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Valorization of porcine by-products: a combined process for protein hydrolysates and hydroxyapatite production

Valorization of porcine by-products: a combined process for protein hydrolysates and... Introduction more water-soluble peptides and free amino acids. With The agri-food industry generates massive quantities of the hydrolysis, there is an increase in protein recovery; by-products, which can be an environmental issue and moreover, valuable compounds such as protein hydro- should be properly addressed. By-products from slaugh- lysates are produced. ter and processing of pigs represent approximately 44% Protein hydrolysis can be a suitable method to extract of the total live weight of the animal. These by-products proteins from meat residues; the process can be made are commonly used as animal feed, fertilizers and also more efficient if performed with appropriate enzymes in the production of biogas; these applications, however, (Toldrá et  al. 2016). Enzymatic hydrolysis can be per- have a relatively low economic value (Lapeña et al. 2018). formed using endogenous enzymes (digestive enzymes) Nevertheless, this residual raw material has a high nutri- or exogenous enzymes (commercially available) (Aspevik tional  value containing large amounts of protein, lipids et  al. 2017). The process, however, is more specific and and minerals, which have potential to generate high reproducible with exogenous enzymes; hence, this rep- value-added ingredients. Therefore, a better exploitation resents a good option to produce food-grade and well- of these meat by-products is crucial for sustainability and defined protein hydrolysates (PH). Despite the additional the circular economy. Such valorization, as an alternative costs of commercial enzymes, the process is still econom- to a simple reuse of the by-products, could also provide ically viable, since the products have potential to achieve novel ingredients and products that innovate the food higher-paying markets compared for example with prod- industry (Fu et al. 2018). ucts based on rendering (Aspevik et al. 2017). Several studies show that meat residues such as trim- Enzymatic hydrolysis has been used to obtain anti- mings and bones contain high quantities of proteins, oxidant compounds from various animal by-products particularly collagen (Toldrá et  al. 2016), whose poten- including duck (Li et  al. 2020), goat (de Queiroz et  al. tial is well known; indeed, collagen, is used in various 2017) and bovine (Zou et  al. 2019). Previous studies fields, including biomedicine and cosmetics (Ferraro also showed that porcine peptides could be an antioxi- et al. 2017). In addition to this, collagen can also be used dant source, namely peptides from porcine hemoglobin as a source of smaller bioactive molecules, which can (Chang et  al. 2007; Álvarez et  al. 2012), skin (Li et  al. be obtained with a process of hydrolysis (Ahmed et  al. 2007), myofibrillar protein (Saiga et  al. 2003) and other 2020), as proteins are broken down into smaller and porcine tissues (colon, appendix, rectum, pancreas, heart, B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 3 of 12 liver, and lung) (Damgaard et  al. 2014). These bioactive extraction of organic and mineral fraction as added-value peptides derived from pork by-products with potential compounds (PH and CaP). health-promoting effects have a wide range of promising The obtained products (PH and CaP) were character - applications, such as nutraceuticals for pets and humans, ized by several analytical techniques, to evaluate their as well as in cosmetic and pharmaceutical formulations composition and to explore their potential to food/feed, (Aspevik et al. 2017). medical and environmental applications. This work Animal by-products, besides being a valuable pro- shows it is possible to perform a simultaneous extraction tein source, can also be an important basis to extract of several high added-value compounds from the same calcium phosphates (CaP), particularly hydroxyapatite by-products of pigs slaughter and processing, usually (HAp). HAp, whose formula is C a (PO ) (OH) , is the discarded. 10 4 6 2 major inorganic component of hard tissues  (Lü et  al. 2007); more specifically, in animal bones its content is Materials and methods over 60%. HAp is widely used in the biomedical area for Materials and reagents bone regeneration due to its excellent properties such Porcine by-products (meat and bones) were obtained as biocompatibility, bioactivity, osteoconductivity and by ETSA (Loures, Portugal), a company specialized in also noninflammatory and nonimmunogenicity behav - the collection of animal by-products from conventional iors (Barakat et  al. 2009). In addition to this, HAp has centers including slaughterhouses. All reagents were other applications; in fact, it can also be used for envi- purchased from Sigma-Aldrich (USA) unless mentioned ronment remediation, as it can remove bivalent heavy otherwise. metals from contaminated wastewaters and soils (Khan et  al. 2020; Nie et  al. 2020; Safavi et  al. 2020). The syn - Combined process of protein and hydroxyapatite thetic HAp involves a chemical reaction between calcium extraction from porcine by‑products and phosphorus in appropriate conditions; this approach, A scheme of the process employed to extract proteins however, is not sustainable in the long term, due to the and CaP is shown in Fig. 1. increasing demand of phosphorus for agriculture (San- A mixture of porcine bones and meat trimmings were tos et  al. 2019). It is therefore important to consider used; they were ground at room temperature, to obtain innovative and sustainable sources of HAp; indeed HAp a pulp-like meat paste, which was then submitted to extraction from food by-products has been explored, hydrolysis in order to extract the PH and CaP. for instance from bovine bones (Barakat et al. 2009) and To extract the proteins by hydrolysis, water was added porcine bones and teeth (Lü et  al. 2007). In other cases, to the meat/bone paste in a ratio of 1:1. Prior to the for instance from fish bones (Piccirillo et al. 2013) a mix- enzymatic hydrolysis, the pH was adjusted to 8.0 with ture of HAp and other CaP compounds was obtained; 1  M NaOH. The substrate was hydrolyzed by alcalase this was because the ratio between Ca and P in the bones at a ratio of enzyme:substrate of 1% (v/w) at 50  °C for was smaller than the stoichiometric one (1.67). Literature 6  h. During the hydrolysis, the pH of the reaction mix- data showed that natural HAp and/or CaP are suitable ture was kept constant by continuous addition of NaOH. for biomedical applications (i.e., bone substitutes, graft- Enzymatic reaction occurred at the optimal pH and tem- ing, etc.). Currently, some bone substitutes of animal ori- perature conditions described for alcalase (Borrajo et  al. gin are commercially available, as is the case of Apatos 2020b; Sousa et  al. 2020). The mixture was then sub - which is derived from a cortical porcine bone in the form mitted to centrifugation (5000g, 5  min) (Gyrozen 1248, of particles. Korea) in order to separate and obtain three phases: the As mentioned above, processes to recover/extract pro- fat in the upper phase, the intermediate water phase con- tein hydrolysates or CaP have been considered; litera- taining the soluble protein, and the lower phase contain- ture, however, does not report on a combined process to ing the mineral part. The upper phase containing the fat extract both compounds from meat by-products. Such a was discarded and the protein fraction was collected and process would be important to have a more complete val- stored at −20  °C for further analysis. The mineral frac - orization of these by-products. tion or inorganic fraction was washed in water, dried at This work explores for the first time a combined pro - 120 °C in an oven, and ground in a coffee mill, obtaining a cess for the valorization of porcine by-products (bone white bone powder. Then, to remove the residual organic residues and meat trimmings), which includes a bio- fraction and obtain pure minerals, the powder was cal- process (enzymatic hydrolysis) followed by a ther- cined at 700 °C. The heating ramp was 5 °C/min and the mal treatment. This approach allows the simultaneous annealing time was 1 h (Piccirillo et al. 2013). Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 4 of 12 Fig. 1 Scheme of the combined process to extract proteins and CaP Characterization of porcine protein hydrolysates Composition analysis Determination of degree of hydrolysis The composition analysis was performed according to The hydrolysis efficiency was determined through the the Association of Official Analytical Chemists pro - degree of hydrolysis (DH), which was assessed by meas- cedures (AOAC 1995). The moisture was determined uring the free amino groups by reaction of 2,4,6-trini- at 105  °C for 24  h. The ash content was determined at trobenzenesulfonic acid solution (TNBS) (Sousa et  al. 550 °C for 5 h. The protein content was measured using 2020). Briefly, a reaction mixture with 50 μL of PH the Kjeldahl method and the nitrogen to protein con- extract, 125 μL of 200  mM sodium phosphate buffer version factor used was 6.25. The protein content was (pH 8.2) and 50 μL of TNBS at 0.025% were placed in a expressed on a dry weight basis. All measurements 96-well microplate (Sarstedt, Germany). The microplate were performed in triplicate. was incubated at 45  °C for 1  h and the absorbance was measured at 340  nm using a Multiskan GO plate reader (Thermo Scientific, USA). L-leucine (0.078–2.5 mM) was Molecular weight distribution used to generate a standard curve. Three replicates were The molecular weight (MW) distribution of porcine recorded. The DH was determined by following formula: PH extract was determined by a fast protein liquid chromatography (FPLC) (Sousa et al. 2020). An aliquot L − L t 0 DH % = 100 ∗ , (100 µL) of filtered samples was injected in a AKTA ( ) L − L max 0 pure 25 L system, from GE Healthcare Life Sciences (Freiburg, Germany), coupled with two gel filtration where L is the