Received: 06/03/2025 Accepted: 30/04/2025 Published: 30/05/2025 1 of 7 https://doi.org/10.52973/rcfcv-e35646 Revista Cientíﬁca, FCV-LUZ / Vol. XXXV ABSTRACT Fish processing produces signiﬁcant amounts of by–products. The disposal of these wastes can lead to environmental and human health problems; however, these by–products can often be transformed into high–value and beneﬁcial products. This study investigated the physicochemical properties of gelatin extracted from the heads and skins of rainbow trout (Oncorhynchus mykiss). The yield of gelatin obtained from the heads and skins were 6.17 and 8.46%, respectively. The protein content of gelatin obtained from the skin (84.68%) was greater than that obtained from the head (81.9%) (P<0.05). Both gelatins exhibited slightly acidic pH values (6.42 for heads and 6.20 for skins). FTIR analysis conﬁrmed typical collagen–specific spectral features in both samples. The gelatin obtained from the skin exhibited a lighter and more reddish–yellow color compared to the gelatin obtained from the head (P<0.05). The sensory analysis results indicated that both gelatin samples had a mild odor; however, the gelatin obtained from the head exhibited a easily more perceptible odor compared to that extracted from the skin (P<0.05). These ﬁndings suggest that the heads and skins of rainbow trout are appropriate sources for gelatin production and support the sustainable use of wastes from ﬁsh processing. Key words: Gelatin; trout by–products; physicochemical properties; FTIR analysis; sustainability RESUMEN El procesamiento del pescado produce cantidades signiﬁcativas de subproductos. La disposición de estos desechos puede provocar problemas ambientales y de salud humana; sin embargo, estos subproductos a menudo pueden ser transformados en productos mas beneficiosos y de alto valor. Este estudio investigó las propiedades ﬁsicoquímicas de la gelatina obtenida de las cabezas y pieles de la trucha arcoíris (Oncorhynchus mykiss). El rendimiento de gelatina obtenida de las cabezas y pieles fue del 6,17 y 8,46 %, respectivamente. El contenido de proteína en la gelatina obtenida de la piel (84,68 %) fue mayor que el de la gelatina obtenida de la cabeza (81,9 %) (P<0,05). Ambas gelatinas exhibieron valores de pH ligeramente ácidos (6,42 para las cabezas y 6,20 para las pieles). El análisis FTIR conﬁrmó las características espectrales típicas especíﬁcas del colágeno en ambas muestras. La gelatina obtenida de la piel exhibió un color más claro y más rojizo– amarillo en comparación con la gelatina obtenida de la cabeza (P<0,05). Los resultados del análisis sensorial indicaron que ambas muestras de gelatina tenían un olor suave; sin embargo, la gelatina obtenida de la cabeza exhibió un olor fácilmente más perceptible en comparación con el derivado de la piel (P<0,05). Estos resultados indican que las cabezas y pieles de trucha arco íris son fuentes adecuadas para la producción de gelatina y promueven la utilización sostenible de los subproductos generados durante el procesamiento del pescado. Palabras clave: Gelatina; subproductos de trucha; propiedades ﬁsicoquímicas; análisis FTIR; sostenibilidad Collagen extraction from rainbow (Oncorhynchus mykiss) trout heads and skins Extracción de colágeno de cabezas y pieles de trucha arcoíris (Oncorhynchus mykiss) Muhsine Duman Fırat University, Faculty of Fisheries, Department of Fish Processing Technology. Elazig, Türkiye. Corresponding author: mduman@ﬁrat.edu.tr

Collagen from trout heads and skins / Duman __________________________________________________________________________________________ 2 of 7 INTRODUCTION Global fish production, especially aquaculture, has shown signiﬁcant growth in recent years. In parallel with this increase, the ﬁsh processing industry is also expanding, and by–products from processed ﬁsh are rapidly accumulating [1]. Among these, rainbow trout, which is a high–value and widely consumed aquatic species, produces large amounts of waste during processing. These wastes are generally composed of heads, bones, skins, and other body parts. These by–products, which are often of low economic value, also pose an environmental issue [2, 3]. Lately, investigations into the potential value of ﬁsh processing waste has signiﬁcantly increased. These studies have demonstrated that ﬁsh wastes can be transformed into high quality products through biotechnological processes. In particular, collagen, one of the biologically signiﬁcant compounds that can be obtained from these wastes, has