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Effect of Autoxidation on the Nutritive Value of Acidulated Soybean Soapstock in Chicks1
Effect of Autoxidation on the Nutritive Value of Acidulated Soybean Soapstock in Chicks1 BlANKA LlPSTEIN, P . BUDOWSKI AND S. BORNSTEIN National and University Institute of Agriculture, Rehovot, Israel (Received for publication April 13., 1965)
1
Contribution from the National and University Institute of Agriculture, Rehovot, Israel. 1965 series, No. 849-E. Part of a Ph.D. thesis to be submitted by the senior author to the Faculty of Agriculture, Hebrew University of Jerusalem.
cause undesirable effects in experimental animals (Schultz et al., 1962). In feed fats of animal origin, this difficulty has been largely overcome by the addition of antioxidants, and furthermore, it has recently been reported that beef tallow caused no detrimental effects in rats after it had been oxidized until its iodine value had fallen by 4 units (Lea et al., 1964). The object of the present work was to investigate the effect of autoxidation of ASS on its nutritional value in chicks. EXPERIMENTAL
PROCEDURE
Trial 1. The purpose of this trial was to ascertain the effect of aerobic heating of ASS on its carbonyl value, iodine number and oxy-fatty acid content, and to study the influence of these changes on the performance of chicks. ASS was heated in an open aluminum pot placed on an electric plate inside a well-ventilated hood. During the first 20 days of heating the temperature of ASS ranged from 90° to 105°C, and from 115° to 135°C. during the following 11 days. Samples of the heated ASS were withdrawn at frequent intervals, and iodine number and oxy-fatty acids were determined by methods of the American Oil Chemists' Society (1961). Carbonyl values were determined according to Henick et al. (1954). On the basis of the results of these chemical tests, ASS samples in quantities sufficient for feeding trials with chicks were withdrawn after heating for 22, 24 and 31 days, respectively. One-day-old Leghorn cockerels, obtained from a commercial hatchery, were raised in
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ACIDULATED soybean-oil soapstock 1\- (ASS) has recently become the main fat supplement for poultry feeds in Israel. This by-product of the edible oil industry is obtained from the alkali-neutralization of the crude oil in the form of raw soapstock (a mixture of soaps, neutral oil, water, sterols, pigments and other minor constituents). This rather unstable product is converted to acidulated soapstock by treatment with an excess of hot aqueous sulfuric acid. Most of the "impurities" which are removed from the crude oil by alkali-refining accumulate in the acidulated soapstock. Compared to crude soybean oil (CSO), ASS contains high levels of free fatty acid (over 50%), unsaponifiable matter and oxy-fatty acids (Bornstein and Lipstein, 1963), and is also rich in carotenoids (Budowski et al., 1964). Previous work of this labortory (Bornstein and Lipstein, 1961, 1963), as well as that of Sibbald et al. (1962), has shown that ASS is comparable to tallow in its effect on growth and feed efficiency of young chicks. Little is known, however, about the stability of ASS toward oxidative rancidity and about possible detrimental effects on chicks due to oxidation of ASS. It is well known that extensive autoxidation of polyunsaturated oils is liable to
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NUTRITIVE VALUE OF SOAPSTOCK TABLE 1.—Composition of diets Trials Ingredients F a t supplement Ground sorghum (milo) Soybean meal (49% protein) Fish meal (60% protein) Alfalfa meal (dehydr.) Oyster shell flour Dicalcium phosphate Mineral supplement 1 Vitamin supplement 1 DL-methionine Coccidiostat Calculated Analysis Crude protein (%) Prod. Energy (Cal./lOO gm.) C / P ratio*
—
•— 1
2
3
10.0 44.6 36.0 3.0 2.5 1.4 1.6 0.35 0.35 0.10 0.10
8.0 48.83 34.0 3.0 2.5 1.5 1.5 0.30 0.25 0.12 0.10
8.0 50.08 35.0 3.0
24.5 228 9.3
24.0 224 9.3
—
1.4 1.6 0.35 0.35 0.12 0.10 23.7 220 9.3
1 Detailed composition described in another paper (Bornstein and Lipstein, 1963). 2 Cal. (PE) per 100 gm./% protein ratio.
macromethod of Kjeldahl (A.O.A.C., 1960). Dietary fat was determined by the Soxhlet extraction procedure (A.O.A.C, 1960) and excreta fat by the hydrolysis procedure of Van de Kamer (1949). Metabolizable energy values per gram of diet were calculated as described by Sibbald et al. (1960), except for collecting total excreta rather than representative samples marked by chromic oxide. Trial 3. The objectives of this trial were: a) to determine the effect of heating ASS on its chemical composition; b) to follow subsequent changes in composition after the heated ASS samples were incorporated into the diet and latter stored under various conditions; and c) to investigate the biological effects of these latter feeds in chicks. The heating of ASS was carried out as in the previous trials, the temperature ranging from 115° to 140°C. Two samples of ASS, heated for 3 and IS days, respectively, as well as an unheated sample, were added to the diet (Table 1). The three feeds were stored for 70 days under the following three conditions: a) at 3 ° C ; b) at room temperature in open cans, frequently mixed and exposed to sunlight most of the days; and c) at 34°C. The three diets stored at room temperature
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battery-brooders equipped with raised wire floors and thermostatically controlled electric heating units. They were fed a commercial starter ration for one week, at which time they were individually weighed; about 60% of them, belonging to the middle of the weight distribution, were divided into 4 lots of 15 chicks each on the basis of body weight, equalizing both mean weight and weight distribution among the lots. The diet described in Table 1 was fed ad libitum during a 4-week experimental period, the fat supplement being either an unheated, or one of the above 3 heated samples of ASS. Individual weights and group feed consumption were recorded weekly. Trial 2. The purpose of this trial was to compare the effects of heating ASS and CSO on the metabolizable energy of the diets, the performance of the chicks and their apparent protein and fat retention. The heating procedure was similar to that used in Trial 1, except that the heating period lasted only 7 days. The temperature ranged from 130° to 150°C. The composition of the basal diet used in this trial is presented in Table 1. The experimental design was a 2 X 2 factorial, with type of oil and heat treatment as the variables. Leghorn cockerels were raised and distributed into 16 lots as described for the first trial. Each of the 4 diets was fed to 4 replicates of 10 chicks each during a 4-week period, beginning at one week of age. Beginning with the 12th day of the experiment, feed consumption was measured and droppings were collected daily from each group of chicks during four consecutive days. The droppings were dried at 70°C. in a forced-draft oven for 24 hours, after which they were ground and stored at room temperature in closed glass containers. Feed and excreta samples were analyzed for gross energy, using a Parr oxygen bomb calorimeter, and for nitrogen by the
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B . LlPSTEIN, P . BUDOWSKI AND S. BORNSTEIN
ol f
I
I »
I
1 20 t
i t
L_ t
30
Days of heating
FIG. 1. Chemical changes in acidulated soybean oil soapstock during heating at 90-135°C. in open pans (Trial 1). Carbonyl value (0); Oxy fatty acids (A); and Iodine value (•). Arrows indicate sampling for chick experiments.