amount of amino groups released after columns: Superdex 200 increase10/300 GL and Super- a hydrolysis time equal to t, L is the amount of amino dex peptide, 10/300 GL. The eluent used was 0.025  M groups in the sample at initial hydrolysis time (blank) phosphate buffer (pH 7.0), 0.15 M sodium chloride and and L is the maximum amount amino groups exist- max 0.2 g/L of sodium azide. The flow rate was 0.5 mL/ min ing in porcine by-products. The L was obtained by max and elution was monitored at 280 nm. A MW standard acid hydrolysis of porcine by-products with 6  M HCl at curve was established using thyroglobulin (669  kDa), 105 ºC for 24  h. Then, the acid-hydrolyzed sample was ferritin (440  kDa), aldolase (158  kDa), conalbumin filtered and the supernatant was neutralized with 6  M (75  kDa), ovalbumin (44  kDa), carbonic anhydrase NaOH before amino group acids assessment. B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 5 of 12 Determination of Ca and P (29 kDa), ribonuclease A (13.7 kDa) and a whey peptide Powders were dissolved in HNO (Merck, Germany) to (1.2 kDa). The analysis was performed in duplicate and determine calcium and phosphorus concentrations. the results were expressed in milli Absorbance Units Calcium content was measured by flame atomic (mAU) per eluted volume (mL). The software used to absorption spectrometry (Solaar 969 AA Spectrometer, evaluate the results was UNICORN 7.0. Unicam, UK). A La solution (Spectrosol, England; 4 g/L) was added to the samples acid solution to prevent ioniza- Analysis of antioxidant activity tion interference. A calibration curve of Ca (0.5–2.0 mg/ ABTS scavenging assay The ability of free radical-scav - mL) was prepared by dilution of the respective atomic enging by porcine PH extract was evaluated through absorption standard solution (Spectrosol, England). 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid Phosphorus concentration was measured by a spectro- (ABTS) radical decolourization assay (Re et al. 1999). The photometric method, using a Spectroquant phospho- radical cation was formed by reacting ABTS with potas- rus reagent kit (Merck, Germany). A calibration curve sium persulfate. Then, 1 mL of ABTS solution was reacted of standard K HPO was used and all measurements with the sample for 6  min and then the absorbance was 2 4 were performed at 400  nm. The assays were performed measure at 734 nm. A calibration curve was prepared with in duplicate. The results were expressed in % (g of Ca or ascorbic acid in the range of 0.063–0.250 mg/mL and all P/100 g of sample); Ca/P molar ratio was also calculated. the determinations performed in triplicate. Results were expressed as mg ascorbic acid equivalent/g of dry extract. Phase analysis ORAC assay The measurement of oxygen radical absorb - Phase analysis of the inorganic residues and of the cal- ance capacity (ORAC-FL) was performed (Ou et al. 2001). cined powder was determined by X-ray diffraction The porcine PH sample were dissolved in 75  mM phos - (XRD). A X’Pert PRO MRD diffractometer was used, phate buffer (pH 7.4) and the solution was placed in a black with CuKα radiation; the diffraction patterns were 96-well microplate (Nunc, Denmark), mixed with 120 μL acquired with a step size of 0.005° and a count time of of fluorescein (70 nM) and incubated at 40 °C for 10 min. 100  s; an interval between 20 and 60° was considered. Then, 60 μL of 2,2’-azobis(2-amidinopropane) dihydro - The registered patterns were compared with the JCPDF chloride (AAPH) solution (14 mM) was added to the mix- standard file 01-072-1234 for HAp. ture, and the fluorescence was recorded using a micro - Samples were also analyzed by Fourier transformed plate reader (Synergy H1, USA) at excitation and emission infrared spectroscopy (FTIR) in a spectrum series Perkin wavelengths of 485 and 528 nm, respectively, for 140 min Elmer spectrometer (ABB, Switzerland) equipped with at intervals of 1 min. The area under curve (AUC) was cal - an attenuated total reflectance (ATR) sampling accessory culated for each sample by integrating the relative fluo - (PIKE technologies, USA) and a diamond/ZnSe crystal. −4 −3 −1 rescence curve. Trolox (9.98 × 10 –7.99 × 10   μmol/ All spectra were acquired between 500 and 4000  cm . mL) was used as the standard and regression equations for Trolox and samples were calculated. The ORAC values Sample morphology were determined by the ratio of sample slope to the trolox The morphology of the samples was analyzed with the slope obtained in the same assay. Final ORAC values were scanning electron microscopy (SEM) technique, using a expressed as mg Trolox equivalent/g of dry extract. Carl Zeiss Merlin instrument, equipped with a Gemini II column and an integrated high efficiency In-lens for sec - Characterization of the inorganic fraction—CaP ondary electrons. Before the analysis, the samples were The inorganic fraction, separated and calcined as sputtered with gold to prevent charge accumulation. described above, was characterized with the following techniques. Results and discussion Yield of the process Figure  1 shows the scheme of the process, as well as the Thermal analysis (TGA) yield for each step. It can be seen that, starting from The thermogravimetric analysis of the inorganic residues 1  kg of material, the amount of PH extracts is about (prior to calcination) was performed using SDT Q600 135  g–13.5%; the inorganic residues, on the other hand, (TA Instruments) TGA equipment, with an air flow rate are about 205 g–20.5%. of 100 ml/min and a heating ramp of 5 °C/min. Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 6 of 12 Porcine protein hydrolysates DH of 17.6%. Chang and collaborators (Chang et al. 2007) Characterization of porcine PH performed the proteolytic reaction of porcine hemo- Proteins of animal origin are known for their nutritional globin with 2.0% alcalase, and after 6  h obtained hydro- properties as a crucial source of amino acids; in fact, lysates with a DH less than 10%. Verma and collaborators these are released upon digestion or industrial processing (Verma et  al. 2017) carried out the proteolytic reaction from the parent protein. Meat is one of the most stud- of porcine liver with 1% (w/w) alcalase over 6 h, and the ied sources for the production of bioactive peptides due hydrolysates had a DH of 23.56%. to the presence of high-quality proteins (Albenzio et  al. Regarding the composition, the dry matter of PH 2017). Some industrial food-grade proteinases, namely extracts was 10.3 ± 0.0%; this is in agreement with lit- alcalase, flavourzyme, bromelain and papain, have been erature, which reports that the dry matter content in the used for the generation of hydrolysates of porcine pro- porcine hydrolysates can vary between 5.9 and 13.8%, teins (Chang et al. 2007; Liu et al. 2010; Wang et al. 2008; depending on pork tissue types used for hydrolysis (Dam- López-Pedrouso et al. 2020). gaard et  al. 2014). As expected, this fraction is protein- Alcalase is very noteworthy from an industrial stand- rich, showing a content of 70.4 ± 2.4% (w/w dry basis), point, because of its activity/stability at alkaline pH val- which is within the values described for porcine hydro- ues, having a wide application. Alcalase has been used as lysates; for instance, hydrolyzed swine mucus protein has additive in detergent formulations, it can be employed in approximately 59% crude protein and hydrolyzed swine meat tenderizing, dehairing and bating leather, cheese liver has ca. 78% crude protein (dos Santos Cardoso et al. flavor improvement, baked manufacture, or enhanc - 2020). The enzymatic hydrolysis is able to produce pep - ing digestibility of animal feeds. The reaction of protein tides which are more water-soluble than the intact pro- hydrolysis catalyzed by alcalase has a strong tendency teins, so it was possible to obtain a high protein recovery. to develop a hydrolysate with many peptides of small PH extracts showed an ash content of 13.9 ± 0.4% (w/w size, due to the extensive range of amino acids that this dry basis), which indicates a large amount of miner- enzyme can recognize. Therefore, the broad enzyme als. Minerals are essential for human and animal health selectivity and specificity allows the use of alcalase in because they are important for several functionalities, a variety of protein substrates, yielding a high protein such as building strong bones, imparting nerve impulses, hydrolysis degree (Tacias-Pascacio et al. 2020). Moreover, producing different hormones and also regulating the there is growing evidence that alcalase on its own shows heartbeat (Gharibzahedi et  al. 2017). The proportion of a higher ability for hydrolysis in comparison with other the remaining components was calculated by difference; commercial enzymes. As demonstrated in the work of it is likely that some lipids are present in the extracts. Ahmadifard et al. (2016), the enzymatic hydrolysis of rice Considering these results, this porcine-derived PH bran protein concentrate and soybean protein showed extract showed a high nutritive quality. that alcalase presented a higher capability for hydrolysis of approximately 10 times higher than other enzymes. Peptide profile analysis Based on these data, the hydrolysis of porcine by-prod- Besides DH measurement, an extremely important ucts was accomplished by alcalase, a serine endopepti- parameter used for characterization of PH extracts is the dase from Bacillus licheniformis. molecular weight distribution of the peptides. This is a A complete characterization of the PH extract was per- direct analysis of the peptides and protein content, unlike formed; the results are reported in Table 1. the DH which is a measure relative to the raw material. The DH is an indicator widely used to compare hydrol - Enzymatic hydrolysis decreases the molecular weight of ysis efficiency among different protein hydrolysates. intrinsic protein and increases the number of ionizable The DH of PH extracts from these food by-products groups, resulting in novel peptides. Porcine PH extracts was 53.3 ± 5.1%. This value is higher than achieved for were analyzed by gel filtration chromatography