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Author(s): Nora Pap, Sari Mäkinen, Ulla Moilanen, Marjatta Vahvaselkä, Jyri Maunuksela, 
Maritta Kymäläinen & Anne Pihlanto 
Title: Optimization of Valorization of Chicken MDCM to Produce Soluble Protein and 
Collagen Peptides 
Year: 2022 
Version: Published version 
Copyright:   The Author(s) 2022 
Rights: CC BY 4.0 
Rights url: http://creativecommons.org/licenses/by/4.0/ 
 
Please cite the original version: 
Pap, N.; Mäkinen, S.; Moilanen, U.; Vahvaselkä, M.; Maunuksela, J.; Kymäläinen, M.; Pihlanto, A. 
Optimization of Valorization of Chicken MDCM to Produce Soluble Protein and Collagen Peptides. 
Appl. Sci. 2022, 12, 1327. https://doi.org/10.3390/app12031327. 






Citation: Pap, N.; Mäkinen, S.;
Moilanen, U.; Vahvaselkä, M.;
Maunuksela, J.; Kymäläinen, M.;
Pihlanto, A. Optimization of
Valorization of Chicken MDCM to
Produce Soluble Protein and
Collagen Peptides. Appl. Sci. 2022, 12,
1327. https://doi.org/10.3390/
app12031327
Academic Editor: Vincenza Ferraro
Received: 3 December 2021
Accepted: 17 January 2022
Published: 26 January 2022
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Licensee MDPI, Basel, Switzerland.
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distributed under the terms and
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Attribution (CC BY) license (https://
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4.0/).
applied  
sciences
Article
Optimization of Valorization of Chicken MDCM to Produce
Soluble Protein and Collagen Peptides
Nora Pap 1, Sari Mäkinen 1, Ulla Moilanen 2 , Marjatta Vahvaselkä 3, Jyri Maunuksela 3, Maritta Kymäläinen 2
and Anne Pihlanto 1,*
1 Natural Resources Institute Finland, Production Systems, Myllytie 1, 31600 Jokioinen, Finland;
nora.pap@luke.fi (N.P.); sari.makinen@luke.fi (S.M.)
2 HAMK Bio Research Unit, Häme University of Applied Sciences, Visamäentie 35 B, 13100 Hämeenlinna, Finland;
ulla.moilanen@hamk.fi (U.M.); maritta.kymalainen@hamk.fi (M.K.)
3 Natural Resources Institute Finland, Production Systems, Latokartanonkaari 9, 00790 Helsinki, Finland;
marjatta.vahvaselka@luke.fi (M.V.); jyri.maunuksela@luke.fi (J.M.)
* Correspondence: anne.pihlanto@luke.fi; Tel.: + 35-82-9532-6438
Abstract: This study aimed to utilize enzymatic treatment and pressurized hot water extraction
(PHWE) to recover soluble food-grade protein and collagen peptides from mechanically deboned
chicken meat (MDCM), a side-stream from the meat industry. Food-grade enzyme blends Ermitase 1
and Ermitase 2 were used to fractionate the mechanically deboned meat into fat, soluble protein,
and solids. Response surface methodology was utilized to optimize treatments to maximize the
protein yield. At the optimum conditions (hydrolysis time 240 min, E:S 0.27%, and a hydromodule
1 L/kg), the enzymatic treatment produced high protein yield, approximately 90%. The protein
hydrolysates showed a good solubility index, but weak gelling properties. The PHWE of the bones
resulted in a high nitrogen yield, approximately 87%, at the optimum conditions of 190 ◦C and
83 min. Peptides in the bone extract were in the range of 0.5–13.7 kDa. Overall, our study highlights
the importance of response surface methodology to optimize parameters for mechanically deboned
chicken meat enzymatic and PHWE treatments to achieve high yields of protein for food applications
and low-molecular-weight collagen peptides for cosmetic applications. The crucial role of protein
and peptide prices was observed in preliminary profitability analysis.
Keywords: MDCM; enzymatic treatment; pressurized hot water extraction; soluble protein;
collagen peptides
1. Introduction
The production and consumption of poultry products have increased globally. Statis-
tics from poultry industry have shown that the United States, China, and Brazil have
maintained their positions as the largest producers of poultry meat. With this high pro-
duction, thousands of tons of byproducts in the form of viscera, feet, head, bones, blood,
and feathers are generated [1]. The viscera constitute about 30% of these wastes, while
feathers can be up to 10% [2,3]. The filleting step generates a byproduct known as keel
bone cartilage during the processing of the chicken carcass, which is defined as a flexible
cartilage that connects the breast muscle of the chicken to the tip of the sternum. This
cartilage is simply discarded from the carcass after removal of the breast fillets, even though
it is rich in collagen and an added value compound for biochemical and biomedical applica-
tions [4,5]. MDCM is a paste-like meat product produced by forcing ground chicken under
high pressure through a sieve to separate the bone from the edible meat tissue [6]. The
resulting product is a blend primarily consisting of tissues not generally considered meat,
such as bone, cartilages, skin and the scraps of meat remaining on the bones. Generally,
MDCM is high in lipid and ash content, and it contains more free heme groups than the
Appl. Sci. 2022, 12, 1327. https://doi.org/10.3390/app12031327 https://www.mdpi.com/journal/applsci
Appl. Sci. 2022, 12, 1327 2 of 15
fresh meat [7]. Due to the inexpensive cost and high nutritional value, MDCM is a potential
sustainable source of meat protein for food applications.