fats of the above chicks. Liver vitamin A was assayed according to Ames et al. (1954)). Results were analyzed by the analysis of variance (Snedecor, 1956) and treatment means were separated according to Duncan's (1955) multiple range test. All interpretations of the data are based on the 1 percent level of probability. RESULTS
Trial 1. Some of the chemical changes occurring in heated ASS are presented graphically in Fig. 1. While the iodine value of the oil dropped during heating, the oxy-fatty acid level and carbonyl values rose. These changes were slow during the first two weeks and became increasingly more rapid thereafter. This acceleration was probably due to the rise in the temperature, although both the carbonyl value and oxyfatty acids levels seemed to increase even before the stepped up heating. Samples were selected for feeding tests when the iodine value had dropped by 9, 11 and 23 units. Table 2 summarizes the performance of growing chicks receiving four samples of ASS. For unknown reasons' chicks fed the moderately oxidized
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were used for a chick experiment after 42 days of storage, at which time a fourth diet was prepared for this purpose from the same ingredients and with an unheated sample of ASS, which had been kept in the meanwhile in cold storage. One-day-old male crossbred (Cornish X White Rock) chicks were randomly divided into 4 experimental groups of 14 chicks each, for which the experimental diets were randomly assigned. Feed and water were supplied ad libitum during the S-week experimental period. Individual body weights were recorded weekly, and feed consumption on a group basis was determined at 3, 4 and 5 weeks of age. Metabolizable energy values and apparent protein and fat retentions were determined similarly to the previous trial, beginning with the 28th day. Ten chicks per group (those closest to the average weight) were killed at 35 days of age. Adipose tissue and livers were removed, placed in plastic containers and frozen. Soapstocks and feed lipids (ether extracts) were analyzed for iodine and peroxide values and total and oxy-fatty acids (all according to A.O.C.S., 1961). Total carotenoids were measured by passing an ether solution of the lipid through a column of alumina containing 5% water, and measuring the absorbency of the eluate in a Bausch and Lomb "Spectronic" colorimeter at 450mpi. (3-carotene was used as a reference standard. This rapid method gave similar results to one in which the unsaponifiable matter was chromatographed on magnesium oxide and eluted with a 1:1 mixture of petroleum ether and ethanol. Fatty acid composition of feed lipids was determined by GL chromatography of the methyl esters, using a column of diethylene-glycol adipate polyester at a temperature of 210°C. Peak areas were measured by triangulation. Fatty acid composition was also determined for the depot and liver
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NUTRITIVE VALUE OF SOAPSTOCK
TABLE 2.—Performance of chicks receiving diets containing 10% fresh or heated acidulated soybean soapstock from 1 to 5 weeks of age (15 chicks per diet; Trial 1) Days of heating
Drop in IV 1
Body weight (g-)
Feed intake (g-)
Feed/gain ratio
0 22 14 31
0 9 11 23
453 408 435 440
735 687 737 754
1.62 1.68 1.69 1.71
1
IV=iodine value.
crude oil to be significantly higher than that of the heated soapstock. Encephalomalacia (diagnosed on the basis of pathognomonic symptoms and macroscopic and histological lesions) was observed among the chicks fed the diet containing oxidized soapstock. Apparent protein retention and growth rate remained unaffected by dietary treatments. Trial 3. This trial involved fresh, mildly oxidized and strongly oxidized ASS. Chemical characteristics of these samples are given in Table 4. The fall in iodine value, due to heating, is accompanied by a rise in oxy-fatty acids, which, in turn, corresponds exactly to the decrease in total (non-oxy) fatty acids. Carotenoids are largely destroyed by the mild treatment and disappear completely upon further oxidation of
TABLE 3.—Characteristics of the oils used as 8% of the diets, and the performance of chicks from 1 to 5 weeks of age (4 replicates of 10 chicks each; Trial 2) Oil supplements 3
CSO CSO.heated ASS3 ASS,heated S.E. 1
IV 1
124.6 118.0 124.9 118.6
OFA, 1
% 1.08 3.05 4.12 6.21
Retention
ME cal./g. diet
Body weight (g-)
Feed intake (g.)
Feed/gain ratio
3.24 a" 3.26 a 3.19 ab 3.13 b 0.02
655 633 668 605 —
942 932 982 990 —
1.47 a 1.47 a 1.47 a 1.60 b 0.01
Protein
Mortality 2
Fat
%
%
62.7 62.0 62.2 60.8 —
89.4 86.7 86.6 85.3 0.78
a ab ab b
0/40 0/40 0/40 3/40
IV=iodine value; OFA=oxy-fatty acids. No. of dead chicks/total no. of chicks. Death was due to encephalomalacia and occurred at the age of three weeks. 3 CSO = crude soybean oil; ASS = acidulated soybean-oil soapstock. 4 Within any one column, means which are not followed by the same letter are significantly (P<0.01) different from each other. 2
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sample (drop in iodine value of 9 units) ate less feed and hence gained less weight than the other groups. However, these differences were not significant. The feed/ gain ratio increased with the length of the heating period, indicating decreased feed utilization. There was no mortality in this trial. Trial 2. In the second experiment the oils were heated for only 7 days, hence the iodine values decreased by only 6.6 and 6.3 units in the cases of CSO and ASS, respectively, and the oxy-fatty acids increased by about 2 percentage units in both cases (Table 3). The heated soapstock lowered feed conversion significantly compared to unheated soapstock and both of the crude oils. These results parallel the metabolizable energy contents of the diets, in which respect the heated soapstock differed significantly from both crude oils, but not from the unheated soapstock. The energy data, in turn, can be partially explained on the basis of apparent fat retention, which was slightly (but not significantly) decreased by the heating of either oil. This together with the slightly lower energy content of unheated ASS as compared to unheated CSO, caused the metabolizable energy level of unheated
1484
120
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acidulated soybean soapstock used in trial 3
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soap- Pd£ ' °sd . stock ay
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IVi
%
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TABLE 4.—Characteristics of fresh and heated
1
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110
AND S .
123.9 118.3 87.3
1.4 1.9 0.8
4.5 7.6 24.8
91.8 87.9 70.5
156.8 17.2 0.0
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1 IV=iodine values; PV=peroxide value; OFA =oxy-fatty acids; TFA = total fatty acids (excluding unsaponifiable matter)
1
1
0 3 15
1
80 020 Days of storage
40
60
80
FIG. 2. Effect of storage on the iodine value and carotenoid content of ether extractable feed lipids. The feeds contained 8% ASS (Table 4) either unheated ( ), slightly heated ( ), or very heated ( ). Storage was at 3° (A), at room temperature (•) or at 34° (0).
the soapstock. Peroxides did not accumulate under the conditions of the heating. When the above oil samples were incorporated into the diets, and stored for 70 days under three different conditions (at 3°C, room temperature and 34°C), the changes were most pronounced in the iodine value and carotenoid content, which decreased during storage (see Fig. 2). The drop in iodine value for the unheated soapstock A was slowest at the highest storage temperature, and the reverse was true for the strongly heated soapstock C. For sample B, no effect of storage temperature was indicated. Destruction of carotenoids proceeded more rapidly the higher the storage temperature. The carotenoid contents of the diets containing heated soapstocks B and C were too low to be measured and only the data for the unheated soapstock are given in Fig. 2. Peroxides increased only slightly for all samples, the highest value, 8.6 [i.equ./g., being reached by the feed containing mildly oxidized oil and stored at 34°C. for 70 days.
The iodine value and fatty acid composition of the lipids extracted from the four diets used in the chick experiment are presented in Table 5. Storing the feed containing the unheated ASS slightly reduced its iodine value and caused only very slight changes in fatty acid composition. On the other hand, the fats extracted from the diets to which had been added the heated samples of ASS (especially the stronger heated sample C) exhibited lower linoleic and linolenic acids levels and higher saturated and monounsaturated fatty acid contents, apparently due to the heating rather than to the storage. Generally, the differences in iodine value of the feed lipids paralleled the drop in iodine value induced in the soapstock by the heat treatments (see Table 4). Performance of the chicks, metabolizable energy values of the experimental diets and the retention of protein and fat are presented in Table 6. Storage of the diet containing fresh ASS did not appear to affect TABLE 5.—Iodine value and fatty acid composition of
the lipids extracted from the experimental diets used in trial 3 Experimental diet 1
IV!
