1. Introduction
In the European Union (EU), nearly 59 million tons of food waste (corresponding to 131 kg/inhabitant) from households, retail establishments, and the food service industry are generated annually with an associated market value estimated at 132 billion euros [
1].
Food waste (FW) is addressed in the EU Plan for the Circular Economy, including a FW reduction target under the Strategy of Farm-to-Fork within the European Green Deal [
2]. The EU and EU countries are committed to meeting the Sustainable Development Goal 12.3 target [
3] to halve per capita food waste at the retail and consumer stages by 2030 [
4].
At the retail period, the main food waste produced is meat waste: around 70% of retail waste generated every day corresponds to meat waste [
5]. This enormous amount of waste, currently mostly landfilled, is a valuable resource which, for both economic and ecological reasons, should be kept within the production cycle.
Retail meat waste is constituted by the products for sale in the butcher shops that was originally destined for human consumption but that has not reached this purpose either because they have not been sold, or because they are remains from cleaning and conditioning the pieces sold. Meat waste is included in Category 3 of Regulation (EC) No 1069/2009 of the European Parliament and of the Council laying down health rules as regards to animal by-products and derived products [
6]. Products in Category 3 are low risk. They include carcasses or parts of slaughtered animals that are suitable for human consumption, but are not intended for this purpose, such as bones, hides, skins, heads of poultry, etc.; that is, food products from animal origins not intended for human consumption due to commercial motives, manufacturing or packaging defects, or other defects which do not imply any health risk. Category 3 products can be processed for feeding purposes, for the manufacture of cosmetics, for the manufacture of pharmaceutical products, etc. [
6], so there is no impediment to using retail meat waste as raw material for obtaining products with a higher added value.
Meat waste is mainly composed of proteins and lipids [
7,
8]. These essential nutrients can be recovered from meat waste via enzymatic hydrolysis.
Enzymatic hydrolysis is regarded as a green technology [
9,
10]. The use of enzymes in this process is considered environmentally friendly because it can be performed under mild conditions, reducing the need for harsh chemicals (such as strong acids or strong alkalis) and high temperatures that can harm the environment [
11,
12]. The use of hydrolases enzymes to hydrolyze proteins (proteases) or lipids (lipases) is considered a gentler alternative method to alkaline or acidic approaches to protein and lipid solubilization and extraction [
13].
During enzymatic hydrolysis with proteases, proteins are cleaved into smaller peptides and free amino acids [
14], thus increasing their solubility [
15]. Protein hydrolysates containing mainly di- and tripeptides has been proved to be superior to whole proteins and free amino acids in terms of being applied to different applications such as nutrition [
16], biotechnology [
17], and cosmetic industries [
18].
The native structure of proteins is crucial in its recognition via proteases [
19]. In meat waste, two types of native structures of proteins can be found: globular-type and fibrous-type [
20]. The main protein in retail meat waste is collagen, an insoluble fibrous-type protein [
21]. Collagen is less susceptible to hydrolysis than the globular-type proteins found in this animal waste [
22]. But the reduced hydrolysis of collagen has the advantage of making collagen recovery possible for commercial use, including biotechnological and biomedical uses, cosmetics manufacturing, etc. [
23].
Meat lipids are mainly composed of triglycerides, corresponding to about 95% of meat lipids [
24]. In lipid hydrolysis, the triglyceride molecule is degraded into three separate fatty acid chains [
25]. The addition of specific lipases is needed to catalyze the lysis of fatty acids from the triglyceride [
26]. Fatty acids are long hydrocarbon chain molecules with a carboxyl-terminal group [
27] that can be widely used as versatile intermediates and chemicals in industrial applications such as, among others, cosmetics [
28], pharmaceutical manufacturing [
29], or surfactants [
30].
In accordance with the above mentioned, the enzymes’ capacity to hydrolyze proteins and lipids enables the production of short peptides and the release of collagen and fatty acids from retail meat waste.
