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Chemical and nutritive characteristics of fish silage produced by biological fermentation
Biological Wastes 20 (1987) 187-201
Chemical and Nutritive Characteristics of Fish Silage Produced by Biological Fermentation* T. E. Hassan & J. L. Heath Department of Poultry Science, University of Maryland, College Park, MD 20742, USA (Received 14 February 1986; accepted 8 August 1986) ABSTRACT Fish silage was compared before and after biological fermentation with L. plantarum. When lactose remaining after fermentation was accounted for, no ( P < 0.05) differences in proximate composition were found. Trout had a larger per cent protein and fat and less ash than white perch both before and after fermentation. Total amino acids increased ( P < 0.05) as a result of fermentation and the increase was reflected in changes in the amino acid profile. A decrease ( P < 0.05) in moisture and an increase ( P < 0"05) in fat were found after the silage was stored at both ambient temperature and 37° C for 35 days. The decrease in moisture was due to condensation on the fermentation vessels and the extraction of lactic acid with the fat resulted in higher fat values. All amino acids increased during storage to a small degree and the increase was attributed to the decrease in moisture. The p H of the silage did not change during storage at either ambient temperature or 37°C but the silage stored at ambient temperature was more acidic than that stored at 37°C. Silage at both temperatures had a sufficiently low p H to maintain a successful fermentation. There was an increase ( P < 0"05) in water-soluble nitrogen at both temperatures as storage time increased. This change should improve the digestibility of the silage. Samples stored at 37°C had a higher Total Volatile Bases value than samples stored at ambient temperature. Total Volatile Bases increased ( P < 0.05) as storage times increased up to 18 days and then started to decline. * Scientific Article No. A4340, Contribution No. 7329 of the Maryland Agricultural Experiment Station (Department of Poultry Science). 187 Biological Wastes 0269-7483/87/$03-50 © Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain
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T. E. Hassan, J. L. Heath
No trends were established when oxidative rancidity measurements were considered. The TBA values were high and could indicate a problem with rancidity which may require the addition of antioxidants. Biologically fermenting fish and storage of the silage under the conditions used in this experiment will result in an increase in free fatty acids due to lipolysis. I f the oil is extracted it will yield an inferior product by commercial standards. Broiler chicks fed a ration containing 5% and 10% fish silage had better ( P < 0.05) feed efficiency than did birds fed a ration with no silage. Results indicate that up to lO% fish silage could be included in broiler rations without adversely affecting feed efficiency or body weight.
INTRODUCTION A method of biological fermentation of fish waste to produce a product (fish silage) that could be used as a protein source in animal and poultry feeds has been described in previous work (Hassan & Heath, 1986). If it is used in animal rations, nutritive characteristics and chemical composition must be determined on the fresh and stored product. Most of the research on chemical changes and nutritive characteristics has been conducted on fish silage produced by adding mineral or organic acids. Windsor (1974) investigated proximate composition of fish silage and monitored certain chemical changes in the protein and oil fractions of the silage which was prepared by adding formic acid to six types of fish. The protein content of all six fish samples fell within a range of 15%-17%. The per cent soluble nitrogen increased sharply to 75% after 10 days and continued to increase to 85% after 30 days. After 50 days there was a negligible change in soluble nitrogen when compared with the level found after 30 days. Moisture and ash contents were at the same levels found in the raw material before the acid was added. Windsor's results showed that the free fatty acid content increased due to lipolysis and that oxidative changes caused the oil to darken in color. The increase in fatty acids was shown to be temperature dependent and was most rapid during the first few days of storage. Krishnaswamy et al. (1965) evaluated the nutritive value of ensiled, fresh water fish prepared by biological fermentation. The ensiled fish was incorporated into a diet fed to rats and compared with a skim milk standard. There was no significant difference found when 10% ensiled fish was incorporated into the diet and compared with the diet containing skim milk powder. In another study, March & Bierly (1961) compared experimentally prepared liquid fish made from British Columbia herring with press cake herring meal, whole herring meal and condensed herring solubles as a source
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of supplementary protein for chicks. Two samples of the liquid fish made by enzymatic and by high pressure acid treatment were used to supply 3 % of the protein in the diet. The comparison showed that the two preparations were similar in protein value and gave a growth response intermediate between that obtained with herring meal and with condensed herring solubles. The effect of fish silage on the quality of hen's eggs and broiler meat was studied by Wirahadikusumah (1969). Eggs from hens fed the silage had thicker and more dense shells than eggs from hens fed fish meal. Egg yolks from hens fed fish silage had a less intense yellow color and a higher iodine value than the yolks from hens fed the fish meal. Feeding up to 40 g per hen per day of wet fish silage did not impair the flavor of the eggs. Feedi~ng broilers from 14 to 60 g of fish silage each day resulted in off flavors after 7 weeks but, when the fish silage was omitted from the ration 1 week prior to slaughter, no off flavor was observed. The TBA and peroxide values were low. The objectives of this research were to determine the composition and chemical characteristics of fresh and stored fish silage produced by biological fermentation and to evaluate it nutritionally in a broiler chick feeding study. METHODS
Fish White perch and trout were used to provide a low-oil fish that would represent some of the underutilized species that could be used in animal feeds. The fish were obtained locally on the day of catch, placed in ice and transported to the laboratory for silage production. Whole fish (including the viscera and heads) were used to produce samples for study.
Silage The fish were ground, lactose added, inoculated with Lactobacillus plantarum and fermented as described in Hassan & Heath (1986). Trout and white perch were fermented separately to compare their chemical characteristics before and after fermentation.
Analytical methods The pH of the supernatant was measured after 5 g of fish silage was mixed with distilled water to a volume of 50ml and centrifuged for 10min at 8000 rev m i n - ~ using a Sorvall RC-2 refrigerated centrifuge.
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T. E. Hassan, J. L. Heath
Soluble nitrogen was determined according to the method of Barbour (1962, unpublished work in: A collection o f methods used by Pacific fisheries technologists) and expressed as per cent of total nitrogen. Protein was determined on 20-ml aliquots of the filtrate using AOAC (1975) methods. Moisture, fat, protein and ash were measured using methods described in AOAC (1975). Thiobarbituric acid value (TBA) was determined using the method of Tarladgis et al. (1960) and free fatty acids as an index of hydrolytic rancidity were determined according to the method of Wirahadikusumah (1968). Total Volatile Bases (TVB) were measured using the procedures described by Pearson (1970). The amino acid profile of the fish silage was determined using a Durrum D-500 amino acid analyzer (Durrum Instrument Corp., Palo Alto, CA). Three 100-200 mg samples of the fish silage were hydrolyzed under nitrogen in 4 ml of 6N HC1, two for 24 h and one for 72 h, in sealed vacuum tubes at 110°C. Samples were distilled until completely dry, after which 20 ml of a 2.2 pH sodium citrate buffer was added. Preparation of a sample for cystine analysis required the conversion of cystine to cysteic acid. To accomplish this, 2 ml performic acid was added to 100-200 mg of the silage. The solution was refrigerated for 24 h, 0.3 ml hydrobromic acid was added and the solution distilled. The preparation was then hydrolyzed with HC1 as described previously. The sample was injected onto the amino acid analyzer ion exchange column.
