Stability of crystalline and coated vitamins duringmanufacture and storage of ®sh feeds
M. MARCHETTI & N. TOSSANI Vitamin Research Centre, Department of Biochemistry G. Moruzzi, University
of Bologna, Bologna, Italy
S. MARCHETTI Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Bologna,
Italy
G. BAUCE Hendrix Research Centre, Mozzecane (VR), Italy
Abstract
The stability of two forms of vitamins, crystalline and fat-
coated, during manufacturing processes and storage of ®sh
feeds was investigated. After extrusion, vitamin recovery was
generally lower than that found after pelleting. The recovery
of coated forms after pelleting was higher than that of
crystalline ones, especially for ascorbic acid, menadione,
pyridoxine and folic acid. Coated forms were also found to
be more resistant to extrusion than crystalline forms. No
signi®cant di�erences in vitamin losses during storage were
found between pelleted and extruded feeds. However, losses
of ascorbic acid, menadione and pyridoxine in both manu-
factured feeds were much greater in crystalline than in coated
forms.
KEY WORDS:KEY WORDS: extrusion, ®sh feed, pelleting, storage, vitamins
Received 17 February 1998, accepted 25 November 19981
Correspondence: Prof. M. Marchetti, Department of Biochemistry
G. Moruzzi, Via Irnerio, 48, 40126 Bologna, Italy2
Introduction
Vitamin supplementation of feeds is essential to ensure that
farmed animals receive an adequate and balanced supply of
these micronutrients to achieve optimal performance; in feed
ingredients, vitamins occur in small and sometimes variable
quantities. Particularly important is vitamin supplementation
of aquaculture feeds, since the dietary vitamin requirements
of ®sh, in general, and some other species in particular are
much higher than those of other animals (Halver 1989;
Woodward 1994; De Silva & Anderson 19953 ). However,
proper integration is di�cult to achieve owing to the great
instability of many vitamins with physical and chemical
agents. The presence of trace-minerals4 and choline (March-
etti et al. 1995b5 ) and, above all, stress during manufacturing
processes and storage cause serious losses of vitamin contents
in feeds (Lovell 1989; Sandnes & Utne 1991; Gadient et al.
1992; McGinnis 1994; Gadient & Fenster 1994; Li et al.
1996).
Although over6 forti®cation of vitamins is often made to
compensate for expected losses (Robison 1991), this practice
does not always ensure that farmed animals receive adequate
and balanced supplies of these micronutrients.
We therefore carried out studies aimed at ascertaining
whether the use of vitamins in forms unlike those normally
used in feed supplements could reduce the processing and
storage losses that occur to such a great extent in ®sh feeds.
In a previous work, we studied the behaviour of the fat-
soluble vitamins retinol, cholecalciferol and tocopherol
coated with fat and compared it with that of commercial
forms of the same vitamins in ®sh feeds during pelleting,
extrusion and storage (Marchetti et al. unpubl. obs.).
In the present study, we compared the stability of
B-complex vitamins, ascorbic acid and menadione (as men-
adione sodium bisulphite), in coated and crystalline forms
during manufacturing processes and storage.
Materials and Methods
Chemicals
All reagents were analytical grade; HPLC grade chemicals
were used for liquid chromatography. Assay media
supplied by Difco Laboratories (Detroit, MI, USA) and
microorganisms supplied by American Type Culture Collec-
tion (Rockville, MD, USA) were used in microbiological
determinations.
115
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Ó 1999 Blackwell Science Ltd
Crystalline vitamins to be incorporated into feeds were
purchased from Ho�man-La Roche, Basle, Switzerland and
coated vitamins from Trouw Nutrition-Nutreco, Verona,
Italy.
Feeds
Two commercial ®sh feeds (produced by Hendrix, Mozze-
cane, Verona, Italy) with the same composition (Table 1)
were used in the study. One was forti®ed with crystalline
vitamins, the other with vitamins coated with fat (for fatty
acid composition, see Table 2). Both feeds were processed by
either extrusion or pelleting. Processing parameters used to
produce the pellets and physical characteristics of experi-
mental feeds are given in Table 3.