Protein hydrolysates containing mostly di- and tripeptides, collagen, and fatty acids have a growing demand in recent years. In the year 2022, the global protein hydrolysate market size was valued at USD 583.30 million; the size of this market is expected to increase to USD 1.14 billion by the year 2030, while growing at a compound annual growth rate (CAGR) of 8.8% [
31]. The global collagen market size was accounted for at USD 9.12 billion in 2022 and is expected to expand at a CAGR of 10.2% from 2023 to 2030 [
32]. The fatty acids market size is estimated to be worth USD 29.52 billion in 2022 and is forecast to a readjusted size of USD 45.56 billion by 2030 with a CAGR of 4.9% during the forecast period of 2023–2030 [
33].
This increase in demand for protein hydrolysates, collagen, and fatty acids means that more sources of proteins and lipids are needed. But this increase in demand must be environmentally sustainable, so that sources such as meat waste are an alternative option to the typical raw materials used in the production of these products.
Therefore, the aim of this work is the study of enzymatic hydrolysis of retail meat waste to generate added-value products and to fulfill the requirements in the EU Circular Economy Package. The study consists of three parts: (i) to optimize the production of high-quality protein hydrolysates and collagen from hydrolysis of meat waste using a protease, (ii) to optimize the production of fatty acids from hydrolysis of meat waste using a lipase, and (iii) to optimize the simultaneous production of protein hydrolysates, collagen, and fatty acids from hydrolysis of meat waste with a combination of protease and lipase. To accomplish these goals, hydrolysis of retail meat waste was carried out at different pHs, temperatures, and enzyme/substrate ratios.
2. Materials and Methods
2.1. Materials
Meat waste was gathered from a local retail meat store in Salamanca (Spain). This waste was mainly composed of flesh, bones, fat, kidney, tongue, heart, brain, lungs, and liver from pigs, lambs, cattle, rabbits, and poultry. Waste was ground in a cutting mill (Restch SM 2000 model), homogenized at 1 mm of the particle diameter and freeze-stored in small portions at −20 °C. Prior to experimentation, the portions were defrosted overnight in the refrigerator at 4 °C.
Two food grade enzymes were employed in the study:
Alcalase 2.4 L, a proteolytic enzyme obtained from
Bacillus licheniformis, with a declared activity of 2.4 AU/g and a density of 1.17 g/mL [
34], whose optimal conditions are temperatures between 30 °C and 65 °C, and its pH values are between 7.0 and 9.0 [
34].
Resinase
® HT, a lipase originating from
Aspergillus sp., is highly effective for triglyceride hydrolysis with a declared activity of 50 KLU/g [
35] and a density of 1.05 g/mL [
34], whose optimal conditions are temperatures of 50–70 °C (although it is stable up to temperatures of 90 °C), and its pH is between 5 and 8 [
35].
To select the best protease and lipase to hydrolyze meat waste, the material was independently hydrolyzed in previous studies [
5] using the proteases: Alcalase 2.4 L, Flavourzyme 1000 L, Neutrase 0.8 L, Pancreatic Trypsin 6.0 S, and Protamex; and the lipases: Lipozyme CALB L, Lipozyme TL 100 L, Novocor AD L, Novozym 51032, and Resinase HT (all enzymes from Novozymes A/S, Bagsvaerd, Denmark). Alcalase and Resinase showed the highest capacity to generate large amounts of protein hydrolysates and free fatty acids, respectively, and were therefore chosen for this study.
Analytical-grade chemicals were employed in all experiments.
2.2. Analytical Methods
Chemical composition of raw material and products was determined based on the appropriate methods [
36].
Moisture was determined in waste samples. Meat waste and hydrolyzed samples were analyzed for protein, collagen, lipids, and fatty acids. Total N content was analyzed using the Kjeldahl method; the protein content was subsequently calculated from the Kjeldahl N by multiplying it by the nitrogen-to-protein conversion factor of 6.25 [
37]. Total collagen was measured using a hydroxyproline assay. Lipid content was assayed via petroleum ether extraction. The fatty acids profile of the isolated lipids was determined using hydrolytic extraction, methylation, and a capillary gas chromatography-flame ionization detection (GC-FID) analysis of the resulting fatty acid methyl esters (FAMEs).
