Effect of Dietary Proline on the Growth Performance, Collagen Deposition, and Texture Quality of Sea Cucumbers’ Body Wall (Apostichopus japonicus)

Abstract

Sea cucumber (Apostichopus japonicus) is an important economically cultured species in the northern coastal regions of China. Its body wall is rich in collagen, which directly determines product quality and market value. However, with the expansion of aquaculture scale, issues such as insufficient collagen deposition have led to inconsistent quality among cultured individuals. Therefore, there is an urgent need to improve growth performance and body wall quality through nutritional regulation. As functional nutrients, amino acids play key roles in collagen synthesis, yet relevant research on A. japonicus remains limited. This study was conducted to investigate the effects of dietary proline on the growth performance, body wall collagen deposition and organoleptic quality of sea cucumber (initial body weight: 30.20 ± 2.02 g). Three kinds of feed with equal levels of nitrogen and other lipids, and supplemented with different concentrations of proline (0%, 1.5%, 3%) in the basal diet, were named P0, P1.5, and P3, and the experiment was conducted for 60 days. The results showed that supplementation with 3% proline significantly increased final body weight and weight gain rate (p < 0.05), reaching 66.39 g and 115.30%, respectively. Collagen content in the body wall increased by approximately 18.5% compared to the control group. Histological analysis of the body wall showed that the collagen fibers in the P1.5 and P3 groups were thicker, with an increased proportion of type I collagen. Texture profile analysis indicated that hardness, cohesiveness, and chewiness were significantly improved in the P3 group (p < 0.05). In summary, supplementation with 1.5% and 3% proline effectively enhanced growth, collagen deposition, and body wall quality. Compared to the P0 group, the relative expression levels of collagen type I alpha 2 chain (COL1A1), Sma- and Mad-related protein 1(SMAD1), and sp-smad2/3 (SMAD2/3) in the body wall tissue were significantly upregulated in both the P1.5 and P3 groups (p < 0.05).

1. Introduction

Sea cucumber (Apostichopus japonicus) is an invertebrate belonging to the class Holothuroidea of the echinoderms, naturally distributed in the northern part of the West Pacific, including the Far East coast of Russia, Japan, Korea, and the Yellow and Bohai Seas of China [1,2]. In 2022, A. japonicus production in China reached 248,508 tons, making it one of the most valuable mariculture species in coastal provinces such as Liaoning, Shandong, and Fujian, with significant contributions to local economies [3]. A. japonicus, as a premium sea food product, is characterized by high protein content and rich bioactive compounds including collagen, polysaccharides, and saponins, which has been proven to possess immunomodulatory, anti-inflammatory, antioxidant, and health-promoting properties [4,5]. Due to its high nutritional value, it is gradually receiving more attention. With the enhancement of people’s health and wellness awareness, the demand for sea cucumbers is increasing, and sea cucumber farming has become an important part of China’s aquaculture industry [6].

The body wall is the main edible part of the sea cucumber, with collagen being the primary component of the body wall [7]. Collagen, as the main component of connective tissue, affects the functional and structural characteristics of muscles [8]. The content and structure of collagen in the body wall not only affect its texture but also determine the soaking ratio of the sea cucumbers. Nutritional regulation technology has proven to be an efficient and effective solution to enhance the quality of fresh and processed aquatic products. It was found that appropriate supplementation of certain amino acids effectively increased the collagen content and texture quality of muscle in several fish species. The addition of taurine in feeds can improve the growth performance, muscle composition, digestive enzyme activity, and stress resistance of white shrimp (Litopenaeus vannamei) [9]. The addition of hydroxyproline in feeds can improve the growth performance and muscle hardness of Nibe croaker (Nibea diacanthus) [10]. Proline is the only α-imino acid that constitutes collagen and plays an important role in maintaining the structural integrity of the protein framework [11]. Studies have reported that proline, as a functional amino acid, can regulate the synthesis of collagen and promote the repair of damaged tissues of animals [12]. Studies have shown that the combination of proline and vitamin C in feeds can improve the hardness and collagen content in the muscles of the large yellow croaker (Larimichthys crocea). Rong et al. [13,14] found that adding proline to the feeds improved the crude protein content and antioxidant capacity in the body of the Yellow Drum (Nibea coibor), and affected the expression of genes related to collagen synthesis in the swim bladder tissue, involving type I collagen and prolyl 4-hydroxylase (P4H). Deng et al. [15] found that adding proline to the feeds improved the growth performance and collagen synthesis of juvenile Chinese soft-shelled Turtles (Pelodiscus sinensis).

