Enzyme conditioning of chicken collagen and taguchi design of experiments enhancing the yield and quality of prepared gelatins

Citation: Mokrejš, P.; Gál, R.; Pavlaˇcková, J. Enzyme Conditioning of Chicken Collagen and Taguchi Design of Experiments Enhancing the Yield and Quality of Prepared Gelatins. Int. J. Mol. Sci. 2023,24, 3654. https://doi.org/10.3390/ ijms24043654 Academic Editor: Andreas Taubert Received: 27 January 2023 Accepted: 9 February 2023 Published: 11 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Enzyme Conditioning of Chicken Collagen and Taguchi Design of Experiments Enhancing the Yield and Quality of Prepared Gelatins Pavel Mokrejš 1,* , Robert Gál2and Jana Pavlaˇcková3 1Department of Polymer Engineering, Faculty of Technology, Tomas Bata University in Zlín, Vavreˇckova 275, 760 01 Zlín, Czech Republic 2Department of Food Technology, Faculty of Technology, Tomas Bata University in Zlín, Vavreˇckova 275, 760 01 Zlín, Czech Republic 3 Department of Lipids, Detergents and Cosmetics Technology, Faculty of Technology, Tomas Bata University in Zlín, Vavreˇckova 275, 760 01 Zlín, Czech Republic *Correspondence: mokr[email protected]; Tel.: +42-05-7603-1230 Abstract: During the production of mechanically deboned chicken meat (MDCM), a by-product is created that has no adequate use and is mostly disposed of in rendering plants. Due to the high content of collagen, it is a suitable raw material for the production of gelatin and hydrolysates. The purpose of the paper was to process the MDCM by-product into gelatin by 3-step extraction. An innovative method was used to prepare the starting raw material for gelatin extraction, demineralization in HCl, and conditioning with a proteolytic enzyme. A Taguchi design with two process factors (extraction temperature and extraction time) was used at three levels (42, 46, and 50 ◦ C; 20, 40, and 60 min) to optimize the processing of the MDCM by-product into gelatins. The gel-forming and surface properties of the prepared gelatins were analyzed in detail. Depending on the processing conditions, gelatins are prepared with a gel strength of up to 390 Bloom, a viscosity of 0.9–6.8 mPa · s, a melting point of 29.9–38.4 ◦ C, a gelling point of 14.9–17.6 ◦ C, excellent waterand fat-holding capacity, and good foaming and emulsifying capacity and stability. The advantage of MDCM by-product processing technology is a very high degree of conversion (up to 77%) of the starting collagen raw material to gelatins and the preparation of 3 qualitatively different gelatin fractions suitable for a wide range of food, pharmaceutical, and cosmetic applications. Gelatins prepared from MDCM by-product can expand the offer of gelatins from other than beef and pork tissues. Keywords: biomaterials; by-product; enzyme conditioning; collagen; gelatin; mechanically deboned chicken meat; Taguchi design; zero-waste 1. Introduction Gelatin is one of the most versatile biopolymers, and due to its unique film, gel, and surface properties, it is widely used in the food, pharmacy, cosmetics, and photography industries, as well as in the production of packaging materials and encapsulates and in a number of technical applications [ 1 – 3 ]. This is evidenced by the global production of gelatin, which represented approximately 700 kilotons in 2021; the total turnover in terms of raw material represents approximately 3500 million USD. Of this amount, approximately 30% was consumed in the production of food and beverages, 25% in nutraceuticals, 19% in pharmaceuticals, 14% in photography, 7% in personal care products, and 5% in other applications. A further increase in gelatin production is expected for 2025 by approximately 6.0% compared to 2019 [ 4 ]. Gelatin can be made from any animal tissue that contains collagen. Currently, approximately 95% of all gelatin is produced industrially from beef and pork tissues. The rest consists of alternative sources of collagen which have gained importance in the last 20 years not only due to the growing demand for gelatin but also Int. J. Mol. Sci. 2023,24, 3654. https://doi.org/10.3390/ijms24043654 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023,24, 3654 2 of 22 due to special consumer requirements [ 5 ]. Some examples are religious or cultural reasons for rejecting pork or beef products or consumer preferences for fish or poultry products over beef and pork. It is also necessary to mention the socially changing attitudes towards handling animal by-products and the possibilities of their use (philosophy of the circular economy). Gelatins can be prepared from various unused parts of poultry, most commonly chicken feet and skin [ 6 , 7 ], duck feet and skin [ 8 , 9 ], and chicken bones [ 10 , 11 ]; other types of poultry are less common [ 12 ]. From fish (both freshwater and marine), gelatins are most often prepared from skin, bones, scales, fins, or heads [ 13 – 17 ]. The conditions for preparing gelatin from frog skin are also known [ 18 ]. However, the disadvantage of alternative raw material sources containing collagen is their non-standard parameters, which significantly complicates their processing into gelatins with properties suitable for specific applications. For example, gelatins prepared from cold-water fish species are less stable and have worse rheological properties, which also complicates their processing. There are also fundamental differences in the properties of gel formation (gel strength, gelling, and melting point) between gelatins prepared from cold and warm water fish [19–22]. When processing collagen raw materials (mainly skin and tendons) into gelatins, it is necessary to remove accompanying components (most often fat, globular proteins, and glycoproteins) from the starting raw material and to prepare the raw material in a suitable way for controlled extraction. For this purpose, traditional or alternative procedures are used—namely conditioning in an acidic or alkaline environment and rarely the use of enzymes [ 2 , 23 ]. The exception is for bones for which demineralization is necessary. This is done in an acidic environment [ 24 ]. Gelatin extraction is carried out with hot water (depending on the type of raw material at a temperature of 40 ◦ C minimum) in extractors of various designs. In the industrial production of beef and pork gelatin, multistage extraction is used to efficiently convert collagen into gelatin [2]. Mechanically deboned meat can be obtained from all animals, with the exception of ruminants, which have been banned as a raw material since 2011 due to concerns about the possible disease of bovine spongiform encephalopathy (BSE). Mechanically deboned chicken meat (MDCM) is