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A Comparison of Four Methods for Determining Available Lysine in Animal Protein Meals1,2
A Comparison of Four Methods for Determining Available Lysine in Animal Protein Meals1'2 J. P. NORDHEIM and C. N. COON Department of Animal Sciences, Washington State University, Pullman, Washington 99164 (Received for publication July 1, 1983)
INTRODUCTION Because lysine is often the first or second limiting amino acid in poultry feeds, the amount of dietary lysine biologically available is of extreme importance. The main factor regulating the total lysine content of animal protein meals is the composition of raw materials rendered (Wilder, 1972). Skurray and Herbert (1974) reported meat meals prepared from sheep and calves heads contained 5.9 to 6.4 g lysine/16 g of nitrogen compared to 7.2 to 8.8 g lysine/16 g of nitrogen in meals composed primarily of sheep and calves stomachs and intestines. The researchers reported no changes in total lysine following three different batch rendering process conditions; however, the nutritional qualities of the meals were improved as demonstrated with chick feeding studies. Heat processing of many protein feeds
1 Scientific Paper No. 6587. College of Agriculture, Agricultural Research Center, Washington State University, Pullman, WA 99164. Project 0308. 2 Research supported in part by a grant from the Fats and Protein Research Foundation, 2250 East Devon Avenue, Des Plaines, IL 60018. 3 Durrum D-500, Durrum, 3950 Fabian Way, Palo Alto, CA 94303.
is required, because the heat eliminates microorganisms such as Salmonella, nutritional inhibitors, and improves overall digestibility by altering protein structure to allow enzymatic cleavage of peptide bonds by animals. The epsilon amino group of lysine may chemically react with carbohydrates or oxidized lipids (Carpenter and Booth, 1973) and free carboxyl groups of glutamic and aspartic acid (Bjarnason and Carpenter, 1970) during heat processing of protein feedstuffs, thus decreasing the digestion of processed protein and the biological availability of lysine. Animal proteins such as meat and bone meal are protected during heat processing with large proportions of animal fat, which keep the protein from becoming dry and scorched (Wilder, 1973). Meat and bone meals contain very small quantities of carbohydrate and a higher proportion of saturated lipids which would have less tendency to oxidize than unsaturated lipids. Raw feather and hair are almost pure keratin protein that is second limiting in lysine for poultry (Moran and Summers, 1967; Moran et al, 1967; Luong and Payne, 1977) and relatively nondigestible (Moran et al., 1966). Hydrolyzed feather and hair meals are cooked by high pressure steam heat to allow optimum utilization by poultry (Moran and Summers,
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ABSTRACT The available lysine in 7 fish, 10 meat and bone, and 6 keratin meals was determined with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and l-fluoro-2,4-dinitrobenzene (FDNB) chemical procedures, chick bioassay (CBA), and digestible lysine (DL) technique. The percent available lysine and total lysine (TL) in test meals is reported as grams of lysine per 100 g dry weight. The available lysine means determined by the TNBS, FDNB, CBA, and DL procedures were 5.28, 4.34, 4.50, and 4.36% for fish meals; 2.48, 2.53, 2.71, and 2.61% for meat and bone meals; and 2.33, 2.74, 1.50 and 1.92% for keratin meals, respectively. The correlation coefficients (r) for CBA versus TNBS lysine, FDNB lysine, DL, and TL are .90, .83, .97, and .93, respectively, for all animal proteins tested. The available lysine determined by the DL procedure correlated best with available lysine determined with the CBA for each type of animal protein meal and for all test meals collectively. The DL technique is a logical alternative to the CBA, FDNB, and TNBS procedure for determining available lysine in animal proteins because the DL procedure is simpler, less expensive, and quicker than the CBA procedure and more accurate than the colorimetric chemical procedures. (Key words: lysine availability, chick bioassay, l-fluoro-2, 4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), meat and bone meal, fish meal, keratin meal) 1984 Poultry Science 63:1040-1051
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
MATERIALS AND METHODS
General. Analyses for dry matter and nitrogen were conducted on triplicate samples according to the methods of the Association of Analytical Chemists (1975). Total lysine (TL), available lysine, and crude protein (CP) values are expressed on a dry weight basis. Lysine availability is defined as the proportion of TL that potentially can be absorbed and utilized by the animal. Available lysine is the quantitative amount of lysine (g lysine/100 g sample) in a sample that can be used by an animal for its metabolic functions. The terms availability and available are linked together as in the equation: Available lysine = lysine availability X total lysine The mean values for available lysine and lysine availability for the different animal by-products were statistically compared by analysis of variances and Duncan's new multiple range test (Steel and Torrie, 1960). Digestible Lysine. Twenty Single Comb White Leghorn roosters, weighing 1.5 to 2.0 kg, were randomly assigned to individual wire cages containing automatic water cups and separate fecal collection trays. Within experiment the 20 roosters were divided into groups of 6 for each
by-product material with the remaining 2 roosters being starch-fed controls. The roosters were fasted for 36 hr and then were force-fed 30 g of each test material to determine true DL as described by Sibbald (1979). Excreta from test roosters were collected for 36 hr following the force-feeding of test samples. Endogenous lysine losses were determined by collecting excreta from control roosters fed 30 g of corn starch. Excreta from roosters fed the test material and control roosters were individually weighed and lyophilized. Inidividual excreta samples were arbitrarily paired within each test group of 6 roosters. Triplicate samples of lyophilized excreta from by-product-fed and starch-fed control roosters and duplicate feed samples were hydrolyzed with 6 N HC1 for 24 hr and analyzed for amino acid content by AAA Laboratory of Mercer Island, WA. The samples were analyzed with an automatic amino acid analyzer following the manufacturer's procedures. 3 The lysine values are based on the molecular weight of lysine (146.2 g) instead of the residue molecular weight (128.2 g). The residue molecular weight is the molecular weight of the amino acid in the protein prior to hydrolysis and the addition of water. Chick Bioassay. Two-thousand-two-hundred and twenty male Hubbard broiler chicks, one day of age, were placed in electrically-heated brooders with raised wire floors and automatic water cups and ad libitum fed a low lysine basal diet (Combs et al, 1968; Coon et al, 1978) containing .65% available lysine for a 10-day feeding period. Chicks were randomly selected on the 10th day and weighed in replicate groups of 10 chicks each. The total weight of each replicate group was adjusted for uniformity by chick substitution. Chicks were then provided with water and experimental diets ad libitum for a 14-day feeding period. Reference diets (Coon et al, 1978) containing .49, .57, .65, .73, and .79% available lysine were each fed to four replicate groups. Each test diet, containing 5% of the animal by-product meals, was fed to four replicate groups. The diets were made isocaloric and isonitrogenous by adding corn oil and glutamic acid, respectively. Calcium, phosphorus, and amino acids other than lysine were adjusted to National Research Council (NRC, 1977) levels or were in excess. The calculated lysine content in all test diets containing the 5% animal protein meal was adjusted with crystalline lysine to contain approximately .65% available lysine to assure
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1967). Keratin proteins that have been undercooked have lower protein quality, because the nonhydrolyzed disulfide bonds cross-linking the protein make it unavailable for maximum enzymatic digestion by animals. Wilder (1972) reported overcooking feather meal produces a loss of cystine but not other amino acids. A fast, simple, inexpensive, and accurate method for determining available lysine would enable manufacturers of animal protein meals to control quality and to produce protein meals with consistent nutritional quality. The chick bioassay is a standard technique for determining biologically available lysine (Combs et al, 1968; Coon et al, 1978), but the method is slow and costly. Alternative chemical and short-term digestibility methods may produce numerically different available lysine values that are still highly indicative of protein quality. The objective of the present study was to compare digestible lysine (DL), chick bioassay (CBA), l-fluoro-2,4-dinitrobenzene (FDNB), and 2,4,6-trinitrobenzene sulfonic acid (TNBS) methods for determining available lysine in animal protein meals.
