Agricultural Wastes 15 (1986) 1-15

Biological Fermentation of Fish Waste for Potential Use in Animal and Poultry Feeds*

T. E. Hassan & J. L. Heath~"

Department of Poultry Science, University of Maryland, College Park, Maryland 20742, USA

ABSTRACT

Biological fermentation of whole fish, viscera and heads using Lactobacillus plantarum was evaluated and the minimum lactose necessary for a successful fermentation under pilot conditions was found to be 5 %. Preheating the fish before fermentation decreased the amount of soluble nitrogen substances both before and after fermentation. The relationships between lactic acid bacteria growth, yeast and mold growth and pH indicated that it may be necessary to add an antimycotic agent to achieve and maintain sufficiently low pH values for successful fermentation and storage. Fermentation temperatures of 25 and 35°C and inoculum size of 103 organisms g - l offish produced successful fermentations.

INTRODUCTION

Fish protein is used extensively in poultry and swine production as a source of high-quality protein. Because the production of fish meal has levelled off in the past years and available fish stocks are not sufficient to sustain increased production levels, supplies have not been adequate (Anon., 1979). Limited supplies and the demand for fish meal as a source

* Scientific Article No. A-3918, Contribution No. 6899 of the Maryland Agriculture Experiment Station (Department of Poultry Science). t To whom correspondence and requests for reprints should be addressed.

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Agricultural Wastes 0141-4607/86/$03-50 © Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain

2 T .E . Hassan, J. L. Heath

of human food has made it necessary to look for alternative sources offish protein for animal feed.

Waste products from fish-processing operations and the use of underutilized species of fish to produce fish protein for animal feed have been studied. The use of fermentation or acid addition to produce fish silage has been studied as a way of using these sources to produce fish protein for use in animal feed. Fish silage has been defined by Tatterson & Windsor (1974) as a liquid product from whole or parts offish to which no materials are added other than acid, and in which liquefaction of the fish mass is carried out by enzymes present in the fish. Fish silage has become of interest to the poultry industry as a feed ingredient and this interest has been directed toward methods of production using either direct acid addition or biological fermentation. Most of the information is available on the direct acid addition method with comparatively little information on the biological fermentation method. Biological fermentation has been used to produce a fish silage product by using microorganisms to produce the acid (Peterson, 1953). Stanton & Yeoh (1977) investigated the use of lactic acid fermentation at low salt concentration to produce fish silage under South-East Asian conditions. They used a local source of carbohydrate, tapioca starch, as an energy source. Results showed that fish silage can be successfully produced using a 1:1 fish/starch ratio and adding ragi and lactic acid starter cultures. March (1962) indicated that the application of molasses and inoculation with lactic acid bacteria was the basis of a patented Danish method.

The effect offish silage (14-60 g hen- 1 day- 1) on the quality of hen's eggs and broiler meat was studied by Wirahadikusumah (1969). He found that the eggshells from hens fed fish silage were thicker and denser than shells from hens fed fish meal. He theorized that the lactic acid present in the fish silage apparently facilitated the uptake of calcium. The yolks of eggs from hens fed with fish silage were less yellow and had a higher iodine value than yolks from hens fed fish meal. Stability of the egg foam did not differ between the two treatments. Feeding 40 g of wet fish silage per hen per day did not impair the taste or flavor of the eggs. Feeding broilers at the same level resulted in off-taste and flavor within 7 weeks, but when silage was removed 1 week prior to slaughter no off-taste and flavor were observed. It was concluded that fish silage can be incorporated into the diets of both broilers and hens if precautions are observed to prevent off- taste or flavor. Norwegian research has demonstrated that fish silage is equal to herring meal for growth and egg production in hens; superior

Fermentation oj~sh waste 3

hatchability was also reported. Laying birds may be fed about 250 g of fish silage per 12 birds without producing tainted eggs, and breeding fowl may be fed 50 % more than this (Windsor, 1974).

The objective of this research was to provide information to facilitate production of fish silage from waste and underutilized fish by biological fermentation for potential use in animal and poultry feed. To provide this information, levels of carbohydrate necessary for successful fermen- tation, and the effect(s) on the silage product of using whole fish or offal, were evaluated. The effects of preheating the fish, yeast and mold contamination, fermentation temperature and inoculum size on the fermentation process were also investigated.

