The research has tested the hypothesis that the thermal stability of gelatine gels decreases with rising temperature and humidity.

Stevens LFRA Texture Analyser for measuring gelatine gel strength (Leonard Farnell and Co Ltd., England), P 98 meat mincer (Brather, Spain), Memmert ULP 400 drying device (Memmert GmbH + Co. KG, Germany), LT 43 shaker (Nedform, Czech Republic), Kern 440 – 47 electronic scale, Kern 770 electronic analytical scale (Kern, Germany), A 10 labortechnik analytical mill (IKA-Werke, Germany), ULP 400 drying oven (Memmert GmbH+Co. KG, Germany), SLR heating board (Schott Gerate GmbH, Germany), Whatman No. 1 paper (Sigma Aldrich, UK), a metal filter sieve with the size of pores 1 and 2 mm (Labor-komplet, Czech Republic). Chemicals: NaCl, NaOH, petroleum ether, ethanol, and chloroform (Verkon, Czech Republic); all chemicals were of analytical grade. Proteolytic enzyme Polarzyme 6.0 T – serine endoprotease manufactured by fermentation of microorganisms that are not present in the final product (Novozymes, Denmark) with the declared enzyme activity of 6 KPU per g (kilo protease unit per g). Commercial mammalian gelatines: pork gelatines with the gel strength of 212 and 288 Bloom, beef gelatines with the gel strength of 266 and 273 Bloom.

Chicken feet were purchased in Raciola Uherský Brod, Czech Republic, and processed to chicken feet gelatine according to the method described by Mokrejš et al. (2019). Two types of pork gelatines and two types of beef gelatines were purchased as well and used for the comparison.

Gelatine gels were prepared following GMIA – Standard Testing Methods for Edible Gelatin (2019) so that 7.5 g of gelatine was mixed with 105 mL distilled water and placed into a standardized bloom jar with a volume of 150 mL and dimensions as follows: overall height of 85 mm, inside diameter of 59 mm, outside diameter of 66 mm, neck inside diameter of 41 mm, and a shoulder height of 65 mm. The mixture was allowed to swell at room temperature for 1 – 3 h. Afterward, the bloom jar (Figure 1) was heated in a water bath at 65 °C to prepare a gelatine solution which was cooled at room temperature and maintained in a cooling box for 16 – 18 h to form gelatine gel. Gel strength (or Bloom value) was measured by Stevens LFRA Texture Analyser (Leonard Farnell and Co Ltd., England, Figure 1). Gelatine gel strength is defined as a force (weight in g) required to depress a measuring probe by specific penetration to a definite area of the gelatine surface to a particular distance.

Studies examining gelatine gels thermal stability mostly used methods of thermal analysis (DSC), rheological testing, or determination of activating energy. Considering a wide range of applications of gelatines in the food industry assessing the quality of gelatines based on their gels strength, this study has employed a method of measuring gel strength according to GMIA - Standard Testing Methods for Edible Gelatin (2019). It has monitored a decline of gel strength in time which is in contrast with other studies that have not considered testing of gelatines on a long-term time scale. The thermal stability of gelatine gels was expressed as a percentage change (decline) of gelatine gel strength during the time period of 5 days. Gelatine gel strength was measured every hour within the first 8 hours of the experiment, and then after 16, 23, 87, 93, 111, and 120 hours. Experiments were performed at three different temperatures of 23, 29, and 35 °C with the relative humidity of 60 and 80%. In total, six series of experiments were performed in which 30 gelatine samples were analysed.

1-sample and 2-sample standard deviation tests on the significance level of p = 0.05 were applied to all results using Minitab 18 statistical software for Windows (Minitab, Ltd., USA). All analyses were performed in triplicate and arithmetic means and standard deviations were calculated.

