Physicochemical Properties of Various Types of 3D Bio-ink with Animal-based Gelatin Powder

Physicochemical Properties of Various Types of 3D Bio-ink with Animal-based Gelatin Powder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Physicochemical Properties of Various Types of 3D Bio-ink with Animal-based Gelatin Powder Kyu-Min Kang, Sol-Hee Lee, Hack-Youn Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8770112/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract In this study, we analyzed the physical and chemical properties of bio-ink prepared using various types and ratios of animal-based gelatin. The pH analysis revealed that as the gelatin ratio increased, the pH decreased, with fish gelatin samples showing the highest pH values (p < 0.05). The color measurements indicated that the lightness (CIE L*) of the 4% gelatin sample was higher than that of the other ratios (p < 0.05), while redness (CIE a*) increased with higher gelatin ratios, and yellowness (CIE b*) was significantly higher in beef gelatin samples (p < 0.05). Viscosity measurements showed that initial viscosity increased with higher gelatin ratios, with pork gelatin exhibiting the highest viscosity. Printability results demonstrated that 4%, 12%, and 20% gelatin samples could not form structures; however, only pork gelatin formed scaffolds up to 0.5 cm in height. Structural property analysis revealed that pork gelatin samples had rough surfaces, and the 20% pork gelatin sample showed significantly higher grid size and gel strength (p < 0.05). Pearson correlation analysis indicated a strong positive correlation between gel strength and viscosity (correlation coefficient 0.88) as well as between grid size and gel strength (correlation coefficient 0.89). In conclusion, the type and ratio of gelatin significantly influence the pH, color, viscosity, printability, and structural properties of bio-ink, suggesting that using pork gelatin powder is suitable for 3D bio-ink production considering stable structure formation. Animal-based Gelatin powder Bio-ink Physicochemical properties 3D bio-print Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The world population exceeded 7.6 billion in 2018 and continues to increase annually, with projections estimating it will reach 9.2 billion by 2050. This growth is expected to increase food demand by 59–102% [1]. As food demand rises, global meat consumption is also anticipated to increase annually, reaching 4.55 million tons by 2050. Developing countries are expected to account for 56% of feed grain consumption [2]. Consequently, while meat production is increasing worldwide, the need for alternative meat sources is also growing due to various factors such as environmental concerns, animal welfare, and health issues [3]. Among these alternatives, cultured meat, produced by cultivating livestock cells, stands out in the meat industry due to its potential to conserve finite resources and minimize the killing of animals [4]. Cultured meat produced using 3D bio-printing has recently garnered attention because it can be manufactured in various shapes that resemble or exceed the quality of traditional meat. In contrast, conventional cultured meat, which uses only existing cells, has been limited to forms such as patties [5]. Additionally, by optimizing the cell growth environment, production time can be shortened, raw material usage minimized, and cost-effectiveness improved [6]. Furthermore, 3D bio-printing allows for the development of customized meat that can be tailored to individual consumers' dietary preferences or health needs [7]. These 3D bio-printed cultured meats are produced based on bio-ink, which is made from various raw materials such as alginate and gelatin. The quality of the final product is determined by the characteristics of these raw materials [8]. Gelatin, an animal-based raw material, plays a crucial role in the production process of cultured meat as it is necessary for cell attachment and proliferation [9]. Gelatin is a degradation product of collagen, exhibiting excellent biocompatibility and suitability for providing a conducive cellular environment, making it widely used in bio-ink for 3D bio-printing [10]. Furthermore, gelatin promotes cell growth and differentiation during cultured meat production, facilitating more natural and efficient tissue formation [11]. Although gelatin shares similar properties, its characteristics may vary depending on its source. Therefore, it is essential to compare and analyze gelatin from different sources [12]. This study aims to manufacture various animal gelatin bio-inks using bovine, porcine, and fish gelatin powder and to analyze the qualitative and mechanical properties of each gelatin type to identify the appropriate ratio for scaffold production. 2. Materials and Methods 2.1 Preparation of animal-based gelatin bio-ink Beef (Holstein, Bos taurus taurus; molecular weight, 50–100 kDa), pork (LYD, Landrace×Yorkshire×Duroc; molecular weight, 50–100 kDa), and fish (Salmon, Oncorhynchus keta; molecular weight, 10–80 kDa) skin gelatin powder were obtained from Pureundeulpan Co., Ltd. (Seoul, Korea). The animal-based gelatin (AG) was prepared by dissolving animal-based gelatin powders in distilled water (DW) at a concentration of 4, 12, 20, 32, and 40% (w/v) at 45℃. And the sodium alginate (SA) was prepared by dissolving sodium alginate powder (Sigma-Aldrich, St. Louis, MO, USA) in DW at a concentration of 6% (w/v) at 70℃. As shown in Fig. 7, each concentration of AG was mixed with SA with a 1:1 ratio filled into a printing cartridge (Cellink, Göteborg, Sweden), and stored at 4℃ for the experiments. 2.2 pH Four grams of sample was mixed with 16 mL of distilled water using Ultra Turrax homogenizer (HMZ-20DN, Poonglim Tech, Seongnam, Korea) for 1 min at 6991 хg. The pH was measured using a glass electrode pH meter (Model S220, Mettler-Toledo, Schwerzenbach, Switzerland). 2.3 Color The surfaces of the samples were randomly evaluated using a colorimeter, adjusted to operate with an aperture of 8 mm, 2° standard observer, illuminant D65, and pulsed xenon lamp as a default light source. Before measuring, the device was calibrated with a white plate, CIE L*: +97.83, CIE a*: -0.43, and CIE b*: +1.98 (CR-10, Minolta, Tokyo, Japan), and the lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) were recorded. Hue angle and chroma values were calculated using the following formula: $$\:\text{Hue\:angle=}{\text{tan}}^{\text{-1}}{\text{b}}^{\text{*}}\text{/}{\text{a}}^{\text{*}}\text{,\:Chroma=}{\left({\text{a}}^{\text{*2}}\text{+}{\text{b}}^{\text{*2}}\right)}^{\frac{\text{1}}{\text{2}}}$$ 2.4 Viscosity The samples' viscosity (i.e., the flow behavior) and time dependency were measured using a viscometer (Merlin VR, Rheosys, Hamilton Township, NJ, USA). The viscometer was attached to a 30-mm cone, and the sample was placed in a 25-mm coaxial cylinder. The viscometer conditions were as follows: measurement time 30 s at 10 ℃ with a head speed of 20 rpm. The values are presented in Pa⋅s. 2.5 Printability The printability was performed using a 3D bio-printer (BIO-X, Cellink) with a motorized Z-axis. The bio-inks were printed with a square shape, 20% infill density, 1 × 1 cm size, and varying heights (0.1, 0.3, and 0.5 cm). The printing cartridge filled with samples was attached with sterile high-precision conical bioprinting nozzles (27G-200 µm) and installed in a 3D bio-printer. The installed printing cartridge was extruded and the forms were observed. The printing conditions were as follows: print-bed of 10 ± 1 ℃, print speed of 5 mm/s, print temperature 30 ± 1 ℃, print pressure 150 ± 1 kPa. After printing, the printed structures were dipped into a 100 mM CaCl 2 for ionic cross-linking and the forms were observed again. 2.6 Microphotograph, Grid thickness, and Pore size The microphotograph of the samples was observed and scanned using an Eclipse Ci-L microscope (Nikon, Tokyo, Japan) with a × 40 scope, and grid thickness and pore size of the samples were determined using NIS-Elements imaging software (Nikon, Tokyo, Japan). 2.7 Gel strength The gel strength of the samples was determined using the method by Park and Kim [13] with slight modifications. The samples (1 × 1 × 0.3 cm) were stored at 10 ℃ for 24 h and then used for experiment. The gel strength of the prepared samples was measured using a texture analyzer (TA 1, Ametek Inc., Berwyn, PA, USA) at room temperature, with the following settings: a cylinder probe of 10 cm with a test speed of 2.0 mm/s, distance of 8.0 mm, and a force of 5.6 N. The measured values were expressed in gf. 2.8 Statistical analysis All experimental data in this study were analyzed after a minimum of three repeated trials and the results are presented as mean, standard deviation (SD). The statistical analyses of pH, color, printability, grid thickness, grid size, and gel strength were verified using one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test in the GraphPad Prism 10 for windows (version 10.4.0; GraphPad Prism Software Inc., San Diego, CA, USA) at a significant level of p < 0.05. The Pearson correlation analysis was performed using matrix of heat-map and scatter plot in the R-studio for windows (version 4.4.2; RStudio Inc., Boston, MA, USA). 