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Article

Influence of the Silkworm-Derived (Bombyx mori) Functional Substance (Silkrose-BM) on the Fish Meat Quality of Yellowtail (Seriola quinqueradiata)

by
Athira Athira
1,2,
Haruki Nishiguchi
2,
Daichi Hayashi
2,
Yuki Otsu
2,
Chiemi Miura
2,
Ibnu Bangkit Bioshina Suryadi
2,3,
Muhammad Fariz Zahir Ali
2,4 and
Takeshi Miura
2,*
1
Department of Fisheries, Faculty of Marine Science and Fisheries, Universitas Hasanuddin, Jl. Perintis Kemerdekaan km 10, Makassar 90245, Indonesia
2
Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan
3
Department of Fisheries, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jl. Ir. Soekarno km 21, Sumedang 45363, Indonesia
4
Research Center for Marine and Land Bio Industry, National Research and Innovation Agency, Jl. Raya Senggigi, Kodek Bay, Pemenang 83352, Indonesia
*
Author to whom correspondence should be addressed.
Fishes 2025, 10(3), 130; https://doi.org/10.3390/fishes10030130
Submission received: 4 February 2025 / Revised: 5 March 2025 / Accepted: 11 March 2025 / Published: 17 March 2025
(This article belongs to the Special Issue Advances in Aquaculture Feed Additives)
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Abstract

Popular foods such as sushi and sashimi depend on the quality of raw fish meat to maintain consumer satisfaction. Recently, dietary insect meal and insect-derived substances have been extensively studied for application in aquaculture as a protein alternative or immunostimulant. However, the impact of insect functional substances on the fish meat quality of teleosts remains unclear. Here, we investigated the influence of dietary inclusion of silkrose-BM, a novel bioactive polysaccharide derived from the silkworm, Bombyx mori, on the meat quality of yellowtail (Seriola quinqueradiata). This study was conducted by comparing two groups given different feeds, commercial EP and feeds containing Silkrose-BM (0.1%), after a culture period of six months in separate floating-net cages. The yellowtail were specifically cut into loins and several meat quality parameters were observed, including proximate, meat color changes, total collagen, drip loss, muscle histology, and gene expression (qRT-PCR). The results of the color-change analysis showed that discoloration of red muscle in the EP feed group occurred faster than in the silkrose-BM group, indicating an antioxidant property of silkrose-BM. Dietary silkrose-BM also significantly reduced drip loss and increased the total collagen content of yellowtail meat. Furthermore, qRT-PCR analysis showed that genes related to lipid and protein degradation were downregulated in the muscles of fish fed on silkrose-BM. In contrast, proximate analysis indicated no significant change in the nutritional composition of the meat between the groups. Taken together, our results suggest that dietary silkrose-BM could improve fish meat quality by minimizing protein denaturation and inhibiting lipid oxidation during fish meat storage.
Keywords:
fish meat quality; insects; silkworm; silkrose; polysaccharide
Key Contribution: Bioactive polysaccharide from silkworm: oral administration of silkrose-BM was shown to be effective in improving the meat quality of cultured yellowtail.

1. Introduction

Maintaining the quality of fish meat is crucial, as it is a perishable food. Quality indicators of fish meat can have a major impact on consumer preferences, so it is essential to maintain its quality at the highest possible level [1]. Raw fish meat is highly valued worldwide, for example in popular foods such as sushi and sashimi. In Japan, these dishes traditionally use yellowtail (Seriola quinqueradiata), specifically ‘hamachi’ (young yellowtail) and ‘buri’ (mature yellowtail) as the main ingredients. Fish meat is highly nutritious, containing macronutrients and micronutrients that are important for humans, such as protein and fat, as well as some carbohydrates, vitamins, and minerals. However, the overall quality of fish meat, including its nutritional composition, can be influenced by various factors such as the type of feed and the farming methods used to rear the fish. These factors also impact texture, flavor, and freshness—key attributes that determine the desirability of fish meat that is to be consumed raw. Quality parameters of fish meat that are commonly considered critical in assessing the suitability of raw fish for sushi or sashimi include its color, overall appearance, aroma, wateriness, fattiness, taste, flavor, and texture [2,3].
The use of functional compounds or substances from insects are being increasingly widely studied, and insects are considered to be a promising source of various beneficial compounds to optimize fish health and growth during cultivation and culture [4,5,6,7]. The pupae of silkworm (Bombyx mori) contain many potentially useful compounds, including a bioactive polysaccharide known as silkrose-BM [8]. The use of silkrose-BM in the aquaculture industry has recently been investigated due to its unique advantage in terms of availability as a by-product of silk production. This suggests there may be potential synergies between the aquaculture and sericulture industries in the future [8,9].
Silkrose-BM was initially studied as a feed supplement to improve the resistance of farmed fish to bacterial diseases and Ectoparasites by stimulating their immunity. However, further analysis revealed that its effects go beyond immune stimulation, inducing changes to muscle fiber structure that affect meat quality and enhance stress tolerance, including tolerance to high water temperatures. Silkrose-BM consists of the monosaccharides 1-rhamnose, 1-fucose, 1-arabinosa, d-glucuronic acid, d-mannose, d-glucose, d-galactosa, N-acetyl-D-glucosamine (GlcNAc), and N-acetyl-D-galactosamine (GalNAc) [10,11]. GlcNAc and GalNAc are bioactive monosaccharides that belong to the group collectively known as glycosaminoglycans (GAGs). GAGs are important components of connective tissue structure, including in collagen, which forms the structure of muscle tissue [12,13]. It is likely that GAGs support collagen, which influences the quality of fish meat, particularly in terms of texture and muscle tissue structure.
The application of silkroses (including silkrose-BM and silkrose-AY, the latter derived from the Japanese oak silkmoth, Antheraea yamamai) as feed additives in aquaculture has proven effective in enhancing the immune systems and survival rates of various fish species, such as white trevally (Pseudocaranx dentex) [5], paneids (Litopenaeus vannamei and Marsupenaeus japonicus) [8], and Japanese medaka fish (Oryzias latipes) [14]. However, studies of the effects of a silkrose-BM diet on fish meat quality remain limited. In this study, we aim to investigate the impact of a silkrose-BM feeding treatment on fish meat quality indicators in yellowtail (Seriola quinqueradiata), such as nutritional value, appearance, texture, and gene expression. This study provides important insights into the potential of silkworm-derived functional substances to enhance the quality and market value of aquaculture products.

