Aromatic hydrocarbons removal by immobilized bacteria (Pseudomonas sp. Staphylococcus sp.) in fluidized bed bioreactor

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Characterization of fermented fish waste used in feeding trials with broilers

A. H a m m o u m i , a M. Faid, b* M. E1 yachioui c and H. A m a r o u c h "

"Facult6 des Sciences Ain Chok, Route dl~l Jadida Km 8, BP 5366, Casablanca, Morocco bInstitut Agronomique et V6t6rinaire Hassan II BP 6202 Rabat-lnstituts, Morocco

~Facult6 des Sciences Kdnitra, BP. 133, K6nitra, Morocco

(Received 17 March 1997; revised version received 8 September 1997; accepted 19 October 1997)

Abstract

Chopped pilchard waste was mixed with 15% molasses and inoculated with a starter culture made of Lactobacillus plantarum. The silage was incubated at 22°C for 20 days. Changes in the nutritional quality and biochemical properties (pH, dry matter, minerals, total and non-protein nitrogen, lipids and fiber) were monitored during a fermentation period of 15 days. The product obtained by fermentation was incorporated with bran and barley to make three formulas, which were then fed to broilers in four trials using five broilers each. The weight gain in broilers was recorded for 20 days. Results indicated that pH decreased con- siderably in the fermenting product and then remained constant at 4.2 and 4.5 in the two trials. Total nitrogen decreased slightly, whereas non-protein nitrogen increased significantly. The nutritional assays showed a net increase in the broiler weight relatively to the control diet. All the formulas made with the combinations of different proportions of ingredients resulted in similar growth of broilers compared with the commercial control-feed formula. © 1998 Elsevier Science Ltd. All rights reserved

Keywords: fish, fermentation, feed, nutrition, biopreservation, biotechnology.

Introduction

Animal feed production increases year after year, and new protein resources are needed throughout the world, especially in countries where proteins from vegetable origin are in short supply due to drought.

Solid wastes from food industries are the most interesting resource for proteins to be incorporated into feed formulation. A m o n g these, fish waste may represent an available ingredient of high protein content.

Fish waste ensilation is an old technique for pre- serving organic matter against spoilage, but may lead to the loss of this important potential feed ingredient.

A newer technology adopted for the preservation of fish waste is drying, so that the fish meal obtained is easy to transport and to store. However, some prob- lems may occur in feed formulas made with the fish

*Author to whom correspondence should be addressed.

423

meal because of p o o r preservation before or after drying.

Ensilage of fish waste was practiced in some coun- tries several years ago, but is not widely used now because of the water content, which may render trans- portation expensive. Moreover, fish waste silage is smelly and this may limit its use in a high proportion of feed formulations.

Chemical and nutritional characteristics of fish wastes have been investigated by several authors [1-10]. So far, fish silage is still unknown to Moroccan industry and to farmers, despite the scarcity of animal- feed ingredients.

With more biotechnological research into feed products, new techniques may improve fish waste silage by monitoring fermentation with an appropriate micro- bial starter. These would play a role in the:

- - preservation of fish waste by fermentation;

- - removal of fish-odor; and

- - probiotic effect by inhibiting pathogens in poultry.

424 Faid et al.

In the present work, fish wastes were transformed into a stable ingredient that was used in feed formula- tions for assays on broilers.

Materials and methods Fish waste~molasses preparation

Fish wastes of the species Sardina pilchardus were obtained from the fish market in Rabat. Wastes were chopped and mixed with 15% cane molasses. Then, 2 kg of the mixture was introduced into a disinfected 5-liter plastic container. The starter culture of Lactoba- cillus plantarum was grown on molasses supplied with yeast extract (0.3%) and casein peptone (1%). This culture was incubated for 48 h at 30°C. The fish waste/

molasses mixture was inoculated with 5% of the starter culture and incubated at 22 + 2°C.