3. Results and Discussion 3.1 pH Table 1 shows the pH of bio-ink with various types and ratios of animal-based gelatin. As the ratio of gelatin increased, the pH tended to decrease in all gelatin types. When collagen breaks down into gelatin, the presence of acidic amino acids, ionization reactions, and the structural properties of gelatin all contribute to a slightly acidic pH [14]. Accordingly, as the concentration of gelatin increases, the content of acidic amino acids also rises, leading to an increase in hydrogen ion concentration and resulting in weak acidity [15]. Notably, all fish gelatin samples exhibited significantly higher pH values compared to samples from other gelatin types (p < 0.05). The observed differences in pH depending on the type of gelatin are thought to be influenced by the vitamins, minerals, and amino acids present in the livestock species from which the gelatin was derived [16]. Specifically, beef and pork gelatin are believed to have lower pH values than fish gelatin due to their higher content of acidic amino acids such as aspartic acid and glutamic acid [17]. Furthermore, because higher pH levels are associated with increased metabolic activity in cells, bio-ink with elevated pH is considered suitable for use as scaffold material in the production of cultured meat [18]. Table 1 pH of 3D bio-ink with various types and ratios of animal-based gelatin powder Traits Gelatin (%) 4 12 20 32 40 pH Beef 6.03 ± 0.01 aC 5.91 ± 0.02 bC 5.89 ± 0.01 bC 5.81 ± 0.01 cC 5.80 ± 0.02 cC Pork 7.24 ± 0.05 aB 6.93 ± 0.19 bB 6.66 ± 0.25 bcB 6.41 ± 0.20 cB 6.04 ± 0.08 dB Fish 7.52 ± 0.03 aA 7.42 ± 0.07 aA 7.10 ± 0.06 bA 7.04 ± 0.07 bA 6.90 ± 0.03 cA All values are mean ± SD. a−c Means in the same row with different letters are significantly different ( p -value < 0.05). A−C Means in the same column with different letters are significantly different ( p -value < 0.05). 3.2 Color The color results of bio-ink with various types and ratios of animal-based gelatin are shown in Table 2 and Fig. 2 . In the lightness (CIE L*), 4% gelatin samples were significantly higher than the other gelatin ratio samples (p < 0.05). And as the ratio of gelatin increased, the lightness tended to decrease in all gelatin types. In the redness (CIE a*) as the ratio of gelatin increased, the redness tended to increase in all gelatin types. And fish gelatin samples showed the lowest values in all gelatin ratios. In the yellowness (CIE b*), beef gelatin samples showed significantly higher values than the other gelatin type samples (p < 0.05). And as the ratio of gelatin increased, the yellowness tended to increase in all gelatin types. Sinthusamran et al. [19] reported that the lightness decreased and the redness and yellowness increased as the gelatin concentration increased in all bio-inks made from beef, pork, and fish gelatin, showing similar results to this study. This is because as the concentration of gelatin increases, the degree to which the particles in the bio-ink scatter light changes, which affects the brightness, and as the concentration of the pigment contained increases, more light of a specific wavelength is absorbed, which increases the intensity of the color [20]. In addition, gelatin is a protein derived from collagen, and as the concentration increases, the intermolecular interaction is strengthened to form a photonic structure, and this structural change affects the transmission and scattering of light, thereby affecting color [21]. Hanani et al. [22] reported that the color difference of the final product by the type of gelatin is greatly affected by the color of the gelatin powder itself used, and that bovine gelatin powder has a higher yellowness than other gelatin powders, resulting in a greater color difference. In addition, during the powderization process of gelatin, it goes through a drying process, during which it reacts with oxygen in the atmosphere to cause the Maillard reaction, which increases the yellowness of the gelatin powder [23]. In particular, beef gelatin has a higher content of amino acids that are sensitive to the Maillard reaction, such as lysine, than gelatin from other livestock species, resulting in higher yellowness and redness values [24]. Since the yellowness of the beef gelatin powder used in this study can be seen to be high even with the naked eye, it is judged that it affected the redness and yellowness of the final bio-ink produced. In hue angle and chroma, 20%, 32%, and 40% beef gelatin samples showed different chroma values than the other samples. Choma represents a relative color effect based on the brightest point in the color space, and it was reported that as the concentration of gelatin increases, the saturation also increases, showing similar results to this study [25]. In the color of gelatin, Chroma is positively correlated with redness, and chroma tends to increase mainly as redness increases [26]. And since chroma is also affected by the Maillard reaction, a non-enzymatic browning reaction between amino acids and reducing sugars, it is thought that the chroma value differs depending on the type of extracted gelatin powder or extraction method [27]. Although the color of gelatin used in bio-ink does not significantly affect the functional properties, it does have an aesthetic impact on consumers, so it is thought that gelatin with low yellowness or redness would be advantageous in order to impart a color similar to meat [28]. Table 2 Color of 3D bio-ink with various types and ratios of animal-based gelatin powder Traits Gelatin (%) 4 12 20 32 40 L * Beef 58.00 ± 0.56 aB 55.20 ± 0.20 bC 55.07 ± 0.15 bB 54.70 ± 0.10 bB 53.93 ± 0.21 cA Pork 61.20 ± 0.26 aA 57.03 ± 0.15 bB 55.23 ± 0.31 cB 51.20 ± 0.36 dC 46.93 ± 0.35 eB Fish 61.77 ± 0.31 aA 57.77 ± 0.15 bA 56.97 ± 0.21 cA 55.83 ± 0.42 dA 53.60 ± 0.53 eA a * Beef 1.67 ± 0.06 dA 1.73 ± 0.07 dA 1.87 ± 0.06 cA 2.20 ± 0.10 bA 2.43 ± 0.04 aA Pork 1.17 ± 0.04 cB 1.30 ± 0.10 bB 1.37 ± 0.06 bB 1.53 ± 0.05 aB 1.63 ± 0.06 aB Fish 1.13 ± 0.06 cB 1.27 ± 0.05 bB 1.33 ± 0.08 bB 1.37 ± 0.06 bC 1.50 ± 0.10 aB b * Beef 9.40 ± 0.46 eA 10.10 ± 0.10 dA 12.00 ± 0.10 cA 13.93 ± 0.15 bA 14.97 ± 0.15 aA Pork 6.63 ± 0.21 eB 7.33 ± 0.21 dB 7.70 ± 0.10 cB 8.07 ± 0.25 bB 8.60 ± 0.10 aB Fish 5.87 ± 0.15 dC 6.73 ± 0.21 cC 7.80 ± 0.10 bC 8.10 ± 0.10 bB 8.63 ± 0.25 aB All values are mean ± SD. a−e Means in the same row with different letters are significantly different ( p -value < 0.05). A−C Means in the same column with different letters are significantly different ( p -value < 0.05). 3.3 Viscosity Figure 3 shows the viscosity of bio-ink with various types and ratios of animal-based gelatin. As shown in Fig. 3 D, as the gelatin ratio increased, the initial viscosities were increased in all gelatin types. Also pork gelatin samples showed the highest initial viscosities in all gelatin ratios. In Fig. 3 A and 3 C, the viscosities of all ratio beef and fish gelatin samples tended to increase as the time increased. This is believed to be affected by the structure, amino acid composition, intermolecular bonding, polydispersity, and thermal stability of the gelatin used in bio-ink production, and among them, the differences between gelatin types are believed to be mainly due to the amino acid composition and intermolecular bonding [29,30]. Compared to gelatin extracted from warm-blooded animals, fish gelatin has a looser structure and weaker intermolecular bonds, resulting in a relatively low initial viscosity and maintaining low viscosity even at high concentrations [31]. On the other hand, pork gelatin has higher structural integrity of β- and γ-chains than beef and fish gelatin, which causes physical constraints and entanglements that impede the flow of the polymer, resulting in a high initial viscosity [32]. In terms of amino acid composition, the content of proline and hydroxyproline, which are the main factors in the gelation and melting point of gelatin, is important. In the case of fish gelatin, the content is lower than that of beef and pork, so it is sensitive to heat and dissolves easily, making it difficult to increase initial viscosity even at high concentrations [33]. However, in Fig. 3 B, 32% and 40% pork gelatin samples tended to decrease as the time increased. This is thought to be because as the gelatin concentration became very high, intermolecular interactions occurred excessively, preventing the formation of a stable gel network [34]. Especially, under high concentration conditions such as 32% and 40%, the internal stress increases, and the structural stability is not maintained, so the viscosity decreases over time [35]. The higher the concentration of the gelatin solution, the more non-Newtonian the flow characteristics become, and the viscosity changes with external force or over time. At this time, the flow and molecular motion of the solution are restricted, so the gel structure is easily destroyed [36]. Therefore, considering the viscosity results, it is thought that it would be appropriate to use less than 32% of porcine gelatin when manufacturing bioink. 