2. Materials and Methods

2.1. Experimental Animals

One-year-old yellowtail (S. quinqueradiata) were used in this study. They were fed on commercial extruded pellets (EP) until they reached an average body weight of 1.4–1.9 kg (young yellowtail), before being subjected to the silkrose-BM diet treatment.

2.2. Preparation of Experimental Diets

The silkrose-BM was provided by Shintoa Co., Ltd. (Tokyo, Japan). The basal feed used in this experiment was commercial extruded pellets (EP) produced by Sakamoto Feed Co., Ltd. (Choshi, Japan). For the treated group, silkrose-BM was added to the EP feed at a concentration of 0.1% at the time of pellet preparation on consignment by Sakamoto Feed Co., Ltd. The concentration of silkrose-BM at 0.1% was chosen based on the previous studies [5,8]. The feed composition and nutritional profiles are shown in Table 1.

2.3. Fish Culture

The fish were cultured in two separate floating-net cages (10 × 10 × 6 m), in Uwajima, Japan. The number of fish in each cage ranged from approximately 3080 to 3100 individuals. The fish were maintained for six months (March to September), with an average sea water temperature 14 °C in the spring and 24 °C in the summer [15]. The fish were fed twice daily, once in the morning and once in the afternoon, until satisfied.

2.4. Sampling Techniques

At the end of the feeding trial, five fish from each group were randomly selected for sampling. The sampled fish were all females to minimize bias in the interpretation of results due to mixing samples from both sexes, especially for molecular analyses such as qRT-PCR. Different sexes of fish can express distinct genes, which made the determination of the sex of the fish crucial for this study.
Yellowtail are large fish that are typically divided into several sections for consumption. Their ordinary muscle (OM) is categorized by type into dorsal ordinary muscle (DOM), ventral ordinary muscle (VOM), and caudal ordinary muscle (COM) regions. These sections are further processed into loins and then distributed in the marketplace [16]. The sampling techniques used in this study were modified from the methodologies described by Ando et al. [17] and Shioya et al. [18]. The modifications were implemented to align with the specific observed parameters, as shown below (Figure 1).
The proximate composition of yellowtail fish meat varies according to the specific part of the body. Therefore, to analyze the proximate composition of yellowtail fish meat, previous studies have sampled meat from the dorsal, ventral, and caudal sections of the fish and then combined them [19,20,21]. Shioya et al. [18] stated that although the proximate composition in different parts of farmed yellowtail fish varies, sampling the proximate composition from the 7th to the 10th internal vertebrae (musculus latero-dorsalis) provides a comprehensive overview of the overall proximate content. As nutrient content varies in fish meat obtained from different sections of the fish, this sampling technique was adapted to ensure more accurate results and minimize bias from sampling inconsistent or diverse parts of the fish.

2.5. Proximate Analysis

The proximate composition (moisture, crude fat, crude protein, and ash) was analyzed based on the Association of Analytical Communities (AOAC) method [22]. Briefly, the content of crude protein was analyzed with the Kjeldahl method [22]. “Kjeltab” (containing K2SO4) was added to the samples, and the samples were digested in a block heater (TecatorTM Digestion Systems 2520, FOSS, Hilleroed, Denmark). The nitrogen content was analyzed using an auto analyzer (KjeltecTM 8400, FOSS, Hilleroed, Denmark). The crude protein was obtained based on the calculation of the nitrogen content with the nitrogen–protein conversion factor, 6.25. Crude fat was analyzed with the Soxhlet extraction method [22]. Extraction from the samples was conducted with petroleum ether in an Automated extractor (SoxtecTM 8000, FOSS, Hilleroed, Denmark). The content of ash was analyzed with an electric furnace (MMF-1, AS ONE, Osaka, Japan).

