A Green Technology Approach Using Enzymatic Hydrolysis to Valorize Meat Waste as a Way to Achieve a Circular Economy

Abstract

The retail meat industry produces a significant amount of waste, containing proteins, lipids, and other elements that could serve as the basis for other products. This work presents the results of research on the enzymatic hydrolysis of meat waste as a green technology to obtain products with added value as a substitute for other raw materials. pH, temperature, the protease/proteinic substrate ratio (Eo/So), and the lipolase/lipidic substrate ratio (Eo’/So’) were studied as process variables for hydrolysis of proteins and lipids, respectively. Hydrolysis for the recovery of proteins (as protein hydrolysates or collagen) was carried out with the protease Alcalase; pH around 8.0, temperature around 50 °C, and Eo/So around 0.16 AU/g were the optimum process variables’ values for obtaining high amounts of recovered proteins and peptides that are easily digestible and have a pleasant taste. The lipase Resinase was used to hydrolyze the lipids; a clear relationship was observed between Eo’/So’ and the amounts of recovered fatty acids. The optimum process variables’ values were found to be Eo’/So’ around 0.83 kLU/g, pH around 8.0 and temperature around 50 °C. Unsaturated fatty acids prevailed in the final product. For the simultaneous recovery of protein hydrolysates, collagen, and fatty acids, a combination of Alcalase and Resinase was used; the process variables examined included the optimal range of values for Eo/So and Eo’/So’, as well as pH and temperature that were suggested in research for both Alcalase and Resinase, separately. The results showed that the simultaneous process was mainly influenced by the Eo/So and Eo’/So’ ratios, instead of being influenced by the pH and temperature values which were less influential. For Eo/So = 0.16 AU/g, Eo’/So’ = 1.11 kLU/g, pH = 7.5, T = 50 °C, the maximum amounts of products (0.8 kg by kg of dry meat waste) were obtained more economically, where the whole of the proteins and lipids in meat waste were practically recovered. Therefore, in order to preserve a circular economy for retail meat waste, enzymatic hydrolysis is appealing and environmentally friendly.

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:

$$MM=130·PCL$$

(1)

and the PCL from the hydrolysis degree (DH) [39] was calculated as shown below:

$$PCL=\frac{100}{DH}$$

(2)

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:

$$DH \left(\%\right)=\frac{h}{h_{tot}}=\frac{B·N_b}{M_p· \alpha · h_{tot}}\times 100$$

(3)

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 (h_tot = 7.6 eqv/kg for meat protein [41]). B and N_b refer to the alkali consumption during hydrolysis and its normality, M_p is the initial mass of protein in the reactor, and α represents the average degree of dissociation of the α-NH₂ 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.

3. Results and Discussion

3.1. Basic Compositional Analysis of Meat Waste

The moisture content of meat waste used in this investigation was 45.04 ± 1.21%. On a dry weight basis, meat waste had a protein content (proteinic substrate contained in meat waste) of 19.03 ± 0.42%, and a lipid content (lipidic substrate contained in meat waste) of 68.91 ± 1.14%. These results agree with those presented by García et al. [43], who found contents of 24.6 ± 10.3% and 69.9 ± 13.7% for proteins and lipids, respectively, in research describing the characteristics of meat waste produced by 208 butchers.

The results obtained for total collagen showed a content of 67.91 ± 0.14 mg/g, representing a percentage of 35.7% of the total protein present in waste. This is coherent with the fact that collagen is the most plentiful protein in mammalians, accounting for 25% of the total body protein [44], 95% of the fibrous elements in connective tissue, and 90% of the bones [45]. It is not unexpected that the percentage of collagen obtained somewhat surpasses the typical 25% in body protein given that the bones and connective tissue in meat waste are a larger percentage than usual in the body of an animal.

The analysis of the fatty acid profile in meat waste (

Table 1. Fatty acids profile in lipids from meat waste.

Fatty Acid	Meat Waste Mean ± SD (%)	Bibliographic Values Minimum–Maximum (%)	Sources
Saturated fatty acid
C14:0	4.25 ± 0.09	1.70–30.20	[46,47,48]
C15:0	0.27 ± 0.03	0.00–0.60	[49,50,51]
C16:0	25.45 ± 0.26	2.28–36.89	[46,48,52]
C17:0	3.48 ± 0.10	0.00–3.90	[47,48,53]
C18:0	12.85 ± 0.24	0.40–32.73	[46,50,53]
C20:0	0.34 ± 0.02	0.06–4.69	[46,51,54]
Unsaturated fatty acids
C14:1	0.00 ± 0.00	0.00–1.16	[50,51,55]
C15:1	0.00 ± 0.00	0.00–1.70	[47,51,56]
C16:1	0.30 ± 0.01	0.00–37.9	[48,55,57]
C17:1	0.32 ± 0.02	0.00–0.90	[47,52,58]
C18:1n9c + t	24.64 ± 0.34	0.84–46.29	[46,52,59]
C20:1	0.00 ± 0.00	0.00–2.78	[51,53,58]
C18:2n6c + t	10.50 ± 0.27	0.49–38.28	[46,48,54]
C20:2	1.76 ± 0.05	0.00–2.57	[51,52,60]
C18:3	3.31 ± 0.06	0.03–4.71	[46,54,60]
C20:3n3	4.86 ± 0.05	0.07–4.82	[47,51,53]
C20:3n6 + c21:0	3.88 ± 0.08	0.07–4.82	[47,51,53]
C20:4n6	3.78 ± 0.07	0.03–6.16	[52,53,61]

