
saccharose to complete lo00 g. When raw and processed sardines were used as protein source, fat was
included simultaneously in the diet. Thus, the amount
of olive oil included in the diet was reduced to compensate for the amount of fat supplied by the sardine.
The macronutrient content of the sardines is given in
Table 1.
Wistar rats were used, 54 males and 54 females, from
the Animals Service (Department of Nutrition, Faculty
Nutritive utilisation of canned sardine protein in olive oil
Moisture (g kg-') and protein, fat and ash (g kg-' dry
matter) contents" of raw and processed sardines
ANCOVA) was used to determine the differences
between experimental groups. By means of statistical
tests 'F' and 'Tukey', the difference was checked and
quantified in the control lots (casein DL-methionine)
in the three periods. The significance of the results was
established at the P < 0-05 level. All the statistical estimations were carried out with the BMDP 7D program
(University of California 1981).
630 f 0.5
567 f 1.0
559 f 3.0
541 & 0.2
476 k 49.6
508 f 4.4
601 f 8.7
537 f 16.4
531 f 5.5
527 f 3.9
406 f 8.8
315 f 1.1
359 f 9.4
362 f 4.1
365 f 7.3
92 f 3.8
88 f 1.8
84 f 1.3
84 +_ 1.8
84 f 1.6
Values are means f standard deviations.
sardines; CS, just canned
sardines; 6MS, 6 months maturation sardines; 5YS, 5 years
maturation sardines.
* RS, raw sardines; PS, precooked
of Pharmacy, Complutense University, Madrid, Spain),
selected from the same colony with an initial mean
body weight of 58 g and maximum weight differences of
2 g. During the experimental periods the animals were
placed in individual metabolism cages as Dra Schiller
model, in an environmentally controlled room maintained at 22 & 2°C with a 12 h light-12 h dark cycle
and properly ventilated by means of an air renewal
system. The evaluation of the protein nutritive value
was carried out by the nitrogen balance technique
according to Miller and Bender (1955), allowing estimation of the digestible and metabolic utilisation of the
nitrogen. The experimental period was 10 days, the first
3 days were for the adaptation to the diet and the 7
remaining ones were to determine the digestibility of the
protein. Animals were fed ad libitum. The weight and
the intake between the third day and the tenth day were
monitored, also during this period the faeces were collected, weighed and stored in plastic individual bags at
- 18°C until analysed. On the tenth day, the rats were
killed with chloroform and immediately each one was
put into an 1000 ml flask with 400 ml of 6 M HCl and
kept for 24 h in a bath at 100°C. The resultant liquid
from the acid hydrolysis of the rat was filtered and
made up to 1000 ml with distilled water. The whole
body and faecal nitrogen were determined in this liquid
and in faeces respectively by the Kjeldahl procedure
(AOAC 1975).
The following indexes were calculated :
Food efficiency ratio (FER) = weight increase in g/
ingested dry food in g
True digestibility coefficient (TD) = (N ingested--(N
excreted, corrected for endogenous N))/N ingested
Net protein utilisation (NPU) = whole body N, corrected for endogenous N)/ingested N
Biological value (BV) = NPU/TD.
Statistical analysis
An analysis of variance and co-variance (ANOVA-
Quantitatively, during the canning process there was a
dehydration and defatting in the precooking and this
corresponded to an increase in the protein level of the
fish (Table 1).
Intake and weight data of the rats is shown in Table
2. The summary of this is included in the calculated
indexes mentioned now. With our procedures the FER
(Table 3) was lower in the precooking sample
(0.19 k 0.05) than in the raw sardines (0-21k 0.06) and
the just canned sardines (0.23 f 0.03), although there
were no significant differences between these samples.
However, there was a significant increase in the 6
months canned sardines (0.31 & 0.05) and the value at 5
years was 0.21 k 0.03, the same as the CS.
The TD (Table 3) increased significantly from
0.91 k 0-02 for raw to 0.94 f 0.01 for just canned sardines. There were no further significant changes during
the 6 months of storage (0.93 & 0.01) but a significant
reduction at 5 years of storage (0.91 f 0.02). This value,
although significantly lower than just canned sardines,
was high and the same level as the raw sardines.