A(0) A (42) B (42) C(42)
121.5 111.0 104.0 78.6
% fatty acid composition 3 16:0 12.3 13.5 15.3 19.3
16:1
18:0
18:1
18:2
18:3
1.1 0.8 1.3 2.2
4.8 5.0 4.1 6.1
2 2 . 1 51.0 19.6 52.8 23.4 50.1 26.0 43.9
8.8 8.3 5.8 2.5
1 The diet is designated by the soapstock it contains (A, B and C—see Table 4) and the number of days (in parentheses) it has 2 been stored at room temperature. IV=iodine value. 3 Chain length:no. of double bonds.
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_
A B C
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NUTRITIVE VALUE OF SOAPSTOCK
TABLE 6.—Performance of chicks receiving the experimental diets of trial 3 until the age of 5 weeks (14 chicks per diet)
g-
Feed/gain ratio
ME cal./g. feed
Protein
%
%
776 765 763 793
1,357 1,294 1,503 1,583
1.78 1.69 1.97 1.97
3.12 3.02 2.97 2.77
46.3 47.8 47.6 44.5
83.6 85.5 79.8 73.0
1 2
^
Fat
Mortality 2
0/14 0/14 1/14 3/14
See footnote 1 to Table 5. From encephalomalacia; no. of dead chicks/total no. of chicks.
the performance of the chicks. Feed intake was decidedly higher in the groups receiving diets containing heated ASS, whereas weight gains were similar for all groups. This resulted in an unfavorable feed/gain ratio for the diets containing the heated soapstock. Apparent protein retention was not affected by the dietary treatments, but fat retention was markedly lower in the chicks receiving the diet with the strongly oxidized fat. This low retention also caused the latter diet to have the lowest metabolizable energy content. Encephalomalacia developed in a few chicks receiving the diets containing the oxidized soapstocks. Table 7 describes the fatty acid composition of the abdominal fat of chicks receiving the experimental diets. The changes in fatty acid composition reflect those observed in the dietary lipids (see Table 5): there is a decrease in polyunsaturated fatty acids (PUFA) in the fat of chicks fed the diets containing the oxidized oils, as compared to the levels observed in the other two groups. This is accompanied by a corTABLE 7.—Fatty acid composition of the abdominal fat of 5-week old chicks raised on the experimental diets {trial 3) % fatty acid composition2
Experimental diet'
14:0
16:0
16:1
18:0
18:1
18:2
18:3
A(0) A (42) B(42) C(42)
0.7 0.5 0.5 0.6
20.8 21.8 20.6 24.3
1.3 2.0 2.8 2.5
2.9 2.5 4.0 2.9
30.1 30.4 31.6 38.3
39.6 38.3 36.2 28.4
4.8 4.4 4.2 2.4
i.« See footnotes 1 and 3, Table 5.
responding increase in oleic and palmitic acids. These changes become pronounced only in the case of the diet containing the strongly oxidized ASS. In the liver lipids, the picture is similar, except that the fatty acid composition is less sensitive to dietary manipulation (Table 8). In fact, arachidonic acid remains the same for all groups, while the changes in oleic, linoleic and linolenic acids are relatively slight, although they again parallel the dietary variations. The vitamin A values, also presented in Table 8, show that using heated ASS had an appreciable effect on the vitamin reserves in the liver, whereas storage of the feed was apparently without effect. DISCUSSION
The data obtained in these experiments indicate that heat-induced autoxidation of ASS may result in decreased feed utilization (Tables 1, 3 and 6) and cause mortality in growing chicks fed diets containing such ASS (Tables 3 and 6). This poorer performance of chicks fed heated soapstock seems to be due to the lower metabolizable energy content of the diets containing this type of ASS, thus resulting in higher feed intake. It is interesting to note that growth rate and protein retention were apparently unaffected by dietary heated ASS (Tables 3 and 6), although a slight trend toward lower weight gains is apparent in trial
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A(0) A (42) B (42) C(42)
Retention
Feed intake, g-
Experimental
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B . LlPSTEIN, P . BUDOWSKI AND S. BOENSTEIN
TABLE 8.-—Fatty
acid composition and vitamin A values of the livers of 5-week-old chicks raised on the experimental diets (trial 3) % fatty acid1 composition2
Vitamin A
16:0
16:1
18:0
18:1
18:2
18:3
20:4
Unknown3
I.U./ liver
Storage 4 %
A(0) A (42) B(42) C(42)
18.4 17.6 22.1 22.0
1.0 0.6 1.3 0.9
22.8 21.7 21.7 21.9
12.9 14.1 15.1 16.1
26.8 26.9 24.6 23.3
2.1 1.7 1.3 0.8
12.9 12.9 11.3 12.2
3.1 4.5 2.6 2.8
5,178 4,959 4,115 4,189
47.7 47.8 34.2 33.1
1,2 3 4
See footnotes 1 and 3, Table 5. Two minor peaks were eluted between linolenic and arachidonic acids. Liver vitamin A expressed as percentage of total intake.
2 (Table 3). The metabolizable energy, in turn, seems to be dependent on the retention of fat, which was lower in the case of oxidized soapstock (Tables 3 and 6). The lowered fat retention may be caused by the presence of increased levels of oxy-fatty acids (Table 3 and 4) and possibly by polymeric material which remains mostly unabsorbed (Kaunitz et al., 1956). Before that Lassen et al. (1949) had shown a decrease in digestibility of a highly unsaturated oil with an increase in polymerization, the latter being expressed as percentage drop in the iodine value. In trials 2 and 3, feeding oxidized ASS produced encephalomalacia in some of the chicks. It had been shown previously (Mokadi and Budowski, 1963), that safflowerseed oil, oxidized to a drop in iodine value of 13 units and fed at 20% level in a commercial-type diet, caused encephalomalacia in all the chicks to which it was fed. Oxidized ASS, which is more saturated than safflowerseed oil and which was fed as 8% of the diet, still produced some encephalomalacia in the present trials. On the other hand, no mortality was found by Lea et al. (1964) in rats fed 5% oxidized beef fat which is poor in PUFA. A comparison between ASS and CSO in trial 2 (Table 3) shows that the latter, when oxidized under the same conditions and to the same extent as the ASS, caused
no detrimental effects at all in the chicks. ASS initially contains more oxy-fatty acids than crude soybean oil, so that upon aerobic heating, the soapstock ends up with a higher oxy-fatty content than the oil, the drop in iodine value and increase in oxy-fatty acids due to heating being similar for both materials. It has been shown that certain types of long-chain keto acids have pronounced pro-oxidant activity (Bhalerao et al., 1962) and may cause encephalomalacia in chicks (Kokatnur et al., 1960). It is possible that, upon heating, such compounds form in ASS from oxyfatty acids present initially, or that such a process is favored by the high free fatty acid content of ASS. When the feed containing 8% unheated or heated ASS was stored at room temperature for 42 days, no additional peroxides or oxy-fatty acids accumulated in the feed lipids. In the other hand the iodine value dropped slightly (Table 5). In the case of the unheated soapstock, storage produced no detrimental effect on chick performance (Table 6). Therefore, it can be assumed that the detrimental effects of diets B (42) and especially C (42) on chick performance (Table 6) are due to the use of heated oils, and not due to the storage of the feed. Moreover, the decrease observed in carotenoids upon storage (Fig. 2) may cause lesser pigmentation of skin and
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Experimental diet 1
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NUTRITIVE VALUE OF SOAPSTOCK
dized soybean oil, and by Lea et al. (1964), who fed autoxidized beef tallow. As shown in Table 8, no detrimental effect in this respect was obtained by the storage of the feed containing 8% fresh ASS. SUMMARY