) clearly showed the predominance of unsaturated fatty acids (53.36 ± 0.33) over saturated fatty acids (46.64 ± 0.19). Due to the lack of information in the literature on the content of fatty acid in butcher waste, a review of the literature on the fatty acid profile in different animal fats and meats was carried out. The survey showed that the fatty acid concentration changes significantly depending on whether the sample is meat or lard and on the sample’s origin (lamb, beef, chicken, rabbit, etc.). A range of values were established based on all these data; the outcomes are shown in Table 1. The percentages of fatty acids acquired in the current investigation were found to fall within the range established in the literature.

3.2. Protein Hydrolysis

3.2.1. Influence of pH and Temperature

The Eo/So ratio used in this study was 0.16 AU/g (5.7% g of Alcalase/g proteins in meat waste) according to Schmidt and Salas-Mellado [62], who used the ratios from 2.9 to 9.1% for meat hydrolysis with Alcalase. The tests were performed under different conditions with respect to temperature (40–60 °C at a pH of 8.0) and pH (7.0–9.0 at a temperature of 50 °C). The pH and temperature were selected in accordance with the optimal values for Alcalase (see Section 2.1). The proteins were recovered as peptides in the forms of hydrolysates and collagen.

The results obtained show how high pHs and low temperatures favor hydrolysis, producing hydrolysates with a higher percentage of hydrolyzed protein and a lower percentage of unhydrolyzed collagen (

Table 2. Recovery of proteins at different pHs.

pH	Hydrolyzed Protein (%)	Unhydrolyzed Collagen (%)
7.0	40.30 ± 0.14	48.18 ± 0.71
7.5	41.05 ± 0.11	45.41 ± 0.94
8.0	45.03 ± 0.20	43.10 ± 0.10
8.5	58.65 ± 0.38	42.18 ± 0.45
9.0	59.30 ± 0.62	38.18 ± 0.44

and

Table 3. Recovery of proteins at different temperatures.

T (°C)	Hydrolyzed Protein (%)	Unhydrolyzed Collagen (%)
40	46.76 ± 0.20	35.94 ± 0.70
45	45.82 ± 0.27	39.23 ± 0.08
50	45.03 ± 0.20	43.10 ± 0.10
55	41.62 ± 0.38	43.17 ± 0.34
60	40.83 ± 0.62	47.47 ± 0.73

).

The effect of pH on protein hydrolysis is justified by the increase of enzymatic activity with increasing the pH and the stability of the enzyme in the pH range of 7–10 [63].

At first glance, it might seem that the effect of temperature on protein hydrolysis disagrees with the literature where a higher enzymatic activity is achieved when the temperature rises [63]. However, the Alcalase inactivation over time with an increase of temperature is so high that the efficiency of the process is greater at 40 °C for 240 min of reaction time [63].

According to the results obtained, within the analyzed pH and temperature ranges, lower pHs or higher temperatures are recommended to recover unhydrolyzed collagen, while higher pHs or lower temperatures are more adequate to obtain protein hydrolysates. The selection of the operating conditions of pH and temperature will depend on the size of the peptides whose obtainment is desired in the hydrolysates.

Table 4. Average peptide chain length (PCL) and molecular weight (MM) of the hydrolysates obtained under different pHs and temperatures (T).

pH	T (°C)	PCL	MM (Daltons)
7.0	50	4.74	616.55
7.5	50	3.90	506.43
8.0	50	3.63	471.95
8.5	50	3.34	434.13
9.0	50	2.82	366.77
8.0	40	2.96	385.13
8.0	45	3.25	422.28
8.0	50	3.63	471.95
8.0	55	3.86	501.16
8.0	60	4.51	586.77

shows the average peptide chain lengths and the average molecular weights of the peptides present in the hydrolysates obtained under different operating conditions.

To use protein hydrolysates in the food industry, they must be easily digestible and palatable, which is only reached when the molecular weight of peptides makes up the hydrolysate ranges in a certain interval because:

• Peptides with a high molecular weight (over 6000 Dalton) can cause allergies [64].
• Peptides with a molecular weight range from 1000 to 6000 Dalton have bitter taste [65].
• Free amino acids (molecular weight under 200 Dalton) are hyperosmotic and cause diarrhea [66].

So, to avoid the problems of allergenicity, bitterness, and hyperosmoticity, the hydrolysates should mainly consist of di- and tripeptides and have a range of molecular weight distribution as narrow and as feasible with an average molecular weight near 500 Dalton and, in any case, between 200 and 1000 Dalton [16].

