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A BIOPROCESS
FOR TRANSFORMING FISH INTO FEED INGREDIENTS
This process
is patented
Introduction
Fish waste may designate the offals in the canning industry (viscera, heads, damaged and small fishes, strange species etc...). Fish waste may inlude also wastes of fish markets. In this case deteriorated fishes may also be considered as well as altered cans and altered frozen fishes.
Large amounts are discarded every day in the canning industry and/or in the fish markets throughout the world. These amounts can be recycled as a source of high nitrogen content ingredients to be used in feeds. Only part of these wastes is transformed into fish meal by a drying process. The fish meal process cannot be applied to small plants because of the equipment and energy cost, and even if it is used in large production plants, it cannot treat all the wastes available. The energy consumption of the drying process may increase the price of the fish meal and consequently the price of the feed made of this ingredient.
It is assumed that fish wastes from the canning industry constitute a potential protein source for animal feedstuffs. Large quantities of this potential protein source are produced daily and large proportions are discarded throughout the world. The need for animal feeds is increasing, especially for breeding animals and poultry. The agricultural crops to be used in animal feeds are in shortness. So, to balance the lack of vegetable proteins (soya, peanuts etc..) which are imported, the local fish wastes can be enhanced by a low cost process that can be applied to all the canning factories and/or feeds industry.
New techniques using biotechnological processes are more interesting for the control of food and feed systems. Not only wastes are preserved but they are also transformed into a new ingredient. Poultry may require ingredients different to those that would be used for fish or for breeding animals. The biotransformation technology can be monitored in such a way that an ingredient can be made for every kind of animals. Poultry, fish, bovine, dogs and cats etc...
Fish silage was investigated by several workers in the scandinavian countries. They showed various aspects of fish silage techniques and in different diets (Raa et al, 1983; Jackson et al, 1984; Haaland and Njaa 1989, 1990; Espe et al 1992a, 1992b; Espe and Haaland 1992; Faid et al, 1994). So far, The fish silage is still not known to the industrials and/or farmers despite the low cost of the installation for a high scale production and despite the severe lack of animal feed ingredients.
Biotechnological transformation and preservation through a microbial fermentation by acid producing microorganisms (lactic acid bacteria), or a chemical acidification by inorganic and/or organic acids addition, would be the most suitable procedure for recycling wastes from food industry.
Why Lactic acid Bacteria
Biotransformation and/or biopreservation is now being more and more used for several food and feed systems. These techniques are not new to man since fermentation had been used before the discovery of microorganisms. Indeed
food preservation by mean of
salting and drying (olives pickles, meat, dairy and fish products)
dated back to several centuries ago. All the biological processes involving fermentation are almost similar and the most important points in the occurrence of fermentation are:
- Inhibition of undesirable microorganisms in the product
Hazardous species (safety)
Spoilage species (preservation)
- Transformation of some compounds to improve the nutritional quality.
- Improvement of the aroma and flavour.
These properties are due mainly to lactic acid bacteria naturally present in the raw material or brought out as pure starter cultures and which may resist high salt concentrations and overgrow the other microorganisms during fermentation at relatively low temperatures (less than 30°C).
Lactic acid bacteria (LAB) are involved in several food fermentations as a natural microbiota in fermented foods such as olives, pickles, meat and fish products or brought out as commercial starter cultures and used according to a well monitored technology. The activity of lactic acid bacteria in food fermentation is complex but the main metabolism would lead to the production of organic acids (lactic acid), aroma compounds and bacteriocins. All the properties occur naturally in some fermented foods and the self-preservation is due to the activity of these micro-oganisms.
LAB are among the natural microbiota of fish and shell fish but in low numbers so a natural fermentation would take much time and the undesirable micro-organisms can grow while the environment is still suitable for their growth (pH, near neutral, and absence of inhibitors). Inoculation by commercial starter cultures of LAB would be more suitable for a safe product. Acidification of fish waste close to pH 5, prior to fermentation, with mineral acids (sulfuric acid) or organic acids (formic, acetic etc..) may help preventing the early growth of undesirable micro-organisms and may also encourage the LAB growth.