In three trials the chemical changes occurring during the heating of acidulated soybean soapstock (ASS), and in one case crude soybean oil (CSO), were investigated. These changes were related to the performance of chicks the dietary metabolizable energy level and the apparent retention of fat and protein. In one trial the chemical changes occurring in stored diets containing heated ASS were followed, and the effect of these feeds on chick performance, on the fatty acid composition of liver and depot fat, and on liver vitamin A were determined. Heating ASS decreased the iodine value, increased the carbonyl value, increased the concentration of oxy-fatty acids and destroyed most of the carotenoids. These heatinduced autoxidative changes may result in a decreased feed utilization by growing chicks receiving a commercial-type diet supplemented with the oxidized oils. Heating of ASS also lowered the metabolizable energy content of the diets. The lower metabolizable energy resulted from decreased fat retention, which was definitely lower in the case of oxidized soapstock. Protein retention, on the other hand, was not affected by autoxidation of ASS, nor was growth rate (or only slightly so). Feeding oxidized ASS also produced encephalomalacia in some of the chicks. The changes in depot fat composition reflect those found in the feed lipids, i.e., there is a decrease in polyunsaturated fatty acids with a corresponding increase in saturated and monounsaturated fatty acids. The fatty acid composition of the liver lip-
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yolks. The failure of lipid oxidation products to accumulate during storage of feed has recently been emphasized by Lea et al. (1964). The effect of autoxidized ASS on the fatty acid composition of depot and liver fat, as shown in Tables 7 and 8, can be explained by the changes induced in the ASS as the result of heating. An additional reason for the lowered PUFA content of chicks receiving heated soapstocks may be the interference with absorption and deposition of PUFA caused by fat oxidation products (Lea et al., 1964). Perkins et al. (1961) have shown that dietary hydroxy acids cause an increase in carcass monoenoic acids. By itself, such an alteration in fatty acid composition of carcass fat is not detrimental ; in fact, it may increase the stability of this fat (Lea et al., 1964), provided sufficient dietary a-tocopherol is available to the chick. The fatty acid composition of the liver lipids is affected in a similar fashion, except for arachidonic acid, which remains constant whatever the state of autoxidation of the dietary ASS. This acid is formed from linoleic acid in vivo, and it is, therefore, surprising that no drop in arachidonic acid is observed upon feeding autoxidized ASS. The explanation may possibly be found in the recent work by Bernhard et al. (1964), in which it was shown that the extent of conversion of linoleic to arachidonic acid in the livers of rats is inversely related to the amount of a-tocopherol supplied. The lowered tocopherol level of autoxidized ASS would, therefore, cause increased conversion, thus counteracting the effect from the decrease in linoleic acid. Vitamin A storage was depressed as the result of feeding autoxidized ASS to chicks. A similar effect was found in rats by Reporter and Harris (1961), who fed autoxi-
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B . LlPSTEIN, P . BUDOWSKI AND S. BORNSTEIN
ACKNOWLEDGMENTS
The authors are indebted to M. Mosheh and C. Genige for technical assistance during the courst of these studies. REFERENCES Ames, S. R., H. A. Risley and P. L. Harris, 1954. Simplified procedure for extraction and determination of vitamin A in liver. Anal. Chem. 26: 1378-1381. American Oil Chemists Society, 1961. Official and Tentative Methods of Analysis. 3rd Ed. Rev. to 1961, Chicago, 111. Association of Official Agricultural Chemists, 1960. Methods of Analysis, 9th Ed. Wash., D.C. Bernhard, K., S. Leisinger and W. Pedersen, 1964. Vitamin E und Arachidonsaure-Bildung in der Leber. Helv. Chim. Acta, 46: 1767-1772. Bhalerao, V. R., M. G. Kokatnur and F. A. Kummerow, 1962. The effect of 12, oxo-cis, 9-octadecenoic acid on the autooxidation of corn oil. J. Am. Oil. Chem. Soc. 39: 28-30. Bornstein, S., and B. Lipstein, 1961. By-products of soybean oil as fat supplements in practical broiler rations. World's Poultry Sci. J. 17: 167178. Bornstein, S., and B. Lipstein, 1963. Some unusual waste vegetable oils as fat supplements in practical broiler rations. World's Poultry Sci. J. 19: 172-184.
Budowski, P., J. Ascarelli, J. Gross, J. Nir and A. Bondi, 1964. Vitamin A activity of acidulated soybean soapstock in chicks. J. Am. Oil. Chem. Soc. 4 1 : 441-445. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Henick, A. S., M. F. Benca and J. H. Mitchell, 1954. Estimating carbonyl compounds in rancid fats and foods. J. Am. Oil. Chem. Soc. 34: 88. Kaunitz, H., C. A. Slanetz, R. E. Johnson, H. B. Knight, D. H. Saunders and D. Swern, 1956. Biological effects of the polymeric residues isolated from autoxidized fats. J. Am. Oil. Chem. Soc. 33: 630-634. Kokatnur, M. G., S. Okui, F. A. Kummerow and H. M. Scott, 1960. Effect of long-chain ketoacids on encephalomalacia in chicks. Proc. Soc. Expt. Biol. Med. 104: 170-171. Lassen, S., E. K. Bacon and H. J. Dunn, 1949. The digestibility of polymerized oils. Arch. Biochem. 23: 1-7. Lea, C. H., L. T. Parr, J. L. L'Estrange and K. T. Carpenter, 1964. Nutritional effects of autoxidized fats in animal diets. Beef fat in the diet of rats. Brit. J. Nutr. 18: 369-384. Mokadi, S., and P. Budowski, 1963. Relationship between vitamin E and encephalomalacia in chicks. Brit. J. Nutr. 17: 347-355. Perkins, E. G., J. G. Enders and F. A. Kummerow, 1961. Effect of ingested thermally oxidized corn oil on fat composition in the rat. Proc. Soc. Expt. Biol. Med. 106: 370. Reporter, M. C. and R. S. Harris, 1961. Effects of oxidized soybean oil on the vitamin A nutrition of the rat. J. Am. Oil. Chem. Soc. 38: 4757. Schultz, H. W., E. A. Day and R. O. Sinnhuber, editors, 1962. Lipids and Their Oxidation. The Avi Publ. Co., Westport, Conn. Sibbald, I. R., W. F. Pepper and S. J. Slinger, 1962. A comparison of feed grade tallow and acidulated soapstocks in practical chick starter rations. Poultry Sci. 4 1 : 120-124. Sibbald, I. R., J. D. Summers and S. J. Slinger, 1960. Factors affecting the metabolizable energy content of poultry feeds. Poultry Sci. 39: 544-556. Snedecor, G. W., 1956. Statistical Methods, Sth Ed., Iowa State College Press, Ames, Iowa. Van De Kamer, 1949. Rapid method for the determination of fat in feces. J. Biol. Chem. 177: 347-355.
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ids is affected in a similar manner, but to a lesser extent. Moreover, the arachidonic acid content of liver lipids was not affected by the state of oxidation of the diet fat. Vitamin A storage was decreased as the result of feeding autoxidized ASS to chicks. Feeding chicks a diet containing oxidized CSO had no detrimental effect, unlike a sample of ASS which had been oxidized to the same extent. Whereas thermally induced autoxidation of ASS decreased its nutritional value for growing chicks, storage of feed containing either unheated or heated 8% ASS caused no detrimental effects, other than a lowering of the carotenoids found in the soapstock.