to deter - other pork tissue hydrolysates also produced by alcalase. mine the peptide length distribution (Fig.  2). The chro - Liu and colleagues (Liu et  al. 2010) hydrolyzed porcine matogram revealed that alcalase produced hydrolysates plasma protein with 2% (w/w) alcalase for 5 h, showing a with small peptides. According to the calibration curve of Table 1 Composition of the PH extracts DH (%) Dry matter (% w/w) Protein content (% Ashes (% w/w dry Other components (% Yield of the process (%) w/w dry basis) basis) w/w dry basis) PH extracts 53.1 ± 5.1 10.3 ± 0.0 70.4 ± 2.4 13.9 ± 0.4 5.4 13.5 B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 7 of 12 Fig. 2 Size-exclusion FPLC profile of porcine protein hydrolysates obtained upon hydrolyzing with alcalase. Molecular weight markers of 13.7 kDa and 1.2 kDa are indicated standards, PH contained peptides with molecular weight Table 2 Antioxidant activity of the protein hydrolysates lower than 13.7 kDa, with a high contribution of peptides ABTS (mg ascorbic acid ORAC (mg Trolox with molecular weight smaller than 1.2  kDa. This result equivalent/g dry extract) equivalent/g dry extract) confirmed that proteins in porcine by-products were PH extracts 21.1 ± 0.5 87.7 ± 6.3 degraded by alcalase into low MW peptides or free amino acids. Fu and co-authors (Fu et al. 2019) also showed that porcine hemoglobin and whole blood treated with differ - ent proteases, such as alcalase, generated peptide frac- capacity of porcine splenic hydrolysate produced using tions with low MW, most of them below 1 kDa. alcalase, suggested that porcine splenic hydrolysates This is expected to be beneficial for the antioxidant improve the antioxidant status in rats by increasing activity, since small peptides have been revealed to have hepatic catalase and glutathione peroxidase activities higher activity than peptides with high MW (Ajibola (Han et al. 2014). et al. 2011; Irshad et al. 2015). uTh s, the antioxidant capacity of porcine protein hydrolysates prepared with alcalase was evaluated by ABTS and ORAC assays; results are reported in Table 2. Antioxidant activity of porcine protein hydrolysates The ABTS method evaluates the ability of an antioxi - The search for bioactive protein hydrolysates from meat dant compound to transfer electrons or donate hydrogen by-products has been instigated by the growing inter- atoms to a preformed ABTS radical cation, whose change est in the development of functional foods, along with of color causes a decrease in absorbance (Re et al. 1999). the control of food lipid oxidation. These valuable com - The value of the radical scavenging activity of porcine pounds could revalue the by-products, while mitigating PH extract was 21.1 ± 0.5  mg ascorbic acid equivalent/g the environmental and economic issues caused by the of dry extract (55.16 ± 1.34 in terms of % radical scav- meat industry. Furthermore, the antioxidant properties enging activity—%RSA). This antioxidant activity is in related with these compounds offers an alternative to agreement with the values observed for other porcine synthetic additives, which are linked with adverse effects hydrolysates extracted with alcalase. Porcine liver protein on human health (Borrajo et al. 2020a). hydrolysates showed a RSA ranged from 38.43 to 74.62% Some animal proteins have been used as substrates for 0–6  h reaction time (Verma et  al. 2017). Damgaard for alcalase hydrolysis, such as sheep visceral protein, et  al. (2015) tested the antioxidant capacity of different which produced a PH with an antioxidant activity of 68% porcine tissue hydrolysates (heart, colon and neck) using (Meshginfar et al. 2014). In vivo and in vitro antioxidant Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 8 of 12 Inorganic fraction: CaP‑based compounds a mixture of alcalase and protamex; they registered val- From inorganic residues to CaP: thermal treatment ues of RSA between 37.9 and 49.6%. To extract CaP phases from natural sources, a thermal The activity of hydrolysates to scavenge ABTS radicals treatment (calcination) is generally performed; this is is affected by several factors such as enzymes, DH, solu - done to remove possible organic fragments still present bility of hydrolysates and MW of peptides. The ORAC-FL in the residues, as well as increasing the crystallinity of method evaluates the scavenging capacity due to a hydro- the obtained CaP (Piccirillo et al. 2014). gen-atom transfer mechanism. The antioxidant com - To understand the changes taking place during the pound is exposed to a peroxyl radical generator (AAPH) heating and, therefore, to choose the best temperature for and the oxidative degradation of fluorescein is meas - the calcination, a thermogravimetric analysis was carried ured (Ou et al. 2001). This assay uses a biological radical out on the mineral residues; Fig.  3a shows the results, source, so is considered the most relevant method from a while Fig.  3b reports the first derivative of the curve, to biological point of view, integrating the degree and time better visualize the different steps. of antioxidant reaction (López-Pedrouso et al. 2020). The The first weight loss (about 8%, T < 200  °C) is due to value of the peroxyl radical scavenging activity of por- the removal of water, either adsorbed on the surface or cine PH extracts was 87.7 ± 6.3  mg Trolox equivalent/g included in the structure of the powder. A slightly larger of dry extract. Indeed, porcine liver protein hydrolysates loss (about 10%) is observed for 200 < T < 600 °C; losses in from enzymatic hydrolysis with several enzymes such as this temperature interval are associated with the burning alcalase, bromelain, flavourzyme and papain have shown of the residual organic fragments present in the mate- antioxidant capacity using ORAC-FL method (López- rial. This weight loss is much smaller to that previously Pedrouso et  al. 2020; Borrajo et  al. 2020b). Our results observed for porcine bones—about 30% (Figueiredo corroborated this, proving that antioxidant peptides from et  al. 2010); this difference confirming that a significant porcine by-products can protect cells from oxidative amount of organic matter was removed from the pow- damage. der during the enzymatic hydrolysis. For higher tem- u Th s, these antioxidant peptides can be employed to peratures, a further decrease in weight can be observed, maintain human health and also food safety and quality, although it is small (< 3%); this could be due to the by mitigating oxidative stress and lipid peroxidation trig- removal of the carbonate present in the material (Figue- gered by free radicals produced during oxidation reac- iredo et al. 2010). tions of the human body and food products. Thereby, antioxidant peptides have received noteworthy attention CaP characterization in the food industry as functional ingredients and food Based on these results, it was decided to perform the cal- additives. The use of synthetic antioxidants agents in the cination of the material at 700 °C; this value was chosen food industry is under strict regulation due to their side- as the organic fragments were already removed but some effects on human health, namely induction of DNA dam - carbonate ions were still present in the material—indeed, age and toxicity. Consequently, substituting synthetic literature reports that the presence of such ions can be antioxidants by natural antioxidants has become required beneficial for bone-like cellular growth and bioactivity (Tadesse and Emire, 2020). (a) 0.00 (b) -0.05 -0.10 -0.15 0200 400600 8001000 0200 400 600 800 1000 Temperature ( C) Temperature ( C) Fig. 3 a Thermogravimetric analysis ( TGA) of the inorganic residues; b first derivative of the curve Residualweight(%) Δm/ΔT(a.u.) B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 9 of 12 Table 3 Calcium and phosphorus content (wt %) and Ca/P inorganic residues molar ratio for the non-calcined powder and CaP sample OH Sample Ca (%) P(%) Ca/P Yield of the CO process (%) PO 4 OH Non- 34.40 ± 0.28 15.25 ± 0.07 1.74 ± 0.01 20.5 calcined PO 4 PO powder PO CaP 40.30 ± 0.28 18.90 ± 0.42 1.65 ± 0.03 17.0 CaP PO 4000 3600 1500 1200 900600 -1 Wavenumber (cm ) CaP Fig. 5 FTIR of the inorganic residues (i.e., non-calcined powder) and of the calcined CaP sample inorganic residues 100020 30 40 50 60 HAp 20 30 40 50 60 2θ (degrees) Fig. 4 XRD data for the inorganic residue (non-calcined powder) and the CaP sample. The patterns are compared to the 01-072-1234 standard for HAp (Nakamura et  al. 2016). Calcination at this temperature led to a yield of about 170 g, i.e., 17%. Elemental analysis was performed to determine the content of calcium and phosphorus, as well as the Ca/P molar ratio—see Table 3. It can be seen that, although both calcium and phos- Fig. 6 SEM micrograph of the calcined powder CaP phorus show higher relative content after the calcination, the increase is different for the two elements; in fact, the Ca/P ratio decreases slightly—from 1.74 to 1.65. This more crystalline than the starting non-calcined powder indicates that a small quantity of calcium is lost during (sharper peaks). the calcination; this behavior was previously observed for FTIR spectra of the samples are shown in Fig.  5. It CaP derived from natural sources (Aydin et al. 2020). The can be seen that the signals are much sharper and more Ca/P ratio for the CaP powder is very close to the stoi- resolved for the calcined CaP sample, due to its higher chiometric one, that is 1.67. crystallinity. Peaks belonging to the HAp phosphate ions Figure  4 shows the XRD patterns for the inorganic −1 can be observed at 1090, 1040, 960, 603, 568  cm (Pic- powder, prior the calcination and after (CaP). It can be cirillo et  al. 2013); it is interesting to note that no peak seen that in both cases the only phase present is HAp; no −1 is present at 1122  cm . This signal corresponds to the other phosphate compounds, for instance β-tricalcium β-TCP (Piccirillo et  al. 2013); its absence confirms that phosphate (β-TCP), are present. This was expected, the this phase is not formed in the CaP sample, in agree- Ca/P ratio being not statistically