Research Article pubs.acs.org/journal/ascecg
Practical Limitations of the Dilute Acid Hydrolysis Method for Solubilizing Meat and Bone Meal Protein Matthew Essandoh,* Rafael A. Garcia, Christine M. Nieman, Lorelie P. Bumanlag, George J. Piazza, and Congmu Zhang United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Biobased and Other Animal Coproducts Research Unit, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, United States S Supporting Information *
ABSTRACT: A previous study reported conditions that hydrolyze meat and bone meal (MBM) protein in a sitespecific manner, transforming insoluble MBM protein into large, soluble protein fragments with good flocculant functionality. It is not clear, however, that this phenomenon can be adapted for industrial-scale processing of MBM at reasonable expense. The present study examines whether the desirable characteristics of the reaction product are retained when hydrolysis is conducted using much higher MBM concentration, MBM with relatively large solid particles, and in the presence of fat and bone. The results showed that bone mineral depressed the conversion rate progressively as the MBM concentration increased. An increase in acid concentration reversed the rate depression, and good conversion rates were achieved for up to 200 g of substrate per liter. Particle size and fat showed no consistent effect on the conversion rate. The reaction was shown to retain its site specificity under all conditions tested. Higher MBM concentration and longer reaction times both favored the production of relatively large peptides (MW >5 kDa). Finally, the protein hydrolysate was shown to retain flocculant properties comparable to those of water-extracted MBM protein. These results indicate that the reaction under consideration could be used to convert MBM protein into a more valuable functional product. KEYWORDS: Rendered protein, Meat and bone meal, Bioflocculant, Amino acid analysis, Size exclusion chromatography, Protein Extraction, Protein hydrolysis
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INTRODUCTION Meat and bone meal (MBM) is a product from the rendering of slaughterhouse bones and offal.1 In the US, MBM is used almost exclusively as an ingredient in livestock and companion animal feed;2 in other countries, some MBM is used for fertilizer or fuel.3 Each of these application types are relatively low value; the rendering industry would benefit significantly if MBM application could be diversified into higher value uses. Such uses might include the production of flocculants,4 adhesives,5 films,6 or bioactive peptides.7 Most opportunities to use protein in higher value applications require the protein to be soluble. MBM is typically ≥50% protein; however, the majority of this protein is insoluble under mild conditions.8 Various authors have employed hydrolysis as a way to increase the functionality and solubility of proteins including fish,9 casein,10 and soy protein.11 Previously, our research group worked on solubilizing MBM protein through both chemical and enzymatic means for the purposes of flocculant production.12−14 Through a series of studies, it was shown that high proportions of MBM protein could be solubilized through hydrolysis with strong alkali or various proteases, but the small size of the resulting protein fragments limited the flocculant activity. Although acid hydrolysis is widely recognized to lyse polypeptides more or less randomly, protein structural analysis © 2017 American Chemical Society
literature of the 1960s contains references to reaction conditions under which acid hydrolysis is highly specific, breaking peptide bonds only at aspartic acid (Asp) and asparagine (Asn) residues.15−17 All reports of this reaction were related to structural analysis of pure, soluble proteins. As the field of protein structural analysis progressed, this technique apparently became obsolete and fell out of use; we were unable to find any reference to its use after 1967. Recently, Zhang et al.18 tested this dilute acid hydrolysis reaction on MBM protein. The reaction increased protein solubility 3−4-fold and produced protein fragments that were much larger and had much better flocculant properties compared to those of other hydrolysis methods. Application of the reaction to MBM showed that it could be used with heterogeneous mixtures of insoluble proteins. Despite these results, it is unclear whether this reaction will provide a practical approach for transforming MBM into a more useful protein hydrolysate. The experiments were conducted using conditions that might be unnecessarily expensive to apply at large scale. In particular, fat had been extracted from the MBM; the defatted material had been milled down to a small particle size, and very low substrate Received: August 31, 2017 Revised: October 19, 2017 Published: October 26, 2017 11652
DOI: 10.1021/acssuschemeng.7b03050 ACS Sustainable Chem. Eng. 2017, 5, 11652−11659
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Free Amino Acid Analysis. Acid hydrolysates and water extracts were treated with 4 volumes of acetone and centrifuged to precipitate intact proteins and large peptides. Subsamples of the supernatant were dried under vacuum and derivatized with AccQFluor reagent (Waters Corp.) according to the manufacturer’s directions. Chromatography was performed using an HPLC method described as “system 1” in the reference22 with α-aminobutyric acid as an internal standard. Separation was achieved using an AccQTag C18 reverse phase column (Waters Corp.); detection by fluorescence used excitation with 250 nm light and measured emission at 395 nm. For the release of free amino acids due to hydrolysis to be quantified, analytical results from acid hydrolysates were compared to water extracts produced using the same substrate type, concentration, and reaction time according to
concentrations were used (2 mg of protein substrate/mL). The present study examines whether the defatting, milling, and low substrate concentrations are actually required for achieving high yield, high specificity, and good flocculation characteristics.
