Today a serious form of malnutrition in the world is protein malnutrition . Fish Protein Concentrate (FPC) presents a potentially invaluable addition to the available world protein supply . It is an economical source of high grade animal protein in areas of the world where protein malnutrition and poverty go hand in hand . FPC is a stable wholesome supplement of high nutritive value and low caloric content , prepared from whole fish and can be used as a protein supplement , it improves the nutritive quality of cereal based foods such as flour and bread (Kvitka and Chen,1982) . Bread , in all its various form ,is eaten in most countries and the quantitative consumption of bread is not easily surpassed by any other single food product . This is the reason why bread has been used as the main food item in several studies , involving the improvement of the nutritional status of many people (Nikkila et al,1972) . For many years food scientist have been striving to improve the nutritional status of developing countries through fortifying grain products with essential nutrients or by supplementing wheat flour with high-protein additives (Lorenz and Maga,1972). So far studies have been carried out where bread has been enriched with oil seed such as soybean and fish protein concentrate . Among plant protein concentrate soy protein has been the most widely used , giving the best result . However , in comparative studies , the biological value of soy protein concentrates has been found to be inferior to FPC. Also several baking studies with FPC have been reported in the literature . Generally , Yanez et al (1969) enriched bread with 3 , 6 , 9 and 12 % of FPC . This research has shown that the flour samples with FPC levels of 3-9 % have given acceptable result . Also in bread with 6% of FPC and high level , the darkness of bread was reported . In research by Nikkila et al (1976) , the effect of FPC on Arabic and Indian bread was studied . Result of taste panel test shown that supplemented breads were well accepted when 5% of wheat flour was replaced with FPC and the supplementation of 10% of the flour didn´t alter the characteristics of Arabic bread texture and also fishy test was observed by any of the panelists . Such a supplementation with the best test FPC used elevated the protein efficiency ratio (PER) in rats feeding of enriched bread (in 10%) . In research by Kvitka and Chen (1982) the effect of FPC on the quality and acceptability of bread was studied at two levels of FPC (5 and 10%) supplementation . The quality and acceptability of the bread were evaluated by both subjective and objective methods. Result of research has shown that the color of the samples supplement with FPC was darker than the control . Incorporation of FPC into bread also decreased the loaf volume . So the results showed that bread can be supplement with 5-10 % of FPC as a replacement for flour result in standard quality as well as consumer acceptability . The aim of the present investigation is to report on the effect of enrichment of wheat flour with fish protein concentrate prepared from silver carp (Hypophthalmichthyes molitrix) on organoleptic , chemical composition , amino acids and shelf life of brotchen bread .
Materials and methods : Preparation of FPC : FPC was prepared in Iranian fish processing research center laboratory in Bandar Anzali (winter 2009 ) . The internal organs of fresh silver carp were removed and the fish washed and deboned with de boner (SEPAmatic model) . So the fish meat was passed through a 3 stage extraction with 2-propanol . After de fatting ( including removing of smell and color ) , the product was dried in oven with 100 °C to 12 hour . So the product ground and packed . Determination of protein , moisture, ash, fat , TVN and PV in FPC was carried out by A.O.A.C methods (1995) .
Bread baking : the samples was ordinary brotchen bread prepared in a bakery according to the following formula : wheat flour : 4 kg , yeast powder : 24g , sodium chloride : 40g , vegetable oil : 20g , water : 200 litter . The bread enriched with FPC contained 2.5 , 5 , 7 and 10 percent . The dough was prepared using the straight dough method . After first fermentation , it was divided into 60g portion and allowed to rise for 40 minute in ferment room with 30 °C and 65% humidity . Then the dough pieces moving into oven at 220 °C . The total baking time was 20 minute .
Sensory evolution : A test panel consisting of 10 professional panelists evaluated the quality and acceptability of the breads . During each tasting session , the panelists received a reference sample made without FPC followed by four samples consisting of samples fortified with 2.5 , 5 , 7 and 10 percent of FPC . The panelists were instructed to compare the enriched samples with the reference sample and to note any difference by checking a nine point numerical scale to indicate a quantitative difference (Jellinek,1985) . In addition to the multiple comparison test and flavor , texture , color , odor and overall quality of each enriched samples were also rated by the panelists as acceptable or unacceptable . The results of sensory evolution showed that the enriched samples by 5 and 7% of the FPC accepted . Therefore , these two products were chosen for study and the other samples were rejected as the results of flavor , texture , color , odor and overall quality (Table 2) .