different from the stoi - ment with XRD data (Fig.  4). Other weaker peaks pre- chiometric one; literature reports the formation of β-TCP sent in the spectrum correspond to the OH ions at 3570 for smaller Ca/P ratios, i.e., values close to 1.5 (Piccirillo −1 −1 and 634  cm ; moreover, signals at 1412 and 1450  cm et  al. 2014). It can also be observed that CaP is much Intensity(a.u.) Transmittance (%) Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 10 of 12 belong to the carbonate group (Figueiredo et  al. 2010). the first step to extract different parts from the residues; These signals confirm that, after a treatment at 700  °C other step(s) for other fraction(s) could be added. carbonate ions are still present in the HAp lattice. The extraction of PH was performed with an enzy - Figure  6 shows a SEM micrograph of the CaP sample. matic bioprocess, without employing toxic solvents, It can be seen that the powder has a nanometric struc- according to the principles of the green chemistry, as an ture; indeed particles with average size of about 50  nm aqueous solution was used. For CaP, on the other hand, can be observed. Literature data report that HAp from no solvent was necessary, but a simple thermal process natural sources can be nanometric or not, depending was performed. Although energy is used for this treat- on the sources (Barakat et  al. 2009; Santana et  al. 2019). ment, with an associated impact on the environment, it The use of HAp in the form of nanoparticles has recently has to be highlighted that the conventional routes for gained increasing attention, as it can show enhanced sin- CaP production are likely to have a greater environ- tering properties, if compared to the micrometric pow- mental impact. With these, in fact, chemical reactions der (György et  al. 2019), due to a higher specific surface between Ca and P are performed; in addition to the and consequently a higher powder reactivity. Moreo- energy cost linked to this, the use of Ca- and P-contain- ver, nano-HAp was also preferred for the preparation of ing reagents, derived from non-renewable sources, has composites with other compounds (i.e., biopolymers) to be considered. Both elements, in fact, are obtained (Turon et  al. 2017). Compared with micrometric HAp, from mining activities, whose impact is known to be the nanoscale one induces better cellular functions like quite high (Dubsok and Kittipongvieses 2016; Petrov osteoblast responses (such as adhesion, proliferation, and and Danilov 2020). Considering this, by-product valori- differentiation) (Li et  al. 2013). Also, for its application zation surely offers a more sustainable solution. as a heavy metal remover, nano-HAp showed enhanced From the economic point of view, both PH and CaP performance (Kowthaman and Varadappan 2019). have high market value. Considering CaP, it is a good Based on these results, it can be stated that HAp with quality powder, with high purity and high level of crys- interesting properties and potential for application in tallinity for biomedical applications, can cost up to tens biomedicine (for instance, as bone substitute) and as a of euros per gram. PH, for feed applications, is about 5x heavy metal remover was successfully extracted in this more valuable than a non-hydrolyzed protein. This makes combined process. The use of natural HAp has been the process profitable. This makes the process profitable. explored instead to synthetic HAp, because it has com- parable metabolic activity, preserves chemical composi- tion and structure of the precursor material (Boutinguiza Conclusions et  al. 2012). In addition, there is a growing concern to Porcine by-products (meat and bones) are a valuable develop clean, non-toxic and environmentally friendly source to produce natural value-added compounds for procedures for HAp synthesis (with lower impact on different markets. The combined process described in the environment). It is required to reuse waste not only this work shows that it is possible to extract different because waste materials are accumulating, but also compounds, both organics and minerals—indeed pro- because natural raw materials are being exhausted. tein hydrolysates and hydroxyapatite were obtained. Overall, about 30% of the by-products were converted into valuable compounds—13.5% and 17% of protein Evaluation of the process hydrolysates and hydroxyapatite, respectively. A full environmental and economic assessment of the Protein hydrolysates were rich in low MW pep- process is beyond the scope of this work; some general tides and showed significant antioxidant properties. comments, however, can already be made. Hydroxyapatite, on the other hand, was shown to be As reported in Sect. 3.1, the overall yield is about 30%; single-phase and with a nanometric structure. this value is well below the 100% associated with the full The proposed combined process is quite simple, cheap valorization. It is worth highlighting, however, that with- and easily scalable; all these features make it applicable at out performing this combined process, only PH or CaP industrial scale, to achieve a more complete valorization would be extracted, with an overall minor yield and a of porcine by-products. Moreover, in principle the pro- fewer effective by-products valorization. Moreover, the cess could be applied also to by-products of other meat residues also contain other organic fractions, such as or fish industries. As future work, other steps could be lipids; these phases should also be considered to achieve added to the process, for the extraction of other phases a full valorization. The process presented here represent (i.e., lipids) and to achieve a more complete valorization. B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 11 of 12 Abbreviations Álvarez C, Rendueles M, Díaz M (2012) Production of porcine hemoglobin AAPH: 2,2’-Azobis(2-amidinopropane) dihydrochloride; ABTS: 2,2-Azino-bis- peptides at moderate temperature and medium pressure under a nitro- 3-ethylbenzothiazoline-6-sulphonic acid; ATR : Attenuated total reflectance; gen stream. Functional and antioxidant properties. J Agric Food Chem CaP: Calcium phosphates; DH: Degree of hydrolysis; FPLC: Fast protein liquid 60:5636–5643 chromatography; FTIR: Fourier transformed infrared spectroscopy; HAp: AOAC (1995) Official methods of analysis 16th Ed. Association of official ana- Hydroxyapatite; mAU: Milli absorbance units; MW: Molecular weight; ORAC-FL: lytical chemists. Washington DC, USA Oxygen radical absorbance capacity; PH: Protein hydrolysates; RSA: Radical Aspevik T, Oterhals Å, Rønning SB, Altintzoglou T, Wubshet SG, Gildberg A, scavenging activity; SEM: Scanning electron microscopy; TCP: Tricalcium Afseth NK, Whitaker RD, Lindberg D (2017) Valorization of proteins from phosphate; TGA : Thermal analysis; TNBS: Trinitrobenzenesulfonic acid solution; co-and by-products from the fish and meat industry. Chem Chem Tech- XRD: X-ray diffraction. nol Waste Valoriz 1:123–150 Aydin G, Terzioğlu P, Öğüt H, Kalemtas A (2020) Production, characterization, Acknowledgements and cytotoxicity of calcium phosphate ceramics derived from the bone Not applicable. of meagre fish, Argyrosomus regius. J Austr Ceram Soc 1:1–10 Barakat NAM, Khil MS, Omran AM, Sheikh FA, Kim HY (2009) Extraction of pure Authors’ contributions natural hydroxyapatite from the bovine bones bio waste by three differ - SB: methodology, investigation, writing—original draft, preparation. CP: inves- ent methods. J Mater Process Technol 209:3408–3415 tigation, writing- reviewing and editing. FS: investigation, visualization and Borrajo P, López-Pedrouso M, Franco D, Pateiro M, Lorenzo JM (2020a) data curation. RM: investigation. AR: investigation. JAC: writing—reviewing Antioxidant and antimicrobial activity of porcine liver hydrolysates using and editing. AA: conceptualization, resources. MP: conceptualization, project flavourzyme. Appl Sci 10:3950 administration, writing—reviewing and editing. All authors read and approved Borrajo P, Pateiro M, Gagaoua M, Franco D, Zhang W, Lorenzo JM (2020b) the final manuscript. Evaluation of the antioxidant and antimicrobial activities of porcine liver protein hydrolysates obtained using alcalase, bromelain, and Funding papain. Appl Sci 10:2290 This work was supported by National Funds from project MOREPEP (POCI-01– Boutinguiza M, Pou J, Comesaña R, Lusquiños F, De Carlos A, León B (2012) 0247-FEDER-017638) funded by Fundo Europeu de Desenvolvimento Regional Biological hydroxyapatite obtained from fish bones. Mater Sci Eng, C (FEDER), under Programa Operacional Competitividade e Internacionalização 32:478–486 (POCI) and from FCT – Fundação para a Ciência e a Tecnologia through project Chang C-Y, Wu K-C, Chiang S-H (2007) Antioxidant properties and protein UIDB/50016/2020. Clara Piccirillo and Francesca Scalera thank Fondazione con compositions of porcine haemoglobin hydrolysates. Food Chem il Sud for funding the HApECOrk project (Grant Number 2015–0243). 100:1537–1543 Damgaard TD, Otte JAH, Meinert L, Jensen K, Lametsch R (2014) Antioxidant Availability of data and materials capacity of hydrolyzed porcine tissues. Food Sci Nutr 2:282–288 All data supporting this article’s conclusion are available. Damgaard T, Lametsch R, Otte J (2015) Antioxidant capacity of hydrolyzed animal by-products and relation to amino acid composition and pep- tide size distribution. J Food Sci Technol 52:6511–6519 Declarations de Queiroz ALM, Bezerra TKA, de Freitas PS, da Silva MEC, de Almeida GCA, Gadelha TS, Pacheco MTB, Madruga MS (2017) Functional protein Ethics approval and consent to participate hydrolysate from goat by-products: Optimization and characterization Not applicable. studies. Food Biosci 20:19–27 dos Santos Cardoso M, Godoy AC, Oxford JH, Rodrigues R, dos Santos Consent for publication CM, Bittencourt F, Signor A, Boscolo WR, Feiden A (2020) Apparent Not applicable. digestibility of protein hydrolysates from chicken and swine slaughter residues for Nile tilapia. 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Valorization of porcine by-products: a combined process for protein hydrolysates and hydroxyapatite production