Statistical analysis Data were analyzed using the analysis of variance programs BMDP2V and BMD1R at the University of Maryland Computer Science Center. Significantly different means were separated using the Student-NewmanKeul test as described in Sokal & Rholf (1969). Experiment 1 This experiment was conducted to determine the effect of fermentation on composition of ground fish. White perch or trout (100g) were ground separately in a meat grinder using plates with 5mm, 2 m m and 1.5mm diameter pores. Samples were ground through the plates in order of size starting with the largest pore size. Each 100-g sample was placed in a glass screw-capped jar, lactose was added and the mixture inoculated with 1 ml of a culture of L. plantarum grown for 18 h in lactobacilli MRS broth (Difco Laboratories, Detroit). White perch samples were fermented with 5% lactose and trout was fermented with 3% lactose. The difference in the amount of lactose added was based on previous work (Hassan & Heath,
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TABLE 1
Proximate Composition of Whole White Perch and Whole Trout Before and After Fermentation with L. plantarum at 35°C
White perch
Moisture Protein Fat Ash Carbohydrate*
Trout
Before (%)
After (%)
Before (%)
After (%)
73.19 + 3"6c 15"99 ___0"7a 4.57 ___0-3° 6'25 __0"4b --
70-22 _ 5.0b 15-91 + 1.0a 4-99 + 0-4a 6"11 __0'5b 2.77
70-61 ___4.3 b 18"23 _ 1'0 b 8-63 + 0"6b 2"53+0'2 a --
68-56 ___5"6a 17"96 _ 1.5b 8-84 + 0'8 b 2"83__ 0.2 a 1"81
* Carbohydrate was determined by difference. abcMeans ___Istandard deviation with unlike superscripts for each parameter are significantly (P < 0"05) different. 1986) w h e r e it was f o u n d t h a t as ash c o n t e n t o f the sample increased, m o r e acid w o u l d have to be p r o d u c e d to lower the p H to the desired level. P r o x i m a t e c o m p o s i t i o n o f g r o u n d fish was d e t e r m i n e d (Table 1) a n d the a m o u n t o f lactose necessary for a successful f e r m e n t a t i o n calculated. T h e p r e p a r a t i o n was t h o r o u g h l y mixed, sealed and i n c u b a t e d in a w a t e r b a t h at 35°C for 4 days. Samples were r e m o v e d b e f o r e a n d after f e r m e n t a t i o n for p r o x i m a t e c o m p o s i t i o n (n = 10) a n d a m i n o acid (n = 5) analyses. L a c t o s e used in this and s u b s e q u e n t e x p e r i m e n t s was o b t a i n e d f r o m F o r e m o s t F o o d s , Inc., San Francisco.
Experiment 2 W h o l e g r o u n d trout, p o t a s s i u m s o r b a t e (0.05%), c o m m e r c i a l lactose (4%) a n d i n o c u l u m were mixed in a H o b a r t electric m i x e r for 3 min a n d used to d e t e r m i n e if storage after f e r m e n t a t i o n affected the c o m p o s i t i o n o f fish silage. P o t a s s i u m s o r b a t e was mixed first with lactose to ensure u n i f o r m d i s t r i b u t i o n p r i o r to a d d i n g the m i x t u r e to g r o u n d fish a n d f u r t h e r mixing the c o m b i n e d material. U p o n c o m p l e t i o n o f mixing, the c o m p o s i t e was divided into 100-g p o r t i o n s a n d placed in screw c a p p e d jars a n d i n c u b a t e d at 35°C for 3 days. F e r m e n t a t i o n jars were r e m o v e d f r o m the i n c u b a t o r a n d s e p a r a t e d into two equal groups. T h e first g r o u p was stored in an i n c u b a t o r at 37°C while the second g r o u p was m a i n t a i n e d at a m b i e n t t e m p e r a t u r e (23-25°C). A f t e r f e r m e n t a t i o n a n d b e f o r e storage, two samples were r e m o v e d f r o m each j a r a n d a n a l y z e d for p r o x i m a t e c o m p o s i t i o n (n = 10) a n d a m i n o acid (n = 10) content. Samples were r e m o v e d f r o m the jars after storage for 35 d a y s a n d the same analyses c o n d u c t e d .
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T. E. Hassan, J. L. Heath
Experiment 3 This experiment was a repeat of Experiment 2 except that samples were taken from each fermentation jar after 0, 6, 12, 18 and 35 days' storage at either 37°C or ambient temperature. Each sample (n = 8) was analyzed for free fatty acids, TBA, TVB, soluble nitrogen and pH to determine any changes in the chemical characteristic of the silage after storage.
Experiment 4 Broiler chicks were used to compare the nutritive value of commercially formulated diets containing fish silage with diets without fish silage. Weight gain and feed conversion (feed consumed/weight gain) were monitored. Day old chicks were obtained from a commercial hatchery, placed in electrically heated Petersime brooder units and fed a commercially formulated and mixed starter ration for one week. At the end of the first week chicks were divided into treatment groups with each group having six replicates of nine chicks. Feed and water were available ad libitum and feed wasted was determined and used to correct feed consumption. The treatments consisted of three levels of trout s i l a g e ~ % , 5 % and 10% (Table 6). The experiment was terminated on the twenty-first day. The diets were formulated to be isonitrogenous and isocaloric and to provide 80% of the nutritive requirements of the chick. This provided nutrients at a level calculated not to meet 100% of the nutritive requirements of the chick so that improvement, if any, by the silage could be noted. Diets, both basal and experimental, were formulated with a least cost computer program. The least cost computer program is used by nutritionists in the broiler industry to determine ration ingredients for optimum growth at the least cost. The computer program used the chemical analyses of fish silage from the preceding experiments.
RESULTS A N D DISCUSSION
Experiment 1 Proximate composition of white perch and trout were compared befo(e and after fermentation (Table 1). Differences (P < 0.05) in composition were noted because of the addition of lactose to ground fish to facilitate fermentation. Ground fish analyzed before fermentation did not have lactose added and samples analyzed after fermentation had lactose remaining after fermentation. Lactose was not completely metabolized by L. plantarum and, as a result, had a dilution effect on the fractions. When the lactose remaining in silage was taken into account no differences (P > 0"05)
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in moisture, protein, fat and ash were found. The two types of fish, white perch and trout, had different compositions. Trout had more (P < 0.05) protein and fat and less (P < 0.05) ash than white perch. The difference in carbohydrate can be attributed to the different amounts of lactose added at the start of fermentation. Total amino acid content was compared before and after fermentation and tended to increase (P < 0.05) as a result of fermentation, 0.9% for white perch and 3"8 % for trout (Table 2). This increase could partially be explained by the decrease in moisture (Table 1) during fermentation of 3 % and 2% for white perch and trout, respectively, which would increase the relative amount of protein in the silage. The amino acid content of ground fish analyzed after fermentation was diluted by lactose added to the mixture and not metabolized by L. plantarum. The explanation is further complicated because per cent protein decreased during fermentation by 0.08% and 0.27%, respectively, for white perch and trout (Table 1). The change in the per cent of each amino acid was not uniform as would be expected if the TABLE 2 Comparison of the Amino Acid Content of White Perch and Trout Before and After Fermentation at 35°C by L. plantarum
Amino acid
Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total
Before fermentation
After fermen tat ion
White perch
Trout
White perch
Trout
(%)
(%)
(%)
(%)
1'31 0'57 0'59 1'87 0.79 1'26 0'98 0'65 0.40 0.54 0.98 0.43 0.53 0.27 1.01 0'95 13-13 ___0"7°
1-20 0.61 0-54 1"75 0"65 0-98 0"89 0.61 0.42 0-55 0"99 0.44 0-53 0"31 1"04 0"90 12"41 _+0'7 °
1.36 0.61 0.57 1-73 0"99 1-50 1-19 0.72 0.44 0'57 1.05 0'36 0.57 0.29 1' 15 0'95 14.05 + 1.0 ~
1-62 0.74 0-64 2'29 0"92 1"50 1-23 0.79 0'53 0'66 1.25 0.49 0.62 0'39 1"40 1"17 16"24 ___0'9 c
abe Means +__standard deviation with different superscripts are significantly (P<0.05) different.
194
T. E. Hassan, J. L. Heath
increase was caused completely by a decrease in moisture. Apparently, differences in amino acid content before and after fermentation were due to a combination of factors associated with chemical and physical changes during fermentation and cannot be explained simply by concentration due to moisture loss. These changes could affect the availability of the protein for animal metabolism and result in differences in feed conversion and growth. Percentages of individual amino acids did not change (P < 0.05) when the samples before and after fermentation were compared (Table 2). This leads to the conclusion that fermentation of white perch and trout did not change the amino acid quality of the product.