Despite its good nutritive properties being suitable for people on gluten-free and keto
diets, MDCM is still underutilized [8]. The same authors described high-pressure homoge-
nization as a novel trend for meat and fish byproduct valorization for functional ingredients.
However, further processing is needed to meet the requirements of regulation and
sensory properties regarding food use. Collagen is a major structural protein in connective
tissues of bone, tendon, and skin. In the form of small peptides, it is commonly used in
cosmetic applications and nutraceuticals [9].
Enzymatic hydrolysis is a promising technology for converting the meat industry
byproducts into food-grade ingredients through producing hydrolysates rich in nutrients
and suitable functional properties. Mokrejš et al. [10] described MDCM as a potential
material for producing gelatin through enzymatic treatment as a conditioning step, and by
implementing experimental design, these authors could describe the optimum conditions of
processing to achieve high conversion rates (30–32%) and functionality, such as gel strength.
The properties of the hydrolysates can be adjusted by choosing appropriate enzymes
and varying processing parameters such as the enzyme–substrate ratio, pH, temperature,
and time of hydrolysis [11]. Commercial microbial (Alcalase®, Protamex®, Flavourzyme®,
and Neutrase®) or plant (papain, bromelain, actinidain) proteases have been used to
hydrolyze beef, chicken, and pig bones, pigskin, and marine fish to produce hydrolysates
of increased value [12–16]. For example, hydrolysates of chicken bone extract, made using
Protamex® or Flavourzyme® [14,17], and veal bone extract, made using Neutrase® [18],
were found to be nutritional and flavorful. This made them potential ingredients as natural
meat flavor enhancers for food products including soups and sauces.
To this end, the aim of this study was (i) to produce functional protein hydrolysates
from mechanically deboned chicken meat (MDCM) using noncommercial protease enzyme
blends (Meatco, the Netherlands), (ii) to gather multidisciplinary information about the
properties of the hydrolysates, (iii) to further process the residual bone material to extract
collagen with pressurized hot water and optimize the process, (iv) to determine the molec-
ular weight distribution and techno-functional properties of the collagen peptide fractions,
and (v) to preliminarily assess the techno-economic feasibility of the developed processes.
2. Materials and Methods
2.1. Chicken MDCM Fractions Enzymatic Processing
The MDCM was provided by HKScan in Eura in Finland. The byproduct was trans-
ported under ice and stored at −20 ◦C to ensure good microbiological quality.
The MDCM fractions were treated with enzymes to separate the soluble and non-
soluble protein, fat, and the residual clean bones. Two different enzyme preparations were
used in the studies: (i) Ermitase 1 (ERM 1; endocut in bone treatment) alone, or (ii) ERM 1 in
combination with Ermitase 2 (ERM 2; exocut in bone treatment) (Meatco, The Netherlands).
Ermitase is a proteolytic enzyme product that contains exclusively endo-proteinase with an
activity of 840 UHb·g−1 and is obtained from Bacillus subtilis cultures. The hydrolyses were
carried out at 55 ◦C with continuous stirring for 60, 150, or 240 min after the addition of
the enzyme. Different solid–liquid ratios (hydromodule 1–3 L/kg) and enzyme–substrate
ratios (E:S, 0.05–0.5%) were used in the experimental design. The enzyme was inactivated
at the end of the processing by heating to 85 ◦C and keeping for 15 min. The mixture was
then left to rest for 30 min to separate the non-soluble protein and bone fraction at the
bottom of the reactor from the liquid phase containing the soluble protein and the lipids.
The lipid fraction was further separated by a separator (Frau CN 3/A, Italy) to obtain
high-quality protein while preventing quick deterioration due to the oxidation of the oil.
The protein hydrolysate was thereafter dehydrated by freeze-drying until a dry matter
content of >96% was reached. Figure 1 is a schematic illustration of the processing.
Appl. Sci. 2022, 12, 1327 3 of 15
Figure 1. Chicken mechanically deboned meat processing flow chart to produce soluble protein and
collagen peptides.
2.2. Bone Treatment
To study the bone fraction, 15 kg of MDCM and 15 kg of water were mixed constantly
with 15 g of Endocut and 15 g of Exocut enzymes at 55 ◦C for 4 h. The enzymes hydrolyzed
the protein from the bone surfaces to generate an almost clean bone fraction. The enzy-
matically treated bone fractions were separated from the liquid fraction, rinsed clean, and
dried in an oven at 58 ◦C for about 2 days. The bone yield of the chicken was 45% (450 g
dried bone/kg dry second stage MDCM fraction). The dried bones were ground with a
hammer mill.