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Abstract

Introduction more water-soluble peptides and free amino acids. With The agri-food industry generates massive quantities of the hydrolysis, there is an increase in protein recovery; by-products, which can be an environmental issue and moreover, valuable compounds such as protein hydro- should be properly addressed. By-products from slaugh- lysates are produced. ter and processing of pigs represent approximately 44% Protein hydrolysis can be a suitable method to extract of the total live weight of the animal. These by-products proteins from meat residues; the process can be made are commonly used as animal feed, fertilizers and also more efficient if performed with appropriate enzymes in the production of biogas; these applications, however, (Toldrá et  al. 2016). Enzymatic hydrolysis can be per- have a relatively low economic value (Lapeña et al. 2018). formed using endogenous enzymes (digestive enzymes) Nevertheless, this residual raw material has a high nutri- or exogenous enzymes (commercially available) (Aspevik tional  value containing large amounts of protein, lipids et  al. 2017). The process, however, is more specific and and minerals, which have potential to generate high reproducible with exogenous enzymes; hence, this rep- value-added ingredients. Therefore, a better exploitation resents a good option to produce food-grade and well- of these meat by-products is crucial for sustainability and defined protein hydrolysates (PH). Despite the additional the circular economy. Such valorization, as an alternative costs of commercial enzymes, the process is still econom- to a simple reuse of the by-products, could also provide ically viable, since the products have potential to achieve novel ingredients and products that innovate the food higher-paying markets compared for example with prod- industry (Fu et al. 2018). ucts based on rendering (Aspevik et al. 2017). Several studies show that meat residues such as trim- Enzymatic hydrolysis has been used to obtain anti- mings and bones contain high quantities of proteins, oxidant compounds from various animal by-products particularly collagen (Toldrá et  al. 2016), whose poten- including duck (Li et  al. 2020), goat (de Queiroz et  al. tial is well known; indeed, collagen, is used in various 2017) and bovine (Zou et  al. 2019). Previous studies fields, including biomedicine and cosmetics (Ferraro also showed that porcine peptides could be an antioxi- et al. 2017). In addition to this, collagen can also be used dant source, namely peptides from porcine hemoglobin as a source of smaller bioactive molecules, which can (Chang et  al. 2007; Álvarez et  al. 2012), skin (Li et  al. be obtained with a process of hydrolysis (Ahmed et  al. 2007), myofibrillar protein (Saiga et  al. 2003) and other 2020), as proteins are broken down into smaller and porcine tissues (colon, appendix, rectum, pancreas, heart, B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 3 of 12 liver, and lung) (Damgaard et  al. 2014). These bioactive extraction of organic and mineral fraction as added-value peptides derived from pork by-products with potential compounds (PH and CaP). health-promoting effects have a wide range of promising The obtained products (PH and CaP) were character - applications, such as nutraceuticals for pets and humans, ized by several analytical techniques, to evaluate their as well as in cosmetic and pharmaceutical formulations composition and to explore their potential to food/feed, (Aspevik et al. 2017). medical and environmental applications. This work Animal by-products, besides being a valuable pro- shows it is possible to perform a simultaneous extraction tein source, can also be an important basis to extract of several high added-value compounds from the same calcium phosphates (CaP), particularly hydroxyapatite by-products of pigs slaughter and processing, usually (HAp). HAp, whose formula is C a (PO ) (OH) , is the discarded. 10 4 6 2 major inorganic component of hard tissues  (Lü et  al. 2007); more specifically, in animal bones its content is Materials and methods over 60%. HAp is widely used in the biomedical area for Materials and reagents bone regeneration due to its excellent properties such Porcine by-products (meat and bones) were obtained as biocompatibility, bioactivity, osteoconductivity and by ETSA (Loures, Portugal), a company specialized in also noninflammatory and nonimmunogenicity behav - the collection of animal by-products from conventional iors (Barakat et  al. 2009). In addition to this, HAp has centers including slaughterhouses. All reagents were other applications; in fact, it can also be used for envi- purchased from Sigma-Aldrich (USA) unless mentioned ronment remediation, as it can remove bivalent heavy otherwise. metals from contaminated wastewaters and soils (Khan et  al. 2020; Nie et  al. 2020; Safavi et  al. 2020). The syn - Combined process of protein and hydroxyapatite thetic HAp involves a chemical reaction between calcium extraction from porcine by‑products and phosphorus in appropriate conditions; this approach, A scheme of the process employed to extract proteins however, is not sustainable in the long term, due to the and CaP is shown in Fig. 1. increasing demand of phosphorus for agriculture (San- A mixture of porcine bones and meat trimmings were tos et  al. 2019). It is therefore important to consider used; they were ground at room temperature, to obtain innovative and sustainable sources of HAp; indeed HAp a pulp-like meat paste, which was then submitted to extraction from food by-products has been explored, hydrolysis in order to extract the PH and CaP. for instance from bovine bones (Barakat et al. 2009) and To extract the proteins by hydrolysis, water was added porcine bones and teeth (Lü et  al. 2007). In other cases, to the meat/bone paste in a ratio of 1:1. Prior to the for instance from fish bones (Piccirillo et al. 2013) a mix- enzymatic hydrolysis, the pH was adjusted to 8.0 with ture of HAp and other CaP compounds was obtained; 1  M NaOH. The substrate was hydrolyzed by alcalase this was because the ratio between Ca and P in the bones at a ratio of enzyme:substrate of 1% (v/w) at 50  °C for was smaller than the stoichiometric one (1.67). Literature 6  h. During the hydrolysis, the pH of the reaction mix- data showed that natural HAp and/or CaP are suitable ture was kept constant by continuous addition of NaOH. for biomedical applications (i.e., bone substitutes, graft- Enzymatic reaction occurred at the optimal pH and tem- ing, etc.). Currently, some bone substitutes of animal ori- perature conditions described for alcalase (Borrajo et  al. gin are commercially available, as is the case of Apatos 2020b; Sousa et  al. 2020). The mixture was then sub - which is derived from a cortical porcine bone in the form mitted to centrifugation (5000g, 5  min) (Gyrozen 1248, of particles. Korea) in order to separate and obtain three phases: the As mentioned above, processes to recover/extract pro- fat in the upper phase, the intermediate water phase con- tein hydrolysates or CaP have been considered; litera- taining the soluble protein, and the lower phase contain- ture, however, does not report on a combined process to ing the mineral part. The upper phase containing the fat extract both compounds from meat by-products. Such a was discarded and the protein fraction was collected and process would be important to have a more complete val- stored at −20  °C for further analysis. The mineral frac - orization of these by-products. tion or inorganic fraction was washed in water, dried at This work explores for the first time a combined pro - 120 °C in an oven, and ground in a coffee mill, obtaining a cess for the valorization of porcine by-products (bone white bone powder. Then, to remove the residual organic residues and meat trimmings), which includes a bio- fraction and obtain pure minerals, the powder was cal- process (enzymatic hydrolysis) followed by a ther- cined at 700 °C. The heating ramp was 5 °C/min and the mal treatment. This approach allows the simultaneous annealing time was 1 h (Piccirillo et al. 2013). Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 4 of 12 Fig. 1 Scheme of the combined process to extract proteins and CaP Characterization of porcine protein hydrolysates Composition analysis Determination of degree of hydrolysis The composition analysis was performed according to The hydrolysis efficiency was determined through the the Association of Official Analytical Chemists pro - degree of hydrolysis (DH), which was assessed by meas- cedures (AOAC 1995). The moisture was determined uring the free amino groups by reaction of 2,4,6-trini- at 105  °C for 24  h. The ash content was determined at trobenzenesulfonic acid solution (TNBS) (Sousa et  al. 550 °C for 5 h. The protein content was measured using 2020). Briefly, a reaction mixture with 50 μL of PH the Kjeldahl method and the nitrogen to protein con- extract, 125 μL of 200  mM sodium phosphate buffer version factor used was 6.25. The protein content was (pH 8.2) and 50 μL of TNBS at 0.025% were placed in a expressed on a dry weight basis. All measurements 96-well microplate (Sarstedt, Germany). The microplate were performed in triplicate. was incubated at 45  °C for 1  h and the absorbance was measured at 340  nm using a Multiskan GO plate reader (Thermo Scientific, USA). L-leucine (0.078–2.5 mM) was Molecular weight distribution used to generate a standard curve. Three replicates were The molecular weight (MW) distribution of porcine recorded. The DH was determined by following formula: PH extract was determined by a fast protein liquid chromatography (FPLC) (Sousa et al. 2020). An aliquot L − L t 0 DH % = 100 ∗ , (100 µL) of filtered samples was injected in a AKTA ( ) L − L max 0 pure 25 L system, from GE Healthcare Life Sciences (Freiburg, Germany), coupled with two gel filtration where L is the amount of amino groups released after columns: Superdex 200 increase10/300 GL and Super- a hydrolysis time equal to t, L is the amount of amino dex peptide, 10/300 GL. The