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NORHEIM AND COON
l-Fluoro-2,4-Dinitrobenzene Available Lysine. The FDNB method used for chemically determining available lysine was essentially that of Carpenter (1960) as modified by Booth (1971). Triplicate samples of .5 g animal by-product meal were weighed into 125-ml glass-stoppered, flat-bottomed flasks and buffered with 10 ml of 8% (w/v) sodium bicarbonate. The samples were mixed with 15 ml of a 2.5% FDNB-ethanol (v/v) solution for 2 hr on a mechanical shaker. The protein samples were digested by adding 30 ml of 8.1 N HC1 and refluxing for 16 hr. The refluxed samples were filtered, and then digestion flask and residue were washed thoroughly with hot water until the filtrate volume was 500 ml. Booth (1971) modified the Carpenter (1960) procedure by using hot water washings instead of water at room temperature and the filtrate volume was increased to 250 ml instead of 200 ml for a more complete extraction of the e-DNP-lysine. The characteristic yellow color was still present in the washings of the animal protein samples when the filtrate volume was 250 ml; therefore, the total filtrate volume was increased to 500 ml for a complete extraction of e-DNP-lysine. The samples were extracted twice with ethyl ether to remove interfering a-DNP-amino acids and dinitrophenol, and the absorbance was determined at 435 nm. A correction factor of
1.08 for meat and bone meal and 1.05 for fish and keratin meals was used to account for e-DNP-lysine loss during the 16-hr digestion of protein samples as suggested by Booth (1971). 2,4,6-Trinitrobenzene Sulfonic Acid Available Lysine. Available lysine was determined by the TNBS method of Kakade and Liener (1969) as modified by Ousterhout and Wood (1970). Ousterhout and Wood (1970) used 100 mg of animal by-product meal for each sample to increase precision compared to 1 mg of purified protein in the original assay of Kakade and Liener (1969). Lysine-TNBS derivatives are formed by shaking test samples with 10 ml 4% NaHC0 3 (pH 8.5) for 30 min and then adding 10 ml of 1% TNBS solution and continue to shake for 2 hr more. The test proteins containing the lysine-TNBS derivatives are hydrolyzed by adding 30 ml of concentrated HCl and autoclaving for 1 hr at 115 C (15 psi). The 125-ml flasks containing the animal protein samples were covered with inverted beakers and set in a tray of water during autoclaving in order to minimize destruction of the autoclave by the hydrochloric acid. Triplicate 1-ml aliquots were extracted twice with 5 ml of ethyl ether, which was removed with a suction line. Residual ether was removed by use of a hot water bath. The intensity of the yellow color was determined by measuring absorbance at 346 nm. RESULTS AND DISCUSSION
Available Lysine in Fish Meals. Available lysine in Alaska herring meal was higher than in other fish meals for all of the methods studied (Table 1). The total lysine in Alaska herring (6.77%) was higher than other fish meal samples and is comparable to the NRC, (1977) value of 6.13%. Alaska herring meal containing 74.0% CP had the highest protein level compared to other fish meals and is consistent with previous reports of 72 and 73% (Tarr and Biely, 1973). Peruvian anchovy, California anchovy, and British Columbia scrap were the next highest in available lysine. California and Peruvian anchovy meals with 71 and 71.4% protein contained 5.61 and 6.01% total lysine, respectively. The four available lysine techniques give lower lysine values for Peruvian anchovy and California anchovy (4.10 to 6.38 and 5.08 to 5.98%, respectively) (Table 1) than the range of 5.65 to 7.05% reported by Bacigalupo (1973). Peruvian anchovy fish meal may be lower in available lysine than California anchovy meal
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weight gains within the range of weight gains from chicks fed reference diets. The chicks were weighed in replicate groups on the 24th day and feed consumption recorded. The biologically available lysine in each animal by-product test diet was determined by comparing the weight gain and feed consumption of chicks receiving reference diets as reported by Combs et al. (1968). Values reported were averaged from duplicate experiments with eight total replicates. The biologically available lysine in corn gluten meal used in the test and reference diets was previously determined by feeding chicks a crystalline amino acid diet containing two levels of corn gluten meal (5 and 14.2%) substituting for crystalline lysine. The biologically available lysine in corn gluten meal was verified by feeding increasing increments of corn gluten meal and decreasing levels of crystalline lysine. Each lysine-limiting ration was calculated as providing equal amounts of total available lysine. The differences in weight gain would indicate a disparity between the actual lysine value in the corn gluten meal and the value used to calculate the rations.
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
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Available Lysine in Meat and Bone Meals. The four methods of determining available lysine in meat and bone meals did not consistently rank the samples the same (Table 2). The available lysine in meat and bone meal No. 3 was higher (P<.05) than other meat and bone meals as determined by the CBA and TNBS lysine procedures, whereas meat meal No. 10 contained the highest.FDNB available lysine. Meat and bone meal No. 3 and meat meal No. 10 have been previously determined to be superior to seven other meat and bone meals in six protein quality assays (Johnston and Coon, 1979). Meat meal No. 9 contained the largest amount of TL (3.47%) and DL (2.96%). The quantitative amount of available lysine for each meat and bone meal sample was different for
the four methods. Meat and bone meal No. 3 has a higher TNBS lysine value (3.20%) than TL (2.98%) (Table 2). The DL, FDNB lysine, and CBA lysine values for the meat and bone meal samples were consistently below the TL values (Table 2). The TL in meat and bone meal ranged from 2.47 to 3.19% with a mean of 2.92% TL. Meat meal No. 10 had 3.13% TL and meat meal No. 9 had 3.47% TL (Table 2). The NRC (1977) value for lysine in dry matter is 2.80% for meat and bone meals and 3.26% for meat meals. Average lysine availability values for all meat and bone meals were 83, 85, 87, and 91% (Table 5) as indicated by TNBS, FDNB, DL, and CBA techniques, respectively. The CBA technique did not show significantly different available lysine values in the meat and bone meal samples compared to other methods. The correlations between CBA and other available lysine methods are much lower for meat and bone meal samples than they were for the fish meals and keratin protein samples (Table 7). The low correlation for CBA lysine and available lysine as determined by DL, FDNB, and TNBS techniques may be due to the narrow range of TL in the meat and bone meals. The CBA method correlated best (P< .05) with the digestible lysine technique for meat and bone meal samples (r = .67). Available Lysine in Keratin Meals. Feather mean No. 2 contained the highest available lysine and TL among feather meals (Table 3) for all lysine analyses (P<.05). Two samples are higher (2.17 and 2.73%) and two samples have less or equal TL (1.65 and 1.75%) than 1.80 and 1.75% lysine values for feather meal reported by NRC (1977) and the National Renderers Association, Inc. (1975), respectively. Feather meal No. 4 had the lowest values for TNBS, FDNB, and CBA available lysine (1.42, 1.71, and 1.11%, respectively) compared to other feather meals. The protein quality of feather meal No. 4 has been previously determined to be lower than five other feather meal samples (Johnston and Coon, 1979). Hair meal No. 1 was higher in available lysine than hair meal No. 2, but No. 2 contains more TL. Both hair meals clearly contain more available lysine and TL than all feather meals except feather meal No. 2. The chemical assays show hair meals and feather meals to have high lysine availability, whereas the bioassay methods do not confirm the high availability of lysine for either hair or feather meals (Table 6). Feather meals have been reported by Moran et al.
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(Table 1), because Peruvian anchovy fish meal often undergoes more extensive heat processing to ensure a Salmonella-free meal (Lawrence R. Berg, 1975, personal communication). Total lysine content for anchovy samples is higher (5.61 to 6.01%) than the NRC (1977) value of 5.33%. In general, Seattle rock cod, shrimp meal, and tuna meal were lower in available lysine than other fish meal samples. Tuna meal was not significantly lower (P>.05) in available lysine than Peruvian anchovy and British Columbia scrap as determined by the CBA method. Correlations between the CBA and FDNB, TNBS, and DL techniques for determining available lysine in fish meals are high (r = .89, r = .80, and r = .97, respectively) (Table 7), although the quantitative amount of lysine in fish meals are different for the four methods. The TNBS lysine values for fish meals exceed the TL values of the meals (Table 2). Average lysine availability values of 104% (Tables 4 and 8) were based on TNBS available lysine divided by total lysine (TL) and multiplied by 100. The DL and FDNB technique produced the lowest average lysine availability values for the fish meals, with 86% of the total being available. The chick bioassay method showed an average availability of 90% for all fish meals. These data are similar to a report suggesting about 90% lysine availability for fish meals as determined by FDNB lysine procedure (Carpenter and Woodham, 1974). The lysine versus CP correlation (r = .76) for fish meals is high (P<.05) (Table 7). This is probably from the herring and anchovy fish meals containing large amounts of available lysine and CP and the remaining fish meals with lower protein containing less available lysine (Table 1).
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
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1.38 b 1.92 a 1.23b l.llc 1.86a 1.50a
CBA
+ ± ± ± ± ±
Crude protein (Kjeldahl) analysis, dry matter basis.
Total lysine from amino acid analyzer, dry matter basis.
Digestible lysine.
Chick bioassay.
l-Fluoro-2,4-dinitrobenzene.
2,4,6-Trinitrobenzene sulfonic acid.
Available lysine values represent mean ± SEM on a dry matter basis (g lysine/100 g dry matter).
' ' Means that do not share a common letter within a column are significantly different (P<.05).