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MATERIALS AND METHODS
Milling and Extraction of Meat and Bone Meal (MBM). Approximately 50% crude protein, mixed-species MBM was donated by Darling Ingredients (Irving, TX). Experiments used normal MBM (as received), as well as MBM that had been defatted (MBM-D), MBM that had been milled into fine powder (MBM-M), or MBM that had been defatted and milled (MBM-DM). MBM-M and MBM-DM were ground into a fine powder using a cryogenic mill (model 6800, Spex Certiprep, Metuchen, NJ). MBM-D and MBM-DM (5g), which were previously dried, were defatted through Soxhlet extraction using hexane (∼160 mL); after extraction for a period of approximately 5 h, the samples were dried of both solvent and residual water. Fat content was calculated by comparing the mass of the extracted fat with initial mass of the sample. Proximate Analysis. Nitrogen analyses were used to estimate protein content; solid MBM and liquid extracts were analyzed using different methods. MBM and its various preparations were analyzed by Kjeldahl nitrogen determination according to a standard method.19 Analyses were performed in quadruplicate using a system comprised of a Tecator Digestor Auto (Foss, Eden Prairie, MN) and a Kjeltec 8100 Distillation Unit (Foss). Extracts and hydrolysates were analyzed using a TNT 880 s-TKN kit (Hach, Loveland, CO) following manufacturer’s directions; this kit is designed to produce values equivalent to standard total Kjeldahl nitrogen (TKN) analysis of water. Analyses were performed in triplicate. For both types of nitrogen analysis, a nitrogento-protein conversion factor of 5.37 was used based on the results of Sriperm et al.20 Moisture contents of the different substrate samples were determined by decrease in mass after incubating at 105 °C for 24 h. Ash content was found by heating the moisture-free samples overnight in a muffle furnace at 600 °C until a constant weight was obtained. Particle Size Distribution. Particle size distribution of MBM (as received) was investigated through a standard sieve stack method.21,17 In brief, each empty sieve was weighed; the sieves were stacked in order with the largest opening size sieves on top, and then ∼50 g of the sample was placed on the top of the stack. The stack was shaken for 10 min, and then each sieve was weighed to determine the mass of sample retained. The geometric mean particle size (dgw) and the lognormal geometric standard deviation (Slog) were then computed. Particle size distribution of MBM-M was carried out using a laser diffraction method (Mastersizer 3000, Malvern Instruments, Worcestershire, UK). A suspension of MBM-M in water was used in the analysis. Measurements were taken when obscuration was ∼18%. Analyses were conducted in triplicate. Morphology Studies. Images before and after defatting the MBM were carried out under a vacuum using a scanning electron microscope (FEI Quanta 200 F, Hillsboro, OR, USA). The accelerating voltage used in this study was 10 kV. Protein Extraction and Hydrolysis. Water extractions and acid hydrolyses were conducted in glass vials fitted with valve caps. MBM and water or a 0.03 N HCl solution were added to the vials. pH was adjusted to 1.6−1.9 if necessary, and then the final volume was brought to 15 mL. The MBM substrate concentrations used in the experiment included 0, 25, 50, 100, and 200 g/L. A PicoTag workstation (Waters Corp., Milford, MA, USA) was used to evacuate the vial headspace, purge with nitrogen, and re-evacuate; this process was repeated one or more times, particularly when higher MBM loadings were used, due to the increased bubbling and foaming observed with these experiments. Vials were placed on a rotator in a 105 °C oven. Rotation was adequate to keep MBM well mixed in the suspension. The reaction time was 1 h except where noted otherwise. After the reaction was completed, the vials were cooled; the contents were centrifuged (15000g for 30 min), and the supernatant was diluted and stored at 4 °C.
