Effects of morphometric and biochemical parameters on collagen and pepsin-solubilized collagen yields of Holothuria tubulosa (Gmelin, 1790) and Holothuria (Roweothuria) poli (Delle Chiaje, 1823)

Sea cucumber is a benthic marine echinoderm species that occurs in most seas of the world. There are about 1500 sea cucumber species, and about 60 of them are used commercially (Purcell 2010). While some of these economic species such as Stichopus japonicus are cultivated in Asian countries, most of them are intensively exploited from natural sources due to high market demand and commercial value, which ranges from USD 15 to USD 385 (Purcell et al. 2013; Purcell 2014). The main reason for the high market demand for sea cucumbers is related to their unique biochemical composition, which includes high amounts of collagen, glycosides, chondroitin sulfate, and a low fat to high protein ratio, making them a perfect choice as a valuable functional food for consumers (Saito et al. 2002; Bordbar et al. 2011; Shi et al. 2016; Senadheera et al. 2020). The sea cucumber is mainly known in its commercial form as “beche-de-mer”, which is prepared basically through processes of salting, boiling, and drying (Çaklı et al. 2004; Ferdouse 1999; Ferdouse 2004). Due to the long shelf life of the end product and production greater than the existing end-product stocks, overexploitation in most regions, including the Mediterranean, reaches maximum levels in each season.

Global production of sea cucumber ranged from 227 to 274 gross tons in 2015–2018 (FAO 2020). Apostichopus japonicus is the most produced species, but the production volume of this species decreased from 86.37% to 78.54%, following the increase in the production of other Holothurians from 13.58% to 21.40% in global sea cucumber production between 2015 and 2018 (FAO 2020). This may also lead to increased production of species from the Mediterranean such as H. tubulosa, H. poli, H. forskali, H. mammata, H arguinensis, H. sanctori, and Parastichopus regalis (Ramon et al. 2010; Antoniadou & Vafidis 2011; Borrero-Perez et al. 2011; Mezali & Thandar 2014; Gonzalez-Wangüemert et al. 2016). Of these species, Holothuria tubulosa and Holothuria poli are Mediterranean endemic species that occur in higher densities down to a depth of 30 m on a soft benthic substrate consisting of sand, mud, and sea meadows (Coulon & Jangoux 1993; Kazanidis et al. 2010; Gonzelez-Wangüemert et al. 2016; Tolon & Engin 2019). Their colors range from light brown (H. tubulosa) to blackish brown with white podia (H. poli; Aydın & Erkan 2015). The length of these species can reach up to 35 cm, however, well-represented sizes range from 8–10 cm to 23–26 cm, and commercial interest starts mainly above 10–12 cm (Gonzalez-Wangüemert et al. 2018; Aydın 2019). Both of these holothurians constitute an important proportion of sea cucumber production in Mediterranean countries, mainly Italy, Spain, Greece and Turkey (Mezali et al. 2006; Antoniadou and Vafidis 2011; Valente et al. 2015; Gonzalez-Wangüemert et al. 2016; Künili & Colakoglu 2018; Aydın 2019). The average total production of these species in Turkey was reported as 419 tons for the last seven years and 855 tons for 2017 (Aydın 2020). The marketing of these economic species was generally in the form of fresh frozen and beche-de-mer, as is usual in these countries (Aydın 2008; Antoniadou & Vafidis 2011; Aydın et al. 2011; Sicuro & Levine 2011; Gonzalez-Wangüemert et al. 2016; Aydın 2017; Künili & Colakoglu 2019).

Collagen is an important fibrous protein found in almost all living organisms, as it is the main component of structural tissues (Regenstein & Zhou 2007). In marine animals, collagen is also an important contributor to total proteins in the whole body as it is a major constituent in scales, bones, skin, and fins (Gómez-Guillén et al. 2011; Coppola et al. 2020). However, collagen and collagenous tissues are of particular importance in echinoderms, such as sea urchins, starfishes, and sea cucumbers, because they protect their coelom and body shape, while also playing a key role in motion and defense systems by being the major constituent of their skin, podia, and spines (Santos et al. 2005; Wilkie 2005; Senadheera et al. 2020).

