Impact of Different Combinations of Processing Steps on Product Quality and Proximate Composition of Bêche-de-mer: A Case Study from Sri Lanka

Bêche-de-mer (dried sea cucumbers) is considered as one of the most luxurious seafood in the world. The market demand for bêche-de-mer mainly depends on species and product quality. This study is aimed at assessing the effect of different processing methods on the product quality and nutritional composition of bêche-de-mer. Three sea cucumber species, Stichopus naso, Holothuria spinifera, and Bohadschia vitiensis, dominant in the commercial catches of Sri Lanka, were processed by following widely practiced local processing methods where 24, 48, and 24 different processing combinations were tested for each species, respectively. Bêche-de-mer satisfying the export quality were selected by considering their appearance and overall ranking made by exporters (9 individuals per each combination). The proximate composition of these products was assessed using standard methods. According to the exporters' prioritization, very few processing methods used in this study resulted in the export-quality bêche-de-mer. Both product quality and proximate composition showed considerable variations with respect to the processing methods. It is proved that even a very small change in one processing step could lead to produce poor-quality products. Processing methods are species specific, and it is important to select the most appropriate method for each species to produce high-quality bêche-de-mer.


Introduction
Sea cucumbers belonging to the class Holothuroidea of the phylum Echinodermata are soft-bodied, worm-like marine benthic invertebrates found at all latitudes and all depths [1,2]. The exploitation history of sea cucumbers dates to several centuries, and sea cucumbers first became commodities in China and India more than 1000 years ago [3,4]. As there was a lucrative market for sea cucumber products throughout the world, fishing activities were rapidly expanded into new fishing grounds and currently more than 70 countries are engaged in sea cucumber fishing worldwide, targeting around 66 species [5][6][7].
The sea cucumber fishery has been providing an important means of livelihood for the coastal fishing communities in Sri Lanka for centuries. Although around 21 sea cucumber species are commercially exploited in the coastal waters of Sri Lanka, there is no tradition of consuming them locally and the entire harvest is exported to Singapore, Taiwan, and China as bêche-de-mer [15]. In Sri Lanka, sea cucumbers are processed into bêche-de-mer by both domestic and industrial level processors, mainly following the major processing steps of grading, evisceration, first boiling, salting, second boiling, and drying [12] but with their own modifications to one or many steps according to their traditional knowledge and hands-on experience. Most of these modifications have been passed from generation to generation and remain undisclosed, while others are ad hoc practices done by domestic level processors.
It is well documented that the market demand for bêchede-mer fluctuates with species and product quality. Appearance, color, odor, and moisture content are the key determinants of bêche-de-mer product quality, and these parameters are mainly influenced by processing methods [12]. According to Wen et al. [16], the nutritional composition of bêche-de-mer is significantly affected by the way they are processed. Although many attempts have been made to study sea cucumber processing practices, their advantages, and drawbacks [6,12,14,17], only a few studies have focused on assessing the impact of different processing practices on the product quality and nutritional composition [1,18]. Thus, this study is aimed at filling this research gap by providing a detailed analysis on how different combinations of processing steps affect the product quality and nutritional composition of three sea cucumber species, i.e., Stichopus naso, Holothuria spinifera, and Bohadschia vitiensis, dominating the commercial catches in Sri Lanka.

Materials and Methods
2.1. Sample Collection. Fresh samples of S. naso (n = 360), H. spinifera (n = 720), and B. vitiensis (n = 360) were collected from major sea cucumber landing sites in the north and northwest coasts of Sri Lanka ( Figure 1). All collected sea cucumbers were properly washed using clean seawater to remove slime and sand on the body surface and transported to the processing plants located in Colombo (transportation time = 06 − 07 hours) and Jaffna (transportation time = 01 − 02 hours), Sri Lanka under chilled conditions. As S. naso autolyzes its body tissue immediately after taking it out of the water, they were transported to the processing plants in seawater.

Experimental
Design for Processing. The individual weight of fresh sea cucumbers was measured at the processing plants, and they were processed into bêche-de-mer following different combinations of processing steps used by local processors for each species. Accordingly, forty-eight alternative methods were used to process H. spinifera and 24 processing methods were applied to B. vitiensis and S. naso samples (Table 1 and Figure 2).