%FAA i =
H̅ i − Ei̅ × 100% Ei̅
(1)
where % FAAi = percent increase in the amount of free amino acid i in hydrolysate compared to extract, H̅ i = mean concentration of amino acid i in hydrolysate, and E̅ i = mean concentration of amino acid i in water extract. Standard deviations of results were estimated using an appropriate propagation of error formula19,23 according to
sFAA i =
H̅ i Ei̅
sE2 Ei̅ 2
+
s H2 H̅ i2
(2)
where SFAAi = standard deviation of % FAAi and sE and sH = standard deviations of amino acid in extract and hydrolysate, respectively. Molecular Weight Distribution. The molecular weight distribution of the soluble proteins were studied using high-performance size exclusion chromatography (HP-SEC). Chromatography was carried out on an HPLC system (Alliance 2695 Separations Module, Waters Corp.) with a photodiode array detector (model 996, Waters Corp.). The column used was the BioSep SEC-s3000 (Phenomenex, Torrance, CA, USA). This prepacked column is designed for the separation of peptides, proteins, and other biological molecules with molecular weight between 5 and 700 kDa. Chromatography experiments were run for 26 min using 50 mM ammonium bicarbonate (pH 7) as the eluting solvent. UV absorbance at a wavelength of 254 nm was used to detect all eluted compounds. Protein standard mixtures were also run under the same experimental condition for calibration graph preparation. All data generated were analyzed with Empower Pro software (Waters Corp.). Flocculation Effectiveness. The flocculant properties of extracts and hydrolysates were determined according to a published method24 with modifications. Briefly, tests were conducted using a model suspension of 1 g/L of kaolin clay in 25 mM Malic-MES-Tris buffer, pH 5.5.25 Extract or hydrolysate was added to the suspension and shaken for 1 min at 400 rpm, followed by another 15 min of shaking at 200 rpm. The turbidity of the suspension was measured at 10 and 30 min postshaking. The initial turbidity (Ti) and final turbidity (Tf) of the suspension are used to determine the kaolin clarification effectiveness (KCE), which is computed according to
⎛T ⎞ KCE(s.c.) = log10⎜ i ⎟ ⎝ Tf ⎠
(3)
KCE is a log scale quantity, where a result of 1 represents a 90% reduction in turbidity, a result of 2 represents a 99% reduction, and so on. Statistical Analysis. All experiments were carried out using a minimum of triplicate samples unless otherwise specified. The results obtained are reported as the mean ± standard deviation (SD). We used IBM SPSS Statistics software (SPSS Inc., Chicago, IL, USA) to carry out analysis of variance (ANOVA) and Tukey’s Honest Significant Difference (HSD) test for multiple comparisons among treatments. Results were considered significant at p < 0.05. 11653
DOI: 10.1021/acssuschemeng.7b03050 ACS Sustainable Chem. Eng. 2017, 5, 11652−11659
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RESULTS AND DISCUSSION MBM Properties and the Effects of Defatting and Milling. In this study, it was hypothesized that the large size of the MBM particles and the presence of fat could limit protein extraction or hydrolysis. The MBM used in this study had a broad particle size distribution (Figure 1a), and a considerable
Table 1. Proximate Analysis of MBM, MBM-M, MBM-D, and MBM-DMa crude protein (%, w.b.) (N × 5.37) MBM milled MBM defatted MBM defatted milled MBM
44.7 44.1 47.9 48.2
± ± ± ±
0.9a 0.2a 0.7b 0.3b
moisture content (%) 4.2 4.6 3.8 1.3
± ± ± ±
0.1a 0.0a 0.5a 0.2b
ash (%, w.b.) 29.7 30.8 33.6 33.8
± ± ± ±
0.5a 0.1b 0.3c 0.1c
Reported values are mean ±1 standard deviation. A minimum of triplicate samples was used. Different lowercase letters are significantly different (p < 0.05) according to Tukey’s HSD mean separation test. a