The protein , fat , ash , moisture and carbohydrate percentage were determined according to A.O.A.C methods (1995) for reference sample and enriched samples with 5 and 7% of FPC . Essential and nonessential amino acids were determined by High - Pressure Liquid Chromatography or HPLC (Aligent 1046A model) were carried out according to the method of A.O.A.C (1998) . Shelf life were determined according to the method of Piggot (1984) . During testing to determined the minimum and maximum shelf life for reference sample and enriched one according their freshness and staleness .
Result and discussion: The results of chemical analysis of silver carp FPC composition have shown this product contains 91.5 % protein , 0.03% fat , 3.3% ash and 1.3% moisture . Its TVN and PV content were 12 mg/1000g and 0 meqO2/1000g respectively . In Table1 a comparison between nutritional composition of the FPC-A (FAO) standards , egg , milk powder , milk and soybean powder which usually used for enrichment of bread and FPC of silver carp made (Taha et al .,1982.,El-Badawy,1997) . As one can see from Table 1 the protein content of silver carp (91.5%) is much higher than FPC-A of FAO ( >75) , soybean powder (32.4%) , milk powder (26.2%) , egg (11.8%) and milk (3.3%) . On the other hand its fat (0.03%) , moisture (1.3%) and ash content (1.3%) is lower than FPC-A (FAO standard) . The fat , moisture and ash content of FPC-A are <0.5% , < 10% and <15% respectively . As can be seen from Table1, between egg , milk powder , milk and soybean powder only milk powder with 26.2% protein and 30.6% fat and soybean powder with 32.4% protein and 28.5% fat after de fatting may be useful for bread enrichment .
Table 1 : Comparison of the standard FPC ,trial FPC and four kinds of food
Ash
Moisture
Fat
Protein
Name
< 15
< 10
< 0.5
> 75
FPC-A of FAO
3.3
1.3
0.03
91.5
Silver carp FPC
-----
70.8
15.0
11.8
Egg
-----
2.0
30.6
26.2
Milk powder
-----
86.7
4.2
3.3
Milk
-----
3.7
28.5
32.4
Soybean powder
FAO,1986
The sensory acceptability of enriched bread by silver carp FPC based on its flavor , texture , color , odor and overall quality is given in Table 2 . By increasing the supplementation level silver carp FPC from 5% to 7% and 10% , the score of the overall quality of enriched bread decreased from 28.4 (for 5%) , 26.6 (for 7%) and 20 (for 10%) .
Table 2 .Sensory evolutiona of enriched bread with FPC
Overall scores
Odor
Color
Texture
Flavor
Sample
26.4
6.8
6.7
6.5
6.4
Reference sample
26.2
6.7
6.7
6.4
6.4
Bread with 2.5 % FPC
28.4
6.9
7.7
7.1
6.7
Bread with 5% FPC
26.6
5.9
7.5
6.9
6.3
Bread with 7% FPC
20
4.3
4.8
6.2
4.7
Bread with 10% FPC
a : very good sample = 9 , very bad sample = 1
The interesting outcome of this sensory investigation on level of supplementation was that sensory panelists who were living near sea not only do not have any objection to sensory properties of bread which has been enriched by 10% silver carp FPC , but on the contrary prefer this bread to the other ones . The major factors contributing to the significant sensory differences found by the taste panel were flavor and texture (Table 2) . The scores of taste panel for bread enriched by 5% and 7% FPC for flavor and texture were 6.7 , 6.3 and 7.1 , 6.9 respectively (P<0.05) . The increase of supplementation of FPC from 7% to 10% caused a decrease in score for flavor from 6.7 to 4.7 and for texture from 7.7 to 4.8 (P<0.05) . The flavor and texture scores of the taste panel for reference sample and bread which had been enriched by 2.5% FPC did not change much (Table 2) . So many researchers tried to enrich bread by FPC which had been made from different species of fish . As early as 1969 Yanez et al , enriched bread with 3 , 6 , 9 and 12% of FPC , Sidwell (1970) tried to change the physical and sensory characteristics of dough and bread by adding FPC and Lysine in different amounts to dough which was used for production of bread . Also in 1976 Nikkila et al enriched Arabic and Indian breads with 5 to 10 % of FPC made from different species . Kvitka and Chen (1982) tried to enrich four types of bread with FPC . The results which were obtained by all these researchers have shown clearly that supplementation of bread by FPC which was obtained from different species of fish can be successfully done at level of 