obtained most often and used for the production of meat products [ 25 , 26 ]. It is obtained by mechanical separation of the remaining parts of the meat, which are found in the bones and ribs after the meat has been cut and can make up to 30% of the muscle content. To obtain MDCM, a traditional separation procedure is used, which is based on pressing bone raw materials; a continuous filling and pressing method or a separate filling and pressing process can be applied. During the pressing technique, the muscle with the fatty and connective parts is separated from the bones and rough connective tissues. When the MDCM separation decantation procedure is applied, bone raw materials are ground with the addition of flake ice and a sodium nitrite curing salt mixture. The resulting liquid homogenate is continuously centrifuged based on the principle of decantation and immediately frozen. Screw conveyors, hydraulic pistons, or drum separators are used for separation; the yield and quality of MDCM can be regulated, for example, by the size of the holes in the separation sieves or the flow rate of crushed meat and bone raw material [ 27 ]. In the MDCM separation process, higher pressures are sometimes used to increase the yield, resulting in a higher Ca content in the MDCM. Due to the presence of a higher amount of mineral substances, MDCM has good water binding capacity and is suitable as an addition to sausages, pâtés or poultry semi-products; additions up to 10% do not negatively affect the properties of the final products [ 28 , 29 ]. MDCM has a limited shelf life, which is related to the possibility of microbial contamination, the increase in temperature during the separation process, and the higher pH value due to the Ca 3 (PO 4 ) 2 content. The solid residue after MDCM production is characterized by a high content of proteins (up to 40% in dry matter), fats (25–30% in dry matter), and minerals (approximately 30% in dry matter) and thus represents an important source of raw materials rich in nutrients. In addition to the basic physicochemical properties of gelatin (composition, swelling, solubility, color, clarity, odor, and taste), the main attributes that best define the commercial Int. J. Mol. Sci. 2023,24, 3654 3 of 22 quality of gelatin include gel strength and viscosity [ 30 ]. However, the complex quality of gelatins is determined by a set of gel-forming and surface properties. These are important not only for the application of gelatin in final products but also for the choice of a suitable processing technology (extrusion, casting, dipping, injection). The gel-forming properties also include the gelling point (GP), melting point (MP), water holding capacity (WHC), and fat binding capacity (FBC). Surface properties include foaming capacity (FC) and foaming stability (FS), emulsifying capacity (EC), emulsion stability (ES), film-forming ability, and adhesive and cohesive properties. The properties of gelatins depend on many factors, especially the type of collagen (beef, pork, fish, poultry), the conditions of collagen processing (acidic, alkaline, enzyme, combined), the conditions of gelatin extraction (especially temperature, pH, time), and the methods of processing the extracted gelatin (especially the choice of drying method) [ 31 ]. The type of collagen and the processing conditions affect the amino acid composition of gelatin and the distribution of molecular weights [ 32 , 33 ]. The representation and ratio between α -, β -, and γ -chains in gelatin affects the viscosity of gelatin (viscosity increases as the amount of β - γ -chains increases) [ 34 ]; it also affects the GP and MP of gelatin (a higher representation of α -chains shifts both temperatures to higher values) [ 31 ]. The structural stability of gelatin is mainly due to the content of the amino acids proline and hydroxyproline, which contribute to the stabilization of the structure by means of hydrogen bridges [ 35 ]. A higher content of these amino acids will be reflected in an increase in the GP and MP of gelatin [ 36 ]. More detailed information on the structure of gelatin is provided by rheological measurements [ 37 ], scanning and transmission electron microscopy (SEM and TEM) [ 38 ], Fourier transform infrared (FTIR) spectroscopy [39], and differential scanning calorimetry (DSC) [40]. In our previous study devoted to the preparation of gelatin from the MDCM by-product, a two-level factorial experiment with three studied process factors was used [ 41 ]. Compared to studies devoted to the preparation of gelatin from the same raw material [42–44] , in our work higher gelatin yields were achieved. In our study, the basic properties of gelatin (gel strength, viscosity, ash content) were determined. It is clear that all studies showed important results in regard to the processing of previously unused MDCM by-products into gelatins. Considering the great potential of this raw material source, it would be advisable to deal with a more detailed optimization of the gelatin preparation procedure, a thorough characterization of gelatin, and the proposal of its applications with regard to their properties. The objectives of the current study are as follows: (1) Optimize the process of preparing gelatin from the MDCM by-product to achieve the maximum degree of conversion of the starting raw material to gelatins without a negative effect on their quality. For this purpose, we propose an innovative process and Taguchi design of experiments: demineralization of the MDCM by-product, enzyme conditioning of the purified collagen, and 3-stage gelatin extraction; (2) design the processing technology so as to limit the number of by-products created; (3) perform a comprehensive assessment of the quality of prepared gelatins by determining their gel-forming and surface properties; (4) propose potential industrial applications of the prepared gelatins. Scientific hypotheses: By adjusting the process conditions during the processing of collagen from MDCM by-product into gelatins, gelatins are prepared with a higher yield than using standard technological procedures. The higher yields of gelatin will not have a negative effect on their properties. 2. Results The results of processing the MDCM by-product into three fractions of gelatins are presented in the following four subsections. 