a range of 18 different MBM samples that were studied that have ash compositions between 20.7 and 39.9%.1 This high ash content is mainly due to the presence of bone particles in the MBM samples. Differences in ash contents were not statistically significant (p = 0.835) for MBM-D vs MBM-DM. In the case of moisture content, no statistically significant (p < 0.05) results were obtained except when any of the substrate is being compared to MBM-DM. As expected, milling did not show any statistical difference in crude protein content. However, the crude protein content of defatted materials (MBM-D and MBM-DM) was significantly increased (p < 0.05) compared to those of full fat materials (raw MBM and MBM-M). This result is consistent with a prior report in which MBM crude protein content increased from 54.5 to 61.2% after defatting.26 Changes in fat content significantly affected the morphology of the MBM samples (Figure 1c and d). Compared to raw MBM, the particles of defatted MBM (MBM-D) are smaller and more homogeneous. The fat in MBM is known to make the particles cohesive,27 so it may be defatting frees particles that had previously been adhered to one another. Other authors have also shown through SEM micrographs that MBM contains irregularly shaped particles.28 MBM Protein Solubilization. Under the conditions used, MBM protein had limited solubility in hot water (no acid added). Figure 2 shows that, although the amount of protein extracted increases with time, after 2 h only ∼20% of the total
Figure 1. Particle size distribution of the (a) raw MBM and (b) MBMM. The raw MBM sample particle size distribution was determined using a sieve stack technique, and the MBM-M was characterized using laser diffraction. SEM images of (c) MBM and (d) defatted MBM.
proportion of the mass was particles greater than 100 μm. The geometric mean particle size (dgw) was 455.62 μm, and the lognormal geometric standard deviation (Slog) was calculated to be 0.29 μm. Cryogenic milling was used to prepare MBM with much smaller particles (MBM-M). Particle size distribution of MBM-M (Figure 1b) was also broad, but the particles were generally much smaller compared to those of the unmilled MBM. The volume weighted mean diameter of the MBM-M particle was found to be 42.53 μm. Milling was therefore found to reduce the mean particle size to approximately one-tenth of the unmilled sample. The very broad range of particle sizes in these materials (submicrometer to greater than 1 mm) precludes using the same technique to characterize both sample types. It is also important to recognize that both techniques are based on the assumption that the particles are spherical. However, this assumption is significantly violated, as will be shown later through microscopic imaging, and presumably results in some bias in the measurements. The fat content (dry basis) of raw MBM was found to be 9.14%. Comparing this result to a report in which the median fat content of MBM from multiple sources was found to be 12.2%, the fat content of the MBM used in the present study is on the low side of the typical range.1 Fat extraction resulted in products (MBM-D and MBM-DM) with fat contents too low to measure. As expected, the removal of fat increased the proportion of protein and ash in the remaining materials (Table 1). The ash content for all the samples ranged from 29.7 to 33.8%. The ash content in all four samples studied were within
Figure 2. Percent of total protein solubilized from MBM and MBMDM by water extraction (MBMw and MBM-DMw) and dilute acid hydrolysis (MBMa and MBM-DMa) at 25 g/L for different reaction times. Error bars represent ±1 standard deviation. 11654
DOI: 10.1021/acssuschemeng.7b03050 ACS Sustainable Chem. Eng. 2017, 5, 11652−11659
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Figure 3. Effect of MBM concentration (g/L) on the initial pH (red, square) and percent of total MBM protein solubilized (blue, triangle) in hydrolysis reactions. MBM was suspended in 0.03 M HCl (a) without pH adjustment or (b) with the addition of 6 M HCl to bring the pH to 1.6− 1.9. Error bars represent ±1 standard deviation and are present for all data points.
Figure 4. Concentrations of select amino acids and ammonia (NH3) in (a) water extracts and (b) hydrolysates of raw MBM over the reaction duration. Error bars representing ±1 standard deviation are present for all data points but in most cases are too small to be visible.
MBM protein had been solubilized (MBMw). Somewhat more protein was extracted from MBM that had been defatted and milled prior to extraction (MBM-DMw). Dilute acid hydrolysis brings a much greater proportion of the MBM protein into solution, up to ∼63% after 2 h of hydrolysis (MBM). Thus, dilute acid hydrolysis solubilizes more protein than water extraction. Similar patterns were seen when comparing MBMDMw and MBM-DMa. The MBM concentration, in prior reports as well as our results presented up to this point, was 2 g/L. This concentration, however, is too low for a practical process unless the end product has a very high value. A series of experiments involving increasing the MBM concentration in the reaction by up to 100-times (to 200 g/L) was therefore carried out. It was found that, when the MBM was used at greater concentrations, the reaction pH increased and the proportion of MBM protein solubilized decreased sharply (Figure 3a). Prior reports on this hydrolysis method specify 0.03 M HCl, which will theoretically produce a pH of 1.52. Hydroxyapatite (Ca10(PO4)6(OH)2), the predominant mineral in bone, is highly soluble at pH 1.5,29 and its dissolution yields phosphate ions. At low pH, phosphate ions absorb hydrogen ions and raise the solution pH. This presumably accounts for the trend of increasing reaction pH when using greater MBM concentrations (Figure 3b). In all further acid hydrolyses, MBM was first suspended in 0.03 M HCl, and then 6 M HCl was added until the suspension equilibrated within the pH range 1.6−1.9. This practice substantially enhanced the proportion of
protein solubilized when MBM was added at elevated concentrations (Figure 3b); at 100 g/L, 57% of MBM protein was solubilized compared to 22% in the absence of pH adjustment. Effect of Different Substrate Types and Concentrations. A central concern of the present study is whether the presence of fat, or the relatively large particle size MBM, would interfere with the hydrolysis. Contrary to the hypothesis that MBM-DM would be solubilized most readily, there was no consistent difference in hydrolysis between the different MBM preparations (Figure S1a). Increasing substrate concentration does, however, depress the proportion of the MBM protein that is solubilized. At the lowest MBM concentrations tested, 65− 75% of the protein was solubilized, whereas at the highest concentrations tested, 46−52% of the protein was solubilized. It is likely that the MBM concentration could be increased further, although care would be required to ensure that mixing at such high concentrations is adequate. Other authors have also observed a reduced rate of solubilization at higher fish protein concentrate.30 Interestingly, the trend of higher substrate concentration depressing the rate of protein solubilization does not apply to water extraction, where the percent of protein solubilized remains approximately constant over the MBM concentration range tested (Figure S1b). Hydrolysis Reaction Specificity. Free amino acids were measured to evaluate whether the reaction was producing the hydrolysis site specificity that has been reported in the literature. Ideally, the reaction conditions deamidate asparagine 11655
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Figure 5. Percent difference in concentration of free amino acids in acid hydrolysates relative to water extracts prepared using the same conditions. (a) Results obtained using defatted and milled MBM reacted for 1 h over a range of substrate concentrations. (b) Results obtained using the various MBM preparations at 25 g/L and reacted for 1 h. Error bars represent ±1 standard deviation.
Figure 6. Effects of (a) 1 h hydrolysis at 25 g/L concentration for different MBM preparations, (b) concentration on defatted milled MBM using 1 h hydrolysis, and (c) time on raw MBM at 25 g/L concentration on the molecular weight distribution profile.