Pressurized hot water extraction (PHWE) was tested to produce and separate collagen
peptides. The PHWE treatment was performed in pressure vessels (working volume
30 mL). The treatment temperature was controlled with a laboratory oven. The variables for
optimizing the conditions were temperature (140–190 ◦C), treatment time (15–100 min), and
dry matter content (5–30%). To analyze the effect of temperature on the molecular weight
of the collagen peptides, a PHWE test series was performed in which the temperature was
varied between 110 and 190 ◦C, and dry matter content (20%) and treatment time (53 min)
were kept constant.
2.3. Response Surface Methodology (RSF) for Process Optimization
To identify the optimal process parameters in the enzymatic hydrolyses, Modde 8.1.
(Umetrics, Sweden) was used to design the experiments according to CCF design (star
distance 1); CCF is composed of a full or fractional factorial design and star points placed on
the surfaces of the sides. The design included three factors: hydrolysis time, hydromodule,
and E:S ratio. Protein concentration was used as a response function. The design included
a total of 17 test runs, with 14 design runs and three center points to validate the model.
The experimental design is summarized in Table 1.
Appl. Sci. 2022, 12, 1327 4 of 15
Table 1. CCF design for optimization of MDCM hydrolysis parameters.
Experiment
Number
Factors Response
Time Hydromodule(L:S) E:S Protein Yield g/kg
(h) L/kg % ERM 1 ERM 1 + 2
N1 1 1 0.05 60.21 72.13
N2 4 1 0.05 51.20 94.69
N3 1 3 0.05 47.90 53.04
N4 4 3 0.05 45.50 66.53
N5 1 1 0.5 81.86 86.87
N6 4 1 0.5 121.12 155.24
N7 1 3 0.5 50.91 74.81
N8 4 3 0.5 64.07 88.61
N9 1 2 0.275 67.62 90.09
N10 4 2 0.275 70.44 80.17
N11 2.5 1 0.275 105.79 28.64
N12 2.5 3 0.275 72.27 78.27
N13 2.5 2 0.5 58.40 77.45
N14 2.5 2 0.5 56.95 104.37
N15 2.5 2 0.275 77.77 100.11
N16 2.5 2 0.275 70.40 101.89
N17 2.5 2 0.275 77.78 74.48
The protein yield was calculated as follows:
Protein yield (%)
=
Protein concentration in the protein hydrolysate (%) × Weight of protein hydrolysate (g)
Protein concentration in the raw material × Weight of raw material (g) .
To optimize collagen peptide production, a CCF design was used, and the results were
modeled with response surface modelling with Modde 10 (Umetrics, Sweden). The factors
were temperature, time, and dry matter content, and the responses were the nitrogen yield
of the solution (describes the protein yield) and the mineral content of the solid residue.
The design included a total of 20 test runs, with six center points. Table 2 summarizes
the design.
The experiments were conducted in random order to avoid systematic error, and the
optimal processing parameters were determined using the software’s optimizer function.
All the experiments were included in the final modeling, as none of the measurements were
indicated to be a statistical outlier. New test runs using the determined optimal conditions
were performed to verify the model’s validity. The soluble protein fraction was analyzed for
proximate composition, techno-functional properties, and molecular weight distribution.
Appl. Sci. 2022, 12, 1327 5 of 15
Table 2. CCF design for optimization of bone treatment for collagen peptide production.
Exp Name Run Order Temperature ◦C Dry Matter Time (min) Protein Yield (%) MineralConcentration (g/kg)
N1 13 140 5 15 33.5 69.91
N2 15 190 5 15 63.75 81.12
N3 20 140 30 15 59.06 69.84
N4 2 190 30 15 73.68 79.42
N5 16 140 5 100 60.82 79.2
N6 17 190 5 100 87.06 85.68
N7 5 140 30 100 76.74 77.77
N8 9 190 30 100 80.59 84.32
N9 10 140 17.5 57.5 66.74 77.26
N10 12 190 17.5 57.5 79.53 83.13
N11 11 165 5 57.5 67.8 81.31
N12 18 165 30 57.5 83.59 80.64
N13 3 165 17.5 15 53.26 74.56
N14 14 165 17.5 100 69.32 81.03
N15 19 165 17.5 57.5 71.65 80.76
N16 6 165 17.5 57.5 66.9 81.17
N17 7 165 17.5 57.5 67.48 80.02
N18 8 165 17.5 57.5 64.76 79.94
N19 4 165 17.5 57.5 70.18 79.98
N20 1 165 17.5 57.5 71.53 80.58
2.4. Proximate Analysis
Protein, fat, moisture, ash, and carbohydrate content were determined or calculated
using the procedures described below.
The moisture content was determined by drying the samples at 105 ◦C until a constant
final weight was reached. Measurements were taken using a thermogravimetric analyzer
(LECO TGA 701, Geleen, The Netherlands).
