Protein and Amino Acid Quality of Meat and Bone Meal

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1 Protein and Amino Acid Quality of Meat and Bone Meal C. M. PARSONS,1 F. CASTANON, and Y. HAN Department of Animal Sciences, University of Illinois, Urbana, Illinois ABSTRACT The in vivo protein quality of 14 meat and bone meals (MBM) was evaluated in three chick growth assays and a 48-h excreta collection assay using conventional and cecectomized roosters. In addition, in vitro evaluation of protein quality was assessed using pepsin N digestibility (0.2, 0.002, or % pepsin), KOH protein solubility, and multi-enzyme ph change. Crude protein, lysine, and SAA in the MBM varied from 48 to 56, 2.32 to 3.01, and 1.0 to 2.13%, respectively. Protein efficiency ratio (weight gain:protein intake) estimated from feeding chicks diets containing 9% protein from a MBM ranged from 0.61 to 2.89 and averaged Lysine bioavailability determined by slope-ratio chick assay ranged from 43 to 89%. True amino acid digestibility and TME n values determined in cecectomized roosters were generally lower (P < 0.05) than those determined in conventional roosters. True digestibility of amino acids (percentage) also varied among MBM, with the mean (and range) for lysine, methionine, and cystine in cecectomized birds being 81 (73 to 88), 85 (77 to 91), and 58% (37 to 72%), respectively. Pepsin N digestibility values determined using or % pepsin were positively correlated (P < 0.05) with lysine digestibility. Pepsin N digestibility determined using 0.2% pepsin, KOH protein solubility, and multi-enzyme ph change were not significantly correlated with in vivo protein quality. Ash content was negatively correlated ( 0.80, P < 0.05) with protein efficiency ratio. These results indicated that there is substantial variation in protein quality among commercial MBM and that pepsin N digestibility and ash content are correlated with some in vivo protein quality measurements. (Key words: meat and bone meal, protein quality, metabolizable energy, poultry) 1997 Poultry Science 76: INTRODUCTION Large amounts of meat and bone meal (MBM) are produced in the U.S. annually. Johnston and Coon (1979a) and Nordheim and Coon (1984) showed that high quality MBM can be produced from rendered materials. However, because of the nature of the raw materials and processing methods, the quality of animal protein meals can vary markedly (Wilder, 1973; Skurray, 1974; Johnston and Coon, 1979a). The variability in protein quality of MBM is one of the most important concerns, and often limitation, in its use in poultry and livestock rations. There is a definite need for research data that better characterize or quantify the variability in in vivo protein quality among commercial MBM. In addition, there is a further need for rapid inexpensive and accurate methods for assessing quality of MBM in vitro, so that manufacturers and nutritionists can more consistently monitor quality control of this ingredient. Johnston and Coon (1979b) reported that reducing the pepsin concentration from 0.2 to 0.002% increased the Received for publication June 6, Accepted for publication September 28, To whom correspondence should be addressed: 284 Animal Sciences Laboratory, 1207 West Gregory Drive, Urbana, IL accuracy of the pepsin N digestibility assay as a predictor of in vivo MBM protein quality; however, lower pepsin levels were not evaluated. The KOH protein solubility assay (Araba and Dale, 1990; Parsons et al., 1991) and multi-enzyme ph change assay (Hsu et al., 1977) have been shown to be useful in vitro assays for many feed ingredients but these assays have not been evaluated for MBM. Consequently, the objective of the current study was to determine the variability in in vivo protein quality among commercial samples of MBM and to evaluate several in vitro assays as predictors of in vivo protein quality. MATERIALS AND METHODS Meat and Bone Meals Approximately 50 kg of 16 MBM were obtained from various suppliers in the U.S. and Canada. All samples were analyzed for DM, gross energy, CP (N 6.25), ether extract, ash, Ca, and P according to the procedures of the Association of Official Analytical Chemists (AOAC, 1980). Upon completion of the latter assays, MBM 3 and 15 were found to contain in excess of 60% CP and less than 15% ash. It was concluded that the latter samples were probably poultry by-product meals, and they were 361

