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Functional Additives
Thursday, November 25, 2004 7:12:23 PM
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Antioxidants: Why You Need Them and How They Work

 
Christopher E. Nelson, ph.D.

Vice President, Research & Development

Kemin Industries, Inc.

Des Moines, Iowa U.S.A.

 

 

The oxidation of fats, oils and other oxygen labile substrates is a phenomena that has challenged food and feed technologists since the beginning of their respective sciences. Normally, oxidation is thought of as "rancid odour" development that is a characteristic of oxidized fat, or the browning process that occurs with fresh fruits upon cutting. The problems of oxidation in feeds encompasses a wide variety of both nutritional and tactile qualities, many of which have yet to be fully quantitated.

 

In most simple terms, autoxidation can be said to be "the process by which oxygen adds irreversibly to feed at room temperature". Importantly, in this definition is the irreversible nature of this degradation which implies a loss of feed quality that cannot be reclaimed. It is also a process that occurs at room temperature in sharp contrast to many chemical reactions. Heat will greatly accelerate this process. This reaction can also take place without water being present which is contrary to microbial forms of degradation.

 

Autoxidation takes place in a very short amount of time once feed is made. Normally, the protective cuticle of the seed grain prevents oxidation of natural seed oils by effectively excluding oxygen. Grinding of grains exposes fats to air whereupon the oxidation process begins immediately.

 

While fat content greatly influences the degree of threat in a given feed, it may not predict the immediacy to which oxidation occurs. A recent survey of beef feedlot feeds showed how quickly oxidation could occur. Feeds were sampled from bunks some 4 to 8 hours after mixing. Fat was extracted using the method of Bailey, the solvent evaporated and the peroxide value determined by AOCS method. The results of this survey is shown in Table 1.

 

TABLE 1

PEROXIDE DEVELOPMENT IN BEEF FEEDLOT RATIONS
 

 

 

The data indicates that there was no significant peroxide problems in any of the individual ingredients with the exception of the supplement. It is especially noteworthy that the fat was of good quality. When the ingredients were combined, oxidation began immediately. Further data (not shown) indicated that the problem increased exponentially when the feed was actually placed in the bunk.

 

Oxidative destruction is a highly complex series of chemical reactions that only in the past few years has begun to be characterized. The problems of autoxidation however, can be summarized perhaps into four distinct areas as is shown in Table 2.

 

TABLE 2

PROBLEMS ASSOCIATED WITH AUTOXIDATION OF FEEDSTUFFS

 

 

 

These four problems lead to a lowering of the overall quality of feed. This process is strictly chemical and, perhaps for this reason, its significance has been consistently understated in animal feeds.

 

Destruction of fat soluble vitamins frustrates the very purpose of vitamin/mineral premix addition into feeds. Often, feed manufacturers feel that in purchasing high quality sources of fat-soluble vitamins (such as gelatin/antioxidant coated vitamin A esters), there is little reason for additional antioxidant addition into their feeds.

 

Work done by Farmland Cooperative in Kansas City, Kansas and reported in Feedstuffs in 1982, showed that just the opposite was true. These studies noted that vitamin A loss is dependent on two factors; time and temperature. In a three-dimensional representation shown in Figure I, only 64% of the vitamin A can be retained in a 67 day period when finished feed is kept between 75 - 95¡ã F.

 

FIGURE I

TIME AND TEMPERATURE EFFECT ON PERCENTAGE RETENTION
OF VITAMIN A IN FEEDS

 

                                      From: Shields, R.G., D.R.Campbell, D.M. Hughes and D.A. Dillingham (1982)

                                      Feedstuffs, November 15, pp 22-28

 

 

Importantly, over 10% of the vitamin A is lost in as little as a seven day storage period at temperatures of less than 65¡ã F (18.3¡ã C). Higher temperatures result in more loss.

 

Since vitamin A is a critical part of any animal ration, its preservation should be insured through the use of antioxidants. Normally, when antioxidants are included in a feed it can be calculated that degradation will decrease by some 66%. This means that a premix which is degrading at a rate of 10% per month will degrade at a rate of 3.3% per month when adequately treated with antioxidant.

 

To summarize the effect of oxidation and antioxidant protection on vitamins A and E, the following table could be constructed (Table 3).