Taking this into account, the most appropriate operating conditions, according to our study, should be between 7.5 and 8.5 for pH and 45 and 55 °C for temperature because the obtained hydrolysates have average molecular weights closest to 500 Dalton, as shown by the lower relative deviation (see Table S1 in Supplementary Materials).

3.2.2. Influence of Protease/Proteinic Substrate Ratio (Eo/So)

To check the influence of the Eo/So ratio on product recovery, experiments were carried out at different ratios, from 0.09 to 0.27 AU/g (3.2–7.5% g of Alcalase/g proteins in meat waste). This range was chosen based on literature [62] where, as previously said, the ratios from 2.9 to 9.1% are used for meat hydrolysis with Alcalase. The temperature (50 °C) and pH (8.0) were selected at the midpoint of the optimal values obtained previously (see Section 3.2.1). Again, the proteins were recovered as peptides in the forms of hydrolysate and collagen.

As expected, the results obtained indicate that an increase in the protease/proteinic substrate ratio causes a higher percentage of hydrolyzed protein and a lower percentage of unhydrolyzed collagen (

Table 5. Recovery of proteins at different protease/proteinic substrate ratios (Eo/So).

Eo/So (AU/g)	Hydrolyzed Protein (%)	Unhydrolyzed Collagen (%)
0.09	29.79 ± 0.17	59.37 ± 0.11
0.11	35.42 ± 0.40	53.85 ± 0.06
0.16	45.03 ± 0.16	43.26 ± 0.11
0.21	49.93 ± 0.26	34.39 ± 0.05
0.27	64.42 ± 0.24	31.54 ± 0.06

).

The results in Table 5 show that a greater amount of protease in the reactor for the same concentration of proteinic substrate (higher Eo/So) allows the increase in the number of enzyme–substrate bonds formed, promoting a high number of broken peptide bonds, favoring the hydrolysis, and generating an increase in the amount of the hydrolysates produced.

However, collagen is a fibrous-type protein whose behavior against hydrolysis differs from that of globular-type proteins. Whereas globular-type proteins are more easily hydrolyzed given their characteristics, collagen is less soluble and more difficult to hydrolyze in its native state, which means that a higher concentration of protease is necessary for its hydrolysis [67]. Therefore, for a greater recovery of collagen for industrial applications, it would be convenient to work with near zero initial concentrations of Alcalase.

The choice of the Eo/So operating conditions will rely on the required size of the peptides in the hydrolysates.

Table 6. Average peptide chain length (PCL) and molecular weight (MM) of the hydrolysates obtained under different protease/proteinic substrate ratios (Eo/So).

Eo/So (AU/g)	PCL	MM (Daltons)
0.09	4.35	566.08
0.11	4.17	542.34
0.16	3.63	471.95
0.21	3.45	449.05
0.27	3.08	401.05

shows the average length of the peptide chain and the average molar mass for the hydrolysates obtained using different Eo/So ratios.

As previously said, to use protein hydrolysates in the food industry, the molecular weight of the hydrolysates should be of the order of 500 Dalton and, in any case, between 200 and 1000 Dalton [16]. Bearing this in mind, the best quality hydrolysates would be those obtained for the ratios of protease/protein substrate concentrations between 0.11 and 0.21 AU/g since, with these ratios, the average molar mass of the final products is closest to 500 Dalton, as shown by the lower relative deviation (see Table S2 in Supplementary Materials).

3.3. Lipid Hydrolysis

3.3.1. Influence of pH and Temperature

For this study, the Eo’/So’ ratio used was 0.83 kLU/g (1.5% g Resinase/g lipids in meat waste). This ratio agrees with the experiments by Song et al. [68] where 1.0 to 1.5% g of lipase/g lipids were used for hydrolysis of lard that was catalyzed by lipase.

The experiments were conducted at the same pHs (7.0–9.0 at a temperature of 50 °C) and temperatures (40–60 °C at a pH of 8.0) as experiments with Alcalase. These values of pHs and temperatures are within or very close to the optimal range for Resinase (see Section 2.1).

The results achieved (

Table 7. Recovery of lipids at different pHs.

pH	Recovered Lipids (%)
7.0	77.36 ± 0.01
7.5	91.31 ± 0.12
8.0	95.40 ± 0.33
8.5	96.45 ± 0.08
9.0	97.68 ± 0.02

and

Table 8. Recovery of lipids at different temperatures.

T (°C)	Recovered Lipids (%)
40	98.89 ± 0.05
45	97.29 ± 0.06
50	95.39 ± 0.34
55	87.01 ± 0.01
60	68.12 ± 0.10

) indicate that hydrolysis of lipids is favored by lowering the temperature or by increasing the pH. Table 7 shows that the most suitable pH seems to be around 8.0 because, for higher pHs, there are no significant variations in the percentages of recovered lipids that justify its use. Table 8 indicates that the percentage of recovered lipids in the range from 40 to 55 °C are relatively close to each other while, for 60 °C, this percentage is significantly reduced. This suggests that lipolytic hydrolysis should not be carried out at temperatures above 55 °C.