1
a
Means
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2.23 b 3.15 a 2.43t> 1.71 c 3.66 a 3.23 a
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1.92 b 2.77 a 1.60 bc 1.42C 2.97a 2.75 a
1. 2. 3. 4. 5. 6.
Feather meal Feather meal Feather meal Feather meal Hair meal No. 1 Hair meal No. 2
FDNB
TNBS
.19 .16 .04 .07 .30 .25
D
FDNB,3 CBA, and DLs), total lysine, and crude p
Sample
TABLE 3.. Percent available lysine1 (TNBS,
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AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
FDNB methods (Table 8) are because the TNBS and FDNB reagents bind with compounds other than lysine. The lack of complete specificity of TNBS and FDNB methods shows up in the colorimetric analysis as a higher lysine value. Chemical assays for available lysine usually show higher lysine concentrations than do animal or microbiological assays (McBee and Marshall, 1978). Lysine may be bound in the protein in such a way that it is not capable of being utilized by the animal. Lysine that is not biologically available may be released by acid hydrolysis, thus causing erroneously high available lysine values when assayed by chemical methods (Woodham, 1969). All available lysine assays purport to measure lysine with unbound epsilon amino groups. The FDNB available lysine procedure has been reported to measure only lysine with a reactive epsilon amino group bound in a protein chain and not free lysine or lysine at the N-terminal protein end (Carpenter and Booth, 1973; Carpenter and Woodham, 1974). Carpenter and Booth (1973) suggest that TNBS reacts with Maillard compounds containing nutritionally unavailable lysine, free lysine, and some hexosamines, and, thus, the TNBS procedure in theory should indicate higher available lysine than the FDNB method. The TNBS procedure produced higher lysine availability values for fish meals than the FDNB
TABLE 4. Percent lysine availability' in fish meals determined by TNBS, Sample
Peruvian anchovy California anchovy Alaska herring Seattle rock cod British Columbia scrap Shrimp meal Tuna meal
1. 2. 3. 4. 5. 6. 7.
Means
FDNB
TNBS
106 a b 107ab 100 a b 103ab 116 a 109ab 93b
104
±2 ±7 ±3 ±3 t 2 ±3 ±5
79d 91b 82cd 88bc 96a 87 b cd 81cd
86
± 4
±2 ±2 ±1 ±1 +3 +2
2
FDNB,3 CBA/ '
andDL5
CBA
DL
69b + 3 + 7 91a 96a + 2 89 a + 1 98ab' + 8 95a + 8 + 12 92a
74 c 93a 93a 82b 91a 87ab 83b
90
86
+ + + ±
1 0 2 1
±0 ± 5 ± 3
' 'cMeans that do not share a common letter within a column are significantly different (P<.05). Lysine availability percentage values are means ± SEM determined by dividing percent available lysine (Table 1) by percent total lysine and multiplied by 100. 2 2,4,6-Trinitrobenzene sulfonic acid. 3 l-Fluoro-2,4-dinitrobenzene. 4 Chick bioassay. 5 Digestible lysine. 1
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(1966) as having extremely imbalanced amino acid levels and poor protein digestibility, which suggests that chemical methods are overrating the available lysine content of keratin meals. Average lysine availability of keratin meals is 115% of TL as determined by the FDNB method. Lysine availabilities for keratin meals are 93, 64, and 80% as indicated by the TNBS, CBA, and DL methods, respectively (Table 6). Correlation coefficients between CBA lysine and the lysine from the three remaining available lysine assays (FDNB, TNBS, and DL) are high (r = .87, r = .92, and r = .94, respectively) (Table 7) for keratin meals; however, the quantitative amount of available lysine determined by each method is different. The variation in TL values for feather meals may reflect different amounts of blood in the raw material since processed blood meal contains a larger amount of lysine (7.5 to 9.5%) than average feather meals (NRC, 1977). The variation in available lysine may also reflect the processing conditions of the hair and feather meals because previous research suggests that the amino acids in feather proteins can be made almost completely available to a chick by appropriate treatment (Burgos et al, 1974). Comparison of Methods to Determine Available Lysine. The high lysine availabilities found in some samples analyzed by TNBS and
1047
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NORHEIM AND COON
TABLE 5. Percent lysine availability1 in meat and bone meals determined by TNBS,2 FDNB,3 CBA,4 and DLS Sample
Meat Meat Meat Meat Meat Meat Meat Meat Meat Meat
and bone and bone and bone and bone and bone and bone and bone and bone meal meal
meal meal meal meal meal meal meal meal
Means
87bc 87bc 107 a 84bc gibed 75cd 72cd 77Cd 62d 100 a b
±12 ± 7 ± 0 ± 3 + i + 2 ± 3 + 9 + 12 ± 4
83
72 e 92abc 85bcd 97a g7bcd 81de 78de 83cd 79de 96 a b
+ + + + + ± + ± + ±
2 2 10 2 2 1 1 2 4 3
85
92ab 89ab 99& 96ab 9iab 89ab 95ab 92ab 73b 93ab
DL
± 5 ±12 ±11 ± 10 + 7 ± 6 ± 3 ± 3 ± 5 + 7
83c
±1
86^° ± 2 bc
85 86ab 90 a 90a 88 a b 90a 85bd 89ab
±2 ±1 +1 ±1 ±l +0 + i ±1
87
91
ii h f d p
' ' ' ' Means that do not share a common letter within a column are significantly different (P<.05).
1
Lysine availability percentage values are means ± SEM determined by dividing percent available lysine (Table 2) by percent total lysine and multiplied by 100. 2 3
2,4,6-Trinitrobenzene sulfonic acid. l-Fluoro-2,4-dinitrobenzene.
"Chick bioassay. s
Digestible lysine.
p r o c e d u r e (Table 4 ) ; however, t h e o p p o s i t e was t r u e for m e a t and b o n e and keratin meals (Tables 5 and 6). T h e correlation coefficients (r) of CBA versus TNBS lysine, F D N B lysine, DL, and T L for all animal b y - p r o d u c t samples were .90, . 8 3 ,
.97, a n d . 9 3 , respectively (Table 7 ) . T h e CBA lysine versus F D N B lysine correlation (r = .83) ( P < . 0 5 ) for all animal b y - p r o d u c t s was lower t h a n o t h e r assay correlations. T h e r e is a significant correlation ( P < . 0 5 ) between t h e CBA available lysine and protein d e t e r m i n e d b y t h e
TABLE 6. Percent lysine availability1 in keratin meals determined by TNBS,2 FDNB,3 CBA," and DLS Sample
TNBS
1. 2. 3. 4. 5. 6.
89ab 101 a b 97ab 81b 105 a 84b
Feather meal Feather meal Feather meal Feather meal Hair meal No. 1 HairmealNo. 2
Means
93
±1 ±0 ±6 ±7 ±8 ±0
FDNB
CBA
97d 115 c 148 a 97d 129 b 98d
64ab ± 70ab ± 75 a ± 64ab ± 66ab± 45b ±
115
±2 ± 2 ±8 ± 7 ±4 ± 2
64
DL
9 6 1 4 11 8
77b 89 a 83ab 82ab 84ab 67c
±1 ±1 ±6 ±4 +5 ± 7
80
' ' ' Means that do not share a common letter within a column are significantly different (P<.05). 1
Lysine availability percentage values are means ± SEM determined by dividing percent available lysine (Table 3) by percent total lysine and multiplied by 100. 2 3
2,4,6-Trinitrobenzene sulfonic acid. l-Fluoro-2,4-dinitrobenzene.
4
Chick bioassay.
5
Digestible lysine.
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1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
CBA
FDNB
TNBS
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
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TABLE 7. Correlation for CBA1 available lysine versus TNBS,* FDNB,3 DL," TL,5 or CP6
X
Y
Fish meals
Meat and bone meals
Keratin meals
All samples
CBA vs. CBA vs. CBAra. CBA vs. CBA vs.
TNBS FDNB DL TL CP
.80 .89 .97 .83 .76
.66 .44 .67 .63 .33*
.92 .87 .94 .73 .20*
.90 .83 .97 .93 -.39*
1
Chick bioassay.
2
2,4,6-Trinitrobenzene sulfonic acid.
3
l-Fluoro-2,4-dinitrobenzene.
4
Digestible lysine.
s
Total lysine from amino acid analyzer.
6
Crude protein (Kjeldahl) analysis.