2.1. Mass Balance of the Process The schedule of experiments and results of the processing of the MDCM by-product into three gelatin fractions are presented in Table 1. Table 2shows the results of the analysis of variance for the gelatin yields. Int. J. Mol. Sci. 2023,24, 3654 4 of 22 Table 1. The experimental design and the results of the process mass balance. Exp. No. Factor A (◦C) Factor B (min) YH (%) YG1 (%) YG2 (%) YG3 (%) UR (%) MBE (%) YG∑ (%) 1 42 20 10.6 6.2 39.7 8.0 32.6 2.9 53.9 2 42 40 12.0 22.5 44.8 3.2 14.2 3.3 70.5 3 42 60 11.1 25.4 49.3 2.1 8.9 3.2 76.8 4 46 20 12.4 45.4 24.2 2.9 11.8 3.3 72.5 5 46 40 11.7 38.4 30.4 4.0 12.2 3.3 72.8 6 46 60 11.0 19.2 49.8 3.9 14.6 1.5 72.9 7 50 20 12.1 29.1 30.8 7.2 17.8 3.0 67.1 8 50 40 11.6 30.5 27.1 7.3 21.1 2.4 64.9 9 50 60 10.8 32.8 22.4 7.2 21.9 4.9 62.4 10 * 46 40 3.3 1.3 2.4 4.4 86.9 1.7 8.1 Factor A—extraction temperature at 1st extraction step; Factor B—extraction time at 1st extraction step; Y H —the yield of collagen hydrolysate; Y G1 —the yield of the 1st gelatin fraction; Y G2 —the yield of the 2nd gelatin fraction; Y G3 —the yield of the 3rd gelatin fraction; UR—an undissolved residue; MBE—the mass balance error; Y G∑ —total gelatin extraction yield; * Exp. No. 10—a blind experiment (no enzyme conditioning). Table 2. Analysis of variance of the experimental design for gelatin yields. Degree of Freedom Sum of Squares Mean Squares F-Value p-Value Response: The yield of the 1st gelatin fraction, YG1 (%) = −44.6 + 1.60A −0.028B Regression 2 246.30 123.148 0.94 0.441 Factor A (Extraction temperature) 1 244.48 244.482 1.87 0.220 Factor B (Extraction time) 1 1.82 1.815 0.01 0.910 Error 6 784.12 130.686 Total 8 1030.42 Response: The yield of the 2nd gelatin fraction, YG2 (%) = 129.0 −2.229A + 0.223B Regression 2 596.7 298.37 5.59 0.043 Factor A (Extraction temperature) 1 477.0 477.04 8.94 0.024 • Factor B (Extraction time) 1 119.7 119.71 2.24 0.185 Error 6 320.2 53.36 Total 8 916.9 Response: The yield of the 3rd gelatin fraction, YG3 (%) = −9.4 + 0.350A −0.0408B Regression 2 15.762 7.881 1.79 0.246 Factor A (Extraction temperature) 1 11.760 11.760 2.67 0.153 Factor B (Extraction time) 1 4.002 4.002 0.91 0.377 Error 6 26.407 4.401 Total 8 42.169 •statistically significant factor (p-value ≤0.05). Figure 1shows the relationship between a response variable (gelatin yields) and two predictor variables (extraction temperature and extraction time) using contour plots. Depending on the values of both studied process factors, the yield of the first gelatin fraction (Y G1 ) ranges from less than 10% to more than 40%. The highest Y G1 yields were achieved at extraction temperatures of 45–49 ◦ C with extraction time < 35 min (see Figure 1a); both studied process factors were not found to be significant at the monitored level of Int. J. Mol. Sci. 2023,24, 3654 5 of 22 significance (p-value ≤ 0.05). The second gelatin fraction (Y G2 ) is among the dominant gelatin fractions in terms of percentage representation, with yields of approximately 22 to 50%; gelatins from the second fractions show the best gel-forming and surface properties (see Section 2.3). From Figure 1b, there is an obvious trend of Y G2 yield growth, especially with increasing extraction time (Factor B). The extraction time is a statistically significant factor with a p-value = 0.024, see Table 1. On the contrary, it is evident from the contour position that the extraction temperature (Factor A) has a smaller effect on Y G2 ; the p-value is higher than 0.05. The third gelatin fractions, with their approximate yield (Y G3 ) of 2–8%, have the lowest representation of extracted gelatins, see Figure 1c. Neither of the two monitored process factors is statistically significant (p-values are > 0.05). Figure 1d then shows the total yield of extracted gelatin, Y G∑ (sum of Y G1 , Y G2 , and Y G3 ). It is obvious that at an appropriately chosen extraction temperature (42–44 ◦ C) and an extraction time of 50–60 min, the degree of collagen-to-gelatin conversion is very high, up to approximately 75%. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 23 Figure 1 shows the relationship between a response variable (gelatin yields) and two predictor variables (extraction temperature and extraction time) using contour plots. Depending on the values of both studied process factors, the yield of the first gelatin fraction (YG1) ranges from less than 10% to more than 40%. The highest YG1 yields were achieved at extraction temperatures of 45–49 °C with extraction time < 35 min (see Figure 1a); both studied process factors were not found to be significant at the monitored level of significance (p-value ≤ 0.05). The second gelatin fraction (YG2) is among the dominant gelatin fractions in terms of percentage representation, with yields of approximately 22 to 50%; gelatins from the second fractions show the best gel-forming and surface properties (see Section 2.3). From Figure 1b, there is an obvious trend of YG2 yield growth, especially with increasing extraction time (Factor B). The extraction time is a statistically significant factor with a p-value = 0.024, see Table 1. On the contrary, it is evident from the contour position that the extraction temperature (Factor A) has a smaller effect on YG2; the p-value is higher than 0.05. The third gelatin fractions, with their approximate yield (YG3) of 2–8%, have the lowest representation of extracted gelatins, see Figure 1c. Neither of the two monitored process factors is statistically significant (p-values are > 0.05). Figure 1d then shows the total yield of extracted gelatin, YG∑ (sum of YG1, YG2, and YG3). It is obvious that at an appropriately chosen extraction temperature (42–44 °C) and an extraction time of 50–60 min, the degree of collagen-togelatin conversion is very high, up to approximately 75%. Figure 1. The influence of extraction temperature at 1st extraction step and extraction time at 1st extraction step on gelatin yields: (a) the yield of the 1st gelatin fraction; (b) the yield of the 2nd gelatin fraction; (c) the yield of the 3rd gelatin fraction; (d) the total yield of gelatins. If we compare the yields of gelatins (YG1, YG2, and YG3) prepared according to our proposed procedure consisting of demineralization of the starting raw material, enzyme conditioning of collagen, and 3-stage gelatin extraction according to Taguchi design (see Exp. Nos. 1–9 in Table 1) with a blind experiment under conditions corresponding to the mean values of the monitored factors (extraction temperature 46 °C and extraction time 40 min) without enzyme collagen conditioning (see Exp. No. 10 in Table 1), it is evident Figure 1. The influence of extraction temperature at 1st extraction step and extraction time at 1st extraction step on gelatin yields: ( a ) the yield of the 1st gelatin fraction; ( b ) the yield of the 2nd gelatin fraction; (c) the yield of the 3rd gelatin fraction; (d) the total yield of gelatins. If we compare the yields of gelatins (Y G1 , Y G2 , and Y G3 ) prepared according to our proposed