BIOTRANSFORMATION PROCESS
Principle
Fish wastes can be supplied with some other by products from food industry as a source of energy to encourage microbial growth. The mixture can be then inoculated with a starter culture composed of strains of lactic acid bacteria and belonging to the genera Lactobacillus and Pediococcus. The study of the biotransformation should be based on the chemical and physico-chemical changes and these may include pH, dry matter, ash, total and volatile nitrogen, lipids and trimethylamine during a fermentation period of 15 days. In parallel, microbiological determinations must also be carried out to control the course of the fermentation. The microbiological determinations (plate count, coliforms, Clostridium, lipolytics, proteolytics and molds) should also be followed up during the biotransformation period (15 days). These determinations would constitute data on the product for it can be used properly in feeds.
Description
The research works was focused on the preservation of fish wastes by decreasing the pH by the use of lactic acid bacteria. Fish wastes were supplemented with molasse to encourage the fermentation process by microbial strains and inoculated mixture with mixed strains of lactic acid bacteria. The strains tested were isolated locally from some fermenting products.
The role of the lactic acid bacteria strains is to decrease the pH as well as other properties that are not well known. Results of some preliminary assays indicated that the reached pH after 10 days is around 4.2-4.4.
The product is not only preserved against spoilage by proteolytic and lipolytic microorganisms but also may acquire desirable organoleptic characteristics to be incorporated in feeds at proportions higher than those of the fish meal. Moreover the prehydrolysed product is suitable for several feeds.
Strains selection
Screening of lactic acid bacteria strains in single starter cultures and in combinations. This part had been realized in our laboratory during the last two years. 12 strains of lactic acid bacteria isolated from different fermenting materials in Morocco, were identified and characterized for their activities (pH decrease) in the fermentation of fish waste supplemented with beet molasse as a source of carbon. Our collection is now ready for more research on biotechnological processes for fish waste and/or whole deteriorated fish.
The suitable starter culture was added to 10 kg of fish waste in a plastic container to control the fermentation parameters inluding pH, temperature, aeration, agitation and substrate concentration. These parameters are important for a further pilot production and also for the design of a high scale production at the factory. The determination of the suitable conditions for a succeded fermentation was done before any experiment on the biotransformation. This step is the key for an efficient process.
Growth parameters
The parameters studied were pH, molasses proportions and temperature (table 1). The most suitable pH for the fermentation was between 5 and 5.4. This was adjusted by a 50 % sulfuric acid solution. The pH of the silage may decrease during the first phase of fermentation due to the growth of L. plantarum to reach a final pH of 4.31. This value can increase in the product during storage due to protein degradation and also to the buffering capacity of these proteins. The use of L. plantarum as an acidifying agent in the starter culture may maintain the acidity at a low level in the product to prevent some alterations by the undesirable microorganims and/or the contamination by the toxigenic bacteria such as Clostridium or Salmonella. Not only the acid produced by L. plantarum may have a role in the preservation but also some antimicrobial metabolites (bacteriocins) can be produced by this specie and may contribute to the preservation of the silage against pathogens.
Table 1 : Growth parameters for the starter
culture
The addition of sulfuric acid to the initial mixture may increase the acidity for the undesirable microorganims that would induce some alteration reactions during the beginning of fermentation and may prevent some toxins and/or biogenic amines formation. Molasses are added to induce fermentation which may result in lactic acid and some metabolites production that may hide the fish odor in the product. The proportion of 30 % (w/w) results in a best fermentation. This is evaluated by the fish odor disappearence and its substitution by the alcohol odor in the obtained product.
The temperature was also set at 26-28 °C. A good fermentation had been observed at 26°C in a short incubation time. This would be very convenient for the industry because it may correspond to the ambient temperature during peak production of fish wastes. Fermentation by lactic acid bacteria would be done at 30°C.
Chemical and microbiological determinations are carried out on the raw material and on the product during the fermentation and also on the fish product during storage.
- Chemical analyses includes the following parameters:
- pH.
- Non Volatile Dry Matter (NVDM).
- Ash.
- Nitrogen (Total nitrogen TN; Non Proteic Nitrogen NPN; Total Volatile Nitrogen TVN).
Trimetylamine TMA.
Histamine and other biogenic amines.
Carbohydrates (Reducing sugars and sucrose).
Lipids (content, acid degree value and the carbonyls).
- Microbiological determinations include the following microorganisms:
Microorganisms of hygienic significance.