1
Contribution from the National and University Institute of Agriculture, Rehovot, Israel. 1965 series, No. 849-E. Part of a Ph.D. thesis to be submitted by the senior author to the Faculty of Agriculture, Hebrew University of Jerusalem.
cause undesirable effects in experimental animals (Schultz et al., 1962). In feed fats of animal origin, this difficulty has been largely overcome by the addition of antioxidants, and furthermore, it has recently been reported that beef tallow caused no detrimental effects in rats after it had been oxidized until its iodine value had fallen by 4 units (Lea et al., 1964). The object of the present work was to investigate the effect of autoxidation of ASS on its nutritional value in chicks. EXPERIMENTAL
PROCEDURE
Trial 1. The purpose of this trial was to ascertain the effect of aerobic heating of ASS on its carbonyl value, iodine number and oxy-fatty acid content, and to study the influence of these changes on the performance of chicks. ASS was heated in an open aluminum pot placed on an electric plate inside a well-ventilated hood. During the first 20 days of heating the temperature of ASS ranged from 90° to 105°C, and from 115° to 135°C. during the following 11 days. Samples of the heated ASS were withdrawn at frequent intervals, and iodine number and oxy-fatty acids were determined by methods of the American Oil Chemists' Society (1961). Carbonyl values were determined according to Henick et al. (1954). On the basis of the results of these chemical tests, ASS samples in quantities sufficient for feeding trials with chicks were withdrawn after heating for 22, 24 and 31 days, respectively. One-day-old Leghorn cockerels, obtained from a commercial hatchery, were raised in
1480
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ACIDULATED soybean-oil soapstock 1\- (ASS) has recently become the main fat supplement for poultry feeds in Israel. This by-product of the edible oil industry is obtained from the alkali-neutralization of the crude oil in the form of raw soapstock (a mixture of soaps, neutral oil, water, sterols, pigments and other minor constituents). This rather unstable product is converted to acidulated soapstock by treatment with an excess of hot aqueous sulfuric acid. Most of the "impurities" which are removed from the crude oil by alkali-refining accumulate in the acidulated soapstock. Compared to crude soybean oil (CSO), ASS contains high levels of free fatty acid (over 50%), unsaponifiable matter and oxy-fatty acids (Bornstein and Lipstein, 1963), and is also rich in carotenoids (Budowski et al., 1964). Previous work of this labortory (Bornstein and Lipstein, 1961, 1963), as well as that of Sibbald et al. (1962), has shown that ASS is comparable to tallow in its effect on growth and feed efficiency of young chicks. Little is known, however, about the stability of ASS toward oxidative rancidity and about possible detrimental effects on chicks due to oxidation of ASS. It is well known that extensive autoxidation of polyunsaturated oils is liable to
1481
NUTRITIVE VALUE OF SOAPSTOCK TABLE 1.—Composition of diets Trials Ingredients F a t supplement Ground sorghum (milo) Soybean meal (49% protein) Fish meal (60% protein) Alfalfa meal (dehydr.) Oyster shell flour Dicalcium phosphate Mineral supplement 1 Vitamin supplement 1 DL-methionine Coccidiostat Calculated Analysis Crude protein (%) Prod. Energy (Cal./lOO gm.) C / P ratio*
—
•— 1
2
3
10.0 44.6 36.0 3.0 2.5 1.4 1.6 0.35 0.35 0.10 0.10
8.0 48.83 34.0 3.0 2.5 1.5 1.5 0.30 0.25 0.12 0.10
8.0 50.08 35.0 3.0
24.5 228 9.3
24.0 224 9.3
—
1.4 1.6 0.35 0.35 0.12 0.10 23.7 220 9.3
1 Detailed composition described in another paper (Bornstein and Lipstein, 1963). 2 Cal. (PE) per 100 gm./% protein ratio.
macromethod of Kjeldahl (A.O.A.C., 1960). Dietary fat was determined by the Soxhlet extraction procedure (A.O.A.C, 1960) and excreta fat by the hydrolysis procedure of Van de Kamer (1949). Metabolizable energy values per gram of diet were calculated as described by Sibbald et al. (1960), except for collecting total excreta rather than representative samples marked by chromic oxide. Trial 3. The objectives of this trial were: a) to determine the effect of heating ASS on its chemical composition; b) to follow subsequent changes in composition after the heated ASS samples were incorporated into the diet and latter stored under various conditions; and c) to investigate the biological effects of these latter feeds in chicks. The heating of ASS was carried out as in the previous trials, the temperature ranging from 115° to 140°C. Two samples of ASS, heated for 3 and IS days, respectively, as well as an unheated sample, were added to the diet (Table 1). The three feeds were stored for 70 days under the following three conditions: a) at 3 ° C ; b) at room temperature in open cans, frequently mixed and exposed to sunlight most of the days; and c) at 34°C. The three diets stored at room temperature
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battery-brooders equipped with raised wire floors and thermostatically controlled electric heating units. They were fed a commercial starter ration for one week, at which time they were individually weighed; about 60% of them, belonging to the middle of the weight distribution, were divided into 4 lots of 15 chicks each on the basis of body weight, equalizing both mean weight and weight distribution among the lots. The diet described in Table 1 was fed ad libitum during a 4-week experimental period, the fat supplement being either an unheated, or one of the above 3 heated samples of ASS. Individual weights and group feed consumption were recorded weekly. Trial 2. The purpose of this trial was to compare the effects of heating ASS and CSO on the metabolizable energy of the diets, the performance of the chicks and their apparent protein and fat retention. The heating procedure was similar to that used in Trial 1, except that the heating period lasted only 7 days. The temperature ranged from 130° to 150°C. The composition of the basal diet used in this trial is presented in Table 1. The experimental design was a 2 X 2 factorial, with type of oil and heat treatment as the variables. Leghorn cockerels were raised and distributed into 16 lots as described for the first trial. Each of the 4 diets was fed to 4 replicates of 10 chicks each during a 4-week period, beginning at one week of age. Beginning with the 12th day of the experiment, feed consumption was measured and droppings were collected daily from each group of chicks during four consecutive days. The droppings were dried at 70°C. in a forced-draft oven for 24 hours, after which they were ground and stored at room temperature in closed glass containers. Feed and excreta samples were analyzed for gross energy, using a Parr oxygen bomb calorimeter, and for nitrogen by the
1482
B . LlPSTEIN, P . BUDOWSKI AND S. BORNSTEIN
ol f
I
I »
I
1 20 t
i t
L_ t
30
Days of heating
FIG. 1. Chemical changes in acidulated soybean oil soapstock during heating at 90-135°C. in open pans (Trial 1). Carbonyl value (0); Oxy fatty acids (A); and Iodine value (•). Arrows indicate sampling for chick experiments.