procedure consisting of demineralization of the starting raw material, enzyme conditioning of collagen, and 3-stage gelatin extraction according to Taguchi design (see Exp. Nos. 1–9 in Table 1) with a blind experiment under conditions corresponding to the mean values of the monitored factors (extraction temperature 46 ◦ C and extraction time 40 min) without enzyme collagen conditioning (see Exp. No. 10 in Table 1), it is evident that the innovative method of collagen conditioning has a fundamental effect on gelatin yield. The total yield of gelatin (Y G∑ ) in the blind experiment is only 8.1%, which is approximately 9 times less than that for gelatin extracted under the same process conditions (Exp. No. 5) with enzyme collagen conditioning. Compared with the yield of gelatins prepared under different conditions (Exp. Nos. 1–9), the Y G∑ in the blind experiment is 6.7–9.5 times lower. Int. J. Mol. Sci. 2023,24, 3654 6 of 22 2.2. First Gelatin Fractions The results of the properties analysis of the first gelatin fractions prepared from the MDCM by-product are shown in Table 3. Table 3. Results of the analysis of the properties of the first gelatin fractions. Process Factors Gelatin Properties Exp. No. Factor A (◦C) Factor B (min) Ash (%) υ (mPa·s) WHC (%) FBC (%) FC (%) FS (%) EC (%) ES (%) 1 42 20 1.17 1.7 220 840 8 0 47 93 2 42 40 0.97 1.6 220 900 8 2 47 92 3 42 60 1.23 1.5 230 920 7 2 48 93 4 46 20 0.88 1.6 230 1090 6 2 48 93 5 46 40 0.96 1.5 230 1090 7 2 48 94 6 46 60 1.43 1.5 240 1110 7 2 47 93 7 50 20 1.39 1.5 240 1140 8 3 47 93 8 50 40 1.02 1.4 250 1210 8 4 48 95 9 50 60 1.16 1.4 240 1210 7 3 46 94 10 * 46 40 1.02 1.6 240 1150 7 4 48 93 Factor A—temperature at 1st extraction step; Factor B—extraction time at 1st extraction step; υ —viscosity; WHC—water holding capacity; FBC—fat binding capacity; FC—foaming capacity; FS—foaming stability; EC— emulsifying capacity; ES—emulsion stability; * Exp. No. 10—a blind experiment (no enzyme conditioning). None of the gelatins obtained in the first extraction step formed measurable gels; therefore, these are zero Bloom value gelatins. The zero Bloom value is also related to the viscosity of gelatin, which reaches very low values (1.4–1.7 mPa · s), regardless of the changing extraction conditions. Similarly, it is with WHC, where no significant difference between gelatins is apparent; depending on extraction conditions, WHC = 220–250%. For FBC, a slight growth trend is evident with increasing extraction temperature and, at the same time, prolonging extraction time; from values slightly exceeding 800% at the minimum values of both monitored factors to approximately 1200% at the upper limits of the factors. Foaming properties, FC and FS, are very low, 6 to 8% or 0 to 4%, respectively; temperature and extraction time do not fundamentally affect these parameters. It is similar to the emulsifying properties, EC and ES, for which process conditions do not affect their changes. However, all gelatins have very good EC values (46–48%) and excellent ES (92–95%). The properties of gelatin prepared under the conditions of a blind experiment (without enzyme conditioning) under conditions corresponding to the mean values of the monitored factors (extraction temperature 46 ◦ C and extraction time 40 min)–see Exp. No. 10 in Table 3–do not fundamentally differ from the properties of gelatin prepared in Exp. Nos. 1–9. 2.3. Second Gelatin Fractions The results of the properties analysis of the second gelatin fractions prepared from the MDCM by-product are shown in Table 4. Table 5shows the results of the analysis of variance for the strength of the gelatin gel, the viscosity, the melting point, and the gelling point. The ash content is very low in all gelatins prepared according to the Taguchi design (Exp. Nos. 1–9); it varies between 0.34–0.70%. Fundamental differences were not found in the water holding capacity (930–1090%) and fat binding capacity (980–1470%). Gelatin prepared according to the conditions of Exp. No. 9 has a significantly higher foaming capacity (36%) than the other gelatins (18–22%); there is a similar difference in foaming stability (24% versus 8–18%). In terms of emulsifying capacity and emulsion stability, there are no fundamental differences between the gelatins prepared according to experiments 1–9. Int. J. Mol. Sci. 2023,24, 3654 7 of 22 Table 4. Results of the analysis of the properties of the second gelatin fractions. Process Factors Gelatin Properties Exp. No. Factor A (◦C) Factor B (min) GS (Bloom) MP (◦C) GP (◦C) υ (mPa·s) Ash (%) WHC (%) FBC (%) FC (%) FS (%) EC (%) ES (%) 1 42 20 174 35.3 16.6 2.2 0.66 1010 1310 20 16 48 93 2 42 40 80 28.9 15.0 1.6 0.35 930 980 18 8 46 93 3 42 60 125 32.3 15.3 1.8 0.34 970 1390 18 10 47 93 4 46 20 143 32.8 15.5 1.9 0.43 950 1070 22 18 48 93 5 46 40 105 30.4 14.9 2.0 0.36 960 1170 20 8 51 90 6 46 60 262 36.8 17.1 2.9 0.45 960 1230 20 16 48 93 7 50 20 284 37.9 16.7 2.7 0.46 980 1250 20 16 47 93 8 50 40 269 35.1 16.4 2.6 0.43 990 1470 20 18 49 90 9 50 60 290 38.4 17.6 3.8 0.70 1090 1460 36 24 52 92 10 * 46 40 460 35.1 26.8 6.8 0.54 1320 1540 40 28 54 93 Factor A—temperature at 1st extraction step; Factor B—extraction time at 1st extraction step; GS—gel strength; MP—melting point; GP—gelling point; υ —viscosity; WHC—water holding capacity; FBC—fat binding capacity; FC—foaming capacity; FS—foaming stability; EC—emulsifying capacity; ES—emulsion stability; * Exp. No. 10—a blind experiment (no enzyme conditioning). Table 5. Analysis of variance of the experimental design for gelatin gel strength, gelatin viscosity, melting point, and gelling point. Degree of Freedom Sum of Squares Mean Squares F-value p-Value Response: Gel strength (Bloom) = −723 + 19.33A + 0.64B Regression 2 36,870.8 18,435.4 5.69 0.041 Factor A (Extraction temperature) 1 35,882.7 35,882.7 11.07 0.016 • Factor B (Extraction time) 1 988.2 988.2 0.30 0.601 Error 6 19,451.2 3241.9 Total 8 56,322.0 Response: Viscosity (mPa·s) = −4.89 + 0.1458A + 0.01417B Regression 2 2.5233 1.2617 5.98 0.037 Factor A (Extraction temperature) 1 2.0417 2.0417 9.68 0.021 • Factor B (Extraction time) 1 0.4817 0.4817 2.28 0.182 Error 6 1.2656 0.2109 Total 8 3.7889 Response: Meting point (◦C) = 5.2 + 0.621A + 0.0125B Regression 2 37.3767 18.6883 2.21 0.191 Factor A (Extraction temperature) 1 37.0017 37.0017 4.37 0.082 Factor B (Extraction time) 1 0.3750 0.3750 0.04 0.840 Error 6 50.8322 8.4720 Total 8 88.2089 Response: Gelling point (◦C) = 8.44 + 0.1583A + 0.0100B Regression 2 2.6467 1.3233 1.60 0.277 Factor A (Extraction temperature) 1 2.4067 2.4067 2.92 0.138 Factor B (Extraction time) 1 0.2400 0.2400 0.29 0.609 Error 6 4.9489 0.8248 Total 8 7.5956 •statistically