Effect of Processing on Product
Quality. The quality of the processed samples was initially assessed based on their appearance, mainly considering the percentages of damaged, deformed (with bent and misshaped), patched, and oversalted individuals. Then, these products were packed in polythene bags according to the assigned code number and anonymously presented to exporters (n = 9). They were asked to select export-quality products and line them up from the highest to the least quality, respectively, by assigning numbers in ascending order starting from 1. Factors considered to select export-quality products and rank them were reported by interviewing exporters. The results were recorded separately for each species using the code number assigned to each product ( Figure 2).
By considering the initial assessment and selection made by exporters, the export-quality bêche-de-mer were selected and ranked by averaging the individual ranking given by exporters to understand the effect of different processing procedures on the final product quality and to select the most appropriate processing practices for each species.
2.4. Effect of Processing on Proximate Composition. To assess the effect of different processing practices on the nutritional composition of bêche-de-mer, the proximate composition of the processed products was analyzed. Due to practical limitations, only the selected export-quality bêche-de-mer from each species were considered for this analysis. Representative samples (n = 5) from each selected product were taken and ground using a grinder to obtain a homogenous sample. Each sample was analyzed for percentage moisture and dry matter percentages of crude ash, crude fat, and crude protein. The moisture content was determined by drying the samples in a thermostat oven at 100 ± 5°C until a constant weight was obtained [19]. Crude ash content was determined by incinerating the samples in a muffle furnace at 550°C for 24 h [19]. The micro-Kjeldahl method with acid digestion was used to determine the crude protein content, and conversion factor 6.25 was used to convert total nitrogen to crude protein [10]. Bligh and Dyer's method was used to determine the crude fat content [20]. Each experiment was carried out in triplicate.

Statistical
Analysis. The mean (±SD) weight of the fresh and processed samples, percentage weight loss during processing, percentages of damaged, deformed, patched, and oversalted individuals, and the exporters' ranking of each product were calculated using the MS Excel software. All proximate components, except moisture, were determined on a dry weight basis. Variations in the proximate composition of the selected products of each species were compared using analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Differences were considered significant when p < 0:05. The statistical tests were performed with Minitab 18 for the Windows statistical package.

Weight Changes during
Processing. According to the initial assessment and overall ranking of exporters, differently processed 24 products, representing 8 products from each species, were selected as the export-quality bêche-de-mer. Fresh weight, processed weight, and percentage weight loss of these selected products are summarized in Table 2, and the same results obtained for H. spinifera (n = 48), B. vitiensis (n = 24), and S. naso (n = 24) under different processing methods are summarized in Supplementary Tables 1, 2, and 3, respectively.

2
International Journal of Food Science The percentage weight loss of H. spinifera ranged from 85.89 to 94.11%, while it was from 89.45 to 93.26% and 86.54 to 93.51%, respectively, for B. vitiensis and S. naso. The results indicated that considerable weight loss of fresh sea cucumbers can occur during processing, and this mainly depends on the target species and processing practices. The processing processes like long-time boiling and drying with electric dryers have made more contribution to the weight loss of the processed products.