2.6. Color Change Analysis

Color changes or discoloration in the red muscle of fish were measured using a chroma meter (CR-400, Konica Minolta Inc., Tokyo, Japan). Color changes were observed for 0, 24, 48 h after storage at 4 °C. Analysis of red muscle color changes in fish using a chromameter (CR-400, Konica Minolta) based on the CIELAB color space (L*a*b*), which is a color space defined by the International Commission on Illumination (abbreviated as CIE) in 1976. CIELAB expresses color as three values: L* for perceptual brightness, a* and b* for the unique colors of human vision: red, green, blue, and yellow. CIELAB is intended to be a perceptually uniform space, where a given numerical change corresponds to an observed color change. Images of each sample were taken using a mobile phone camera (iPhone 6 Plus type, Apple Inc., Zhengzhou, China) during measurement using a chromameter instrument as supporting data for statistical analysis of fish meat color change. Image imaging was performed using the remove-background feature on the hardware (MacBook Air 13, Apple Inc., Zhengzhou, China). The data obtained from the chromameter instrument was then calculated using the formula below:
Δ E * a b = ( ( L 1 − L 0 ) 2 + ( a 1 − a 0 ) 2 + ( b 1 − b 0 ) 2 )
note:
  • ΔE*ab = total color difference (calculated for each observation 0, 24, and 48 h)
  • L1 = mean L* value of silkrose-BM
  • L0 = mean L* value of EP feed
  • a1 = mean a* value silkrose-BM
  • a0 = mean a* value EP feed
  • b1 = mean b* value of silkrose-BM
  • b0 = mean b* value of EP feed

2.7. Total Collagen Determination

Total collagen content testing was performed using the Total Collagen Assay kit (QuickZyme Biosciences, Leiden, The Netherlands), with the testing principle based on the detection of hydroxyproline. This assay kit measures the total amount of hydroxyproline formed covering all types of collagen present in the sample including pro-collagen, moisture collagen, and collagen degradation products. The absorbance of the assay is read using a microplate reader at 570 nm. The reading results of the assay kit plate were then calculated based on the manufacturer’s protocol. Calculation of total collagen content was done by entering the absorbance data into the standard curve obtained as shown in the Figure 2 below:

2.8. Drip Loss Analysis

Drip loss was assessed by placing the fish fillet between drip sheets and observing after 24 h storage at 4 °C. Before that, the sheets were weighed one by one and marked, and the filleted fish meat was weighed separately. After 24 h of storage time, the sheets were weighed again one by one, as well as the fillet. The calculation of drip loss used the formula below:
D r i p   l o s s   % = W 0 − W 1 W 0 × 100
note:
  • W0 = weight of fillet before storage time (g)
  • W1 = weight of fillet after storage time (g)

2.9. Histological Analysis

The samples for histological analysis were fixed immediately following their collection during the rigor mortis phase. The sample was fixed in a 100 mL bottle containing 70 mL of 12% formaldehyde. The fixation solution was refreshed daily for 3–5 days, after which it was replaced with 70% histol. Under these conditions, the samples could be stored and preserved for up to one year for further analysis.
The samples were embedded in paraffin blocks and, following fixation, sectioned to a thickness of 0.3 mm using a microtome. The specimens were stained using a modified Gomori trichrome method, incorporating aniline blue to highlight collagen fibers, which subsequently appeared stained blue in the fish meat. The staining method using Histological Method for CNS modified with Gomori’s One Step Trichrome (S3). The histological samples were then observed under a microscope at 40× magnification.

2.10. Analysis of Gene Expression (Total RNA Isolation and qRT-PCR)

Total RNA was extracted from the yellowtail muscle using ISOGEN II (Nippon Gene, Toyama, Japan), according to the manufacturer’s protocol. The total RNA yield was assessed spectrophotometrically using a Nanophotometer P330 (Implen, Munich, Germany). RNA from n = 5 individuals sampled from each group was used for qRT-PCR analysis following cDNA synthesis. First-strand cDNA synthesized from the total RNA was generated using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA), according to the manufacturer’s protocol. Gene-specific primers used for qRT-PCR were Ampd1, hao1, mecr, acadm, mstn, and fbxo32 (Table S1). Melting curve analyses were performed following the amplification of each gene to confirm that only a single product had been amplified. The thermocycling process was conducted in a 96-well white Multiplate PCR plate (Bio-Rad Laboratories, Hercules, CA, USA) using a quantitative RT-PCR detection system (Bio-Rad Laboratories) with the following cycling conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 55 °C for 5 s. The relative gene expression was calculated using the comparative threshold (CT) cycle method described by Livak and Schmittgen [23], with 18s rRNA as an endogenous reference.

2.11. Statistical Analysis

Each item measured was expressed as mean ± standard error (SE). Statistical analysis of proximate composition and total collagen was performed using Mann–Whitney U test, color changes data were analyzed using a two-way ANOVA followed by a Duncan post-hoc test (p < 0.05). Statistical analysis of drip loss, histology, and gene expression was performed using the Shapiro–Wilk for normality test and Levene’s test of variance, and continued using a t-test to compare the differences between each group. The level of significance was 0.05 (95%). The statistical analyses were performed using Rstudio (R 4.3.3 GUI 1.80 Big Sur ARM build (8340)).