The metabolic utilisation of the sardine protein indicated by the BV (Table 3) was reduced in the CS
(0-55 k 0.04) and precooked (0-55& 0.08) samples in
respect of raw sardines (0.64 k 0-19) and was significantly different to the BV of the casein (0.71 & 0.08).
However, at 6 months storage the initial value
(0.65 k 0.07) was observed. Maturation for 5 years did
not influence significantly the BV of the protein
although a falling trend was observed (0.62 f 0.05)
compared with 6 months, but it was the same level as
the RS.
The NPU, is a combined measure of digestibility and
metabolic utilisation. A reduction in NPU of precooked
sardines (0.50 & 0.08) and in just canned sardines
(0.52 & 0.04) was observed which were statistically different to the control value (067 f 0.07). Afterwards, the
control value was almost reached at 6 months
(0.61 k 0.07) but it was reduced at 5 years (0.56 k 0.05),
although this value was similar to raw sardines
(0.58 & 0.17) (Table 3). In spite of these changes the
NPU remained high and nutritionally adequate compared with the casein + DL-methionhe control.
B Ruiz-Roso et a1
Our results show that the first stage of the canning
process, the precooking, induced significant losses of
FER, digestibility and BV of sardine protein (Table 3).
Other authors report that excessive heat treatment leads
to transformations in the proteins that reduce their
digestibility (Neucere and Cherry 1982). In accordance
with this, there are some studies carried out by Seet and
co-workers (1983, 1985) and Tanaka and Kimura (1988)
that report slight but significant losses of digestibility in
tuna after the canning process. Other studies report that
baking does not modify the digestibility of the fish, particularly ‘bonito from the north’ (Garcia Arias 1989).
Thus, Banga et al(1992) and Aitken and Connell(l979)
concluded also that the thermal process of the canned
tuna or other kind of fish did not affect to the nutritional parameters significantly. This is also reported by
different authors (Varela et a1 1963; Sidwell 1976;
Udarbe et a1 1985).
The losses in digestibility, food efficiency and biological value that we found, can be the result of the precooking, since this is the only stage in which heat is
applied with the can open. Heating the fish in these conditions gives rise to complex changes in the muscular
proteins such as denaturation, involving their molecular
aggregation with products of lipid oxidation
(Synowiecki and Shahidi 1991). It is well known that
lipid hydroperoxides can induce oxidative changes in
the sulphoamino acids and in this way can cause nutri-
Daily intake and body weight increase in rats fed on diets with and without protein
(casein and raw or processed sardines)”
Number of
initial weight
(y as is d a y - ’ )
62.3 f 1.35
62.0 f 1.26
62-2 f 1.78
63.4 i-1.28
62.6 f 1.69
5.17 & 0.73
8.35 _+ 0.61
9.28 2 1.16
10.11 & 1.16
11.37 _+ 0.86
- 1.3 & 0.18
Assay 11
53.5 F 1.69
53.4 1.11
53.6 i 1.74
5.11 k 0.57
9.95 5 1.50
10.5 & 1.04
- 1.26 f 0.13
Assay i I i
58-0 1.25
57.8 f 1.93
58.1 f 1.30
4.2 5 0.50
9.1 k 0.92
8.64 1.00
-1.37 5 0.22
3.51 & 0.55
2.36 f 0.45
Assay I
Weight increase
(g d a y - ’ )
2.51 f 0.40
1.71 f 0.46
1.70 _+ 0.59
2.37 f 0.45
2.75 2 0.49
2.83 _+ 0.73
” Values are means
standard deviations.
CS, just canned sardines; PS, precooked sardines; RS, raw sardines; 6MS, 6
months maturation sardines; 5YS, 5 years maturation sardines.
The protein nutritive value of casein and raw or processed sardines“
0.21 f 0.06ae
0.91 f 0.02ad
0.64 f 0.19
0.58 +_ 0.17
0.19 0.05ae
0.90 f O.02ade
0.55 f 0.08a
0.50 f 0.08a
0.23 k 0.03ae
0.94 f OeOlbcf
0.55 f 0.04a
0.52 f 0.04a
0.31 f O.OSbcdf
0.93 f 0.01acf
0.65 k 0.07
0.61 f 0.07
0.21 f 0.03ae
0.91 f O.02ade
0.62 & 0.05
0.56 0.05a
0.33 f 0.04bcdf
0.95 0.01bcef
0.71 f 0.08cd
0.67 f 0.07cdf
Values are means & standard deviations.