Experiment 2 Significant (P < 0.05) differences were found when the proximate compositions of fish silage were compared before and after storage for 35 days at ambient temperature and 37°C (Table 3). A decrease in moisture content and an increase in fat content were found. The decrease in moisture content was attributed to condensation on the inside of the fermentation vessels and loss during sampling. This decrease resulted in relative increases in some of the other fractions. The amount of fat increased when stored silage was compared with the samples taken before storage. The larger amount of fat found in samples stored at 37°C for 35 days could be the result of analytical procedures used. Since lactic acid is soluble in ether (Merck Index, 1968), it is reasonable to expect that lactic acid was removed during fat extraction. The amount of carbohydrate remaining in the sample after storage is consistent with this explanation. A significant (P < 0.05) difference was found when the TABLE 3 Proximate C o m p o s i t i o n o f T r o u t Silage Before a n d After 35 Days' Storage at A m b i e n t T e m p e r a t u r e a n d at 37°C
Start
35 days storage at
(%) Ambient
37°C
69-76 _+ 5.7 b 16-89 _+ 1'1 a 8-11 _+ 0.8 a 2.60 _ 0.1" 2-64
68"30 _+ 6"2 a 17.44 _+ 1.4 a 10-96 _+ 0'9 b 3-00 _+ 0'2" 0-30
(%)
Moisture Protein Fat Ash Carbohydrate*
70'83 _ 3"6 c 17'10 _+ 1"0a 7"64 _+ 0"4 ~ 2-61 _+ 0.2" 1.82
(%)
* C a r b o h y d r a t e determined by difference. QbcM e a n s _+ s t a n d a r d deviation with unlike superscripts are significantly ( P < 0"05) different.
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carbohydrate level in fish silage before storage and silage stored at ambient temperature was compared with the silage stored at 37°C. There were no differences (P > 0"05)i between the carbohydrate levels of silage before storage and that stored at ambient temperature. Obviously, the lactose was more completely metabolized by L. p l a n t a r u m and consequently more acid was produced in the silage stored at 37°C.
TABLE 4
Amino Acid Content of Trout Silage Before and After 35 Days' Storage at 37°C and at Ambient Temperature Amino acid
Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total
Start
1.50 0-66 0.54 2.20 0.80 1-27 1.13 0.71 0.53 0.66 1"18 0.43 0.60 0-43 1.30 1'07 15.01 ___0-8a
35 days storage at Ambient
37°C
1"60 0"71 0'57 2-32 0.85 1"35 1"19 0.76 0.54 0.70 1.23 0'46 0-63 0.50 1.38 1.13 15-92 + 0.8 ~
1-64 0.74 0"61 2.37 0.89 1'43 1"25 0-78 0"55 0-73 1"26 0"45 0-64 0.48 1.41 0'93 16.16 _ 1.2a
a Means _+standard deviation with the same superscript are not significantly (P > 0"05) different.
Total amino acids did not (P > 0.05) change during storage (Table 4). With the exception of the a m o u n t o f arginine in the silage after storage at 37°C, all amino acids increased to a small extent. This increase was attributed to the decrease in moisture since the per cent amino acids were calculated on a wet weight basis. Storage under the conditions of this experiment had a minor,effect on the proximate and amino acid composition of the silage. The differences noted would not be expected to adversely affect the inclusion of stored, biologically fermented fish silage in animal rations.
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T. E. Hassan, J. L. Heath
Experiment 3 The pH of silage did not change (P > 0.05) during storage for 35 days at either ambient temperature or 37°C (Table 5). The pH of the sample stored at ambient temperature was more acidic (pH 4.43) than that of the samples stored at 37°C (pH4.55) after 12 days' storage and this relationship continued to the end of storage at 35 days. Both treatments had a pH low enough to maintain a successful fermentation. There was an increase (P < 0-05) in water-soluble nitrogen as the storage time increased at both storage temperatures and, after 6 days, those stored at 37°C increased more rapidly than those stored at ambient temperatures. This agreed with work reported by Windsor (1974). Water-soluble nitrogen increased from 58.7% to 71.8% and 77-5% for samples stored at ambient and 37°C, respectively. A relationship was observed between the amount of soluble nitrogen and the consistency of the preserved mass. The silage became more liquefied as storage time and water-soluble nitrogen increased. Since the growth of lactic acid bacteria and the decline in pH prevented other bacterial growth (Hassen & Heath, 1986), it can be assumed that liquefaction was due, almost entirely, to autolytic proteolysis. Tatterson & Windsor (1974) pointed out that little is known about the mechanism involved in liquefaction but it is assumed that gut enzymes which can be spread throughout the mass are responsible. They reported that fillets alone do not liquefy nor do whole fish or parts of fish which have been cooked for 15min at 100°C. The increase in soluble nitrogen is the result of degradation of the complex protein structure and would be expected to increase levels of free amino acids and short chained peptides. These changes should improve the digestibility of silage. Samples stored at 37°C had higher (P < 0"05) TVB values than samples stored at ambient temperature. TVB values increased (P < 0"05) in samples stored at both temperatures as storage time increased up to 18 days and then declined. TVB are related to trimethylamines (TMA), ammonia and other bases. It is known that TMA-oxide can be reduced to TMA by several mechanisms such as by bacterial and endogenous enzymes. Ammonia can come from several reactions such as deamination of amino acids, degradation of nucleic bases and oxidation of amines by bacterial aminooxidases and results in an increase in TVB. Other bases come from the decarboxylation of amino acids. An increase in TVB was reported during the storage of marinated herring prepared by adding acetic acid and NaC1. The increase in TVB was related to the decarboxylation of amino acids by certain strains of lactic acid bacteria (not L. plantarum) isolated from the product (Meyer, 1965).
15.6 _ 1-2 ~b
14.5 _ 1.0 °
67"7 + 3"8 c 81 _ 4.3 b
21.3 ___0.9 ~
16.9 _ 1.1 b
6'5 + 0"6 ~b
152 _ 7"0 e 10.0 _ 0.9 b~a
69"8 _ 4-4 ae 91 _ 5"7 b~
65"5 _ 4"1 b
4"43 _ 0"16f' 4.55 _ 0.17 b~
12
Storage time (days)
a-h M e a n s + - s t a n d a r d d e v i a t i o n w i t h u n l i k e s u p e r s c r i p t s a r e s i g n i f i c a n t l y ( P < 0"05) different.
S o l u b l e N is e x p r e s s e d as p e r c e n t o f t o t a l n i t r o g e n . T V B is t h e m i l l i g r a m s o f n i t r o g e n p e r 100 g silage. T B A is t he m i l l i g r a m s o f m a l o n a l d e h y d e p e r k i l o g r a m .