The nitrogen content of the samples was determined with an in-house Kjeldahl method
according to the Association of Official Analytical Chemists (AOAC) method 2001.11, SFS
EN ISO 20483:2013 and EN ISO5983-2. A correction factor of 6.25 was used in protein
content calculations.
The total fat content of the samples was determined using the SoxCap TM 2047 in
combination with the Soxtec TM 2050 extraction system with a preparatory acid hydrolysis
step and diethyl ether extraction (Foss A/B, Hillerød, Denmark) according to ISO 6492.
The total carbohydrate (TC) content was calculated with the following formula and
expressed as g/100g FW:
TC (%) = 100−moisture (%)− protein (% FW)− crude fat (% FW)− ash (% FW).
The ash content was analyzed by weighing the samples before and after burning at
550 ◦C until a constant weight was reached. Measurements were taken with a thermogravi-
metric analyzer (LECO TGA 701, Geleen The Netherlands). The ash content of the bone
fractions was analyzed by weighing the samples before and after burning at 550 ◦C for 2 h.
Appl. Sci. 2022, 12, 1327 6 of 15
2.5. Protein Molecular Weight Distribution Analysis
Protein hydrolysates produced with PHWE were subjected to size exclusion chro-
matography to analyze the protein fragmentation during the PHWE treatments. As a
first step, the PHWE extracts were purified from non-proteinaceous components with
solid-phase extraction using Sep-Pak C18 cartridges. The molecular weight distribution of
the purified hydrolysates was then analyzed with a UPLC method, using an Acquity UPLC
system (Waters Corporation, USA) equipped with an Acquity BEH125 SEC column, 1.7 um
particles, 4.6 × 150 mm (Waters Corporation, Milford, MA USA). Proteins and peptides
were eluted with 100 mM sodium phosphate buffer pH 6.8 with a flowrate of 0.3 mL/min.
2.6. Functional Properties
The MDCM hydrolysates produced at the optimal processing conditions were tested
for their functionality in terms of nitrogen solubility and gelling properties.
Protein solubility was determined using an in-house method. Protein dispersions
(1%, w/v) were prepared in water; the pH was adjusted to 8 with 0.5 M or 0.1 M NaOH,
and the samples were stirred at room temperature for 2 h. The suspension was divided
into two parts: one part was stored at −20 ◦C until further analysis, while the remainder
was centrifuged at 3000× g for 10 min. The supernatants were filtered through a Whatman
42 filter, collected, and stored at −20 ◦C until analysis. The nitrogen content of the suspen-
sion and the filtered supernatant was determined using the Kjeldahl protocol described
above. The nitrogen solubility index was expressed as
NSI (%) =
concentration of nitrogen in supernatant
total nitrogen concentration
× 100.
Gel hardness was measured from protein hydrolysate gels with a protein content of
5% at pH 5.5, pH 6.0, and pH 6.5, and a 10% solution at pH 5.5 was prepared for the gel
hardness analysis, and the results were compared between samples. The pH was adjusted
with 0.1 M HCl, the solution was stirred at room temperature with 150 rpm for 1 h, and
the pH was measured and adjusted if required. The solutions were transferred to stirring
bottles and filled with MilliQ water to reach a volume of 50 mL, and 1.9 mL was transferred
to 2.5 mL Eppendorf tubes. One Eppendorf tube was filled with water for temperature
control. The tubes were incubated for 45 min at 80 ◦C, transferred to an ice bath, and left
for 16 h at 6 ◦C. The samples were tempered to room temperature for 30 min before the gel
hardness measurement. The hardness was measured with a Lloyd texture analyzer (Lloyd
LR, 10K, Worthing UK) with a depression limit of 8 mm and a trigger of 0.001 kgf, and the
gel hardness value data were collected.
2.7. Profitability Assessment for MDCM Sidestream Valorization
The process diagram for MDCM side-stream valorization (Figure 1) developed in this
study was used as the starting point for the profitability assessment. The process yields
food-grade chicken protein hydrolysate, chicken bone collagen peptides, chicken fat, and
calcium- and phosphate-rich minerals. The assessment was performed for a hypothetical
processing plant treating 1.0 × 107 kg of chicken MDCM annually.
The values used as the input in calculating the material balance were as follows: dry
matter content of the MDCM, 39%; yield of pure bones after protease treatment, 45%;
mineral fraction in chicken bones, 61%; calcium and phosphorus content in the mineral
fraction, 80%. Chicken bone proteins account for 70% of bone organic fraction, and collagen
accounts for 90% of bone protein [19].
3. Results and Discussion
3.1. MDCM Protein Hydrolysate Production
The volume and protein concentrations of the produced protein fraction were collected,
and the protein yield was calculated to establish the statistical model between processing
Appl. Sci. 2022, 12, 1327 7 of 15
factors and the response. The model was fitted with multiple linear regression, and ANOVA
was performed. The resulting regression coefficients for MDCM meat hydrolyzed with
ERM 1 were as follows: R2 = 0.888; Q2 = 0.451. ANOVA showed that the model was
statistically significant (p = 0.001), and no lack of fit was observed (p = 0.166). Similarly, in
the second set of experiments when both ERM 1 and ERM 2 were used in the hydrolyses
process, these values were R2 = 0.687 and Q2 = 0.615. ANOVA showed that the model was
statistically significant (p = 0.001), and no lack of fit was observed (p = 0.620) in this case.