Table 1 displays initial Bloom values of commercial mammal and prepared chicken feet gelatines ranging in all samples between 220 and 300 which means all tested gelatines showed a significant gel strength (Johnston-Bank, 1983). The highest gel strength was recorded in Pork288 and the lowest in Pork212. The gel strength of prepared chicken feet gelatine was 240 Bloom. Gel strength of commercial pork and beef gelatine gels ranging from 100 to 300 Bloom, whereas 200 – 250 Bloom is most preferred (Holzer, 1996). Several studies discussing the preparation and testing of the strength of chicken feet gelatine gels were published. Taufik et al. (2010) examined the effect of age and extraction temperature on characteristics of chicken feet skin gelatine and reported Bloom values from 112 to 125 which is significantly lower than the values of chicken feet gelatine prepared in this study. Rahman and Jamalulail (2012) performed extractions of chicken feet gelatine, inspected its physicochemical characteristics and sensory quality, and recorded the value of 264 Bloom which is slightly higher than the value of chicken feet gelatine established in this study. Widyasari and Rawdkuen (2014) described gelatine obtained from chicken feet by acid and ultrasound-assisted extraction and reported Bloom values of 185 and 79 which is less than this study provided. Chakka et al. (2016) extracted chicken feet gelatine using food-grade acids and monitored the gel strength in the range from 119 to 204 Bloom which is less than it was recorded in this study. Almeida and Lannes (2013) extracted gelatine from chicken feet and characterized its physicochemical properties. They confirmed the gel strength of 295 Bloom which is more than it was measured in this study. Sompie and Triasih (2018) reported very low gel strength of chicken leg skin gelatine (78 Bloom). On the contrary, in the case of gelatine extracted from residues after mechanical processing of poultry meat, very high Bloom values were recorded: 309-318 Bloom (Fonkwe and Singh, 1997) and 374-380 Bloom (Rafieian et al., 2013) and (Rafieian et al., 2015). High Bloom values have also been reported for poultry gelatine by other researchers: 294 Bloom (Almeida, Calarge and Santana, 2013), 338 Bloom (Du, et al., 2014) and 355 Bloom (Ee et al., 2019) and (Sarbon et al., 2013). Such differences in results may stem from different extraction conditions, particularly time and temperature, as well as from the applied pre-treatment method. The age and gender of the animal could also affect the quality of prepared gelatines.

The results of determination of the thermal stability of chicken feet, beef, and pork gelatine gels tested at the temperatures of 23, 29, and 35 °C and relative humidity of 60 and 80% are shown in Tables 2 – 7. Initial Bloom values of gelatines are expressed as 100%. Changes in gelatine gel strength (declines) are expressed as Bloom index in %.

Table 2 shows the results of the thermal stability of chicken feet, pork, and beef gelatines at a temperature of 23 °C and humidity of 60%. Gel strength gradually decreased as expected. After one hour of measurement, the slightest decline was recorded in CFG240 and the highest in Beef273. A drop of gel strength in other types of gelatines was approximately 25%; differences were not statistically significant (p >0.05). Similar trends were observed also in the following measurements with declines of gel strength between 30 and 65%. The smallest decrease of gel strength was recorded in chicken feet gelatine while the most significant drop was determined in Beef273 (3 – 5 h of measurement) followed by Pork212 (87 – 120 h of measurement). Within the last two measurements (after 111 and 120 h), no further changes in gel strength were established. The deepest decline in gel strength in the final measurement was monitored in Beef266 (more than 90%) while the smallest drop was identified in prepared gelatine CFG240 (approximately 75%); this gelatine showed this trend for the whole testing period. The reduction of gel strength in other types of gelatines was approximately 85% with statistically insignificant differences (p >0.05).

Table 3 summarizes the measurements of the thermal stability of chicken feet, pork and beef gelatines at the temperature of 23 °C and humidity of 80%. Prepared chicken feet gelatine CFG240 provided values with statistically insignificant differences (p >0.05) recorded at both levels of humidity – 60 and 80%.

However, within commercial gelatines in the first 6 hours of measurement, higher declines of gelatine gel strength with statistically significant differences (p <0.05) were recorded at the humidity of 80% than 60 %. In the following measurements, statistically significant (p <0.05) smaller declines of commercial gelatine gel strength were recorded at the humidity of 80% than 60%. After 1 h of measurement, the slightest drop of gelatine gel strength was identified in Pork288 and the most considerable in Beef273. In all the following measurement CFG240 showed the smallest decline. After two hours of testing, the fall of gelatine gel strength ranged from 33 to 64%. The deepest decline in 2, 3, and 4 h of measurement were recorded in Beef273 whereas in further measurements the most significant decrease was determined in Pork212. Nevertheless, within the measurement in 111 and 120 h, no further changes in gelatine gel strength were identified with the final declines of the strength of approximately 80%. Prepared chicken feet gelatine performed either comparable or even better thermal stability at a humidity of 60 and 80% if compared with commercial beef and pork gelatine.