3 to 5% of FPC , but supplementation of bread more than that causes rejection by consumer due to flavor , texture , odor and color changes (Yanez et al , 1969.,Kvitka and Chen ,1982) .On the other hand the supplementation of bread by 3 to 5% causes an increase of 1.5 to 3% of protein and 1 to 2% of fat (sidwell,1970 ., Taha et al ., 1982) . The protein in FPC made from silver carp is higher (91.5%) in comparison to that made from other species ( Yanez et al .,1969., Nikkila et al .,1972 ., Kvitka and Chen , 1982) . The supplementation of brotchen bread by 5% and 7% of silver carp FPC result in an increase of protein content compared to reference bread of 11.4% to 15.36% and 17% respectively (Table 3) . This means an increased of 3.93 to 5.61% protein over the other supplementation which has been reported so far . The moisture of supplemented brotchen bread also has shown an increase from 26.69% to 34.27% and 36.25% , also the result have shown (Table 3) a decrease for carbohydrate . The carbohydrate of reference bread was 58.74% which decreased to 47.01% and 43.18% for bread which had been enriched by 5% and 7% of silver carp FPC respectively (P<0.05) .
Table 3 . Chemical composition of sample & enriched bread with FPC of silver carp
Carbohydrate(%)
Ash(%)
Moisture(%)
Fat(%)
Protein (%)
Sample
58.74±0.252
2.014±0.027
26.69±0.024
1.13±0.016
11.43±0.051
Reference sample
47.01±0.137
2.22±0.05
34.27±0.028
1.14±0.016
15.36±0.051
Bread with 5% FPC
43.18±0.154
2.42±0.042
36.25±0.056
1.15±0.022
17±0.042
Bread with 7% FPC
Mean ± standard deviation
In this research work the effect of supplementation of bread on essential amino acids content of bread enriched by 5% and 7% of silver carp FPC also has been investigated (Table 4) . The increase in amount of essential amino acids in bread which has been enriched by 5% and 7% of silver carp FPC in comparison to the reference sample (Table 4) : Histidine (from 0.1% to 0.15 and 0.21%) , Tryptophane (from 0.55% to 10.3 and 15%) , Methionine (from 1.06% to 1.5 and 2.1%) , Leucine (from 0.68% to 0.71 and 1%) , Isoleucine (from 0.38% to 0.42 and 0.65%) , Phenylalanine (from 0.4% to 0.5 and 0.7%) , Lysine (from 0.4% to 0.9 and1.25%) , Threonine (from 0.3% to 0.42 and 0.65%) and Valine (from 0.13% to 0.53 and 0.75%) (P<0.05) . Some of the non essential amino acids such as Argenine , Aspartic acid , Glutamic acid ,Serine , Glycine and Tyrosine+Alanine have also been identified (Table 4) . Result which have been presented in Table 4 clearly show that the essential and non essential amino acids have increased as the result of enrichment of bread by silver carp FPC . Madden (1944) reported that parenterally administered amino acids are able to maintain nitrogen balance and sustain weight in man for as long as75 days . Another function of amino acids in the body that has been studied , is that of regeneration of plasma proteins and hemoglobin ( Davidson et al .,2000) . West et al (2000) have reported on the morphological findings in rats subjected to a deficiency of each of three amino acids . On a deficiency of either Phenylalanine or Leucine for one month , rats showed at autopsy thymic atrophy , atrophy and decreased lipid content of adrenal cortex , degeneration and atrophy of seminiferous tubular epithelium , and
Table 4. Comparision of amino acids composition in reference & enriched bread with FPC (%)
Bread enriched with
5% FPC 7% FPC
Reference sample
Amino acid
7
5.2
0.4
Argenine
0.21
0.15
0.1
Histidine
15
10.3
0.55
Tryptophane
2.1
1.5
1.06
Methionine
1
0.71
0.68
Leucine
0.65
0.42
0.38
Isoleucine
1.1
0.78
0.5
Aspartic acid
3.3
2.35
2.1
Glutamic acid
0.7
0.5
0.4
Phenylalanine
0.66
0.47
0.45
Serine
0.45
0.3
0.2
Glycine
1.25
0.9
0.4
Lysine
0.65
0.42
0.3
Threonine
0.5
0.35
0.3
Tyrosine+Alanine
0.75
0.53
0.13
Valine