The results indicated that there was a difference in the dominant factors in the ERM 1
and ERM 1 + 2 treatments. Hydromodule and the enzyme–substrate ratio as main factors
played a significant role in protein recovery with the single ERM 1 enzyme hydrolyses,
while the hydrolysis time did not play significant role. The interaction terms were also
investigated, and hydrolysis time, the hydromodule, and the enzyme–substrate ratio,
therefore, played an important role in the model. Furthermore, the second orders of the
hydromodule and the enzyme-to-substrate ratio were also significant in the modeling. On
the basis of the results, the second-order polynomial equation to describe the model is
as follows:
Y = 8.766X1 − 27.90X2 + 22.34X3 + 23.41X22 − 39.29X32 + 15.99X1X2 − 15.95X2X3,
where Y(%) is protein recovery, X1 = time, X2 = hydromodule (L/kg), and X3 = E:S ratio.
In the optimization function, a hydrolysis time of 4 h, an E:S ratio of 0.4325%, and a
hydromodule of 1 L/kg were selected. In these conditions, protein recovery was shown to
be above 114.03 g/kg protein stock (yield of approximately 90%) with ERM 1.
The model was shown to be simpler in the case of hydrolyses with both ERM 1 and
ERM 2, in which case only the major terms, the time of the hydrolyses, the hydromodule,
and the enzyme–substrate ratio, played a significant role in the protein recovery, although
the model fitting showed lower R2 and Q2 values than those described above.
The second-order polynomial equation based on the results to describe the model is
as follows:
Y = 21.66X1 − 35.62X2 + 21.66 X3.
In the optimization function, a hydrolysis time of 4 h, an E:S ratio 0.2709% for both
enzymes, and a hydromodule of 1 L/kg were selected. In these conditions, the protein
concentration was above 118.03 g/kg protein stock (yield of approximately 90%) with ERM
1 + 2 enzymes. The longer enzymatic treatment (72 h) of MDCM was found also more
favorable to achieve higher hydrolysate yield (6.2–6.8%); correspondingly, the total gelatin
recovery was also significantly influenced by the extraction temperature and time with
close to a final recovery yield of 46%. Lindberg et al. [20], on the other hand, determine that
the optimal combination of different proteases might be a good way toward more profitable
processing with an increase in yield of 15% in poultry byproduct valorization, making
the process even more profitable. The model was verified by repeating the experiments
twice within the optimal process parameters, and similar results were found for the protein
concentration and recovery values.
Table 3 shows the proximate composition of fractions produced in optimal conditions
after 4 h of hydrolysis with one enzyme and the combination of enzymes using the optimal
process conditions. Lindberg et al. [20] also found that the recovery yield was higher
when the time of hydrolyses was increased from 1 h to 3 h; however, little improvement in
recovery was observed beyond the 3 h time. There were no considerable differences in pro-
tein concentrations between the hydrolysate fractions prepared with ERM1 or ERM 1 + 2,
and ERM 1 alone was effective. The fat concentrations showed more differences between
treatments that might be due to an incomplete separation in the 4 h ERM 1 + 2 treatments
rather than analytical error.
Appl. Sci. 2022, 12, 1327 8 of 15
Table 3. The ash, dry matter, protein, and fat content of the soluble protein fraction after treatment
with ERM 1 and ERM 1 + 2 for 4 h.
Enzyme Time of Hydrolysis Ash (% DW) Dry Matter Protein (% DW) Fat (% DW)
Ermitase 1 4 h 9.90 93.31 87.01 0.74
Ermitase 1 + 2 4 h 11.58 95.16 86.17 2.44
The protein yield depends on the extraction method, raw material type, and the
deboning machine [21]. Studies have shown that, depending on the extraction method,
6.7–17.6% of total protein from chicken bone may be recovered. Kijowski and Ntewtarow-
icz [21] extracted chicken bone with a water solution of NaCl with the addition of NaNO2
and ascorbate, and they obtained extracts containing 3.4% of protein, which constituted
18% of total chicken bone protein. Young [22] reported a yield of 11–17% from total protein
content in animal bone, while Lawrence and Jelen [23] reported that more than 18% of the
protein in chicken bone was alkali-extractable. Dong et al. [14] observed that 80.25% of the
protein in the chicken bone could be extracted using the hot-pressure extraction method.