In recent years, increasing attention has been paid to the molecular regulatory mechanisms underlying collagen synthesis in the body wall of sea cucumbers. The biosynthesis of collagen involves the coordinated regulation of key enzymes such as prolyl 4-hydroxylase (P4H) and lysyl oxidase (LOX), which catalyze hydroxylation and cross-linking of procollagen, playing a decisive role in collagen fiber formation and deposition [16]. Among the signaling pathways involved, the transforming growth factor-β (TGF-β)/Smads pathway is considered a central regulatory axis. Activation of TGF-β receptors promotes the phosphorylation of Smad2/3, which subsequently translocate into the nucleus to regulate the transcription of collagen synthesis-related genes, thereby facilitating collagen deposition [17,18]. For example, Wang et al. reported that dietary supplementation with vitamin E significantly increased collagen content in the body wall of A. japonicus by enhancing antioxidant capacity, upregulating the expression of TGF-β1, Smad2, and Smad3, and downregulating the inhibitory factor Smad7, indicating that vitamin E promotes collagen synthesis and deposition through activation of the TGF-β/Smads pathway [19]. These findings not only reveal the molecular mechanisms of collagen deposition but also provide a theoretical basis for nutritional regulation of body wall quality in sea cucumbers. However, there is currently a lack of studies elucidating the effects of dietary proline on collagen synthesis and the transcriptional regulation of collagen-related genes in A. japonicus.

Although numerous studies have been conducted extensively on the role of proline in collagen synthesis in aquatic animals, the effects of proline on the quality of the body wall of sea cucumbers are not yet fully understood. Therefore, this experiment aimed to investigate the effects of adding different concentrations of proline to the feeds on the growth performance of sea cucumbers, the content and structure of collagen, the organoleptic quality, and the expression of relevant collagen synthesis genes in the body wall of sea cucumbers.

2. Materials and Methods

2.1. Ethics Statement

In this study, sea cucumbers were successfully bred in a controlled hatchery environment. All experimental procedures strictly adhered to China’s national ethical guidelines and the research protocols established by Dalian Ocean University.

2.2. Experimental Diets and Feeding Experiment

In this experiment, defatted fish meal, Sargassum thunbergii meal, and fermented soybean meal were used as the main sources of protein. Different ratios of L-Proline and L-Serine (provided by Shanghai Sangon Biotechnology, Shanghai, China purity ≥ 98.5%) were added to make three experimental feeds with graded levels of proline (0, 1.5%, and 3%). The feed formula and nutritional composition are presented in

Table 1. Ingredients and proximate analysis of experimental diets (%).

Ingredient	Dietary Proline Levels (% Dry Diet)
	0		1.5		3
Skim Fish Meal 1	2		2		2
Sargassum Thunbergii Meal 2	28		28		28
Fermented Soybean Meal 3	12		12		12
Proline	0		1.5		3
Serine	3		1.5		0
Vitamin Premix 4	0.5		0.5		0.5
Mineral Premix 5	0.5		0.5		0.5
Ca(H2PO4)2 6	2		2		2
Sea Mud	52		52		52
Proximate composition
Crude Protein (%)	9.52		9.56		9.48
Crude lipid (%)	0.12		0.11		0.13

Note: ¹ Skim fish meal: crude protein 68.7% dry matter, crude lipid 2.6% dry matter. Dalian Xinyulong Marine Biological Seed Technology Co., Ltd. (Dalian, Liaoning Province, China). ² Sargassum thunbergii meal: crude protein 16.8% dry matter, Dalian Xinyulong Marine Biological Seed Technology Co., Ltd. (Dalian, Liaoning Province, China). ³ Fermented Soybean meal: crude protein 51.56% dry matter, crude lipid 0.9% dry matter. Dalian Xinyulong Marine Biological Seed Technology Co., Ltd. (Dalian, Liaoning Province, China). ⁴ Vitamin premix: provided by Dalian Xinyulong Marine Biological Seed Technology Co., Ltd. (Dalian, Liaoning Province, China). ⁵ Mineral premix: provided by Dalian Xinyulong Marine Biological Seed Technology Co., Ltd. (Dalian, Liaoning Province, China). ⁶ Ca(H₂PO4)₂: Source of available phosphorus and calcium.

.

All solid ingredients were carefully ground into a fine powder and then passed through an 80-mesh sieve. L-proline and L-serine were first proportionally mixed. Then, other feed ingredients were weighed, added sequentially and mixed evenly with the mixture of proline and serine. Subsequently, the experimental feeds were sealed in plastic bags and stored at −20 °C until use. A measured portion of the feed was mixed with 30% distilled water to achieve the right consistency. The mixture was then kneaded into spherical solid pellets, each approximately 1.5 cm in diameter.