fats of the above chicks. Liver vitamin A was assayed according to Ames et al. (1954)). Results were analyzed by the analysis of variance (Snedecor, 1956) and treatment means were separated according to Duncan's (1955) multiple range test. All interpretations of the data are based on the 1 percent level of probability. RESULTS
Trial 1. Some of the chemical changes occurring in heated ASS are presented graphically in Fig. 1. While the iodine value of the oil dropped during heating, the oxy-fatty acid level and carbonyl values rose. These changes were slow during the first two weeks and became increasingly more rapid thereafter. This acceleration was probably due to the rise in the temperature, although both the carbonyl value and oxyfatty acids levels seemed to increase even before the stepped up heating. Samples were selected for feeding tests when the iodine value had dropped by 9, 11 and 23 units. Table 2 summarizes the performance of growing chicks receiving four samples of ASS. For unknown reasons' chicks fed the moderately oxidized
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were used for a chick experiment after 42 days of storage, at which time a fourth diet was prepared for this purpose from the same ingredients and with an unheated sample of ASS, which had been kept in the meanwhile in cold storage. One-day-old male crossbred (Cornish X White Rock) chicks were randomly divided into 4 experimental groups of 14 chicks each, for which the experimental diets were randomly assigned. Feed and water were supplied ad libitum during the S-week experimental period. Individual body weights were recorded weekly, and feed consumption on a group basis was determined at 3, 4 and 5 weeks of age. Metabolizable energy values and apparent protein and fat retentions were determined similarly to the previous trial, beginning with the 28th day. Ten chicks per group (those closest to the average weight) were killed at 35 days of age. Adipose tissue and livers were removed, placed in plastic containers and frozen. Soapstocks and feed lipids (ether extracts) were analyzed for iodine and peroxide values and total and oxy-fatty acids (all according to A.O.C.S., 1961). Total carotenoids were measured by passing an ether solution of the lipid through a column of alumina containing 5% water, and measuring the absorbency of the eluate in a Bausch and Lomb "Spectronic" colorimeter at 450mpi. (3-carotene was used as a reference standard. This rapid method gave similar results to one in which the unsaponifiable matter was chromatographed on magnesium oxide and eluted with a 1:1 mixture of petroleum ether and ethanol. Fatty acid composition of feed lipids was determined by GL chromatography of the methyl esters, using a column of diethylene-glycol adipate polyester at a temperature of 210°C. Peak areas were measured by triangulation. Fatty acid composition was also determined for the depot and liver
1483
NUTRITIVE VALUE OF SOAPSTOCK
TABLE 2.—Performance of chicks receiving diets containing 10% fresh or heated acidulated soybean soapstock from 1 to 5 weeks of age (15 chicks per diet; Trial 1) Days of heating
Drop in IV 1
Body weight (g-)
Feed intake (g-)
Feed/gain ratio
0 22 14 31
0 9 11 23
453 408 435 440
735 687 737 754
1.62 1.68 1.69 1.71
1
IV=iodine value.
crude oil to be significantly higher than that of the heated soapstock. Encephalomalacia (diagnosed on the basis of pathognomonic symptoms and macroscopic and histological lesions) was observed among the chicks fed the diet containing oxidized soapstock. Apparent protein retention and growth rate remained unaffected by dietary treatments. Trial 3. This trial involved fresh, mildly oxidized and strongly oxidized ASS. Chemical characteristics of these samples are given in Table 4. The fall in iodine value, due to heating, is accompanied by a rise in oxy-fatty acids, which, in turn, corresponds exactly to the decrease in total (non-oxy) fatty acids. Carotenoids are largely destroyed by the mild treatment and disappear completely upon further oxidation of
TABLE 3.—Characteristics of the oils used as 8% of the diets, and the performance of chicks from 1 to 5 weeks of age (4 replicates of 10 chicks each; Trial 2) Oil supplements 3
CSO CSO.heated ASS3 ASS,heated S.E. 1
IV 1
124.6 118.0 124.9 118.6
OFA, 1
% 1.08 3.05 4.12 6.21
Retention
ME cal./g. diet
Body weight (g-)
Feed intake (g.)
Feed/gain ratio
3.24 a" 3.26 a 3.19 ab 3.13 b 0.02
655 633 668 605 —
942 932 982 990 —
1.47 a 1.47 a 1.47 a 1.60 b 0.01
Protein
Mortality 2
Fat
%
%
62.7 62.0 62.2 60.8 —
89.4 86.7 86.6 85.3 0.78
a ab ab b
0/40 0/40 0/40 3/40
IV=iodine value; OFA=oxy-fatty acids. No. of dead chicks/total no. of chicks. Death was due to encephalomalacia and occurred at the age of three weeks. 3 CSO = crude soybean oil; ASS = acidulated soybean-oil soapstock. 4 Within any one column, means which are not followed by the same letter are significantly (P<0.01) different from each other. 2
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sample (drop in iodine value of 9 units) ate less feed and hence gained less weight than the other groups. However, these differences were not significant. The feed/ gain ratio increased with the length of the heating period, indicating decreased feed utilization. There was no mortality in this trial. Trial 2. In the second experiment the oils were heated for only 7 days, hence the iodine values decreased by only 6.6 and 6.3 units in the cases of CSO and ASS, respectively, and the oxy-fatty acids increased by about 2 percentage units in both cases (Table 3). The heated soapstock lowered feed conversion significantly compared to unheated soapstock and both of the crude oils. These results parallel the metabolizable energy contents of the diets, in which respect the heated soapstock differed significantly from both crude oils, but not from the unheated soapstock. The energy data, in turn, can be partially explained on the basis of apparent fat retention, which was slightly (but not significantly) decreased by the heating of either oil. This together with the slightly lower energy content of unheated ASS as compared to unheated CSO, caused the metabolizable energy level of unheated
1484
120
s
B.
1
!
LlPSTEIN,
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acidulated soybean soapstock used in trial 3
^"\,
^,
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soap- Pd£ ' °sd . stock ay
PVi /iequ./g.
OFAi,
TFA,i
IVi
%
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Hi
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TABLE 4.—Characteristics of fresh and heated
1
fc.^
110
AND S .
123.9 118.3 87.3
1.4 1.9 0.8
4.5 7.6 24.8
91.8 87.9 70.5
156.8 17.2 0.0
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1 IV=iodine values; PV=peroxide value; OFA =oxy-fatty acids; TFA = total fatty acids (excluding unsaponifiable matter)
1
1
0 3 15
1
80 020 Days of storage
40
60
80
FIG. 2. Effect of storage on the iodine value and carotenoid content of ether extractable feed lipids. The feeds contained 8% ASS (Table 4) either unheated ( ), slightly heated ( ), or very heated ( ). Storage was at 3° (A), at room temperature (•) or at 34° (0).
the soapstock. Peroxides did not accumulate under the conditions of the heating. When the above oil samples were incorporated into the diets, and stored for 70 days under three different conditions (at 3°C, room temperature and 34°C), the changes were most pronounced in the iodine value and carotenoid content, which decreased during storage (see Fig. 2). The drop in iodine value for the unheated soapstock A was slowest at the highest storage temperature, and the reverse was true for the strongly heated soapstock C. For sample B, no effect of storage temperature was indicated. Destruction of carotenoids proceeded more rapidly the higher the storage temperature. The carotenoid contents of the diets containing heated soapstocks B and C were too low to be measured and only the data for the unheated soapstock are given in Fig. 2. Peroxides increased only slightly for all samples, the highest value, 8.6 [i.equ./g., being reached by the feed containing mildly oxidized oil and stored at 34°C. for 70 days.
The iodine value and fatty acid composition of the lipids extracted from the four diets used in the chick experiment are presented in Table 5. Storing the feed containing the unheated ASS slightly reduced its iodine value and caused only very slight changes in fatty acid composition. On the other hand, the fats extracted from the diets to which had been added the heated samples of ASS (especially the stronger heated sample C) exhibited lower linoleic and linolenic acids levels and higher saturated and monounsaturated fatty acid contents, apparently due to the heating rather than to the storage. Generally, the differences in iodine value of the feed lipids paralleled the drop in iodine value induced in the soapstock by the heat treatments (see Table 4). Performance of the chicks, metabolizable energy values of the experimental diets and the retention of protein and fat are presented in Table 6. Storage of the diet containing fresh ASS did not appear to affect TABLE 5.—Iodine value and fatty acid composition of
the lipids extracted from the experimental diets used in trial 3 Experimental diet 1
IV!