RS, raw sardines; PS, precooked sardines; CS, just canned sardines; 6MS, 6 months maturation sardines; 5YS, 5 years
maturation sardines; FER, food efficiency ratio; TD, true digestibility coefficient; BV, biological value; NPU, net protein
utilisation. The value against which a letter is entered differs significantly ( P < 0.05) from the value for casein a, RS b, PS c,
CS d, 6MS e, 5YS f.
Nutritive utilisation of canned sardine protein in olive oil
tional losses (Nawar 1984). The lipid oxidation can lead
to other nutritional changes that include losses of essential fatty acids, liposoluble vitamins and essential
aminoacids (Eriksson 1987) and also toxicity development (Ames 1983; Pearson et a1 1983). This has important consequences for such processes as fish drying and
salting (Smith and Hole 1991) where lipid oxidation
induces a colour change (to brown) as well as a flavour
change of the fish. This can also reduce the protein
nutritional value (Maruf et al 1990). Astawan et a1
(1994) proved that dried-salted fish stored for 3 months
had lower values of protein quality compared with
either casein or fresh fish, but the nutritional quality
was still high; although this quality was reduced after 6
months of storage.
All of this would contribute to the fall in the protein
nutritional value that is caused in the precooking with
consequent dehydration of the canned sardines.
However, we were surprised to observe that of all the
nutritive value parameters recovered after the coating
oil addition and can sterilisation, except only the precooked sardines (Table 3). Of course, the coating oil
addition, closing and sterilisation modify the sardine
texture as reported by some authors (Adrian 1974;
Bender 1978; Raughunath et a1 1995) and would affect
positively the protein digestibility. This fact does not
explain the recovery of the nutritive value because the
protein degradation is irreversible (Bender 1978) thus
the recovery of the sardine protein NPU could be due
to the elimination from the food of the products of oxidative alteration along with the oil as these products,
could contribute to the reduction in protein bioavailability when the can is closed (Lea et a1 1960;
Desai and Tappel 1963; Andrews et a1 1965; Miller et a1
1970; Nawar 1984; Mauron 1986). It should be noted
that Navarro and Garcia Arias (1992) report that sterilisation of canned tuna without oil for 90 min caused a
significant reduction in animal growth.
This possible elimination of oxidation products, due
to the interchange between the coating oil and the food
would make possible a restoration of protein quality
which was maximum for all the quality indicators
studied at 6 months after canning (Table 3). Strangely,
this is the approximate period after which the product
appears in the market, namely after the variable maturation period, sometimes not well known, at room temperature. Garcia Arias et a1 (1994) also verify the
negative effect of the thermal treatment of raw tuna on
nutrient absorption that is minimised when the canned
fish is held for 1 and 3 years in storage. Furthermore, in
a study carried out by Casales et aE (1991) it was reported that the maturation of canned mackerel produces to
a product with a higher quality in terms of its appearance and flavour. In contrast, other authors (Cameron
et a1 1949; Hellendoorn et al 1969; Kramer 1986) report
that during storage, due to the same chemical reactions,
a negative change in the sensory and nutritional proper-
ties can arise. In our results, with 5 years storage (Table
3) there was a slight, although significant, fall in the
nutritive quality of the sardine protein. Nevertheless,
the product continued to exhibit a high digestive and
metabolic utilisation of the protein. In contrast, Garcia
Arias (1989) reported that steam-baked canned bonito
had a falling trend in its nutritive value at the end of
one year of storage that was more pronounced at the
end of 3 years. At this time the protein changes did not
modify the digestibility, but the biological value had
In conclusion our results show that the canning of
sardines is a process that leads to stability of the
product for 5 years without any significant reductions in
protein quality in practical terms. This is maintained at
a high level, the maximum value occurring at 6 months.
We think that these findings need to be taken into
account, to determine the so-called dates of preferred
This work was supported in part by Alfageme SA.
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