2 362 deleted from the study. Amino acids were analyzed by ion-exchange chromatography (Spackman et al., 1958) following hydrolysis of samples in 6 N HCl under N for 24 h at 110 C. Analyses of Met and Cys were performed separately after performic oxidation by the method of Moore (1963), with the exception that the excess performic acid was removed by lyophilization after dilution with water. Chick Assays for Protein Efficiency Ratio and Lys Bioavailability One-week-old male chicks resulting from the cross of New Hampshire males and Columbian Plymouth Rock females were used in all chick assays. The chicks were fed a 24% CP corn-soybean meal pretest diet during the 1st wk posthatching. Following an overnight period without feed, the chicks were weighed, wing-banded, and allotted to dietary treatments as described by Sasse and Baker (1973). Triplicate groups of five chicks were assigned to each dietary treatment and each assay lasted for 9 d. All chicks were housed in thermostatically controlled starter batteries with raised wire floors in an environmentally regulated room. Feed and water were consumed ad libitum, and light was provided 24 h daily. A N-free basal diet (Willis and Baker, 1980) was used in the first two chick assays for determining protein quality of the MBM. Dietary treatments consisted of the N-free basal diet or the basal diet plus 9% CP provided solely by one of the MBM, which replaced some of the cornstarch and dextrose. The protein efficiency ratio (PER) and net protein ratio (NPR) were calculated as follows with all measurements in grams: and PER = BW gain/cp intake NPR = [BW gain BW gain of chicks fed N-free diet]/cp intake. PARSONS ET AL. True Digestibility Assay Mature Single Comb White Leghorn roosters approximately 50 wk of age were used. The birds were housed in an environmentally regulated room and kept in individual cages with raised wire floors and subjected to a photoperiod of 16 h light and 8 h dark daily. Feed and water were supplied for ad libitum access before the start of the experiments. Cecectomy was performed according to the procedure of Parsons (1985) when the birds were 25 wk of age. The CONV roosters received a sham operation within a similar time period. All roosters were given at least 8 wk to recover from surgery prior to being used in experiments. The assay procedure was that described by Sibbald (1979), with some minor modifications. Following a 24-h period without feed, CEC and CONV roosters were given 30 g of a MBM via crop intubation. Additional roosters of each type were deprived of feed throughout the experimental period to measure endogenous excretion of DM, energy, N, and AA. Three roosters were assigned to all treatments. A plastic tray was placed under each cage and excreta were collected quantitatively for 48 h after crop intubation. The excreta samples were lyophilized, weighed, and ground to pass through a 60-mesh screen. Gross energy, N, and AA concentrations were determined on at least two replicates of each individual sample of excreta according to the procedures described previously. True digestibilities of AA were calculated according to the method of Sibbald (1979), and TME n was calculated by the method of Parsons et al. (1982). In Vitro Assays Pepsin digestible N in each MBM sample was determined according to the procedure of the AOAC (1980) using 0.2, 0.002, or % pepsin solutions. Samples were ground to pass through a 20-mesh screen prior to digestion. The samples were not defatted. Protein solubility in 0.2% KOH was performed