The feeding trial was conducted at the Key Laboratory of Mariculture and Stock Enhancement in the North China Sea, Ministry of Agriculture and Rural Affairs, Dalian Ocean University. Experimental A. japonicus were obtained from Dalian Xinyulong Marine Biotechnology Co., Ltd. Prior to the trial, individuals were acclimated for two weeks in a controlled aquaculture system and provided with a standard basal diet to ensure uniform health status and adaptation to experimental conditions. Following a 36 h fasting period, 180 healthy individuals were randomly assigned to nine 160 L square tanks (20 per tank), with an initial total biomass of approximately 500 ± 1 g per group. The rearing experiment lasted 80 days. A. japonicus were fed twice (07:00 and 17:00) every day. For each tank, feeds were allocated to animals at the amount of 2% of their body weight. The A. japonicus in each tank were weighed and the feeding amount was accordingly adjusted every week. During the experiment, the water temperature was maintained at 19-21 °C, salinity at 30 ± 1 ‰, pH at 8.0 ± 0.1, dissolved oxygen above 8.0 mg/L, ammonia concentration below 0.2 mg/L, and nitrite concentration below 0.1 mg/L. One-third of the water volume was exchanged daily, and residual feed and feces were removed every other day. Tanks were maintained under dim illumination to simulate the natural habitat.

2.3. Sample Collection

Upon completion of the feeding trial, A. japonicus species were subjected to a 48 h fasting period. All individuals in each tank were then counted, measured, and weighed to calculate survival rate, weight gain rate, and body wall mass. Coelomic fluid was extracted from six randomly selected specimens per tank. For histological examination, the excised body wall tissues were fixed in 4% paraformaldehyde. A section from the mid-dorsal region of the body wall was trimmed into approximately 1.5 cm × 1.5 cm × 1.5 cm cubes for texture profile analysis. Body wall and intestinal samples were collected, placed in 1.5 mL RNase-free tubes (Axygen, Union City, CA, USA), and stored at −80 °C for subsequent proximate composition analysis.

2.4. Feed Composition Analysis

The proximate composition of the experimental diets was analyzed according to AOAC (1995) [20]. The crude protein (CP) content of the diets was determined according to the Kjeldahl method. Briefly, approximately 1.0 g of finely ground sample was digested with sulfuric acid. The digest was then neutralized with sodium hydroxide, and the released ammonia was distilled into a boric acid solution and subsequently titrated with standardized hydrochloric acid. The nitrogen content was calculated, and the crude protein was expressed as nitrogen × 6.25. The crude fat (ether extract, EE) was measured following the Soxhlet extraction method. About 2.0 g of dried sample was wrapped in filter paper and extracted with petroleum ether in a Soxhlet apparatus for 6 h. After extraction, the solvent was removed by evaporation, and the residue was dried to a constant weight at 105 °C. The crude fat content was calculated as the percentage of residue weight relative to the sample weight.

2.5. Collagen Contents Analysis

Hydroxyproline (Hyp) content in the body wall was quantified using a commercial assay kit (Art. No. A030-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Absorbance was measured at 550 nm using a microplate reader (Infinite^® Pro 200, Tecan, Männedorf, Switzerland). Collagen content was calculated as Hyp × 8, as described by AOAC (2002) [21].

2.6. Amino Acid Contents Analysis

Amino acid composition of the body wall was determined following the AOAC (2002) standard procedure with minor modifications. Briefly, freeze-dried body wall samples (approximately 100 mg) were hydrolyzed with 6 mol/L HCl at 110 °C for 24 h under a nitrogen atmosphere. After hydrolysis, the samples were neutralized, filtered, and diluted to a fixed volume with ultrapure water. The amino acid contents were analyzed using an automatic amino acid analyzer (e.g., Hitachi L-8900, Tokyo, Japan) [21]. Based on the results, amino acids were classified as essential amino acids (EAAs) or non-essential amino acids (NEAAs). EAAs—including lysine, methionine, threonine, valine, isoleucine, leucine, phenylalanine, and histidine—must be supplied through the diet, as they cannot be synthesized de novo. NEAAs, such as glycine, glutamic acid, aspartic acid, alanine, and proline, can be synthesized endogenously. Arginine was considered a conditionally essential amino acid in aquatic animals, particularly during early developmental stages or under certain stress conditions.

2.7. Histological Staining of the Body Wall

Body wall tissues were fixed in 4% paraformaldehyde at room temperature for 24 h, dehydrated through a graded ethanol series, cleared with xylene, embedded in paraffin, and sectioned using a rotary microtome (Leica RM2016, Leica Microsystems GmbH, Wetzlar, Germany). Hematoxylin–eosin (HE) staining was conducted according to standard protocols, and sections were mounted with neutral balsam. Van Gieson and Sirius Red staining followed identical sample preparation steps. Tissue morphology was examined under an upright light microscope (ECLIPSE E100, Nikon, Japan), and collagen distribution after Sirius Red staining was visualized using a polarizing microscope (ECLIPSE Ci, Nikon, Japan).