METHODS

Fish

White perch were used in this study to provide a low-oil fish that would be representative of some of the underutilized species and would provide material that was representative of waste material from commercial processing operations.

Sample preparation

Whole fish and viscera and heads were required for the studies. Whole fish were defined as the entire fish as they came from the water. Viscera and heads were obtained by making a longitudinal cut from the back of the head to the anus. Whole fish and viscera and head samples were ground separately in a meat grinder using plates with 5 mm, 2 mm and 1.5 mm diameter pores. The samples were ground through the plates in order of size starting with the largest pore size.

Inoculum

Lactobacillus plantarum was used to inoculate the fish for fermentation because it had been proved to ferment fish efficiently under the conditions used in the present experiments (Hassan, 1982). The organism was obtained from the culture collection of the Food Science Program at the University of Maryland and maintained in litmus milk at 4 °C. Prior to

4 T .E . Hassan, J. L. Heath

use, it was activated by three successive transfers into MRS broth (Difco Laboratory, Detroit, Michigan). Inoculation was made from a culture grown for 18 h.

Analytical methods

5 g offish were mixed with distilled water to a volume of 50 ml, centrifuged at 8000 rev min- 1 for 10 min using a Sorvall RC-2 refrigerated centrifuge, and pH was measured on the supernatant.

Titratable acidity was measured by titrating 10 ml of the supernatant obtained as described for pH determination with 0"IN NaOH using phenolphthalein as the indicator. Acidity was expressed as ~ lactic acid by applying the following formula:

(Vol. NaOH) (N of NaOH) (eq. wt of lactic acid) (100) ~o Lactic acid --

1000 (g of sample)

Percent developed acidity was determined by subtracting the ~o titratable acidity of fish before fermentation from the ~ titratable acidity of the fermented sample.

Soluble nitrogen was determined according to the method of Barbour (1962, unpublished work). Moisture, fat, protein and ash were measured using AOAC (1975) methods.

Microbiological examination

The sample was prepared by homogenizing l0 g of fermented fish in 90 ml of peptone water (0.1 ~) for 2 min, and dilutions of 10- 2 to 10- s were prepared. Total viable counts were determined using the procedure of Gilliland et al. (1976) and total mold and yeast counts were determined as outlined by Koburger (1976).

Statistical analysis

Analysis of variance was calculated using the computer program BMDP2V and BMD 1R at the University of Maryland Computer Science Center. Significant differences between means were determined by the Student-Newman-Keul test as described in Sokal & Rohlf (1969).

Fermentation offish waste 5

Experiment 1

100 g of either ground fish or ground viscera and heads were placed in glass, screw-capped jars. To each jar 2, 3, 4 or 5 ~ lactose was added, then the sample was inoculated with 1 ml of a culture of L. plantarum grown for 18 h in MRS broth. The preparation was mixed thoroughly, sealed and incubated in a water bath at 35 °C. After 2 and 7 days, 5 g offish were aseptically removed and mixed with 45 ml of distilled water. Titratable acidity and pH were measured on each sample.

Samples were removed from the whole fish and the viscera and head samples prior to fermentation for proximate composition analysis.

The lactose used in this and subsequent experiments was obtained from Foremost Foods, Inc. (San Francisco, California).

Experiment 2

Four groups of 18 sterilized, screw-capped jars were used. To each of the jars, 20 g of ground, whole fish was added. The first group was autoclaved at 109°C for 15 min and then cooled (preheated fish). 5 ~o lactose and 0-2 ml of L. plantarum were added to each of these jars. To the remainder of the jars either 5, 7 or l0 ~ lactose was added and inoculated with 0.2 ml of L. plantarum. These jars contained fish that had not been preheated. All jars were mixed, sealed and incubated in a water bath at 35 °C.

At intervals of 6, 12, 24, 48, 72 and 168 h, three jars were removed at random from each treatment and pH, titratable acidity and total soluble nitrogen were measured. Titratable acidity was also determined on the fresh and preheated fish before fermentation so that developed acidity could be calculated.

Experiment 3

To study relationships between lactic acid bacterial growth, yeast and mold growth and pH offish silage, a total of 100 g offish and either 3 ~o or 5 ~o lactose were added to glass jars and each was inoculated with I ml of L. plantarum. Each jar was mixed, sealed and incubated at 35 °C. Samples were removed from each jar before incubation and after 24, 48, 96 and 168 h and yeast and mold counts, lactic acid bacteria counts and pH were determined.