emerged as an important source from an industrial perspective [4, 5, 6, 7, 8, 9, 10, 11, 12, 13]. Gelatin is a biopolymer protein obtained through the partial hydrolysis of collagen, a basic component of animal bone, connective tissue and skin, and has a wide range of uses in many areas, especially in the food, pharmaceutical and photography industries [14, 15]. Gelatin is traditionally sourced from the skin and bones of pigs and cattle. However, due to the rising demand for halal food and concerns about health risks, there has been growing interest in alternative gelatin sources derived from seafood by–products. Fish waste is a highly abundant source of collagen and has the potential to yield high–quality gelatin production. Gelatin obtained from ﬁsh is more soluble and has a wider range of applications than gelatin obtained from traditional sources such as pigs and cattle [16, 17, 18]. Rainbow trout (Oncorhynchus mykiss) is an important freshwater ﬁsh species both economically and ecologically, and holds a signiﬁcant place in aquaculture. Due to its rapid growth rate, high protein content, and excellent taste, rainbow trout has become an attractive option for both consumers and producers. Rainbow trout accounted for 744,000 tons of total aquaculture production in 2021. The leading exporting countries are Türkiye, Chile, and Norway [19]. According to the 2023 data, trout production in Türkiye, which was 134.174 thousand tons in 2021, has exceeded 200 thousand tons [20]. However, this level of production results in the generation of a signiﬁcant amount of ﬁsh by–products during processing. It has been determined that about 35% of the total amount of ﬁsh is wasted [19, 21]. Several studies have been conducted on gelatin production from rainbow trout skins [10, 22, 23]. However, based on the available literature, no research have been reported on gelatin production from rainbow trout heads. Therefore, this study aims to assess the efﬁciency and quality of gelatin extracted from rainbow trout heads and skins, as well as to explore the potential of converting trout processing waste into valuable products. In this context, gelatin was extracted from raw materials, and its chemical composition, pH value, color, turbidity, FTIR spectroscopy, and sensory properties were analyzed. The study seeks to contribute to the sustainable utilization of ﬁsh processing waste by transforming it into high–value products. MATERIALS AND METHODS Raw materials Rainbow trout, weighing an average of 713.285 ± 197 g, were obtained from domestic markets and transported to the laboratory. The ﬁsh samples were placed in ice boxes and transferred to the laboratory within 1 hour (h). On the same day (d), the heads and skins of the ﬁsh were manually removed using appropriate knives. The weight measurements were conducted using a KERN CB 6K 1N digital scale (Germany). The ﬁsh heads and skins were washed with cold water, placed into plastic bags, and stored in a deep freezer at -18 ± 2°C (Arçelik 5287 NFI, Türkiye). Extraction of gelatin from ﬁsh heads and skins Gelatin was extracted from ﬁsh heads and skins using a slightly modiﬁed version of the method described by Boran and Regenstein [24]. Initially, frozen ﬁsh heads and skins were thawed under running tap water. Subsequently, both materials were manually cut into small pieces and processed separately in a household food processor (Arçelik, Robomaster, Türkiye) until a homogeneous paste was obtained. The resulting paste was mixed with drinking water at a paste–to–water ratio of 1:3 and agitated for 15 min to remove water–soluble proteins.The mixture was then ﬁltered through a nylon sieve to separate the liquid from the solid components. The remaining solid mass was washed with a 0.55 N NaOH solution for 30 min, with the NaOH solution replaced every 3 h. After these treatments, the material was rinsed with distilled water and added to a 0.1 N hydrochloric acid (HCl) solution at a 1:5 (w/v) ration, then left at 25 ± 1 C for 1.5 h. Following the acidic and alkaline washings, the materials were washed three times with pure water at a ratio of 5:1 (w/v) and ﬁltered through two layers of muslin cloth, followed by manual squeezing. Finally, the materials were subjected to extraction in a water bath at 50°C for 3 h at a ratio of 4:1 (water:head), until a gelatin solution was obtained. The solution was then ﬁltered again using a double layer of muslin cloth and subsequently dried in an oven at 60°C for approximately 72 h. Once dried, the