significant factor (p-value ≤0.05). Int. J. Mol. Sci. 2023,24, 3654 8 of 22 Properties of gelatin prepared under the conditions of a blind experiment (without enzyme conditioning) under conditions corresponding to the mean values of the monitored factors (extraction temperature 46 ◦ C and extraction time 40 min)–see Exp. No. 10 in Table 4– differs significantly in some parameters from the properties of the gelatins prepared in Exp. Nos. 1–9. In particular, this is a very high gel strength value, which is 1.6 to 5.8 times higher compared to gelatins prepared from Exp. Nos. 1–9; for viscosity, the value is 1.8–4.3 times higher. WHC (1320% versus 930–1090%) and FBC (1540% versus 980–1470%) are also higher. This is also true for FC (40% versus 18–36%) and FS (28% versus 8–24%). There are no fundamental differences in EC and ES for gelatin from Exp. No. 10 compared to gelatins prepared according to Exp. Nos. 1–9. Figure 2shows the relationship between the response variables and two predictor variables (extraction temperature and extraction time) by contour plots. From Figure 2a, the trend of increase in gel strength is evident, especially with increasing extraction temperature; extraction temperature is a statistically significant factor (p-value of 0.016; see Table 5). Lower gel strength values (up to 200 Bloom) are achieved at temperatures < 48 ◦ C and extraction times up to 50 min. Very good gel strength values (200–250 Bloom) are achieved at extraction temperatures close to the upper limit of the observed temperature (50 ◦ C), while the extraction time does not have a significant effect on the gel strength value. A very similar trend of influence of extraction temperature and extraction time on gelatin viscosity can be seen in Figure 2b. Gelatins with a lower viscosity (2.0–2.5 mPa · s) are prepared at an extraction temperature <42.5 ◦ C regardless of the extraction time; increasing the extraction temperature to 50 ◦ C while simultaneously shortening the extraction time has the same effect. The highest viscosity (3.0–3.5 mPa · s) was achieved at extraction temperatures of 49–50 ◦C with extraction times >55 min. The extraction temperature is a statistically significant factor (p-value = 0.021), see Table 5. An almost identical effect of both process factors, as with gel strength, was recorded on the MP; see Figure 2c. The melting point ranges from relatively lower values (around 30–32 ◦ C) at lower extraction temperatures (<47 ◦ C) without a significant influence on extraction time. A very high MP (35–38 ◦ C) is achieved at extraction temperatures above 49 ◦ C; the extraction time has no significant effect on the change in MP values. The GP is not fundamentally affected by changes in the monitored process conditions; it ranges from 15.0 to 17.5 ◦ C, with lower GP values corresponding to lower extraction temperatures and shorter extraction time, and higher GP values to extraction temperatures >49 ◦ C. Both monitored process factors are statistically insignificant (p-values > 0.05, see Table 5). 2.4. Third Gelatin Fractions The results of the properties analysis of the third gelatin fractions prepared from the MDCM by-product are shown in Table 6. From the results of the third gelatin fraction properties, gelatins prepared under Taguchi design conditions (Exp. Nos. 1–9) can be divided into 3 groups; the first group consists of gelatins prepared at the lowest extraction temperature (42 ◦ C, Experiments 1–3), the second gelatins prepared at medium extraction temperature (46 ◦ C, Experiments 4–6) and the third gelatins prepared at the highest extraction temperature (50 ◦ C, Experiments 7–9); see Table 6. The most fundamental is the difference in the strength of the gels. While gelatins prepared at 46 ◦ C did not form gels at all and gelatins prepared at 42 ◦ C formed weak gels (80–88 Bloom), gelatins prepared at 50 ◦ C had very high gel strengths ( 223–230 Bloom ). The differences between MP and GP are not fundamental between gelatins with the ability to form gels. However, for gelatins prepared at 50 ◦ C, the MP (33.9–34.8 ◦ C) is higher than for gelatins prepared at 42 ◦ C (29.2–30.8 ◦ C); for GP, there is a difference between these two groups of gelatins, 16.0–16.5 ◦ C versus 14.9–15.3 ◦ C. The group of gelatins prepared at 46 ◦ C did not form gels; therefore, it was not possible to determine MP and GP for these gelatins. For viscosity, the trend is analogous to that of gel strength; the highest (2.4–2.6 mPa · s) in gelatins prepared at 50 ◦ C, followed by gelatins prepared at 42 ◦ C (1.7–1.8 mPa · s), with a slight decrease in gelatins prepared at 46 ◦ C. Int. J. Mol. Sci. 2023,24, 3654 9 of 22 The ash content of all 9 prepared gelatins is very low and ranges from 0.48 to 0.96%. The water holding capacity is 2.6 to 3.2 times lower for gelatins prepared at 46 ◦ C than for gelatins prepared at 42 ◦ C and even 3.2 to 3.5 times lower than for gelatins prepared at 50 ◦ C; 210–220% versus 550 to 680% versus 680 to 730%. There are no significant differences in FBC between the three groups of gelatin; FBC = 990–1220%. On the other hand, in FC, gelatins prepared at 46 ◦ C outperform both gelatins prepared at 50 ◦ C (18–20% versus 16–17%) and gelatins prepared at 42 ◦ C, which have a very low FS (6–8%). For gelatins prepared at 42 and 50 ◦ C, there is zero FS, while for gelatins prepared at 46 ◦ C, it is 11–12%. There are no significant differences in EC and ES between gelatins prepared according to experiments 1–9. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 23 Figure 2. The influence of extraction temperature and extraction time on second gelatin fractions properties: (a) gel strength; (b) viscosity; (c) melting point; (d) gelling point. 2.4. Third Gelatin Fractions The results of the properties analysis of the third gelatin fractions prepared from the MDCM by-product are shown in Table 6. Table 6. Results of the analysis of the properties of the third gelatin fractions. Process Factors Gelatin Properties Exp. No. Factor A (°C) Factor B (min) GS (Bloom) MP (°C) GP (°C) υ (mPa·s) Ash (%) WHC (%) FBC (%) FC (%) FS (%) EC (%) ES (%) 1 42 20 80 29.2 14.9 1.7 0.67 550 1190 7 0 48 95 2 42 40 82 30.1 15.0 1. 