according to the procedure of Parsons et al. (1991), and multi-enzyme ph change by the procedure of Hsu et al. (1977). A purified crystalline AA diet (Baker et al., 1979) was used to determine bioavailability of Lys in the third chick assay. Graded levels of crystalline test Lys were added to a basal diet deficient in Lys to produce a standard growth curve. A single level of MBM was added to the basal diet at the expense of cornstarch to provide an amount of Lys that would fall within the boundaries of the reference curve. In addition, one level of crystalline AA mixture simulating the average AA profile of the 14 MBM was also included as a dietary treatment to study the effects of MBM AA pattern on bioavailability estimates. The AA mixture included all dietary essential (indispensable) and nonessential (dispensable) AA, which were based on AA analyses except for Trp. Because the latter AA was not analyzed, the NRC (1984) value of 0.28% for Trp in MBM was used. Statistical Calculations and Analyses In the digestibility assay, data were analyzed by ANOVA for a completely randomized design with a factorial arrangement of treatments (14 MBM 2 bird types). In the protein quality assay, differences among treatments were assessed by the least significant difference calculated from the pooled SEM from the ANOVA for a completely randomized design (Steel and Torrie, 1980). Data from the chick growth assays for Lys bioavailability were initially subjected to ANOVA for a completely randomized design (Steel and Torrie, 1980). Slope ratio methodology (Finney, 1978) was then used to estimate bioavailability of AA. Multiple regression equations were calculated with chick weight gain (grams) as the dependent variable and the intake of supplemental crystalline Lys or feedstuff Lys (milligrams) as the

3 PROTEIN QUALITY OF MEAT AND BONE MEAL 363 TABLE 1. General nutrient composition of meat and bone meals (MBM) 1 MBM Sample Crude Ether number Moisture protein extract Ash Ca P (%) Mean Pooled SEM Values are means of duplicate analyses expressed on an air-dry basis. independent variables. The latter were based on the individual feed intakes for the three replicate groups of chicks fed each MBM because only one level of MBM was added to the basal diet (Boebel and Baker, 1982). The bioavailability of Lys in each MBM was estimated by the ratio of its slope to that of the crystalline test Lys, which was assumed to be 100% bioavailable. Correlations between the in vivo (PER, NPR, Lys bioavailability, true Lys, Met, and Cys digestibility in CEC and CONV birds) and in vitro assays (pepsin, KOH, and multi-enzyme ph change) were assessed using Pearson s linear test (Steel and Torrie, 1980). Correlations between the proximate components (moisture, CP, ether extract, and ash) and the in vivo and in vitro assays were also calculated. In addition, the correlation between PER and digestible sulfur AA (CEC roosters) per unit of MBM CP was computed, because the sulfur-containing AA are the most limiting AA in MBM CP (Kratzer and Davis, 1959; Castanon et al., 1988). RESULTS The chemical compositions of the MBM are presented in Tables 1 and 2. Concentrations of CP, ether extract, ash, Ca, P, and AA varied substantially among samples. Chick performance data and calculated PER and NPR values for the protein quality assay are presented in TABLE 2. Amino acid composition of meat and bone meals (MBM) 1 MBM sample number Arg Cys His Ile Leu Lys Met Phe Thr Val (%) Mean Pooled SEM Values are means of duplicate analyses expressed on an air-dry basis.