2.8. Characterization of Water Absorption of the Body Wall

Water absorption, also referred to as the soaking ratio, represents the percentage increase in body wall weight following soaking under standardized conditions and serves as an indicator of the water-holding capacity of the sea cucumber body wall. The soaking ratio of sea cucumbers is a crucial parameter reflecting the extent of expansion of dried sea cucumbers upon rehydration, and it directly indicates their quality and economic value. Sea cucumbers with higher soaking ratios generally exhibit plump flesh and abundant collagen, making them more desirable in the market, while also affecting processing costs and sensory attributes [22]. Fresh body wall samples were boiled in tap water at a ratio of 1:4 (w:w) for 30 min, cooled, and weighed. They were then immersed in deionized water at a ratio of 1:10 (w:w) and stored at 4 °C for 48 h. Final weights after soaking were recorded, and water absorption capacity was calculated accordingly.

2.9. Texture Profile Analysis (TPA)

TPA was performed using a texture analyzer (TMS-Pro, Beijing Yingsheng Hengtai Technology Co., Ltd., Beijing, China) equipped with a P20 cylindrical probe. Tests were conducted at room temperature with pre-test, test, and post-test speeds of 0.50 mm/s, a compression ratio of 75%, and a trigger force of 0.5 g.

2.10. Real-Time PCR (RT-PCR) of Collagen Synthesis-Related Genes

The expression pattern of collagen synthesis-related genes in the body wall was detected with RT-PCR using the specific primers. The sequences of specific primers are present in

Table 2. Real-time PCR primers used in the present study.

Genes	Gene ID	Annealing Temperature (°C)	Primer Sequences (5′-3′)
Col1A2 1	c54738.graph_c0	56	F:5′-CGGACTTTTACTTTGGCGTTAT-3′ R:5′-TTTCTGGCGGTCTGCCTAT-3′
TGF-β 2	BSL78_20284	59	F:5′-ACCGCTCCTCACCCTTTAACAC-3′ R:5′-CCACTAGCACTAAGCAGCATATCAG-3′
SMAD1 3	BSL78_15508	58	F:5′-GGCATACTCGCAGCAGTCTAAAG-3′ R:5′-AGGTGTCTCATCGGAAAGGTCTAC-3′
SMAD2/3 4	BSL78_11878	59	F:5′-AGAACCACCACGAACTCAAACATG-3′ R:5′-GCAGACAGCAGCAGGGATAAAC-3′

Note: ¹ Col1A2, collagen type I alpha 2 chain. ² TGF-β, transforming growth factor beta. ³ SMAD1, Sma- and Mad-related protein 1. ⁴ SMAD2/3, sp-smad2/3.

. The detailed procedures are described in the study by Zuo et al [23].. Briefly, total RNA was extracted and examined for concentration and integrity of RNA. Then, Total RNA was reverse transcribed using a Commercial kit (TaKaRa, Beijing, China). LightCycler^® 96 real-time PCR system was used, and the relative gene expression was determined by using the 2^−ΔΔCT [24].

2.11. Calculations and Statistical Analysis

$$\mathrm{Survival}\mathrm{rate}(\mathrm{SR},\%)=N_f/N_i\times 100$$

$$\mathrm{Weight}\mathrm{growth}\mathrm{rate}(\mathrm{WGR},\%)=\left(W_f-W_i\right)/W_i\times 100$$

$$\mathrm{Body}\mathrm{wall}\mathrm{index}(\mathrm{BWI},\%)=P_W/W\times 100$$

$$\mathrm{Expansion}\mathrm{multiple}\left(\mathrm{EM}\right)=W_r/W_0$$

where $N_i$ and $N_f$ represent the initial and final numbers of A. japonicus in each tank, respectively; $W_i$ and $W_f$ denote the initial average weight and final average weight; $P_W$ is the wet weight of the body wall, and $W$ is the total wet body weight; $W_0$ and $W_r$ correspond to the wet mass of the body wall before and after soaking in water, respectively.

Statistical analyses were performed using SPSS 22.0 (IBM, Armonk, NY, USA). Data were subjected to one-way analysis of variance (ANOVA), and significant differences among dietary treatments (p < 0.05) were identified using Tukey’s post hoc test.

3. Results

3.1. Growth Performance

There were no significant differences in survival rates among the different dietary groups (p > 0.05). As the dietary proline level increased, the WGR and body wall weight significantly increased (p < 0.05). When the dietary proline supplementation level increased to 3%, the WGR of A. japonicus reached 115.30%, which was significantly higher than those of the other two groups (p < 0.05). As the dietary proline level increased, the body length of A. japonicus significantly decreased (p < 0.05), while the weight of the body wall significantly increased (p < 0.05) (

Table 3. Effects of proline addition levels on the growth performance of sea cucumber (Apostichopus japonicus).

