A Study on the Formation and Change of Yesso Scallop Odor Characteristics by GC-O-MS, Sensory Evaluation, and Flavor-Precursor Analysis

Scallops are exposed to drying and hypoxia for a long duration in the supply chain, leading to quality deterioration and changes in odor characteristics. In this study, quality deterioration of Yesso scallop was simulated under diferent conditions to explore the formation and change of its unique aroma. Its volatile components were analyzed by partial least squares (PLS) regression, GC-O, and smell threshold, and its odor characteristics were analyzed with reference to favor precursors. Te sensory descriptors selected comprehensively described the odor characteristics of Yesso scallop. Te 16 most relevant volatile components for each odor characteristic were identifed, and a new star-mark grading method was established to evaluate the role of volatile components in the formation of odor characteristics. Te possible odor precursors were determined, providing preliminary information on the generation of pleasant and unpleasant odor characteristics by Yesso scallops. Tus, our results provide a scientifc basis for the further study and control of the odor characteristics of aquatic products.


Introduction
Yesso scallops (Mizuhopecten yessoensis) are one of the most important economic shellfsh in China's aquaculture industry and naturally distribute along the coastlines of the Far East of Russia, northern Japan, and the northern part of the Korean Peninsula. As one of the major cultured shellfsh groups, Yesso scallops provide abundant minerals, essential amino acids, and high-quality protein in addition to its characteristic favor, making it highly attractive to customers [1].
Fresh live Yesso scallops have a faint smell of seafood and develop a strong unique favor during the cooking process. However, scallops are exposed to drying and hypoxia for a long duration in the supply chain, leading to quality deterioration and changes in odor characteristics; specifcally, the pleasant odor characteristics of cooked scallops are lost, while their unpleasant odor characteristics increase. Several studies have been conducted to evaluate the efects of environmental stress (dryness, hypoxia, and air exposure) on the immune system of scallops; these studies indicate that environmental stress infuence the activity of scallops, which may cause changes in odors [2].
At present, research on the mechanisms by which the aromas of aquatic products form and change is mainly focused on the application of GC-MS or GC-O in combination with sensory evaluation, favor precursor determination, and other technical means to analyze their volatile components [3]. Te volatile components of Yesso scallops are mainly aldehydes, ketones, alcohols, aromatics, and sulfur-containing compounds, as well as many other small-molecule compounds [4]. By establishing a reliable sensory analysis method, the quantitative sensory evaluation results can be used for chemometric analysis along with instrumental measurements of various physical and chemical indicators of scallops, including volatile components [5].
Te formation of unpleasant odors from animal-origin food is mainly due to degradation by endogenous enzymes or microorganisms, oxidation of lipids and fatty acids, diet, and environmental pollution. For example, the lipids of aquatic products are easy to oxidize during processing and storage, generate hydroperoxides, and then decompose into small molecules such as aldehydes and ketones, thus leading to changes in the favor, texture, and color of aquatic products [6]. Te components produced by diferent formation mechanisms are also diferent and are related to the type and content of favor precursors in food raw materials [7]. Water-soluble favor precursors, such as free amino acids, free fatty acids, and reducing sugars, have been identifed [8]. Tese favor precursors produce various favor substances under the action of microorganisms, enzymes, oxygenation, and the Maillard reaction [9]. Changes of favor precursors lead to changes in food favor.
In this study, the odor characteristics of Yesso scallops were divided into pleasant and unpleasant odors, and the volatile components mostly related to these two odor characteristics were identifed. Ten, the correlation, GC-O smell, and odor threshold were combined to establish a standard star-mark method to further classify the volatile components. Finally, on the basis of the abovementioned results, two types of possible favor precursors were analyzed to complete a preliminary exploration of the mechanisms by which odor characteristics form and change upon quality deterioration in the supply chain. Te results provide a scientifc basis for further research into favor loss and favor-precursor removal, as well as a method for studying the odor characteristics of aquatic products.

Materials and Methods
2.1. Sample Treatment. At 5:00 am in Huangsha Port, Sheyang County, Yancheng City, Jiangsu Province, China, 100 live scallops, 8-10 cm in size, were purchased and transported to the laboratory in crushed ice. Among them, 25 scallops (adductor muscle and main edible parts) were taken for immediate treatment and analysis (alive in excellent condition) and 25 scallops were taken for analysis after steaming (cooked in excellent condition). Te remaining 50 were subjected to simulated environmental stress. After dry exposure (anoxic) was placed at room temperature (∼25°C on average) for 24 h, 25 shellfsh columns were taken for direct analysis (alive in poor condition) and 25 shellfsh columns were taken for analysis after steaming (cooked in poor condition).

Quantitative Description of Sensory Analysis.
We referred to the Chinese National Standard (GB/T 16291 Sensory Analysis-General Guidance for Selection, Training, and Monitoring of Assessors) to establish, select, and train sensory teams. With reference to the sensory descriptors of scallops, which were established by Yang et al. [10], the sensory team fnally selected six odor descriptors, among which "stench," "earthy-musty," and "sulfur" described unpleasant odors, while "scallop aroma," "cooked meat," and "potato mud" described pleasant odors, as shown in Table 1.
After training, the sensory team analyzed the sensory characteristics of scallops in diferent conditions using the quantitative description analysis method, and scored each sensory description quantitatively by formulating olfactory reference materials. For example, the scale for quantitative description and analysis of "stench" with reference materials is shown in Figure 1.