Kjeldahl p r o c e d u r e for fish meals, b u t t h e same correlation for all animal p r o t e i n sources is negative (r = - . 3 9 ) (Table 7). T h e highest CBA lysine values were f o u n d in t h e highest p r o t e i n meals (Alaska herring, California anchovy, and Peruvian anchovy) (Table 1), which p r o b a b l y c o n t r i b u t e d t o t h e high correlation ( P < . 0 5 ) (r = .76). T h e CBA has low precision in measuring available lysine (Miller, 1 9 6 7 ) , b u t it is still t h e standard available lysine m e t h o d t o which all other available lysine techniques are c o m p a r e d . T h e t w o bioassays for available lysine (CBA and
D L ) have t h e highest correlation over all animal proteins (r = .97) (Table 7). Sibbald ( 1 9 8 0 ) c o n c l u d e d t h a t t h e D L m e t h o d is a practical measure of available lysine. T h e validity of t h e DL m e t h o d m a y be questioned, since a m i n o acid o u t p u t m a y b e influenced b y q u a n t i t y a n d t y p e of feed c o n s u m e d . Sibbald ( 1 9 8 0 ) s h o w e d t h a t w h e n d e x t r o s e was fed to roosters, a m i n o acid excretion was unchanged from values o b t a i n e d from unfed birds. Microbial action in t h e alimentary canal m a y also affect a m i n o acid availability, although extensive literature review (Sibbald, 1979) reveals little o r n o
TABLE 8. Comparison of lysine availability1 in different animal meals Sample
TNBS2
1. 2. 3. 4.
105a 83c 92 b 94b
FDNB 3
CBA4
DL 5
86b 86 b 116a 117a
90* 9ia 68b 57b
86 a 87 a 83 b 75 c
(%) Fish meals Meat and bone meals Feather meals Hair meals
' ' Means that do not share a common letter within a column are significantly different (P<.05). 1
Lysine availability values are means of percent available lysine for animal protein feedstuff divided by percent total lysine for the feedstuff and multiplied by 100. 2 3
2,4,6-Trinitrobenzene sulfonic acid. l-Fluoro-2,4-dinitrobenzene.
4
Chick bioassay.
5
Digestible lysine.
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*These values are nonsignificant (P<.05); all other values are significant.
NORHEIM AND COON
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REFERENCES Association of Official Analytical Chemists, 1975. Official Methods of Analysis. 12th ed. Washington, DC. Bacigalupo, P. A., 1973. Effect of processing on the nutritional value of feeds in Peru. Pages 4 0 7 - 4 2 7 in Effect of Processing on the Nutritional Values of Feeds. Natl. Acad. Sci., Washington, DC. Bjarnason, J. and K. J. Carpenter, 1970. Mechanism of heat damage in proteins. 2. Chemical changes in pure protein. Br. J. Nutr. 24:313-329. Booth, V. H., 1971. Problems in the determination of FDNB-available lysine. J. Sci. Food Agric. 22: 658-666. Burgos, A., J. I. Floyd, and E. L. Stephenson, 1974. The amino acid content and availability of different samples of poultry by-product meal and feather meal. Poultry Sci. 53:198-203. Carpenter, K. J., 1960. The estimation of the available lysine in animal-protein foods. Biochem. J. 77:604-610. Carpenter, K. J., and V. H. Booth, 1973. Damage to lysine in food processing: its measurement and its significance. Nutr. Abstr. Rev. 43:423—451. Carpenter, K. J., and A. A. Woodham, 1974. Protein quality of feedstuffs: Comparison of the results of collaborative biological assays for amino acids with those of other methods. Br. J. Nutr. 32: 647-660. Combs, G. F., E. H. Bossard, and G. R. Childs, 1968. Improved chick bioassays for available lysine and available methionine. Feedstuffs 40(8);36—37. Coon, C. N., J. P. Nordheim, D. C. McFarland, and D. E. Gould, 1978. Nutritional quality of processed poultry waste for broilers. Poultry Sci. 57:1002—
1007. Johnston, J., and C. N. Coon, 1979. A comparison of six protein quality assays using commercially available protein meals. Poultry Sci. 58:919-927. Kakade, M. L., and Liener, 1969. Determination of available lysine in proteins. Anal. Biochem. 27:273-280. Likuski, H.J.A., and H. G. Dorrell, 1978. A bioassay for rapid determination of amino acid availability values. Poultry Sci. 57:1658-1660. Luong, V. B., and C. G. Payne, 1977. Hydrolyzed feather protein as a source of amino acids for laying hens. Br. Poult. Sci. 18:523-526. McBee, L. E., and R. T. Marshall, 1978. Enzymatic estimation of available lysine. J. Food Sci. 43:1355-1356. Miller, E. L., 1967. Methods of evaluating the amino acid content of feedingstuff and their limitations. Pages 3—15 in Protein Utilization by Poultry. R. A. Morton and E. C. Amoroso, ed., Oliver and Boyd, Edinburgh. Moran, E. T., Jr., and J. D. Summers, 1967. Feather meal and other processed keratins as dietary sources of protein for poultry production. Feedstuffs 39(50): 5 0 - 5 1 . Moran, E. T., Jr., J. D. Summers, and S. J. Slinger, 1966. Keratin as a source of protein for the growing chick. 1. Amino acid imbalance as the cause for inferior performance of feather meal and the implications of disulfide bonding in raw feathers as the reason for poor digestibility. Poultry Sci. 45:1257-1266. Moran, E. T., Jr., J. D. Summers, and S. J. Slinger, 1967. Keratins as sources of protein for the growing chick. 2. Hog hair, a valuable source of protein with appropriate processing and amino acid balance. Poultry Sci. 46:456—465. Muztar, A. J., and S. J. Slinger, 1980. Apparent amino acid availability and apparent metabolizable energy values of tower and candle rapeseeds and rapeseed meals. Poultry Sci. 59:1430-1433. National Renderers Association, Inc., 1975. Feather meal. Internal Rep., Natl. Renderers Assoc, Inc., DesPlaines, IL 60018. National Research Council, 1977. Nutrient Requirements of Poultry. No. 1. In Nutrient Requirements of Domestic Animals. 7th ed., Natl. Acad. Sci., Washington, DC. Ousterhout, L. E., and E. M. Wood, 1970. Available lysine in fish meals: chemical (TNBS) method compared with a chick assay. Poultry Sci. 49: 1423. (Abstr.) Skurray, G. R., and L. S. Herbert, 1974. Batch dry rendering: influence of raw materials and processing conditions on meat meal quality. J. Sci. Food Agric. 25:1071-1079. Sibbald, I. R., 1979. A bioassay for available amino acids and true metabolizable energy in feedingstuffs. Poultry Sci. 58:668-673. Sibbald, I. R., 1980. The effects of dietary cellulose and sand on the combined metabolic plus endogenous energy and amino acid outputs of adult cockerels. Poultry Sci. 59:836—844. Steel, R.G.D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc. New York, NY. Tarr, H.L.A., and J. Biely, 1973. Effect of processing
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discernible effect from microflora. Digestible lysine, calculated from fecal collection and analysis, is part of t h e multi-assay t e c h n i q u e suggested b y Sibbald, ( 1 9 7 9 , 1 9 8 0 ) . T h e D L t e c h n i q u e allows t h e d e t e r m i n a t i o n of t h e availability of all amino acids and t r u e m e t a b o lizable energy. This c o m b i n e d assay t e c h n i q u e has also been suggested b y M u z t a r and Slinger ( 1 9 8 0 ) . T h e Muztar and Slinger assay loses s o m e of its c o m p a r a t i v e advantage over t h e chick bioassay due t o a 14-day feeding period before fecal collection. T h e meaning and a m o u n t of available lysine is different for each available lysine t e c h n i q u e , although lysine d e t e r m i n e d b y several t e c h n i q u e s , such as t h e TNBS and t h e DL assay, correlate well with t h e CBA lysine. T h e DL t e c h n i q u e of Sibbald ( 1 9 7 9 ) is relatively rapid and less expensive t h a n o t h e r biological p r o c e d u r e s (Likuski and Dorrell, 1978). M u l t i - n u m b e r e d analyses possible from each force-feeding increases t h e potential value of t h e D L t e c h n i q u e as an i m p r o v e m e n t o n t h e s t a n d a r d chick bioassay for determining available lysine.
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS on the nutritional value of fish meal and related products. Pages 252—281 in Effect of Processing on the Nutritional Value of Feeds. Natl. Acad. Sci., Washington, DC. Wilder, O.H.M., 1972. Effects of processing on the quality of animal by-products with special reference to feather meal. Pages 83—85 in Maryland Nutr. Conf. Proc. Wilder, O.H.M., 1973. Effect of processing on meat
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meal and other meat products. Pages 282—285 in Effect of Processing on the Nutritional Value of Feeds. Natl. Acad. Sci., Washington, DC. Woodham, A. A., 1969. Protein quality for nonruminants. Pages 261—294 in Nutrition of Animals of Agricultural Importance. Part 1. The Science of Nutrition of Farm Livestock. Sir David Cuthbertson, ed., Pergamon Press, New York, NY.