13-9 _ 0.9 °
37°C
6"3 _ 0"5 ~b
13.9 _ 0.9 ~
11-7 ___ 1.0 ca
37° C
T B A v a l u e (m g pe r 1 0 00 g)
Ambient
37°C Ambient
T V B ( m g p e r 100g)
F F A ( % O l e i c acid)
95 _+ 5"2 c 10'5 _ 1'0 boa
58 ___3-2 a 11"7 + 1"0 ~a
37°C Ambient
64"6 + 3"9 b
58"7 _ 3"2 ~ 58"7 + 3"2 a 58 _+ 3-2 a
Ambient
4'53 -t- 0.14 ab 4.53 _ 0.13 ab
S o l u b l e N (% )
4-50 _ 0"11 ab 4.50 + 0.11 °b
6
Ambient 37°C
0
pH
Storage
23.7 + 1.3 a
22.1 _ 1.4 ca
5'0 _ 0"4 ~
268 _ 16.6 h 4.4 _ 0.3 ~
75-5 + 4.8 y 186 _ 8.0 f
68"0 + 4.4 ca
4-50 _ 0.15 ~b 4'65 _ 0"16 c
18
TABLE 5 Chemical Characteristics of Fish Silage Stored at Ambient Temperaaure and at 37°C
32.8 _ 2.0 y
12.8 _ 1.0 a 27.3 ___ 1-5 e
259 _ 19.5 h 8.2 _ 0.6 ab~
77.5 _ 5.1 f 115 _+ 8.3 d
71-8 _ 4-7 e
4.44 + 0.14 ° 4.55 _ 0.16 bc
35
198
T. E. Hassan, J. L. Heath
The decrease in TVB values from 18 to 35 days of storage could be attributed to volatilization or to reactions of amines with unfermented sugars. Since there was no decrease in amino acids during storage (Table 4), it can be assumed that most of the TVB comes from TMA-oxide or other bases produced as a result of nucleic acid degradation and not from decarboxylation or deamination of amino acids. Oxidative rancidity in the fish silage was measured during storage and reported as TBA values. Results showed a relatively high TBA value in the freshly prepared silage when compared with the samples stored at ambient temperature for 18 days and at 37°C for 6, 12 and 18 days. No clear trends were observed but the high values found indicate that rancidity could be a problem and it may be necessary to add antioxidants to the silage. The free fatty acid content of the fish oil extracted from silage was used to measure the extent to which the glycerides in the oil had been decomposed by lipases. Per cent free fatty acids are often used as a criterion for judging the condition and edibility of oil for commercial purposes and a level of less than 4% free fatty acids is required for high quality oils (Tatterson & Windsor, 1974). To prevent decomposition of the oil, it is best to store it under conditions that are free from moisture and protein particles. Based on this, the fish oil in the silage produced in this experiment was under far from ideal conditions. Free fatty acid content increased (P < 0.05) as storage time increased from 13.9% in the freshly prepared silage to 27.3% and 32.8% in samples stored for 35 days at ambient temperature and 37°C, respectively. Silage stored at 37°C had higher values than those stored at ambient temperature. This agreed with Tatterson & Windsor (1974) who reported that sprat silage stored at a number of temperatures showed rapid increase in free fatty acids during the first 40 days and the increase was temperature dependent. Reece (1980) studied the factors responsible for free fatty acid production in the oil fraction of silage made from sprat. He found that increases in free fatty acid content were partially due to the release of free fatty acids from their watersoluble salts after acidification and to the carry over of certain organic acid acidulants. Reece (1981) indicated that much of the initial free fatty acids were present in the digestive tract of the fish prior to acidification. He also reported that increases in free fatty acids in oils from oily fish silate were principally associated with the free fatty acids that came from the solid material during liquefaction of the fish. The increase in free fatty acids reported in this experiment was found in samples that also had an increase in soluble nitrogen and the increase in soluble nitrogen was observed to be associated with increased liquefication of the silage. Biologically fermenting fish and storage of the silage under conditions used in this experiment will result in an increase in free fatty acids and, if the oil is extracted, it will yield an inferior product by commercial standards.
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Experiment 4 Broiler chicks fed a ration (Table 6) containing 5% and 10% fish silage had better (P < 0.05) feed efficiency (feed/gain) than did birds fed a ration with no silage (Table 7). No difference in feed efficiency was found when broilers fed the diets containing 5% fish silage were compared with those fed 10% silage. Broilers fed the ration with 5% silage weighed more (P < 0.05) at the end of the experimental period than those fed the ration containing 0% or 10% silage. These results indicate that up to 10% fish silage produced by biological fermentation could be included in broiler rations without adversely affecting weight gain or feed efficiency. Evidence indicated that feed efficiency would be improved but since these rations were balanced to provide only 80% of the chicks' nutrient requirements, additional studies will be necessary to verify that feed efficiency is improved. TABLE 6 Composition of Diets Used in the Feeding Study
Ingredients
Basal
5% Silage
10% Silage
(%)
(%)
(%)
Corn Soybean meal 49% Fish silage Dical 18.5/22 Fat blend 3700 Limestone Salt Choline CI 50 Fixed ingredientsa oL-Methionine 99% Trace mineral mix b Vitamin mix broiler c L-Lysine HCl 98% Water
64.90 21.79 -1.67 2-78 1-02 0.44 0-13 0-10 0.11 0"05 0"05 0.07 6"89
66.56 19.73 5.00 1.54 1'83 0-98 0"43 0-13 0-10 0.10 0"05 0"05 0"05 3-45
67.42 18.38 10.00 1.41 1-00 0-94 0"42 0-12 0"10 0.09 0-05 0-05 0'02 --
a Amprol Antioxidant Potassium Sorbate (g per 50 kg feed)
0"05 0.02 19
0"05 0-02 17"8
0"05 0.02 16.5
Sorbate was added as a preservative to provide 0.04%. The silage contained 0'05% sorbate. bTo supply the following per kilogram of diet: Vitamin A, 5511-5 IU; Vitamin D3, 2204.6 IU; Vitamin E, 4-41 IU; Vitamin B12, 13 mcg; Riboflavin, 6-614 mg; Niacin, 33-07 mg; d-Calcium pantothenate, ll-98mg; menadione sodium bisulfate, 4.41mg; Folic acid, 0.22mg; Pyridoxine, 1.35 mg. cTo supply the following per kilogram of diet: Manganese, 150 mg; Zinc, 120 mg; Iron, 40 mg; Copper, 6 mg; Iodine, 1-5 mg.
T. E. Hassan, J. L. Heath
200
TABLE 7
Weight Gain and Feed Efficiency of Broiler Chicks Fed Fish Silage
Diet Control 5% silage 10% silage
Weight gain +_SE
Feed efficiency
358"7 _ 18.8a 373.7 _ 23.3b 363"1 _ 21.5a
1.80 _ 0.1 ° 1.74 _ 0-1 b 1.75 _ 0.1 b
o.b Means ___standard deviation, with different superscripts are significantly different (P < 0.05), n = 54.
It was n o t e d t h a t w h e n the rations were mixed, the fish silage b l e n d e d well with c o r n at b o t h the 5 % a n d 10% levels. It was n o t possible to i n c o r p o r a t e m o r e t h a n 10% fish silage in the feed because o f the high m o i s t u r e c o n t e n t o f the silage.
REFERENCES AOAC (1975). Official methods of analysis (12th edn), Association of Official Analytical Chemists, Washington, DC. Hassan, T. E. & Heath, J. L. (1986). Biological fermentation of fish waste for potential use in animal and poultry feeds. Agric. Wastes, 15, 1-15. Krishnaswamy, A. M., Kadkob, B. S. & Revonkar, D. G. (1965). Nutritional evaluation of an ensiled product from fish. Canadian J. Biochem., 43, 1879-83. March, E. B. & Bierly, J. (1961). The protein nutritive value of liquid herring preparations. J. Fish. Res. Bd. Canada, 18, 113-16. Merck Index (1968). Merck and Co., Inc., Rahway, NJ. Meyer, V. (1965). Marinades. In: Fish as food (Borgstom, G. (Ed.)), Academic Press, New York. Pearson, D. (1970). Determination of total volatile bases in fresh foods. In: The chemical analysis of foods. J. and A. Churchill, London. Reece, P. (1980). Control and reduction of free fatty acid concentration in oil recovered from fish silage prepared from sprat. J. Sci. Food Agric., 31, 147-55. Reece, P. (1981). Recovery of high quality oil from mackerel and sprat by the silage process. J. Sci. Food Agric., 32, 531-8. Sokal, R. R. & Rholf, F. J. (1969). Biometry. W. H. Freeman and Co., San Francisco. Tarladgis, G. B., Watts, M. B. & Younathan, T. M. (1960). A distillation method for the quantitative determination of malonaldehyde in rancid food. J. Am. Oil Chemist Soc., 37, 44-8. Tatterson, N. I. & Windsor, L. M. (1974). Fish silage. J. Sci. Food Agric., 25, 369-79. Windsor, L. M. (1974). Production of liquid fish silage for animal feed. In: Fishery product (Kreizer, R. (Ed.)), published by arrangement with the Food and
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Agriculture Organization of the United Nations by Fishing News Ltd, Surrey, UK, 140-3. Wirahadikusumah, S. (1968). Preventing clostridium botulinum type E poisoning and fat rancidity by silage fermentation. Lantbr. Hogsk. Ann., 34, 551-689. Wirahadikusumah, S. (1969). The effect of fish silage on the quality of hen eggs and meat of broiler. Lantbr. Hogsk. Ann., 35, 823-35.