Plate Count, coliforms, Staphylococci, Salmonella and Clostridium.
Microorganisms related to spoilage.
Lipolytic microorganisms.
Proteolytic microorganisms.
Chemical and microbiological
changes during fermentation
Chemical changes
pH variations are shown in figure 2. A regular decrease from 6.7 to 4 after 13 days of fermentation was observed. These values remained almost constant during the last phase of fermentation. The pH decrease in the product gives evidence of a good acidification through lactic acid fermentation by the starter culture of Lactobacillus plantarum.
The most important factor to control during the biotransformation processis the pH decrease which must be slown down as quickly as possible to inhibit the growth of spoilage microorganisms in the product. Moreover, lactic acid fermentation is usually accompanied by the formation of some inhibiting metabolites (bacteriocins) which may help in preservation of fermented foods.
The content in non volatile dry matter (NVDM) are shown in figure 3. The decrease observed during the first stage of fermentation may be due to the production of volatile compounds during the fermentation process. These compounds evaporate with water at 105°C during the determination of dry matter and for this reason was designated as "non volatile dry matter". The breakdown of the organic matter by microoganisms and/or their enzymes is unavoidable during the process and it may result in relatively high amounts of volatile compounds. In some cases and at the end of the process a slight increase - due to water evaporation - of the NDVM was observed.
An increase in the acid degree value (ADV) expressed as of the fat in the product was observed during the initial stage of the fermentation (figure 4). The ADV increase may be due to lipid breakdown by lipolytic microorganisms and/or their lipases. This phenomenon is likely to occur during the first stage of the fermentation while the pH is still about neutral, so lipolytic microorganisms can grow and consequently release their lipases. The ADV in the fermenting product reached 13.2 mg/g and 13.7 mg/g for trials 1 and 2 respectively, and remained almost constant during the fermentation period. This may be explained by lipolysis being stopped by the inhibition of the lipolytic microbiota or by the inhibition of the lipolysis reaction by the free fatty acids released in the medium during the fermentation or by the environmental conditions made by the fermentation (pH aw, inhibitors).
The TVN pattern in the product showed a slight increase during fermentation (figure 5). Haaland and Njaa (1990) found higher values of TVN in capelan stored 1 day before ensiling and in a silage stored for 7 days. These values were 3.1 and 2 % of the TN respectively. The rate of protein liquefaction is faster in acid silage than in fermented silage (Raa and Gildberg, 1982). In the present study, the TVN increased from 39.9 mg/100g (raw material) to reach 95.4 mg/100g in the fermented silage and from 44.5 to 140.6 mg/100g for the acidified silage.
The high level of teh TVN in the acidified silage compared to the fermented silage can be explained by the occurrence of poten proteolysis under a low pH. In fact, endogenous proteolytic enzymes coming from viscera may occur more extremely in acidic environment. This phenomenon may tell about a severe liquefaction of acidified silage due to self autolysis by endogenous enzymes.
The NPN increased also notably in during 13 days of fermentation (figure 6) to reach 1.3 % and remained constant for the remaining period. The increase pattern was controlled by the occurrence of lactic acid fermentation. In the acidified silage, the level is higher relatively to the fermented silage and the two curves in figure 6 show a net difference at the end of the fermentation in the NPN content in the product. A slight increase was observed throughout the period of fermentation in fermented silage. The NPN would indicate proteins breakdown to release amino acids and other metabolites originating from proteins.
The TMA pattern in the product during fermentation is plotted in figure 7 and shows a decrease of TMA in fermented silage from 8.08 mg/100g to 6.76 mg/100g during the first 5 days followed by a rapid increase between day 5 and 9 to reach a maximum of 9.4 mg/ 100g. Lower values (from 4.5 mg/100 to 3.6 mg/100g) are observed in the case of acidified silage. Only slight variations were observed during the last phase of the fermentation. This seems to be due to the removal of the produced TMA continuously by self evaporation which is facilitated by gas production by the yeast culture or to a delaying of its formation by the conditions established in the product which are unfavourable for microorganisms involved in proteins transformation into such compounds.