Papain was used for enzymatic hydrolysis for its highest protein extraction rate (46%) of
chicken bone protein compared to other enzymes (trypsin 44%, protamex 40%, alkaline
protease 35%, neutral protease 32%, pepsin 28%, flavored proteinase 19%). Additionally,
ultrasonic treatment was shown to gradually increase the protein yield as the processing
temperature increased, reaching the highest yield (56.49%± 0.67%) at 45 ◦C [24]. In a recent
study, Lindberg et al. [25] investigated the impact of three commercial proteases, Alcalase
2.4 L, Corolase 2TS, and Flavourzyme, on the quality of chicken and turkey carcasses and
MDCM. The results showed that the choice of protease and industrially relevant variations
in poultry raw material composition both had a major effect on product composition and
protein yield, nutrient and amino-acid composition, degree of hydrolysis, size distribution
of peptides, and rheological properties. Alcalase was the most efficient protease, while
Flavourzyme hydrolysis resulted in substantially lower nitrogen yields [25]. According to
the present study’s findings, around 87% of the proteins from the chicken MDCM were
extracted with the Ermitase enzymes, indicating their superiority to other enzymes.
3.2. Properties of Extracted Protein Hydrolysates
Enzymatic hydrolysis treatments in the optimal process conditions produced protein
fragments with a broad molecular weight range. The smallest fragments were observed at a
molecular weight of 50 Da, and the largest proteins were observed at 45 kDa. The majority
(approximately 70%) of the proteins were around 10 kDa, indicating that the hydrolysis
treatments were rather mild.
The nitrogen solubility index (NSI%), which describes the proportion of nitrogen that
is water-soluble compared with total nitrogen content, is relevant to the evaluation of
protein quality. The NSI values of the hydrolyzed chicken MDCM protein fractions were
determined at pH 5–8 and are shown in Figure 2.
Appl. Sci. 2022, 12, 1327 9 of 15
Figure 2. Nitrogen solubility index of chicken MDCM protein fractions obtained with ERM1 or ERM1 + 2
after 4 h hydrolysis.
The measurements indicated that a large proportion of the samples had a high solu-
bility of up to 90%, and no clear trend in the pH change was observed. Using ERM 1 and
2 enzymes increased solubility at pH 6. The protein fraction from MDCM seemed to have
much lower solubility at pH 6 after four hydrolyses when the hydrolysis was carried out
only using ERM 1, but the solubility increased remarkably when both ERM 1 and ERM 2
were used.
The gelling property and gel hardness were evaluated at concentrations of 5%, 10%,
and 15% and at pH values of pH 6.0 and pH 6.5. Furthermore, gelatin gels made by
adjusting the protein content to 6.67% were used as a reference. Gel hardness was measured
with a Lloyd’s instrument (Lloyd LR 10K, Geleen UK). The results showed that gels from
MDCM fractions were remarkably weaker than the reference gelatin. It was also revealed
that there was no major difference in gel hardness whether ERM 1 or ERM 1 + 2 enzymes
were used in the hydrolysis. At pH 6.0 and 5% protein content, the ERM 1 gel hardness
was 0.0145 ± 0.01 N, and the ERM 1 + 2 showed a hardness of 0.01 ± 0.0061 N. The effect of
the pH on gel hardness was somewhat more visible in the results. The gel with 10% protein
content treated in ERM 1 treatment showed a gel hardness of 0.014 ± 0.0022 N at pH 5.5.
At pH 6, the value was 0.036 ± 0.016 N. To this end, a considerable difference in hardness
was observed at the same 10% protein content but different pH in ERM 1 treatment; at
pH 6, the gel had 20 times higher hardness compared to pH 5.5.
The gel hardness of protein fraction is an important factor to determine its applicability
in foods. In many meat-related products, the gelling property is important to obtain the
proper structure and functionality. If the protein derived from the mechanically deboned
meat through enzymatic hydrolyses has no good gelling property, the amount added to
the product should be carefully calculated to avoid having a negative impact on the final
hardness, chewiness, and taste of the product. For example, Cavalheiro et al. [26] observed
that up to 10% of chicken meat could be replaced with MDCM hydrolysates with an effect
on the organoleptic and physicochemical properties of Mortadella-type sausage.
It is also important to mention that this study’s aim was not to produce a protein
isolate but to develop a simple and affordable technology for the better valorization of
mechanically deboned meat.
Appl. Sci. 2022, 12, 1327 10 of 15
3.3. Residual Bone Treatment
3.3.1. Treatments and Optimization Parameters
According to preliminary experiments, pressurized hot water extraction proved the
most promising way to separate collagen peptides. The variables in optimizing the condi-
tions were temperature (140–190 ◦C), treatment time (15–100 min), and dry matter content
(5–30%). The responses were the nitrogen yield of the solution (describes protein yield)
and the mineral content of the solid residue. A statistical test design (CCF) was used in
the series of experiments, and the results were modeled by response surface modeling.
Good, reproducible, and predictive models were obtained for both responses. The nitrogen
yield indices were R2 = 0.96, Q2 = 0.86, validity = 0.77, and reproducibility = 0.94. Corre-
spondingly, R2 = 0.98, Q2 = 0.96, validity = 0.68, and reproducibility = 0.98 were obtained
for the mineral content. The results show that a high temperature and long exposure time
significantly improved the yield (Figure 3).