Analytical methods
The feeds were analysed for their vitamin contents before and
after two processes by the following methods: thiamin and
Table 1 Ingredients and proximate composition of experimental feed
Ingredient g kg)1
Bloodmeal 50Choline chloride (50%) 15Dried whey 50Fish meal 420Fish oil 150Magnesium sulphate 3Meat meal 50Mineral premix1 10Sodium chloride 4Soy lecithin 13Soybeanmeal 160Vitamin premix2 10Wheat starch 80
Approximate composition
Dry matter 891
On dry matter basis28 :Ash 102Fibre 11Lipid 210Protein 510
1 The mineral premix provides the following mg kg)1 of feed: ironsulphate (FeSO4.7H2O),150; zinc sulphate (ZnSO4.7H2O), 240; manganesesulphate (MnSO4.4H2O), 180; copper sulphate (CuSO4. 5H2O), 25; cobaltsulphate (CoSO4.7H2O), 18; calcium iodate (CaIO3.6H2O), 12; sodiumselenite (Na2S2O4), 2.3.2 The vitamin premix provides the following mg kg)1 of feed: retinylpalmitate (500.000 IU g)1), 40; DLDL-cholecalciferol (100.000 IU g)1), 10; DLDL-a-tocopheryl acetate (50%),120; menadione sodium bisulphite (MSB), 60;thiamin hydrochloride, 30; ribo£avin, 40; pyridoxine hydrochloride, 80;nicotinamide, 400; ascorbic acid, 200; DD-calcium pantothenate (91%),250; folic acid (90%), 7.5; DD-biotin (2%), 7.5; cyanocobalamin (1& ), 300.
Table 2 Fatty acid composition and chemical-physical characteris-
tics of fat used for vitamin coating
Fatty acid
8:0^12:0 < 1 %14:0 2^3 %16:0 28^30 %18:0 60^66 %18:1 0.5^1 %Iodine number (g I100 g)1) < 1Acidity number (mg KOH g)1) < 8Saponification number (mg KOH g)1) 190^198Melting point (C°) 62^65
Table 3 Processing parameters and physical characteristics of
experimental feeds
Extrusion Pelleting
Parameters
Machine type Wenger X/185 CPM 7000Added water and steam (%) 16 5Pressure (bar) 12 2.5Conditioning time (s) 25 10Temperature at intake (°C) 85 55Temperature at outlet (°C) 96 68Drying temperature (°C) 95 öDrying time (min) 20 öTemperature at outlet of dryer (°C) 48 öPhysical characteristics
Diameter (mm) 5.0 4.8Bulk density (kg L)1) 0.54 0.68Moisture content (g kg)1) 8 9Moisture content after30 min leaching (g kg)1) 26 18
Table 429,30: Levels of vitamins in crystalline form after manufacturing
process of feed
Feed vitamin content (mg kg)1)
Vitamin Before After pelleting After extrusion
Ascorbic acid 207 þ 16a 107 þ 12b 40.8 þ 5.4c
Biotin 1.47 þ 0.2 1.39 þ 0.2 1.38 þ 0.2Cyanocobalamin 0.26 þ 0.03 0.23 þ 0.04 0.22 þ 0.04Folic acid 6.36 þ 0.8a 4.05 þ 0.4b 3.05 þ 0.5b
Menadione 31.6 þ 3.6a 16.1 þ 3.1b 10.9 þ 2.2c
Nicotinamide 396 þ 35 380 þ 42 364 þ 44Pantothenic acid 226 þ 21 201 þ 19 194 þ 18Pyridoxine 79.7 þ 5.6a 60.2 þ 7.9b 53.0 þ 6.2b
Riboflavin 38.7 þ 4.5 33.8 þ 4.6 33.3 þ 5.6Thiamin 32.2 þ 2.5 26.3 þ 2.9 28.3 þ 3.5
Values are given as means (SEM) of results of assays carried out on threereplicate feed preparations. Means within a row with differentsuperscript letters are significantly different (P < 0.05).
M. Marchetti et al.
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Ó 1999 Blackwell Science Ltd Aquaculture Nutrition 5;115^120
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ribo¯avin by ¯uorimetric methods (AOAC 1995);
ascorbic acid (Ho�man et al. 1992) and menadione (La�
et al. 19887 ) by liquid chromatography. Other vitamins were
assayed by microbiological methods (AOAC 1995) using the
following test microorganisms: Saccharomyces8,9 uvarum 9080
for pyridoxine; Lactobacillus casei8,9 7469 for folic acid;
Lactobacillus10 plantarum 8014 for nicotinamide, pantothenic
acid and biotin; and Lactobacillus11 leichmanni 7830 for
cyanocobalamin.