It would be more interesting to learn about the mechanism by which fish waste can be not only preserved but also the fish odor is removed. The chemical change during the fermentation process would tell about the disappearance of the fish like odor, so the ingredient can be used at high proportions in animal feeds without any artifact. This could reduce the problem of protein sources for animal feeds by encouraging the conversion of fish waste into a suitable ingredient by a low cost process such as lactic acid fermentation. A high increase in the TVN and the NPN was observed while the TMA was reduced considerably in the product. This may be explained by the effect of microorganisms during the fermentation process. Liquefaction may occur by the action of tissue degrading enzymes (Raa and Gildberg, 1982) and the level of autolysis is related to activities of the digestive enzymes either present in the tissue or released by the contaminating proteolytic microorganisms .
Histamin formation in the product was also studied for both silages and results are reported in figure 8. One can figure out that there is net difference between the histamin formation pattern in fermented silage and acidified silage. Preservation by acid addition directly to fish waste would prevent the growth of microoorganisms involved in histamin formation such as most species of Enterobactereaceae and consequently the histamin level may stay almost constant during the storage period.
The fermented silage is susceptible to high levels of histamin because of the long pH decrease process. Lactic acid bacteria may grow first and generate lactic acid from reducing sugars leading to a biological preservation by the pH decrease and some antimicrobial susbtances such as bacteriocins.
It should be enphasized here that histamin formation is widely related to the microbial growth in fish wastes. Even if the phenomenon is accelerated in fermented silage the level stay bellow the average accepted in feeds. Moreover, fish silage is used as an ingredient to be mixed with other constituents and the level of histamin even relatively high should decrease.
Microbiological changes
Microorganisms associated with food hygiene were monitored during fermentation by counting coliforms and Clostridium. The microbial profiles are plotted in figure 9 for coliforms and figure 10 for Clostridium. Coliforms show a net decrease during fermentation to reach a minimum of < 1 cfu/g after 8 days. Counts remained constant for the following days of fermentation. The reduction of coliform numbers may ensure a good biopreservation against undesirable and/or hazardous microorganisms. Clostridium was not detected at high levels in the raw material (<10 cfu/g) and numbers did not increase. The low Clostridium counts may indicate unfavourable conditions made by lactic acid fermentation.
Coliforms were eliminated in the product after 3 days of fermentation. This could be due to the acidification and/or to some inhibitory compounds formed by the lactic acid bacteria. Owens and Mendoza (1985) reported that pathogens (Salmonella) and toxigenic microorganisms (Clostridium and Staphylococcus) are sensitive to a low pH. Moreover fermentation by lactic acid bacteria can result in some inhibitors formed by these microorganisms and may ensure the safety of the product (this hypothesis has not been studied yet in the case of fish silage).
Results concerning the spoilage microorganisms are reported in figures 9 respectively for proteolytics and lipolytics. Proteolytics show a first phase with a rapid slow down to reach low levels after 5 days of fermentation. The same pattern was also observed for the lipolytics but the low level was reached after 8 days. The low level reached for both groups of microoganisms remained constant during the last period of fermentation (from the 8th day to the 17th day) for both trials. The constant low level reached during fermentation may indicate a regular stability at this level and the success of the biopreservation of the fish waste against the undesirable biochemical breakdown of the organic matter leading to a putrefaction of the product during storage.
Proteolytic and lipolytic microorganisms inhibition in the product seems to be due to low pH values and probably to the inhibitory metabolites formed by lactic acid bacteria in the medium during fermentation. It can be deduced that all microorganisms studied showed an asymptotic growth at the end of the process. This is because the method used started with a 1/10 dilution and no colony forming units were detected in 10 ml of this dilution at the end of the process. Numbers were reported as <1 ufc/g but counts could be lower than reported.
Proteolysis may result in high amounts of TVN, NPN and TMA. and high levels of hydrolysis may lead to severe losses in the nutritional compounds in the product obtained. However, liquefaction due to the proteolytic enzymes may produce a good product that would contain avaulable ingredients that could be used by the digestive system of the animal fed on the product.
CHARACTERIZATION OF THE OBTAINED PRODUCT
Microbiological
The microbial profiles reported in table 2 show a good effect of the fermentation on the initial microbiota. The pathogens are eliminated in a short time and less than 1 CFU/g was reached after 10 days. The microorganisms including lipolytic and proteolytic species were reduced to low levels (102 CFU/g) that would not yield severe hydrolysis of the organic matter (proteins and fat) during the storage of the product.