Figure 3. Factors affecting nitrogen and mineral yield in the processing of chicken MDCM clean bones.
The equation to describe the model in the case of collagen yield is
Nitrogen yield = 8.78X1+ 6.07X2 + 9.13X3 + 5.73X22 − 8.68X3 2 − 4.75X1X2 −
1.85X1X2 − 3.26X2X3 + 69.87,
where X1 = temperature, X2 = dry matter (%), and X3 = treatment time (min).
The equation to describe the model in the case of mineral content is
Mineral content = 3.97X1 − 0.52X2 + 3.32X3 + 0.62X22 − 2.56X32 − 0.97 X1X3+
80.26.
The values of the optimal point variables predicted by the model were 190 ◦C, 83 min,
and 5% dry matter content. The model predicted a nitrogen yield of 86% for the extract
and a mineral content of 86% for the remaining bone fraction. The model was validated
by performing an extraction experiment in optimal conditions. The measured nitrogen
yield was 86.9% ± 2.3%, and the mineral content was 84.5% ± 0.2%. The measured values
were very close to the values predicted by the model; thus, the modeling was successful.
In addition, the yields were high; as such, PHWE appears to be a promising method for
isolating collagen peptides from chicken bones. In the second test series, PHWE treatment
was performed with constant dry matter content (20%) and extraction time (53 min), and
the temperature was varied (110–190 ◦C).
Appl. Sci. 2022, 12, 1327 11 of 15
The samples were analyzed for nitrogen content and mineral content. The results
confirmed the observation of a previous series of experiments that raising the temperature
increased the yield of collagen peptides (Figure 4). The purpose of this series of experiments
was to further investigate the peptide composition of the extracted fractions and the effect
of temperature on the composition.
Figure 4. The response surface of the enzymatic hydrolysis model of MDCM clean bones showing
the relationships between nitrogen and mineral yield, temperature, and hydrolysis time.
3.3.2. Molecular Weight Distribution and Peptide Profiles
The molecular size distribution was determined from the residual bone PHWE ex-
tracts to assess the effect of temperature on collagen cleavage. According to the results
of size exclusion chromatography, the majority of the proteinaceous compounds in the
PHW extracts of chicken bones were in the range of 0.5–13.7 kDa in MW with all extraction
temperatures applied (Figure 5). The proportion of MW range of 0.5–13.7 kDa compounds
varied from 61% (110 ◦C) to 75% (190 ◦C) between the applied temperatures. The results
showed that an increase in the extraction temperature resulted in increased protein frag-
mentation. In particular, the proportion of proteins with MW higher than 44.2 kDa was
reduced by increasing the extraction temperature. At an extraction temperature of 190 ◦C,
the degradation products were the smallest; the proportion of compounds with MW smaller
than 0.5 kDa was higher in comparison to the extracts produced at the lower temperatures.
This indicates that the free amino acids and di- and tripeptides were more abundant in the
PHW extract produced at 190 ◦C than in extracts produced at lower temperatures.
Appl. Sci. 2022, 12, 1327 12 of 15
Figure 5. Molecular weight distribution of the proteinaceous compounds in chicken bone PHW extracts.
The skin is the largest organ of the human body and acts as a barrier that protects us
against various external damages. It is also an important organ, regulating temperature and
fluid–electrolyte balance and metabolism, for example [27]. Collagen and its hydrolysates
have attracted particular attention because of their skin-enhancing properties. Cosmetic
and edible cosmetic formulations use hydrolyzed collagen for its superior solubility at a
neutral pH, its easy dermis penetration, and its water-binding properties [28]. Recently,
many studies have reported skin antiaging and healthcare properties in small collagen
peptides [29]. Chicken bones, as sustainable residual materials, have received special
attention as a source of collagen (reviewed by Salvatore et al. [30]).
The present study shows that PHWE treatment is a promising method for producing
collagen peptides with a low molecular weight from chicken bones, a sustainable residual
material from food processing. The nitrogen yield from the purified bones was high (87%),
indicating that most of the chicken bone protein was extracted. The results are in line
with the findings of Wang et al. [31], who studied PHWE treatment with chicken bones
at 120–135 ◦C. They also found that increasing the treatment temperature improved the
recovery of collagen. In addition to PHWE treatment, other extraction methods have
been studied to recover collagen from chicken bones; attempts have been made with an
NaCl solution containing small amounts of NaNO2, ascorbate, and Na4P2O7, combined
with homogenization [21] and alkali extraction [32]. Chicken foot collagen extraction
has been reported with papain and pepsin enzymes in an acetic acid solution [33] and
with dilute acid treatment [34]. Moreover, extraction studies of turkey bones have been
performed with dilute alkaline NaCl solutions [35] and acid and alkaline treatment [36].
These methods have yielded 46% collagen peptides at best. It can, therefore, be stated
that, among the methods studied so far, PHWE seems the most efficient way of extracting
chicken collagen peptides.