A(0) A (42) B (42) C(42)
121.5 111.0 104.0 78.6
% fatty acid composition 3 16:0 12.3 13.5 15.3 19.3
16:1
18:0
18:1
18:2
18:3
1.1 0.8 1.3 2.2
4.8 5.0 4.1 6.1
2 2 . 1 51.0 19.6 52.8 23.4 50.1 26.0 43.9
8.8 8.3 5.8 2.5
1 The diet is designated by the soapstock it contains (A, B and C—see Table 4) and the number of days (in parentheses) it has 2 been stored at room temperature. IV=iodine value. 3 Chain length:no. of double bonds.
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_
A B C
1485
NUTRITIVE VALUE OF SOAPSTOCK
TABLE 6.—Performance of chicks receiving the experimental diets of trial 3 until the age of 5 weeks (14 chicks per diet)
g-
Feed/gain ratio
ME cal./g. feed
Protein
%
%
776 765 763 793
1,357 1,294 1,503 1,583
1.78 1.69 1.97 1.97
3.12 3.02 2.97 2.77
46.3 47.8 47.6 44.5
83.6 85.5 79.8 73.0
1 2
^
Fat
Mortality 2
0/14 0/14 1/14 3/14
See footnote 1 to Table 5. From encephalomalacia; no. of dead chicks/total no. of chicks.
the performance of the chicks. Feed intake was decidedly higher in the groups receiving diets containing heated ASS, whereas weight gains were similar for all groups. This resulted in an unfavorable feed/gain ratio for the diets containing the heated soapstock. Apparent protein retention was not affected by the dietary treatments, but fat retention was markedly lower in the chicks receiving the diet with the strongly oxidized fat. This low retention also caused the latter diet to have the lowest metabolizable energy content. Encephalomalacia developed in a few chicks receiving the diets containing the oxidized soapstocks. Table 7 describes the fatty acid composition of the abdominal fat of chicks receiving the experimental diets. The changes in fatty acid composition reflect those observed in the dietary lipids (see Table 5): there is a decrease in polyunsaturated fatty acids (PUFA) in the fat of chicks fed the diets containing the oxidized oils, as compared to the levels observed in the other two groups. This is accompanied by a corTABLE 7.—Fatty acid composition of the abdominal fat of 5-week old chicks raised on the experimental diets {trial 3) % fatty acid composition2
Experimental diet'
14:0
16:0
16:1
18:0
18:1
18:2
18:3
A(0) A (42) B(42) C(42)
0.7 0.5 0.5 0.6
20.8 21.8 20.6 24.3
1.3 2.0 2.8 2.5
2.9 2.5 4.0 2.9
30.1 30.4 31.6 38.3
39.6 38.3 36.2 28.4
4.8 4.4 4.2 2.4
i.« See footnotes 1 and 3, Table 5.
responding increase in oleic and palmitic acids. These changes become pronounced only in the case of the diet containing the strongly oxidized ASS. In the liver lipids, the picture is similar, except that the fatty acid composition is less sensitive to dietary manipulation (Table 8). In fact, arachidonic acid remains the same for all groups, while the changes in oleic, linoleic and linolenic acids are relatively slight, although they again parallel the dietary variations. The vitamin A values, also presented in Table 8, show that using heated ASS had an appreciable effect on the vitamin reserves in the liver, whereas storage of the feed was apparently without effect. DISCUSSION
The data obtained in these experiments indicate that heat-induced autoxidation of ASS may result in decreased feed utilization (Tables 1, 3 and 6) and cause mortality in growing chicks fed diets containing such ASS (Tables 3 and 6). This poorer performance of chicks fed heated soapstock seems to be due to the lower metabolizable energy content of the diets containing this type of ASS, thus resulting in higher feed intake. It is interesting to note that growth rate and protein retention were apparently unaffected by dietary heated ASS (Tables 3 and 6), although a slight trend toward lower weight gains is apparent in trial
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A(0) A (42) B (42) C(42)
Retention
Feed intake, g-
Experimental
1486
B . LlPSTEIN, P . BUDOWSKI AND S. BOENSTEIN
TABLE 8.-—Fatty
acid composition and vitamin A values of the livers of 5-week-old chicks raised on the experimental diets (trial 3) % fatty acid1 composition2
Vitamin A
16:0
16:1
18:0
18:1
18:2
18:3
20:4
Unknown3
I.U./ liver
Storage 4 %
A(0) A (42) B(42) C(42)
18.4 17.6 22.1 22.0
1.0 0.6 1.3 0.9
22.8 21.7 21.7 21.9
12.9 14.1 15.1 16.1
26.8 26.9 24.6 23.3
2.1 1.7 1.3 0.8
12.9 12.9 11.3 12.2
3.1 4.5 2.6 2.8
5,178 4,959 4,115 4,189
47.7 47.8 34.2 33.1
1,2 3 4
See footnotes 1 and 3, Table 5. Two minor peaks were eluted between linolenic and arachidonic acids. Liver vitamin A expressed as percentage of total intake.
2 (Table 3). The metabolizable energy, in turn, seems to be dependent on the retention of fat, which was lower in the case of oxidized soapstock (Tables 3 and 6). The lowered fat retention may be caused by the presence of increased levels of oxy-fatty acids (Table 3 and 4) and possibly by polymeric material which remains mostly unabsorbed (Kaunitz et al., 1956). Before that Lassen et al. (1949) had shown a decrease in digestibility of a highly unsaturated oil with an increase in polymerization, the latter being expressed as percentage drop in the iodine value. In trials 2 and 3, feeding oxidized ASS produced encephalomalacia in some of the chicks. It had been shown previously (Mokadi and Budowski, 1963), that safflowerseed oil, oxidized to a drop in iodine value of 13 units and fed at 20% level in a commercial-type diet, caused encephalomalacia in all the chicks to which it was fed. Oxidized ASS, which is more saturated than safflowerseed oil and which was fed as 8% of the diet, still produced some encephalomalacia in the present trials. On the other hand, no mortality was found by Lea et al. (1964) in rats fed 5% oxidized beef fat which is poor in PUFA. A comparison between ASS and CSO in trial 2 (Table 3) shows that the latter, when oxidized under the same conditions and to the same extent as the ASS, caused
no detrimental effects at all in the chicks. ASS initially contains more oxy-fatty acids than crude soybean oil, so that upon aerobic heating, the soapstock ends up with a higher oxy-fatty content than the oil, the drop in iodine value and increase in oxy-fatty acids due to heating being similar for both materials. It has been shown that certain types of long-chain keto acids have pronounced pro-oxidant activity (Bhalerao et al., 1962) and may cause encephalomalacia in chicks (Kokatnur et al., 1960). It is possible that, upon heating, such compounds form in ASS from oxyfatty acids present initially, or that such a process is favored by the high free fatty acid content of ASS. When the feed containing 8% unheated or heated ASS was stored at room temperature for 42 days, no additional peroxides or oxy-fatty acids accumulated in the feed lipids. In the other hand the iodine value dropped slightly (Table 5). In the case of the unheated soapstock, storage produced no detrimental effect on chick performance (Table 6). Therefore, it can be assumed that the detrimental effects of diets B (42) and especially C (42) on chick performance (Table 6) are due to the use of heated oils, and not due to the storage of the feed. Moreover, the decrease observed in carotenoids upon storage (Fig. 2) may cause lesser pigmentation of skin and
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Experimental diet 1
1487
NUTRITIVE VALUE OF SOAPSTOCK
dized soybean oil, and by Lea et al. (1964), who fed autoxidized beef tallow. As shown in Table 8, no detrimental effect in this respect was obtained by the storage of the feed containing 8% fresh ASS. SUMMARY