	Dietary Proline Levels (% Dry Diet)
	0	1.5	3
Survival Rate (%)	100.00	100.00	100.00
Initial Body Weight (g)	30.40 ± 2.03	30.02 ± 2.06	30.35 ± 3.49
Final Body Weight (g)	55.54 ± 12.46	60.92 ± 11.08	66.39 ± 19.85
Weight Growth Rate (%)	82.20 ± 34.57 a	101.76 ± 25.09 ab	115.30 ± 42.34 b
Body Wall Weight (g)	34.53 ± 9.16 a	40.34 ± 8.85 ab	42.16 ± 10.89 b
Body Wall Index (%)	69.36 ± 6.37	69.80 ± 5.94	67.93 ± 6.59

Note: Data with different lowercase letters in the same horizontal column are significantly different between the various diet groups (p < 0.05).

).

3.2. Collagen Content and Structure

The supplementation of proline in diets had a significant effect on collagen deposition in the body wall of A. japonicus. As the dietary proline levels increased, the collagen content in the body wall of sea cucumbers initially increased and then decreased. However, the collagen content in the two experimental groups was significantly higher than that in the control group (p < 0.05) (Figure 1).

Following staining, the body wall tissues of A. japonicus were observed under a polarized light microscope. Type I collagen fibers exhibited strong birefringence, appearing as thick red or yellow fibers, whereas Type III collagen fibers displayed weaker birefringence, appearing as thin green fibers. In the proline-supplemented groups, the collagen fibers in the body wall of sea cucumbers were markedly thicker, with a significant increase in the number of Type I collagen fibers (Figure 2).

After staining, collagen fibers appear pink, while muscle fibers are displayed in yellow. Proline supplementation in diets induced notable structural changes in the body wall tissue of A. japonicus. Compared to the control group, the collagen fiber layer in the group with 1.5% proline was thicker and more densely arranged. In contrast, the A. japonicus in the group with 3% proline showed a reduction in the thickness of both the cuticle and epidermis layers compared to those in the other two groups (Figure 3).

3.3. Amino Acid Profile

After appropriate supplementation of proline in the diet, the content of proline, a characteristic amino acid of collagen, in the body wall of A. japonicus showed significant changes. When the dietary proline supplementation level reached 3%, the contents of proline, glycine, and alanine in the body wall of A. japonicus significantly increased (p < 0.05), reaching 9.67%, 13.96%, and 6.90%, respectively. Overall, glutamic acid was the most abundant amino acid in the body, followed by glycine (

Table 4. Effects of proline addition levels on amino acid contents of sea cucumber (Apostichopus japonicus) (% dry matter basis).

Amino Acid	Dietary Proline Levels (% Dry Diet)
	0	1.5	3
Aspartic	8.17 ± 0.47	8.52 ± 0.26	8.15 ± 0.50
Glutamic	15.47 ± 0.44 b	15.63 ± 0.75 b	14.35 ± 0.62 a
Serine	4.90 ± 0.08	4.83 ± 0.31	4.69 ± 0.17
Glycine	11.49 ± 1.66 a	9.92 ± 0.93 a	13.96 ± 1.87 b
Histidine	1.99 ± 0.17 b	2.07 ± 0.14 b	1.71 ± 0.05 a
Argnine	10.14 ± 0.45	9.81 ± 0.53	9.57 ± 0.34
Threonine	5.23 ± 0.19	5.15 ± 0.37	5.21 ± 0.12
Alanine	6.76 ± 0.30 a	6.37 ± 0.18 a	6.90 ± 0.44 b
Proline	8.67 ± 0.37 a	8.40 ± 0.50 a	9.67 ± 0.54 b
Tyrosine	3.40 ± 0.16 b	3.53 ± 0.12 ab	3.25 ± 0.09 a
Valine	4.26 ± 0.07	4.41 ± 0.04	4.36 ± 0.19
Methionine	1.49 ± 0.44 a	2.12 ± 0.41 ab	1.76 ± 0.56 b
Isoleucine	3.76 ± 0.12 b	3.96 ± 0.10 c	3.57 ± 0.18 a
Leucine	0.30 ± 0.12 b	0.29 ± 0.12 b	0.44 ± 0.18 a
Phenylalanine	3.09 ± 0.12 ab	3.21 ± 0.08 b	2.98 ± 0.19 a
Lysine	5.18 ± 0.44 b	5.74 ± 0.52 b	4.40 ± 0.64 a

Note: Data with different lowercase letters in the same horizontal column are significantly different between the various diet groups (p < 0.05).