Vitamin assays were repeated in feeds, stored in paper bags
at room temperature, 30, 90 and 180 days after manufacture.
Experiments were carried out on three replicates of feeds. On
every sample analyses were carried out in duplicate or
triplicate.
Statistical analysis
All the results were processed by analysis of variance (ANOVAANOVA)
using the general linear procedure of SPSS/PC (1988).
Di�erences were considered signi®cant when P < 0.05.
Results and Discussion
The results of vitamin assays in two types of feeds before and
after manufacturing processes (Tables 4, 5)12 show that vita-
min losses caused by the two processes were generally similar,
except for ascorbic acid and menadione, for which losses
caused by extrusion were greater than those caused by
pelleting (P < 0.05). Folic acid and pyridoxine levels also
su�ered to a greater extent than the others in both processes.13
However, the extent of the losses of these four most
vulnerable vitamins (ascorbic acid, menadione, folic acid and
pyridoxine) caused by the two processes di�ers according to
the form used: losses from coated forms were signi®cantly
lower than those from crystalline ones (ascorbic acid and
menadione P < 0.01, folic acid and pyridoxine P < 0.05,
respectively).
The results of vitamin assays in processed feeds stored for
various periods of times (Tables 6, 7, 8, 914 ) show no
signi®cant di�erences between vitamin losses from extruded
and pelleted feeds. Losses were of the same magnitude as
those observed in meal feeds not subjected to extrusion or
pelleting processes (Marchetti et al. unpubl. obs.).
Signi®cant di�erences were found between losses of
crystalline and coated vitamins for ascorbic acid, menadione
and, only in extruded feed, for pyridoxine in both types of
manufactured feed. When used in a coated form, after
180 days of storage these vitamins su�ered signi®cantly
lower losses than those in crystalline form (in pelleted feed:
Table 531,32: Levels of vitamins in coated form after manufacturing
process of feed
Feed vitamin content (mg kg)1)
Vitamin Before After pelleting After extrusion
Ascorbic acid 191 þ 14a 166 þ 20a 100 þ 14b
Biotin 1.67 þ 0.2 1.62 þ 0.3 1.64 þ 0.3Cyanocobalamin 0.27 þ 0.03 0.25 þ 0.03 0.25 þ 0.03Folic acid 6.18 þ 0.8 5.81 þ 0.6 5.56 þ 0.7Menadione 32.3 þ 4.1a 28.1 þ 3.1a 21.8 þ 3.3b
Nicotinamide 424 þ 43 403 þ 52 399 þ 50Pantothenic acid 236 þ 20 222 þ 26 215 þ 24Pyridoxine 76.6 þ 6.9 72.9 þ 7.8 69.9 þ 6.2Riboflavin 38.5 þ 3.3 37.3 þ 5.2 36.2 þ 4.8Thiamin 28.4 þ 2.5 27.0 þ 3.9 25.6 þ 3.5
Values are given as means (SEM) of results of assays carried out on threereplicate feed preparations. Means within a row with differentsuperscript letters are significantly different (P < 0.05).
Table 6 Levels33 of vitamins in crystalline form after storage of
pelleted feed
Feed vitamin content (mg kg)1) at time
Vitamin 0 days 30 days 90 days 180 days
Ascorbic acid 112 þ 13a 83.0 þ 9.6a 48.9 þ 7.6b 24.7 þ 1.9c
Biotin 1.53 þ 0.2 1.50 þ 0.2 1.46 þ 0.2 1.36 þ 0.3Cyanocobalamin 0.29 þ 0.03 0.27 þ 0.04 0.24 þ 0.04 0.22 þ 0.03Folic acid 4.76 þ 0.4 4.56 þ 0.6 4.26 þ 0.5 3.94 þ 3.8Menadione 21.8 þ 2.1a 18.6 þ 2.7a 15.0 þ 2.3b 9.17 þ 1.5c
Nicotinamide 410 þ 37 403 þ 37 395 þ 35 378 þ 37Pantothenic acid 205 þ 18 194 þ 23 174 þ 23 150 þ 21Pyridoxine 65.6 þ 5.0a 60.6 þ 4.8a 52.1 þ 7.2a 46.3 þ 5.4b
Riboflavin 33.5 þ 3.3 32.4 þ 2.8 31.9 þ 3.4 29.8 þ 4.0Thiamin 26.2 þ 3.9 25.4 þ 3.7 26.9 þ 2.7 23.1 þ 2.7
Values are given as means (SEM) of results of assays carried out on threereplicate feed preparations. Means within a row with differentsuperscript letters are significantly different (P < 0.05).