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INTRODUCTION Because lysine is often the first or second limiting amino acid in poultry feeds, the amount of dietary lysine biologically available is of extreme importance. The main factor regulating the total lysine content of animal protein meals is the composition of raw materials rendered (Wilder, 1972). Skurray and Herbert (1974) reported meat meals prepared from sheep and calves heads contained 5.9 to 6.4 g lysine/16 g of nitrogen compared to 7.2 to 8.8 g lysine/16 g of nitrogen in meals composed primarily of sheep and calves stomachs and intestines. The researchers reported no changes in total lysine following three different batch rendering process conditions; however, the nutritional qualities of the meals were improved as demonstrated with chick feeding studies. Heat processing of many protein feeds
1 Scientific Paper No. 6587. College of Agriculture, Agricultural Research Center, Washington State University, Pullman, WA 99164. Project 0308. 2 Research supported in part by a grant from the Fats and Protein Research Foundation, 2250 East Devon Avenue, Des Plaines, IL 60018. 3 Durrum D-500, Durrum, 3950 Fabian Way, Palo Alto, CA 94303.
is required, because the heat eliminates microorganisms such as Salmonella, nutritional inhibitors, and improves overall digestibility by altering protein structure to allow enzymatic cleavage of peptide bonds by animals. The epsilon amino group of lysine may chemically react with carbohydrates or oxidized lipids (Carpenter and Booth, 1973) and free carboxyl groups of glutamic and aspartic acid (Bjarnason and Carpenter, 1970) during heat processing of protein feedstuffs, thus decreasing the digestion of processed protein and the biological availability of lysine. Animal proteins such as meat and bone meal are protected during heat processing with large proportions of animal fat, which keep the protein from becoming dry and scorched (Wilder, 1973). Meat and bone meals contain very small quantities of carbohydrate and a higher proportion of saturated lipids which would have less tendency to oxidize than unsaturated lipids. Raw feather and hair are almost pure keratin protein that is second limiting in lysine for poultry (Moran and Summers, 1967; Moran et al, 1967; Luong and Payne, 1977) and relatively nondigestible (Moran et al., 1966). Hydrolyzed feather and hair meals are cooked by high pressure steam heat to allow optimum utilization by poultry (Moran and Summers,
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ABSTRACT The available lysine in 7 fish, 10 meat and bone, and 6 keratin meals was determined with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and l-fluoro-2,4-dinitrobenzene (FDNB) chemical procedures, chick bioassay (CBA), and digestible lysine (DL) technique. The percent available lysine and total lysine (TL) in test meals is reported as grams of lysine per 100 g dry weight. The available lysine means determined by the TNBS, FDNB, CBA, and DL procedures were 5.28, 4.34, 4.50, and 4.36% for fish meals; 2.48, 2.53, 2.71, and 2.61% for meat and bone meals; and 2.33, 2.74, 1.50 and 1.92% for keratin meals, respectively. The correlation coefficients (r) for CBA versus TNBS lysine, FDNB lysine, DL, and TL are .90, .83, .97, and .93, respectively, for all animal proteins tested. The available lysine determined by the DL procedure correlated best with available lysine determined with the CBA for each type of animal protein meal and for all test meals collectively. The DL technique is a logical alternative to the CBA, FDNB, and TNBS procedure for determining available lysine in animal proteins because the DL procedure is simpler, less expensive, and quicker than the CBA procedure and more accurate than the colorimetric chemical procedures. (Key words: lysine availability, chick bioassay, l-fluoro-2, 4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), meat and bone meal, fish meal, keratin meal) 1984 Poultry Science 63:1040-1051
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
MATERIALS AND METHODS
General. Analyses for dry matter and nitrogen were conducted on triplicate samples according to the methods of the Association of Analytical Chemists (1975). Total lysine (TL), available lysine, and crude protein (CP) values are expressed on a dry weight basis. Lysine availability is defined as the proportion of TL that potentially can be absorbed and utilized by the animal. Available lysine is the quantitative amount of lysine (g lysine/100 g sample) in a sample that can be used by an animal for its metabolic functions. The terms availability and available are linked together as in the equation: Available lysine = lysine availability X total lysine The mean values for available lysine and lysine availability for the different animal by-products were statistically compared by analysis of variances and Duncan's new multiple range test (Steel and Torrie, 1960). Digestible Lysine. Twenty Single Comb White Leghorn roosters, weighing 1.5 to 2.0 kg, were randomly assigned to individual wire cages containing automatic water cups and separate fecal collection trays. Within experiment the 20 roosters were divided into groups of 6 for each
by-product material with the remaining 2 roosters being starch-fed controls. The roosters were fasted for 36 hr and then were force-fed 30 g of each test material to determine true DL as described by Sibbald (1979). Excreta from test roosters were collected for 36 hr following the force-feeding of test samples. Endogenous lysine losses were determined by collecting excreta from control roosters fed 30 g of corn starch. Excreta from roosters fed the test material and control roosters were individually weighed and lyophilized. Inidividual excreta samples were arbitrarily paired within each test group of 6 roosters. Triplicate samples of lyophilized excreta from by-product-fed and starch-fed control roosters and duplicate feed samples were hydrolyzed with 6 N HC1 for 24 hr and analyzed for amino acid content by AAA Laboratory of Mercer Island, WA. The samples were analyzed with an automatic amino acid analyzer following the manufacturer's procedures. 3 The lysine values are based on the molecular weight of lysine (146.2 g) instead of the residue molecular weight (128.2 g). The residue molecular weight is the molecular weight of the amino acid in the protein prior to hydrolysis and the addition of water. Chick Bioassay. Two-thousand-two-hundred and twenty male Hubbard broiler chicks, one day of age, were placed in electrically-heated brooders with raised wire floors and automatic water cups and ad libitum fed a low lysine basal diet (Combs et al, 1968; Coon et al, 1978) containing .65% available lysine for a 10-day feeding period. Chicks were randomly selected on the 10th day and weighed in replicate groups of 10 chicks each. The total weight of each replicate group was adjusted for uniformity by chick substitution. Chicks were then provided with water and experimental diets ad libitum for a 14-day feeding period. Reference diets (Coon et al, 1978) containing .49, .57, .65, .73, and .79% available lysine were each fed to four replicate groups. Each test diet, containing 5% of the animal by-product meals, was fed to four replicate groups. The diets were made isocaloric and isonitrogenous by adding corn oil and glutamic acid, respectively. Calcium, phosphorus, and amino acids other than lysine were adjusted to National Research Council (NRC, 1977) levels or were in excess. The calculated lysine content in all test diets containing the 5% animal protein meal was adjusted with crystalline lysine to contain approximately .65% available lysine to assure
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1967). Keratin proteins that have been undercooked have lower protein quality, because the nonhydrolyzed disulfide bonds cross-linking the protein make it unavailable for maximum enzymatic digestion by animals. Wilder (1972) reported overcooking feather meal produces a loss of cystine but not other amino acids. A fast, simple, inexpensive, and accurate method for determining available lysine would enable manufacturers of animal protein meals to control quality and to produce protein meals with consistent nutritional quality. The chick bioassay is a standard technique for determining biologically available lysine (Combs et al, 1968; Coon et al, 1978), but the method is slow and costly. Alternative chemical and short-term digestibility methods may produce numerically different available lysine values that are still highly indicative of protein quality. The objective of the present study was to compare digestible lysine (DL), chick bioassay (CBA), l-fluoro-2,4-dinitrobenzene (FDNB), and 2,4,6-trinitrobenzene sulfonic acid (TNBS) methods for determining available lysine in animal protein meals.