The results of the thermal stability of chicken feet, pork, and beef gelatines at the temperature of 29 °C and humidity of 60% are displayed in Table 4. In contrast with the measurements at the temperature of 23 °C and humidity of 60%, a significantly deeper (p <0.05) decline of gel strength in all gelatines was monitored in 2-h measurement and further. After 1 h, the smallest decrease was monitored in Beef266 and both pork gelatines, while in CFG240 and Beef273 the deepest reduction of gel strength was recorded. In further measurements, the smallest drop was identified in Beef266 and Pork288 (63%), slightly deeper in Pork212 and CFG240 (65%), and the most significant decline in Beef273 (72%). After 3 and 4 h of measurement, the smallest decrease was recorded in Beef266, Pork288, and CFG240 and the deepest fall in Beef273 and Pork212. After 5, 6, 7, and 8 h, gelatine CFG240 showed the slightest decline of approximately 90% while other types of gelatines performed slightly higher, yet statistically insignificant (p >0.05) declines of gelatine gel strength. A similar trend was observed in further measurements and after 87 h of measurement, no gelatine showed a change in gel strength. The final decline of gel strength was almost 100%.

Table 5 shows the results of the thermal stability of chicken feet, pork, and beef gelatines at a temperature of 29 °C and humidity of 80%. At this humidity, a slightly higher decline of gel strength was monitored in commercial gelatines than at the humidity of 60%; while in chicken feet gelatine no statistically significant differences (p >0.05) were recorded, similarly as at the temperature of 23 °C. After 1 h of measurement, the smallest decline was recorded in Beef266, slightly higher in pork gelatines and the deepest decrease in Beef273 a CFG240. In contrast, after 2 h of measurement, the smallest drop of approximately 70% was in CFG240, slightly deeper in Beef266 and Pork288, and the most significant reduction of approximately 80% in Beef273. After 3 h, the last drop was in CFG240, similarly to further measurements, while commercial gelatines performed slightly deeper reduction of gel strength. Further measurements showed a trend of a gradual decline of gel strength; importantly, commercial gelatines performed statistically significant (p <0.05) higher decrease of gel strength if compared with chicken feet gelatine. After 87 h, almost 100% declines in gel strength in all gelatines were monitored. It has been proved that chicken gelatine performs comparable or even better properties than commercial gelatines considering the thermal stability of gelatine gel strength.

Table 6 summarizes the results of the thermal stability of chicken feet, pork, and beef gelatines at the temperature of 35 °C and humidity of 60%. All gelatines performed a steep decline of gelatine gel strength at this temperature. After 1 h of measurement, the decrease ranged between 38 and 43% with the smallest drop in CFG240 and the highest in Beef266 and Pork212. A dramatic decline of gel strength from 83 to 90% was recorded in further measurements. The smallest reduction was determined in Beef266 and the highest in Beef273. After 3 h of measurement, the smallest decline of more than 90% was in CFG240 while all commercial gelatines showed at a drop of 97%. After 4 h, almost all gelatines performed a decrease of nearly 100% and no gelatine formed a gel after 5 h of measurement.

The results of the thermal stability of chicken feet, pork, and beef gelatines at the temperature of 35 °C and humidity of 80% are shown in Table 7. The figures do not differ much from the results at the same temperature at the humidity of 60%. After 1 h of measurement, the decline of gel strength varied from 36 to 42% which proves no statistically significant differences (p >0.05) between the experiments at the humidity of 80 and 60%. The smallest decrease of gel strength was established in CFG240 and Pork288 while the deepest drop was recorded in Beef266, equally as at the humidity of 60%. After 2 h of measurement, chicken feet gelatine showed the smallest decline (75%), and Pork212 together with other beef gelatines the biggest fall (85%). After 3 h, CFG240 performed a decline of gel strength of more than 91% and commercial gelatines of 97%. Nearly 100% decline of gel strength in all gelatines was recorded after 4 h of measurement. Equally, as in previous experiments, comparable declines of gelatine gel strength were recorded in both chicken feet and commercial gelatines at the temperature of 35 °C.