thinning of epiphyses of long bone . Certain ocular changes were noted in the Leucine-deficient animals . Plasma proteins and hemoglobin were reduce in animals on a Phenylalanine deficient diet . After 28 days of a Histidine-deficient diet , rats showed decreased hemoglobin , atrophy of the thymus gland , and vascularization and metaplasia of the cornea . These result are cited in order to indicate the diversity of pathological finding from a single essential amino acid the deficiency . The possible relations of these results to human deficiencies offer a field for speculation and study . If the supply of dietary protein is insufficient , the cells lack amino acids for their synthetic activities . The first effect of this on a young child is that growth slows down or stops . The effect on the organs and tissues is related to the speed of turn over of proteins . In consequence a failure to digest and absorb the food , leading to diarrhoena and loss of water and electrolytes , is an early feature of protein deficiency . There is also a failure of the liver to maintain its normal structure and function . Fat accumulates in liver cells . The liver also fails to synthesise plasma albumin and this is liable to load to edema . Later , protein deficiency leads to a failure to maintain the structure of skeletal muscle and the production of red blood corpuscles . Muscle wasting and anemia results . As the result of severe amino acids shortage in diet could lead to physiological deficiency and disturbances of mental function (Davidson et al .,2006) . Certainly few, if any persons in Iran obtain their total daily protein requirement from red or white meat . The daily diet of Iranian people and most probably that in the developing countries consists of food which originated from carbohydrate , such as wheat bread and rice . Consider also that a great many individuals ingest perhaps only 30g to 40g of animal protein daily . In then become imperative that a variety of different proteins be included in the diet and especially some animal protein , if amino acid deficiencies are to be avoided (West et al .,2000) . Thus in this work brotchen bread was enriched by 5% and 7% silver carp FPC . The result of this work has shown that all of the amino acids especially essential amino acids have increased (Table 4) , and there was no off flavor or texture changes in comparison to reference bread (Table 2). So by adding FPC which is produced by extraction by Isopropanol method , one could prevent malnutrition in developing countries especially among children , young people and save elderly from physiological deficiency and disturbances of mental function due to aging .
The result of determination of shelf life in enriched bread by 5 and 7% silver carp FPC is given in Table 5 .
Table 5. Average scores a for shelf life test after 72 hour
72 h
48 h
24 h
Sample
0.6
3.4
4.2
Reference sample
0.6
2.2
3.4
Bread with 5% FPC
0.6
2
2.6
Bread with 7% FPC
a: very fresh sample = 6 , very stale sample = 1
As one can see the scores of taste panel for reference sample after 24 h , 48 h and 72 h were 4.2 , 3.4 and 0.6 , but for the same period of storage time at ambient temperature , the scores for enriched bread by 5% and 7% FPC were (3.4 , 2.2 and 0.6) and (2.6, 2 and 0.6) respectively. These result show there is a relationship between concentration of FPC and shelf life which demands more research in order to make the shelf life longer . The reasons for shortage shelf life of enriched bread may be attributed to the undesirable effect of FPC on wheat gluten which causes the enriched bread to go stale more quickly in comparison to reference sample (Sidwell,1970) .Thus as these result indicated , the shelf life of enriched bread by 5% and 7% FPC of silver carp is recommended to be 48 h at ambient temperature .
Conclusion : The extraction of silver carp meat with Isopropanol has resulted in a higher grade FPC in comparison to FPC-A as defined by FAO (1986) . The silver carp FPC composition contents were 91.5% protein , 0.03 % fat , 1.3% moisture , 3.3% ash without any off smell or sensory defects . As the result of high nutritional value and easy production FPC of silver carp appears to be a suitable source for enrichment of bread . A consistent acceptable enrichment level in this study was 5 to 7% . In enriched bread the quality most affected by adding of FPC is its essential amino acid content , such as Methionine Tryptophane , Valine and Lysine which can help to maintain nitrogen balance , the growth in young and school age children and sustain the weight in adult persons . Thus as the result of this study the enrichment of brotchen bread with 5-7% of silver carp FPC for people living far from sea and with 10% for those who live near the sea is recommended .