6 T.E. Hassan, J. L. Heath

Experiment 4

The culture jars were prepared as described in Experiment 3 except that only the 5 ~ lactose level was used and the lactose and culture were added after the fish reached the water bath temperature. The jars were divided into 3 groups and incubated at either 25, 35 or 45°C. After 6, 12, 24, 48, and 72h, samples were removed and pH determined. Samples were removed before incubation and after 72 h and the number of lactic acid bacteria and total viable counts were determined.

Experiment 5

Fermentation jars were prepared as described in Experiment 4 except that two levels of inoculum were used. The jars were divided into three groups and inoculated with either 103 or 105 organisms g- 1 offish. At intervals of 6, 12, 24, 48, 72, 96 and 120, a sample was removed from each jar and the pH was determined.

The inoculum was prepared by spreading prepared MRS agar plates with a culture of L. plantarum. The streaked plates were incubated under CO 2 for 48 h at 35 °C. A suspension of the organisms was prepared by removing colonies from the plates and suspending them in 0" 1 ~/o sterile peptone water. The suspension was shaken until a homogeneous suspension was obtained. The ~o transmissions of the stock and diluted suspension were measured at 540 nm and the total numbers of organisms were determined by direct microscopic count using a counting chamber (Hausser Scientific No. 900). These determinations were found to be accurate and reproducible.

RESULTS AND DISCUSSION

Experiment I

The amount of carbohydrate necessary to ferment fish successfully with L. plantarum was evaluated using lactose. Lactose was chosen because it is readily fermentable by the organism and the experiment would not be confounded by a more complex carbohydrate or by-product source. Also the use of lactose would provide a standard to which other carbohydrates could be compared when determining amounts to be added to produce a

Fermentation offish waste 7

successful fermentation. Whole fish and viscera and heads were compared to determine if the two potential sources of material for fermentation gave different results.

All lactose levels tested resulted in a decrease (P < 0-05) in pH after 2 days of incubation at 35 °C in both whole fish and in viscera and head samples (Table 1). A minimum of 4 % lactose was needed to obtain a successful fermentation to pH 4.5 (Disney & Tatterson, 1977) after 2 days. The pH levels after 7 days showed the same trend as that found after 2 days, which was a decreasing pH as more lactose was added, but the samples taken after 7 days did not have as low a pH as those taken after 2 days with the exception of whole fish with 5 % lactose. This increase in pH after the initial decrease could cause stability problems.

Percent titratable acidity tended to agree with the pH values but was considered to be an overestimation of lactic acid produced because of the influence of proteins (amino acids) on the titrations.

Proximate analysis of the whole fish and viscera and head samples

TABLE 1 Effect of Lactose Level and Length of Fermentation on pH Decline in Whole Perch and

Viscera and Heads During Fermentation by L. plantarum at 35°C

pH* Titratable acidity (°/0)

Treatments 2 days 7 days ApH 2 days 7 days

Whole fish: Initial pH 6.96" 2 ~o lactose 3 ~o lactose 4 ~o lactose 5 ~ lactose

5.20 b 5-78 b 0.58 2-29 1.80 4.75 c 5.14 c 0.39 3-58 3.60

4'49 a 4-64 ~ 0.15 4.81 5.57 4'44 a 4-41 d - 0 . 0 3 5.22 6-68

Viscera and heads: 2 ~o lactose 4-92 b 6.50 b 1.58 1.94 1-22

3 ~ lactose 4.60 ¢ 5.22 c 0.62 3.09 1-56 4 ~,, lactose 4.35 a 4.90 a 0.55 3.96 3-47 5 ~o lactose 4-202 4.78 a 0.58 4.37 3-09

.,b,c.d,e Means within columns for each fish portion with the same superscripts are not significantly (P < 0.05) different. * pH was significantly (P < 0.05) different when 2 and 7 days were compared for both whole fish and viscera and heads samples. The individual differences are not noted by superscripts because significant (P < 0-05) interactions were found.