4 364 PARSONS ET AL. TABLE 3. Evaluation of protein quality of meat and bone meals (MBM) by chick assay Treatment Weight gain 1 Gain:feed 1 PER 2 NPR 2 (g) (g:g) (g) Experiment 1 1. N-free diet d c As 1 + MBMI at 9% CP 4.7 c b 0.62 b 1.76 b 3. As 1 + MBM4 at 9% CP 23.6 b a 2.28 a 3.06 a 4. As 1 + MBM5 at 9% CP 36.5 a a 2.89 a 3.52 a 5. As 1 + MBM7 at 9% CP 7.9 c b 0.98 b 2.00 b 6. As 1 + MBM11 at 9% CP 27.0 ab a 2.31 a 3.00 a 7. As 1 + MBM13 at 9% CP 31.1 ab a 2.78 a 3.51 a 8. As 1 + MBM14 at 9% CP 9.0 c b 1.06 b 2.07 b Pooled SEM Experiment 2 1. N-free diet d d As 1 + MBM2 at 9% CP 11.1 bc bc 1.17 bc 2.47 bc 3. As 1 + MBM6 at 9% CP 22.7 a a 2.26 a 3.50 a 4. As 1 + MBM8 at 9% CP 15.1 b b 1.63 b 2.97 b 5. As 1 + MBM9 at 9% CP 4.8 c c 0.61 c 2.21 c 6. As 1 + MBM10 at 9% CP 28.0 a a 2.45 a 3.55 a 7. As 1 + MBM12 at 9% CP 9.4 bc bc 1.08 bc 2.51 bc 8. As 1 + MBM16 at 9% CP 27.6 a a 2.45 a 3.55 a Pooled SEM a dmeans within columns and experiment with no common superscript differ significantly (P < 0.05). 1Means of three groups of five male chicks each from 8 to 17 d posthatching. Average initial weight was 81.6 g in Experiment 1 and 79.5 g in Experiment 2. 2PER = protein efficiency ratio; NPR = net protein ratio. 3Composition (percentage) was cornstarch:dextrose (2:1), 89.23; corn oil, 5; mineral premix, 5.37; vitamin premix, 0.20; choline chloride, 0.20; a-tocopheryl acetate, 0.002; and ethoxyquin, From Willis and Baker (1980). TABLE 4. Determination of Lys bioavailability in meat and bone meals (MBM) 1 Weight Gain: Supplemental Lys Lys Treatment gain feed intake bioavailability 2 (g) (g:g) (mg) (%) 1. Basal diet (0.55% L-Lys) As % L-Lys As % L-Lys As 1 + AASIM MBM ± 6 5. As % MBM ± 6 6. As % MBM ± 6 7. As % MBM ± 6 8. As % MBM ± 5 9. As % MBM ± As % MBM ± As % MBM ± As % MBM ± As % MBM ± As % MBM ± As % MBM ± As % MBM ± As % MBM ± As % MBM ± 8 Pooled SEM Means of three replicate groups of five chicks each from 8 to 17 d posthatching. Average initial weight was 83.1 g. 2Calculated by the slope-ratio procedure. The multiple regression of gain on supplemental Lys intake from the different sources was: Weight gain = Lys AASIM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM16; R 2 = Crystalline amino acid mixture that simulates the average analyzed amino acid profile of 10% MBM.

5 Table 3. Chicks fed the N-free diet lost weight during the assay period, whereas chicks fed MBM had positive responses in weight gain. Calculated PER values averaged 1.78 with a range of 0.61 to As expected, NPR values were greater than the corresponding PER values. Chicks responded linearly in weight gain and gain: feed ratio to Lys supplementation of the basal diet in the Lys bioavailability assay (Table 4). Supplementation of the basal diet with 10% of a MBM or of an AA mixture simulating MBM protein significantly improved weight gain for all MBM. The Lys bioavailability values of MBM based on weight gain varied from 43% for MBM 14 to 89% for MBM 5. Bioavailability of crystalline L-Lys in the simulated MBM AA mixture was 85%. This value was significantly (P < 0.01) less than that of crystalline L- Lys (100%) when supplemented alone. True digestibility of 10 AA determined with CONV and CEC cockerels and differences in digestibility values between bird types are summarized in Table 5. Digestibility of AA differed among MBM and between bird types (P < 0.05) but the interaction between MBM and bird types was not significant (P > 0.05). Digestibility was generally highest for MBM 5 and MBM 13 and lowest for MBM 12 and MBM 14. Cecectomized birds generally yielded lower digestibility values than CONV birds, although the latter effect was not consistent in all cases. The largest difference between bird type was observed for Cys. The TME n content of the MBM varied among samples (P < 0.05), and values determined in CEC cockerels were generally lower than those determined in CONV cockerels (Table 6). The difference was significant (P < 0.05) when averaged over all MBM samples. The results of the in vitro pepsin, KOH protein solubility, and multi-enzyme ph change assays are shown in Table 7. Decreasing the pepsin concentration from 0.2 to and % yielded incremental reductions in N digestibility. The variation in pepsin N digestibility among MBM samples also increased markedly as pepsin concentration decreased. The KOH protein solubility values were low and did not differ greatly among MBM samples. Multi-enzyme ph change values varied from 0.19 to 1.07 among MBM samples. Correlation analyses between protein quality assays indicated that PER values were significantly (P < 0.05) negatively correlated with ash, and positively correlated with CP, digestible SAA as a percentage of CP, Lys bioavailability, and Cys and Met digestibility determined in CEC roosters and Lys digestibility determined in CONV roosters (Table 8). The correlation coefficients for NPR values (not shown) were almost identical to those for PER. True Lys digestibility determined in CONV and CEC roosters was also positively correlated (P < 0.05) with Lys bioavailability. Nitrogen digestibility determined with or % pepsin was positively correlated (P < 0.05) with Lys bioavailability and Lys digestibility determined in CEC and CONV roosters. No other significant (P > 0.05) correlations were found PROTEIN QUALITY OF MEAT AND BONE MEAL 365

6 366 PARSONS ET AL. TABLE 6. The TME n content of meat and bone meals (MBM) determined in conventional and cecectomized cockerels 1 MBM sample number Conventional Cecectomized (kcal/g DM) Mean SEM Values are means of three individual roosters. Main effects of MBM sample and bird type were significant (P < 0.05). between in vivo and in vitro protein quality measurements or proximate components. DISCUSSION It has been previously documented that the nutritional quality of MBM is variable (Wilder, 1973; Johnston and Coon, 1979a). The results reported herein support the latter finding and indicate that variability is even greater than reported in the earlier studies. For example, the PER values of MBM varied from 0.6 to 2.9 in our study compared with 1.7 to 3.0 in the study of Johnston and Coon (1979a). The exact reasons for such variability in protein quality are not known but are probably due mainly to variation in raw material composition and processing conditions (Batterham et al., 1986). Variation in protein quality is due both to concentration and digestibility of the AA in MBM. Both the Lys bioavailability chick assay and the rooster digestibility assays indicated considerable variation in AA availability or digestibility. The Lys bioavailability values were generally lower and more variable than the Lys digestibility values, although they were correlated with one another. Nordheim and Coon (1984) also reported that the variability in chick assay Lys bioavailability values for MBM was higher than for Lys digestibility values; however, in contrast with our findings, the bioavailability values were higher than the digestibility values. The lower bioavailability values in our study were probably, at least partially, due to the adverse effects of the MBM AA pattern. The bioavailability of the Lys in the MBM-simulated mixture was only 85% even though this was the same source of Lys as used in the diets for the Lys reference curve. The adverse effects of ingredient AA pattern on AA bioavailability have been reported for other ingredients (Hirakawa and Baker, 1986; Han and Parsons, 1991). If the Lys bioavailability values for the MBM are ratioed to that of the Lys in the MBM-AA mixture, the bioavailability values are still generally lower but more similar to the values produced in the rooster digestibility assay. Amino acid digestibility and TME n values for CEC roosters were generally lower than values for CONV roosters; however, this response was not consistent for all samples. The reason for the variation in response is unknown. The observation that CEC birds often yield lower AA digestibility values than CONV birds has TABLE 7. Protein quality assessment of meat and bone meals (MBM) using several in vitro assays MBM sample Pepsin concentration 0.2% 0.002% % KOH Multi-enzyme (% soluble N) (ph change 2 ) MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM MBM Mean Pooled SEM Values are means of duplicate analyses. 2pH change after 20 min of incubation with trypsin, chymotrypsin, and peptidase.