The average molar mass of protein hydrolysates (
MM) was calculated from the average peptide chain length of the hydrolysate (
PCL) [
38] as shown below:
and the
PCL from the hydrolysis degree (
DH) [
39] was calculated as shown below:
Protein hydrolysis was performed using the pH-stat method described by Mat et al. [
40] and the
DH (%) was calculated from the molarity and the volume of alkali used to keep the pH constant (see
Section 2.3) as shown below:
where
DH is the percent ratio of the number of peptide bonds cleaved during hydrolysis (
h) to the total number of peptide bonds in the protein substrate studied (
htot = 7.6 eqv/kg for meat protein [
41]).
B and
Nb refer to the alkali consumption during hydrolysis and its normality,
Mp is the initial mass of protein in the reactor, and
α represents the average degree of dissociation of the α-NH
2 groups in the protein substrate (
α varies with pH and temperature [
41]).
Each measurement was performed three times and averaged. Mean and standard deviation were used to express the results.
2.3. Enzymatic Hydrolysis Procedure
Hydrolysis in a batch mode was performed in a 0.5 L cylindrical jacketed glass reaction vessel using the pH-stat method in controlled hydrolysis conditions (pH, temperature, enzyme concentration, and stirring speed). A suspension was made in the reactor by mixing 50 g of meat waste with distilled water to obtain concentrations of 17.45 g/L and 63.14 g/L for proteinic and lipidic substrates, respectively. Before adding the enzymes, the suspension was first adjusted to the proper pH and temperature; temperature and pH were selected based on the optimal values for the hydrolases (see
Section 2.1). Once enzymes were added to the reaction vessel, the reaction pH was continuously monitored; to maintain the pH at a constant value, 2 N of NaOH was added whenever the pH decreased 0.1 units from the target value.
The time of hydrolysis was set at 240 min since product recovery does not increase significatively with longer durations of treatment. To maintain the homogeneity of the reaction mixture and prevent vortices, a 300 rpm stirring speed was chosen for all the experiments.
2.4. Sample Treatment
The resulting sample was heated for 20 min at 95–97 °C to inactivate the enzymes and pasteurize the mixture; afterwards, the sample was centrifuged for 15 min at 9000 rpm to separate three fractions: a lipid-liquid fraction containing the separated lipids, a water-liquid fraction containing the solubilized protein, and a solid fraction containing the collagen.
All the experiments were carried out in duplicate. Student’s test was applied to evaluate the significant differences (p < 0.05).
2.5. Experimental Strategy
So far, enzymatic hydrolysis of meat waste has not been employed to simultaneously generate protein hydrolysates, fatty acids, and collagen. Nevertheless, many authors have studied hydrolysis of food proteins and, therefore, the factors influencing the reaction kinetics are well known and they are: pH, temperature, and initial enzyme concentration/initial substrate concentration ratios [
42]. Accordingly, in this work, the influence of these factors on the efficiency of product recovery was studied.
Three groups of analyses were made to evaluate the effect of different pHs, temperatures, and initial enzyme concentration/initial substrate concentration ratios in the reactor: hydrolysis with Alcalase to hydrolyze the proteins, hydrolysis with Resinase to hydrolyze the lipids, and hydrolysis using a combination of Alcalase and Resinase to simultaneously hydrolyze the proteins and the lipids.
4. Conclusions
Enzymatic hydrolysis process can be effectively used as a green technology to recover products like protein hydrolysate, collagen, and fatty acids from meat wastes, thus reducing the organic load caused by meat retail stores, solving the pollution problem, and supporting the circular economy of the sector.
Enzymatic hydrolysis has the advantage that it can recover the proteins (hydrolysate and collagen) and the fatty acids either separately or simultaneously, using a protease, a lipase, or a combination of protease and lipase. In this work, the protease Alcalase and the lipase Resinase were used separately or combined to hydrolyze meat waste. The use of a combination of Alcalase and Resinase for the hydrolysis has the additional advantage of recovering all the products in a sole stage.
The protein hydrolysates obtained, which are rich in low molecular weight peptides (average molecular weight near 500 Dalton), are suitable for the food industry.
The lipids recovered are rich sources of unsaturated fatty acids which have several health benefits. Additionally, recovered collagen could be used in a variety of fields, including biomedical and aesthetic ones. In any case, the recovered products have potential application in cosmetic and biotechnology applications.