).

With the increasing dietary proline levels, both the content of EAAs in the body wall of A. japonicus and the ratio of EAAs to NEAAs (EAA/NEAA) initially increased and then decreased. Compared with the other two groups, the P3 group exhibited a significant reduction in both EAA content and the EAA/NEAA (Figure 4). The EAA/NEAA ratio first slightly increased at proline levels below 1.5%, likely due to adequate support for collagen synthesis without altering other amino acids. At higher levels (>1.5%), excess proline was primarily used for collagen production, causing accumulation of non-essential amino acids and a relative decrease in essential amino acids, thereby significantly lowering the EAA/NEAA ratio [25].

3.4. Organoleptic Quality

Dietary proline supplementation had a significant effect on the water adsorption of A. japonicus after boiling. As the level of proline addition in the feed increased, the water adsorption of the sea cucumber body wall initially increased and then decreased. The water adsorption of the body wall in the experimental groups was significantly higher than that in the control group (p < 0.05) (Figure 5).

Dietary proline supplementation had a significant effect on the textural properties of A. japonicus. With increasing levels of proline in the diet, the hardness, cohesiveness, chewiness, and adhesiveness of the sea cucumber body wall were all significantly enhanced (p < 0.05), reaching maximum values at a 3% proline supplementation level. In addition, no significant differences were observed in elasticity among the three dietary groups (p > 0.05) (

Table 5. Effects of proline addition levels on the texture profile analysis of sea cucumber (Apostichopus japonicus).

	Dietary Proline Levels (% Dry Diet)
	0	1.5	3
Hardness 1	2.80 ± 0.08 a	3.32 ± 0.73 ab	3.76 ± 0.55 b
Springiness 2	2.64 ± 0.33	2.84 ± 0.29	2.78 ± 0.27
Cohesiveness 3	0.80 ± 0.05 a	0.83 ± 0.03 ab	0.85 ± 0.03 b
Chewiness 4	5.86 ± 1.03 a	7.79 ± 2.44 ab	8.90 ± 1.88 b
Gumminess 5	2.21 ± 0.16 a	2.72 ± 0.70 ab	3.18 ± 0.46 b

Note: ¹ Hardness (N) was defined as the maximum peak value when compressing the sample for the first time. ² Springiness (mm) was defined as the degree to which the sample can be recovered after the first compression. ³ Cohesiveness (Ratio)was defined as the adhesion within the sample. ⁴ Chewiness (mJ) was defined as the work required to chew a solid sample (springiness × gumminess). ⁵ Gumminess (N) was defined as the viscosity characteristics of semi-solid samples (hardness × cohesiveness). Data with different lowercase letters in the same horizontal column are significantly different between the various diet groups (p < 0.05).

).

3.5. Expression of Collagen Synthesis-Related Genes

Compared to the P0 group, the relative expression levels of collagen type I alpha 2 chain (COL1A1), Sma- and Mad-related protein 1(SMAD1), and sp-smad2/3 (SMAD2/3) in the body wall of A. japonicus were significantly upregulated in both the P1.5 and P3 groups (p < 0.05). However, compared to the P1.5 group, the relative expression levels of COL1A1, transforming growth factor beta (TGF-β), SMAD1, and SMAD2/3 were significantly reduced in the P3 group (p < 0.05) (Figure 6).

4. Discussion

Proline, as a non-polar, neutral amino acid with a cyclic structure, is widely present in both plant and animal organisms and serves as an important component of proteins. Traditionally, proline has been classified as a non-essential amino acid in most animals, including aquatic species. However, recent studies have indicated that endogenously synthesized proline could not sufficiently meet the physiological demands of animals, especially during rapid growth, tissue repair, or under the state of environmental stress [26,27]. Therefore, proline is now considered a member of “conditionally essential amino acids”. Increasing evidence suggests that dietary proline supplementation can significantly promote growth in aquatic animals.