7 PROTEIN QUALITY OF MEAT AND BONE MEAL 367 TABLE 8. Significant (P < 0.05) correlation coefficients between assays 1 Assay r Protein efficiency ratio vs ash 0.80 Protein efficiency ratio vs CP 0.68 Protein efficiency ratio vs DSAA/CP Protein efficiency ratio vs Lys bioavailability 0.61 Protein efficiency ratio vs Cys digestibility in CEC roosters Protein efficiency ratio vs Met digestibility in CEC roosters 0.70 Protein efficiency ratio vs Lys digestibility in CONV roosters 0.54 Lys bioavailability vs Lys digestibility in CONV roosters 0.64 Lys bioavailability vs Lys digestibility in CEC roosters % pepsin N digestibility vs Lys digestibility in CEC roosters % pepsin N digestibility vs Lys digestibility in CONV roosters % pepsin N digestibility vs Lys digestibility in CEC roosters % pepsin N digestibility vs Lys digestibility in CONV roosters % pepsin N digestibility vs Lys bioavailability Values are Pearson correlation coefficients. 2DSAA/CP = digestible sulfur amino acids (cecectomized roosters) per unit of MBM CP; CEC = cecectomized; CONV = conventional. 3Correlations between 0.2% pepsin N digestibility and Lys digestibility in CEC and CONV roosters were 0.25 and 0.40, respectively, and not significant (P < 0.1). been reported previously (Parsons, 1985, 1986; Johns et al., 1986; Han and Parsons, 1991; Green and Kiener, 1989). The large difference between bird types for Cys digestibility in MBM was also observed by Han and Parsons (1991) for feather meal and may be due to microbial metabolism of Cys in the ceca. Correlation analyses indicated that PER values were significantly correlated with several MBM parameters. Interestingly, PER was negatively correlated with ash. As discussed by Partanen (1994), ash has been suggested to be a good indicator of MBM protein quality because it reflects the bone and collagen (poor quality protein) content of the meal. However, little or no data have been published to support the latter hypothesis, and Partanen (1994) found no relationship between ash content of MBM and apparent CP digestibility and N retention in pigs. Our results indicate that ash may, indeed, be a useful indicator of MBM protein quality; however, further work is needed to verify these initial results. The positive correlation between PER and CP content may be largely due to the ash effect, because the samples containing the least ash also contained the highest CP. As expected, there was a high positive correlation between PER and digestible SAA as a percentage of MBM CP. This high correlation resulted because the SAA (or specifically Cys) and Trp are the first limiting AA in MBM protein due to their low concentration and low digestibility (Wang et al., 1996). There would probably also have been a high correlation between PER and digestible Trp as a percentage of MBM CP. The positive correlation between PER and Cys and Met digestibility (CEC roosters) indicated that digestibility of the SAA, in addition to total content, contributed substantially to the differences in PER among MBM. The pepsin digestibility assay is one of the most commonly used tests for monitoring protein quality of MBM. Our results support those of Johnston and Coon (1979b), who indicated that 0.002% pepsin is superior to the AOAC (1980) recommended pepsin level of 0.2% for MBM. Correlation of 0.2% pepsin values with all in vivo protein quality measurements was not significant (P < 0.10) and generally very low. Our results further show that there is little or no advantage to reducing the pepsin concentration from to % for MBM assays. Although there was a positive correlation between pepsin values and Lys digestibility and bioavailability, the correlations did not exceed 0.70 and there were no significant correlations between pepsin values and other protein quality measurements (e.g., PER or SAA digestibility). Thus, although the pepsin digestibility assay is useful for predicting differences in Lys digestibility among MBM, there is a continued need for in vitro assays that are better predictors of in vivo quality than the pepsin assay. Although the KOH protein solubility and multienzyme ph change assays have been shown to be useful in vitro assays for many ingredients (Hsu et al., 1977; Araba and Dale, 1990; Parsons et al., 1991), we found that they were not useful for predicting variation in MBM protein quality. Modifying the multi-enzyme ph change assay to minimize the buffering effects of ingredients (known as the ph stat assay; Pedersen and Eggum, 1983) has been shown to improve its sensitivity. Thus, perhaps the ph stat assay may have been more sensitive than the multi-enzyme assay used herein. However, our correlations between the multi-enzyme assay and in vivo MBM protein quality measurements were very low and often negative. Thus, it is doubtful that the ph stat assay would have been sensitive. ACKNOWLEDGMENT Appreciation is expressed to the Fats and Proteins Research Foundation, Inc. for their support of this study.

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