3.4 Printability The printability results of bio-ink with various types and ratios of animal-based gelatin are shown in Fig. 4 . In Fig. 4 A, 4% (beef, pork, and fish) gelatin, 12% (beef and fish) gelatin, and 20% (beef, pork, and fish) gelatin could not form the structure. And 32% and 40% pork gelatin samples could not print the structure. This is thought to be due to the molecular weight and concentration that affect the physical (hydrogen bonding and van der Waals) bonding involved in the gelatin gelation process [37]. Van der Waals forces are attractive or repulsive forces between molecules, and are very weak bonds to form a gel structure, so low-concentration gelatin has difficulty forming a gel structure because strong bonds such as hydrogen bonds are necessary to maintain the gel structure [38]. On the other hand, in the case of high-concentration gelatin, the elastic restoring force is strong due to strong hydrogen bonds, so the property to resist external stimuli is increased, so it has properties closer to solid rather than flow characteristics [39]. As a result, high extrusion pressure and large-gauge tips are used during the printing process, which reduces the advantages of 3D printing [40]. Also shown in Fig. 4 B, only pork gelatin samples can form the scaffold structure up to 0.5 cm in height. Pork gelatin has a higher content of amino acids, such as proline and hydroxyproline, than bovine or fish gelatin. These amino acids can form strong hydrogen bonds between gelatin molecules to form a stable three-dimensional network structure [41]. This stable three-dimensional network structure exhibits high internal binding strength against gravity resistance [42]. Therefore, considering the printability results, it is expected that using porcine gelatin will enable more stable printing of bio-ink. 3.5 Structure properties Figure 5 shows the structure properties of bio-ink with various types and ratios of animal-based gelatin. As shown in Fig. 5 A, pork gelatin samples showed rough surface than the other gelatin type samples. A rough surface can improve cell adhesion by providing microscopic grooves and protrusions on which cells can adhere and strengthening cell-support interactions [43]. And in Fig. 5 B, 32% and 40% fish gelatin sample showed significantly higher grid thickness than the other samples (p < 0.05). Such a high lattice thickness can enhance structural stability, but an excessively thick structure may have limitations in accuracy [44]. Therefore, fish gelatin can provide structural stability by providing a thick lattice at high concentrations, but excessive thickness may act as a disadvantage in certain applications. However, in Fig. 5 C and 5 D, 20% pork gelatin samples showed significantly higher grid size and gel strength than the other samples (p < 0.05). This indicates the possibility that cell density may decrease due to a decrease in the total area to which cells can attach, and the surface area to which cells can attach may be limited in the early stages of culture [45]. However, the large grid size can create an environment favorable for intercellular material exchange and nutrient supply, providing an advantage in that cells can be uniformly distributed throughout the entire structure in the mid- to late-stage culture as cell proliferation and growth progress [46]. The results of the structure properties are thought to be influenced by the same mechanism as the results of the printability, and it is thought that the strength of the hydrogen bond between molecules affects the density of gelatin, resulting in differences in the structure properties depending on the type and concentration of gelatin [41]. 3.6 Pearson correlation analysis heatmap The pearson correlation analysis heatmap of bio-ink with various types and ratios of animal-based gelatin is shown in Fig. 6 . Gel strength and viscosity have a correlation coefficient of 0.88, showing a strong positive correlation. This means that as the gelatin concentration increases or the intermolecular bonds become stronger, the gel strength and viscosity increase together. In bioink, the higher the gel strength and viscosity, the greater the structural stability, so when used as a cell support, the better the resistance to external pressure, which can provide an environment in which cells can stably attach and grow [47]. And grid size and gel strength also show a strong positive correlation (correlation coefficient 0.89). This suggests that gel strength tends to increase as the lattice size increases. Large grid sizes can provide a favorable environment for cell culture by increasing the space for cells to attach and expand, and increasing gel strength plays an important role in maintaining this structure [48]. In particular, the combination of lattice size and gel strength as a cultured meat support is important in that it can facilitate cell distribution and material exchange while ensuring the stability of the three-dimensional structure [41]. The correlation coefficient between CIE a * and CIE b * is 0.99, showing a very high positive correlation. This suggests that color changes can occur while maintaining the color properties of the bioink uniformly, and that it is possible to maintain a specific color tone depending on the concentration and type of gelatin. This color consistency can serve as an important factor in the appearance quality control of commercial cultured meat [28]. 4. Conclusion In this study, we analyzed the physical and chemical properties of bio-ink formulated with various types and ratios of animal gelatin. The pH analysis revealed that as the gelatin ratio increased, the pH decreased, with fish gelatin samples exhibiting the highest pH values (p < 0.05). Color measurements indicated that the lightness (CIE L*) of the 4% gelatin sample was significantly higher than that of other ratios (p < 0.05), while redness (CIE a*) increased with higher gelatin ratios. Yellowness (CIE b*) was notably higher in the beef gelatin samples compared to others (p < 0.05). Viscosity measurements demonstrated that initial viscosity increased with gelatin concentration, with pork gelatin exhibiting the highest viscosity among the samples. Printability tests indicated that the 4%, 12%, and 20% gelatin samples could not form stable structures; however, only the pork gelatin was able to create scaffolds up to 0.5 cm in height. Structural characterization showed that pork gelatin samples possessed a rough surface, and the 20% pork gelatin sample had significantly larger lattice size and gel strength (p < 0.05). Furthermore, Pearson correlation analysis revealed a strong positive correlation between gel strength and viscosity (correlation coefficient 0.88) and a strong positive correlation between lattice size and gel strength (correlation coefficient 0.89). In conclusion, the type and ratio of gelatin significantly influence the pH, color, viscosity, printability, and structural properties of bio-ink. Given the favorable formation of stable structures, the use of porcine gelatin is deemed suitable for bio-ink production. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding statement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2024–00460285). This work was also supported by the National University Development Project of the Ministry of Education, Korea in 2025. Author Contribution Conceptualisation was conducted by Kang KM and Lee SH. Data curation and formal analysis were performed by Kang KM. Funding acquisition and resources were provided by Lee SH and Kim HY. Investigation was carried out by Kang KM. Methodology was developed by Kang KM and Kim HY. Project administration was managed by Kang KM. Software was prepared by Kang KM. Supervision and validation were provided by Kang KM and Kim HY. Visualisation was undertaken by Kang KM. The original draft of the manuscript was written by Kang KM. Kang KM, SH Lee, and Kim HY contributed to review and editing of the manuscript. References 1. Godara AS, Saresh NV, Bijarnia AL, Godara RS, Kumar D, Meena D, Kumar M. Integrating livestock with crops and forestry for sustainability. Int J Environ Clim Change. 