1041
1042
NORHEIM AND COON
l-Fluoro-2,4-Dinitrobenzene Available Lysine. The FDNB method used for chemically determining available lysine was essentially that of Carpenter (1960) as modified by Booth (1971). Triplicate samples of .5 g animal by-product meal were weighed into 125-ml glass-stoppered, flat-bottomed flasks and buffered with 10 ml of 8% (w/v) sodium bicarbonate. The samples were mixed with 15 ml of a 2.5% FDNB-ethanol (v/v) solution for 2 hr on a mechanical shaker. The protein samples were digested by adding 30 ml of 8.1 N HC1 and refluxing for 16 hr. The refluxed samples were filtered, and then digestion flask and residue were washed thoroughly with hot water until the filtrate volume was 500 ml. Booth (1971) modified the Carpenter (1960) procedure by using hot water washings instead of water at room temperature and the filtrate volume was increased to 250 ml instead of 200 ml for a more complete extraction of the e-DNP-lysine. The characteristic yellow color was still present in the washings of the animal protein samples when the filtrate volume was 250 ml; therefore, the total filtrate volume was increased to 500 ml for a complete extraction of e-DNP-lysine. The samples were extracted twice with ethyl ether to remove interfering a-DNP-amino acids and dinitrophenol, and the absorbance was determined at 435 nm. A correction factor of
1.08 for meat and bone meal and 1.05 for fish and keratin meals was used to account for e-DNP-lysine loss during the 16-hr digestion of protein samples as suggested by Booth (1971). 2,4,6-Trinitrobenzene Sulfonic Acid Available Lysine. Available lysine was determined by the TNBS method of Kakade and Liener (1969) as modified by Ousterhout and Wood (1970). Ousterhout and Wood (1970) used 100 mg of animal by-product meal for each sample to increase precision compared to 1 mg of purified protein in the original assay of Kakade and Liener (1969). Lysine-TNBS derivatives are formed by shaking test samples with 10 ml 4% NaHC0 3 (pH 8.5) for 30 min and then adding 10 ml of 1% TNBS solution and continue to shake for 2 hr more. The test proteins containing the lysine-TNBS derivatives are hydrolyzed by adding 30 ml of concentrated HCl and autoclaving for 1 hr at 115 C (15 psi). The 125-ml flasks containing the animal protein samples were covered with inverted beakers and set in a tray of water during autoclaving in order to minimize destruction of the autoclave by the hydrochloric acid. Triplicate 1-ml aliquots were extracted twice with 5 ml of ethyl ether, which was removed with a suction line. Residual ether was removed by use of a hot water bath. The intensity of the yellow color was determined by measuring absorbance at 346 nm. RESULTS AND DISCUSSION
Available Lysine in Fish Meals. Available lysine in Alaska herring meal was higher than in other fish meals for all of the methods studied (Table 1). The total lysine in Alaska herring (6.77%) was higher than other fish meal samples and is comparable to the NRC, (1977) value of 6.13%. Alaska herring meal containing 74.0% CP had the highest protein level compared to other fish meals and is consistent with previous reports of 72 and 73% (Tarr and Biely, 1973). Peruvian anchovy, California anchovy, and British Columbia scrap were the next highest in available lysine. California and Peruvian anchovy meals with 71 and 71.4% protein contained 5.61 and 6.01% total lysine, respectively. The four available lysine techniques give lower lysine values for Peruvian anchovy and California anchovy (4.10 to 6.38 and 5.08 to 5.98%, respectively) (Table 1) than the range of 5.65 to 7.05% reported by Bacigalupo (1973). Peruvian anchovy fish meal may be lower in available lysine than California anchovy meal
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weight gains within the range of weight gains from chicks fed reference diets. The chicks were weighed in replicate groups on the 24th day and feed consumption recorded. The biologically available lysine in each animal by-product test diet was determined by comparing the weight gain and feed consumption of chicks receiving reference diets as reported by Combs et al. (1968). Values reported were averaged from duplicate experiments with eight total replicates. The biologically available lysine in corn gluten meal used in the test and reference diets was previously determined by feeding chicks a crystalline amino acid diet containing two levels of corn gluten meal (5 and 14.2%) substituting for crystalline lysine. The biologically available lysine in corn gluten meal was verified by feeding increasing increments of corn gluten meal and decreasing levels of crystalline lysine. Each lysine-limiting ration was calculated as providing equal amounts of total available lysine. The differences in weight gain would indicate a disparity between the actual lysine value in the corn gluten meal and the value used to calculate the rations.
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
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NORHEIM AND COON
Available Lysine in Meat and Bone Meals. The four methods of determining available lysine in meat and bone meals did not consistently rank the samples the same (Table 2). The available lysine in meat and bone meal No. 3 was higher (P<.05) than other meat and bone meals as determined by the CBA and TNBS lysine procedures, whereas meat meal No. 10 contained the highest.FDNB available lysine. Meat and bone meal No. 3 and meat meal No. 10 have been previously determined to be superior to seven other meat and bone meals in six protein quality assays (Johnston and Coon, 1979). Meat meal No. 9 contained the largest amount of TL (3.47%) and DL (2.96%). The quantitative amount of available lysine for each meat and bone meal sample was different for
the four methods. Meat and bone meal No. 3 has a higher TNBS lysine value (3.20%) than TL (2.98%) (Table 2). The DL, FDNB lysine, and CBA lysine values for the meat and bone meal samples were consistently below the TL values (Table 2). The TL in meat and bone meal ranged from 2.47 to 3.19% with a mean of 2.92% TL. Meat meal No. 10 had 3.13% TL and meat meal No. 9 had 3.47% TL (Table 2). The NRC (1977) value for lysine in dry matter is 2.80% for meat and bone meals and 3.26% for meat meals. Average lysine availability values for all meat and bone meals were 83, 85, 87, and 91% (Table 5) as indicated by TNBS, FDNB, DL, and CBA techniques, respectively. The CBA technique did not show significantly different available lysine values in the meat and bone meal samples compared to other methods. The correlations between CBA and other available lysine methods are much lower for meat and bone meal samples than they were for the fish meals and keratin protein samples (Table 7). The low correlation for CBA lysine and available lysine as determined by DL, FDNB, and TNBS techniques may be due to the narrow range of TL in the meat and bone meals. The CBA method correlated best (P< .05) with the digestible lysine technique for meat and bone meal samples (r = .67). Available Lysine in Keratin Meals. Feather mean No. 2 contained the highest available lysine and TL among feather meals (Table 3) for all lysine analyses (P<.05). Two samples are higher (2.17 and 2.73%) and two samples have less or equal TL (1.65 and 1.75%) than 1.80 and 1.75% lysine values for feather meal reported by NRC (1977) and the National Renderers Association, Inc. (1975), respectively. Feather meal No. 4 had the lowest values for TNBS, FDNB, and CBA available lysine (1.42, 1.71, and 1.11%, respectively) compared to other feather meals. The protein quality of feather meal No. 4 has been previously determined to be lower than five other feather meal samples (Johnston and Coon, 1979). Hair meal No. 1 was higher in available lysine than hair meal No. 2, but No. 2 contains more TL. Both hair meals clearly contain more available lysine and TL than all feather meals except feather meal No. 2. The chemical assays show hair meals and feather meals to have high lysine availability, whereas the bioassay methods do not confirm the high availability of lysine for either hair or feather meals (Table 6). Feather meals have been reported by Moran et al.
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(Table 1), because Peruvian anchovy fish meal often undergoes more extensive heat processing to ensure a Salmonella-free meal (Lawrence R. Berg, 1975, personal communication). Total lysine content for anchovy samples is higher (5.61 to 6.01%) than the NRC (1977) value of 5.33%. In general, Seattle rock cod, shrimp meal, and tuna meal were lower in available lysine than other fish meal samples. Tuna meal was not significantly lower (P>.05) in available lysine than Peruvian anchovy and British Columbia scrap as determined by the CBA method. Correlations between the CBA and FDNB, TNBS, and DL techniques for determining available lysine in fish meals are high (r = .89, r = .80, and r = .97, respectively) (Table 7), although the quantitative amount of lysine in fish meals are different for the four methods. The TNBS lysine values for fish meals exceed the TL values of the meals (Table 2). Average lysine availability values of 104% (Tables 4 and 8) were based on TNBS available lysine divided by total lysine (TL) and multiplied by 100. The DL and FDNB technique produced the lowest average lysine availability values for the fish meals, with 86% of the total being available. The chick bioassay method showed an average availability of 90% for all fish meals. These data are similar to a report suggesting about 90% lysine availability for fish meals as determined by FDNB lysine procedure (Carpenter and Woodham, 1974). The lysine versus CP correlation (r = .76) for fish meals is high (P<.05) (Table 7). This is probably from the herring and anchovy fish meals containing large amounts of available lysine and CP and the remaining fish meals with lower protein containing less available lysine (Table 1).
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
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(%)
1.50
1.38 b 1.92 a 1.23b l.llc 1.86a 1.50a
CBA
+ ± ± ± ± ±
Crude protein (Kjeldahl) analysis, dry matter basis.
Total lysine from amino acid analyzer, dry matter basis.
Digestible lysine.
Chick bioassay.
l-Fluoro-2,4-dinitrobenzene.
2,4,6-Trinitrobenzene sulfonic acid.
Available lysine values represent mean ± SEM on a dry matter basis (g lysine/100 g dry matter).
' ' Means that do not share a common letter within a column are significantly different (P<.05).
1
a
Means
2.33
2.23 b 3.15 a 2.43t> 1.71 c 3.66 a 3.23 a
± .03 ± .01 + .10 ± .13 + .21 ± .02
1.92 b 2.77 a 1.60 bc 1.42C 2.97a 2.75 a
1. 2. 3. 4. 5. 6.