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Figure 7. Effect of time and flocculant dose on kaolin clarification efficiency at pH 5.5. Both the water extract and acid hydrolysate flocculants were prepared using a substrate concentration of 50 g/L and reaction time of 1 h. Data points shown are mean ± standard deviation. All flocculation experiments were carried out using triplicate samples. Error bars are present for all the data points shown, but in most cases they are too small to be seen.
(Asn) residues, yielding aspartic acid (Asp) residues and free ammonia (NH3), and hydrolyze the peptide chain on both sides of Asp residues, yielding free Asp. This should result in increased concentrations of free Asp and NH3, and no more than slight increases in the concentrations of other free other amino acids (these would result from the hydrolysis of sites such as -Asp-X-Asp- or -Asn-X-OH). The analytical method resolved and quantified 16 types of free amino acids as well as NH3. All water extracts contained some measurable amount of each type of free amino acid (data not shown), which is to be expected in physiological samples.31,32 In water extracts, none of the amino acid concentrations changed with increasing reaction duration, although the concentration of NH3 roughly doubled between 30 min and 2 h. Figure 4a shows the concentrations of NH3, Asp, and four other representative amino acids in extracts of raw MBM. These results suggest the free amino acids are extracted into water completely within 30 min and that hydrolysis is not occurring. In acid hydrolysates, only concentrations of Asp and NH3 increase with reaction time (Figure 4b). Comparison of the results from 1 and 2 h shows that the hydrolysis is not complete at 1 h. For all substrate concentrations (Figure 5a) and MBM preparations (Figure 5b) tested, hydrolysates had greatly increased concentrations of free Asp and NH3 compared to those of the corresponding water extracts. These results show that the level of nonspecificity of the hydrolysis is low. The methods used, though, cannot differentiate between complete specificity and a low level of nonspecificity. The lack of any
obvious trends related to substrate concentration or MBM preparation type suggest the MBM can be hydrolyzed without preparation, in high concentration, without a major compromise in specificity. Molecular Weight Distribution. The molecular weight distribution of hydrolyzed MBM protein is expected to be in a lower range compared to that for water-extracted MBM protein. Analysis of the experimental samples, however, shows that the MW distributions of MBM protein prepared through extraction or hydrolysis are not different (Figure S2). The chromatograms of the extract and hydrolysate are very similar, differing mostly in the relative size of the three major peaks. Bioactive peptides are generally in the range of 3−20 amino acids33 or approximately 330−2200 Da. Unfortunately, the method chosen to study MW distribution was not very effective at separating MW 0.05). The KCE increased from 0 to ∼0.8 with a flocculant dose of 40 mg of protein/g of kaolin for the water extract samples (Figure 7a and b). Increasing the flocculant dose beyond this value saw a dramatic decrease in KCE values. At the highest dose (200 mg of protein/g of kaolin) employed, there was no clarification in the kaolin suspension. Testing flocculation with acid hydrolysates (Figure 7c and d) showed little difference when comparing substrate types, but otherwise, the results were very different (statistically significant) from those generated using the water extracts (p < 0.05). At most doses, the suspensions continued to clarify between 10 and 30 min. Compared to the water extracts, the acid hydrolysates had to be used at higher doses to achieve peak clarification, but the peak clarification itself was substantially better. The percentage increase in peak KCE values in switching from water extract to acid hydrolysate was ∼69 to 73%. Flocculation with hydrolysate is also notably less sensitive to overdosing; that is, the drop in KCE due to using a dose greater than the optimum is very modest compared to the sharp drop off observed in the case of water extract.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b03050. Effect of substrate concentration on protein solubilization (Figure S1) and size exclusion chromatography analysis of dilute acid and MBM water extract hydrolysate (Figure S2) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: (215) 836-6909; fax: (215) 233-6795; e-mail: fl
[email protected]. ORCID
Matthew Essandoh: 0000-0002-3117-9141 Notes
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. The authors declare no competing financial interest.