gelatin was carefully removed from the containers and ground to a ﬁne powder using a household blender. The resulting gelatin was placed in plastic storage containers and stored in desiccators to protect it from moisture [5]. These procedures were applied in the same manner for both ﬁsh heads and skins. Gelatin yield determination Gelatin yield was determined as the ratio of the dry weight of the obtained gelatin to the weight of the unprocessed raw material (ﬁsh heads or skins). This calculation was performed using the following formula: Proximate analysis The proximate analysis of the raw samples (heads and skins) and the extracted gelatin was performed by determining moisture, ash,

_________________________________________________________________________________________________Revista Cientiﬁca, FCV-LUZ / Vol.XXXV 3 of 7 and protein content, following the AOAC [25] methods. Moisture content was measured by drying the samples in an oven (FN–500, Nuve, Türkiye) at 105 ± 2°C until a constant weight was achieved. The ash content was measured by subjecting the samples to incineration in a muffle furnace (M 1813 model, Elektro–Mag, Türkiye) at 550°C for 4 h. The protein content was calculated by measuring the total nitrogen content using the Kjeldahl method. This process was carried out using a Kjeldahl digestion and distillation apparatus (Gerhardt Vap–40, Germany). The protein content of the raw materials was calculated by multiplying the nitrogen values by a conversion factor of 6.25, while the protein content of the gelatin was determined using a conversion factor of 5.55 [26]. The crude fat content was determined using the Soxhlet extraction method with an SER 148 (Velp Scientiﬁca, Italy). Each analysis was carried out in triplicate. pH The pH value of the gelatin was measured by preparing a 6.67% (w/w) gelatin solution. The pH measurements were taken at room temperature using a Thermo Scientiﬁc Orion 3–Star Benchtop model pH meter (Thermo Scientiﬁc, USA), with ﬁve independent measurements taken for each sample. FTIR spectroscopy analysis Fourier Transform Infrared (FTIR) spectroscopy was performed to identify the chemical components of the samples. The analyses were carried out using a Thermo Scientiﬁc Nicolet IS5 FT–IR spectrometer (Thermo Nicolet Corporation, USA) within the spectral range of 4000–450 cm - ¹. Color and turbidity analysis The color of the gelatin samples was analyzed using a 3nh NR200 portable colorimeter (3nh Ltd., Shenzhen, China). Color measurements were obtained based on the L* (lightness, ranging from 0 for black to 100 for white), a* (redness/greenness), and b* (yellowness/blueness) parameters. Each sample was measured by collecting color data from three distinct points at room temperature. Turbidity analysis was performed using a 6.67% (w/v) gelatin solution. For the preparation of the solution, 6.67 g of gelatin powder was added to distilled water, and the total volume was adjusted to 100 mL with additional distilled water. The mixture was then stirred in a water bath at 60°C for 30 min to ensure complete dissolution and achieve a homogeneous solution. The turbidity of the resulting gelatin solution was determined at a wavelength of 620 nm using a UV–VIS spectrophotometer (UV mini-1240, Shimadzu, Japan) [27]. Sensory evaluation The sensory evaluation was carried out by a panel of 10 individuals capable of perceiving and describing odors. Initially, 6.67% gelatin solutions were prepared in screw–capped tubes, and the caps of the tubes were loosely closed to allow dissolution in a water bath at 60°C. Once the dissolution process was ﬁnished, the samples were presented to the panelists, who were asked to open the screw caps of the test tubes, smell the contents, and describe the odor perceived. Additionally, the panelists were asked to evaluate the intensity of the odor using a six–point scale. This scale was deﬁned based on the perceivability of the odor as follows: 5 = very strong and very unpleasant, 4 = strong and unpleasant, 3 = strong but not unpleasant, 2 = faint but easily perceptible, 1 = very faint and perceptible only with careful attention, 0 = no odor [28]. Statistical analysis Data analysis was performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Differences between gelatin samples obtained from heads and skins were evaluated using the independent samples t–test. The statistical signiﬁcance level was set at P<0.05, and the data are presented as mean ± standard deviation (SD). All analyses were conducted