Chemical and Nutritive Characteristics of Fish Silage Produced by Biological Fermentation* T. E. Hassan & J. L. Heath Department of Poultry Science, University of Maryland, College Park, MD 20742, USA (Received 14 February 1986; accepted 8 August 1986) ABSTRACT Fish silage was compared before and after biological fermentation with L. plantarum. When lactose remaining after fermentation was accounted for, no ( P < 0.05) differences in proximate composition were found. Trout had a larger per cent protein and fat and less ash than white perch both before and after fermentation. Total amino acids increased ( P < 0.05) as a result of fermentation and the increase was reflected in changes in the amino acid profile. A decrease ( P < 0.05) in moisture and an increase ( P < 0"05) in fat were found after the silage was stored at both ambient temperature and 37° C for 35 days. The decrease in moisture was due to condensation on the fermentation vessels and the extraction of lactic acid with the fat resulted in higher fat values. All amino acids increased during storage to a small degree and the increase was attributed to the decrease in moisture. The p H of the silage did not change during storage at either ambient temperature or 37°C but the silage stored at ambient temperature was more acidic than that stored at 37°C. Silage at both temperatures had a sufficiently low p H to maintain a successful fermentation. There was an increase ( P < 0"05) in water-soluble nitrogen at both temperatures as storage time increased. This change should improve the digestibility of the silage. Samples stored at 37°C had a higher Total Volatile Bases value than samples stored at ambient temperature. Total Volatile Bases increased ( P < 0.05) as storage times increased up to 18 days and then started to decline. * Scientific Article No. A4340, Contribution No. 7329 of the Maryland Agricultural Experiment Station (Department of Poultry Science). 187 Biological Wastes 0269-7483/87/$03-50 © Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain
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T. E. Hassan, J. L. Heath
No trends were established when oxidative rancidity measurements were considered. The TBA values were high and could indicate a problem with rancidity which may require the addition of antioxidants. Biologically fermenting fish and storage of the silage under the conditions used in this experiment will result in an increase in free fatty acids due to lipolysis. I f the oil is extracted it will yield an inferior product by commercial standards. Broiler chicks fed a ration containing 5% and 10% fish silage had better ( P < 0.05) feed efficiency than did birds fed a ration with no silage. Results indicate that up to lO% fish silage could be included in broiler rations without adversely affecting feed efficiency or body weight.
INTRODUCTION A method of biological fermentation of fish waste to produce a product (fish silage) that could be used as a protein source in animal and poultry feeds has been described in previous work (Hassan & Heath, 1986). If it is used in animal rations, nutritive characteristics and chemical composition must be determined on the fresh and stored product. Most of the research on chemical changes and nutritive characteristics has been conducted on fish silage produced by adding mineral or organic acids. Windsor (1974) investigated proximate composition of fish silage and monitored certain chemical changes in the protein and oil fractions of the silage which was prepared by adding formic acid to six types of fish. The protein content of all six fish samples fell within a range of 15%-17%. The per cent soluble nitrogen increased sharply to 75% after 10 days and continued to increase to 85% after 30 days. After 50 days there was a negligible change in soluble nitrogen when compared with the level found after 30 days. Moisture and ash contents were at the same levels found in the raw material before the acid was added. Windsor's results showed that the free fatty acid content increased due to lipolysis and that oxidative changes caused the oil to darken in color. The increase in fatty acids was shown to be temperature dependent and was most rapid during the first few days of storage. Krishnaswamy et al. (1965) evaluated the nutritive value of ensiled, fresh water fish prepared by biological fermentation. The ensiled fish was incorporated into a diet fed to rats and compared with a skim milk standard. There was no significant difference found when 10% ensiled fish was incorporated into the diet and compared with the diet containing skim milk powder. In another study, March & Bierly (1961) compared experimentally prepared liquid fish made from British Columbia herring with press cake herring meal, whole herring meal and condensed herring solubles as a source
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of supplementary protein for chicks. Two samples of the liquid fish made by enzymatic and by high pressure acid treatment were used to supply 3 % of the protein in the diet. The comparison showed that the two preparations were similar in protein value and gave a growth response intermediate between that obtained with herring meal and with condensed herring solubles. The effect of fish silage on the quality of hen's eggs and broiler meat was studied by Wirahadikusumah (1969). Eggs from hens fed the silage had thicker and more dense shells than eggs from hens fed fish meal. Egg yolks from hens fed fish silage had a less intense yellow color and a higher iodine value than the yolks from hens fed the fish meal. Feeding up to 40 g per hen per day of wet fish silage did not impair the flavor of the eggs. Feedi~ng broilers from 14 to 60 g of fish silage each day resulted in off flavors after 7 weeks but, when the fish silage was omitted from the ration 1 week prior to slaughter, no off flavor was observed. The TBA and peroxide values were low. The objectives of this research were to determine the composition and chemical characteristics of fresh and stored fish silage produced by biological fermentation and to evaluate it nutritionally in a broiler chick feeding study. METHODS
Fish White perch and trout were used to provide a low-oil fish that would represent some of the underutilized species that could be used in animal feeds. The fish were obtained locally on the day of catch, placed in ice and transported to the laboratory for silage production. Whole fish (including the viscera and heads) were used to produce samples for study.
Silage The fish were ground, lactose added, inoculated with Lactobacillus plantarum and fermented as described in Hassan & Heath (1986). Trout and white perch were fermented separately to compare their chemical characteristics before and after fermentation.
Analytical methods The pH of the supernatant was measured after 5 g of fish silage was mixed with distilled water to a volume of 50ml and centrifuged for 10min at 8000 rev m i n - ~ using a Sorvall RC-2 refrigerated centrifuge.
190
T. E. Hassan, J. L. Heath
Soluble nitrogen was determined according to the method of Barbour (1962, unpublished work in: A collection o f methods used by Pacific fisheries technologists) and expressed as per cent of total nitrogen. Protein was determined on 20-ml aliquots of the filtrate using AOAC (1975) methods. Moisture, fat, protein and ash were measured using methods described in AOAC (1975). Thiobarbituric acid value (TBA) was determined using the method of Tarladgis et al. (1960) and free fatty acids as an index of hydrolytic rancidity were determined according to the method of Wirahadikusumah (1968). Total Volatile Bases (TVB) were measured using the procedures described by Pearson (1970). The amino acid profile of the fish silage was determined using a Durrum D-500 amino acid analyzer (Durrum Instrument Corp., Palo Alto, CA). Three 100-200 mg samples of the fish silage were hydrolyzed under nitrogen in 4 ml of 6N HC1, two for 24 h and one for 72 h, in sealed vacuum tubes at 110°C. Samples were distilled until completely dry, after which 20 ml of a 2.2 pH sodium citrate buffer was added. Preparation of a sample for cystine analysis required the conversion of cystine to cysteic acid. To accomplish this, 2 ml performic acid was added to 100-200 mg of the silage. The solution was refrigerated for 24 h, 0.3 ml hydrobromic acid was added and the solution distilled. The preparation was then hydrolyzed with HC1 as described previously. The sample was injected onto the amino acid analyzer ion exchange column.