8 T .E. Hassan, J. L. Heath

provided data on composition of the fish used for fermentation (Table 2). The sa/nples consisting of viscera and heads had a larger ~ ash than the whole fish samples (7-6 ~o vs. 5.6 ~) which could account for some of the titratable acidity differences between the two samples. It was reported by Olsson (1942), Peterson (1953) and Lagunov et al. (1958) that ash, which was composed of salts of phosphates, carbonates, etc., was directly related to the amount of added (not produced as a result of fermentation) acid required to lower pH in fish silage. Peterson (1953) reported that there was also a direct relationship between protein and the amount of acid required to lower pH. In this experiment, both types of samples had pH values that increased (P < 0.05) as the fermentation continued from 2 to 7 days and the samples composed of viscera and heads had larger increases than the whole fish samples. At least part of the differences between the two types offish samples could be explained by differences in ash content. The increase could also be caused by protein breakdown, insutticient fermentable sugar or not enough viable organisms to resume acid production after the pH increased, and yeast or mold growth that would use lactic acid as a carbon source.

Experiment 2

The level of lactose was increased to determine if additional fermentable sugar would allow acid production to continue or would permit growth of other organisms after L. p l a n t a r u m stopped growing. Also whole fish samples with 5 ~ lactose added were preheated (109°C, 15 min) in an effort to inactivate enzymes responsible for increasing total volatile bases, decrease flora on the fish prior to fermentation that mightcompete for the added sugar, and reduce total volatile nitrogen substances that are present in fish.

During the first 24 h of fermentation, the preheated treatment showed a

TABLE 2 Proximate Composition of Whole Perch and Viscera and Heads

Moisture Protein Fat Ash

t Vo) ~ %) I °/o) ~ "/o)

Whole perch 73.9 15.9 4.6 5.6 Viscera and heads 71.0 15.9 5.5 7.6

Fermentation offish waste 9

TABLE 3 A Comparison of pH Levels after Fermentation of Preheated Fish Containing 5 %

Lactose and Fish Containing 5, 7 and 10 ~o Lactose

Treatment Fermentation time (h)

6 12 24 48 72 96 168

Preheated ° (5 % lactose) 6-40 4.73 4.70 4-45 4.43 4.45 4.32 ¢

Fresh b (5 % lactose) 5.57 4.62 4.67 4.50 4-50 4.52 4.43 ~

Fresh b (7 ~o lactose) 5.62 4.60 4.62 4.47 4.42 4.42 4.37 cd

Fresh b (10% lactose) 5.58 4-67 4.65 4.47 4.40 4.40 4-33 c

° Autoclaved at 5 Ib pressure (109°C) for 15 min. b Not preheated. c,d Means within columns with different superscripts are significantly (P < 0"05) different.

o

o t_

I-- 4

Fig. 1.

d,5 Yo L a c t o l l •

Hours Titratable acidity due to acid production by L. plantarum grown on preheated

fish with 5% lactose and on fresh fish with 5, 7 and 10% lactose.

10 T. E. Hassan, J. L. Heath

slower rate of pH reduction than the fresh fish treatments (Table 3). The pH values for the preheated fish were significantly (P < 0.05) lower than those for fresh fish with 5 % lactose after 168 h. Titratable acidity was also much lower for the preheated samples when compared to all of the fresh fish samples (Fig. 1). The increase in pH between 2 and 7 days for 1-4% lactose samples (whole fish) in the first experiment was not found at these lactose levels (5, 7 and 10 %) or in preheated fish. This verified the finding in Experiment 1 that 5 ~o lactose in whole fish did not result in a pH increase as the silage aged. The addition of higher levels of lactose did not result in lower pH values because L. plantarum will not grow and produce acid below a pH range of 4-4.5 according to Jay (1978). No evidence of increased yeast, mold or other microorganism growth was detected when samples from the various treatment groups were compared.

The amount of soluble nitrogen compounds was determined to see if they were reduced by preheating. This was done by measuring levels in

60

5¢

40

c- o 3 ¢

Z ~

o V .~ 2c

o o9

Fig. 2.

, • i i Z4 48 72 96

Hours

Soluble nitrogen ( ~ of total N) produced during fermentation at 35°C using fresh white perch and preheated white perch with 5 ~o lactose.

Fermentation offish waste 11

preheated and fresh samples before and after fermentation with 5 % lactose (Fig. 2). Preheating reduced the initial amount of soluble nitrogen substances and substantially reduced the amount released during fermentation. After 24h of fermentation only small amounts were released up to 96 h.