Collagen and its properties in sea cucumber species have been extensively studied in the last decade, however, there is a significant lack of knowledge about collagen and its properties in both H. tubulosa and H. poli. Although these are economically important sea cucumber species, with collagen being one of the main reasons for their importance, its relationship with morphometric and biochemical characteristics is still unclear. Moreover, the yield of collagen as a function of morphometric and biochemical parameters has not been investigated in any of the sea cucumber species, and this is an important issue for the optimum utilization of sea cucumbers. For this reason, the present study was designed to investigate the collagen yields from H. tubulosa and H. poli as economic species. In addition, the objective was to determine the yield of collagen hydrolysates, which are indicative of the physiological utility of collagen in the body and in many industries due to their increased solubility and purity (Coppola et al. 2020). By determining the amount of collagen and hydrolysates obtained from both species, we hoped to determine the optimum utilization sizes for both species, while filling the gap in the literature by comparing changes in collagen yields as a function of morphometric and biochemical parameters.

Research materials of Holothuria tubulosa and Holothuria poli were collected by hand during SCUBA diving from depths ranging from 1 to 15 m along the coasts of the southern Marmara Sea and the northern Aegean Sea. A total of 200 specimens were collected for each species between March 2018 and July 2020 at different time intervals from four locations indicated in Fig. 1. Live specimens individually packed in polyethylene bags were transported to the laboratory using ice-cooled insulated boxes within 4 h. Upon arrival at the laboratory after each sampling, the specimens were divided into five length frequencies according to the distribution of their minimum and maximum lengths. After morphometric measurements were performed, they were stored at −20°C until the number of specimens was sufficient for all analyses in each relevant length frequency. All chemicals used in this study were of analytical grade.

Figure 1

Morphometric data for both sea cucumber species used in this study are summarized in for H. tubulosa and in Table 2 for H. poli.

The minimum and maximum lengths of H. tubulosa and H. poli were measured as 10.60–25.00 and 8.90–22.40 cm, respectively. The median lengths of H. tubulosa and H. poli populations were determined as 15.88 ± 1.02 cm and 14.58 ± 0.82 cm, respectively. The minimum and maximum weight and gutted weight were in the range of 26.02–118.08 g and 22.56–59.14 g for H. tubulosa, and 26.42–95.70 g and 21.12–55.43 g for H. poli, respectively. A total of 68 specimens (34%) of H. tubulosa and 44 specimens (22%) of H. poli were identified as being in the reproductive period.

3.1

Relationship between collagen yield and length and biochemical composition

In this study, the relationship between the yield of collagen, as the major component of the edible part (body wall) of sea cucumbers, and the length and biochemical composition was investigated. Due to limited sex differentiation in both species, biochemical composition was determined from 100 g randomly subsampled fresh body wall for each sex. The mean content of moisture, proteins, ash, fat, carbohydrates, and the yield of collagen by species in relation to length frequencies are summarized in Fig. 2 and Fig. 3.

Figure 2

Figure 3

For all length frequencies, mean percentage levels of biochemical composition in H. tubulosa and H. poli ranged from 83.88 ± 1.03 to 84.47 ± 1.09 and from 82.66 ± 0.29 to 83.00 ± 0.28 for moisture, from 8.44 ± 0.99 to 8.66 ± 1.01 and from 8.32 ± 0.24 to 8.54 ± 0.25 for proteins, from 4.66 ± 0.45 to 4.76 ± 0.47 and from 6.59 ± 0.78 to 6.68 ± 0.88 for ash, from 1.75 ± 0.35 to 1.80 ± 0.44 and from 1.51 ± 0.09 to 1.55 ± 0.11 for fat, and from 0.53 ± 0.12 to 1.09 ± 0.66 and from 0.40 ± 0.11 to 0.68 ± 0.15 for carbohydrates, respectively.

In H. tubulosa samples, mean percentage levels of collagen yield for all length frequencies ranged from 5.34 ± 0.98 to 5.81 ± 0.84% of wet weight. The minimum yield was determined from samples belonging to 16.7–17.4 cm length frequency, whereas the maximum yield was obtained from samples belonging to 10.6–13.2 cm length frequency (Fig. 2).