eluent used was 0.025  M groups in the sample at initial hydrolysis time (blank) phosphate buffer (pH 7.0), 0.15 M sodium chloride and and L is the maximum amount amino groups exist- max 0.2 g/L of sodium azide. The flow rate was 0.5 mL/ min ing in porcine by-products. The L was obtained by max and elution was monitored at 280 nm. A MW standard acid hydrolysis of porcine by-products with 6  M HCl at curve was established using thyroglobulin (669  kDa), 105 ºC for 24  h. Then, the acid-hydrolyzed sample was ferritin (440  kDa), aldolase (158  kDa), conalbumin filtered and the supernatant was neutralized with 6  M (75  kDa), ovalbumin (44  kDa), carbonic anhydrase NaOH before amino group acids assessment. B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 5 of 12 Determination of Ca and P (29 kDa), ribonuclease A (13.7 kDa) and a whey peptide Powders were dissolved in HNO (Merck, Germany) to (1.2 kDa). The analysis was performed in duplicate and determine calcium and phosphorus concentrations. the results were expressed in milli Absorbance Units Calcium content was measured by flame atomic (mAU) per eluted volume (mL). The software used to absorption spectrometry (Solaar 969 AA Spectrometer, evaluate the results was UNICORN 7.0. Unicam, UK). A La solution (Spectrosol, England; 4 g/L) was added to the samples acid solution to prevent ioniza- Analysis of antioxidant activity tion interference. A calibration curve of Ca (0.5–2.0 mg/ ABTS scavenging assay The ability of free radical-scav - mL) was prepared by dilution of the respective atomic enging by porcine PH extract was evaluated through absorption standard solution (Spectrosol, England). 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid Phosphorus concentration was measured by a spectro- (ABTS) radical decolourization assay (Re et al. 1999). The photometric method, using a Spectroquant phospho- radical cation was formed by reacting ABTS with potas- rus reagent kit (Merck, Germany). A calibration curve sium persulfate. Then, 1 mL of ABTS solution was reacted of standard K HPO was used and all measurements with the sample for 6  min and then the absorbance was 2 4 were performed at 400  nm. The assays were performed measure at 734 nm. A calibration curve was prepared with in duplicate. The results were expressed in % (g of Ca or ascorbic acid in the range of 0.063–0.250 mg/mL and all P/100 g of sample); Ca/P molar ratio was also calculated. the determinations performed in triplicate. Results were expressed as mg ascorbic acid equivalent/g of dry extract. Phase analysis ORAC assay The measurement of oxygen radical absorb - Phase analysis of the inorganic residues and of the cal- ance capacity (ORAC-FL) was performed (Ou et al. 2001). cined powder was determined by X-ray diffraction The porcine PH sample were dissolved in 75  mM phos - (XRD). A X’Pert PRO MRD diffractometer was used, phate buffer (pH 7.4) and the solution was placed in a black with CuKα radiation; the diffraction patterns were 96-well microplate (Nunc, Denmark), mixed with 120 μL acquired with a step size of 0.005° and a count time of of fluorescein (70 nM) and incubated at 40 °C for 10 min. 100  s; an interval between 20 and 60° was considered. Then, 60 μL of 2,2’-azobis(2-amidinopropane) dihydro - The registered patterns were compared with the JCPDF chloride (AAPH) solution (14 mM) was added to the mix- standard file 01-072-1234 for HAp. ture, and the fluorescence was recorded using a micro - Samples were also analyzed by Fourier transformed plate reader (Synergy H1, USA) at excitation and emission infrared spectroscopy (FTIR) in a spectrum series Perkin wavelengths of 485 and 528 nm, respectively, for 140 min Elmer spectrometer (ABB, Switzerland) equipped with at intervals of 1 min. The area under curve (AUC) was cal - an attenuated total reflectance (ATR) sampling accessory culated for each sample by integrating the relative fluo - (PIKE technologies, USA) and a diamond/ZnSe crystal. −4 −3 −1 rescence curve. Trolox (9.98 × 10 –7.99 × 10   μmol/ All spectra were acquired between 500 and 4000  cm . mL) was used as the standard and regression equations for Trolox and samples were calculated. The ORAC values Sample morphology were determined by the ratio of sample slope to the trolox The morphology of the samples was analyzed with the slope obtained in the same assay. Final ORAC values were scanning electron microscopy (SEM) technique, using a expressed as mg Trolox equivalent/g of dry extract. Carl Zeiss Merlin instrument, equipped with a Gemini II column and an integrated high efficiency In-lens for sec - Characterization of the inorganic fraction—CaP ondary electrons. Before the analysis, the samples were The inorganic fraction, separated and calcined as sputtered with gold to prevent charge accumulation. described above, was characterized with the following techniques. Results and discussion Yield of the process Figure  1 shows the scheme of the process, as well as the Thermal analysis (TGA) yield for each step. It can be seen that, starting from The thermogravimetric analysis of the inorganic residues 1  kg of material, the amount of PH extracts is about (prior to calcination) was performed using SDT Q600 135  g–13.5%; the inorganic residues, on the other hand, (TA Instruments) TGA equipment, with an air flow rate are about 205 g–20.5%. of 100 ml/min and a heating ramp of 5 °C/min. Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 6 of 12 Porcine protein hydrolysates DH of 17.6%. Chang and collaborators (Chang et al. 2007) Characterization of porcine PH performed the proteolytic reaction of porcine hemo- Proteins of animal origin are known for their nutritional globin with 2.0% alcalase, and after 6  h obtained hydro- properties as a crucial source of amino acids; in fact, lysates with a DH less than 10%. Verma and collaborators these are released upon digestion or industrial processing (Verma et  al. 2017) carried out the proteolytic reaction from the parent protein. Meat is one of the most stud- of porcine liver with 1% (w/w) alcalase over 6 h, and the ied sources for the production of bioactive peptides due hydrolysates had a DH of 23.56%. to the presence of high-quality proteins (Albenzio et  al. Regarding the composition, the dry matter of PH 2017). Some industrial food-grade proteinases, namely extracts was 10.3 ± 0.0%; this is in agreement with lit- alcalase, flavourzyme, bromelain and papain, have been erature, which reports that the dry matter content in the used for the generation of hydrolysates of porcine pro- porcine hydrolysates can vary between 5.9 and 13.8%, teins (Chang et al. 2007; Liu et al. 2010; Wang et al. 2008; depending on pork tissue types used for hydrolysis (Dam- López-Pedrouso et al. 2020). gaard et  al. 2014). As expected, this fraction is protein- Alcalase is very noteworthy from an industrial stand- rich, showing a content of 70.4 ± 2.4% (w/w dry basis), point, because of its activity/stability at alkaline pH val- which is within the values described for porcine hydro- ues, having a wide application. Alcalase has been used as lysates; for instance, hydrolyzed swine mucus protein has additive in detergent formulations, it can be employed in approximately 59% crude protein and hydrolyzed swine meat tenderizing, dehairing and bating leather, cheese liver has ca. 78% crude protein (dos Santos Cardoso et al. flavor improvement, baked manufacture, or enhanc - 2020). The enzymatic hydrolysis is able to produce pep - ing digestibility of animal feeds. The reaction of protein tides which are more water-soluble than the intact pro- hydrolysis catalyzed by alcalase has a strong tendency teins, so it was possible to obtain a high protein recovery. to develop a hydrolysate with many peptides of small PH extracts showed an ash content of 13.9 ± 0.4% (w/w size, due to the extensive range of amino acids that this dry basis), which indicates a large amount of miner- enzyme can recognize. Therefore, the broad enzyme als. Minerals are essential for human and animal health selectivity and specificity allows the use of alcalase in because they are important for several functionalities, a variety of protein substrates, yielding a high protein such as building strong bones, imparting nerve impulses, hydrolysis degree (Tacias-Pascacio et al. 2020). Moreover, producing different hormones and also regulating the there is growing evidence that alcalase on its own shows heartbeat (Gharibzahedi et  al. 2017). The proportion of a higher ability for hydrolysis in comparison with other the remaining components was calculated by difference; commercial enzymes. As demonstrated in the work of it is likely that some lipids are present in the extracts. Ahmadifard et al. (2016), the enzymatic hydrolysis of rice Considering these results, this porcine-derived PH bran protein concentrate and soybean protein showed extract showed a high nutritive quality. that alcalase presented a higher capability for hydrolysis of approximately 10 times higher than other enzymes. Peptide profile analysis Based on these data, the hydrolysis of porcine by-prod- Besides DH measurement, an extremely important ucts was accomplished by alcalase, a serine endopepti- parameter used for characterization of PH extracts is the dase from Bacillus licheniformis. molecular weight distribution of the peptides. This is a A complete characterization of the PH extract was per- direct analysis of the peptides and protein content, unlike formed; the results are reported in Table 1. the DH which is a measure relative to the raw material. The DH is an indicator widely used to compare hydrol - Enzymatic hydrolysis decreases the molecular weight of ysis efficiency among different protein hydrolysates. intrinsic protein and increases the number of ionizable The DH of PH extracts from these food by-products groups, resulting in novel peptides. Porcine PH extracts was 53.3 ± 5.1%. This value is higher than achieved for were analyzed by gel filtration chromatography to deter - other pork tissue hydrolysates also produced by alcalase. mine the peptide length distribution (Fig.  2). The chro - Liu and colleagues (Liu et  al. 