2024;14(11):83–90. https://doi.org/10.9734/ijecc/2024/v14i114530 2. 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Food Hydrocolloids. 2019;92:163–172. https://doi.org/10.1016/j.foodhyd.2019.01.059 30. Zandi M, Mirzadeh H, Mayer CH. Effects of concentration, temperature, and pH on chain mobility of gelatin during the early stages of gelation. Iran Polym J. 2007;16(12):861–870. https://www.scopus.com/inward/record.uri?eid=2-s2.0-38949195298&partnerID=40&md5=8ca050e83b27d53ba5a27b08bb26afce 31. Gómez-Guillén MC, Turnay J, Fernández-Dıaz MD, Ulmo N, Lizarbe MA, Montero P. Structural and physical properties of gelatin extracted from different marine species: a comparative study. Food Hydrocolloids. 2002;16(1):25–34. https://doi.org/10.1016/S0268-005X(01)00035-2 32. De Farias BS, Rizzi FZ, Ribeiro ES, Diaz PS, Sant’Anna Cadaval Junior TR, Dotto GL, Khan MR, Manoharadas S, de Almeida Pinto LA, Dos Reis GS. Influence of gelatin type on physicochemical properties of electrospun nanofibers. Sci Rep. 2023;13(1):15195. https://doi.org/10.1038/s41598-023-42472-9 33. Sezer P, Okur I, Oztop MH, Alpas H. Improving the physical properties of fish gelatin by high hydrostatic pressure (HHP) and ultrasonication (US). Int J Food Sci Technol. 2020;55(4):1468–1476. https://doi.org/10.1111/ijfs.14487 34. Michon C, Cuvelier G, Launay B. Concentration dependence of the critical viscoelastic properties of gelatin at the gel point. Rheol. Acta. 1993;32(1):94–103. https://doi.org/10.1007/BF00396681 35. Gregory T, Benhal P, Scutte A, Quashie Jr D, Harrison K, Cargill C, Grandison S, Savitsky MJ, Ramakrishnan S, Ali J. Rheological characterization of cell-laden alginate-gelatin hydrogels for 3D biofabrication. J Mech Behav Biomed Mater. 2022;136:105474. https://doi.org/10.1016/j.jmbbm.2022.105474 36. Kokol V, Pottathara YB, Mihelčič M, Perše LS. Rheological properties of gelatine hydrogels affected by flow-and horizontally-induced cooling rates during 3D cryo-printing. Colloids Surf A Physicochem Eng Asp. 2021;616:126356. https://doi.org/10.1016/j.colsurfa.2021.126356 37. Abedinia A, Nafchi AM, Sharifi M, Ghalambor P, Oladzadabbasabadi N, Ariffin F, Huda N. Poultry gelatin: Characteristics, developments, challenges, and future outlooks as a sustainable alternative for mammalian gelatin. Trends Food Sci Technol. 2020;104:14–26. https://doi.org/10.1016/j.tifs.2020.08.001 38. Pakseresht S, Mazaheri Tehrani M. Advances in multi-component supramolecular oleogels-a review. Food Rev Int. 2022;38(4):760–782. https://doi.org/10.1080/87559129.2020.1742153 39. Zhang HJ, Wang X, Wang L, Sun TL, Dang X, King DR, You X. Dynamic bonds enable high toughness and multifunctionality in gelatin/tannic acid-based hydrogels with tunable mechanical properties. Soft Matter. 2021;17(41):9399–9409. https://doi.org/10.1039/D1SM01201K 40. Chee HL, Koo JW, Sim EEI, Zhu Q, Gao X, Ramli MFH, Young JL, Holle AW, Wang F. Hofmeister Ions-Induced Thinning of Gelatin to Enhance 3D Printing Precision. Adv Mater Technol. 2024;2302230. https://doi.org/10.1002/admt.202302230 41. Geonzon LC, Takagi H, Hayano Y, Draget KI, Nordgård CT, Matsukawa S. Elucidating the rheological and thermal properties of mixed fish and pork skin gelatin gels: effects of cooling conditions and incubation times. Food Hydrocolloids. 2024;110317. https://doi.org/10.1016/j.foodhyd.2024.110317 42. Cheng Y, Fu Y, Ma L, Yap PL, Losic D, Wang H, Zhang Y. Rheology of edible food inks from 2D/3D/4D printing, and its role in future 5D/6D printing. Food Hydrocolloids. 2022;132:107855. https://doi.org/10.1016/j.foodhyd.2022.107855 43. Kunzler TP, Huwiler C, Drobek T, Vörös J, Spencer ND. Systematic study of osteoblast response to nanotopography by means of nanoparticle-density gradients. Biomaterials. 2007;28(33):5000–5006. https://doi.org/10.1016/j.biomaterials.2007.08.009 44. Kakarla AB, Turek I, Kong C, Irving H. Printable gelatin, alginate and boron nitride nanotubes hydrogel-based ink for 3D bioprinting and tissue engineering applications. Mater Des. 2022;213:110362. https://doi.org/10.1016/j.matdes.2021.110362 45. Carletti E, Motta A, Migliaresi C. Scaffolds for tissue engineering and 3D cell culture. 3D Cell Cult Methods Protoc. 2011;17–39. https://doi.org/10.1007/978-1-60761-984-0_2 46. Zhang Z, Feng Y, Wang L, Liu D, Qin C, Shi Y. A review of preparation methods of porous skin tissue engineering scaffolds. Mater Today Commun. 2022;32:104109. https://doi.org/10.1016/j.mtcomm.2022.104109 47. Ashammakhi N, Ahadian S, Xu C, Montazerian H, Ko H, Nasiri R, Barros N, Khademhosseini A. Bioinks and bioprinting technologies to make heterogeneous and biomimetic tissue constructs. Mater Today Bio. 2019;1:100008. https://doi.org/10.1016/j.mtbio.2019.100008 48. Li Z, Zhang Y, Zhao Y, Gao X, Zhu Z, Mao Y, Qian T. Graded-three-dimensional cell-encapsulating hydrogel as a potential biologic scaffold for disc tissue engineering. Tissue Eng Regen Med. 2022;19(5):1001–1012. https://doi.org/10.1007/s13770-022-00480-2 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Feb, 2026 Reviews received at journal 20 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviews received at journal 10 Feb, 2026 Reviewers agreed at journal 09 Feb, 2026 Reviewers invited by journal 09 Feb, 2026 Editor assigned by journal 04 Feb, 2026 Submission checks completed at journal 03 Feb, 2026 First submitted to journal 02 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8770112","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589617070,"identity":"66a6e04e-719f-4788-ba87-c38bdd5b20b8","order_by":0,"name":"Kyu-Min Kang","email":"","orcid":"","institution":"Kongju National University","correspondingAuthor":false,"prefix":"","firstName":"Kyu-Min","middleName":"","lastName":"Kang","suffix":""},{"id":589617073,"identity":"11a8beff-b4e2-4531-9b31-be1eede98709","order_by":1,"name":"Sol-Hee Lee","email":"","orcid":"","institution":"National Institute of Animal Science","correspondingAuthor":false,"prefix":"","firstName":"Sol-Hee","middleName":"","lastName":"Lee","suffix":""},{"id":589617077,"identity":"7ef1bbc5-5e82-4cb8-9c6c-9ab1c74d9ded","order_by":2,"name":"Hack-Youn Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACCWYGNgaGigQQOwEiQpyWMyRpYQBqYWxLQBHBDyTbudMe885LkzPnX/BMgqHGjkFy9gH8WqSZebcb827LMbac8SBNguFYMoM0XwJ+LXLMvNukebdVJG64cQCohe0AgxwPAYdBtMyBaflHhBZpsJaGnMQN5xvSJBjbDjBIE9Ii2cy7TXLOsTRjgxsMyRaJfck8kj0EtEicP7tN4k1NspzB+TOJNz58s5OTOENACwgwgZ0ikZMAikxCzoIAxh8gkv/4AaJUj4JRMApGwcgDALJqOz2GuoiEAAAAAElFTkSuQmCC","orcid":"","institution":"Kongju National University","correspondingAuthor":true,"prefix":"","firstName":"Hack-Youn","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2026-02-03 02:38:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8770112/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8770112/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102520517,"identity":"1c8fc7c6-0099-4a29-885d-808bf70a28ff","added_by":"auto","created_at":"2026-02-12 14:30:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":661337,"visible":true,"origin":"","legend":"\u003cp\u003eFormulation of 3D bio-ink with various types and ratios of animal-based gelatin powder.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8770112/v1/2541d7f6dd32f80b2d547d1e.png"},{"id":102746499,"identity":"682e8874-6ff9-485a-94dd-c51f727993da","added_by":"auto","created_at":"2026-02-16 08:57:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66161,"visible":true,"origin":"","legend":"\u003cp\u003eHue angle and chroma of 3D bio-ink with various types and ratios of animal-based gelatin powder.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8770112/v1/01f7ea5ad0e805c6943a6447.png"},{"id":102520512,"identity":"57bce8b3-91b8-4c82-96ba-e149b2911506","added_by":"auto","created_at":"2026-02-12 14:30:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101415,"visible":true,"origin":"","legend":"\u003cp\u003eViscosity of 3D bio-ink with various types and ratios of animal-based gelatin powder. (A) Viscosity of the beef gelatin-based 3D bio-ink with various ratios. (B) Viscosity of the pork gelatin-based 3D bio-ink with various ratios. (C) Viscosity of the fish gelatin-based 3D bio-ink with various ratios. (D) Combination of viscosity of animal-based gelatin 3D bio-ink.