 

TABLE 3
THE EFFECT OF ANTIOXIDANTS ON THE RELATIVE RATES OF VITAMINS A AND E DEGRADATION IN VITAMIN/MINERAL PREMIXES AND FINISHED FEEDS

 


 

When feed or premix is treated with antioxidant, there is a dramatic reduction in vitamin loss. The relative cost/return of antioxidant treatment when such degradation is occurring indicates that antioxidant supplement is always profitable even without consideration of other nutritional components.

 

Palatability of animal feed rations is an important parameter to be considered in feed formulation. In most cases, animal feeds when freshly manufactured from good quality ingredients, have a high degree of palatability. This "fresh feed" factor is always emphasized as an optimum ideal for feeding commercial animals. The practicalities of animal feed manufacture often require that feed be stored before it is given to animals. When antioxidants are not included, oxidation inevitably results. This oxidation can result in the creation of unpalatable side products. By monitoring headspace volatiles in stored milk powder, the creation of such unpalatable volatiles can be monitored (Figure II).

 

figure ii

gas chromatograms of headspace volatiles
from reconstituted fresh and stored whole milk powder.
all chromatograms represent volatiles from sample
with no antioxidant added and stored in air

 

                    From: Hall, G., J. Andersson, H. Lingnert and B. Olofsson (1985) J. of Food Qual. 7: 153-190.

 

 

In the experiment delineated above, milk powder was stored for a 6 week period of time after which gas chromatographs of the headspace volatiles were again taken after the milk powder had been reconstituted. A large number of compounds appeared in the aged milk as compared to the fresh milk. Inevitably, these compounds are non-palatable and can be identified as aldehydes. Their relative level of flavour threshold is extremely low as demonstrated in Table 4.

 

table 4

carbonyls from fat

                           From:   Frankel, E.N. (1984) "Recent advances in the chemistry of rancidity of fats"

                           Spec. Pub R.Soc. Chem. (47) 87-118

 

 

The data illustrates that as little as 1 ppb of these aldehydes can be sensed by the animal, thereby indicating a stale or off-tasting feed. The staleness can directly result in lower consumption and consequently nutritional losses.

 

The effect of an antioxidant on feed palatability preservation was demonstrated in work conducted by Kemin some 10 years ago. In this experiment, feed was freshly made and compared to feed which had been stored with no antioxidant added (Table 5).

 

table 5

effects of an antioxidant on feed palatability

 

                From:   Research & Development Department, Kemin Industries, Inc. (1976)

                "Correlation of the thiobarbituric acid assay to the palatability of pig diets". in The endox® Research

                Kit, Kemin Europa, N.V., 1981.

 

 

When stored feed with antioxidant was compared to stored feed without antioxidant, palatability preferences were switched dramatically to the feed with antioxidant. Thiobarbituric acid values (TBA) demonstrated as well as peroxide values that relative effects of the antioxidant could be quantitated.

 

Recently Kemin researchers verified these data in a highly palatable diet shown in Table 6.

 

table 6

COMPOSITION OF PIG STARTER FEED USED IN PALATABILITY TRIAL

 

                           From: Black, D. and C.E. Nelson (1988)

                          "Retention of palatability in pig feed by endox®, Kemin Industries, Inc.

                           Publication No. 06749, pp 1-3

 

 

This diet was blended with 2.9% additional fat in order to provide additional stress. Environmental stress was realized by heat treating some of the feed. The protocol for this and the resulting palatability preference test is shown in Figure III.

 

figure iii

preparation of feed used in palatability trial

    

            From:     Black, D. and C.E. Nelson (1988)

           "Retention of palatability in pig feed by endox®, Kemin Industries, Inc.

            Publication No. 06749, pp 1-3

 

 

When subjected to a 14 day two-choice trial, it was discovered that stressed antioxidant treated feed is undistinguishable from fresh frozen feed. Fresh frozen  feed was shown preferable to untreated stressed feed. These results are shown in Table 7.

 

table 7

preference in feed as a function of three pen average

 

      From:   Black, D. and C.E. Nelson (1988)

      "Retention of palatability in pig feed by endox®, Kemin Industries, Inc.