The change in pH due to use of lactic acid as a carbon source is still a possibility since yeast and molds could have been present in samples from Experiment 1 but absent in samples from this experiment.

Experiment 3

The relationships between lactic acid bacteria growth, yeast and mold growth and pH offish silage with 3 % and 5 % added lactose are presented

,o 1

--Q-- 3 % Lactose - - o - 5 % Lactose

o ®8" O0 7" Mold and

(h

z

i 7

16 O)

-l- td.

3 -

2- ' 4

Fig. 3.

0 24 48 96 168 H o u r s

Changes in lactic acid bacteria counts, mold and yeast counts and pH during fish silage fermentation at 35°C with 3 % and 5 % lactose added.

12 T. E. Hassan, J. L. Heath

in Fig. 3. The 3 ~ lactose level was used to produce a pH above 4.5 to provide a comparison for the 5 % lactose samples.

Lactic acid bacteria demonstrated an increase in growth to a maximum at 48 h followed by a decline in numbers until 168 h was reached. As expected, the lactic acid bacteria in the 3 ~ lactose samples decreased (P < 0.05) in numbers at a faster rate than those in the 5 % lactose samples. Correspondingly the pH values declined to between 4 and 5, with the 5 ~ lactose samples producing the lowest pH. This agreed with the previous experiments where 5 °//o lactose was required to reduce the pH to 4-5 and lower. As the lactic acid bacteria numbers and pH decreased, the yeast and mold counts increased. Between 96 and 168 h the pH tended to increase in both the 3 ~o and 5 ~o lactose samples, which partially agreed with Experiment 1 (Table 1) where an increase was observed between 2 and 7 days for 3 ~o whole fish samples. This increase in pH value occurred during the period when the yeast and mold counts were reaching their maximum. The yeast and mold growths on the plates were microscopic- ally examined and most of the growth was in samples with 3 % lactose, which were the ones in which the lactic acid bacteria numbers were lowest.

This study indicated that an antimycotic agent may be required to control mold, and possibly yeast, growth to achieve and maintain sufficiently low pH values for a successful fermentation and storage. Addition of agents to reduce mold and yeast growth may allow for a reduction in the amount of carbohydrate required since less lactic acid would have to be produced to maintain the required pH.

Experiment 4

This experiment was conducted to determine the effect of fermenting fish at temperatures other than those considered to be optimum for L. plantarum.

Results of fermenting whole fish at 25, 35 and 45 °C are shown in Fig. 4. Analysis of variance showed a significant (P<0.01) fermentation time × fermentation temperature interaction. The fermentations at 25 and 35 °C were considered to be successful because the pH was reduced to 4.40 and 4.45, respectively. The fermentation at 45 °C produced a pH of 5.03. The successful fermentation at lower temperatures indicated that a wide range of temperatures will produce a satisfactory silage product and this could result in substantial energy and equipment savings.

At 25 °C lactic acid bacteria increased from an initial count of 16 x 105 to 13 x 10 s organismsg -1 fish in 72h, at 35°C the increase was from

Fermentation of .[ish waste 13

22 x 10 6 to 45 x 10 a and at 45°C the increase was from 42 x 10 6 to 27 x 107. The total viable counts were also monitored and indicated that essentially all of the growth was accounted for by lactic bacteria. This was expected from the work of Price & Lee (1970) and Wirahadikusumah et al. (1971), who found that lactobacilli were capable of inhibiting the growth of other organisms.

7

6

¢

0 0 25°C P

~ : 55oc

Fig. 4.

3 i I I I I I I 0 6 12 24 48 72

Time (hrs)

Effect of incubation temperature on pH decline during fermentation of white perch.

Experiment 5

The effects of L. plantarum inoculum sizes of 10 3 and 105 organisms g- 1 fish on fermentation were evaluated. The change in pH after 6, 12, 24, 48, 72, 96 and 120h was used to determine any differences in the rate of fermentation and the time required for successful fermentation to be achieved.