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8770112/v1/dacd918d97ed1a26a070a0a4.png"},{"id":102520515,"identity":"fb4b1d7a-b06a-48f8-b466-e474b1857fa6","added_by":"auto","created_at":"2026-02-12 14:30:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1686767,"visible":true,"origin":"","legend":"\u003cp\u003ePrintability of 3D bio-ink with various types and ratios of animal-based gelatin powder. (A) Single-layer printing of animal-based gelatin 3D bio-ink. (B) Multiple-layer printing of animal-based gelatin 3D bio-ink.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8770112/v1/f14678644ffd3cc662962f4e.png"},{"id":102520513,"identity":"d12e8d3e-630a-49e1-a3f0-6ba8342e8612","added_by":"auto","created_at":"2026-02-12 14:30:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":215263,"visible":true,"origin":"","legend":"\u003cp\u003eStructure properties of bio-ink with various types and ratios of animal-based gelatin 3D bio-ink. (A) Microphotographs of animal-based gelatin scaffolds. (B) Grid thickness of animal-based gelatin scaffolds. (C) Grid size of animal-based gelatin scaffolds. (D) Gel strength of animal-based gelatin scaffolds. The magnification of all the microphotographs is ×40. \u003csup\u003ea-d\u003c/sup\u003e Means in the bars with different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8770112/v1/32a8abcd0491490d9aff128d.png"},{"id":102747340,"identity":"e0dd00d7-c519-41a9-80e8-67da1f14b59f","added_by":"auto","created_at":"2026-02-16 09:04:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119940,"visible":true,"origin":"","legend":"\u003cp\u003ePearson correlation analysis of 3D bio-ink with various types and concentrations of animal-based gelatin. Right upper section means correlation coefficient, diagonal section means histogram, and left lower section means scatter plot.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8770112/v1/e57deeec88654cbc298520b8.png"},{"id":102750707,"identity":"29a10056-b4c3-4d8e-a75d-6a1e46e04eb7","added_by":"auto","created_at":"2026-02-16 09:21:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3677026,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8770112/v1/717e0780-7af9-4d3f-89e5-6839d0e3992e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physicochemical Properties of Various Types of 3D Bio-ink with Animal-based Gelatin Powder","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe world population exceeded 7.6\u0026nbsp;billion in 2018 and continues to increase annually, with projections estimating it will reach 9.2\u0026nbsp;billion by 2050. This growth is expected to increase food demand by 59\u0026ndash;102% [1]. As food demand rises, global meat consumption is also anticipated to increase annually, reaching 4.55\u0026nbsp;million tons by 2050. Developing countries are expected to account for 56% of feed grain consumption [2]. Consequently, while meat production is increasing worldwide, the need for alternative meat sources is also growing due to various factors such as environmental concerns, animal welfare, and health issues [3]. Among these alternatives, cultured meat, produced by cultivating livestock cells, stands out in the meat industry due to its potential to conserve finite resources and minimize the killing of animals [4].\u003c/p\u003e \u003cp\u003eCultured meat produced using 3D bio-printing has recently garnered attention because it can be manufactured in various shapes that resemble or exceed the quality of traditional meat. In contrast, conventional cultured meat, which uses only existing cells, has been limited to forms such as patties [5]. Additionally, by optimizing the cell growth environment, production time can be shortened, raw material usage minimized, and cost-effectiveness improved [6]. Furthermore, 3D bio-printing allows for the development of customized meat that can be tailored to individual consumers' dietary preferences or health needs [7]. These 3D bio-printed cultured meats are produced based on bio-ink, which is made from various raw materials such as alginate and gelatin. The quality of the final product is determined by the characteristics of these raw materials [8].\u003c/p\u003e \u003cp\u003eGelatin, an animal-based raw material, plays a crucial role in the production process of cultured meat as it is necessary for cell attachment and proliferation [9]. Gelatin is a degradation product of collagen, exhibiting excellent biocompatibility and suitability for providing a conducive cellular environment, making it widely used in bio-ink for 3D bio-printing [10]. Furthermore, gelatin promotes cell growth and differentiation during cultured meat production, facilitating more natural and efficient tissue formation [11]. Although gelatin shares similar properties, its characteristics may vary depending on its source. Therefore, it is essential to compare and analyze gelatin from different sources [12].\u003c/p\u003e \u003cp\u003eThis study aims to manufacture various animal gelatin bio-inks using bovine, porcine, and fish gelatin powder and to analyze the qualitative and mechanical properties of each gelatin type to identify the appropriate ratio for scaffold production.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of animal-based gelatin bio-ink\u003c/h2\u003e \u003cp\u003eBeef (Holstein, Bos taurus taurus; molecular weight, 50\u0026ndash;100 kDa), pork (LYD, Landrace\u0026times;Yorkshire\u0026times;Duroc; molecular weight, 50\u0026ndash;100 kDa), and fish (Salmon, Oncorhynchus keta; molecular weight, 10\u0026ndash;80 kDa) skin gelatin powder were obtained from Pureundeulpan Co., Ltd. (Seoul, Korea). The animal-based gelatin (AG) was prepared by dissolving animal-based gelatin powders in distilled water (DW) at a concentration of 4, 12, 20, 32, and 40% (w/v) at 45℃. And the sodium alginate (SA) was prepared by dissolving sodium alginate powder (Sigma-Aldrich, St. Louis, MO, USA) in DW at a concentration of 6% (w/v) at 70℃. As shown in Fig.\u0026nbsp;7, each concentration of AG was mixed with SA with a 1:1 ratio filled into a printing cartridge (Cellink, G\u0026ouml;teborg, Sweden), and stored at 4℃ for the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 pH\u003c/h2\u003e \u003cp\u003eFour grams of sample was mixed with 16 mL of distilled water using Ultra Turrax homogenizer (HMZ-20DN, Poonglim Tech, Seongnam, Korea) for 1 min at 6991 хg. The pH was measured using a glass electrode pH meter (Model S220, Mettler-Toledo, Schwerzenbach, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Color\u003c/h2\u003e \u003cp\u003eThe surfaces of the samples were randomly evaluated using a colorimeter, adjusted to operate with an aperture of 8 mm, 2\u0026deg; standard observer, illuminant D65, and pulsed xenon lamp as a default light source. Before measuring, the device was calibrated with a white plate, CIE L*: +97.83, CIE a*: -0.43, and CIE b*: +1.98 (CR-10, Minolta, Tokyo, Japan), and the lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) were recorded. Hue angle and chroma values were calculated using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{Hue\\:angle=}{\\text{tan}}^{\\text{-1}}{\\text{b}}^{\\text{*}}\\text{/}{\\text{a}}^{\\text{*}}\\text{,\\:Chroma=}{\\left({\\text{a}}^{\\text{*2}}\\text{+}{\\text{b}}^{\\text{*2}}\\right)}^{\\frac{\\text{1}}{\\text{2}}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Viscosity\u003c/h2\u003e \u003cp\u003eThe samples' viscosity (i.e., the flow behavior) and time dependency were measured using a viscometer (Merlin VR, Rheosys, Hamilton Township, NJ, USA). The viscometer was attached to a 30-mm cone, and the sample was placed in a 25-mm coaxial cylinder. The viscometer conditions were as follows: measurement time 30 s at 10 ℃ with a head speed of 20 rpm. The values are presented in Pa\u0026sdot;s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Printability\u003c/h2\u003e \u003cp\u003eThe printability was performed using a 3D bio-printer (BIO-X, Cellink) with a motorized Z-axis. The bio-inks were printed with a square shape, 20% infill density, 1 \u0026times; 1 cm size, and varying heights (0.1, 0.3, and 0.5 cm). The printing cartridge filled with samples was attached with sterile high-precision conical bioprinting nozzles (27G-200 \u0026micro;m) and installed in a 3D bio-printer. The installed printing cartridge was extruded and the forms were observed. The printing conditions were as follows: print-bed of 10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃, print speed of 5 mm/s, print temperature 30\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃, print pressure 150\u0026thinsp;\u0026plusmn;\u0026thinsp;1 kPa. After printing, the printed structures were dipped into a 100 mM CaCl\u003csub\u003e2\u003c/sub\u003e for ionic cross-linking and the forms were observed again.