       Publication No. 06749, pp 1-3

 

 

Oxidation not only affects the vitamin stability and palatability of feeds, real nutritional losses can occur via oxidation. Workers from Germany tested the effects of rancid fat fed to laying hens as is demonstrated in Table 8.

 

TABLE 8

effect of rancid fat on egg production in hens

 

           From: Voreck, V.O. and M. Kirchgessner (1981) Archiv fuer Gelfluegelkunde 45:19-23

 

 

In these experiments, it is noteworthy that a non-oxidized diet resulted in good rates of lay, but when oxidized fat was introduced into the feed, rates of production were dramatically depressed. The researchers attempted to restore production levels by adding various levels of fat soluble vitamins that were thought to be missing in the oxidized ration. Interestingly, vitamin A and E had no effect. When vitamin D was added some increase in production was noted, but this never attained the rate seen in the fresh diet.

 

The reasons for depressed production may not only be due to the loss of nutrients. The creation of toxic metabolites due to oxidation is a new area that has recently been investigated by numerous researchers. In experiments reported by Japanese workers, the effects of autoxidation products of linoleic acid were studied (Figure IV).

 

figure IV. effect of autoxidation products
of linoleic acid on dna
strand breakage

  

                 From: Morita, J., K. Ueda, K. Nakai, Y. Baba and T. Komano (1983) Agric. Biol. Chem. 47:2977-2979.

 

 

In this experiment, an increase of TBA reacting products as well as peroxides was observed when various levels of autoxidized linoleic acid were added to an incubation mixture. An assay was simultaneously utilized that could monitor the amount of cellular DNA breakage. As noted, the amount of DNA left intact decreased linearly as the amount of oxidized products increased. Interestingly, autoxidized fat products are increasingly being linked to the incidence of various cancers. This would be expected when one considers the DNA breakage data shown in Figure IV.

 

From a theoretical aspect, the problems that one may encounter from feed oxidation are quite evident. The combination of these was shown to result in reduced animal performance in a recent broiler trial conducted at the University of Arkansas.

 

In this experiment, fresh poultry fat was obtained and then allowed to oxidize until, when added to the feed, the peroxide level was either 0, 2, 4 or 7 meq/kg. The growth and feed conversion after forty-nine days are shown in Table 9.

 

TABLE 9

the effect of peroxides (meq/kg) on broiler performance after 49 days when fed feed containing oxidized fat

 

 

               From:   Cabel, M. C., P.W. Waldroup, W.D. Shermer and D.F. Calabotta (1989), Poultry Science

               67:1725-1730

 

 

Weight gains were significantly effected (p<0.05) by the inclusion of the oxidized fat in the feed. In addition there were 10 points of feed conversion difference between the group without added oxidized fat and that with 7 meq/kg. Since graduated levels of peroxide containing feed was used in the experiment, it was possible to see that there was approximately a 1 point increase in feed conversion for each point of peroxide increase in the final feed.

 

Given that autoxidation of fats, oils, vitamins and other nutrients is an important measure of feed quality, it would seem of great interest to be able to understand how best to prevent this destructive phenomena. In order to do this, one must begin by studying the chemistry of autoxidation. Autoxidation occurs primarily in molecules which contain double bonds as demonstrated in Figure V.

 

figure v

NUTRIENTS SUSCEPTIBLE TO OXIDATION:COMMON STUCTURAL FEATURES

 

 

 

It is noteworthy that both vitamin A and unsaturated fatty acids contain large numbers of double bonds which provide ideal sites of autoxidation. To understand how autoxidation occurs, the oxidative chemical pathway must be considered (Figure VI).

 

figure vi

pathway of autoxidation

 

   From:   Metzler, D.E. (1977) "Biochemistry: The chemical reactions of living cells",

   Academic Press, New York; and Waters, W.A. (1971) J.Amer. Oil. Chem. Soc. 48:427-433

 

 

The carbon adjacent to the double bonds is the actual site of attack by free radicals. Free radicals can simply be defined as molecules which contain an unpaired electron. These radicals attack the carbon adjacent to the double bond since the double bond causes the carbon to be desired reaction site. The resulting product is actually a free radical in itself. Oxygen takes advantage of this situation by adding to the molecule resulting in the formation of a peroxide. This peroxide still contains an unpaired electron and thus, will look for an opportunity to stabilize itself by forming a hydroperoxide. This results in the propagation of new free radicals and the creation of hydroperoxides. Hydroperoxides are inherently unstable and will rearrange to form aldehyde molecules. Aldehydes are a common feature of oxidized fats and the unpalatable compounds noted earlier.