In H. poli samples, mean percentage levels of collagen yield for all length frequencies ranged from 5.44 ± 0.15 to 5.63 ± 0.17% of wet weight. The minimum yield was determined from samples belonging to 16.0–17.1 cm length frequency, whereas the maximum yield was obtained from samples belonging to 12.5–14.1 cm length frequency (Fig. 3).

Pearson correlation was applied to the data to compare changes in collagen yield as a function of the major determinants (Fig. 4). To determine the correlation, morphometric data were also used with length and biochemical composition. In both sea cucumber species used in the biochemical composition analysis, a negative non-significant correlation was found between length and collagen yield (r −0.437, r −0.321; p > 0.05), whereas the correlation between gutted weight and yield was significantly positive (r 0.069, r 0.239; p < 0.05). The correlation between the collagen yield and biochemical constituents was not significant for both sea cucumber species (r −0.18, r 0.76, p > 0.05; Fig. 4).

Figure 4

3.2

Relationship between length, weight, gutted weight, and maturity and collagen yields

In this study, collagen from mature specimens was isolated separately. Immature specimens with the same length and weight as mature specimens were used as a control group for collagen isolation. The results of collagen yields as per length frequencies along with the comparison of total weight, gutted weight and sexual maturity are summarized for H. tubulosa in Fig. 5 and H. poli in Fig. 6. Sexual maturity was observed at a minimum length of 12.10–12.30 cm and minimum gutted weight of 41.55 ± 2.59 and 33.16 ± 1.75 g for H. tubulosa and H. poli, respectively.

Figure 5

Figure 6

Mature H. tubulosa specimens with GSIs ranging from 2.77% to 12.4% (M/F ratio of 1/7) were used for collagen isolation. On the basis of length frequencies of mature specimens, the minimum yield was determined as 5.25 ± 0.31 g (10.63% based on gutted weight) in the highest length frequencies. The highest collagen yield, on the other hand, was determined as 6.10 ± 0.18 g (14.68% based on gutted weight) in smaller specimens with the lowest length frequency. Collagen yields of immature H. tubulosa specimens of the same length and weight ranged from 6.60 ± 0.27g (16.04%) to 5.69 ± 0.22 g (10.96%), respectively (Fig. 5).

Mature H. poli specimens with GSIs ranging from 4.61% to 6.91% (M/F ratio of 1/4) were used for the collagen isolation. On the basis of length frequencies of mature specimens, the minimum and maximum collagen yields ranged from 5.31 ± 0.04 g (11.90% on the basis of gutted weight) to 5.03 ± 0.11 g (7.12% based on gutted weight) from the smallest to the largest sizes. The collagen yields of immature specimens of the same size ranged from 5.50 ± 0.04 g (13.10%) to 5.09 ± 0.08 g (10.66%), respectively (Fig. 6).

The sex ratio of specimens used for collagen isolation was determined, but differences in the yield between males and females were ignored due to the limited number of male specimens in both species (1/7 M/F of 14 specimens for H. tubulosa and 1/4 M/F of 12 specimens for H. poli) resulting from the inability to identify the sex until breeding. When considering the GSIs of both species, maturity was found to be an insignificant factor affecting the collagen yield in H. tubulosa (r −0.048, p > 0.05), while it was an important determinant for H. poli (r 0.926, p < 0.05). Parallel to this finding, it was found that regardless of the gutted weight, an increase in the number of gonads did not significantly affect the collagen yield of mature specimens (p > 0.05). Among mature specimens of both species, H. tubulosa produced more collagen than H. poli (p < 0.05). In both species, however, the collagen yield among immature specimens with the same length and weight was not significantly different (p > 0.05).

3.3

Relationship between PSC yield and morphometric and biochemical parameters

The hydrolyzation degrees (HD) of collagens isolated from both species ranged from 14% to 32% (Fig. 7a). The maximum HD, which also gave the highest PSC yield, was determined at 5 and 6% enzyme concentrations. The comparison of PSC yield as a function of length and enzyme concentration was limited in the results to 5% pepsin concentration as no significant increase in the HD was observed after 5% concentration (p > 0.05).

