Analyzing the Environmental Impact of Chemically-Produced Protein Hydrolysate from Leather Waste vs . Enzymatically-Produced Protein Hydrolysate from Legume Grains

Protein hydrolysates are largely used as plant biostimulants for boosting crop growth, and improving crop tolerance to abiotic stresses and fruit quality. Protein hydrolysate-based biostimulants are mostly produced by chemical hydrolysis starting from animal wastes. However, an innovative process of enzymatic hydrolysis of legume-derived proteins has been recently introduced by few companies. The objective of this study was to evaluate the energy use and environmental impact of the production processes of enzymatically-produced protein hydrolysate starting from lupine seeds and protein hydrolysate obtained from chemical hydrolysis of leather wastes through the application of life cycle assessment (LCA). The LCA method was applied through the software GEMIS “Global Emission Model for Integrated Systems”, elaborated at L’Oko-Institute in Germany, and the parameters taken into account were: CO2 emissions in g per kg of protein hydrolysate; the consumption of fossil energy expressed in MJ per kg of protein hydrolysate; and water consumption reported in kg per kg of protein hydrolysate. In the case of legume-derived protein hydrolysate, the evaluation of the energy use and the environmental impact started from field production of lupine grains and ended with the industrial production of protein hydrolysate. In the case of animal-derived protein hydrolysate, the LCA method was applied only in the industrial production process, because the collagen is considered a waste product of the leather industry. The type of hydrolysis is the step that most affects the energy use and environmental impact on the entire industrial production process. The results obtained in terms of CO2 emissions, fossil energy consumption and water use through the application of LCA showed that the production process of the animal-derived protein hydrolysate was characterized by a higher energy use (+26%) and environmental impact (+57% of CO2 emissions) in comparison with the enzymatic production process of lupine-derived protein hydrolysate. In conclusion, the production of legume-derived protein hydrolysate by enzymatic hydrolysis is more environmentally friendly than the production of animal-derived protein hydrolysate through chemical hydrolysis.


Introduction
New agricultural strategies, such as the application of natural substances and/or beneficial microbials, have been evaluated as a means for reducing negative environmental impact and improving crop performance and sustainability under adverse ecological conditions [1,2].As defined by the European Biostimulants Industry Council [3], plant biostimulants contain microorganism(s) and/or substance(s) whose function, when applied to plants or the rhizosphere, is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality [4][5][6].Protein hydrolysate-based biostimulants contain free amino acid, oligo and polypeptides achieved through enzymatic and/or chemical hydrolysis of proteins, especially from vegetal or animal sources [7][8][9][10][11][12][13].
Currently, 90% of protein hydrolysate-based biostimulants in the market are obtained from chemical hydrolysis of cattle leather wastes; while only 5-10% is from enzymatic hydrolysis of plant biomass, especially Leguminosae crops.Traditionally, chemical hydrolysis is achieved with strong acids (e.g., chloride acid), and extreme temperatures (>130 • C), and generally yields products with low agronomic quality, since some important amino acids (e.g., tryptophan) and peptides are destroyed during the production process [13].One way of reducing losses of amino acids and peptides during the process would be through digestion of proteins with enzymes [14].An enzymatic hydrolysis system obtains high quality protein hydrolysates [15][16][17] using specific enzymes and low temperatures (<60 • C).The enzymatically-produced protein hydrolysates from plant residues contain not only free amino acids, but also soluble peptides that act as signal molecules regulating a broad spectrum of physiological processes [18][19][20][21][22].
A process-based approach such as life cycle assessment (LCA) provides a methodology for comparing the energy use, greenhouse gas emissions, and water use associated with protein hydrolysate production.The LCA approach applied for the comparison of production processes of hydrolysates from plant and animal origin would be a valuable tool for understanding the real convenience of the different industrial pathways.The results of the LCA will be a beneficial indication for industrial companies in order to evaluate the hydrolysis process efficiency, leading to the identification of more sustainable approaches.LCA method has been used successfully for evaluating the environmental impact of production processes in the field of food production [19,[23][24][25].However, no data are available on the environmental impact of production processes of protein hydrolysate-based biostimulants.Therefore, the aim of this paper was to apply an LCA approach for quantifying the energy use, greenhouse gas emissions, and water consumption associated with protein hydrolysate products starting from leather wastes or lupine seeds.