Feather meal Feather meal Feather meal Feather meal Hair meal No. 1 Hair meal No. 2
FDNB
TNBS
.19 .16 .04 .07 .30 .25
D
FDNB,3 CBA, and DLs), total lysine, and crude p
Sample
TABLE 3.. Percent available lysine1 (TNBS,
d from http://ps.oxfordjournals.org/ at Michigan State University on January 16, 2015
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
FDNB methods (Table 8) are because the TNBS and FDNB reagents bind with compounds other than lysine. The lack of complete specificity of TNBS and FDNB methods shows up in the colorimetric analysis as a higher lysine value. Chemical assays for available lysine usually show higher lysine concentrations than do animal or microbiological assays (McBee and Marshall, 1978). Lysine may be bound in the protein in such a way that it is not capable of being utilized by the animal. Lysine that is not biologically available may be released by acid hydrolysis, thus causing erroneously high available lysine values when assayed by chemical methods (Woodham, 1969). All available lysine assays purport to measure lysine with unbound epsilon amino groups. The FDNB available lysine procedure has been reported to measure only lysine with a reactive epsilon amino group bound in a protein chain and not free lysine or lysine at the N-terminal protein end (Carpenter and Booth, 1973; Carpenter and Woodham, 1974). Carpenter and Booth (1973) suggest that TNBS reacts with Maillard compounds containing nutritionally unavailable lysine, free lysine, and some hexosamines, and, thus, the TNBS procedure in theory should indicate higher available lysine than the FDNB method. The TNBS procedure produced higher lysine availability values for fish meals than the FDNB
TABLE 4. Percent lysine availability' in fish meals determined by TNBS, Sample
Peruvian anchovy California anchovy Alaska herring Seattle rock cod British Columbia scrap Shrimp meal Tuna meal
1. 2. 3. 4. 5. 6. 7.
Means
FDNB
TNBS
106 a b 107ab 100 a b 103ab 116 a 109ab 93b
104
±2 ±7 ±3 ±3 t 2 ±3 ±5
79d 91b 82cd 88bc 96a 87 b cd 81cd
86
± 4
±2 ±2 ±1 ±1 +3 +2
2
FDNB,3 CBA/ '
andDL5
CBA
DL
69b + 3 + 7 91a 96a + 2 89 a + 1 98ab' + 8 95a + 8 + 12 92a
74 c 93a 93a 82b 91a 87ab 83b
90
86
+ + + ±
1 0 2 1
±0 ± 5 ± 3
' 'cMeans that do not share a common letter within a column are significantly different (P<.05). Lysine availability percentage values are means ± SEM determined by dividing percent available lysine (Table 1) by percent total lysine and multiplied by 100. 2 2,4,6-Trinitrobenzene sulfonic acid. 3 l-Fluoro-2,4-dinitrobenzene. 4 Chick bioassay. 5 Digestible lysine. 1
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 16, 2015
(1966) as having extremely imbalanced amino acid levels and poor protein digestibility, which suggests that chemical methods are overrating the available lysine content of keratin meals. Average lysine availability of keratin meals is 115% of TL as determined by the FDNB method. Lysine availabilities for keratin meals are 93, 64, and 80% as indicated by the TNBS, CBA, and DL methods, respectively (Table 6). Correlation coefficients between CBA lysine and the lysine from the three remaining available lysine assays (FDNB, TNBS, and DL) are high (r = .87, r = .92, and r = .94, respectively) (Table 7) for keratin meals; however, the quantitative amount of available lysine determined by each method is different. The variation in TL values for feather meals may reflect different amounts of blood in the raw material since processed blood meal contains a larger amount of lysine (7.5 to 9.5%) than average feather meals (NRC, 1977). The variation in available lysine may also reflect the processing conditions of the hair and feather meals because previous research suggests that the amino acids in feather proteins can be made almost completely available to a chick by appropriate treatment (Burgos et al, 1974). Comparison of Methods to Determine Available Lysine. The high lysine availabilities found in some samples analyzed by TNBS and
1047
1048
NORHEIM AND COON
TABLE 5. Percent lysine availability1 in meat and bone meals determined by TNBS,2 FDNB,3 CBA,4 and DLS Sample
Meat Meat Meat Meat Meat Meat Meat Meat Meat Meat
and bone and bone and bone and bone and bone and bone and bone and bone meal meal
meal meal meal meal meal meal meal meal
Means
87bc 87bc 107 a 84bc gibed 75cd 72cd 77Cd 62d 100 a b
±12 ± 7 ± 0 ± 3 + i + 2 ± 3 + 9 + 12 ± 4
83
72 e 92abc 85bcd 97a g7bcd 81de 78de 83cd 79de 96 a b
+ + + + + ± + ± + ±
2 2 10 2 2 1 1 2 4 3
85
92ab 89ab 99& 96ab 9iab 89ab 95ab 92ab 73b 93ab
DL
± 5 ±12 ±11 ± 10 + 7 ± 6 ± 3 ± 3 ± 5 + 7
83c
±1
86^° ± 2 bc
85 86ab 90 a 90a 88 a b 90a 85bd 89ab
±2 ±1 +1 ±1 ±l +0 + i ±1
87
91
ii h f d p
' ' ' ' Means that do not share a common letter within a column are significantly different (P<.05).
1
Lysine availability percentage values are means ± SEM determined by dividing percent available lysine (Table 2) by percent total lysine and multiplied by 100. 2 3
2,4,6-Trinitrobenzene sulfonic acid. l-Fluoro-2,4-dinitrobenzene.
"Chick bioassay. s
Digestible lysine.
p r o c e d u r e (Table 4 ) ; however, t h e o p p o s i t e was t r u e for m e a t and b o n e and keratin meals (Tables 5 and 6). T h e correlation coefficients (r) of CBA versus TNBS lysine, F D N B lysine, DL, and T L for all animal b y - p r o d u c t samples were .90, . 8 3 ,
.97, a n d . 9 3 , respectively (Table 7 ) . T h e CBA lysine versus F D N B lysine correlation (r = .83) ( P < . 0 5 ) for all animal b y - p r o d u c t s was lower t h a n o t h e r assay correlations. T h e r e is a significant correlation ( P < . 0 5 ) between t h e CBA available lysine and protein d e t e r m i n e d b y t h e
TABLE 6. Percent lysine availability1 in keratin meals determined by TNBS,2 FDNB,3 CBA," and DLS Sample
TNBS
1. 2. 3. 4. 5. 6.
89ab 101 a b 97ab 81b 105 a 84b
Feather meal Feather meal Feather meal Feather meal Hair meal No. 1 HairmealNo. 2
Means
93
±1 ±0 ±6 ±7 ±8 ±0
FDNB
CBA
97d 115 c 148 a 97d 129 b 98d
64ab ± 70ab ± 75 a ± 64ab ± 66ab± 45b ±
115
±2 ± 2 ±8 ± 7 ±4 ± 2
64
DL
9 6 1 4 11 8
77b 89 a 83ab 82ab 84ab 67c
±1 ±1 ±6 ±4 +5 ± 7
80
' ' ' Means that do not share a common letter within a column are significantly different (P<.05). 1
Lysine availability percentage values are means ± SEM determined by dividing percent available lysine (Table 3) by percent total lysine and multiplied by 100. 2 3
2,4,6-Trinitrobenzene sulfonic acid. l-Fluoro-2,4-dinitrobenzene.
4
Chick bioassay.
5
Digestible lysine.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 16, 2015
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
CBA
FDNB
TNBS
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS
1049
TABLE 7. Correlation for CBA1 available lysine versus TNBS,* FDNB,3 DL," TL,5 or CP6
X
Y
Fish meals
Meat and bone meals
Keratin meals
All samples
CBA vs. CBA vs. CBAra. CBA vs. CBA vs.
TNBS FDNB DL TL CP
.80 .89 .97 .83 .76
.66 .44 .67 .63 .33*
.92 .87 .94 .73 .20*
.90 .83 .97 .93 -.39*
1
Chick bioassay.
2
2,4,6-Trinitrobenzene sulfonic acid.
3
l-Fluoro-2,4-dinitrobenzene.
4
Digestible lysine.
s
Total lysine from amino acid analyzer.
6
Crude protein (Kjeldahl) analysis.