Table 7 Levels34 of vitamins in coated form during storage of pelleted
feed
Feed vitamin content (mg kg)1) at time
Vitamin 0 days 30 days 90 days 180 days
Ascorbic acid 161 þ 15a 153 þ 18a 133 þ 15a 122 þ 13b
Biotin 1.62 þ 0.2 1.62 þ 0.2 1.57 þ 0.2 1.51 þ 0.2Cyanocobalamin 0.24 þ 0.04 0.24 þ 0.03 0.23 þ 0.04 0.20 þ 0.04Folic acid 5.83 þ 0.5 5.74 þ 0.7 5.60 þ 0.6 5.48 þ 0.5Menadione 30.2 þ 2.8 28.1 þ 3.6 26.2 þ 2.9 24.2 þ 3.5Nicotinamide 404 þ 38 402 þ 34 398 þ 36 385 þ 44Pantothenic acid 223 þ 20 215 þ 20 207 þ 21 198 þ 18Pyridoxine 70.3 þ 6.2 68.2 þ 5.2 64.3 þ 6.0 62.1 þ 6.8Riboflavin 37.4 þ 4.3 36.7 þ 4.2 36.1 þ 4.3 35.3 þ 4.3Thiamin 27.1 þ 2.4 26.9 þ 3.2 26.5 þ 3.5 25.5 þ 3.2
Values are given as means (SEM) of results of assays carried out on threereplicate feed preparations. Means within a row with differentsuperscript letters are significantly different (P < 0.05).
Vitamin stability in fish feeds
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ascorbic acid P < 0.01, menadione P < 0.05; in extruded
feed: ascorbic acid P < 0.01, menadione and pyridoxine
P < 0.05).
The data obtained in this study agree with the observations
of some authors (Lovell 1989; Sandnes & Utne 199115 ; Gadient
& Fenster 199416 ; Li et al. 1996) on the decay of some water-
soluble vitamins during manufacturing processes and storage
of ®sh feeds. However, our results clearly show the greater
stability of coated vitamins compared to crystalline ones
which are generally more vulnerable to attack by chemical
and physical agents.17
During feed manufacture, the recovery rates of ascorbic
acid, menadione, pyridoxine and folic acid in crystalline form
after pelleting were 52%, 51%, 76% and 64%, respectively,
whereas those in coated form were 87%, 87%, 95% and 94%
(Fig. 1). After extrusion, the recovery rates of the same
vitamins in crystalline form were 20%, 34%, 70% and 61%,
and in coated form 53%, 67%, 91% and 90%, respectively
(Fig. 2).
Our data con®rm previous ®ndings on premix vitamin
stability during the storage (Marchetti et al. 1995a18 ). In
pelleted feed, after 180 days of storage, only 30% of ascorbic
Table 8 Levels35 of vitamins in crystalline form during storage of
extruded feed
Feed vitamin content (mg kg)1) at time
Vitamin 0 days 30 days 90 days 180 days
Ascorbic acid 48.4 þ 4.5a 36.1 þ 3.5a 16.7 þ 1.5b 9.72 þ 1.0c
Biotin 1.34 þ 0.2 1.28 þ 0.1 1.27 þ 0.1 1.22 þ 0.2Cyanocobalamin 0.22 þ 0.04 0.20 þ 0.02 0.17 þ 0.02 0.14 þ 0.03Folic acid 4.38 þ 0.4 3.70 þ 0.4 3.07 þ 0.4 2.95 þ 0.4Menadione 12.9 þ 2.2a 10.6 þ 0.9a 8.04 þ 1.1b 5.16 þ 0.8c
Nicotinamide 392 þ 41 388 þ 42 387 þ 43 380 þ 44Pantothenic acid 194 þ 17 171 þ 20 152 þ 18 142 þ 17Pyridoxine 61.8 þ 5.5a 56.2 þ 5.1a 47.9 þ 4.1a 38.0 þ 3.8b
Riboflavin 35.5 þ 3.7 35.0 þ 3.9 33.2 þ 4.1 29.6 þ 3.9Thiamin 30.1 þ 2.6 29.3 þ 2.4 25.4 þ 1.9 23.8 þ 2.8
Values are given as means (SEM) of results of assays carried out on threereplicate feed preparations. Means within a row with differentsuperscript letters are significantly different (P < 0.05).