in triplicate. RESULTS AND DISCUSSION The gelatin extraction from the heads and skins of rainbow trout was conducted following the same protocol. The yields of the obtained gelatins are given in TABLE I. The gelatin yield was expressed as the percentage of dry gelatin obtained per 100 g of raw material. The gelatin yield obtained from trout heads was found to be 6.17%, while the yield from trout skin was 8.46% (P<0.05). These yields differ from the results reported for other ﬁsh species in the literature. For example, Muyonga et al. [28] stated gelatin yields of 2.4 and 16% from the bones and skin of adult Nile perch, respectively. Ninan et al. [29] found gelatin yields of 12 and 12.93% from the skin of common carp and rohu, respectively. Elavarasan et al. [5] documented a gelatin yield of 1.67% from Tiger Tooth Croaker ﬁsh head waste. Additionally, Valcarcel et al. [10] determined gelatin yields from the skin of rainbow trouta, seabass and gilthead seabream as 1.56, 6.83 and 1.56%, respectively. These differences can be attributed to the biological characteristics of the ﬁsh species used, as well as variations in extraction methods and processing conditions. The proximate composition of rainbow trout head, skin, and the gelatin obtained from these by–products, along with the yield of the gelatin, are presented in TABLE I. The protein content of ﬁsh heads was found to be approximately 21.36%, while the protein content in the skin was approximately 23.52%. The protein content in the heads was lower than in the skins, and additionally, the heads exhibited higher ash and moisture content compared to the skins. The fat content in the skins was higher than in the heads, which is likely related to the accumulation of subcutaneous fat in the ﬁsh. The ﬁsh heads also contained a higher ash content TABLE I Proximate compositions and yield of raw and gelatin extracted from rainbow Trout heads and skins Component Head Skin Head Gelatin Skin Gelatin Moisture (%) 59.98 ± 2.35 A 55.81 ± 2.21 A 9.86 ± 0.46 a 7.85 ± 0.23 b Protein (%) 21.36 ± 1.41 A 23.52 ± 1.78 A 81.9 ± 2.26 a 84.68 ± 2.63 b Fat (%) 5.49 ± 0.91 A 7.68 ± 2.53 B 3.58 ± 0.42 a 3.85 ± 0.81 a Ash (%) 4.74 ± 0.18 A 1.24 ± 0.03 B 0.53 ± 0.43 a 0.45 ± 0.2 b Yield (%) 11.29 ± 1.34 A 9.53 ± 0.83 A 6.17 ± 0.70 a 8.46 ± 0.64 b The data are means ± SD. Diﬀerent uppercase letters (A–B) in the same row indicate a signiﬁcant diﬀerence (P<0.05) for ﬁsh head and skin, while diﬀerent lowercase letters (a–b) indicate a signiﬁcant diﬀerence (P<0.05) for gelatin obtained from the head and skin

Collagen from trout heads and skins / Duman __________________________________________________________________________________________ 4 of 7 than the skins. The proximate composition of ﬁsh heads and skins varies signiﬁcantly among different ﬁsh species. These results are consistent with the proximate composition of Atlantic salmon processing by–products [30] and rainbow trout heads [31]. These ﬁndings indicate that ﬁsh heads and skins possess distinct biochemical compositions and suggest that both by–products hold potential as valuable resources for the food industry. The proximate composition of gelatin was shown to differ based on the type of raw material used. Gelatin extracted from ﬁsh heads exhibited a higher moisture content than gelatin obtained from ﬁsh skins (P<0.05). The quality of gelatin is largely dependent on its protein content. According to the results of the study, the protein content of gelatin extracted from rainbow trout heads was 81.9%, while the protein content of gelatin extracted from the skins was 84.68%. The lipid content in the skins was higher than that in the heads, likely due to the increased accumulation of subcutaneous lipids in the ﬁsh; the fat content of gelatin from ﬁsh heads was measured at 3.58%, while that of gelatin from ﬁsh skins was 3.38%. Ash content was higher in gelatin obtained from ﬁsh heads (0.53%) compared to gelatin extracted from ﬁsh skins (0.45%). These ﬁndings indicate that gelatin extracted from ﬁsh skins has lower ash content and higher protein content (P<0.05). Elavarasan et al. [5] reported that gelatin extracted from the tiger tooth croaker ﬁsh head had a protein content of 83% and a moisture content of 7.83%. Similar results were also reported by Ninan et al. [29] and Rosmawati et al. [32]. The pH value of gelatin plays an important role in determining its applications. In this study, the pH values of the collagen obtained from rainbow trout heads and skins were determined