3. Results and Discussion

The concentration of silkrose-BM (0.1%) was tested at the most effective concentration in accordance with our previous papers [5,8]. Dietary silkrose-BM at a concentration of 0.1% effectively prevents vibriosis in penaeid prawns (Litopenaeus vannamei and Marsupenaeus japonicus) and significantly reduced skin parasitism in yellowtail and white trevally (Pseudocaranx dentex). Furthermore, silkrose-BM also significantly reduced cortisol level and altered expression of various genes, including those involved in immunity, stress response, wound healing, and heat responses, in yellowtail, white trevally, and Japanese medaka (Oryzias latipes) [4,5,8]. In the present study, we used yellowtail fed with 0.1% silkrose-BM for six months to reveal the effect of silkworm-derived polysaccharides on the fish meat quality. Below, we highlighted several key constituents for yellowtail meat quality affected by dietary silkrose-BM.

3.1. Proximate Composition

The proximate composition of fish meat is influenced by various factors such as diet, breeding season, and environmental conditions. Previous studies have shown that the proximate composition, particularly lipid content, of yellowtail fish meat is related to gonadal maturity [24], as well as to the water temperature and the type of feed provided [18]. The water quality data during the feeding period of yellowtail are displayed in Table 2.
In this study, we determined the sampling method from specific parts of the fish meat, such as the proximate samples taken from the inner part of the Musculus latero-dorsalis. The results of the proximate composition analysis are shown in Table 3.
The proximate composition of yellowtail meat was determined in both feeding treatment groups. The mean values for the EP feed group for moisture, crude fat, crude protein, and crude ash were different; fish in the silkrose-BM diet group had higher crude fat and moisture values, but statistical analysis revealed no significant differences between the groups.

3.2. Discoloration in Yellowtail Red Muscle

Color changes in the red muscle of fish are one of the indicators of a decline in meat quality and have a considerable impact on consumer preferences. The color of red muscle in fish from each treatment group (n = 5) was measured using a CR-400 instrument (Konika Minolta Inc., Tokyo, Japan) before being stored for 48 h at 4 °C, with further color measurements taken every 24 h. The total color difference before storage began was ΔE*ab = 0.84, with no significant differences observed between the feeding treatment groups.
Differences in L* values indicate variations in the lightness level of an object. (−L*) value indicates a darker color, while a (+L*) value indicates a lighter color. The a* value indicates differences between redness (+a*) and greenness (−a*), while the b* value indicates differences between yellowness (+b*) and blueness (−b*) in an object. The higher the value of the color index, the more colorful the observed object.
In this study, the L* values in the EP feed group increased drastically (p < 0.05) after 48 h of storage, but the same phenomenon was not observed in the silkrose-BM diet treatment group. A significant decrease (p < 0.01) in the a* value occurred every 24 h in the EP feed group; meanwhile, in the silkrose-BM diet group, the decrease in a* value was not significant at 24 h, and the changes seemed to slow even after the 24 h mark. The changes in b* values in the EP feed group were significantly different every 24 h (p < 0.01), while the b* values in the silkrose-BM diet group only changed after storage for 48 h (p < 0.05). The mean values of the results of this analysis are shown in Table S2. The conclusions we drew from the color changes in yellowtail fish meat are summarized in Table 4.
The extended storage of fish (48 h) from both groups led to considerable color changes of fish meat in values of lightness (L*), decreases in redness (a*), and an increase in yellowness (b*). Color changes in the EP feed group occurred more rapidly, indicating that the oxidation and degradation of pigments and lipids were more intense. Color changes happened more slowly in the silkrose-BM diet group. Overall, the extended storage period led to lipid oxidation and pigment degradation, which caused the fish meat to become darker/more dull (decreases/increases in L*); less red (decreased a*), indicating the degradation of myoglobin; and more yellow (increase in b*), suggesting a loss of quality in terms of pigment and chemical composition due to lipid oxidation.
The lower intensity of the red a* value found in the EP feed treatment group was probably associated with the denaturation of sarcoplasmic proteins, including myoglobin. This denaturation causes increased light scattering and a greater amount of oxidized myoglobin in the muscle. These findings align with those of previous studies by Langer & Zhang [25,26]. Figure 3 shows a comparison of the appearance of yellowtail red muscle between groups.
In addition to the chemical composition of yellowtail fish meat being able to influence color changes, especially in the red muscle, microorganisms that proliferate at specific pH levels are also suspected to have an indirect influence by contributing to the degradation of fish meat quality and altering its color [27]. The nutrients contained in fish meat provide an ideal medium for microbial growth, so the presence of microorganisms in fish meat accelerates the deterioration and spoilage process through biochemical reactions once the fish have been killed, which can directly affect the quality of fish meat.
Lipid oxidation and myoglobin oxidation in meat lead to the development of off-flavor and discoloration, respectively, and they are influenced by both external environmental factors and endogenous substances [28]. These processes often appear to be linked, and the oxidation of one can lead to the formation of chemical species that can exacerbate the oxidation of the other [29]. Oxidation can generate harmful substances that pose health risks to humans. Lipid oxidation is thus the primary factor in quality degradation, a process mediated by myoglobin [30,31].
When alive, fatty fish have an antioxidant system that stabilizes their high content of unsaturated lipids. This endogenous antioxidant system includes compounds that can capture free radicals and enzymes that eliminate reactive oxygen species, such as superoxide radicals, hydrogen peroxide, and lipid peroxides. Following death, these endogenous antioxidants are sequentially consumed, and several studies have linked the depletion of these antioxidants to the development of oxidation. Antioxidants are molecules that can inhibit damage in food products by minimizing lipid oxidation. They can perform various actions in food, including capturing free radicals, binding metal ions, and neutralizing singlet oxygen. Biological tissues have a highly efficient endogenous antioxidant system, which arises from the need to protect aerobic organisms from oxygen and its reactive species [30,32,33]. Several investigators have reported the preservation of color in fresh meat following the inclusion of antioxidant ingredients [29].
The bioactive monosaccharides present in silkrose-BM mainly play a role in biological activities related to enhanced antioxidant activity, via protein and cell modification. These bioactive compounds, including GlcNAc and GalNAc, are involved in modifying proteins via glycation, thereby affecting cellular activity and responses to oxidative stress. According to Chatham et al. [34], protein modification by sugars is one of the most common post-translational modifications of protein. In addition, L-fucose, a unique monosaccharide used by cells in a fucosylation process, is also present in silkrose-BM. Fucosylation is a process used to post-translationally modify and regulate the behavior and function of proteins. It plays an important role in various cellular processes, including cell signaling, immune responses, and the regulation of protein interactions. Adding fucose residues to proteins enables cells to alter the stability, localization, and activity of proteins, influencing various biological functions, including antioxidant defense and stress responses [35].
The mechanisms via which bioactive polysaccharides from silkrose-BM affect color changes in fish meat, and their molecular pathways, remain unclear. However, feeding fish a silkrose-BM diet likely plays a role in slowing the color changes seen in the red muscle compared with the speed of the color changes seen in the EP feed group. This phenomenon, which leads to the production of antibacterial substances, is promoted by silkrose-BM and helps to prevent microbial growth and oxidative stress in the muscle tissue. The effects may occur because the innate immunity of fish fed with silkrose-BM diet was activated. As a result, the oxidative processes that typically accelerate color change and spoilage in fish muscle, such as the oxidation of myoglobin and lipids, may be slowed down, so the color and quality of the fish meat can be preserved during longer storage periods [9]. Thus, the process of quality deterioration in fish meat post-mortem, such as lipid oxidation and protein degradation, slows down.