2010) hydrolyzed porcine matogram revealed that alcalase produced hydrolysates plasma protein with 2% (w/w) alcalase for 5 h, showing a with small peptides. According to the calibration curve of Table 1 Composition of the PH extracts DH (%) Dry matter (% w/w) Protein content (% Ashes (% w/w dry Other components (% Yield of the process (%) w/w dry basis) basis) w/w dry basis) PH extracts 53.1 ± 5.1 10.3 ± 0.0 70.4 ± 2.4 13.9 ± 0.4 5.4 13.5 B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 7 of 12 Fig. 2 Size-exclusion FPLC profile of porcine protein hydrolysates obtained upon hydrolyzing with alcalase. Molecular weight markers of 13.7 kDa and 1.2 kDa are indicated standards, PH contained peptides with molecular weight Table 2 Antioxidant activity of the protein hydrolysates lower than 13.7 kDa, with a high contribution of peptides ABTS (mg ascorbic acid ORAC (mg Trolox with molecular weight smaller than 1.2  kDa. This result equivalent/g dry extract) equivalent/g dry extract) confirmed that proteins in porcine by-products were PH extracts 21.1 ± 0.5 87.7 ± 6.3 degraded by alcalase into low MW peptides or free amino acids. Fu and co-authors (Fu et al. 2019) also showed that porcine hemoglobin and whole blood treated with differ - ent proteases, such as alcalase, generated peptide frac- capacity of porcine splenic hydrolysate produced using tions with low MW, most of them below 1 kDa. alcalase, suggested that porcine splenic hydrolysates This is expected to be beneficial for the antioxidant improve the antioxidant status in rats by increasing activity, since small peptides have been revealed to have hepatic catalase and glutathione peroxidase activities higher activity than peptides with high MW (Ajibola (Han et al. 2014). et al. 2011; Irshad et al. 2015). uTh s, the antioxidant capacity of porcine protein hydrolysates prepared with alcalase was evaluated by ABTS and ORAC assays; results are reported in Table 2. Antioxidant activity of porcine protein hydrolysates The ABTS method evaluates the ability of an antioxi - The search for bioactive protein hydrolysates from meat dant compound to transfer electrons or donate hydrogen by-products has been instigated by the growing inter- atoms to a preformed ABTS radical cation, whose change est in the development of functional foods, along with of color causes a decrease in absorbance (Re et al. 1999). the control of food lipid oxidation. These valuable com - The value of the radical scavenging activity of porcine pounds could revalue the by-products, while mitigating PH extract was 21.1 ± 0.5  mg ascorbic acid equivalent/g the environmental and economic issues caused by the of dry extract (55.16 ± 1.34 in terms of % radical scav- meat industry. Furthermore, the antioxidant properties enging activity—%RSA). This antioxidant activity is in related with these compounds offers an alternative to agreement with the values observed for other porcine synthetic additives, which are linked with adverse effects hydrolysates extracted with alcalase. Porcine liver protein on human health (Borrajo et al. 2020a). hydrolysates showed a RSA ranged from 38.43 to 74.62% Some animal proteins have been used as substrates for 0–6  h reaction time (Verma et  al. 2017). Damgaard for alcalase hydrolysis, such as sheep visceral protein, et  al. (2015) tested the antioxidant capacity of different which produced a PH with an antioxidant activity of 68% porcine tissue hydrolysates (heart, colon and neck) using (Meshginfar et al. 2014). In vivo and in vitro antioxidant Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 8 of 12 Inorganic fraction: CaP‑based compounds a mixture of alcalase and protamex; they registered val- From inorganic residues to CaP: thermal treatment ues of RSA between 37.9 and 49.6%. To extract CaP phases from natural sources, a thermal The activity of hydrolysates to scavenge ABTS radicals treatment (calcination) is generally performed; this is is affected by several factors such as enzymes, DH, solu - done to remove possible organic fragments still present bility of hydrolysates and MW of peptides. The ORAC-FL in the residues, as well as increasing the crystallinity of method evaluates the scavenging capacity due to a hydro- the obtained CaP (Piccirillo et al. 2014). gen-atom transfer mechanism. The antioxidant com - To understand the changes taking place during the pound is exposed to a peroxyl radical generator (AAPH) heating and, therefore, to choose the best temperature for and the oxidative degradation of fluorescein is meas - the calcination, a thermogravimetric analysis was carried ured (Ou et al. 2001). This assay uses a biological radical out on the mineral residues; Fig.  3a shows the results, source, so is considered the most relevant method from a while Fig.  3b reports the first derivative of the curve, to biological point of view, integrating the degree and time better visualize the different steps. of antioxidant reaction (López-Pedrouso et al. 2020). The The first weight loss (about 8%, T < 200  °C) is due to value of the peroxyl radical scavenging activity of por- the removal of water, either adsorbed on the surface or cine PH extracts was 87.7 ± 6.3  mg Trolox equivalent/g included in the structure of the powder. A slightly larger of dry extract. Indeed, porcine liver protein hydrolysates loss (about 10%) is observed for 200 < T < 600 °C; losses in from enzymatic hydrolysis with several enzymes such as this temperature interval are associated with the burning alcalase, bromelain, flavourzyme and papain have shown of the residual organic fragments present in the mate- antioxidant capacity using ORAC-FL method (López- rial. This weight loss is much smaller to that previously Pedrouso et  al. 2020; Borrajo et  al. 2020b). Our results observed for porcine bones—about 30% (Figueiredo corroborated this, proving that antioxidant peptides from et  al. 2010); this difference confirming that a significant porcine by-products can protect cells from oxidative amount of organic matter was removed from the pow- damage. der during the enzymatic hydrolysis. For higher tem- u Th s, these antioxidant peptides can be employed to peratures, a further decrease in weight can be observed, maintain human health and also food safety and quality, although it is small (< 3%); this could be due to the by mitigating oxidative stress and lipid peroxidation trig- removal of the carbonate present in the material (Figue- gered by free radicals produced during oxidation reac- iredo et al. 2010). tions of the human body and food products. Thereby, antioxidant peptides have received noteworthy attention CaP characterization in the food industry as functional ingredients and food Based on these results, it was decided to perform the cal- additives. The use of synthetic antioxidants agents in the cination of the material at 700 °C; this value was chosen food industry is under strict regulation due to their side- as the organic fragments were already removed but some effects on human health, namely induction of DNA dam - carbonate ions were still present in the material—indeed, age and toxicity. Consequently, substituting synthetic literature reports that the presence of such ions can be antioxidants by natural antioxidants has become required beneficial for bone-like cellular growth and bioactivity (Tadesse and Emire, 2020). (a) 0.00 (b) -0.05 -0.10 -0.15 0200 400600 8001000 0200 400 600 800 1000 Temperature ( C) Temperature ( C) Fig. 3 a Thermogravimetric analysis ( TGA) of the inorganic residues; b first derivative of the curve Residualweight(%) Δm/ΔT(a.u.) B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 9 of 12 Table 3 Calcium and phosphorus content (wt %) and Ca/P inorganic residues molar ratio for the non-calcined powder and CaP sample OH Sample Ca (%) P(%) Ca/P Yield of the CO process (%) PO 4 OH Non- 34.40 ± 0.28 15.25 ± 0.07 1.74 ± 0.01 20.5 calcined PO 4 PO powder PO CaP 40.30 ± 0.28 18.90 ± 0.42 1.65 ± 0.03 17.0 CaP PO 4000 3600 1500 1200 900600 -1 Wavenumber (cm ) CaP Fig. 5 FTIR of the inorganic residues (i.e., non-calcined powder) and of the calcined CaP sample inorganic residues 100020 30 40 50 60 HAp 20 30 40 50 60 2θ (degrees) Fig. 4 XRD data for the inorganic residue (non-calcined powder) and the CaP sample. The patterns are compared to the 01-072-1234 standard for HAp (Nakamura et  al. 2016). Calcination at this temperature led to a yield of about 170 g, i.e., 17%. Elemental analysis was performed to determine the content of calcium and phosphorus, as well as the Ca/P molar ratio—see Table 3. It can be seen that, although both calcium and phos- Fig. 6 SEM micrograph of the calcined powder CaP phorus show higher relative content after the calcination, the increase is different for the two elements; in fact, the Ca/P ratio decreases slightly—from 1.74 to 1.65. This more crystalline than the starting non-calcined powder indicates that a small quantity of calcium is lost during (sharper peaks). the calcination; this behavior was previously observed for FTIR spectra of the samples are shown in Fig.  5. It CaP derived from natural sources (Aydin et al. 2020). The can be seen that the signals are much sharper and more Ca/P ratio for the CaP powder is very close to the stoi- resolved for the calcined CaP sample, due to its higher chiometric one, that is 1.67. crystallinity. Peaks belonging to the HAp phosphate ions Figure  4 shows the XRD patterns for the inorganic −1 can be observed at 1090, 1040, 960, 603, 568  cm (Pic- powder, prior the calcination and after (CaP). It can be cirillo et  al. 2013); it is interesting to note that no peak seen that in both cases the only phase present is HAp; no −1 is present at 1122  cm . This signal corresponds to the other phosphate compounds, for instance β-tricalcium β-TCP (Piccirillo et  al. 2013); its absence confirms that phosphate (β-TCP), are present. This was expected, the this phase is not formed in the CaP sample, in agree- Ca/P ratio being not statistically different from the stoi - ment with XRD data (Fig.  4). Other weaker peaks pre- chiometric one; literature reports the formation of β-TCP sent in the spectrum correspond to