The behaviour against pH is justified by the great stability of the enzyme in the pH range used [69]. Although the literature indicates that Resinase is active at temperatures up to 90 °C, it has been verified that, in the presence of waste, lipase inactivation occurs over time with increasing temperature and that inactivation is so high at higher temperatures that the efficiency of the process is less at 60 °C given the long reaction time.

The amounts of fatty acids released as a function of pH and temperature are shown in

Table 9. Amounts of fatty acids obtained at different pHs (mg_{fatty acid}/g_lipids).

Fatty Acid	pH
	7.0	7.5	8.0	8.5	9.0
C14:0	20.17 ± 0.12	23.14 ± 0.50	27.08 ± 1.14	28.23 ± 1.26	29.82 ± 0.02
C15:0	0.76 ± 0.00	0.91 ± 0.01	0.97 ± 0.04	0.99 ± 0.03	1.02 ± 0.03
C16:0	171.00 ± 0.05	186.10 ± 0.29	204.22 ± 8.54	212.69 ± 3.56	215.47 ± 1.03
C17:0	17.29 ± 0.20	18.97 ± 0.63	20.80 ± 0.95	22.00 ± 0.13	22.53 ± 0.10
C18:0	101.72 ± 2.49	116.64 ± 1.72	129.86 ± 5.47	135.69 ± 0.09	140.49 ± 0.38
C20:0	0.85 ± 0.01	0.92 ± 0.05	0.99 ± 0.03	1.07 ± 0.01	1.13 ± 0.01
SFA	311.79 ± 2.23	346.68 ± 3.09	383.92 ± 1.18	400.65 ± 2.37	410.46 ± 1.50
C16:1	1.02 ± 0.01	1.08 ± 0.05	1.07 ± 0.00	1.09 ± 0.02	1.15 ± 0.00
C17:1	2.89 ± 0.06	3.05 ± 0.13	3.40 ± 0.13	3.74 ± 0.01	3.84 ± 0.03
C18:1	183.71 ± 2.21	195.39 ± 3.98	225.59 ± 7.82	231.68 ± 0.17	234.26 ± 1.15
C18:2	86.15 ± 0.92	101.76 ± 0.88	121.08 ± 5.10	126.66 ± 0.17	131.24 ± 0.60
C18:3n3	28.79 ± 0.15	30.28 ± 0.13	30.80 ± 1.10	32.40 ± 0.05	33.04 ± 0.17
C18:3n6	1.07 ± 0.03	1.15 ± 0.03	1.09 ± 0.04	1.21 ± 0.06	1.17 ± 0.04
C20:2	10.57 ± 0.04	11.48 ± 0.29	13.09 ± 0.53	13.45 ± 0.03	13.70 ± 0.08
C20:3n3	24.05 ± 0.03	25.48 ± 0.69	29.42 ± 1.07	30.64 ± 0.03	31.44 ± 0.12
C20:3n6 + C21:0	37.45 ± 0.11	39.88 ± 0.10	42.24 ± 0.08	43.05 ± 0.04	43.74 ± 0.13
C20:4n6	17.55 ± 0.41	19.64 ± 0.06	22.46 ± 0.96	23.09 ± 0.07	23.58 ± 0.10
UFA	393.26 ± 2.63	429.19 ± 4.00	490.39 ± 1.61	507.05 ± 0.26	517.17 ± 0.83

SFA: Saturated fatty acids; UFA: unsaturated fatty acids.

and

Table 10. Amounts of fatty acids obtained at different temperatures (mg_{fatty acid}/g_lipids).