Figure 7

It was found that maturity of H. tubulosa had a significant positive effect on the PSC yield, compared to immature specimens (r 0576, p < 0.05; Fig. 7b). On the other hand, maturity had a negative effect on the PSC yield in H. poli (r −0.518, p > 0.05). Among biochemical composition variables, protein proved to be a major determinant for the PSC yield in both species (r from 0.768 to 0.904, p < 0.05), contrary to the collagen yield (r from −0.02 to 0.0, p > 0.05; Fig. 7b).

PSC yields from body walls of H. tubulosa and H. poli as a function of length frequencies and enzyme concentrations were summarized in Fig. 8. In the lowest length frequency of H. tubulosa, the PSC yield ranged from 0.83 ± 0.05 to 1.68 ± 0.11%. In the highest length frequency, the PSC yield ranged from 0.88 ± 0.08 to 1.83 ± 0.12% (Fig. 8a).

Figure 8

In the lowest length frequency of H. poli, the PSC yield ranged from 1.02 ± 0.06 to 1.76 ± 0.11%. For the highest length frequency, the PSC yields ranged from 1.1 ± 0.06 to 1.89 ± 0.12% among these length frequencies (Fig. 8b).

3.4

Molecular weight distribution of collagen hydrolysates

SDS-PAGE was applied to verify whether the isolation and hydrolyzation of collagen in both species was successful. The gel image indicating molecular weight distribution of pepsin-solubilized collagen is presented in Fig. 9.

Figure 9

Clear and characteristic bands of sea cucumber pepsin-solubilized collagen, including β, α-1, and α-2, were visualized from the SDS-PAGE gel. Most of the unbroken polypeptides belonging to pepsin-solubilized collagen fibrils were observed above the β chain (≈ 180 kDa) and γ chain residues (> 205 kDa), while characteristic sea cucumber PSC bands of α-1 and α-2 polypeptide chains were determined at ≈ 100–130 kDa. In both species, the density and distribution of protein bands were similar. This can be considered as an indication that the same procedure for both species provided optimum conditions for collagen isolation and hydrolyzation.

In this study, collagen and collagen hydrolysate yields – one of the main factors affecting the exploitation of sea cucumber species – were determined in two economic sea cucumber species as a function of their length frequencies. As the first study that reports the relationship between morphometric parameters and collagen yield of two sea cucumber species, the effects of maturity and biochemical composition on collagen and collagen hydrolysate yields were also investigated. The relationship between length and weight measurements in sea cucumbers was mainly examined after specimens were gutted because of a possible evacuation of their coelomic fluid and internal organs under stress. For this reason, gutted weight (body wall) of sea cucumber is used for a more accurate determination of morphometric data correlations (Conand 1981; Kazanidis et al. 2010; Gonzalez-Wangüemert et al. 2014). In this study, the correlation between the total weight and length was not clear, however, the gutted weight and total length showed a strong positive correlation for both species (r 0.924 and r 0.966, p < 0.05). The minimum maturity sizes of both species were 10.6 and 8.9 cm for H. tubulosa and H. poli, respectively. Non-significant correlations were found between length and maturity for both species (r 0.677 for H. tubulosa and r −0.609 for H. poli, p > 0.05). Previous studies from Turkey reported length distribution in the range of 4.0–30.3 cm for H. tubulosa and 3.40–18.0 cm for H. poli (Dereli et al. 2016; Gonzalez-Wangüemert et al. 2015; Aydın 2019). The first maturity length of H. tubulosa and H. poli was reported as 8.1–8.4 and < 13.5 cm, respectively (Dereli et al. 2016; Tamacha et al. 2019). In this study, the median lengths of mature specimens were 15.6 and 13.5 for both species, which are considerably lower than the mean lengths of the total sampled population. Along with the first maturity length, gonadosomatic indices for both species were also at lower levels. However, these lengths and GSIs were still in the same ranges compared to the studies mentioned above.