the OH ions at 3570 for smaller Ca/P ratios, i.e., values close to 1.5 (Piccirillo −1 −1 and 634  cm ; moreover, signals at 1412 and 1450  cm et  al. 2014). It can also be observed that CaP is much Intensity(a.u.) Transmittance (%) Borges et al. Bioresources and Bioprocessing (2022) 9:30 Page 10 of 12 belong to the carbonate group (Figueiredo et  al. 2010). the first step to extract different parts from the residues; These signals confirm that, after a treatment at 700  °C other step(s) for other fraction(s) could be added. carbonate ions are still present in the HAp lattice. The extraction of PH was performed with an enzy - Figure  6 shows a SEM micrograph of the CaP sample. matic bioprocess, without employing toxic solvents, It can be seen that the powder has a nanometric struc- according to the principles of the green chemistry, as an ture; indeed particles with average size of about 50  nm aqueous solution was used. For CaP, on the other hand, can be observed. Literature data report that HAp from no solvent was necessary, but a simple thermal process natural sources can be nanometric or not, depending was performed. Although energy is used for this treat- on the sources (Barakat et  al. 2009; Santana et  al. 2019). ment, with an associated impact on the environment, it The use of HAp in the form of nanoparticles has recently has to be highlighted that the conventional routes for gained increasing attention, as it can show enhanced sin- CaP production are likely to have a greater environ- tering properties, if compared to the micrometric pow- mental impact. With these, in fact, chemical reactions der (György et  al. 2019), due to a higher specific surface between Ca and P are performed; in addition to the and consequently a higher powder reactivity. Moreo- energy cost linked to this, the use of Ca- and P-contain- ver, nano-HAp was also preferred for the preparation of ing reagents, derived from non-renewable sources, has composites with other compounds (i.e., biopolymers) to be considered. Both elements, in fact, are obtained (Turon et  al. 2017). Compared with micrometric HAp, from mining activities, whose impact is known to be the nanoscale one induces better cellular functions like quite high (Dubsok and Kittipongvieses 2016; Petrov osteoblast responses (such as adhesion, proliferation, and and Danilov 2020). Considering this, by-product valori- differentiation) (Li et  al. 2013). Also, for its application zation surely offers a more sustainable solution. as a heavy metal remover, nano-HAp showed enhanced From the economic point of view, both PH and CaP performance (Kowthaman and Varadappan 2019). have high market value. Considering CaP, it is a good Based on these results, it can be stated that HAp with quality powder, with high purity and high level of crys- interesting properties and potential for application in tallinity for biomedical applications, can cost up to tens biomedicine (for instance, as bone substitute) and as a of euros per gram. PH, for feed applications, is about 5x heavy metal remover was successfully extracted in this more valuable than a non-hydrolyzed protein. This makes combined process. The use of natural HAp has been the process profitable. This makes the process profitable. explored instead to synthetic HAp, because it has com- parable metabolic activity, preserves chemical composi- tion and structure of the precursor material (Boutinguiza Conclusions et  al. 2012). In addition, there is a growing concern to Porcine by-products (meat and bones) are a valuable develop clean, non-toxic and environmentally friendly source to produce natural value-added compounds for procedures for HAp synthesis (with lower impact on different markets. The combined process described in the environment). It is required to reuse waste not only this work shows that it is possible to extract different because waste materials are accumulating, but also compounds, both organics and minerals—indeed pro- because natural raw materials are being exhausted. tein hydrolysates and hydroxyapatite were obtained. Overall, about 30% of the by-products were converted into valuable compounds—13.5% and 17% of protein Evaluation of the process hydrolysates and hydroxyapatite, respectively. A full environmental and economic assessment of the Protein hydrolysates were rich in low MW pep- process is beyond the scope of this work; some general tides and showed significant antioxidant properties. comments, however, can already be made. Hydroxyapatite, on the other hand, was shown to be As reported in Sect. 3.1, the overall yield is about 30%; single-phase and with a nanometric structure. this value is well below the 100% associated with the full The proposed combined process is quite simple, cheap valorization. It is worth highlighting, however, that with- and easily scalable; all these features make it applicable at out performing this combined process, only PH or CaP industrial scale, to achieve a more complete valorization would be extracted, with an overall minor yield and a of porcine by-products. Moreover, in principle the pro- fewer effective by-products valorization. Moreover, the cess could be applied also to by-products of other meat residues also contain other organic fractions, such as or fish industries. As future work, other steps could be lipids; these phases should also be considered to achieve added to the process, for the extraction of other phases a full valorization. The process presented here represent (i.e., lipids) and to achieve a more complete valorization. B orges et al. Bioresources and Bioprocessing (2022) 9:30 Page 11 of 12 Abbreviations Álvarez C, Rendueles M, Díaz M (2012) Production of porcine hemoglobin AAPH: 2,2’-Azobis(2-amidinopropane) dihydrochloride; ABTS: 2,2-Azino-bis- peptides at moderate temperature and medium pressure under a nitro- 3-ethylbenzothiazoline-6-sulphonic acid; ATR : Attenuated total reflectance; gen stream. Functional and antioxidant properties. J Agric Food Chem CaP: Calcium phosphates; DH: Degree of hydrolysis; FPLC: Fast protein liquid 60:5636–5643 chromatography; FTIR: Fourier transformed infrared spectroscopy; HAp: AOAC (1995) Official methods of analysis 16th Ed. Association of official ana- Hydroxyapatite; mAU: Milli absorbance units; MW: Molecular weight; ORAC-FL: lytical chemists. Washington DC, USA Oxygen radical absorbance capacity; PH: Protein hydrolysates; RSA: Radical Aspevik T, Oterhals Å, Rønning SB, Altintzoglou T, Wubshet SG, Gildberg A, scavenging activity; SEM: Scanning electron microscopy; TCP: Tricalcium Afseth NK, Whitaker RD, Lindberg D (2017) Valorization of proteins from phosphate; TGA : Thermal analysis; TNBS: Trinitrobenzenesulfonic acid solution; co-and by-products from the fish and meat industry. Chem Chem Tech- XRD: X-ray diffraction. nol Waste Valoriz 1:123–150 Aydin G, Terzioğlu P, Öğüt H, Kalemtas A (2020) Production, characterization, Acknowledgements and cytotoxicity of calcium phosphate ceramics derived from the bone Not applicable. of meagre fish, Argyrosomus regius. J Austr Ceram Soc 1:1–10 Barakat NAM, Khil MS, Omran AM, Sheikh FA, Kim HY (2009) Extraction of pure Authors’ contributions natural hydroxyapatite from the bovine bones bio waste by three differ - SB: methodology, investigation, writing—original draft, preparation. CP: inves- ent methods. J Mater Process Technol 209:3408–3415 tigation, writing- reviewing and editing. FS: investigation, visualization and Borrajo P, López-Pedrouso M, Franco D, Pateiro M, Lorenzo JM (2020a) data curation. RM: investigation. AR: investigation. JAC: writing—reviewing Antioxidant and antimicrobial activity of porcine liver hydrolysates using and editing. AA: conceptualization, resources. MP: conceptualization, project flavourzyme. Appl Sci 10:3950 administration, writing—reviewing and editing. All authors read and approved Borrajo P, Pateiro M, Gagaoua M, Franco D, Zhang W, Lorenzo JM (2020b) the final manuscript. Evaluation of the antioxidant and antimicrobial activities of porcine liver protein hydrolysates obtained using alcalase, bromelain, and Funding papain. Appl Sci 10:2290 This work was supported by National Funds from project MOREPEP (POCI-01– Boutinguiza M, Pou J, Comesaña R, Lusquiños F, De Carlos A, León B (2012) 0247-FEDER-017638) funded by Fundo Europeu de Desenvolvimento Regional Biological hydroxyapatite obtained from fish bones. Mater Sci Eng, C (FEDER), under Programa Operacional Competitividade e Internacionalização 32:478–486 (POCI) and from FCT – Fundação para a Ciência e a Tecnologia through project Chang C-Y, Wu K-C, Chiang S-H (2007) Antioxidant properties and protein UIDB/50016/2020. Clara Piccirillo and Francesca Scalera thank Fondazione con compositions of porcine haemoglobin hydrolysates. Food Chem il Sud for funding the HApECOrk project (Grant Number 2015–0243). 100:1537–1543 Damgaard TD, Otte JAH, Meinert L, Jensen K, Lametsch R (2014) Antioxidant Availability of data and materials capacity of hydrolyzed porcine tissues. Food Sci Nutr 2:282–288 All data supporting this article’s conclusion are available. Damgaard T, Lametsch R, Otte J (2015) Antioxidant capacity of hydrolyzed animal by-products and relation to amino acid composition and pep- tide size distribution. J Food Sci Technol 52:6511–6519 Declarations de Queiroz ALM, Bezerra TKA, de Freitas PS, da Silva MEC, de Almeida GCA, Gadelha TS, Pacheco MTB, Madruga MS (2017) Functional protein Ethics approval and consent to participate hydrolysate from goat by-products: Optimization and characterization Not applicable. studies. Food Biosci 20:19–27 dos Santos Cardoso M, Godoy AC, Oxford JH, Rodrigues R, dos Santos Consent for publication CM, Bittencourt F, Signor A, Boscolo WR, Feiden A (2020) Apparent Not applicable. digestibility of protein hydrolysates from chicken and swine slaughter residues for Nile tilapia. 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Journal

Bioresources and Bioprocessing – Springer Journals

Published: Mar 21, 2022

Keywords: Porcine by-products; Bioactive peptides; Enzymatic hydrolysis; Natural hydroxyapatite; Nanomaterial

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