Fatty Acid	Temperature (°C)
	40	45	50	55	60
C14:0	28.26 ± 0.43	27.36 ± 1.01	27.08 ± 1.14	26.70 ± 0.41	24.55 ± 0.72
C15:0	1.10 ± 0.03	1.05 ± 0.03	0.97 ± 0.04	0.90 ± 0.04	0.65 ± 0.01
C16:0	230.67 ± 1.21	216.64 ± 4.08	204.22 ± 8.54	189.61 ± 4.45	149.47 ± 0.55
C17:0	23.14 ± 0.09	22.07 ± 0.59	20.80 ± 0.95	20.03 ± 0.52	15.32 ± 0.07
C18:0	143.01 ± 0.90	135.78 ± 1.00	129.86 ± 5.47	125.41 ± 0.55	97.31 ± 0.59
C20:0	1.13 ± 0.01	1.02 ± 0.03	0.99 ± 0.03	0.90 ± 0.01	0.70 ± 0.00
SFA	427.30 ± 1.60	403.92 ± 4.79	383.92 ± 1.68	363.55 ± 3.03	288.00 ± 0.84
C16:1	1.19 ± 0.01	1.13 ± 0.03	1.07 ± 0.00	1.03 ± 0.05	0.83 ± 0.01
C17:1	4.19 ± 0.01	3.75 ± 0.24	3.40 ± 0.13	2.96 ± 0.08	2.01 ± 0.04
C18:1	233.49 ± 2.65	230.18 ± 0.13	225.59 ± 7.82	215.25 ± 4.58	192.38 ± 4.25
C18:2	135.57 ± 1.70	127.44 ± 0.13	121.08 ± 5.10	114.50 ± 1.80	85.30 ± 0.03
C18:3n3	40.50 ± 0.05	35.89 ± 1.23	30.80 ± 1.10	27.14 ± 0.75	11.72 ± 0.69
C18:3n6	1.19 ± 0.02	1.16 ± 0.01	1.09 ± 0.04	1.11 ± 0.06	0.94 ± 0.02
C20:2	14.11 ± 0.06	13.61 ± 0.22	13.09 ± 0.53	12.67 ± 0.01	10.13 ± 0.22
C20:3n3	33.10 ± 0.16	31.69 ± 0.26	29.42 ± 1.07	28.04 ± 0.07	21.45 ± 0.13
C20:3n6 + C21:0	43.88 ± 0.16	43.36 ± 0.60	42.24 ± 0.08	39.43 ± 1.19	31.71 ± 0.18
C20:4n6	24.98 ± 0.47	23.50 ± 0.01	22.46 ± 0.96	20.62 ± 0.21	13.46 ± 0.07
UFA	532.21 ± 4.18	511.71 ± 2.05	490.24 ± 4.57	462.75 ± 6.73	369.90 ± 4.33

SFA: Saturated fatty acids; UFA: unsaturated fatty acids.

. As can be appreciated, the free fatty acid amounts follow the same trend as the percentage of recovered lipids: the higher the pH and the lower the temperature, the greater the quantity of individual acids obtained. The influence of pH and temperature on the amounts of fatty acids released in the lipid hydrolysis has also been observed by other researchers [70].

The graphical representations of the total amount of fatty acids released by lipid hydrolysis against the pH and the temperature are displayed in Figure 1 and Figure 2, respectively. They allow us to clearly appreciate how, for pH greater than 8.0 at 50 °C or temperatures above 50 °C at pH 8.0, there are no significant variations in the amounts of fatty acids obtained that justify the additional cost of reaching higher quantities of fatty acids.

3.3.2. Influence of Lipase/Lipidic Substrate Ratio (Eo’/So’)

The experiments were conducted at various ratios, ranging from 0.28 and 1.19 kLU/g (0.5–2.1% g of Resinase/g lipids in meat waste), to examine the effect of the Eo’/So’ ratio on product recovery. This range is consistent with the tests by Song et al. [68], which required 1.0 to 1.5% g of lipase per gram of lipids to hydrolyze lard, and the experiments by Sharma et al. [71], which used 2.0% g of lipase per gram of lipids to hydrolyze cod liver oil and obtain fatty acids. The pH (8.0) and the temperature (50 °C) were fixed according to our results from the previous section.

The findings demonstrate how hydrolysis is favored by the high lipase/lipidic substrate ratios, producing a higher percentage of recovered lipids (

Table 11. Recovery of lipids at different lipase/lipidic substrate ratios (Eo’/So’).

Eo’/So’ (kLU/g)	Recovered Lipids (%)
0.28	65.00 ± 0.75
0.55	79.05 ± 0.48
0.83	95.40 ± 0.33
1.11	97.07 ± 0.76
1.19	97.45 ± 0.01

). The results suggest a value around 0.83 kLU/g as the most suitable Eo’/So’ ratio for lipolytic hydrolysis of meat waste because the ratios under 0.55 kLU/g show a significant decrease in the percentage of lipids hydrolyzed and, for the ratios above 1.11 kLU/g, similar percentages (very close to each other) are obtained.

Similarly to what happened in protein hydrolysis, the rise in the relative amount of enzyme in relation to that of the lipidic substrate (higher Eo’/So’) generates a greater number of bonds between the Resinase and the lipid fraction of meat waste, promoting a high number of broken lipidic bonds, favoring the hydrolysis, and generating an increase in the amount of hydrolysate produced. There are no previous studies in the literature on the influence of the Eo’/So’ ratio on lipid hydrolysis; however, from the research carried out, this influence is evident.

This effect was also noted in the amounts of fatty acids obtained.

Table 12. Amounts of fatty acids obtained at different lipase/lipidic substrate ratios (Eo’/So’) (mg_{fatty acid}/g_lipids).