to be measured as 6.42 ± 0.06 and 6.20 ± 0.10, respectively. The observed difference can be attributed to the distinct biochemical compositions of these two tissues. These results indicate that the pH of the gelatin obtained through acidic and alkaline treatments is generally slightly acidic in nature. Adnan et al. [33] reported a pH range of 6.11–6.38 for gelatin isolated from by–products of various freshwater ﬁsh species, including striped catﬁsh, catﬁsh, and tilapia, which is consistent with the present ﬁndings. Similarly, Elavarasan et al. [5] reported comparable ﬁndings for gelatin extracted from the head waste of tiger tooth croaker. Cheow et al. [34] reported pH values ranging from 3.35 to 4.87 for gelatin extracted from the skins of tropical ﬁsh species, Johnius dussumieri and Decapterus macrosoma. These values are significantly lower than those observed for gelatin extracted from rainbow trout heads and skins. FTIR is a fast and effective spectroscopic technique used to identify functional groups in materials and assist in their characterization. This method enables the identification of functional groups and the examination of molecular structures, as each functional group vibrates at a speciﬁc frequency, which appears as characteristic peaks in the FTIR spectrum [35]. The FTIR spectra of gelatin extracted from ﬁsh heads and skins of rainbow trout (Oncorhynchus mykiss) are presented in FIG. 1 and TABLE II. The FTIR spectra of gelatin samples obtained from trout heads and skins show generally similar characteristics. The small differences observed in the spectra may be attributed to slight chemical variations between the samples. However, both samples exhibit similar bands and collagen–speciﬁc features, indicating that the fundamental protein compositions of both types of gelatin are very similar. The major peaks observed in the spectral analysis of gelatin samples obtained from rainbow trout heads and skins were at wavenumbers of 3270.3–3285 cm -1 (Amide A), 1634.7–1643 cm -1 (Amide I), 1535.6–1539 cm -1 (Amide II), and 1237–1156.6 cm -1 (Amide III. These bands correspond to N–H stretching (Amide A), C=O stretching (Amide I), N–H bending and FIGURE 1. Fourier Transform Infrared (FTIR) Spectra of Gelatin Extracted from Fish Heads (a) and Skins (b) TABLE II Fourier Transform InfraRed (FTIR) spectra of gelatin extracted from ﬁsh heads and skin Wavenumber (cm -1 ) Assignment Head Skin 3270.3 3285 N–H stretching 3012 3006.5 Aliphatic C–H stretching 2921.9 – 2852.2 2923.3 – 2852.7 Aliphatic C–H stretching 1745.4 1742.7 C=O stretching 1634.7 1643 Amid I band, Collagen’s α–helix and β– pleated sheet structure 1535.6 1539 The Amide II band, C–N stretching and N–H bending 1458.4 1455.9 C–H bending: Aliphatic group 1401 1397.7 C–H bending, Aliphatic hydrogen bending, alkyl groups presence 1237 and 1159.2 1239.7 and1156.6 The Amide III band, N–H bending and C–N bands

_________________________________________________________________________________________________Revista Cientiﬁca, FCV-LUZ / Vol.XXXV 5 of 7 C–N stretching (Amide II), and C–N stretching and N–H bending (Amide III), respectively. These ﬁndings conﬁrm the presence of polypeptide chains and suggest that the type I collagen–like protein structure is preserved in the gelatin samples [36]. Similar spectral patterns and peaks were also obtained by Sila et al. [37] and Hassan et al. [38] in their analysis of European eel skin and ﬁsh gelatins. This similarity indicates that gelatin extracted from different ﬁsh species shares common structural features and functional groups. Color and turbidity values do not influence the practical characteristics of gelatin, but they play an important role in consumer acceptance and potential applications of gelatin in the food industry [39]. The color and turbidity values of the gelatin obtained from rainbow trout head and skin are given in TABLE III. Turbidity refers to the cloudiness in a liquid caused by particles blocking light. The turbidity values of gelatin from rainbow trout head and skin were determined to be 0.67 and 0.62 (P<0.05), respectively, indicating high turbidity. The turbidity and dark color of gelatin typically result from contaminant substances that were not eliminated during the extraction process [40]. These substances may include residual proteins, minerals, or other organic compounds left in the gelatin after the extraction process. The sensory evaluation was carried out to assess consumer preference regarding the odor of gelatin