In three trials the chemical changes occurring during the heating of acidulated soybean soapstock (ASS), and in one case crude soybean oil (CSO), were investigated. These changes were related to the performance of chicks the dietary metabolizable energy level and the apparent retention of fat and protein. In one trial the chemical changes occurring in stored diets containing heated ASS were followed, and the effect of these feeds on chick performance, on the fatty acid composition of liver and depot fat, and on liver vitamin A were determined. Heating ASS decreased the iodine value, increased the carbonyl value, increased the concentration of oxy-fatty acids and destroyed most of the carotenoids. These heatinduced autoxidative changes may result in a decreased feed utilization by growing chicks receiving a commercial-type diet supplemented with the oxidized oils. Heating of ASS also lowered the metabolizable energy content of the diets. The lower metabolizable energy resulted from decreased fat retention, which was definitely lower in the case of oxidized soapstock. Protein retention, on the other hand, was not affected by autoxidation of ASS, nor was growth rate (or only slightly so). Feeding oxidized ASS also produced encephalomalacia in some of the chicks. The changes in depot fat composition reflect those found in the feed lipids, i.e., there is a decrease in polyunsaturated fatty acids with a corresponding increase in saturated and monounsaturated fatty acids. The fatty acid composition of the liver lip-
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yolks. The failure of lipid oxidation products to accumulate during storage of feed has recently been emphasized by Lea et al. (1964). The effect of autoxidized ASS on the fatty acid composition of depot and liver fat, as shown in Tables 7 and 8, can be explained by the changes induced in the ASS as the result of heating. An additional reason for the lowered PUFA content of chicks receiving heated soapstocks may be the interference with absorption and deposition of PUFA caused by fat oxidation products (Lea et al., 1964). Perkins et al. (1961) have shown that dietary hydroxy acids cause an increase in carcass monoenoic acids. By itself, such an alteration in fatty acid composition of carcass fat is not detrimental ; in fact, it may increase the stability of this fat (Lea et al., 1964), provided sufficient dietary a-tocopherol is available to the chick. The fatty acid composition of the liver lipids is affected in a similar fashion, except for arachidonic acid, which remains constant whatever the state of autoxidation of the dietary ASS. This acid is formed from linoleic acid in vivo, and it is, therefore, surprising that no drop in arachidonic acid is observed upon feeding autoxidized ASS. The explanation may possibly be found in the recent work by Bernhard et al. (1964), in which it was shown that the extent of conversion of linoleic to arachidonic acid in the livers of rats is inversely related to the amount of a-tocopherol supplied. The lowered tocopherol level of autoxidized ASS would, therefore, cause increased conversion, thus counteracting the effect from the decrease in linoleic acid. Vitamin A storage was depressed as the result of feeding autoxidized ASS to chicks. A similar effect was found in rats by Reporter and Harris (1961), who fed autoxi-
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B . LlPSTEIN, P . BUDOWSKI AND S. BORNSTEIN
ACKNOWLEDGMENTS
The authors are indebted to M. Mosheh and C. Genige for technical assistance during the courst of these studies. REFERENCES Ames, S. R., H. A. Risley and P. L. Harris, 1954. Simplified procedure for extraction and determination of vitamin A in liver. Anal. Chem. 26: 1378-1381. American Oil Chemists Society, 1961. Official and Tentative Methods of Analysis. 3rd Ed. Rev. to 1961, Chicago, 111. Association of Official Agricultural Chemists, 1960. Methods of Analysis, 9th Ed. Wash., D.C. Bernhard, K., S. Leisinger and W. Pedersen, 1964. Vitamin E und Arachidonsaure-Bildung in der Leber. Helv. Chim. Acta, 46: 1767-1772. Bhalerao, V. R., M. G. Kokatnur and F. A. Kummerow, 1962. The effect of 12, oxo-cis, 9-octadecenoic acid on the autooxidation of corn oil. J. Am. Oil. Chem. Soc. 39: 28-30. Bornstein, S., and B. Lipstein, 1961. By-products of soybean oil as fat supplements in practical broiler rations. World's Poultry Sci. J. 17: 167178. Bornstein, S., and B. Lipstein, 1963. Some unusual waste vegetable oils as fat supplements in practical broiler rations. World's Poultry Sci. J. 19: 172-184.
Budowski, P., J. Ascarelli, J. Gross, J. Nir and A. Bondi, 1964. Vitamin A activity of acidulated soybean soapstock in chicks. J. Am. Oil. Chem. Soc. 4 1 : 441-445. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Henick, A. S., M. F. Benca and J. H. Mitchell, 1954. Estimating carbonyl compounds in rancid fats and foods. J. Am. Oil. Chem. Soc. 34: 88. Kaunitz, H., C. A. Slanetz, R. E. Johnson, H. B. Knight, D. H. Saunders and D. Swern, 1956. Biological effects of the polymeric residues isolated from autoxidized fats. J. Am. Oil. Chem. Soc. 33: 630-634. Kokatnur, M. G., S. Okui, F. A. Kummerow and H. M. Scott, 1960. Effect of long-chain ketoacids on encephalomalacia in chicks. Proc. Soc. Expt. Biol. Med. 104: 170-171. Lassen, S., E. K. Bacon and H. J. Dunn, 1949. The digestibility of polymerized oils. Arch. Biochem. 23: 1-7. Lea, C. H., L. T. Parr, J. L. L'Estrange and K. T. Carpenter, 1964. Nutritional effects of autoxidized fats in animal diets. Beef fat in the diet of rats. Brit. J. Nutr. 18: 369-384. Mokadi, S., and P. Budowski, 1963. Relationship between vitamin E and encephalomalacia in chicks. Brit. J. Nutr. 17: 347-355. Perkins, E. G., J. G. Enders and F. A. Kummerow, 1961. Effect of ingested thermally oxidized corn oil on fat composition in the rat. Proc. Soc. Expt. Biol. Med. 106: 370. Reporter, M. C. and R. S. Harris, 1961. Effects of oxidized soybean oil on the vitamin A nutrition of the rat. J. Am. Oil. Chem. Soc. 38: 4757. Schultz, H. W., E. A. Day and R. O. Sinnhuber, editors, 1962. Lipids and Their Oxidation. The Avi Publ. Co., Westport, Conn. Sibbald, I. R., W. F. Pepper and S. J. Slinger, 1962. A comparison of feed grade tallow and acidulated soapstocks in practical chick starter rations. Poultry Sci. 4 1 : 120-124. Sibbald, I. R., J. D. Summers and S. J. Slinger, 1960. Factors affecting the metabolizable energy content of poultry feeds. Poultry Sci. 39: 544-556. Snedecor, G. W., 1956. Statistical Methods, Sth Ed., Iowa State College Press, Ames, Iowa. Van De Kamer, 1949. Rapid method for the determination of fat in feces. J. Biol. Chem. 177: 347-355.
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ids is affected in a similar manner, but to a lesser extent. Moreover, the arachidonic acid content of liver lipids was not affected by the state of oxidation of the diet fat. Vitamin A storage was decreased as the result of feeding autoxidized ASS to chicks. Feeding chicks a diet containing oxidized CSO had no detrimental effect, unlike a sample of ASS which had been oxidized to the same extent. Whereas thermally induced autoxidation of ASS decreased its nutritional value for growing chicks, storage of feed containing either unheated or heated 8% ASS caused no detrimental effects, other than a lowering of the carotenoids found in the soapstock.