Research has shown that proline supplementation significantly promotes growth performance in various aquatic species. Taziki et al. [28] demonstrated that dietary supplementation with 5 g/kg of L-proline, in combination with an equal amount of L-alanine, significantly enhanced the growth performance and survival rate of juvenile common carp (Cyprinus carpio). Additionally, supplementation of 1.0% proline in the diets significantly increased the weight gain rate of large yellow croaker (Larimichthys crocea) [28]. In L.vannamei, dietary supplementation with 2.29–2.34% proline has been found to significantly enhance antioxidant capacity, immune function, and stress resistance, thereby providing a robust foundation for subsequent growth [27]. In addition to proline, hydroxyproline―the major metabolite of proline—has also been shown to exert similar growth-promoting effects in aquatic species, further supporting the functional importance of this amino acid family in aquaculture nutrition. Aksnes et al. (2008) found that the addition of 2.9 g/kg crystalline hydroxyproline to high plant protein-based diets significantly promoted growth and vertebral development of Atlantic salmon (Salmo salar) [29]. In the present study, dietary supplementation with 1.5–3% proline significantly improved the weight gain rate of A. japonicus, which differs markedly from previously reported optimal levels in other aquatic species. This discrepancy may be attributed to the fact that collagen synthesis in the body wall of sea cucumbers relies heavily on proline and hydroxyproline as precursors, resulting in a higher dependence on exogenous proline. Additionally, the basal diet used in this experiment was primarily composed of plant-based protein sources, which may have contained lower intrinsic levels of proline, thereby increasing the requirement for dietary supplementation. Moreover, variations in proline utilization efficiency, differences among species, and the growth stage of the animals may also contribute to the observed differences.

Collagen is one of the most abundant structural proteins in living organisms, widely distributed in the skin, bones, blood vessels, and connective tissues [30]. Proline is not only an essential amino acid component of collagen molecules but also plays a critical role in the hydroxylation of collagen precursors [16,31]. Numerous studies have demonstrated that appropriate dietary supplementation of proline can significantly promote collagen deposition in aquatic animals, thereby improving muscle quality and tissue structure. In yellow drum (Nibea coibor), dietary proline supplementation significantly enhanced collagen synthesis and deposition, and muscle elasticity [32]. Similarly, in triploid crucian carp (Carassius auratus), proline supplementation not only significantly promoted body weight gain and improved feed utilization but also notably increased collagen content in the dorsal muscle [33]. In olive flounder (Paralichthys olivaceus), although proline supplementation did not significantly enhance growth performance, it markedly increased collagen content in the muscle tissue [34]. These findings confirm the promoting effect of proline on collagen synthesis in aquatic animals. In shrimp, dietary proline has been shown to significantly increase collagen content in both muscle tissue and the exoskeleton [35]. The body wall of sea cucumbers is primarily composed of collagen and proline, which as a key substrate in collagen biosynthesis, can promote collagen deposition, thereby enhancing body wall hardness and elasticity. In the present study, appropriate proline supplementation in diets significantly increased the collagen content in the body wall of A. japonicus, particularly in the 1.5% proline supplementation group, where collagen deposition was approximately 14% higher than that of the control group. This suggests that proline supplementation can directly promote the synthesis and deposition of collagen in the body wall of A. japonicus.

Proline is one of the primary amino acids constituting collagen and plays a critical role in the process of collagen synthesis [36,37]. As a structural protein, collagen is widely distributed in the skin, bones, muscles, and connective tissues, and its contents directly influence muscle hardness, elasticity, and ultimately, market quality of aquatic animals [38]. Textural properties are essential indicators for evaluating food quality, as texture analysis provides insights into attributes such as mouthfeel, chewiness, and overall sensory experience, which are crucial for quality control and consumer satisfaction [39,40]. Proline not only serves as a necessary precursor for collagen synthesis but also contributes to increasing the degree of intermolecular cross-linking within collagen, thereby enhancing its stability and resistance to degradation. This, in turn, improves muscle hardness and elasticity in aquatic animals, ultimately enhancing flesh firmness and mouthfeel [41]. In the present study, the elasticity of the body wall of A. japonicus in the proline-supplemented groups improved to some extent, although there was no significant difference compared to the control group. Meanwhile, the hardness, cohesiveness, chewiness, and adhesiveness of the body wall increased significantly with the increase of dietary proline. This was consistent with the results of Wang et al. [42] who found that appropriate myo-inositol(MI) levels increased flesh texture quality by increasing muscle hardness, adhesive force, springiness, resilience and chewiness. The proportion of type I and type III collagen fibers could influence the texture characteristics of the body wall of A. japonicus. The hardness of the body wall of A. japonicus is positively correlated with its type I collagen content [43]. In the periodontal ligament, the tissue’s resistance to deformation is positively correlated with its collagen fiber content [44]. Similarly, in bone tissue, mechanical strength and deformation resistance are enhanced when collagen fibers are organized in a denser and more orderly manner [45]. In this study, Sirius Red staining, a technique specific for labeling collagen fibers, demonstrated that the collagen fibers in the experimental groups were markedly thicker than those observed in the control group, accompanied by a significant increase in the proportion of type I collagen fibers. Furthermore, Van Gieson staining corroborated these findings, revealing a denser arrangement of collagen fibers in the experimental groups. Collagen fibers are formed by the self-assembly of collagen triple-helical molecules into microfibrils, which subsequently aggregate into thicker fibers [46]. Therefore, when the concentration of collagen molecules increases or the fibers are more tightly aligned, lateral associations between microfibrils may occur, leading to the formation of thicker fibers. Alternatively, an increase in intra- and intermolecular cross-linking can stabilize the interactions between microfibrils, thereby increasing fiber diameter. In addition, elevated levels of extracellular matrix proteins may promote tighter alignment and aggregation of collagen fibers.