LCA Method
The work was carried out implementing the Life Cycle Assessment (LCA) approach to the two production chains of protein hydrolysate-based biostimulants as reported in Figure 1.
The process to biostimulant production includes the following phases: (1) dry milling and grinding of seeds, or cutting of leather waste; (2) water extraction, where the seed flour or shredded leather waste is dispersed in acidified water (pH 4.5, 50 • C) for 6 hours to extract the soluble compounds; (3) filtration and centrifugation, where the protein concentrate is separated from the other organic compounds in a centrifuge decanter; (4) enzymatic or acid hydrolysis at 60 • C or 130 • C, respectively; (4) centrifugation, where the soluble compounds like amino acids and peptides are separated from the insoluble residual compounds; and (5) product concentration, where the soluble compounds are concentrated ca.6 times through water evaporation in a mechanical vapor recompression evaporator.
With the aim of relating each input datum to the corresponding impact in terms of GHGE and fossil energy requirement, the biostimulant production chains were implemented in Gemis 4.7 software (Öko Institut, Berlin, Germany).The sources of the main up-stream processes are reported in Table 1: the majority of the processes are found in the Gemis database, with the only exception being protease enzyme, where emission factors taken from the scientific literature [20,21] were used.With the aim of relating each input datum to the corresponding impact in terms of GHGE and fossil energy requirement, the biostimulant production chains were implemented in Gemis 4.7 software (Öko Institut, Berlin, Germany).The sources of the main up-stream processes are reported in Table 1: the majority of the processes are found in the Gemis database, with the only exception being protease enzyme, where emission factors taken from the scientific literature [20,21] were used.
Table 1.Up-stream processes used for the LCA implementation of the two production chains of biostimulants.The developed approach for the environmental evaluation of the two production chains is based on the fundamentals of the ISO 14040 and 14044, aiming to promote a simplified comparative and attributional LCA.The LCA is a developed, standardized primary tool for environmental assessments, and LCA evaluates, from the environmental point of view, all the resources and inputs needed for the system studied, and all the outputs from the system, which include emissions to air, water and soil.In this way, first indications about the environmental pressures on the production chains were obtained.Considering the quality of the inventory data and their level of uncertainty, in this phase, the simplified LCA was limited to the calculation of three specific indicators, i.e., the GHGE with unit the CO2 equivalent (CO2eq), the Cumulated Energy Requirement (CER) and the Water Use (WU).In particular, the CO2eq is a metric measure for comparing emissions from various GHGs on the basis of their global-warming potential (GWP) [22], the CER represents the fossil energy required for extracting, manufacturing and disposing raw and auxiliary materials all along each production chain; the WU is the sum of the fresh water used along all the production processes accounted in the LCA.The LCA method was applied for both production processes with reference to an output of 1 kg of protein hydrolysate-based biostimulant.

Up-Stream Production
The LCA was modeled using the input data reported in Table 2: the first one uses lupine seeds as a source of proteins, the second one uses leather wastes.The data for energy, material inputs and outputs for both protein hydrolysate production systems were obtained from the literature [26] and  The developed approach for the environmental evaluation of the two production chains is based on the fundamentals of the ISO 14040 and 14044, aiming to promote a simplified comparative and attributional LCA.The LCA is a developed, standardized primary tool for environmental assessments, and LCA evaluates, from the environmental point of view, all the resources and inputs needed for the system studied, and all the outputs from the system, which include emissions to air, water and soil.In this way, first indications about the environmental pressures on the production chains were obtained.Considering the quality of the inventory data and their level of uncertainty, in this phase, the simplified LCA was limited to the calculation of three specific indicators, i.e., the GHGE with unit the CO 2 equivalent (CO 2 eq), the Cumulated Energy Requirement (CER) and the Water Use (WU).In particular, the CO 2 eq is a metric measure for comparing emissions from various GHGs on the basis of their global-warming potential (GWP) [22], the CER represents the fossil energy required for extracting, manufacturing and disposing raw and auxiliary materials all along each production chain; the WU is the sum of the fresh water used along all the production processes accounted in the LCA.The LCA method was applied for both production processes with reference to an output of 1 kg of protein hydrolysate-based biostimulant.
The LCA was modeled using the input data reported in Table 2: the first one uses lupine seeds as a source of proteins, the second one uses leather wastes.The data for energy, material inputs and outputs for both protein hydrolysate production systems were obtained from the literature [26] and interviews with experts, such as production managers of companies dealing with the production of protein hydrolysates.
The lupine production was assessed based on data collected in [23,26]: an inventory of the agricultural data used in the LCA is reported in Table 2.For the leather wastes, a specific analysis was carried out in order to calculate which impacts could be associated to these wastes.As reported in the COOP Environmental Product Declaration [21], beef meat production in Italy causes 23.8 kg CO 2 eq/kg of bone-free meat.This value is consistent with similar processes modeled in Gemis 4.7 for Southern Europe.Moreover, the amount of leather is equal to 7.9%, whilst the meat and other edible parts are about 92.1%.Then, the leather is tanned and the solid wastes of the process are about 43% of the raw material input.Therefore, considering the above mass allocation ratios in different animal tissues, 1 kg of leather gave a CO 2 emission of 0.958 kg CO 2 eq, an energy consumption of 3864 MJ and water use of 1562 kg.However, the emissions associated with leather waste were set to zero in the LCA analysis because this waste does not have any market value.
Concerning the industrial phases for the production of protein hydrolysates, the inventory data are reported in Tables 3 and 4 were used.