Statistical analysis Data were analyzed using the analysis of variance programs BMDP2V and BMD1R at the University of Maryland Computer Science Center. Significantly different means were separated using the Student-NewmanKeul test as described in Sokal & Rholf (1969). Experiment 1 This experiment was conducted to determine the effect of fermentation on composition of ground fish. White perch or trout (100g) were ground separately in a meat grinder using plates with 5mm, 2 m m and 1.5mm diameter pores. Samples were ground through the plates in order of size starting with the largest pore size. Each 100-g sample was placed in a glass screw-capped jar, lactose was added and the mixture inoculated with 1 ml of a culture of L. plantarum grown for 18 h in lactobacilli MRS broth (Difco Laboratories, Detroit). White perch samples were fermented with 5% lactose and trout was fermented with 3% lactose. The difference in the amount of lactose added was based on previous work (Hassan & Heath,
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TABLE 1
Proximate Composition of Whole White Perch and Whole Trout Before and After Fermentation with L. plantarum at 35°C
White perch
Moisture Protein Fat Ash Carbohydrate*
Trout
Before (%)
After (%)
Before (%)
After (%)
73.19 + 3"6c 15"99 ___0"7a 4.57 ___0-3° 6'25 __0"4b --
70-22 _ 5.0b 15-91 + 1.0a 4-99 + 0-4a 6"11 __0'5b 2.77
70-61 ___4.3 b 18"23 _ 1'0 b 8-63 + 0"6b 2"53+0'2 a --
68-56 ___5"6a 17"96 _ 1.5b 8-84 + 0'8 b 2"83__ 0.2 a 1"81
* Carbohydrate was determined by difference. abcMeans ___Istandard deviation with unlike superscripts for each parameter are significantly (P < 0"05) different. 1986) w h e r e it was f o u n d t h a t as ash c o n t e n t o f the sample increased, m o r e acid w o u l d have to be p r o d u c e d to lower the p H to the desired level. P r o x i m a t e c o m p o s i t i o n o f g r o u n d fish was d e t e r m i n e d (Table 1) a n d the a m o u n t o f lactose necessary for a successful f e r m e n t a t i o n calculated. T h e p r e p a r a t i o n was t h o r o u g h l y mixed, sealed and i n c u b a t e d in a w a t e r b a t h at 35°C for 4 days. Samples were r e m o v e d b e f o r e a n d after f e r m e n t a t i o n for p r o x i m a t e c o m p o s i t i o n (n = 10) a n d a m i n o acid (n = 5) analyses. L a c t o s e used in this and s u b s e q u e n t e x p e r i m e n t s was o b t a i n e d f r o m F o r e m o s t F o o d s , Inc., San Francisco.
Experiment 2 W h o l e g r o u n d trout, p o t a s s i u m s o r b a t e (0.05%), c o m m e r c i a l lactose (4%) a n d i n o c u l u m were mixed in a H o b a r t electric m i x e r for 3 min a n d used to d e t e r m i n e if storage after f e r m e n t a t i o n affected the c o m p o s i t i o n o f fish silage. P o t a s s i u m s o r b a t e was mixed first with lactose to ensure u n i f o r m d i s t r i b u t i o n p r i o r to a d d i n g the m i x t u r e to g r o u n d fish a n d f u r t h e r mixing the c o m b i n e d material. U p o n c o m p l e t i o n o f mixing, the c o m p o s i t e was divided into 100-g p o r t i o n s a n d placed in screw c a p p e d jars a n d i n c u b a t e d at 35°C for 3 days. F e r m e n t a t i o n jars were r e m o v e d f r o m the i n c u b a t o r a n d s e p a r a t e d into two equal groups. T h e first g r o u p was stored in an i n c u b a t o r at 37°C while the second g r o u p was m a i n t a i n e d at a m b i e n t t e m p e r a t u r e (23-25°C). A f t e r f e r m e n t a t i o n a n d b e f o r e storage, two samples were r e m o v e d f r o m each j a r a n d a n a l y z e d for p r o x i m a t e c o m p o s i t i o n (n = 10) a n d a m i n o acid (n = 10) content. Samples were r e m o v e d f r o m the jars after storage for 35 d a y s a n d the same analyses c o n d u c t e d .
192
T. E. Hassan, J. L. Heath
Experiment 3 This experiment was a repeat of Experiment 2 except that samples were taken from each fermentation jar after 0, 6, 12, 18 and 35 days' storage at either 37°C or ambient temperature. Each sample (n = 8) was analyzed for free fatty acids, TBA, TVB, soluble nitrogen and pH to determine any changes in the chemical characteristic of the silage after storage.
Experiment 4 Broiler chicks were used to compare the nutritive value of commercially formulated diets containing fish silage with diets without fish silage. Weight gain and feed conversion (feed consumed/weight gain) were monitored. Day old chicks were obtained from a commercial hatchery, placed in electrically heated Petersime brooder units and fed a commercially formulated and mixed starter ration for one week. At the end of the first week chicks were divided into treatment groups with each group having six replicates of nine chicks. Feed and water were available ad libitum and feed wasted was determined and used to correct feed consumption. The treatments consisted of three levels of trout s i l a g e ~ % , 5 % and 10% (Table 6). The experiment was terminated on the twenty-first day. The diets were formulated to be isonitrogenous and isocaloric and to provide 80% of the nutritive requirements of the chick. This provided nutrients at a level calculated not to meet 100% of the nutritive requirements of the chick so that improvement, if any, by the silage could be noted. Diets, both basal and experimental, were formulated with a least cost computer program. The least cost computer program is used by nutritionists in the broiler industry to determine ration ingredients for optimum growth at the least cost. The computer program used the chemical analyses of fish silage from the preceding experiments.
RESULTS A N D DISCUSSION
Experiment 1 Proximate composition of white perch and trout were compared befo(e and after fermentation (Table 1). Differences (P < 0.05) in composition were noted because of the addition of lactose to ground fish to facilitate fermentation. Ground fish analyzed before fermentation did not have lactose added and samples analyzed after fermentation had lactose remaining after fermentation. Lactose was not completely metabolized by L. plantarum and, as a result, had a dilution effect on the fractions. When the lactose remaining in silage was taken into account no differences (P > 0"05)
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193
in moisture, protein, fat and ash were found. The two types of fish, white perch and trout, had different compositions. Trout had more (P < 0.05) protein and fat and less (P < 0.05) ash than white perch. The difference in carbohydrate can be attributed to the different amounts of lactose added at the start of fermentation. Total amino acid content was compared before and after fermentation and tended to increase (P < 0.05) as a result of fermentation, 0.9% for white perch and 3"8 % for trout (Table 2). This increase could partially be explained by the decrease in moisture (Table 1) during fermentation of 3 % and 2% for white perch and trout, respectively, which would increase the relative amount of protein in the silage. The amino acid content of ground fish analyzed after fermentation was diluted by lactose added to the mixture and not metabolized by L. plantarum. The explanation is further complicated because per cent protein decreased during fermentation by 0.08% and 0.27%, respectively, for white perch and trout (Table 1). The change in the per cent of each amino acid was not uniform as would be expected if the TABLE 2 Comparison of the Amino Acid Content of White Perch and Trout Before and After Fermentation at 35°C by L. plantarum
Amino acid
Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total
Before fermentation
After fermen tat ion
White perch
Trout
White perch
Trout
(%)
(%)
(%)
(%)
1'31 0'57 0'59 1'87 0.79 1'26 0'98 0'65 0.40 0.54 0.98 0.43 0.53 0.27 1.01 0'95 13-13 ___0"7°
1-20 0.61 0-54 1"75 0"65 0-98 0"89 0.61 0.42 0-55 0"99 0.44 0-53 0"31 1"04 0"90 12"41 _+0'7 °
1.36 0.61 0.57 1-73 0"99 1-50 1-19 0.72 0.44 0'57 1.05 0'36 0.57 0.29 1' 15 0'95 14.05 + 1.0 ~
1-62 0.74 0-64 2'29 0"92 1"50 1-23 0.79 0'53 0'66 1.25 0.49 0.62 0'39 1"40 1"17 16"24 ___0'9 c
abe Means +__standard deviation with different superscripts are significantly (P<0.05) different.
194
T. E. Hassan, J. L. Heath
increase was caused completely by a decrease in moisture. Apparently, differences in amino acid content before and after fermentation were due to a combination of factors associated with chemical and physical changes during fermentation and cannot be explained simply by concentration due to moisture loss. These changes could affect the availability of the protein for animal metabolism and result in differences in feed conversion and growth. Percentages of individual amino acids did not change (P < 0.05) when the samples before and after fermentation were compared (Table 2). This leads to the conclusion that fermentation of white perch and trout did not change the amino acid quality of the product.