extracted from the heads and skins of rainbow trout. The gelatins obtained from both the ﬁsh heads and skins were found to have a subtle but noticeable odor. The average sensory scores for the gelatins from the ﬁsh heads and skins were 2.2 and 1.7, respectively. Muyonga et al. [28] mentioned that gelatins made from Nile perch bones and skins did not have a ﬁshy odor but instead had a mild, unpleasant smell, with a mean hedonic score ranging from 2 to 2.5. Moreover, a study by Ninan et al. [29] discovered that gelatins made from the skins of rohu and common carp had a mild but detectable odor, with mean hedonic scores of 2.3 and 2.4, respectively. Although there is no dominant ﬁsh odor in the gelatins, neutralizing the odor completely could increase consumer acceptance and expand its use in the food industry. CONCLUSIONS This study investigated the physicochemical properties of gelatin extracted from rainbow trout (Oncorhynchus mykiss) heads and skins. The gelatin extracted from the skin had a higher protein content compared to the gelatin from the head. Both gelatin samples exhibited mildly acidic pH values, with the skin extracted gelatin displaying a lighter color. The turbidity of both gelatin samples was high, which may be attributed to residual impurities from the extraction process. FTIR analysis conﬁrmed the presence of collagen–speciﬁc spectral features in both samples. Sensory analysis revealed that the skin extracted gelatin had a milder odor compared to the head extracted gelatin. These ﬁndings suggest that both the head and skin of rainbow trout are promising sources of high–quality collagen and can be sustainably utilized. Future research may focus on optimizing extraction methods to improve the yield and quality of gelatin. Gelatin extracted from rainbow trout heads and skins holds signiﬁcant potential as an alternative and sustainable source of gelatin for applications in the food, pharmaceutical, and cosmetic industries. Conflict of Interests The authors declare that there is no conflict of interest. BIBLIOGRAPHIC REFERENCES [1] Food and Agriculture Organization of the United Nations (FAO). The State of World Fisheries and Aquaculture 2024 – Blue Transformation in action. [Internet]. Rome: FAO, 2024 [cited dd/mm/año]. doi: https://doi.org/ppmm [2] Polat A, Tokur B, Buga H. Extraction of protein from fresh rainbow trout (Onchorhynchus mykiss) viscera and smoked trout trimmings using commercial enzymes. Ege J. Fish. Aquat. Sci. [Internet]. 2022; 39(1):71-80. doi: https://doi.org/ppmn The color analysis reveals signiﬁcant differences between the gelatin extracted from the head and that extracted from the skin. The color values for the gelatin derived from the head are L = 7.73, a = 2.05, and b = 2.03, indicating a darker and more yellowish tone. In contrast, the gelatin obtained from the skin exhibits color values of L = 7.81, a = 2.65, and b = 2.97, reflecting a lighter, more reddish, and yellowish tone. The ΔL values are 6.87 for the gelatin extracted from the head and 6.94 for that extracted from the skin. Additionally, the ΔE values are 7.68 for the gelatin derived from the head and 7.33 for the gelatin obtained from the skin. The ΔE values being above 5 for both samples indicate that the color differences are visually noticeable. Gelatin from the head tends to have darker tones with more pronounced red and yellow hues, while the gelatin from the skin is lighter with a more pronounced reddish and yellowish appearance. Similar ﬁndings were obtained by Tabarestani et al. [23] regarding gelatin production from rainbow trout. However, in the research carried out by Ninan et al. [29], it was stated that the gelatin extracted from the skins of common carp and rohu exhibited a pure white appearance. These differences may be linked to variations in the type of raw materials used, extraction conditions, environmental factors, and analysis methods. However, these changes do not affect other functional properties. TABLE III Color and turbidity values of gelatin extracted from rainbow trout head and skin Color Head Skin L* 7.73 ± 0.02 a 7.81 ± 0.01 a a 2.05 ± 0.06 a 2.65 ± 0.07 b b 2.03 ± 0.05 a 2.97 ± 0.04 b ΔL* 6.87 ± 0.02 a 6.94 ± 0.06 a ΔE* 7.68 ± 0.05 a 7.33 ± 0.08 a Turbidity Absorbans (A620) 0.67 ± 0.02 a 0.62 ± 0.02 a Transmittance (%) 21.1 ± 0.3 a 23.9 ± 0.9 a The data are mean ± SD. Different letters in the same row indicate statistically signiﬁcant diﬀerences between groups (P<0.05)

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