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Microphotograph, Grid thickness, and Pore size\u003c/h2\u003e \u003cp\u003eThe microphotograph of the samples was observed and scanned using an Eclipse Ci-L microscope (Nikon, Tokyo, Japan) with a \u0026times; 40 scope, and grid thickness and pore size of the samples were determined using NIS-Elements imaging software (Nikon, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Gel strength\u003c/h2\u003e \u003cp\u003eThe gel strength of the samples was determined using the method by Park and Kim [13] with slight modifications. The samples (1 \u0026times; 1 \u0026times; 0.3 cm) were stored at 10 ℃ for 24 h and then used for experiment. The gel strength of the prepared samples was measured using a texture analyzer (TA 1, Ametek Inc., Berwyn, PA, USA) at room temperature, with the following settings: a cylinder probe of 10 cm with a test speed of 2.0 mm/s, distance of 8.0 mm, and a force of 5.6 N. The measured values were expressed in gf.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data in this study were analyzed after a minimum of three repeated trials and the results are presented as mean, standard deviation (SD). The statistical analyses of pH, color, printability, grid thickness, grid size, and gel strength were verified using one-way analysis of variance (ANOVA) and Tukey\u0026rsquo;s multiple comparisons test in the GraphPad Prism 10 for windows (version 10.4.0; GraphPad Prism Software Inc., San Diego, CA, USA) at a significant level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The Pearson correlation analysis was performed using matrix of heat-map and scatter plot in the R-studio for windows (version 4.4.2; RStudio Inc., Boston, MA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 pH\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the pH of bio-ink with various types and ratios of animal-based gelatin. As the ratio of gelatin increased, the pH tended to decrease in all gelatin types. When collagen breaks down into gelatin, the presence of acidic amino acids, ionization reactions, and the structural properties of gelatin all contribute to a slightly acidic pH [14]. Accordingly, as the concentration of gelatin increases, the content of acidic amino acids also rises, leading to an increase in hydrogen ion concentration and resulting in weak acidity [15]. Notably, all fish gelatin samples exhibited significantly higher pH values compared to samples from other gelatin types (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The observed differences in pH depending on the type of gelatin are thought to be influenced by the vitamins, minerals, and amino acids present in the livestock species from which the gelatin was derived [16]. Specifically, beef and pork gelatin are believed to have lower pH values than fish gelatin due to their higher content of acidic amino acids such as aspartic acid and glutamic acid [17]. Furthermore, because higher pH levels are associated with increased metabolic activity in cells, bio-ink with elevated pH is considered suitable for use as scaffold material in the production of cultured meat [18].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003epH of 3D bio-ink with various types and ratios of animal-based gelatin powder\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eTraits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eGelatin (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eaC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ebC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ebC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePork\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ebcB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ecB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003edB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFish\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eAll values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u0026minus;c\u003c/sup\u003e Means in the same row with different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eA\u0026minus;C\u003c/sup\u003e Means in the same column with different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Color\u003c/h2\u003e \u003cp\u003eThe color results of bio-ink with various types and ratios of animal-based gelatin are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In the lightness (CIE L*), 4% gelatin samples were significantly higher than the other gelatin ratio samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). And as the ratio of gelatin increased, the lightness tended to decrease in all gelatin types. In the redness (CIE a*) as the ratio of gelatin increased, the redness tended to increase in all gelatin types. And fish gelatin samples showed the lowest values in all gelatin ratios. In the yellowness (CIE b*), beef gelatin samples showed significantly higher values than the other gelatin type samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). And as the ratio of gelatin increased, the yellowness tended to increase in all gelatin types. Sinthusamran et al. [19] reported that the lightness decreased and the redness and yellowness increased as the gelatin concentration increased in all bio-inks made from beef, pork, and fish gelatin, showing similar results to this study. This is because as the concentration of gelatin increases, the degree to which the particles in the bio-ink scatter light changes, which affects the brightness, and as the concentration of the pigment contained increases, more light of a specific wavelength is absorbed, which increases the intensity of the color [20]. In addition, gelatin is a protein derived from collagen, and as the concentration increases, the intermolecular interaction is strengthened to form a photonic structure, and this structural change affects the transmission and scattering of light, thereby affecting color [21]. Hanani et al. [22] reported that the color difference of the final product by the type of gelatin is greatly affected by the color of the gelatin powder itself used, and that bovine gelatin powder has a higher yellowness than other gelatin powders, resulting in a greater color difference. In addition, during the powderization process of gelatin, it goes through a drying process, during which it reacts with oxygen in the atmosphere to cause the Maillard reaction, which increases the yellowness of the gelatin powder [23]. In particular, beef gelatin has a higher content of amino acids that are sensitive to the Maillard reaction, such as lysine, than gelatin from other livestock species, resulting in higher yellowness and redness values [24]. Since the yellowness of the beef gelatin powder used in this study can be seen to be high even with the naked eye, it is judged that it affected the redness and yellowness of the final bio-ink produced. In hue angle and chroma, 20%, 32%, and 40% beef gelatin samples showed different chroma values than the other samples. Choma represents a relative color effect based on the brightest point in the color space, and it was reported that as the concentration of gelatin increases, the saturation also increases, showing similar results to this study [25]. In the color of gelatin, Chroma is positively correlated with redness, and chroma tends to increase mainly as redness increases [26]. And since chroma is also affected by the Maillard reaction, a non-enzymatic browning reaction between amino acids and reducing sugars, it is thought that the chroma value differs depending on the type of extracted gelatin powder or extraction method [27]. Although the color of gelatin used in bio-ink does not significantly affect the functional properties, it does have an aesthetic impact on consumers, so it is thought that gelatin with low yellowness or redness would be advantageous in order to impart a color similar to meat [28].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eColor of 3D bio-ink with various types and ratios of animal-based gelatin powder\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eTraits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eGelatin (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eL\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ebC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e53.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePork\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ecB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003edC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eeB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFish\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003edA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e53.