 

Several important features should be understood from studying this pathway. First, oxygen is not the cause of oxidation. Free radicals are the cause of oxidation. Secondly, several important intermediates can be monitored to determine the extent of oxidation of a particular material, namely peroxides and aldehydes. These compounds are simple transient intermediates and not terminal products of autoxidation. The question as to how antioxidants interact in this complex chemical pathway must now be addressed. Shown in Figure VII is the termination of free radicals by an antioxidant.

 

figure vii

termination of free radicals by butylated hydroxyanisole (bha) 

 

 

 

In this particular example, BHA reacts with a free radical to stabilize it and become a free radical in itself (due to the presence of an unpaired electron). Antioxidants are unique in their ability to stabilize these unpaired electrons due to the pi orbitals of the ring structure of the compound. All antioxidants, whether they be ethoxyquin, BHT or BHA. behave in a similar manner. It is noteworthy that antioxidants act rather early in the autoxidation pathway. Once peroxides are formed, it is virtually impossible to reverse the autoxidation process.

 

If one was to design antioxidant systems, it is important to be able to have molecules which react easily with free radicals. In the 1960's, Kemin, while examining this process in detail, felt it may also be advantageous to also study the source of the free radicals within animal feeds. Free radical sources can be summarized as in Table 10.

 

table 10

possible sources of free radicals in feeds 

                        From:   Waters, W.A. (1971) J. Amer. Oil Chem. Soc. 48:427-433; and Schaich, K.M. (1980)

                        CRC Reviews in Food Science and Nutrition, pp 189-244.

 

 

While light is an important source of free radicals to exposed rubber (such as tyres), this is of little concern in the animal feed since feed are seldom packaged in transparent containers. Enzymes are a great problem in fresh meats since they can cause oxidation, such as that which occurs in fish muscle and the resulting "old fish" taste and aroma. These enzymes (such as lipoperoxidase) must have at least 85-95% water to be active. Since most feeds are manufactured at 13% moisture, enzymes are not of real concern.

 

Metal ions are of importance to animal feed manufacturers. Two metal ions (copper and iron) are especially potent in their ability to catalyse the creation of free radicals as shown in Figure VIII.

 

figure Viii

catalysis of free radical formation by metal ions


 

These ions act to help to spontaneously create the free radicals in feeds and once peroxides are created, they catalyse the propagation reaction. Evidence of this catalysis is widespread in the literature. Characteristic of this work by Marcuse and Fredriksson shown in Figure IX.

 

FIGURE IX

oxygen consumption in linoleic acid as a function of

molar copper ion concentration

                                  From: Marcuse, R. and P. Fredriksson (1971) "Fat oxidation at low pressure: III

                                  kinetic studies on linoleic acid oxidation in emulsions in the presence of added

                                  metal salts", JAOCS 48:448-457

 

 

In these experiments, free radicals were not monitored directly, but oxygen consumption was measured over a period of time. By increasing copper concentrations, the amount of oxygen consumption (and thus, the number of free radicals created) is dramatically increased. A logical antioxidant strategy would be to not only absorb free radicals, but also to prevent the creation of free radicals. This can be accomplished through chelation (Figure X).

 

figure x

CHELATION OF METAL IONS

 

 

 

 

 

Through the chelation of copper ions, the catalysis by metal ions of the free radical reaction can be prevented. Chelation also makes the minerals more available for absorption by the animals, as has been commonly experienced in the feed industry for the past 15 years with "chelated mineral sources". It is thus, possible to conclude that an ideal antioxidant system can be summarized as follows (Table 11).

 

table 11

COMPONENTS OF AN IDEAL ANTIOXIDANT SYSTEM

 

 

 

An ideal antioxidant material must be a system that is a combination of materials. It must first contain an excellent quality antioxidant that is able to easily combine with those types of free radicals commonly found in animal feeds. BHA, for instance, is one such antioxidant known for its extremely high efficacy within animal feed systems.