Data about the microbiology of fish silage is not precised relatively to the chemical composition. It would be interesting to know about the microbial load of the silage for more elucidation of the safety (pathogens and toxigenic microorganisms) and the biochemical process (proteolytic lipolytic and lactic acid bacteria). Low levels of pathogens in the silage may indicate a safe product and a succeded silage-process for eliminating or delying undesirable microorganisms. The level of the lipolytic and proteolytic microoganisms decreased notably in the silage (table 2). Values for the lipolytics ranged from 1.103 to 1.106 cfu/g in the raw material to less than 10 cfu/g in the obtained product. proteolytics ranged from 1.5 104 to 1.6 105 cfu/g in the raw material to less that 10 cfu/g in the product. A high level of these microorganisms would tell about the biochemical changes that would occur in the product during the fermentation and also during storage.
Table 2: Microbial profiles in the different trials of fish waste fermentation (incfu/g) after 10 days.
Proteolysis may result in high amounts of TVN, NPN and TMA. and high levels hydrolysis may lead to severe losses in the nutritional compounds in the obtained product. However, liquefaction due to the proteolytic enzymes may constitute a good product that would contain disponible ingredients that would be used by the digestive system of the animal feeded on the product.
Coliforms are eliminated (in a short period and the presence of Proteus and other amines forming species can be prevented by the acidification/fermentation process. Total coliform counts ranged from 4 103 to 1. 106 cfu/g in the raw material to a less than 1 cfu/g in the product. Salmonella was not detected in the obtained product and this may ensure a good safety of the product. Some species of Salmonella like S.pollurum gallinarum are pathogenic for poultry and the obtained product can be used in poultry feeds as a high protein ingredient. Pathogens were eliminated in the product after 3 days of fermentation. This can be due to the acidification and/or to some inhibitory compounds formed by the lactic acid bacteria.
Owens and Mendoza (1985) reported that the pathogens (Salmonella) and the toxigenic microorganisms (Clostridium and Staphylococcus) are sensitive to a low pH. Moreover the fermentation by lactic acid bacteria can result in some inhibitors formed by these microorganisms and which may ensure the safety of the product.
Chemical
The chemical and physico-chemical characteristics of the raw material (initial) and the obtained product are reported in table 3. Only slight variations are observed in the broad composition of the product. The dry matter ranged from 28.25 to 39.9 % in the product for the 4 trials and a decrease of the dry matter was also observed.This is due to the hydrolysis of the organic matter by enzymes and/or microoganisms. Fat content and ash were almost stable in the raw material and in the product. The ash content ranged from 6.84 to 8.68 %. There was wide variation among samples so the fat content values ranged from 3.83 to 8.85 %.
Table 3 : Physico-chemical and chemical composition of fish waste after 10 days fermentation
DM: Dry Matter RS: Reducing Sugar, NPN: Non Proteic Nitrogen
TVN: Total Volatile Nitrogen TMA: Trimethylamine * calculated as TN x 6.25 in %
Our results do not agree with those reported by Espe et al (1989) who found a fat content of 11.5 % after 15 days storage in a formic acid silage. The same authors obtained an ash content of 1.96 % in the silage. This difference can be explained by the nature of the raw material. That in our case the fish wastes contained high proportions of bones and low levels of fat. Tatterson et al (1984) observed a slight variation of the ash content ranging from 2.1 to 2.7 and a wide variation of fat content between 0.5 and 16.3. The authors worked on different fish species.
Wide variations in TVN, TMA and NPN were observed in the obtained product relatively to the raw matter (fish waste). The TVN values ranged from 1.57 to 3.24 in the product and from 1.13 to 1.88 in the raw material. This increase is not unexpected because of the protein metabolism. Haaland and Njaa (1990) found higher values of the TVN in the raw material stored 1 day before ensiling and in a 7 day stored silage. These values were respectively 3.1 and 2 % of the TN. It was reported that the protein liquefaction is more severe in acid silage than in fermented silage (Raa and Gildberg, 1982). The NPN increased also notably in the product relatively to the raw material. The average values of NPN were 23.79 and 54.92 (as % of the TN) respectivly for the raw material and the final product.