7 0.35 560 1180 6 0 47 97 3 42 60 88 30.8 15.3 1.8 0.81 680 1220 8 0 48 97 4 46 20 0 NA NA 1.4 0.96 220 1060 20 12 48 96 5 46 40 0 NA NA 1.5 0.71 210 990 18 11 47 96 6 46 60 0 NA NA 1.5 0.6 7 220 1040 20 12 48 95 7 50 20 223 33.9 16.0 2.4 0.59 680 1100 17 0 47 96 8 50 40 225 34.4 16.2 2.4 0.65 680 1120 16 0 48 96 9 50 60 230 34.8 16.5 2.6 0.48 730 1130 16 0 48 97 10* 46 40 245 34.1 15.2 2.4 0.63 910 1220 19 0 47 91 Factor A—temperature at 1st extraction step; Factor B—extraction time at 1st extraction step; GS— gel strength; MP—melting point; GP—gelling point; υ—viscosity; WHC—water holding capacity; FBC—fat binding capacity; FC—foaming capacity; FS—foaming stability; EC—emulsifying capacity; ES—emulsion stability; * Exp. No. 10—a blind experiment (no enzyme conditioning); NA—not applicable. Figure 2. The influence of extraction temperature and extraction time on second gelatin fractions properties: (a) gel strength; (b) viscosity; (c) melting point; (d) gelling point. Table 6. Results of the analysis of the properties of the third gelatin fractions. Process Factors Gelatin Properties Exp. No. Factor A (◦C) Factor B (min) GS (Bloom) MP (◦C) GP (◦C) υ (mPa·s) Ash (%) WHC (%) FBC (%) FC (%) FS (%) EC (%) ES (%) 1 42 20 80 29.2 14.9 1.7 0.67 550 1190 7 0 48 95 2 42 40 82 30.1 15.0 1.7 0.35 560 1180 6 0 47 97 3 42 60 88 30.8 15.3 1.8 0.81 680 1220 8 0 48 97 4 46 20 0 NA NA 1.4 0.96 220 1060 20 12 48 96 5 46 40 0 NA NA 1.5 0.71 210 990 18 11 47 96 6 46 60 0 NA NA 1.5 0.67 220 1040 20 12 48 95 7 50 20 223 33.9 16.0 2.4 0.59 680 1100 17 0 47 96 8 50 40 225 34.4 16.2 2.4 0.65 680 1120 16 0 48 96 9 50 60 230 34.8 16.5 2.6 0.48 730 1130 16 0 48 97 10* 46 40 245 34.1 15.2 2.4 0.63 910 1220 19 0 47 91 Factor A—temperature at 1st extraction step; Factor B—extraction time at 1st extraction step; GS—gel strength; MP—melting point; GP—gelling point; υ —viscosity; WHC—water holding capacity; FBC—fat binding capacity; FC—foaming capacity; FS—foaming stability; EC—emulsifying capacity; ES—emulsion stability; * Exp. No. 10—a blind experiment (no enzyme conditioning); NA—not applicable. Int. J. Mol. Sci. 2023,24, 3654 16 of 22 secondary source of protein in feed mixtures for farm animals and pets, or due to its high protein content, as a source of nitrogen for the production of plant growth stimulators. 4. Materials and Methods 4.1. Materials, Appliances and Chemicals Mechanically deboned chicken meat (MDCM) by-product (from Ross 708 broiler chicken aged 35 days) was supplied by Raciola, Ltd. (UherskýBrod, Czech Republic). First, by-product material analyses were performed by conventional food methods [ 50 – 52 ]. Dry matter content 38.2 ± 0.7%; in dry matter: protein 40.3 ± 1.2%, collagen (as a part of protein content) 79.9 ± 0.5%, fat 26.0 ± 1.5% and inorganic solids 29.6 ± 3.8%. Each analysis was repeated three times; mean values and standard deviations were calculated. Stevens LFRA texture analyzer (Leonard Farnell and Co Ltd., Liverpool, UK), Ubbelohde viscometer (Technisklo Ltd., Držkov, Czech Republic), Nedform LT 43 shaker (Valašské Meziˇrící, Czech Republic), electronic scale Kern 440-47, electronic analytical balance Kern 770 (Balingen, Germany), analytical mill IKA A 10 labortechnik (Staufen, Germany), Memmert ULP 400 drying oven (B˝uchenbach, Germany), Samsung fridge freezer (Seoul, Republic of Korea), Henkelman Boxer 42 vacuum packaging machine (CK ‘s-Hertogenbosch, Netherlands), IKA T 25 digital Ultra-Turrax (IKA-Werke, Germany), Whatman no. 1 paper (Sigma Aldrich, Gillingham, UK), WTW Multical pH 526 pH meter (Weilheim, Germany), heating board Schott Geräte (Mainz, Germany), a 1 mm pores size metal filter sieve (Laborkomplet, Praha, Czech Republic), ordinary laboratory glass. Chemicals: NaCl, NaOH, HCl, petroleum ether, ethanol (Verkon, Prague, Czech Republic); all chemicals were analytical grade. Protamex ® , Novozymes endopeptidase (Copenhagen, Denmark), used for conditioning purified collagen. It is a Bacillus protease complex with declared activity of 1.5 AU/g; optimal working conditions are at pH 5.5 to 7.5 and temperature 60 ◦C. The enzyme complies with the recommended purity specifications for food-grade enzymes issued by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Food Chemicals Codex (FCC). 4.2. Experimental Design and Statistical Analysis Design of experiments (DOE) is a tool that enables the examination of the influence of independent variables (process factors) on dependent variables. Therefore, it enables the identification of significant factors of the process and its optimization [ 53 ]. Various experiment planning designs are used in practice, e.g., three-level full factorial design, central composite design, Box-Behnken design, or Taguchi design [ 54 ]. The extraction temperature and extraction time proved to be key process factors that influence not only the degree of conversion of collagen to gelatins but also the properties of gelatins. Therefore, these factors were studied using the Taguchi design of the experiments. This will achieve a more effective optimization of the gelatin preparation process from the MDCM by-product. Independent variables with factor levels: factor A (extraction temperature), 42, 46, 50 ◦ C; factor B (extraction time), 20, 40, 60 min. The selected dependent variables were as follows: gelatin yields (Y G1 , Y G2 , Y G3 ), gel strength, viscosity, MP, GP, WHC, FBC, FC, FS, EC, and ES. The gelatin analysis was performed in triplicate; mean values were calculated using Microsoft Office Excel 2013 (Microsoft, Denver, CO, USA). Minitab ® 17.2.1 statistical software for Windows (Fujitsu Ltd., Tokyo, Japan) was used to perform regression analysis of the data obtained. The statistical significance was evaluated using analysis of variance (ANOVA). The level of significance was established at 5% (p-value ≤ 0.05); factors with a value <0.05 have an effect on the process variables evaluated with 95% probability. The same software evaluated the graphical analysis of the data by creating contour plots showing the relationship between the dependent variables and the independent variables by viewing discrete contours of the dependent response variables. Int. J. Mol. Sci. 2023,24, 3654 17 of 22 4.3. Processing of MDCM By-Product into Gelatins The scheme of complex processing of MDCM by-product into three fractions of gelatins, including usable by-products created during processing, is shown in a flow chart in four technological sections, see Scheme 1. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 18 of 23 Scheme 1. In our case, we proposed an innovative process of disrupting the quaternary structure of purified (demineralized) collagen: a proteolytic enzyme conditioning and gelatin extraction in 3 stages. The details of the procedure are given below. I. Separation of organic matter. The thawed raw material was first washed with cold H2O. It was mixed with 0.2 mol/L NaCl in a 1: 6 ratio and shaken at room temperature (22.0 ± 1.0 °C) for 90 min and then washed with cold H2O. It was then mixed with 0.03 mol/L NaOH in a 1:6 ratio and shaken at room temperature for 45 min and, after filtration, washed with cold H2O; this procedure was repeated three more times. Finally, the raw material was washed with cold H2O and dried at 35 ° C for 24 h. This was followed by the defatting step: the raw material was mixed in a 1:9 (w/v) ratio with petroleum ether and ethanol (mixed in a ratio of 1:1, v/v) and shaken for 48 h at room temperature; after 12 h, the solvent was changed. II. Demineralization. The raw material was mixed in a 1:8 ratio with 3.0% HCl and demineralized with gentle shaking at room temperature for 96 h; after 24 h, the acid was replaced with a new one. After filtration (maceration liquor as a by-product of the process), the demineralized collagen was thoroughly washed with cold H2O and dried for 24 h at 35 °C. III. Purified collagen was mixed with H2O in a 1:10 ratio and after shaking for 20 min, the pH was adjusted to 6.5–7.0 (by adding a 5% NaOH solution). The 0.6 % proteolytic enzyme (based on the weight of purified collagen) was then added and the mixture was shaken at room temperature for 24 h; during the first 4 h at 30-min intervals, the pH was checked (and adjusted) to the prescribed range. After filtering off the liquid by-product (collagen hydrolysate), solid collagen was washed thoroughly with cold H2O. Collagen hydrolysate was dried in a thin layer (4 mm) in a circulating air drier at 60.0 ± 0.5 °C for 20 h. IV. 3-step extraction of gelatins. Biotechnologically treated collagen was subjected to 3 separate (sequential) extraction cycles using a batch process extractor. In the first extraction stage, collagen was mixed with H2O in a ratio of 1:20 and the mixture was heated while stirring at a rate of dt/dτ = 10 °C/min to a temperature according to factor A (42.0 ± 0.5, 46.0 ± 0.5, 50.0 ± 0.5 °C), at which point the gelatin extraction lasted for the time according to factor B (20, 40, 60 min). After filtration, the solution of the 1st gelatin fraction was immediately heated to a temperature of 85.0 ± 0.5 °C (dt/dτ = 15 °C/min) and kept at this temperature for 8 min; the residual enzyme was inactivated this way. The gelatin solution was poured into a thin film (4 mm) and dried in a circulating air drier, first at Scheme 1. In our case, we proposed an innovative process of disrupting the quaternary structure of purified (demineralized) collagen: a proteolytic enzyme conditioning and gelatin extraction in 3 stages. The details of the procedure are given below. I. Separation of organic matter. The thawed raw material was first washed with cold H 2 O. It was mixed with 0.2 mol/L NaCl in a 1: 6 ratio and shaken at room temperature ( 22.0 ±1.0 ◦C ) for 90 min and then washed with cold H 2 O. It was then mixed with 0.03 mol/L NaOH in a 1:6 ratio and shaken at room temperature for 45 min and, after filtration, washed with cold H 2 O; this procedure was repeated three more times. Finally, the raw material was washed with cold H 2 O and dried at 35 ◦ C for 24 h. This was followed by the defatting step: the raw material was mixed in a 1:9 (w/v) ratio with petroleum ether and ethanol (mixed in a ratio of 1:1, v/v) and shaken for 48 h at room temperature; after 12 h, the solvent was changed. II. Demineralization. The raw material was mixed in a 1:8 ratio with 3.0% HCl and demineralized with gentle shaking at room temperature for 96 h; after 24 h, the acid was replaced with a new one. After filtration (maceration liquor as a by-product of the process), the demineralized collagen was thoroughly washed with cold H 2 O and dried for 24 h at 35 ◦C. III. Purified collagen was mixed with H 2 O in a 1:10 ratio and after shaking for 20 min, the pH was adjusted to 6.5–7.0 (by adding a 5% NaOH solution). The 0.6 % proteolytic enzyme (based on the weight of purified collagen) was then added and the mixture was shaken at room temperature for 24 h; during the first 4 h at 30-min intervals, the pH was checked (and adjusted) to the prescribed range. After filtering off the liquid by-product (collagen hydrolysate), solid collagen was washed thoroughly with cold H 2 O. Collagen hydrolysate was dried in a thin layer (4 mm) in a circulating air drier at 60.0 ± 0.5 ◦ C for 20 h. IV. 3-step extraction of gelatins. Biotechnologically treated collagen was subjected to 3 separate (sequential) extraction cycles using a batch process extractor. In the first extraction stage, collagen was mixed with H 2 O in a ratio of 1:20 and the mixture was heated while stirring at a rate of dt/d τ = 10 ◦ C/min to a temperature according to factor A ( 42.0 ±0.5 , 46.0 ± 0.5, 50.0 ± 0.5 ◦ C), at which point the gelatin extraction lasted for the time according to factor B (20, 40, 60 min). After filtration, the solution of the 1st gelatin Int. J. Mol. Sci. 2023,24, 3654 18 of 22 fraction was immediately heated to a temperature of 85.0 ± 0.5 ◦ C (dt/d τ = 15 ◦ C/min) and kept at this temperature for 8 min; the residual enzyme was inactivated this way. The gelatin solution was poured into a thin film (4 mm) and dried in a circulating air drier, first at 40.0 ± 0.5 ◦ C for 12 h, and then at 65.0 ± 0.5 ◦ C for 8 h. The resulting gelatin film was scraped, weighed, and ground to a powder. In the second and third extraction stages, the same procedure was followed at extraction temperatures of 65.0 ± 0.5 ◦ C for 30 min and 80.0 ± 0.5 ◦ C for 30 min. The second gelatin fraction was inactivated in the same way as the first gelatin fraction. The undissolved residue (a by-product of the extraction of gelatin) remained after the third extraction cycle and was dried at 103.0 ± 1.0 ◦ C to constant weight and then weighed. The prepared gelatins were subjected to further analysis. 4.4. Analytical Part The hydrolysate yield (Y H ) was calculated from the weight of the hydrolysate prepared after conditioning the purified collagen according to the initial weight of the purified collagen (Equation (6)), the yield of gelatins (Y G1 , Y G2 , Y G3 ) from the weight of extracted gelatins according to the initial weight of the purified collagen (Equation (7)). Furthermore, the total extraction yield of gelatin ( Σ Y G ) and the portion of undissolved residue (UR) was calculated (Equations (8) and (9)). The mass balance error (MBE) is expressed by the percentage difference of the dry matter mass balance between the input (purified collagen) and the output (hydrolysate + gelatins + undissolved residue); see Equation (10). YH= (mH/m0)×100 (6) YG= (mG/m0)×100 (7) ΣYG= YG1+YG2 + YG3 (8) UR = (mUR/m0)×100 (9) MBE = [100 −(YH+ YG1 + Y G2 + Y G3 + UR)] (10) where Y H is the hydrolysate yield (%), Y G1 is the yield of the first gelatin fraction (%), Y G2 is the yield of the second gelatin fraction (%), Y G3 is the yield of the third gelatin fraction (%), UR is an undissolved residue (%), m 0 is the weight of purified collagen (g), m H is the hydrolysate weight (g), m G is the weight of gelatins (g), and m UR is the weight of the undissolved residue (g). Gel strength, viscosity, and ash content were determined according to standard test methods for edible gelatins [ 55 ]. Because these are common gelatin testing methods, we present only their principles. The gel strength was determined from a gel formed from a 6.67 % solution prepared according to prescribed conditions by measuring the force (weight in grams, which is equal to the Bloom value) required to depress a prescribed area of the sample surface to a distance of 4 mm. The viscosity of a 6.67 % gelatin solution was determined by the Ubbelohde viscometer and ash gravimetrically after burning and annealing the sample. The following gelatin properties are not described in standard gelatin testing methods, so a brief test procedure will be provided. Gelatin water holding capacity was determined according to Nasrin et. al. [ 56 ] with slight modifications. In a plastic test tube, 1.0 g of the gelatin sample was mixed with 25.0 mL of distilled H 2 O and then the contents were shaken vigorously for 5 min at room temperature. The contents of the test tube were then centrifuged at 5000 rpm for 30 min and then the supernatant was filtered through Whatman no. 1. filter paper. WHC (%) was calculated from the weight of water absorbed by the gelatin sample, w 1 (g), based on the weight of gelatin weighed, w 0 (g), and multiplied by a coefficient of 100; see Equation (11). WHC = (w1/w0)×100 (11) Gelatin fat binding capacity was determined according to Li et. al. [ 57 ]. In a plastic test tube, 0.1 g of the gelatin sample was mixed with 10.0 mL of sunflower oil, and the Int. J. Mol. Sci. 2023,24, 3654 19 of 22 contents were vigorously shaken for 30 min at room temperature. The contents of the test tube were then centrifuged at 2500 rpm for 30 min and the supernatant was pipetted and weighed. FBC (%) was calculated from the weight of oil absorbed by the gelatin sample, w 2 (g), based on the weight of gelatin weighed, w 0 (g), and multiplied by a coefficient of 1000; see Equation (12). FBC = (w2/w0)×1000 (12) Gelatinfoaming capacity and foamingstabilitywere determined accordingtoSatheet.al.[ 58 ] with slight modifications. The amount of 1.0 g of the gelatin sample was weighed in a graduated cylinder and 50.0 mL of distilled H 2 O was added; the gelatin was dissolved in a water bath at 60.0 ± 1.0 ◦ C while stirring. After dissolving, a dispersing instrument was placed below the level of the resulting solution and the solution was whipped at 10,000 rpm for 5 min. After whipping, the volume of the whipped solution was measured; FC (%) was calculated according to Equation (13). After standing at room temperature for 30 min, the volume of the whipped solution was measured again; FS (%) was calculated according to Equation (14). FC = [(V1−V0)/V0]×100 (13) FS = [(V2−V0)/V0]×100 (14) where V 0 is the original volume of liquid (50 mL), V 1 is the volume of the whipped solution (mL), and V2is the volume of the whipped solution after 30 min (mL). Gelatin emulsifying capacity and the emulsion stability were determined according to Neto et. al. [ 59 ] with slight modifications. In a plastic test tube, 0.01 g of the gelatin sample was mixed with 5.0 mL of distilled H 2 O, and after 10 s of thorough shaking, 5.0 mL of sunflower oil was added and shaken for 1 min at room temperature. The contents of the test tube were then centrifuged at 1000 rpm for 5 min. The heights of the entire volume of liquid in the tube and the emulsion were measured. The tube was then placed in a preheated water bath at 55.0 ± 0.5 ◦ C for 5 min; then, the emulsion height was measured. EC (%) and ES (%) were calculated according to Equations (15) and (16). EC = (h1/h0)×100 (15) ES = (h2/h0)×100 (16) where h 0 is the height of the entire volume of liquid (mm), h 1 is the height of the emulsion after centrifugation (mm), and h 2 is the height of the emulsion after 5 min of heating (mm). The Moosavi-Nasab method [ 60 ] with some modifications was used to determine the melting point; a solution of gelatin at the same concentration (6.67%) as after determination of gel strength and viscosity was used. A gelatin solution was introduced into a glass capillary of 3.0 mm in diameter to form a column at a height of 6.0 ± 1.0 mm. The sample capillary was allowed to cool at 10.0 ± 0.1 ◦ C for 17 h (sol-gel transition). The capillary was then placed in a water bath at 10.0 ± 0.5 ◦ C so it was completely immersed. The water bath was heated at 2 ◦ C/min and the gelatin column in the capillary was monitored. The temperature at which the gelatin column began to move in the capillary (gel-sol transition) was recorded as the MP. The Schrieber and Gareis method [ 2 ] with slight modifications was used to determine the gelling point; a gelatin solution at the same concentration (6.67%) as after determination of gel strength and viscosity was used. The gelatin solution in the test tube was placed in a water bath. After warming to 35.0 ± 0.5 ◦ C, ice water was added to the water bath so that the cooling rate of the gelatin solution in the tube was 2 ◦ C/min. Each time the temperature dropped by 0.5 ◦ C, a 0.10 g metal ball was inserted into the tube. The temperature at which the ball got stuck in or on the gelatin solution layer was recorded as a GP. Int. J. Mol. Sci. 2023,24, 3654 20 of 22 5. Conclusions The work is a contribution to the resolution of issues of environmental aspects of biomaterials. It has been proven that with the appropriate choice of innovative processing technology using Taguchi design as a modern method of experiment planning, it is possible to prepare high-quality gelatins from unused residue arising during the production of mechanically deboned chicken meat (MDCM). A completely new benefit of the work is the very high gelatin yields, which have not been achieved in previous works dealing with MDCM by-product processing. Furthermore, intermediate products formed during processing do not represent residual waste and can be further used; the presented technology belongs to zero waste processing of the MDCM by-product. One of the practical benefits of the work is that, even from a nontraditional source of collagen, gelatins of different quality can be prepared by multistage extraction. These are comparable to traditional pork and beef gelatins. Gelatins prepared from MDCM by-products are suitable for common food and pharmaceutical applications, for cosmetic products, and for production of biomedical matrixes as well. 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