 

The antioxidant system must also contain a chelator. Using these two classes of chemicals, it is possible to construct a system that is able to prevent the initial formation of the free radicals and to absorb the few free radicals that the chelators cannot prevent.

 

This binary system is far superior to one which only utilizes an antioxidant alone. In such a simple system which contained only an antioxidant (such as the addition of only ethoxyquin to a feed), it may be conceivable enough free radicals could be created to "use up" the antioxidant. Thus, prevention and absorption are called for.

 

An antioxidant should also contain surfactants. Antioxidant molecules by nature are normally hydrophobic, while free radicals are usually hydrophilic. To be able to insure that the reaction between these two moieties occurs, the materials must actually come in physical contact with each other. By the addition of a surfactant, this feature is guaranteed. A particle of endox® is represented in Figure XI.

 

figure xi

KEMIN ANTIOXIDANT PRODUCTS

 


 

ENDOX® is a combination of calcium silicate that has been coated, with a synergistic blend of BHA, ethoxyquin, phosphoric acid and EDTA. In addition, mono- and diglycerides have been added to act as surfactants within the antioxidant system.

 

To summarize more practical programs that could be utilized in feed fat stabilization, a program is outlined in Table 12.

 

table 12

a practical program to prevent autoxidation in animal feeds

 

 

 

One hundred twenty-five (125) ppm of antioxidant has almost universally been established as the ideal dosage for final feeds. Given the possible problems that oxidation  can cause in the feeds, this relatively inexpensive ingredient can return its cost many times over to the feed manufacturer when used at these levels.

 

While 125 ppm is a general recommendation for feeds, more specificity to this recommendation can be gained when the amount of fat in the diet and length of storage time. Such specific recommendations are shown in Table 13.

 

table 13

antioxidant concentration (ppm) to be used in feed as a function of fat concentration and storage time(1)

 

                     (1)   This recommendation is only a general guideline.  Other factors, such as the nature of the

                     fat, the storage temperature, and the mineral content (particularly Cu and Fe) etc. should also

                     be considered.

 

The recommendations are based on the total fat content (ether extract) of the feed. As noted, if no fat is contained in the material (or nothing soluble in ether), then no antioxidant is required. As storage time increases or fat level increases, more antioxidant is needed.

 

Vitamin/mineral premixes can contain antioxidants at two levels. They can be placed in the mineral at 500 ppm which is sufficient to simply preserve the vitamins within the premix until they are used at feed manufacture, or higher amounts could also be placed in the premix such that they will affect the final concentration of 125 ppm in the feed. As noted in the example above, a 10% premix thus would need to be fortified with 1,250 ppm of antioxidant.

 

Evaluation of antioxidant programs should be based on a global parameter considering the overall palatability of the feed and the relative stability of the vitamins as they are evaluated in a feed quality program.

 

Feed fats are quite different and must be considered separately. Kemin recommends when fats arrive at the feed mill that the specifications call for the addition of the antioxidant be made at this point once it has been demonstrated that the peroxide value is 2 meq/kg or less. This program gives a number of advantages.

 

Firstly, by adding the antioxidant at the feed mill, we are insured of its addition. The fat will have to be treated very carefully before it reaches the feed mill and not subject to thermal degradation to be able to pass the specification of 2 meq or less.

 

Secondly, this procedure is significantly quicker. Feed millers in the past have subjected fats to such tests as the active oxygen method in order to determine feed fat stability. Unfortunately, this test can take many hours, even days. Fat that is found to be not acceptable by this test usually has been pumped into fat holding tanks and often used in feeds. A simpler test involves determining the peroxide value at the feed mill when it arrives. This assay requires only 5-10 minutes and very simple equipment. Fats then can be accepted or rejected before being unloaded and thus, feed quality insured.

 

It is noteworthy that when calculating the final amount of antioxidant to place in a ration, credit can be taken from both the antioxidant added to the feed and, if the vitamin premix is not fortified completely, credit can be taken from the addition also.

 

Modern feed manufacture requires attention to the feed as manufactured as well as to the state of the finished product as presented to the animal. Prevention of autoxidation perhaps is one of the most certain ways to prevent loss of many of the critical components of nutritious feed.

 

 

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