Experiment 2 Significant (P < 0.05) differences were found when the proximate compositions of fish silage were compared before and after storage for 35 days at ambient temperature and 37°C (Table 3). A decrease in moisture content and an increase in fat content were found. The decrease in moisture content was attributed to condensation on the inside of the fermentation vessels and loss during sampling. This decrease resulted in relative increases in some of the other fractions. The amount of fat increased when stored silage was compared with the samples taken before storage. The larger amount of fat found in samples stored at 37°C for 35 days could be the result of analytical procedures used. Since lactic acid is soluble in ether (Merck Index, 1968), it is reasonable to expect that lactic acid was removed during fat extraction. The amount of carbohydrate remaining in the sample after storage is consistent with this explanation. A significant (P < 0.05) difference was found when the TABLE 3 Proximate C o m p o s i t i o n o f T r o u t Silage Before a n d After 35 Days' Storage at A m b i e n t T e m p e r a t u r e a n d at 37°C
Start
35 days storage at
(%) Ambient
37°C
69-76 _+ 5.7 b 16-89 _+ 1'1 a 8-11 _+ 0.8 a 2.60 _ 0.1" 2-64
68"30 _+ 6"2 a 17.44 _+ 1.4 a 10-96 _+ 0'9 b 3-00 _+ 0'2" 0-30
(%)
Moisture Protein Fat Ash Carbohydrate*
70'83 _ 3"6 c 17'10 _+ 1"0a 7"64 _+ 0"4 ~ 2-61 _+ 0.2" 1.82
(%)
* C a r b o h y d r a t e determined by difference. QbcM e a n s _+ s t a n d a r d deviation with unlike superscripts are significantly ( P < 0"05) different.
195
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carbohydrate level in fish silage before storage and silage stored at ambient temperature was compared with the silage stored at 37°C. There were no differences (P > 0"05)i between the carbohydrate levels of silage before storage and that stored at ambient temperature. Obviously, the lactose was more completely metabolized by L. p l a n t a r u m and consequently more acid was produced in the silage stored at 37°C.
TABLE 4
Amino Acid Content of Trout Silage Before and After 35 Days' Storage at 37°C and at Ambient Temperature Amino acid
Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total
Start
1.50 0-66 0.54 2.20 0.80 1-27 1.13 0.71 0.53 0.66 1"18 0.43 0.60 0-43 1.30 1'07 15.01 ___0-8a
35 days storage at Ambient
37°C
1"60 0"71 0'57 2-32 0.85 1"35 1"19 0.76 0.54 0.70 1.23 0'46 0-63 0.50 1.38 1.13 15-92 + 0.8 ~
1-64 0.74 0"61 2.37 0.89 1'43 1"25 0-78 0"55 0-73 1"26 0"45 0-64 0.48 1.41 0'93 16.16 _ 1.2a
a Means _+standard deviation with the same superscript are not significantly (P > 0"05) different.
Total amino acids did not (P > 0.05) change during storage (Table 4). With the exception of the a m o u n t o f arginine in the silage after storage at 37°C, all amino acids increased to a small extent. This increase was attributed to the decrease in moisture since the per cent amino acids were calculated on a wet weight basis. Storage under the conditions of this experiment had a minor,effect on the proximate and amino acid composition of the silage. The differences noted would not be expected to adversely affect the inclusion of stored, biologically fermented fish silage in animal rations.
196
T. E. Hassan, J. L. Heath
Experiment 3 The pH of silage did not change (P > 0.05) during storage for 35 days at either ambient temperature or 37°C (Table 5). The pH of the sample stored at ambient temperature was more acidic (pH 4.43) than that of the samples stored at 37°C (pH4.55) after 12 days' storage and this relationship continued to the end of storage at 35 days. Both treatments had a pH low enough to maintain a successful fermentation. There was an increase (P < 0-05) in water-soluble nitrogen as the storage time increased at both storage temperatures and, after 6 days, those stored at 37°C increased more rapidly than those stored at ambient temperatures. This agreed with work reported by Windsor (1974). Water-soluble nitrogen increased from 58.7% to 71.8% and 77-5% for samples stored at ambient and 37°C, respectively. A relationship was observed between the amount of soluble nitrogen and the consistency of the preserved mass. The silage became more liquefied as storage time and water-soluble nitrogen increased. Since the growth of lactic acid bacteria and the decline in pH prevented other bacterial growth (Hassen & Heath, 1986), it can be assumed that liquefaction was due, almost entirely, to autolytic proteolysis. Tatterson & Windsor (1974) pointed out that little is known about the mechanism involved in liquefaction but it is assumed that gut enzymes which can be spread throughout the mass are responsible. They reported that fillets alone do not liquefy nor do whole fish or parts of fish which have been cooked for 15min at 100°C. The increase in soluble nitrogen is the result of degradation of the complex protein structure and would be expected to increase levels of free amino acids and short chained peptides. These changes should improve the digestibility of silage. Samples stored at 37°C had higher (P < 0"05) TVB values than samples stored at ambient temperature. TVB values increased (P < 0"05) in samples stored at both temperatures as storage time increased up to 18 days and then declined. TVB are related to trimethylamines (TMA), ammonia and other bases. It is known that TMA-oxide can be reduced to TMA by several mechanisms such as by bacterial and endogenous enzymes. Ammonia can come from several reactions such as deamination of amino acids, degradation of nucleic bases and oxidation of amines by bacterial aminooxidases and results in an increase in TVB. Other bases come from the decarboxylation of amino acids. An increase in TVB was reported during the storage of marinated herring prepared by adding acetic acid and NaC1. The increase in TVB was related to the decarboxylation of amino acids by certain strains of lactic acid bacteria (not L. plantarum) isolated from the product (Meyer, 1965).
15.6 _ 1-2 ~b
14.5 _ 1.0 °
67"7 + 3"8 c 81 _ 4.3 b
21.3 ___0.9 ~
16.9 _ 1.1 b
6'5 + 0"6 ~b
152 _ 7"0 e 10.0 _ 0.9 b~a
69"8 _ 4-4 ae 91 _ 5"7 b~
65"5 _ 4"1 b
4"43 _ 0"16f' 4.55 _ 0.17 b~
12
Storage time (days)
a-h M e a n s + - s t a n d a r d d e v i a t i o n w i t h u n l i k e s u p e r s c r i p t s a r e s i g n i f i c a n t l y ( P < 0"05) different.
S o l u b l e N is e x p r e s s e d as p e r c e n t o f t o t a l n i t r o g e n . T V B is t h e m i l l i g r a m s o f n i t r o g e n p e r 100 g silage. T B A is t he m i l l i g r a m s o f m a l o n a l d e h y d e p e r k i l o g r a m .