Chemical analyses

The pH was determined using a p H - m e t e r (Crison MicropH 2000). Dry matter was determined by oven drying a weighed amount of the product at 105°C until constant weight. Fat content was determined on the dry matter by the Soxhlet method using hexane as solvent. Total nitrogen (TN) was determined by the Kjeldhal method described by the A P H A [11]. The non-protein nitrogen was also determined by the Kjel- dahl method on the filtrate after precipitation with a 10% trichloroacetic acid solution.

Feeding assays

T h r e e diets were made from fermented fish waste silage and the control was a commercial formula, to make a total of four trials as indicated below:

Silage Bran Ground barley CCF

Formula 1 1 0.5 0.5 2

Formula 2 2 0.5 0.5 1

Formula 3 2 1 1 0

Formula 4 0 0 0 4

CCF = commercial control feed; the figures represent parts of the total amount.

The CCF is a commercial formula made for poultry as follow: proteins 14%; fat 3.5%; moisture 14%; crude cellulose 16%; minerals 6%; phosphorus 1%; calcium 2%; vitamins (A:I000000UI; D3: 200000UI; E:

2.000 mg).

No mineral or vitamin mixtures were added to any diet formula other than the CCF. Each diet was given to 30-days old broilers in all experiments (five broilers).

Broilers weighing between 1200 and 1400 g were used.

The experiments were run when the fish waste had been fermented for 20 days and subsequently had been stored at ambient temperature for 2 months. The

broiler lots were fed in separate boxes and diets were fed ad libitum for 21 days. All the birds were weighed just before the start of the experiments and every 2 days for 20 days. The growth curve was established on the basis of weight gain.

Results and discussion Chemical changes

Chemical and physico-chemical characteristics of the raw material (initial) and the product obtained are reported in Table 1. Only slight variations were observed in the broad chemical composition of the product. The pH decreased from an average pH of 6.04 in the raw material to 4.3 in the product after 8 days of fermentation in all the trials, which provides evidence of induction of a low acidity through lactic acid production by lactic acid bacteria. The values then remained constant at around 4.1. The pH is the most important factor to monitor during fermentation because of lactic acid bacteria. Mineral or formic acid addition to lower the pH in fermenting materials is a possible means to prevent growth of the undesirable and hazardous microorganisms before biological acidity reaches an inhibiting level in the product.

Only slight differences were found between the DNVM of the raw material and the silage product. The latter showed a decrease due to the production of vola- tile nitrogen compounds within the transformation process. These compounds evaporated at 105°C during the determination of the dry matter and for this reason we changed the term dry matter to 'dry non-volatile matter'. The organic matter breakdown by micro- organisms and/or their enzymes is unavoidable during the process. This may result in relatively high amounts of volatile compounds.

The dry matter values ranged from 37.56 to 38.93%

Table 1. Physico-chemical properties of fish wastes trans- formed by fermentation

Min Max AV

pH RM 6.13 6.71 6.40

Si 4.05 4.30 4.14

DNVM % RM 38.40 39.83 39.21

Si 37.56 39.02 38.36

Ash % RM 7.03 8.40 7.47

Si 7.63 8.72 7.94

Fat % RM 5.13 6.04 5.58

Si 5.93 6.51 6.12

RS% RM 12.74 14.15 13.61

Si 10.53 11.05 10.79

Prot % RM 10.94 12.78 11.74

Si 10.05 12.15 11.34

Fib % RM 0.06 0.14 0.10

Si 0.05 0.11 0.08

RM = raw material; Si = silage; DNVM = dry non-volatile

matter; RS = reducing sugar; Prot = proteins; Fib = fiber.

in the product obtained from the four trials with an average of 38.36% (Table 1). A decrease in dry matter was observed in the silage product relative to the initial raw material. This is due to the hydrolysis of the protein matter by enzymes and/or microorganisms.

The ash content was almost stable in the raw material and in the silage. Values ranged from 7.63 to 8.72% in the product (Table 1). The high ash content was due to the abundance of bones in the waste and also to the mineral content of molasses. The raw material was rich in minerals and this will be discussed below.