Results and Discussions
The LCA results for the two-production process are reported in Tables 5 and 6.The calculated indicators demonstrated a lower impact for the production process based on the enzymatic hydrolysis of lupine seeds than for that based on chemical hydrolysis of leather waste.This result is mainly due to the different processes of hydrolysis, which requires higher temperature, pressure and chemical inputs in a chemical hydrolysis process in comparison to an enzymatic hydrolysis process.In Figure 2, the CO 2 emission percentage for each production phase of enzymatically-produced protein hydrolysates from lupine grains is reported.The calculated indicators demonstrated a lower impact for the production process based on the enzymatic hydrolysis of lupine seeds than for that based on chemical hydrolysis of leather waste.This result is mainly due to the different processes of hydrolysis, which requires higher temperature, pressure and chemical inputs in a chemical hydrolysis process in comparison to an enzymatic hydrolysis process.In Figure 2, the CO2 emission percentage for each production phase of enzymatically-produced protein hydrolysates from lupine grains is reported.In Figure 3, the CO2 emission percentage for each production phase of chemically-produced protein hydrolysates from leather waste is reported.In Figure 3, the CO 2 emission percentage for each production phase of chemically-produced protein hydrolysates from leather waste is reported.The results of this study showed that the production process based on enzymatic hydrolysis of lupine seeds had the lowest environmental impact.In particular, the greatest differences in CO2 emissions between the production processes were observed in the hydrolysis phase (n.4), with a saving of 57.03% using enzymatic hydrolysis (Figure 4).Concerning the energy consumption (Figure 5), enzymatic hydrolysis of lupine seeds required less energy in phase 4, while the opposite behavior was observed for phase 2 and 6, where the production of leather-derived protein hydrolysate by chemical hydrolysis reduced the energy needed by 8.5 and 13.1%, respectively.The results of this study showed that the production process based on enzymatic hydrolysis of lupine seeds had the lowest environmental impact.In particular, the greatest differences in CO 2 emissions between the production processes were observed in the hydrolysis phase (n.4), with a saving of 57.03% using enzymatic hydrolysis (Figure 4).Concerning the energy consumption (Figure 5), enzymatic hydrolysis of lupine seeds required less energy in phase 4, while the opposite behavior was observed for phase 2 and 6, where the production of leather-derived protein hydrolysate by chemical hydrolysis reduced the energy needed by 8.5 and 13.1%, respectively.

Conclusions
Overall, the results demonstrated that the production of legume-derived protein hydrolysate by enzymatic hydrolysis is more environmentally friendly than the production of protein hydrolysate through chemical hydrolysis of leather waste.

Figure 1 .
Figure 1.Production processes of protein hydrolysates: enzymatically-produced protein hydrolysate from legume grains vs. chemically-produced protein hydrolysate from leather waste.

Figure 1 .
Figure 1.Production processes of protein hydrolysates: enzymatically-produced protein hydrolysate from legume grains vs. chemically-produced protein hydrolysate from leather waste.

Figure 2 .
Figure 2. CO2 emission percentages (on a total basis) for the industrial phases of enzymaticallyproduced protein hydrolysate from lupine grains.

Figure 2 .
Figure 2. CO 2 emission percentages (on a total basis) for the industrial phases of enzymaticallyproduced protein hydrolysate from lupine grains.

Figure 3 .
Figure 3. CO2 emission percentages (on a total basis) for the industrial phases of chemically-produced protein hydrolysate from leather waste.

Figure 4 .
Figure 4. CO2 emissions resulting from the difference between enzymatically-produced protein hydrolysate from lupine grains and chemically-produced protein hydrolysate from leather waste by industrial phase.

Figure 3 .
Figure 3. CO 2 emission percentages (on a total basis) for the industrial phases of chemically-produced protein hydrolysate from leather waste.

Figure 4 .
Figure 4. CO2 emissions resulting from the difference between enzymatically-produced protein hydrolysate from lupine grains and chemically-produced protein hydrolysate from leather waste by industrial phase.

Figure 4 .
Figure 4. CO 2 emissions resulting from the difference between enzymatically-produced protein hydrolysate from lupine grains and chemically-produced protein hydrolysate from leather waste by industrial phase.

Figure 4 .
Figure 4. CO2 emissions resulting from the difference between enzymatically-produced protein hydrolysate from lupine grains and chemically-produced protein hydrolysate from leather waste by industrial phase.

Figure 5 .
Figure 5. Energy consumption resulting from the difference between enzymatically-produced protein hydrolysate from lupine grains and chemically-produced protein hydrolysate from leather waste.

Table 1 .
Up-stream processes used for the LCA implementation of the two production chains of biostimulants.

Table 3 .
Key characteristics of the production process of protein hydrolysate-based biostimulant from lupine seeds (data refer to the production of 1 kg of protein hydrolysate).

Table 4 .
Key characteristics of the production process of protein hydrolysate-based biostimulant from leather waste (data refer to the production of 1 kg protein hydrolysate).

Table 5 .
LCA results for protein hydrolysate production from lupine grains (data refer to the production of 1 kg protein hydrolysate).

Table 6 .
LCA results for protein hydrolysate production from leather wastes (data refer to the production of 1 kg protein hydrolysate).