Biochemical composition, defined as changes in the percentage of moisture, protein, ash, fat, and total carbohydrates in marine organisms, depends and is affected by species differences, feeding regime, geographical location, sex and maturity, seasons, and morphometric characteristics (Colakoglu et al. 2011; Pereira et al. 2014; Khotimchenko 2015; Künili & Colakoglu 2019). In immature specimens of both species, the determined protein amounts and collagen yields were not significantly different across the length frequencies, but moisture and ash showed a strong positive correlation with collagen yields (p < 0.05). In general, the content of moisture, protein, and ash was similar to that reported in the range of 81.2–86.0%, 7.41–8.82%, and 5.13–7.04%, respectively, however, the fat content was at higher levels than values reported for different sea cucumber species (Aydın et al. 2011; Omran 2013; Haider et al. 2015; Roggatz et al. 2016). It is believed that the variation in fat levels may be mainly due to regional changes, which may directly affect the fat content and properties of the species (Zhang et al. 2017).

In this study, the length frequency was linked only with biochemical composition, as the sex of both sea cucumber species cannot be identified until they are in the reproduction cycle. The reproduction cycle can vary in holothurians, even within the same species or in individuals of the same length and weight, because the major regulators of maturity are temperature, food availability, and photoperiod (Harriott 1985; Conand 1993; Asha & Muthiah 2008). Nonetheless, reproduction in both species occurs mainly in warmer months, from May to September (Despalotovic et al. 2004; Aydın 2008; Aydın & Erkan 2015; Dereli et al. 2016). In this study, water temperature of sampling areas can reach 25°C during the reproduction period (TSMS 2020), and this may accelerate changes in biochemical and physiological characteristics of the species as a result of increasing food availability and maturity of sea cucumbers (Coulon & Jangoux 1993; Kazanidis 2010; Günay et al. 2015; Künili & Colakoglu 2019). Therefore, the effect of reproduction on collagen yields in both species was determined by performing sampling until sufficient numbers of both immature and mature specimens in different length frequencies were obtained, and biochemical changes along with collagen yields could be compared regardless of temperature (Figs 5, 6, 7b). Although, the amount of proteins in sea cucumbers may not vary significantly between seasons, except in summer (Künili & Colakoglu 2019), the percentage level of predominant and collagen precursor amino acids in the body wall comprised alanine, glycine, glutamic acid, proline, and hydroxyproline at varying levels (Ciu et al. 2007; Zhong et al. 2007; Gao et al. 2011; Liu et al. 2010; Liu et al. 2011; Bechtel et al. 2012; Sicuro et al. 2012; Omran 2013; Wang et al. 2013; Haider et al. 2015; Zhong et al. 2015; Widianingsih et al. 2016). This may also change during reproduction due to the possible demand of the organism for these amino acids. Thus, the change in the ratio of amino acids to total protein may also lead to changes in collagen synthesis and collagen yield.

In this study, we found that maturity was not a non-significant factor affecting collagen yield in H. tubulosa (r −0.048, p > 0.05), whereas it was an important determinant for H. poli (r 0.926, p < 0.05). Comparing the yields among mature specimens in both species, H. tubulosa had higher collagen yield than H. poli (p < 0.05). The main reasons for the differences in collagen yield between the two species during the maturation period may be related to chemical and physiological changes depending on species-specific characteristics, as many results from previous studies indicate that significant changes may occur in the reproduction cycle of different species of echinoderms (Olive 1995; Singh et al. 2001; Muthiga 2006; Mercier & Hamel 2009; Doyle et al. 2012; Kazanidis et al. 2014). In sea cucumbers, the percentage of collagen can be more than 70% of the total proteins in the body wall (Saito et al. 2002; Chen 2003; Li et al. 2020). Therefore, in addition to maturity, the early stage of the life cycle and the feeding regime may also be the main factors that affect the amount and structural properties of the collagen in the body of sea cucumbers (Miller et al. 1983; Silva et al. 2014).