T h e r e was a wide variation in fat contents among the samples: values ranged from 5.68 to 6.51% in the product obtained (Table 1). O u r results are different from those reported by Espe et al. [6], who found a fat content of 11.5% after 15 days storage of ground whole capelin 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. In our case, the fish wastes contained high proportions of bones and low levels of fat. Tatterson et al. [12] observed a slight variation in ash content that ranged from 2.1 to 2.7% and a wide range of fat content between 11.5 and 16.3%, when the authors worked with several different fish species. Fiber content was the lowest c o m p o n e n t in the product. This is not unexpected because the fish waste tissue is p o o r in fiber. The average content did not exceed 0./18%

(Table 1). The product can be used as an ingredient source of nitrogen and some minerals but it requires modificatory additions, such as fiber, when feeding the formulas to some animals.

The low content of fiber in the product led us to add a vegetable ingredient prior to feeding it to animals, in order to balance the formulas. Barley and bran were used in the formulas that was tested in the next part of our research.

Total nitrogen expressed as crude proteins was satis- factorily high and ranged from 10.85 to 11.68% of the dry matter with an average of 11.34% (Table 1). A decrease of total nitrogen relative to the raw material was observed. This was because of the transformation of protein nitrogen to non-protein nitrogen by the action of microorganisms and/or some endogenous enzymes.

Minerals were determined in the ash in order to obtain greater precision in the chemical composition of the feed ingredients. These are reported in Table 2 and were calcium, phosphorus, iron and zinc. The concen- trations of these minerals indicated a good balance in the minerals of the silage but these results were expected because of thc ash content of the raw material. The levels were sufficient to balance a final diet formula since the silage was blended with other ingredients to balance the fiber content of actual diets.

Concentrations of each mineral were in the same range as those reported by Stcffcns [13]. The most

Table 2. Nitrogen compounds in raw fish waste (RM) and in the fermented silage product (Si)

Min Max A V

TN RM 1.75 2.05 1.88

Si 1.61 1.94 1.78

NPN RM 9.10 10.11 9.74

Si 29.15 36.60 31.68

TVN RM 1.24 1.85 1.49

Si 2.14 2.70 2.32

TMA RM 0.05 0.33 0.20

Si 0.06 0.32 0.21

TN = total nitrogen; NPN = non-protein nitrogen: TVN = total volatil nitrogen; TMA = trimelhyamine.

important mineral is calcium, since in all poultry feed formulas this mineral is usually required in the greatest concentrations. In a commercial premix, the chemical composition is based on phosphorus, calcium, sodium, zinc, manganese, iodine and selenium. In our case we could not determine the other minerals. This may not represent a problem since other ingredients can balance the final formula as required.

Wide variations in TVN, T M A and NPN were observed in the obtained product relatively to the raw matter (fish waste). The T V N values ranged from 2.01 to 2.48 in the product and from 1.19 to 1.52 in the raw material (Table 3). This increase can be cxplained by protein degradation. Haaland and Njaa [5] found higher values of T V N in the raw material stored for 1 day befl)re ensiling than in silage stored for 7 days.

These values were 3.1 and 2%, respectively, of the TN.

It has been reported that the liquefaction of proteins is more severe in acid digested silage than in fermented silage [14]. The NPN also incrcascd notably in the product obtaincd relative to the raw material, the average values of NPN were 9.74 and 31.68 (as the percentage of the TN), respectively, for the raw material and the silage product.

The T M A was reduced considerably in the product relative to the raw material and to thc control. The average values were 0.2 and 0.21 (as the percentage of TN), respectively, for the initial and the product obtained (Table 3). T M A is responsible for the lishy odor in feeds made with fish by-products such as fish meal. Thc fish odor may constitute a problem in for-

Table 3. Chemical composition of the different fi~rmulas and the commercial control feed (in c~)

F¢~rmula I Formula 2 Formula 3 CCF

Moisture 28 32 33 18.7

Total proteins 13 12,2 10.5 12.5

Fat 5.4 5.8 5.2 3.2

Fiber* 15 14 12.5 16.2

Minerals 4.5 4.8 4.8 5.8

*As crudc ccllulosc.