13-9 _ 0.9 °
37°C
6"3 _ 0"5 ~b
13.9 _ 0.9 ~
11-7 ___ 1.0 ca
37° C
T B A v a l u e (m g pe r 1 0 00 g)
Ambient
37°C Ambient
T V B ( m g p e r 100g)
F F A ( % O l e i c acid)
95 _+ 5"2 c 10'5 _ 1'0 boa
58 ___3-2 a 11"7 + 1"0 ~a
37°C Ambient
64"6 + 3"9 b
58"7 _ 3"2 ~ 58"7 + 3"2 a 58 _+ 3-2 a
Ambient
4'53 -t- 0.14 ab 4.53 _ 0.13 ab
S o l u b l e N (% )
4-50 _ 0"11 ab 4.50 + 0.11 °b
6
Ambient 37°C
0
pH
Storage
23.7 + 1.3 a
22.1 _ 1.4 ca
5'0 _ 0"4 ~
268 _ 16.6 h 4.4 _ 0.3 ~
75-5 + 4.8 y 186 _ 8.0 f
68"0 + 4.4 ca
4-50 _ 0.15 ~b 4'65 _ 0"16 c
18
TABLE 5 Chemical Characteristics of Fish Silage Stored at Ambient Temperaaure and at 37°C
32.8 _ 2.0 y
12.8 _ 1.0 a 27.3 ___ 1-5 e
259 _ 19.5 h 8.2 _ 0.6 ab~
77.5 _ 5.1 f 115 _+ 8.3 d
71-8 _ 4-7 e
4.44 + 0.14 ° 4.55 _ 0.16 bc
35
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T. E. Hassan, J. L. Heath
The decrease in TVB values from 18 to 35 days of storage could be attributed to volatilization or to reactions of amines with unfermented sugars. Since there was no decrease in amino acids during storage (Table 4), it can be assumed that most of the TVB comes from TMA-oxide or other bases produced as a result of nucleic acid degradation and not from decarboxylation or deamination of amino acids. Oxidative rancidity in the fish silage was measured during storage and reported as TBA values. Results showed a relatively high TBA value in the freshly prepared silage when compared with the samples stored at ambient temperature for 18 days and at 37°C for 6, 12 and 18 days. No clear trends were observed but the high values found indicate that rancidity could be a problem and it may be necessary to add antioxidants to the silage. The free fatty acid content of the fish oil extracted from silage was used to measure the extent to which the glycerides in the oil had been decomposed by lipases. Per cent free fatty acids are often used as a criterion for judging the condition and edibility of oil for commercial purposes and a level of less than 4% free fatty acids is required for high quality oils (Tatterson & Windsor, 1974). To prevent decomposition of the oil, it is best to store it under conditions that are free from moisture and protein particles. Based on this, the fish oil in the silage produced in this experiment was under far from ideal conditions. Free fatty acid content increased (P < 0.05) as storage time increased from 13.9% in the freshly prepared silage to 27.3% and 32.8% in samples stored for 35 days at ambient temperature and 37°C, respectively. Silage stored at 37°C had higher values than those stored at ambient temperature. This agreed with Tatterson & Windsor (1974) who reported that sprat silage stored at a number of temperatures showed rapid increase in free fatty acids during the first 40 days and the increase was temperature dependent. Reece (1980) studied the factors responsible for free fatty acid production in the oil fraction of silage made from sprat. He found that increases in free fatty acid content were partially due to the release of free fatty acids from their watersoluble salts after acidification and to the carry over of certain organic acid acidulants. Reece (1981) indicated that much of the initial free fatty acids were present in the digestive tract of the fish prior to acidification. He also reported that increases in free fatty acids in oils from oily fish silate were principally associated with the free fatty acids that came from the solid material during liquefaction of the fish. The increase in free fatty acids reported in this experiment was found in samples that also had an increase in soluble nitrogen and the increase in soluble nitrogen was observed to be associated with increased liquefication of the silage. Biologically fermenting fish and storage of the silage under conditions used in this experiment will result in an increase in free fatty acids and, if the oil is extracted, it will yield an inferior product by commercial standards.
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Experiment 4 Broiler chicks fed a ration (Table 6) containing 5% and 10% fish silage had better (P < 0.05) feed efficiency (feed/gain) than did birds fed a ration with no silage (Table 7). No difference in feed efficiency was found when broilers fed the diets containing 5% fish silage were compared with those fed 10% silage. Broilers fed the ration with 5% silage weighed more (P < 0.05) at the end of the experimental period than those fed the ration containing 0% or 10% silage. These results indicate that up to 10% fish silage produced by biological fermentation could be included in broiler rations without adversely affecting weight gain or feed efficiency. Evidence indicated that feed efficiency would be improved but since these rations were balanced to provide only 80% of the chicks' nutrient requirements, additional studies will be necessary to verify that feed efficiency is improved. TABLE 6 Composition of Diets Used in the Feeding Study
Ingredients
Basal
5% Silage
10% Silage
(%)
(%)
(%)
Corn Soybean meal 49% Fish silage Dical 18.5/22 Fat blend 3700 Limestone Salt Choline CI 50 Fixed ingredientsa oL-Methionine 99% Trace mineral mix b Vitamin mix broiler c L-Lysine HCl 98% Water
64.90 21.79 -1.67 2-78 1-02 0.44 0-13 0-10 0.11 0"05 0"05 0.07 6"89
66.56 19.73 5.00 1.54 1'83 0-98 0"43 0-13 0-10 0.10 0"05 0"05 0"05 3-45
67.42 18.38 10.00 1.41 1-00 0-94 0"42 0-12 0"10 0.09 0-05 0-05 0'02 --
a Amprol Antioxidant Potassium Sorbate (g per 50 kg feed)
0"05 0.02 19
0"05 0-02 17"8
0"05 0.02 16.5
Sorbate was added as a preservative to provide 0.04%. The silage contained 0'05% sorbate. bTo supply the following per kilogram of diet: Vitamin A, 5511-5 IU; Vitamin D3, 2204.6 IU; Vitamin E, 4-41 IU; Vitamin B12, 13 mcg; Riboflavin, 6-614 mg; Niacin, 33-07 mg; d-Calcium pantothenate, ll-98mg; menadione sodium bisulfate, 4.41mg; Folic acid, 0.22mg; Pyridoxine, 1.35 mg. cTo supply the following per kilogram of diet: Manganese, 150 mg; Zinc, 120 mg; Iron, 40 mg; Copper, 6 mg; Iodine, 1-5 mg.
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TABLE 7
Weight Gain and Feed Efficiency of Broiler Chicks Fed Fish Silage
Diet Control 5% silage 10% silage
Weight gain +_SE
Feed efficiency
358"7 _ 18.8a 373.7 _ 23.3b 363"1 _ 21.5a
1.80 _ 0.1 ° 1.74 _ 0-1 b 1.75 _ 0.1 b
o.b Means ___standard deviation, with different superscripts are significantly different (P < 0.05), n = 54.
It was n o t e d t h a t w h e n the rations were mixed, the fish silage b l e n d e d well with c o r n at b o t h the 5 % a n d 10% levels. It was n o t possible to i n c o r p o r a t e m o r e t h a n 10% fish silage in the feed because o f the high m o i s t u r e c o n t e n t o f the silage.
REFERENCES AOAC (1975). Official methods of analysis (12th edn), Association of Official Analytical Chemists, Washington, DC. Hassan, T. E. & Heath, J. L. (1986). Biological fermentation of fish waste for potential use in animal and poultry feeds. Agric. Wastes, 15, 1-15. Krishnaswamy, A. M., Kadkob, B. S. & Revonkar, D. G. (1965). Nutritional evaluation of an ensiled product from fish. Canadian J. Biochem., 43, 1879-83. March, E. B. & Bierly, J. (1961). The protein nutritive value of liquid herring preparations. J. Fish. Res. Bd. Canada, 18, 113-16. Merck Index (1968). Merck and Co., Inc., Rahway, NJ. Meyer, V. (1965). Marinades. In: Fish as food (Borgstom, G. (Ed.)), Academic Press, New York. Pearson, D. (1970). Determination of total volatile bases in fresh foods. In: The chemical analysis of foods. J. and A. Churchill, London. Reece, P. (1980). Control and reduction of free fatty acid concentration in oil recovered from fish silage prepared from sprat. J. Sci. Food Agric., 31, 147-55. Reece, P. (1981). Recovery of high quality oil from mackerel and sprat by the silage process. J. Sci. Food Agric., 32, 531-8. Sokal, R. R. & Rholf, F. J. (1969). Biometry. W. H. Freeman and Co., San Francisco. Tarladgis, G. B., Watts, M. B. & Younathan, T. M. (1960). A distillation method for the quantitative determination of malonaldehyde in rancid food. J. Am. Oil Chemist Soc., 37, 44-8. Tatterson, N. I. & Windsor, L. M. (1974). Fish silage. J. Sci. Food Agric., 25, 369-79. Windsor, L. M. (1974). Production of liquid fish silage for animal feed. In: Fishery product (Kreizer, R. (Ed.)), published by arrangement with the Food and
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Agriculture Organization of the United Nations by Fishing News Ltd, Surrey, UK, 140-3. Wirahadikusumah, S. (1968). Preventing clostridium botulinum type E poisoning and fat rancidity by silage fermentation. Lantbr. Hogsk. Ann., 34, 551-689. Wirahadikusumah, S. (1969). The effect of fish silage on the quality of hen eggs and meat of broiler. Lantbr. Hogsk. Ann., 35, 823-35.