3.3. Quantification of Total Collagen

Collagen is the main protein in the extracellular matrix. Collagens are a family of fibrous proteins found in all multicellular animals. The primary types of collagen found in connective tissues are types I, II, III, V, and XI; however, in fish meat, types I and V collagen are more dominant. Other key components of connective tissue include proteoglycans. These are complex multifunctional molecules consisting of a core protein with a molecular weight ranging from 40 to 350 kDa, covalently linked to several dozen GAG chains [36]. GAGs are complex polysaccharides that play an important role in growth, differentiation, morphogenesis, cell migration, and bacterial/viral infections [13]. The main GAG chains in vertebrates consist of amino sugar units and include GlcNAc and GalNAc, which are also present in silkrose-BM. The total collagen content in yellowtail in our study is shown in Figure 4.
Our quantitative analysis of total collagen revealed values of 137.8 μg/mL for the EP feed group and 195.0 μg/mL for the silkrose-BM diet group (Figure 4). Although no statistically significant differences were observed, the collagen content in the fish muscle of the silkrose-BM diet group was notably higher than that of the EP feed group. This difference suggests a potential trend worth further investigation. In addition, distinct visual changes were observed in the muscle fibers of the silkrose-BM diet group. The muscle tissue exhibited a noticeable blue-to-purple hue, with prominent blue lines around the perimysium area, which may reflect alterations in the structural organization or optical properties of the collagen fibers (Figure 4B). The collagen content in fish meat is very important and contributes to the toughness of sliced raw meat [37].
The silkrose-BM diet includes bioactive polysaccharides, so we assume that the provision of this feed has the potential to increase the total collagen content in fish muscle by enhancing protein functions related to the formation of muscle structure and extracellular matrix proteoglycan networks. This hypothesis is supported by the research of Listrat et al. [36], who stated that proteoglycans, one of the main components of connective tissue, are complex multifunctional molecules consisting of a core protein covalently linked to several dozen GAG chains. This structure is essential for the function of proteoglycans in maintaining tissue integrity and modulating various biological processes, including the formation and stabilization of muscle and connective tissue structures [38,39].

3.4. Drip Loss

The drip loss results presented the amount of fluid lost from the yellowtail fish meat that influence the meat weight after 24 h, as shown in Figure 5.
The results of the drip loss analysis showed a significant difference between the groups, with average drip loss values of 2.07% and 1.23% for EP feed and the silkrose-BM diet, respectively. We assumed the reduction in drip loss value in the silkrose-BM diet group was due to the presence of functional compounds in silkrose-BM that may influence protein stability and lipid oxidation. These effects could indirectly contribute to the enhanced quality of yellowtail fish meat in this group compared with the EP feed group, as evidenced by the observed color changes and improved moisture retention.
Drip loss is positively correlated with water-holding capacity [40], and changes in water-holding capacity correlate with lipid oxidation. Wada and Ogawa [41] highlighted this, hypothesizing that water-binding molecules are broken down during lipid oxidation. The denaturation of proteins such as myoglobin, which is related to lipid oxidation, alters the molecular structure and water-holding capacity of fish meat, particularly after it has been stored for two days.
In the EP feed treatment group, the degradation that occurred during storage affected the shrinkage of the sample, as drip loss is generally a continuous process involving the transfer of water from myofibrils in muscle tissue to the extracellular space. Thus, differences in drip loss are associated with changes in protein function in the muscle after it is cut. Protein denaturation reduces the ability of the fish muscle to retain water, resulting in an open structure that increases the amount of drip released from the muscle. The amount of drip lost from the fish tissue can indicate a lack of protein functionality in the binding water. This leads to poor water-holding capacity and facilitates the loss of sarcoplasmic proteins from muscle cells into the extracellular space [40,42,43].