Selection and Ranking of Export-Quality Bêche-de-mer.
The results of the initial assessment (i.e., % damaged, % deformed, % visible patches, and % salted) and the overall ranking for the processed products of H. spinifera, B. vitiensis, and S. naso are summarized in Supplementary Tables 1, 2, and 3, respectively.
Out of the 48 processed products of H. spinifera, the samples coded as F20PST, F15DLT, F10DLT, F10DST, F20PLF, F10PLF, S15DSF, and F20DSF were the selected export-quality products. Among these samples, F20DSF reported the lowest percentage of damaged (0%), deformed (0%), patched (0%), and oversalted (20%) individuals. More than half of the processing practices used to process H. spinifera resulted in a high percentage of damaged individuals ranging from 14 to 50%. The highest percentage of deformed individuals was reported in F15DSF (80%), followed by F20DST (71.43%, Supplementary Table 1). Seven out of the eight selected products were cooked in freshwater with added salts, and half of these products were dried in an electric dryer. Chalky materials in the majority of these products (n = 5) were removed by soaking in freshwater. The high percentage of salted individuals appears to be an issue in most of the processing procedures, and this is especially true when aqueous salt is used (Supplementary Table 1).
The exporters ranked the selected 8 products of H. spinifera as F20DSF, F20PST, F20PLF, S15DSF, F15DLT, F10PLF, F10DST, and F10DLT (Table 2; Supplementary  Table 1) by considering factors such as appearance, dryness, presence of spike-like projections on the body surface, and the absence of CaCO 3 deposits. The way of removing chalky materials and the method of drying are the main differences observed between the F20DSF and F20PST, where chalky materials in the F20DSF were removed by soaking in water and it was dried under sunlight, while papaya leaves were used to remove chalky materials in the F20PST and it was dried in an electric dryer. The results revealed that different combinations of processing steps can be used to produce high-quality bêche-de-mer from H. spinifera. Cooking in freshwater with added salts around 15-20 minutes, removing chalky materials in soaking water, salting with solid salt, and drying under sunlight for a period of 5 days can be considered as a good processing step.
The high-quality processed products of B. vitiensis include the samples labeled as S10ST, F10ST, F15ST, S15LT, F10LT, S20LF, S15LF, and F20LF. Of those, the S10ST sample reported 0% of damaged, deformed, patched, and oversalted individuals. When compared to H. spinifera, B. vitiensis reported a lower percentage of damaged individuals and the two lowest-quality products, F10LF and F15SF, reported only 20% of damaged individuals. However, 20 out of the 24 different processing methods resulted in deformed individuals and their percentage ranged from 0 to 85.71%. The highest percentage of deformed individuals was reported in F20ST (85.7%) followed by S10LT and S15SF (80%). The sample F15LF reported the highest percentage of visible patches (80%). It is noted that B. vitiensis processed using aqueous salt and/or dried under sunlight often results in dull body color than the samples processed using solid salt and electric drying (Supplementary Table 2).
By considering the brightness of the dorsal body surface, the curved nature of the ventral body surface, and the level of drying, exporters lined up the selected 8 products of B. vitiensis from the highest-to least-quality products as F15ST, F10ST, S10ST, S20LF, S15LF, S15LT, F10LT, and F20LF (Table 2; Supplementary Table 2). The least-quality products are characterized by dull-colored body surfaces. The results of this study revealed that B. vitiensis processed by boiling for 15 minutes in freshwater with added salts, salting with solid salts, and drying in an electric dryer for    International Journal of Food Science 15 hours generates higher-quality bêche-de-mer than the other processing methods. The eight samples coded as S6LF, F6LT, F3SF, F6LF, S3LF, F6SF, F9LF, and S3SF of S. naso were identified as the best processed products. Two samples, F3ST and S3SF, showed 0% of damaged, deformed, patched, and oversalted individuals. The lowest quality was evident in the F3LT sample, which reported 25% of damaged and 62.5% of deformed individuals. Similar to B. vitiensis, S. naso processed using aqueous salts resulted in a dull-colored body than the   (Table 2; Supplementary Table 3).
The exporters and processors gave their highest preference to the S3SF, followed by the F3SF (Table 2; Supplementary  Table 3). The adequacy of dryness, free from deformation, and the size of the processed individuals are the primary criteria used by them to rank the processed products of S. naso. Similar to B. vitiensis, the quality products were characterized by a dull-colored external appearance.
The results revealed that different combinations of processing steps can be used to produce high-quality bêche-demer of S. naso. However, a 3-6-minute boiling time, salting with solid salts, and sun drying for a period of 3 days can be considered as a good combination of processing steps.

Proximate Composition of Export-Quality Bêche-de-mer.
The proximate composition of the export-quality bêche-demer of H. spinifera, B. vitiensis, and S. naso is summarized in Table 3.

# Species
Sample code Average weights are given in mean weight in grams ± standard deviation; the highest quality bêche-de-mer was given rank 1 in the exporters' ranking. 6 International Journal of Food Science significantly lower moisture, higher protein, and lower fat compared to other products (ANOVA, p < 0:05).