The TMA was reduced considerably in the product relatively to the raw material and to the control. The average values were 0.13 and 0.04 (as % of TN) respectively for the initial and the obtained product. TMA reduction can be due to a removal during fermentation. This hypothesis is the most probably tangible since the TMA is volatile and since the volatility is accelerated by the gas production by yeasts. TMA can also be metabolized by some microorganisms during the fermentation.
Fermented fish wastes in a feed formula for poultry
A high increase in the TVN and the NPN was observed while the TMA was reduced considerably in the silage. This may be explained by the effect of the microorganisms during the fermentation process. Liquefaction may result from the action of tissue degrading enzymes (Raa and Gildberg, 1982) and the level of autolysis is related to activities of the digestive enzymes either present in the tissue or released by proteolytic microorganisms.
The crude proteins calculated as TN x 6.25 ranged from 8.37 to 12.50 % in the obtained product and only a small part is transformed in NPN, TVN and TMA. In some cases high amount of the TN are transformed into NPN and TVN.
ANIMAL FEEDING ASSAYS
Animal feeding experiments were caried out on rats of Wistar species. The fish ingredient was mixed with starch as an extender and feed to young rats 26 days old. The weight was measured during 56 days. Observations on the animals showed no increase in the weight for rats feeded on fish ingredient alone while there was a net weight increase for animals feeded on fish ingredient supplemented with barley flour. Animals were also slaughtered and the carcass examined for abnormal reactions or pathological development on organs.
The experimental feeding program was first done by feeding rats with fish silage alone for nutritional observations. Symptoms of malnutrition were observed in the first experiments trial. In the second feeding assays, fish silage was supplemented with barley flour at different levels. The following formula were applied to lots of 5 rats:
Fish silage 100 % barley 0 %
Fish silage 75 % barley 25 %
Fish silage 50 % barley 50 %
Fish silage 0 % Barley 100 %
This study showed a net increase in rats weight feed on fish silage supplemented with barley flour. This may tell about a lack of some nutrients in fish silage and the composition must be rectified. Chemical studies showed that not only the nitrogen would be improved but also the extending materials (cellulose, pectins etc..;) should be rectified in the formula. Our experiment are to be carried out on other animals and the lack of some nutritional compounds and or cofactors such as vitamins and minerals.
REFERENCES
1. FAID M. KARANI H. ELMARRAKCHI A. and ACHKARI-BEGDOURI A.1994. Fish waste fermentation by the association of yeast and lactic acid bacteria strains.Bioresource technology .49, 237-241.
Espe M Raa J Njaa L R 1989 Nutritional value of stored fish silage as a protein source for young rats. J Sci Food Agr 49 259 - 270.
Espe M Haaland H 1992 The protein value of fish silage prepared from capelin stored under different conditions before ensiling. Effect of storing the silage for one year. Fisk Dir Skr Ser Ernoering 5 (1) 37 - 44.
Espe M Haaland H Njaa LR 1992 Substitution of fish silage protein and a free amino acid mixture for fish meal protein in a chicken diet. J Sci Food Agric 58 315- 319.
Haaland H and Njaa L R 1990 Fish silages prepared from raw materials of varying quality; Chemical analysis related to balance experiments in rats. Fisk Dir Skr Ser Ernoering III 27-35.
Haaland H and Njaa LR 1989 Effect of temperature on the autolysis of capelin silages stored for one year. Fisk Dir Skr Ser Ernoering III 219-226.
Jackson A S Kerr A K R Cowey C B 1984a Fish Silage as a dietary ingredient for salmon. I-Nutritional and storage characteristics. Aquaculture 38 211-220.
Jackson A.S. Kerr A. K. Cowey C. B. 1984b. Fish Silage as a dietary ingredient for salmon. II Preliminary growth findings and Nutritional pathology. Aquaculture 40 238-291.
Owens J. D. Mendoza L. S. 1985. Enzymically hydrolysed and bacterially fermented fishery products. J. Food Technol 20, 273 - 293.
Raa J. Gildberg A. 1982. Fish silage: A review CRC Critical Review in Food Science and Nutrition. 16 383-419.
Raa J. Gildberg A. Strom T. 1983. Silage production-Theory and pratice in Upgrading waste for feeds and food. eds. Ledward D. A. Taylor A. J. Lawrie R. A (Nottingham)
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