3.5. Histology

In terms of economic value as a food product, the texture of the muscle is important in many types of fish, including yellowtail, when the muscle is eaten raw, such as in sushi and sashimi [18,36]. Microstructural analysis not only allows for the assessment of quality but also enables the determination of the quantitative ratio of the components in a meat product.
Histological examination is one of the methods that enables the constituent components of meat products to be identified, including the degree of autolytic processes in muscle fiber [44]. The histology specimens were stained using the Gomori trichrome special staining method under the same time and conditions between the groups. The blue area in photomicrographs indicates the collagen levels (Figure 4B); cross-sectional areas of yellowtail muscle were observed under a microscope at a magnification of 40× (Figure 6). The muscle condition in the EP feed group indicated unhealthy muscle (atrophy) compared with that seen in the silkrose-BM diet group (Figure 6).
The histological analysis was used to compare the area of the muscle fibers in the perimysium area of the two groups. The area of the muscle fiber is shown in Figure 7.
A significant difference was observed, with a mean muscle fiber area of 228.63/mm2 and 327.48/mm2 for the EP feed and silkrose-BM diet groups, respectively. The greater area observed in the silkrose-BM diet group indicates that the fish possess greater numbers of fiber cells binding the muscle tissue structure, making the muscle in these fish more compact than that of the fish in the EP feed group. This finding suggests that feeding fish with silkrose-BM can enhance the functionality of proteins in their meat, thereby optimizing their role in muscle development and contributing to improving the texture profile as a quality indicator.

3.6. Gene Expression Analysis (qRT-PCR)

Gene expression analysis was conducted using qRT-PCR to detect differences in gene expression between the groups to provide additional data to support our overall analysis. The results are shown in Figure 8.
Figure 8 shows that significant differences in the expression of the acadm and fbxo32 genes were observed between the groups. The average relative gene expression values for acadm were 1.02 and 0.71 and for fbxo32 were 1.07 and 0.36 for the EP feed and silkrose-BM diet groups, respectively. Both genes showed higher relative expression in the EP feed treatment group compared with the silkrose-BM diet group.
An increase in the capacity of the F-box protein atrogin-1/fbxo32 to polyubiquitinate proteins via increases in the expression of ubiquitination machinery is largely responsible for faster rates of protein degradation associated with atrophy [45,46]. Silkrose-BM has pro-inflammatory properties, stimulating the activation of the innate immune system in crustaceans and mammals (as seen in mice RAW264 cells) through its function as a pathogen-associated molecular pattern (PAMP) that is recognized by pathogen recognition receptors (PRRs) [8]. Polysaccharides containing PAMPs affect various types of cells in various animal species, including both vertebrates and invertebrates, as has been reported for lipopolysaccharide (LPS) and β-glucans. According to Tacchi et al. [47], the expression of atrogin-1 (fbxo32) increases following stimulation by LPS, indicating a catabolic process occurs during the inflammatory response. It is possible that feeding silkrose-BM to yellowtail stimulates the formation of cells due to maximal protein catabolism, which then halts, leading to a decrease in the expression of the fbxo32 gene.
The elevated relative expression of these genes in the EP feed treatment group suggested increased lipid metabolism (acadm) [48], and protein degradation (fbxo32) in fish meat [47], which contributed to the reduced overall meat quality compared with the silkrose-BM diet group. The higher expression of the fbxo32 gene in the EP feed group indicated a greater extent of muscle protein breakdown that potentially causes fish muscle to atrophy. This finding aligns with the observed differences in muscle condition, further emphasizing the potential advantages of the silkrose-BM diet in preserving muscle quality vis the minimization of muscle atrophy (Figure 7).

4. Conclusions

A silkrose-BM diet containing bioactive polysaccharides may enhance the quality of fish meat by optimizing protein performance and inhibiting the lipid metabolism that occurs in fish meat during storage. Feeding a silkrose-BM diet to yellowtail had an impact on changes in fish meat color during storage, the total collagen content, the quantity of drip loss, the histological structure, and gene expression. Optimizing protein performance in fish muscle may help to minimize protein denaturation, thereby preserving the functionality of proteins in muscle tissue by promoting the production of muscle cells, as evidenced by the higher muscle density observed in the silkrose-BM diet treatment group. However, the molecular pathways underlying this mechanism remain unclear. Further research is needed to determine relevant RNA sequences and to elucidate the mechanisms and molecular pathways underlying the changes in fish meat quality induced by silkrose-BM.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fishes10030130/s1, Table S1. Gene-specific primers for qRT-PCR of yellowtail muscle (EP feed and silkrose-BM diet treatment groups) [49,50,51,52,53,54,55]; Table S2. Differences in the color changes (ΔE*ab) in the dark flesh of yellowtail fish during storage at 4 °C, observed every 24 h. Table S3. Modified Gomori Thricrome staining method of yellowtail muscle.