Hydrolyzation of collagen by pepsin, also known as pepsin-solubilized collagen (PSC), shows the potential of collagen to be used to some extent in the body and in industries such as food, cosmetics, and pharmacology. For this reason, as the maximum utilization can vary depending on length as well as amounts of protein and collagen, PSC yield was determined in both species according to these parameters. In addition, the effect of enzyme concentration (EC), which is also an important determinant that directly affects the yield of collagen hydrolysates, was also determined for all length frequencies. The EC may be effective until the hydrolysis reaction is saturated, which means the degree of hydrolyzation may not change significantly after the optimum enzyme concentration is reached. The ECs chosen in the study (1–3–5%) were determined from pilot experiments that indicated the hydrolysis process did not change significantly after reaching a 5% (maximum concentration specified in this study) concentration (Fig. 7a). The pepsin-solubilized collagen has been the subject of many studies due to its potential use based on increased solubility and elevated purity of collagen without losing bioactivity and functionality (Liu et al. 2010; Park et al. 2012; Lin et al. 2015). Sea cucumbers have also been the subject of research in the last decade and observations have been made about the superior properties of collagen hydrolysates (Liu et al. 2011; Park et al. 2012; Zhou et al. 2012; Jin et al. 2019; Li et al. 2020). To the best of our knowledge and the available literature, a number of sea cucumber species have been studied to determine characteristics of collagens and hydrolysates. However, collagen and hydrolysates from H. tubulosa and H. poli along with the comparison by length, weight, maturity, and biochemical composition have not been the subject of existing studies. In this study, the maximum PSC yield was determined at 5% EC in both species. The PSC yields for H. tubulosa were determined as 21.09% of the total protein content (dry weight), 32.95% of the total collagen content (dry weight), and 1.83% of the total body wall (wet weight). The PSC yields for H. poli were determined as 34.52% of the total collagen content (dry weight), 22.12% of the total protein content (dry weight), and 1.89% of the total body wall (wet weight). If we consider that moisture levels in both species reach up to 84% (Figs 2 & 3), PSC yields were found to be similar to those reported as 20.8–24.3% (per dry weight of body wall) for Parastichopus californicus (Liu et al. 2010) and 26.6% (dry weight of body wall) for Stichopus japonicus (Park et al. 2012). However, the PSC yields of both species in this study were significantly lower than the yield reported by Lin et al. (2015) for Acaudina leucoproocta as 8.35% (wet weight of body wall). The results obtained in this study for PSC yields in both species were within the range of reported values for some other marine invertebrates. PSC yields on a dry weight basis were reported in the range of 12.3–66% for squids and jellyfishes (Kołodziejska et al. 1999; Barzideh et al. 2013; Khong et al. 2016) and 7–35% for sea urchins (Nagai & Suzuki 2000; Benedetto et al. 2014). However, the determined PSC yields were considerably higher than the levels reported for starfishes as 5.8% for Asterias amurensis (Lee et al. 2009) and 6.1% for Asterina pectinifera (Qi et al. 2017). Differences in collagen and PSC yields in sea cucumber species as well as other invertebrates are attributed primarily to species differences and protocols or handling procedures used during isolation and hydrolyzation of collagen and its hydrolysates. Although these factors can significantly affect the yield, the characteristics of molecular weight patterns of PSCs remain mostly similar. For this reason, as a final product of collagen hydrolyzation, pepsin-solubilized collagen from both species was subjected to SDS-Page analysis to control the isolation and hydrolysis procedures. In this study, PSC bands comprising β, α-1, and α-2 were visualized from SDS-Page gel. The bands of both sea cucumber species were similar to those reported in previous studies (Saito et al. 2002; Cui et al. 2007; Liu et al. 2010; Liu et al. 2011; Adibzadeh et al. 2014; Liu et al. 2019; Zhong et al. 2015; Li et al. 2020).

In this study, the yield of collagen, one of the most important biochemical components of sea cucumbers, was found to be affected by changes in length, weight, and maturity. Length was found to be the most important factor affecting the collagen yields in both species. Moreover, the degree of hydrolyzation in both species, which indicates considerable utilization of collagen, was also found to be affected by length and maturity. These relationships were also found to be significantly affected by species differences.

In conclusion, collagen and hydrolysate yields from economically important and exploited sea cucumber species in Turkey and the Mediterranean showed significant differences depending on maturity, length, weight, and biochemical composition. It is believed that these results can guide future research and sectors to ensure optimum utilization of these species.