Fatty Acid	Eo’/So’ (kLU/g)
	0.28	0.55	0.83	1.11	1.19
C14:0	18.78 ± 0.09	22.46 ± 1.09	27.08 ± 1.14	29.06 ± 0.95	32.49 ± 0.04
C14:1	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
C15:0	0.79 ± 0.01	0.84 ± 0.00	0.97 ± 0.04	1.07 ± 0.01	1.08 ± 0.03
C15:1	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
C16:0	146.43 ± 0.338	164.41 ± 0.81	204.22 ± 8.54	218.87 ± 0.67	222.16 ± 7.27
C16:1	0.77 ± 0.01	0.89 ± 0.01	1.07 ± 0.00	1.15 ± 0.03	1.22 ± 0.03
C17:0	15.46 ± 0.05	16.87 ± 0.02	20.80 ± 0.95	22.56 ± 0.28	23.19 ± 0.76
C17:1	2.72 ± 0.06	2.99 ± 0.01	3.40 ± 0.13	3.60 ± 0.03	3.72 ± 0.13
C18:0	95.90 ± 1.64	105.30 ± 0.52	129.86 ± 5.47	143.38 ± 0.84	140.78 ± 4.61
C18:1n9c + t	160.44 ± 0.70	172.10 ± 0.86	225.59 ± 7.82	257.30 ± 1.34	236.92 ± 8.96
C18:2n6c + t	88.54 ± 1.38	98.91 ± 0.17	121.08 ± 5.10	125.62 ± 2.95	136.27 ± 4.46
C18:3n3	27.90 ± 0.12	29.22 ± 0.09	30.80 ± 1.10	31.84 ± 0.67	33.35 ± 0.28
C18:3n6	0.37 ± 0.00	0.59 ± 0.03	1.09 ± 0.04	1.20 ± 0.01	1.29 ± 0.01
C20:0	0.68 ± 0.01	0.79 ± 0.01	0.99 ± 0.03	1.06 ± 0.04	1.10 ± 0.02
C20:1	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
C20:2	9.36 ± 0.02	10.53 ± 0.25	13.09 ± 0.53	14.14 ± 0.23	14.35 ± 0.87
C20:3n3	21.10 ± 0.23	23.74 ± 0.98	29.42 ± 1.07	32.40 ± 0.13	32.58 ± 0.13
C20:3n6 + c21:0	34.81 ± 0.17	38.19 ± 0.16	42.24 ± 0.08	43.58 ± 0.86	45.47 ± 1.49
C20:4n6	15.69 ± 0.30	17.20 ± 0.22	22.46 ± 0.96	24.05 ± 0.25	24.59 ± 0.10
Total	639.74 ± 0.32	705.02 ± 1.98	874.16 ± 30.75	950.88 ± 3.48	950.57 ± 10.48

shows the quantities of fatty acids released at the different Eo’/So’ ratios tested. It can be seen that the amount of individual fatty acids increases with the enzyme/substrate ratio. Dias [72] observed the same phenomenon in hydrolysis of soybean oil with different lipases, including Resinase HT.

Figure 3 and Figure 4 show the average amounts obtained of the individual fatty acids for the different Eo’/So’ ratios tested.

From these figures, there is a linear dependence between the amount of individual fatty acids (IFA) released and the Eo’/So’ ratio expressed as:

$$IFA=a·Eo’/So’+b$$

(4)

The values of the coefficients a and b were obtained, as well as the corresponding determination coefficient (R²), and they appear in

Table 13. Linear fit of individual fatty acids released in lipidic hydrolysis with Resinase.

Fatty Acid	a	b	R2
C14:0	14.01	14.88	0.97
C15:0	0.47	0.55	0.98
C16:0	87.71	121.75	0.97
C16:1	0.40	0.69	0.98
C17:0	89.30	12.71	0.98
C17:1	10.94	2.42	0.99
C18:0	54.98	79.50	0.96
C18:1	104.69	127.55	0.90
C18:2	50.73	73.90	0.96
C18:3	65.69	26.33	0.98
C20:0	0.17	0.55	0.98
C20:2	5.79	77.08	0.97
C20:3	24.65	49.18	0.99
C20:4	10.47	12.50	0.96

.

Figure 5 shows the fatty acids released categorized according to the number of double bonds they have in their carbon chain: saturated (no double bonds), monounsaturated (one double bond), and polyunsaturated (several double bonds). In this figure, the three types of fatty acids have a linear dependence with the lipase/lipidic substrate ratio.

Saturated fatty acids turned out to be the majority of the recovered acids when compared with monounsaturated and polyunsaturated acids separately but, if the comparison is made between saturated and unsaturated fatty acids (including monounsaturated and polyunsaturated acids), unsaturated fatty acids are predominant. This result is consistent with the fatty acid composition of the lipid fraction of meat waste used (see Table 1), where saturated fatty acids show the highest percentage (46.64%), followed by polyunsaturated (28.09%), and monounsaturated (25.27%) fatty acids. This recovery of fatty acids based on the initial composition of the raw material hydrolyzed was also observed by Morales-Medina et al. [73], who hydrolyzed sardine oil with different lipolytic enzymes such as Lipozyme RM IM (a lipase from Rhizomucor miehei) and Novozyme 435 (a lipase from Candida antarctica).

From a lipase/lipidic substrate ratio of 0.83 kLU/g, the increase in the amounts of released fatty acids is not notable: the maximum increase corresponds to C16:0 and is only 8%, which is far higher than the increase for the other acids released. This suggests that we should work with the Eo’/So’ ratios around 0.83 kLU/g so as not to make the process unnecessarily more expensive.