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REFERENCES
(1) Garcia, R. A.; Rosentrater, K. A.; Flores, R. A. Characteristics of North American meat & bone meal relevant to the development of non-feed applications. Appl. Eng. Agric. 2006, 22, 729−736. (2) Bisplinghoff, F. D. A history of North American rendering industry. In Essential rendering, all about the animal by-products industry; Meeker, D. L., Ed.; National Renderer’s Association: Alexandria, VA, 2006; pp 17−30. (3) Mekonnen, T.; Mussone, P.; Bressler, D. Valorization of rendering industry wastes and co-products for industrial chemicals, materials and energy: review. Crit. Rev. Biotechnol. 2016, 36 (1), 120− 131. (4) Piazza, G. J., Garcia, R. A. Methods for flocculating suspensions using biobased renewable flocculants. US 8,313,654, filed April 19, 2010, and issued November 20, 2012. (5) Mekonnen, T. H.; Mussone, P. G.; Choi, P.; Bressler, D. C. Development of proteinaceous plywood adhesive and optimization of its lap shear strength. Macromol. Mater. Eng. 2015, 300 (2), 198−209. (6) Kaewprachu, P.; Osako, K.; Benjakul, S.; Suthiluk, P.; Rawdkuen, S. Shelf life extension for Bluefin tuna slices (Thunnus thynnus) wrapped with myofibrillar protein film incorporated with catechinKradon extract. Food Control 2017, 79, 333−343. (7) Pleissner, D.; Venus, J. Utilization of protein-rich residues in biotechnological processes. Appl. Microbiol. Biotechnol. 2016, 100 (5), 2133−2140. (8) Garcia, R. A.; Phillips, J. G. Physical distribution and characteristics of meat and bone meal protein. J. Sci. Food Agric. 2009, 89 (2), 329−336. (9) Valencia, P.; Pinto, M.; Almonacid, S. Identification of the key mechanisms involved in the hydrolysis of fish protein by Alcalase. Process Biochem. 2014, 49 (2), 258−264.
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CONCLUSIONS Results showed that although the presence of bone phosphate does interfere with the hydrolysis reaction when the substance concentration is increased, this can be corrected with an increase in the acid concentration. The presence of fat and large particle size do not have major negative impacts on the performance of the reaction, so an industrial process to implement this reaction would not require milling or defatting 11658
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ACS Sustainable Chemistry & Engineering (10) Wang, J.; Su, Y.; Jia, F.; Jin, H. Characterization of casein hydrolysates derived from enzymatic hydrolysis. Chem. Cent. J. 2013, 7, 1−8. (11) de Oliveira, C. F.; Correa, A. P. F.; Coletto, D.; Daroit, D. J.; Cladera-Olivera, F.; Brandelli, A. Soy protein hydrolysis with microbial protease to improve antioxidant and functional properties. J. Food Sci. Technol. 2015, 52 (5), 2668−2678. (12) Garcia, R.; Pyle, D.; Piazza, G.; Wen, Z. Hydrolysis of animal protein meals for improved utility in non-feed applications. Appl. Eng. Agric. 2011, 27 (2), 269−275. (13) Piazza, G.; Garcia, R. Proteolysis of meat and bone meal to increase utilisation. Anim. Prod. Sci. 2014, 54 (2), 200−206. (14) Coll, B. A.; Garcia, R. A.; Marmer, W. N. Diffusion of protease into meat & bone meal for solubility improvement and potential inactivation of the BSE prion. PLoS One 2007, 2 (2), e245. (15) Tsung, C. M.; Fraenkel-Conrat, H. Preferential release of aspartic acid by dilute acid treatment of tryptic peptides. Biochemistry 1965, 4, 793−801. (16) Schultz, J. Cleavage at aspartic acid. Methods Enzymol. 