In the present study, dietary supplementation with 1.5% proline significantly upregulated the expression of COL1A1, SMAD1, and SMAD2/3 genes in the body wall of A. japonicus, thereby promoting collagen synthesis. COL1A1 encodes the α1 chain of type I collagen, a fundamental structural component of collagen fibers that plays a critical role in maintaining tissue integrity and elasticity. Proline contributes to collagen synthesis through dual mechanisms, functioning both as a structural amino acid in procollagen and as a co-substrate for prolyl-4-hydroxylase (P4H) that catalyzes the formation of hydroxyproline, a modification essential for stabilizing the collagen triple-helix structure [19,47]. Previous studies have demonstrated that supplementation with proline or its derivatives can significantly enhance COL1A1 transcription. For instance, in spotted drum, dietary proline has been shown to upregulate COL1A1 expression and facilitate collagen deposition [48]. In the present study, COL1A1 expression in the P1.5 group was significantly higher than that in the control group, supporting the conclusion that proline promotes the transcription and synthesis of procollagen.

It is widely acknowledged that the TGF-β/Smad signaling pathway serves as a central regulatory network governing collagen synthesis [49]. Upon TGF-β activation, receptor-regulated SMAD2/3 are phosphorylated and subsequently form a complex with co-Smad (SMAD4), which translocates into the nucleus to activate target genes such as COL1A1 [18,50]. In fish studies, dietary proline has been reported to activate SMAD2/3 expression and enhance collagen deposition via the TGF-β signaling pathway. For example, in spotted drum, increased SMAD2 expression was associated with elevated collagen content in tendons and the swim bladder. Furthermore, studies suggest that proline not only upregulates SMAD2/3 transcription but may also enhance their activation, thereby facilitating COL1A1 transcription [48]. In the present study, the expression levels of SMAD2/3 in the P1.5 group were significantly higher than those in the control group, accompanied by elevated SMAD1 expression. SMAD1, a receptor-regulated Smad in the BMP signaling pathway, may also participate in collagen gene regulation through crosstalk with the TGF-β pathway. Studies in dermal fibroblasts have shown that SMAD1 and SMAD2/3 can synergistically regulate collagen gene expression downstream of TGF-β receptor activation [51,52,53]. Our findings suggest that the upregulation of SMAD1 and SMAD2/3 in the body wall of sea cucumber under 1.5% proline supplementation may enhance Smad signal transduction, thereby promoting the transcription of collagen-related genes.

In this study, TPA parameters and tissue amino acid contents generally increased with dietary proline levels up to 3.0%, whereas the most pronounced improvements in histological features and gene expression were observed in the 1.5% proline group. Notably, collagen content and gene expression were measured in fresh body wall samples, while TPA parameters were assessed in rehydrated samples after processing. This methodological difference may partly explain the observed divergence, as rehydration can alter water content and the spatial structure of collagen, thereby affecting TPA parameters such as hardness and chewiness [54,55,56]. Furthermore, the disparity between indicators can also be attributed to their underlying physiological response mechanisms. TPA parameters and amino acid contents primarily reflect the accumulation of terminal metabolic products, showing a linear or near-linear response to increasing proline levels [57]. In contrast, tissue morphology and gene expression are subject to physiological regulation and feedback control, reaching optimal activation at specific proline levels, with higher levels potentially causing saturation or negative feedback inhibition [58]. Temporal differences in response also play a role, improvements in TPA parameters and amino acid contents reflect long-term cumulative effects, whereas changes in gene expression and tissue morphology represent more immediate physiological responses.

The findings of this study can provide important insights for the optimization of formulated feeds to produce high quality sea cucumbers characterized by more collagen deposition and better texture quality.

5. Conclusions

In conclusion, dietary supplementation with 1.5% proline in A. japonicus feed upregulated the expression of collagen synthesis-related genes, including COL1A1, SMAD1, and SMAD2/3, thereby enhancing growth performance and promoting hydroxyproline synthesis. Furthermore, the 1.5% proline supplementation facilitated collagen deposition, improved muscle structure, increased collagen fiber density, and enhanced the rehydration ratio of the body wall, ultimately leading to an improvement in the overall quality of A. japonicus. Based on the outcomes related to growth performance, collagen content in the body wall, rehydration ratio, textural properties, and the relative expression levels of collagen-related genes, a dietary proline supplementation level of 1.5% was found to be more beneficial for the growth of A. japonicus.