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eeA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ea\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003edA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003edA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePork\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ecB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFish\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ecB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ebC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eb\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003eeA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003edA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePork\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eeB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003edB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ecB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFish\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003edC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ebC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eAll values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u0026minus;e\u003c/sup\u003e Means in the same row with different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003eA\u0026minus;C\u003c/sup\u003e Means in the same column with different letters are significantly different (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Viscosity\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the viscosity of bio-ink with various types and ratios of animal-based gelatin. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, as the gelatin ratio increased, the initial viscosities were increased in all gelatin types. Also pork gelatin samples showed the highest initial viscosities in all gelatin ratios. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the viscosities of all ratio beef and fish gelatin samples tended to increase as the time increased. This is believed to be affected by the structure, amino acid composition, intermolecular bonding, polydispersity, and thermal stability of the gelatin used in bio-ink production, and among them, the differences between gelatin types are believed to be mainly due to the amino acid composition and intermolecular bonding [29,30]. Compared to gelatin extracted from warm-blooded animals, fish gelatin has a looser structure and weaker intermolecular bonds, resulting in a relatively low initial viscosity and maintaining low viscosity even at high concentrations [31]. On the other hand, pork gelatin has higher structural integrity of β- and γ-chains than beef and fish gelatin, which causes physical constraints and entanglements that impede the flow of the polymer, resulting in a high initial viscosity [32]. In terms of amino acid composition, the content of proline and hydroxyproline, which are the main factors in the gelation and melting point of gelatin, is important. In the case of fish gelatin, the content is lower than that of beef and pork, so it is sensitive to heat and dissolves easily, making it difficult to increase initial viscosity even at high concentrations [33]. However, in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, 32% and 40% pork gelatin samples tended to decrease as the time increased. This is thought to be because as the gelatin concentration became very high, intermolecular interactions occurred excessively, preventing the formation of a stable gel network [34]. Especially, under high concentration conditions such as 32% and 40%, the internal stress increases, and the structural stability is not maintained, so the viscosity decreases over time [35]. The higher the concentration of the gelatin solution, the more non-Newtonian the flow characteristics become, and the viscosity changes with external force or over time. At this time, the flow and molecular motion of the solution are restricted, so the gel structure is easily destroyed [36]. Therefore, considering the viscosity results, it is thought that it would be appropriate to use less than 32% of porcine gelatin when manufacturing bioink.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Printability\u003c/h2\u003e \u003cp\u003eThe printability results of bio-ink with various types and ratios of animal-based gelatin are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, 4% (beef, pork, and fish) gelatin, 12% (beef and fish) gelatin, and 20% (beef, pork, and fish) gelatin could not form the structure. And 32% and 40% pork gelatin samples could not print the structure. This is thought to be due to the molecular weight and concentration that affect the physical (hydrogen bonding and van der Waals) bonding involved in the gelatin gelation process [37]. Van der Waals forces are attractive or repulsive forces between molecules, and are very weak bonds to form a gel structure, so low-concentration gelatin has difficulty forming a gel structure because strong bonds such as hydrogen bonds are necessary to maintain the gel structure [38]. On the other hand, in the case of high-concentration gelatin, the elastic restoring force is strong due to strong hydrogen bonds, so the property to resist external stimuli is increased, so it has properties closer to solid rather than flow characteristics [39]. As a result, high extrusion pressure and large-gauge tips are used during the printing process, which reduces the advantages of 3D printing [40]. Also shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, only pork gelatin samples can form the scaffold structure up to 0.5 cm in height. Pork gelatin has a higher content of amino acids, such as proline and hydroxyproline, than bovine or fish gelatin. These amino acids can form strong hydrogen bonds between gelatin molecules to form a stable three-dimensional network structure [41]. This stable three-dimensional network structure exhibits high internal binding strength against gravity resistance [42]. Therefore, considering the printability results, it is expected that using porcine gelatin will enable more stable printing of bio-ink.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Structure properties\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the structure properties of bio-ink with various types and ratios of animal-based gelatin. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, pork gelatin samples showed rough surface than the other gelatin type samples. A rough surface can improve cell adhesion by providing microscopic grooves and protrusions on which cells can adhere and strengthening cell-support interactions [43]. And in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, 32% and 40% fish gelatin sample showed significantly higher grid thickness than the other samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Such a high lattice thickness can enhance structural stability, but an excessively thick structure may have limitations in accuracy [44]. Therefore, fish gelatin can provide structural stability by providing a thick lattice at high concentrations, but excessive thickness may act as a disadvantage in certain applications. However, in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, 20% pork gelatin samples showed significantly higher grid size and gel strength than the other samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicates the possibility that cell density may decrease due to a decrease in the total area to which cells can attach, and the surface area to which cells can attach may be limited in the early stages of culture [45]. However, the large grid size can create an environment favorable for intercellular material exchange and nutrient supply, providing an advantage in that cells can be uniformly distributed throughout the entire structure in the mid- to late-stage culture as cell proliferation and growth progress [46]. The results of the structure properties are thought to be influenced by the same mechanism as the results of the printability, and it is thought that the strength of the hydrogen bond between molecules affects the density of gelatin, resulting in differences in the structure properties depending on the type and concentration of gelatin [41].