Figure 1 Levels of vitamins retained in crystalline and coated forms
in feed after pelleting process. Data, expressed as percentage of
vitamin retained, are given as mean (SEM) of results of assays
carried out on three replicate feed preparations. Signi®cant
di�erences between two vitamin forms are indicated by asterisks
(*P < 0.05; ** P < 0.01).
Figure 2 Levels of vitamins retained in crystalline and coated forms
in feed after extrusion process. Data, expressed as percentage of
retained vitamin, are given as means (SEM) of results of assays
carried out on three replicate feed preparations. Signi®cant di�eren-
ces between two vitamin forms are indicated by asterisks (* P < 0.05;
** P < 0.01).
Table 9 Levels of vitamins in coated form during storage of extruded
feed
Feed vitamin content (mg kg)1) at time
Vitamin 0 days 30 days 90 days 180 days
Ascorbic acid 89.5 þ 8.01a 85.9 þ 8.25a 77.0 þ 9.27a 71.7 þ 8.46b
Biotin 1.73 þ 0.20 1.66 þ 0.18 1.60 þ 0.19 1.60 þ 0.20Cyanocobalamine 0.29 þ 0.03 0.28 þ 0.03 0.24 þ 0.03 0.22 þ 0.04Folic acid 5.74 þ 0.55 5.42 þ 0.60 5.20 þ 4.89 4.96 þ 0.62Menadione 19.0 þ 2.18a 18.0 þ 2.16a 15.9 þ 2.02a 14.6 þ 2.12b
Nicotinamide 406 þ 37.3 400 þ 36.9 400 þ 43.9 394 þ 43.2Pantothenic acid 205 þ 20.1 198 þ 21.6 194 þ 18.7 190 þ 21.1Pyridoxine 68.1 þ 5.96 67.3 þ 5.88 65.7 þ 5.54 62.9 þ 7.46Riboflavin 38.1 þ 4.21 37.6 þ 4.21 35.9 þ 4.12 34.3 þ 4.25Thiamin 26.2 þ 2.08 25.6 þ 2.36 24.8 þ 2.97 24.4 þ 2.96
Values are given as means (SEM) of results of assays carried out on threereplicate feed preparations. Means within a row with differentsuperscript letters are significantly different (P < 0.05).
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acid and 42% of menadione in crystalline form were found
whereas 76% and 86% were found in the coated form
(Fig. 3). In extruded feed, the quantities found after 180 days
were 20% for ascorbic acid, 40% for menadione and 66% for
pyridoxine in the crystalline form, and 70%, 76% and 92%,
respectively, in the coated form (Fig. 4).
These results show the bene®ts of using coated forms for
vitamin supplementation of feeds in general, but particularly
in aquaculture feeds which cannot be given in meal and
which are processed by pelleting or extrusion.
A further advantage of using coated forms of water-
soluble vitamins in ®sh feed supplementation is their lower
propensity for leaching compared with crystalline forms once
the feed has been immersed in water. Signi®cant di�erences
between the two vitamin forms were observed in both
manufactured feeds for all vitamins but particularly for folic
acid, pantothenic acid, ascorbic acid and menadione (Mar-
chetti et al. 1999).
These two characteristics of coated vitamins Ð greater
stability and lower leaching Ð mean that they may be used
to ensure that farmed ®sh receive better and more19 balanced
supplies of these important micronutrients, for optimal
performance.
Although the cost of coated vitamins is 10±15% higher
than that of ordinary commercial forms, they may prove to
be more economic, since the extra vitamin normally added in
manufacturing ®sh feed, in an attempt to compensate for
losses caused by manufacturing stress, storage and leaching,
may be reduced by at last 60%.
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Figure 3 Levels of vitamins retained in crystalline and coated forms
in pelleted feed after 180 days' storage. Data, expressed as percent-
age of retained vitamin, are given as means (SEM) of results of
assays carried out on three replicate feed preparations. Signi®cant
di�erences between two vitamin forms are indicated by asterisks
(* P < 0.05; ** P < 0.01).
Figure 4 Levels of vitamins retained in crystalline and coated forms
in extruded feed after 180 days' storage. Data, expressed as percent-
age of retained vitamin, are given as means (SEM) of results of assays
carried out on three replicate feed preparations. Signi®cant di�eren-
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** P < 0.01).
Vitamin stability in fish feeds
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