Kjeldahl p r o c e d u r e for fish meals, b u t t h e same correlation for all animal p r o t e i n sources is negative (r = - . 3 9 ) (Table 7). T h e highest CBA lysine values were f o u n d in t h e highest p r o t e i n meals (Alaska herring, California anchovy, and Peruvian anchovy) (Table 1), which p r o b a b l y c o n t r i b u t e d t o t h e high correlation ( P < . 0 5 ) (r = .76). T h e CBA has low precision in measuring available lysine (Miller, 1 9 6 7 ) , b u t it is still t h e standard available lysine m e t h o d t o which all other available lysine techniques are c o m p a r e d . T h e t w o bioassays for available lysine (CBA and
D L ) have t h e highest correlation over all animal proteins (r = .97) (Table 7). Sibbald ( 1 9 8 0 ) c o n c l u d e d t h a t t h e D L m e t h o d is a practical measure of available lysine. T h e validity of t h e DL m e t h o d m a y be questioned, since a m i n o acid o u t p u t m a y b e influenced b y q u a n t i t y a n d t y p e of feed c o n s u m e d . Sibbald ( 1 9 8 0 ) s h o w e d t h a t w h e n d e x t r o s e was fed to roosters, a m i n o acid excretion was unchanged from values o b t a i n e d from unfed birds. Microbial action in t h e alimentary canal m a y also affect a m i n o acid availability, although extensive literature review (Sibbald, 1979) reveals little o r n o
TABLE 8. Comparison of lysine availability1 in different animal meals Sample
TNBS2
1. 2. 3. 4.
105a 83c 92 b 94b
FDNB 3
CBA4
DL 5
86b 86 b 116a 117a
90* 9ia 68b 57b
86 a 87 a 83 b 75 c
(%) Fish meals Meat and bone meals Feather meals Hair meals
' ' Means that do not share a common letter within a column are significantly different (P<.05). 1
Lysine availability values are means of percent available lysine for animal protein feedstuff divided by percent total lysine for the feedstuff and multiplied by 100. 2 3
2,4,6-Trinitrobenzene sulfonic acid. l-Fluoro-2,4-dinitrobenzene.
4
Chick bioassay.
5
Digestible lysine.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 16, 2015
*These values are nonsignificant (P<.05); all other values are significant.
NORHEIM AND COON
1050
REFERENCES Association of Official Analytical Chemists, 1975. Official Methods of Analysis. 12th ed. Washington, DC. Bacigalupo, P. A., 1973. Effect of processing on the nutritional value of feeds in Peru. Pages 4 0 7 - 4 2 7 in Effect of Processing on the Nutritional Values of Feeds. Natl. Acad. Sci., Washington, DC. Bjarnason, J. and K. J. Carpenter, 1970. Mechanism of heat damage in proteins. 2. Chemical changes in pure protein. Br. J. Nutr. 24:313-329. Booth, V. H., 1971. Problems in the determination of FDNB-available lysine. J. Sci. Food Agric. 22: 658-666. Burgos, A., J. I. Floyd, and E. L. Stephenson, 1974. The amino acid content and availability of different samples of poultry by-product meal and feather meal. Poultry Sci. 53:198-203. Carpenter, K. J., 1960. The estimation of the available lysine in animal-protein foods. Biochem. J. 77:604-610. Carpenter, K. J., and V. H. Booth, 1973. Damage to lysine in food processing: its measurement and its significance. Nutr. Abstr. Rev. 43:423—451. Carpenter, K. J., and A. A. Woodham, 1974. Protein quality of feedstuffs: Comparison of the results of collaborative biological assays for amino acids with those of other methods. Br. J. Nutr. 32: 647-660. Combs, G. F., E. H. Bossard, and G. R. Childs, 1968. Improved chick bioassays for available lysine and available methionine. Feedstuffs 40(8);36—37. Coon, C. N., J. P. Nordheim, D. C. McFarland, and D. E. Gould, 1978. Nutritional quality of processed poultry waste for broilers. Poultry Sci. 57:1002—
1007. Johnston, J., and C. N. Coon, 1979. A comparison of six protein quality assays using commercially available protein meals. Poultry Sci. 58:919-927. Kakade, M. L., and Liener, 1969. Determination of available lysine in proteins. Anal. Biochem. 27:273-280. Likuski, H.J.A., and H. G. Dorrell, 1978. A bioassay for rapid determination of amino acid availability values. Poultry Sci. 57:1658-1660. Luong, V. B., and C. G. Payne, 1977. Hydrolyzed feather protein as a source of amino acids for laying hens. Br. Poult. Sci. 18:523-526. McBee, L. E., and R. T. Marshall, 1978. Enzymatic estimation of available lysine. J. Food Sci. 43:1355-1356. Miller, E. L., 1967. Methods of evaluating the amino acid content of feedingstuff and their limitations. Pages 3—15 in Protein Utilization by Poultry. R. A. Morton and E. C. Amoroso, ed., Oliver and Boyd, Edinburgh. Moran, E. T., Jr., and J. D. Summers, 1967. Feather meal and other processed keratins as dietary sources of protein for poultry production. Feedstuffs 39(50): 5 0 - 5 1 . Moran, E. T., Jr., J. D. Summers, and S. J. Slinger, 1966. Keratin as a source of protein for the growing chick. 1. Amino acid imbalance as the cause for inferior performance of feather meal and the implications of disulfide bonding in raw feathers as the reason for poor digestibility. Poultry Sci. 45:1257-1266. Moran, E. T., Jr., J. D. Summers, and S. J. Slinger, 1967. Keratins as sources of protein for the growing chick. 2. Hog hair, a valuable source of protein with appropriate processing and amino acid balance. Poultry Sci. 46:456—465. Muztar, A. J., and S. J. Slinger, 1980. Apparent amino acid availability and apparent metabolizable energy values of tower and candle rapeseeds and rapeseed meals. Poultry Sci. 59:1430-1433. National Renderers Association, Inc., 1975. Feather meal. Internal Rep., Natl. Renderers Assoc, Inc., DesPlaines, IL 60018. National Research Council, 1977. Nutrient Requirements of Poultry. No. 1. In Nutrient Requirements of Domestic Animals. 7th ed., Natl. Acad. Sci., Washington, DC. Ousterhout, L. E., and E. M. Wood, 1970. Available lysine in fish meals: chemical (TNBS) method compared with a chick assay. Poultry Sci. 49: 1423. (Abstr.) Skurray, G. R., and L. S. Herbert, 1974. Batch dry rendering: influence of raw materials and processing conditions on meat meal quality. J. Sci. Food Agric. 25:1071-1079. Sibbald, I. R., 1979. A bioassay for available amino acids and true metabolizable energy in feedingstuffs. Poultry Sci. 58:668-673. Sibbald, I. R., 1980. The effects of dietary cellulose and sand on the combined metabolic plus endogenous energy and amino acid outputs of adult cockerels. Poultry Sci. 59:836—844. Steel, R.G.D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc. New York, NY. Tarr, H.L.A., and J. Biely, 1973. Effect of processing
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discernible effect from microflora. Digestible lysine, calculated from fecal collection and analysis, is part of t h e multi-assay t e c h n i q u e suggested b y Sibbald, ( 1 9 7 9 , 1 9 8 0 ) . T h e D L t e c h n i q u e allows t h e d e t e r m i n a t i o n of t h e availability of all amino acids and t r u e m e t a b o lizable energy. This c o m b i n e d assay t e c h n i q u e has also been suggested b y M u z t a r and Slinger ( 1 9 8 0 ) . T h e Muztar and Slinger assay loses s o m e of its c o m p a r a t i v e advantage over t h e chick bioassay due t o a 14-day feeding period before fecal collection. T h e meaning and a m o u n t of available lysine is different for each available lysine t e c h n i q u e , although lysine d e t e r m i n e d b y several t e c h n i q u e s , such as t h e TNBS and t h e DL assay, correlate well with t h e CBA lysine. T h e DL t e c h n i q u e of Sibbald ( 1 9 7 9 ) is relatively rapid and less expensive t h a n o t h e r biological p r o c e d u r e s (Likuski and Dorrell, 1978). M u l t i - n u m b e r e d analyses possible from each force-feeding increases t h e potential value of t h e D L t e c h n i q u e as an i m p r o v e m e n t o n t h e s t a n d a r d chick bioassay for determining available lysine.
AVAILABLE LYSINE IN ANIMAL PROTEIN MEALS on the nutritional value of fish meal and related products. Pages 252—281 in Effect of Processing on the Nutritional Value of Feeds. Natl. Acad. Sci., Washington, DC. Wilder, O.H.M., 1972. Effects of processing on the quality of animal by-products with special reference to feather meal. Pages 83—85 in Maryland Nutr. Conf. Proc. Wilder, O.H.M., 1973. Effect of processing on meat
1051
meal and other meat products. Pages 282—285 in Effect of Processing on the Nutritional Value of Feeds. Natl. Acad. Sci., Washington, DC. Woodham, A. A., 1969. Protein quality for nonruminants. Pages 261—294 in Nutrition of Animals of Agricultural Importance. Part 1. The Science of Nutrition of Farm Livestock. Sir David Cuthbertson, ed., Pergamon Press, New York, NY.
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