Discussion
The processing of sea cucumbers into bêche-de-mer is a largely unregulated process. However, many attempts are made continuously to study and improve the processing process of sea cucumbers as the end market value of bêchede-mer is mainly dependent on product quality [17,21].
It is reported that many countries, including the Pacific Islands [6,14], Sri Lanka [12], and Madagascar [17], have identified the limitations of their existing processing practices. Although some attempts have been made to improve the processing quality of bêche-de-mer by Chong et al. [22], Duan et al. [18], and Duan et al. [1], there are still many poor-quality products in the international markets. This could be due to many reasons. As reported in this study as well as in many previous studies, processing practices vary from species to species and processors are not well aware of the most appropriate processing method for each species. Due to the lack of species-specific standard processing practices, proper awareness, and training, many different processing practices are currently used by sea cucumber processors in Sri Lanka. However, the results of this study revealed that most of these processing practices contribute to produce inferior-quality products. However, it is also proved that export-quality bêche-de-mer products can be produced following combinations of different processing steps.
Grading and cleaning of sea cucumbers are the first two steps used in the sea cucumber processing chain. Grading is an important activity to separate them into species as well as  [6], different species require different processing methods due to differences in the thickness of their body wall and the extent of water retention, and this study also supports their findings. On the other hand, larger-sized sea cucumbers require longer boiling time than smaller-sized individuals. Thus, processing without grading leads to produce poor-quality products.
Evisceration is a vital step in sea cucumber processing, and it must be carried out with great care [8,12,23]. Evisceration is mainly carried out to remove the internal organs, as the presence of such organs badly affects the quality of bêche-de-mer [14]. Evisceration is mainly done by making a small cut (2.5-4.0 cm) at the posterior end of the body. However, in some species, i.e., S. chloronotus evisceration is done at the ventral body surface, while in teatfish species (H. fuscogilva and H. nobilis), it is commonly done at the dorsal body surface [6,12]. The widely practiced methods were used to eviscerate the experimental animals. The evisceration is generally carried out before the first boiling [12,14,22]; however, highly perishable S. naso was boiled before evisceration to reduce postharvest losses [22].
Boiling or cooking is the most important step in the sea cucumber processing chain as it improves the palatability, the rate of drying, and the odor of the end product [23]. According to Nishanthan et al. [12], the quality of bêchede-mer mainly depends on the boiling medium and boiling time. Previous studies recommended saltwater as the preferred medium for cooking sea cucumbers [22], but this study confirms that the use of freshwater with added salt does not have any significant impact on the final product quality. However, as most of the processors tend to use seawater directly taken from adjacent coastal water bodies contaminated with domestic and industrial waste, it is good to use freshwater with added salt as the boiling medium.
The use of proper boiling time is vital for the final product's quality. The boiling time depends on several factors, such as species, size, cooking pressure, and cooking temperature [1,24]. This study revealed that boiling time varies from species to species and longer or shorter boiling time results in overboiled or partially boiled products with a high percentage of damaged and deformed individuals. As reported in this study, these products normally receive a low market value because the shape and external appearance are some of the major factors used to rank bêche-de-mer [14,18,22,24]. Some species, like H. spinifera and H. scabra have CaCO 3 deposits on their bodies and these should be removed during processing [12,17]. Different methods, including many traditional methods, are used to remove these chalky materials in many countries [6,12,14]. Local processors mainly use two methods to remove the chalky material of sea cucumbers. Although papaya powder is successful in removing chalky materials, as reported in Madagascar and some Pacific Island countries [6,17], local exporters gave less preference for these products due to the lack of spike-like projections on the body surface of H. spinifera. The exact reason for this observation is not very clear, but it could be due to the removal of spike-like projections by the uncontrollable action of papain enzyme on CaCO 3 deposits on the body surface [17].
Salting after the first boiling limits desiccation, minimizes weight and length losses during processing, and preserves the product by limiting microbial growth [6,17,22,25]. According to Purcell [24], poorly salted bêche-de-mer is a regular problem for many exporters in Fiji, Kiribati, and New Caledonia, and many processing methods assessed in this study also resulted in poorly salted products. Although processors use solid salt and aqueous salt as salting media, aqueous salt results in the decolored processed products, especially in B. vitiensis and S. naso. The erosion of the external body surface of sea cucumbers due to long-term contact with an aqueous (water) medium could be a possible reason for this observation.
Although many methods, i.e., smoke drying, freeze drying, microwave drying, and cabinet (electric) drying [14,17,22], are used to dry the processed sea cucumbers, sun drying is the most widely used method [6,12,17,22]. Sri Lankan processors widely practice both sun drying and electric drying methods by considering the cost-effectiveness and practical applicability of these methods. Even though fast and convenient drying can be obtained by electric dryers, the sun-dried products of H. spinifera and S. naso were given high priority by exporters as such products are free from decoloration and unusual shrinkage. However, electric drying seems to be a good method for drying the processed B. vitiensis. Although sun drying is an easy and cost-effective method, there are many disadvantages, such as seasonality due to weather and difficulties in providing optimum drying conditions [22,26]. Such problems can be easily eliminated in cabinet drying, but it is costly and causes some damage to the external appearance of the final products due to overshrinkages of individuals [27].
The proximate compositions of the processed H. spinifera samples were within a similar range to those reported by Nishanthan et al. [12]. As there was no published information on the proximate composition of B. vitiensis and S. naso, it is difficult to compare the present results. This study proved that processing methods have a significant influence on the proximate composition of bêche-de-mer and even a small deviation from the major processing steps is enough to change the proximate composition of the final products. Although exporters' prioritization of bêche-de-mer solely depends on their experience, their selection mostly confirms products with high nutritional value as well as the lowest moisture content.
In conclusion, Sri Lanka uses different combinations of processing steps to process fresh sea cucumbers into bêchede-mer. The results of this study revealed that the product quality and nutritional composition of bêche-de-mer varied with respect to processing practices. Although different combinations of processing steps produce high-quality bêche-de-mer meeting the exporters' satisfaction, most of the practices yield poor-quality products. Therefore, the findings of this study will be useful to understand the effects of different processing practices on product quality and nutritional composition of bêche-de-mer as well as to select suitable processing methods to upgrade the product quality to meet the market demand.