The pH values decreased more rapidly in the samples inoculated with 105 organisms g- 1 of fish than in those inoculated with 10 3 organisms (Table 4). A successful fermentation was achieved between 24 and 48 h in both samples. The pH stabilized at a lower value for the higher inoculum level and both inocula maintained a stable pH for the 72-120 h period. The main effects of inoculum size and time after inoculation produced

14 T. E. Hassan, J. L. Heath

T A B L E 4 Effect of L. plantarum Inoculum Size on Ability to Maintain a Successful Fermentation

when Measured by pH

Inoculum (Organisms g- x) Initial

Fermentation time (h )

6 12 24 48 72 96 120

103 6-86 6.85 6.70 4.93 4.48 4.50 4.50 4.50 105 6.85 6.73 6.18 4-65 4.45 4.40 4.40 4.40

significant (P < 0.01) differences and a time by inoculum size interaction was also found to be significant (P < 0.01).

The fish used were not sterilized and this resulted in the L. plantarum being added to the flora already present in the samples as would be the case in a practical fermentation operation. It appears that 103 organisms g - 1

of fish or more are adequate to give successful fermentation and higher levels may be added to achieve the safety factor of lower pH.

The colonies from the successful fermentations on total plate count agar were homogeneous and identified as L. plantarum. Shewan (1971) and Price & Lee (1970) indicated that peroxide formation by L. plantarum inhibits the growth of organisms such as Bacillus, Pseudomonas and Proteus species which are present on sea-food.

R E F E R E N C E S

Anon. (1979). The state of jood and agriculture 1978. FAO Agriculture Series No. 9, Rome.

A OAC 0975). Official methods of analysis. (12th edn.), Association of Official Analytical Chemists, Washington, DC.

Disney, G. J. & Tatterson, N. I. (1977). Recent developments in fish silage. In: Proceedings of the Conference on the Handling, Processing and Marketing of Tropical Fish. Tropical Products Institute, London.

Gilliland, E. S., Busta, F. F., Brinda, J. J. & Campbell, E. J. (1976). Aerobic plate count. In: Compendium of methods for the microbiological examination of foods (Speck, L. M. (ed.)). American Public Health Association, Washington, DC.

Hassan, T. E. (1982). Fish silage produced by biological fermentation. PhD thesis, University of Maryland, College Park, Maryland.

Jay, M. J. (1978). Modern food microbiology Van Nostrand, Co., New York. Koburger, A. J. (1976). Yeasts and molds. In: Compendium of methods for the

microbiological examination o f foods (Speck, L. M. (ed.)). American Public Health Association, Washington, DC.

Fermentation offish waste 15

Lagunov, L. L., Egorova, L. N., Rekhina, N. I. &-Eremieva, M. N. (1958). Investigation of the acid preservation offish and fish offal. Trudy vsesoyuznozo Nauchno-issledovatelskogo lnstitua Mohskogo Rybnogo Khozyaistua Iokeanograffii ( UNIRO), 35, 115-30.

March, E. B. (1962). Fish meal and condensed fish solubles in poultry and livestock feeding. In: Fish as Food (Borgstrom, G. (ed.)). Academic Press, New York.

Olsson, N. (1942). Experiments concerning the preservation and use of fish products as feeding stuff for hens and chickens. Domestic Animal Research Institute of the Agriculture College, Report No. 7, Lantbrukshagskolans Husdjursfors 9 Ksanstalt, Experimentaltet, G6teborg, Sweden.

Peterson, H. (1953). Acid preservation of fish and fish offal. Technological Laboratory, Ministry of Fisheries, Copenhagen.

Price, R. J. & Lee, J. S. (1970). Inhibition of Pseudomonas species by H20 2 producing lactobacilli. J. Milk Food technol., 33, 13 17.

Shewan, J. M. (1971). The microbiology of fish and fishery products--a progress report. J. Appl. Bacteriol., 34, 299 312.

Sokal, R. R. & Rohlf, F. J. (1969). Biometry. W. H. Freeman and Co., San Francisco.

Stanton, R. W. & Yeoh, L. G. (1977). Low salt fermentation method for conserving trash fish waste under Southeast Asian conditions. In: Proceedings of the Confi, rence on the Handling, Processing and Marketing of Tropical Fish. Tropical Products Institute, London.

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 Agriculture Organization of the United Nations by Fishing News Ltd, Surrey, UK., pp. 140-3.

Wirahadikusumah, S. (1969). The effect of fish silage on the quality of hen eggs and meat of broiler. Lantbr. Hogsk. Ann., 35, 823-35.

Wirahadikusumah, S., Rajala, O., Lindgren, S. & Nilsson, R. (1971). Anti- microbial properties of lactic acid bacteria isolated from fish silage. Swedish J. Agric. Res., 1,225-7.