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Pearson correlation analysis heatmap\u003c/h2\u003e \u003cp\u003eThe pearson correlation analysis heatmap of bio-ink with various types and ratios of animal-based gelatin is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Gel strength and viscosity have a correlation coefficient of 0.88, showing a strong positive correlation. This means that as the gelatin concentration increases or the intermolecular bonds become stronger, the gel strength and viscosity increase together. In bioink, the higher the gel strength and viscosity, the greater the structural stability, so when used as a cell support, the better the resistance to external pressure, which can provide an environment in which cells can stably attach and grow [47]. And grid size and gel strength also show a strong positive correlation (correlation coefficient 0.89). This suggests that gel strength tends to increase as the lattice size increases. Large grid sizes can provide a favorable environment for cell culture by increasing the space for cells to attach and expand, and increasing gel strength plays an important role in maintaining this structure [48]. In particular, the combination of lattice size and gel strength as a cultured meat support is important in that it can facilitate cell distribution and material exchange while ensuring the stability of the three-dimensional structure [41]. The correlation coefficient between CIE a\u003csup\u003e*\u003c/sup\u003e and CIE b\u003csup\u003e*\u003c/sup\u003e is 0.99, showing a very high positive correlation. This suggests that color changes can occur while maintaining the color properties of the bioink uniformly, and that it is possible to maintain a specific color tone depending on the concentration and type of gelatin. This color consistency can serve as an important factor in the appearance quality control of commercial cultured meat [28].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we analyzed the physical and chemical properties of bio-ink formulated with various types and ratios of animal gelatin. The pH analysis revealed that as the gelatin ratio increased, the pH decreased, with fish gelatin samples exhibiting the highest pH values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Color measurements indicated that the lightness (CIE L*) of the 4% gelatin sample was significantly higher than that of other ratios (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while redness (CIE a*) increased with higher gelatin ratios. Yellowness (CIE b*) was notably higher in the beef gelatin samples compared to others (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Viscosity measurements demonstrated that initial viscosity increased with gelatin concentration, with pork gelatin exhibiting the highest viscosity among the samples. Printability tests indicated that the 4%, 12%, and 20% gelatin samples could not form stable structures; however, only the pork gelatin was able to create scaffolds up to 0.5 cm in height. Structural characterization showed that pork gelatin samples possessed a rough surface, and the 20% pork gelatin sample had significantly larger lattice size and gel strength (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, Pearson correlation analysis revealed a strong positive correlation between gel strength and viscosity (correlation coefficient 0.88) and a strong positive correlation between lattice size and gel strength (correlation coefficient 0.89). In conclusion, the type and ratio of gelatin significantly influence the pH, color, viscosity, printability, and structural properties of bio-ink. Given the favorable formation of stable structures, the use of porcine gelatin is deemed suitable for bio-ink production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding statement\u003c/h2\u003e \u003cp\u003eThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2024\u0026ndash;00460285). This work was also supported by the National University Development Project of the Ministry of Education, Korea in 2025.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualisation was conducted by Kang KM and Lee SH. Data curation and formal analysis were performed by Kang KM. Funding acquisition and resources were provided by Lee SH and Kim HY. Investigation was carried out by Kang KM. Methodology was developed by Kang KM and Kim HY. Project administration was managed by Kang KM. Software was prepared by Kang KM. Supervision and validation were provided by Kang KM and Kim HY. Visualisation was undertaken by Kang KM. The original draft of the manuscript was written by Kang KM. Kang KM, SH Lee, and Kim HY contributed to review and editing of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e1. Godara AS, Saresh NV, Bijarnia AL, Godara RS, Kumar D, Meena D, Kumar M. Integrating livestock with crops and forestry for sustainability. Int J Environ Clim Change. 2024;14(11):83\u0026ndash;90. https://doi.org/10.9734/ijecc/2024/v14i114530\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e2. 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Mater Today Commun. 2022;32:104109. https://doi.org/10.1016/j.mtcomm.2022.104109\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e47. Ashammakhi N, Ahadian S, Xu C, Montazerian H, Ko H, Nasiri R, Barros N, Khademhosseini A. Bioinks and bioprinting technologies to make heterogeneous and biomimetic tissue constructs. Mater Today Bio. 2019;1:100008. https://doi.org/10.1016/j.mtbio.2019.100008\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e48. Li Z, Zhang Y, Zhao Y, Gao X, Zhu Z, Mao Y, Qian T. Graded-three-dimensional cell-encapsulating hydrogel as a potential biologic scaffold for disc tissue engineering. Tissue Eng Regen Med. 2022;19(5):1001\u0026ndash;1012. https://doi.org/10.1007/s13770-022-00480-2\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Animal-based, Gelatin powder, Bio-ink, Physicochemical properties, 3D bio-print","lastPublishedDoi":"10.21203/rs.3.rs-8770112/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8770112/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we analyzed the physical and chemical properties of bio-ink prepared using various types and ratios of animal-based gelatin. The pH analysis revealed that as the gelatin ratio increased, the pH decreased, with fish gelatin samples showing the highest pH values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The color measurements indicated that the lightness (CIE L*) of the 4% gelatin sample was higher than that of the other ratios (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while redness (CIE a*) increased with higher gelatin ratios, and yellowness (CIE b*) was significantly higher in beef gelatin samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Viscosity measurements showed that initial viscosity increased with higher gelatin ratios, with pork gelatin exhibiting the highest viscosity. Printability results demonstrated that 4%, 12%, and 20% gelatin samples could not form structures; however, only pork gelatin formed scaffolds up to 0.5 cm in height. Structural property analysis revealed that pork gelatin samples had rough surfaces, and the 20% pork gelatin sample showed significantly higher grid size and gel strength (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Pearson correlation analysis indicated a strong positive correlation between gel strength and viscosity (correlation coefficient 0.88) as well as between grid size and gel strength (correlation coefficient 0.89). In conclusion, the type and ratio of gelatin significantly influence the pH, color, viscosity, printability, and structural properties of bio-ink, suggesting that using pork gelatin powder is suitable for 3D bio-ink production considering stable structure formation.\u003c/p\u003e","manuscriptTitle":"Physicochemical Properties of Various Types of 3D Bio-ink with Animal-based Gelatin Powder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-12 14:30:28","doi":"10.21203/rs.3.rs-8770112/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-24T03:04:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T03:04:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312399034716418455072040300133745850061","date":"2026-02-11T12:35:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-10T07:18:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150568459205277862955121422890958278026","date":"2026-02-10T03:08:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T05:19:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-04T05:04:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-03T09:07:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Science of Animal Resources","date":"2026-02-03T02:23:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-science-of-animal-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)","snPcode":"44463","submissionUrl":"https://submission.springernature.com/new-submission/44463/3?","title":"Food Science of Animal Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c02821ff-c370-462f-985c-298f50ae262d","owner":[],"postedDate":"February 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T01:09:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-12 14:30:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8770112","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8770112","identity":"rs-8770112","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}