Author Contributions

Conceptualization, Y.O., D.H., C.M. and T.M.; methodology, A.A., H.N., D.H., Y.O., M.F.Z.A., C.M., I.B.B.S. and T.M.; formal analysis, A.A., H.N. and D.H.; investigation, D.H., Y.O. and T.M.; writing––original draft, A.A., M.F.Z.A.; writing––review and editing, C.M., I.B.B.S., M.F.Z.A. and T.M.; Supervision, T.M.; Project administration, T.M.; Funding acquisition, T.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the JSPS Kakenhi (20K20574).

Institutional Review Board Statement

Animal experiments were carried out following the guidelines of Ehime University. The study protocol was accepted by the Institutional Animal Care and Use Committee (IACUC) of Ehime University (Permit Number: 08K2-1). Approval date 4 December 2019.

Data Availability Statement

The data presented in this study are available on request from the corresponding author, upon reasonable request.

Acknowledgments

We are grateful to the staff of the Laboratory of Fish Reproductive Physiology, the Graduate School of Agriculture, Ehime University for their assistance in this study. This work was supported by JSPS Kakenhi (20K20574).

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Fishes 10 00130 g001
Figure 1. Muscle parts of yellowtail sampled for this study. (A) Proximate (kept in a sealed plastic bag); (B) histology (kept in fixative in a bottle); RNA (kept in RNAlater solution); (C) total collagen quantification (kept in a sealed plastic bag); (D) discoloration analysis (kept in a sealed plastic bag); and drip loss (dealt with immediately).
Figure 1. Muscle parts of yellowtail sampled for this study. (A) Proximate (kept in a sealed plastic bag); (B) histology (kept in fixative in a bottle); RNA (kept in RNAlater solution); (C) total collagen quantification (kept in a sealed plastic bag); (D) discoloration analysis (kept in a sealed plastic bag); and drip loss (dealt with immediately).
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Figure 2. Absorbance value of total collagen standard solution using total collagen assay kit.
Figure 2. Absorbance value of total collagen standard solution using total collagen assay kit.
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Figure 3. Changes in the appearance of sliced meat prepared from farmed yellowtail during storage at 4 °C. Sliced meat was prepared from fish from both groups and stored on ice for approximately 3 to 5 h after filleting the fish meat.
Figure 3. Changes in the appearance of sliced meat prepared from farmed yellowtail during storage at 4 °C. Sliced meat was prepared from fish from both groups and stored on ice for approximately 3 to 5 h after filleting the fish meat.
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Figure 4. (A) Total collagen in yellowtail muscle from the EP group (dark grey) and the silkrose-BM group (pale grey). Data are presented as the mean ± SE (n = 5). (B) Photomicrographs showing collagen appearance in the perimysium area of yellowtail muscle, 200× magnification.
Figure 4. (A) Total collagen in yellowtail muscle from the EP group (dark grey) and the silkrose-BM group (pale grey). Data are presented as the mean ± SE (n = 5). (B) Photomicrographs showing collagen appearance in the perimysium area of yellowtail muscle, 200× magnification.
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Figure 5. The percentage of drip loss from farmed yellowtail fish meat fed on EP feed (dark grey) and fed on a silkrose-BM diet (pale grey). The asterisk indicates a significant difference between the groups (p < 0.05).
Figure 5. The percentage of drip loss from farmed yellowtail fish meat fed on EP feed (dark grey) and fed on a silkrose-BM diet (pale grey). The asterisk indicates a significant difference between the groups (p < 0.05).
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Figure 6. Cross-sectional areas of yellowtail muscle under a microscope at a magnification of 40×. The sections shown are from the perimysium area of yellowtail muscle from the (A) EP feed group and (B) the silkrose-BM group.
Figure 6. Cross-sectional areas of yellowtail muscle under a microscope at a magnification of 40×. The sections shown are from the perimysium area of yellowtail muscle from the (A) EP feed group and (B) the silkrose-BM group.
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Figure 7. (A) The area of yellowtail muscle fiber (mm2) in cultured yellowtail in the EP feed group (dark grey) and the silkrose-BM diet group (pale grey). The asterisk indicates a significant difference between the groups (* p < 0.05). (B) Images of muscle condition in the two groups.
Figure 7. (A) The area of yellowtail muscle fiber (mm2) in cultured yellowtail in the EP feed group (dark grey) and the silkrose-BM diet group (pale grey). The asterisk indicates a significant difference between the groups (* p < 0.05). (B) Images of muscle condition in the two groups.