3.4. Simultaneous Protein and Lipid Hydrolysis

To obtain protein hydrolysates, unhydrolyzed collagen, and fatty acids in a sole stage, hydrolysis experiments were carried out using a combination of Alcalase and Resinase as enzymatic catalyst. The operating conditions included pH (pH = 7.5, 8.0, and 8.5), temperature (T = 45 °C, 50 °C, and 55 °C), a protease/proteinic substrate ratio (Eo/So = 0.11, 0.16, and 0.21 AU/g), and a lipase/lipidic substrate ratio (Eo’/So’ = 0.55, 0.83, and 1.11 kLU/g). The values of the operating conditions were chosen according to the previous studies with Alcalase and Resinase, separately (Section 3.2 and Section 3.3). To minimize the number of experiments to be carried out, three-factor, three-level Taguchi’s L9 orthogonal array (applied as implemented in the statistical software Minitab version 19) was used as an experimental design (

Table 14. Taguchi L9 experiment design.

Experiment No.	pH	Temperature (°C)	Eo/So (AU/g)	Eo’/So’ (kLU/g)
1	7.5	45	0.11	0.55
2	8.0	50	0.11	0.83
3	8.5	55	0.11	1.11
4	8.0	55	0.16	0.55
5	8.5	45	0.16	0.83
6	7.5	50	0.16	1.11
7	8.5	50	0.21	0.55
8	7.5	55	0.21	0.83
9	8.0	45	0.21	1.11

).

3.4.1. Recovered Products

Meat waste hydrolysis, with a combination of Alcalase and Resinase, fractionated the proteinic and lipidic materials into a solid fraction containing mainly collagen, a water-liquid fraction enriched with solubilized proteins (indicative of the quantity of generated protein hydrolysate according to Latorres et al. [15], and a lipid-liquid fraction that was mainly present as free fatty acids. The percentage recovery was calculated as the recovered compound amount divided by the initial compound amount in meat waste, being these compounds: solubilized proteins, collagen, and lipids.

The recovery of lipids in the lipid liquid fraction after hydrolysis varied between 86% for experiment number 1 (pH = 7.5, T = 45 °C, Eo/So = 0.11 AU/g, and Eo’/So’ = 0.55 kLU/g) and 97% for experiment number 9 (pH = 8.0, T = 45 °C, Eo/So = 0.21 AU/g, and Eo’/So’ = 1.11 kLU/g) (Figure 6). Obviously, higher Eo’/So’ ratios (higher concentrations of Resinase in relation to the lipidic substrate concentration) favor the lipids recovery, but the addition of Alcalase in the reactor also helps to increase the amount of recovered lipids: proteases break the peptide bonds, resulting in the release of lipids from meat waste; the bigger the cleavage of the peptide bond, the higher the amount of lipids released [74].

The variation of collagen recovery in the solid fraction was much higher, between 42% for experiment number 9 (pH = 8.0, T = 45 °C, Eo/So = 0.21 AU/g, and Eo’/So’ = 1.11 kLU/g) and 61% for experiment number 2 (pH = 8.0, T = 50 °C, Eo/So = 0.11 AU/g, and Eo’/So’ = 0.83 kLU/g) (Figure 6). The lower recoveries in hydrolysis with the highest Eo/So ratios (experiments 7, 8, and 9) indicate a significant protein conversion to other products, such as peptides or amino acids. These results are confirmed by the variation of solubilized protein in the water-liquid fraction, which was between 50% for experiment number 2 (pH = 8.0, T = 50 °C, Eo/So = 0.11 AU/g, and Eo’/So’ = 0.83 kLU/g) and 69% for experiment number 9 (pH = 8.0, T = 45 °C, Eo/So = 0.21 AU/g, and Eo’/So’ = 1.11 kLU/g). As expected, hydrolysis of proteins is favored by high Eo/So ratios (higher concentrations of Alcalase in relation to the proteinic substrate concentration); however, the percentage of hydrolyzed protein is also favored by Resinase’s presence in the reactor. The split of the triglyceride bonds via lipases caused the protein release from meat waste; consequently, peptide bonds are more exposed and vulnerable to proteases [5].

The influence of pH and temperature on simultaneous product recovery does not seem to be as significant as the influence of the Eo/So and Eo’/So’ ratios. This makes the enzymatic hydrolysis at high Eo/So and Eo’/So’ ratios, specifically at the conditions of pH = 8.0, T = 45 °C, Eo/So = 0.21 AU/g, and Eo’/So’ = 1.11 kLU/g, are more suitable for achieving high recoveries of the solubilized proteins (that is, protein hydrolysate) and lipids from meat waste. On the contrary, enzymatic hydrolysis at low Eo/So and Eo’/So’ ratios, specifically at the conditions of pH = 8.0, T = 50 °C, Eo/So = 0.11 AU/g, and Eo’/So’ = 0.83 kLU/g, are more suitable to achieve high recovery of collagen.