In this study, the smallest collagen peptides were produced at temperatures above
150 ◦C. However, a large proportion of the total protein in the bone extracts was small
peptides below 10 kDa already at a rather mild temperature between 110 and 140 ◦C. The
results indicate the suitability of the chicken bone PHW extracts for cosmetic use, but more
research is needed to investigate the skin enhancing effects on the cellular level and in vivo.
Appl. Sci. 2022, 12, 1327 13 of 15
3.4. Profitability Assessment
The assessment was performed for a hypothetical fractionation process plant, treating
1.0 × 107 kg of chicken MDCM side-stream annually, and yielding food-grade chicken pro-
tein hydrolysate, chicken bone collagen peptides, chicken fat, and calcium- and phosphate-
rich minerals. The theoretical annual amounts of these products from 1.0 × 107 kg of
chicken MDCM were calculated on the basis of the values given in Section 2.7. A total
yield of fractionation processing estimated as 80% was used to calculate the amounts of
products obtained.
The annual production, prices, and revenues of the chicken MDCM side products are
presented in Table 4. The applied prices were based on expert and industry knowledge.
According to the assessment, the total sales revenues for the side products were about
5.5 × 106 EUR/year. Of this, 62% were derived from collagen peptides, and 31% were
derived from MDCM protein hydrolysate.
Table 4. Annual production and sales revenues of different chicken MDCM side products.
Product Production, 106 kg/year Price, EUR/kg Revenue, 106 EUR/year
Collagen peptides (d.w.) 0.34 10.00 3.4
MDCM protein hydrolysate (d.w.) 1.44 1.20 1.7
Calcium- and phosphate-rich
mineral fraction 0.69 0.18 0.12
Chicken fat 0.27 0.85 0.23
Total 5.5
The main process equipment consisted of a high-pressure extraction unit, filters, cen-
trifuges, dryers, and a packaging unit. The investment cost depended on the existing utili-
ties and production facilities. If the process was integrated with a chicken slaughterhouse,
the investment costs for the MDCM side-stream process were estimated at 1.0 × 107 EUR,
according to expert knowledge.
The operating costs included materials, e.g., food grade protease enzymes, operators’
salary costs, energy consumption, maintenance and repairs, and other variable costs. Op-
erating costs were estimated at 2.0 × 106 EUR/year, and the depreciation of investment
and rate of interest were estimated at 2.0 × 106 EUR/year for 10 years. Therefore, the esti-
mated total annual production costs were 4.0 × 106 EUR. The chicken MDCM fractionation
process was profitable according to this preliminary assessment; the annual profit was
about 1.5 × 106 EUR. This value is in the same range as the profit values calculated for
annual processing of 1.0 × 107 kg of salmon filleting side-streams to various high-value
products [37].
Since prices are the main driver in the protein business case, the price stability is
essential. The price of chicken collagen peptides has decreased in recent years, bringing un-
certainties concerning the process’s profitability. The investment is, therefore, classified as
high-risk. The economic feasibility of the process also depends on how well the additional
processes fit the existing process, and how well the resulting products can be marketed.
The feasibility of a process is always a case-by-case issue, because the skills of the process
designer, marketer, and buyer are essential.
4. Conclusions
Our study highlighted the use of response surface methodology to optimize parame-
ters for mechanically deboned chicken enzymatic and PHWE treatments to achieve high
protein and low-molecular-weight collagen peptide yields. A hydrolysis of 240 min us-
ing noncommercial protease enzyme resulted in high protein concentration and protein
hydrolysates with a good solubility index, but weak gelling properties were produced.
PHWE treatment at 190 ◦C for 83 min resulted in high nitrogen yield and a fraction with
Appl. Sci. 2022, 12, 1327 14 of 15
high content of peptides lower than 13.7 kDa. The results suggest that the profitability of
process should be carefully evaluated, due to uncertainties of the price of proteins and
collagen peptides.
Author Contributions: Data curation, M.V., J.M., M.K.; Investigation, N.P., S.M., U.M., A.P.; Method-
ology, N.P., S.M., U.M., M.V., J.M., M.K. Project administration, M.K., A.P.; Supervision, A.P.; Writing—
original draft, A.P.; Writing—review & editing, A.P., S.M., N.P., U.M., M.V., M.K., J.M. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by Business Finland grant number [899/31/2015].
Institutional Review Board Statement: This study was condacted in accordance to the principles
of good scientific practice of the Research Ethics Advisory Board (TENK). It is the responsibility of
every researcher to follow good scientific practice.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data ise arranged according to FAIR principles and is available upon
request. When opening databases, we shall take into consideration commercial utilisation of the
material and results, as well as the protection of the rights.
Acknowledgments: This study was funded by Business Finland. HKScan is greatly acknowledged
for funding and providing MDCM for this research. The authors thank Antti Järveläinen for helping
with the enzymatic processing of the chicken MDCM fractions and Sara Kujansuu for helping with
the PHWE studies.
Conflicts of Interest: All authors claim that there are no conflicts of interest in this study.
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