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Figure 8. Relative expression of genes in yellowtail muscle of the EP feed (dark grey) and silkrose-BM diet (pale grey) groups. Data are presented as mean ± SE (n = 5). Asterisks indicate a statistically significant difference between the groups (* p < 0.05), (** p < 0.01).
Figure 8. Relative expression of genes in yellowtail muscle of the EP feed (dark grey) and silkrose-BM diet (pale grey) groups. Data are presented as mean ± SE (n = 5). Asterisks indicate a statistically significant difference between the groups (* p < 0.05), (** p < 0.01).
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Table 1. Feed composition and proximate analysis of diets fed to yellowtail during the feeding period.
Table 1. Feed composition and proximate analysis of diets fed to yellowtail during the feeding period.
Ingredients (%)Diet
EP FeedSilkrose-BM diet
Animal meal (fish meal, krill meal)50.050.0
Grain (flour, starch)15.014.9
Vegetable meal (soybean meal, corn gluten meal)9.09.0
Vegetable oil1.01.0
Others (fish meal oil, calcium phosphate, marigold petal extract, licorice extract, rice germ extract)25.025.0
Silkrose-BM 0.1
Proximate analysis (%)
Crude protein±40.0 or more
Crude fat±28.0 or more
Crude fiber±3.0 or less
Ash±13.0 or less
Calcium±1.0 or more
Phosphate±1.0 or more
Total feed intake (kg)11,72612,709
The feed composition and nutritional profile of the diet are provided by Sakamoto Feed Co., Ltd.
Table 2. Overall water quality measurements during the feeding period of yellowtail.
Table 2. Overall water quality measurements during the feeding period of yellowtail.
TimeParameter
Salinity (%)Temperature (°C)DO 1 (%)DOM 2 (mg/L C)Turbidity (ntu 3)
April34.47 ± 0.0216.74 ± 0.02108.55 ± 2.058.68 ± 0.090.36 ± 0.03
May34.48 ± 0.0416.69 ± 0.01113.68 ± 0.178.96 ± 0.010.32 ± 0.01
June34.21 ± 0.0418.20 ± 0.01104.63 ± 0.498.02 ± 0.040.45 ± 0.03
July33.87 ± 0.0220.28 ± 0.11104.11 ± 0.897.70 ± 0.060.32 ± 0.02
1 DO = dissolved oxygen; 2 DOM = dissolved organic matter; 3 ntu = nephelometric turbidity units.
Table 3. The proximate values in yellowtail meat after six months of aquaculture.
Table 3. The proximate values in yellowtail meat after six months of aquaculture.
ParametersCompositions Level (%)p-Value
EP Feed GroupSilkrose-BM Diet Group
Moisture58.39 ± 1.8160.60 ± 4.391
Crude fat20.18 ± 2.0820.59 ± 3.120.84
Crude protein20.07 ± 0.4619.56 ± 1.900.31
Crude ash1.63 ± 0.041.38 ± 0.130.09
Data are presented as mean ± S.E., (n = 5) fish from each group.
Table 4. Color changes in yellowtail fish meat stored at 4 °C, observed after 0, 24, and 48 h.
Table 4. Color changes in yellowtail fish meat stored at 4 °C, observed after 0, 24, and 48 h.
ColorEP Feed GroupSilkrose-BM Diet Group
L*Increased drastically over 48 h of storageDecreased over 48 h of storage
a*Decreased drastically every 24 h during the 48 h storage periodDecreased drastically after 24 h of storage, but the decrease was less drastic after 48 h
b*Increased drastically every 24 h during the 48 h storage periodIncreased drastically after 48 h of storage.
EP feed, commercial extruded pellets feed; *value of L*, a*, b* indicates lightness, redness, and yellowness, respectively.
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Athira, A.; Nishiguchi, H.; Hayashi, D.; Otsu, Y.; Miura, C.; Suryadi, I.B.B.; Ali, M.F.Z.; Miura, T. Influence of the Silkworm-Derived (Bombyx mori) Functional Substance (Silkrose-BM) on the Fish Meat Quality of Yellowtail (Seriola quinqueradiata). Fishes 2025, 10, 130. https://doi.org/10.3390/fishes10030130

AMA Style

Athira A, Nishiguchi H, Hayashi D, Otsu Y, Miura C, Suryadi IBB, Ali MFZ, Miura T. Influence of the Silkworm-Derived (Bombyx mori) Functional Substance (Silkrose-BM) on the Fish Meat Quality of Yellowtail (Seriola quinqueradiata). Fishes. 2025; 10(3):130. https://doi.org/10.3390/fishes10030130

Chicago/Turabian Style

Athira, Athira, Haruki Nishiguchi, Daichi Hayashi, Yuki Otsu, Chiemi Miura, Ibnu Bangkit Bioshina Suryadi, Muhammad Fariz Zahir Ali, and Takeshi Miura. 2025. "Influence of the Silkworm-Derived (Bombyx mori) Functional Substance (Silkrose-BM) on the Fish Meat Quality of Yellowtail (Seriola quinqueradiata)" Fishes 10, no. 3: 130. https://doi.org/10.3390/fishes10030130

APA Style

Athira, A., Nishiguchi, H., Hayashi, D., Otsu, Y., Miura, C., Suryadi, I. B. B., Ali, M. F. Z., & Miura, T. (2025). Influence of the Silkworm-Derived (Bombyx mori) Functional Substance (Silkrose-BM) on the Fish Meat Quality of Yellowtail (Seriola quinqueradiata). Fishes, 10(3), 130. https://doi.org/10.3390/fishes10030130

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