3.4.2. Overall Mass Balance

To obtain a circular economy, it is necessary to recycle materials from meat waste as much as possible to have a material closed loop.

Most past works on enzymatic hydrolysis of meat have only considered the solubilized or hydrolyzed protein to valorize the proteinic fraction) [41,62,75]. However, the valorization opportunities of meat waste are greater if unhydrolyzed collagen is also recovered. Within this concept, the remaining solid fraction, obtained after protein hydrolysis, can also be considered as a source of collagen. Before hydrolysis, the lipids in meat waste had only trace amounts of free fatty acids; but, during hydrolysis of the lipidic fraction, a significant increase in the concentrations was observed; at the end of hydrolysis, the highest concentration of all studied fatty acids was reached [5].

Therefore, an estimate of the material balance after meat waste treatment is essential to supply information on overall recovery of meat waste components: peptides (hydrolyzed protein), collagen, and free fatty acids.

Table 15. Obtention of products after hydrolysis with a combination of Alcalase and Resinase.

Product	Amount of Product (kgprodut/kgdry meat waste)
	Experiment No
	1	2	3	4	5	6	7	8	9
Protein hydrolysate	0.1000	0.0950	0.1010	0.0960	0.1120	0.1280	0.1230	0.1260	0.1320
Unhydrolyzed collagen	0.0047	0.0049	0.0045	0.0048	0.0041	0.0037	0.0038	0.0037	0.0034
C14:0	0.0189	0.0189	0.0201	0.0194	0.0212	0.0215	0.0211	0.0213	0.0209
C15:0	0.0008	0.0008	0.0009	0.0009	0.0009	0.0009	0.0009	0.0009	0.0009
C16:0	0.1303	0.1282	0.1467	0.1442	0.1545	0.1586	0.1591	0.1525	0.1593
C16:1	0.0006	0.0006	0.0007	0.0007	0.0007	0.0007	0.0007	0.0007	0.0007
C17:0	0.0113	0.0120	0.0126	0.0125	0.0134	0.0135	0.0131	0.0135	0.0133
C17:1	0.0026	0.0027	0.0027	0.0028	0.0031	0.0031	0.0030	0.0031	0.0031
C18:0	0.0923	0.0939	0.0947	0.0935	0.0988	0.0975	0.0972	0.0986	0.0981
C18:1	0.1615	0.1641	0.1634	0.1667	0.1664	0.1721	0.1619	0.1660	0.1749
C18:2	0.0899	0.0902	0.0931	0.0942	0.0948	0.0998	0.0995	0.0984	0.0979
C18:3n3	0.0228	0.0244	0.0255	0.0258	0.0273	0.0281	0.0274	0.0277	0.0276
C18:3n6	0.0003	0.0003	0.0003	0.0003	0.0004	0.0004	0.0004	0.0004	0.0004
C20:0	0.0022	0.0022	0.0023	0.0023	0.0024	0.0024	0.0024	0.0023	0.0024
C20:2	0.0194	0.0196	0.0201	0.0202	0.0207	0.0211	0.0206	0.0208	0.0207
C20:3n3	0.0199	0.0202	0.0203	0.0200	0.0210	0.0208	0.0210	0.0208	0.0208
C20:3n6	0.0121	0.0121	0.0132	0.0131	0.0149	0.0152	0.0144	0.0137	0.0147
C20:4n6	0.0113	0.0120	0.0131	0.0132	0.0140	0.0154	0.0145	0.0141	0.0153
Total	0.7009	0.7021	0.7352	0.7306	0.7706	0.8028	0.784	0.7845	0.8064

illustrates the mass balance on a dry basis after enzymatic hydrolysis at the different operating conditions indicated in Table 14. The material balance was calculated for each individual component and included the main components of the solid fraction (collagen), of the water-liquid fraction (protein hydrolysate), and of the lipid-liquid fraction (free fatty acids) with a high added value.

As expected, the increase in the Eo’/So’ ratio significantly affected the total amount of recovered products: the higher that the ratio is, the greater the total amount of products recovered. The same result can be observed with the Eo/So ratio. However, modifications of pH and temperature are the reason why, for different Eo/So and Eo’/So’ ratios, similar total product quantities can be obtained: see experiments no. 1 and 2, 3 and 4, 7 and 8, and 6 and 9.

Based on the results obtained, the operating conditions of experiments nos. 6 and 9 are identified as the most suitable for the circular economy of the process. In these experiments, 0.8 kg of products are obtained for each kg of dry raw material, with the whole of the proteins and lipids in meat waste being practically recovered.

Keeping in mind that the price of the enzymes has the most significant impact on the enzymatic reactor’s running costs [5] and considering exclusively the price of the enzymes, the operating conditions of experiment no. 6 (pH = 7.5, T = 50 °C, Eo/So = 0.16 AU/g, and Eo’/So’ = 1.11 kLU/g) seems to be more advisable for a circular economy.

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.