1967, 11, 255−263. (17) Schultz, J.; Allison, H.; Grice, M. Specificity of the cleavage of proteins by dilute acid. I. Release of aspartic acid from insulin, ribonuclease, and glucagon. Biochemistry 1962, 1 (4), 694−698. (18) Zhang, C.; Garcia, R. A.; Piazza, G. J. Solubilization of meat & bone meal protein by dilute acid hydrolysis for the production of biobased flocculant. Food Bioprod. Process. 2017, 102, 362−366. (19) Horwitz, W. AOAC Official Method 2001.11 Protein (crude) in animal feed, forage (plant tissue), grain and oilseeds. In Official Methods of Analysis of AOAC International, 3rd ed.; Horwitz, W., Latimer, G., Eds.; AOAC International: Gaithersburg, MD, 2000. (20) Sriperm, N.; Pesti, G. M.; Tillman, P. B. Evaluation of the fixed nitrogen-to-protein (N: P) conversion factor (6.25) versus ingredient specific N: P conversion factors in feedstuffs. J. Sci. Food Agric. 2011, 91 (7), 1182−1186. (21) ASTM Standards. D 1921-01. Standard test methods for particle size (sieve analysis) of plastic materials. ASTM International: West Conshohocken, PA, 2001. (22) Cohen, S. A.; De Antonis, K. M. Applications of amino acid derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate: analysis of feed grains, intravenous solutions and glycoproteins. J. Chromatogr. A 1994, 661 (1−2), 25−34. (23) NIST/SEMATECH (2004) Propagation of Error Considerations. In NIST/SEMATECH E-Handbook of Statistical Methods. http://www.itl.nist.gov/div898/handbook/. (24) Garcia, R. A.; Riner, S. A.; Piazza, G. J. Design of a laboratory method for rapid evaluation of experimental flocculants. Ind. Eng. Chem. Res. 2013, 53, 880−886. (25) Newman, J. Novel buffer systems for macromolecular crystallization. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60 (3), 610−612. (26) Chang, M.; Xiao, J.; Liu, R.; Lu, L.; Jin, Q.; Wang, X. Effect of defatting on quality of meat and bone meal. J. Anim. Sci. 2015, 86 (3), 319−324. (27) Garcia, R. A.; Flores, R. A.; Mazenko, C. E. Factors contributing to the poor bulk behavior of meat and bone meal and methods for improving these behaviors. Bioresour. Technol. 2007, 98 (15), 2852− 2858. (28) Staron, P.; Kowalski, Z.; Staron, A.; Seidlerova, J.; Banach, M. Residues from the thermal conversion of waste from the meat industry as a source of valuable macro- and micronutrients. Waste Manage. 2016, 49, 337−345. (29) Kuroda, K.; Okido, M. Hydroxyapatite coating of titanium implants using hydroprocessing and evaluation of their osteoconductivity. Bioinorg. Chem. Appl. 2012, 2012, 1−7. (30) Cheftel, C.; Ahern, M.; Wang, D. I. C.; Tannenbaum, S. R. Enzymic solubilization of fish protein concentrate: batch studies applicable to continuous enzyme recycling processes. J. Agric. Food Chem. 1971, 19 (1), 155−161.
(31) Furst, P.; Pollack, L.; Graser, T.; Godel, H.; Stehle, P. Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials. J. Chromatogr. A 1990, 499, 557−569. (32) Tyler, M. I. Amino acid analysis protocols. In Amino acid analysis: An overview; Cooper, C., Packer, N., Williams, K., Eds.; Humana Press: Totowa, NJ, 2001; pp 1−8. (33) Pihlanto-Leppala, A. Bioactive peptides derived from bovine whey proteins: opioid and ace-inhibitory peptides. Trends Food Sci. Technol. 2000, 11 (9), 347−356. (34) Mekonnen, T. H.; Mussone, P. G.; El-Thaher, N.; Choi, P.; Bressler, D. C. Subcritical hydrolysis and characterization of waste proteinaceous biomass for value added applications. J. Chem. Technol. Biotechnol. 2015, 90, 476−483. (35) Richmond-Aylor, A.; Bell, S.; Callery, P.; Morris, K. Thermal degradation analysis of amino acids in fingerprint residue by pyrolysis GC-MS to develop new latent fingerprint developing reagents. J. Forensic Sci. 2007, 52 (2), 380−382.
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DOI: 10.1021/acssuschemeng.7b03050 ACS Sustainable Chem. Eng. 2017, 5, 11652−11659