426 Faid et al.

mulating feed compositions and can cause fish meal limitations.

A large increase in TVN and NPN was observed, whereas TMA was almost unchanged in the product.

This may be explained by the effect of the micro- organisms during the fermentation process. Liquefac- tion occurs by the action of tissue-degrading enzymes [14], and the level of autolysis is related to the activi- ties of the digestive enzymes that are either present in the tissue or released by proteolytic microorganisms.

Animal-feeding assays

Observation of the broilers showed no mortalities and no symptoms of malnutrition. Results of the feeding assays showed a net increase in the weights of broilers fed on fish silage supplemented with barley flour and bran (Fig. 1). Only slight differences between the trials were observed compared with each other and with the control.

The different formulas were satisfactory in nutri- tional ingredients. It is also interesting to note that no vitamins, minerals or antibiotics were added to the for- mulas. The total chemical composition of the different formulas compared with that made by industry, as reported in Table 3, shows that only slight differences can be observed in the main components. The high moisture contents in the formulas made in laboratory are due to the use of a humid product (fish silage) relatively to the commercial formula.

The present study is the first to be conducted in Morocco on feed from fish waste silage. Not only has weight gain to be considered, but also other observa- tions should be made. In fact, there were no mortalities and no abnormal symptoms (diarrhea, stress, malnutri- tion, drowsiness, feather removal, etc.) observed. All the trials showed an increase in the weights of the poultry (Fig. 1). The same pattern was observed in all the trials, including the animals fed on a conventional

weight (kg)

2,85 . ~ ~

LI

2 , 3 5

1,85

1 , 3 5 i ~ ~ ~

" ~ -

0 5 1 0 1 5 2 0 2 5

days

Fig. 1. Average weights gain in broilers fed on fish waste transformed by fermentation. Formula 1 (e), Formula 2 (+), Formula 3 (*), Formula 4 (D).

retail feed from the 'AI-Atlas Feed Processing Company'.

In formula 2 and to some extent in formula 3, bran and barley were used only as extenders to make the ingredients easy to handle and also so that it would be more readily eaten by the birds. This was because of the basic protein ingredient (fish silage is liquid or viscous). Some of the nutrients, such as minerals and vitamins, may also have been included in these materials. This work suggests that there may be a lack of certain nutrients in diets made from fish silage, which will need to be rectified. The micronutrients (minerals and vitamins) still need to be examined.

Chemical studies showed that not only could nitrogen quality be improved, but also that high-fiber extending materials (cellulose, pectins etc.) can be added to the formulas.

Comparison of the different results from the for- mulas indicates that there is considerable potential for the use of fish silage as a nitrogen source and possibly as a probiotic ingredient for poultry feeding.

References

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2. Jackson, A. S., Kerr, A. K. and Cowey, C. B., Fish silage as a dietary ingredient for salmon. I! Pre- liminary growth findings and nutritional pathology.

Aquaculture 1984, 40, 238-291;

3. Jackson, A. S., Kerr, A. K. R. and Cowey, C. B., Fish silage as a dietary ingredient for salmon I.

Nutritional and storage characteristics. Aquaculture 1984, 38, 211-220.

4. Haaland, H. and Njaa, L. R., Effect of temperature on the autolysis of capelin silages stored for one year. Fisk DirSkr Ser Ernoering 1989, 2, 219-226.

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8. Espe, M., Haaland, H. and Njaa, L. R., Substitu- tion of fish silage protein and a free amino acid mixture for fish meal protein in a chicken diet.

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11. A P H A (American Public Health Association), Standard Methods for Examination of Water and Waste Water (19th). APHA Publishers, Washington, DC.

12. Tatterson, J. N. and Windsor, L. M., Fish silage.

Journal of Science, Food and Agriculture 1984, 25, 369-379.

13. Steffens, W., Replacing fish meal with poultry by-products meal in diets for rainbow trout, Onco- rhynchus mykiss. Aquaculture 1994, 124, 27-34.

14. Raa, J. and Gildberg, A., Fish silage: A review.

Food Science and Nutrition 1982, 16, 383-419.