Analysis of Volatile Components.
Te eluent from the GC capillary tube fows into the MS unit and olfactometer, respectively, at a 1 : 1 split ratio for qualitative, relative quantitative, and olfactory analysis of volatile components. Te specifc methods were as follows:

Qualitative and Quantitative Analysis of GC-MS Volatile Components.
Te method outlined in the study by Wu et al. [11] was used to analyze the volatile components of the scallop column samples. Te equipment used were an Agilent 7890A gas chromatography-mass spectrometer, a MonoTrap RCC18 was used as the solid phase extraction integral collector, and 2,4,6-trimethylpyridine was used as the internal standard. For qualitative analysis, the volatile components were qualitatively determined with reference to the NIST and Wiley spectral libraries, and only the results with positive and negative matching degrees greater than 800 (the maximum value is 1000) were reported. For relative quantitative analysis, 2,4,6-trimethylpyridine was added as an internal standard before sample adsorption, and the relative content of each volatile odor substance was calculated.

GC-O Analysis of the Odor Characteristics of Volatile
Components. Using a Gerstel ODP-2 olfactometer, GC-O olfactory testing was conducted by sensory team members. Each sample was smelled once by each team member, and the odor characteristics and retention time for each volatile component were recorded. Te odor characteristics were limited to the six sensory descriptors described above. At least two sensory ofcials had to identify the same odor characteristic at the same retention time before recording it as a valid result [12,13].

Determination of Free Amino Acids by HPLC.
With reference to the Chinese National Standard GB/T 30987-2020, the method for determination of free amino acids was performed using an Agilent 1260 highperformance liquid chromatography instrument.
Free amino acid content was calculated using the following formula: where W is the target free amino acid content in the sample (mg/kg), C is the concentration of the target substance in the sample determination solution (mg/L), C 0 is the concentration of the target substance in the blank control (mg/L), V is the sample volume (mL), N is the dilution ratio, and M is the sample quantity (g).

Determination of Free Fatty Acids by GC-MS.
Free fatty acids were determined using the methods of Honicky et al. [14] and Sims et al. [15] using an Agilent 7890A gas chromatography-mass spectrometer [14,15]. Free fatty acid content was calculated using the following formula: where W is the content of free fatty acids in the sample (g/ 100 g), C is the concentration of fatty acid methyl ester in the sample determination solution (mg/L), C 0 is the concentration of the target substance in the blank control (μg/L), V is the constant volume (mL), K is the conversion coefcient of fatty acid methyl ester to fatty acid, N is the dilution ratio, 10 −4 is the unit conversion factor, and M is the sample weight (g).

Data Analysis.
Quantitative description of sensory analysis and GC-MS instrument analyses results were analyzed by partial least squares regression (PLSR). Six PLSR analyses were performed for six sensory descriptors, using quantitative results of sensory description as the dependent variable, and relative content of each volatile component as the independent variable. Mintab 14.0 software was used for data analysis. Quantitative results of sensory description were made into a radar chart for better observation. According to the PLSR standardization coefcient and load result analysis, the 16 most relevant volatile components were selected for each descriptor.
Ten, the 16 volatile components mostly related to each descriptor was star rated, combined with the correlation with GC-O odor description and the size of the olfactory threshold. Te number of asterisks obtained is used to grade each volatile component in degrees.

Quantitative Description and Analysis of Odor Characteristic Changes.
In order to determine the formation and change mechanisms of the scallop odor characteristics, they were divided into pleasant and unpleasant odors.
Te sensory team described the pleasant odor characteristics of scallops as "scallop aroma," "cooked meat," and "potato mud." Te basic attributes of scallops are those of a meat product, so the basic odor of scallops is expressed as the aroma of cooked meat. Unlike animal meat, the aroma of scallops is lightly sweet but not greasy, supplemented by the favor of mashed potato. Furthermore, scallops have a unique scallop aroma. Te abovementioned three odor descriptors comprehensively describe the pleasant odor characteristics of scallops. It can be seen from the sensory analysis results (Figure 2) that the pleasant odor scores of samples in the poor alive condition are generally at a low level, while the scallop aroma score for samples in the excellent alive condition is relatively high, but drops rapidly upon quality deterioration. Te quality deterioration has little efect on the "cooked meat" and "potato mud" characteristics of the live samples. After cooking, the pleasant odor characteristics of scallops in both poor and excellent conditions improve compared with those for the alive samples in the same state. After quality deterioration, the scallop aroma and cooked meat scores decrease rapidly, and the potato mud score decreases slightly.
Te sensory team described the unpleasant odor characteristics as "stench," "earthy-musty," and "sulfur." Stench is a typical smell of seafood products after quality degradation. Sulfur is used to describe the pungent odor. Te scallops after quality degradation also produce an unpleasant bad smell similar to that of moldy soil. Tese three descriptors comprehensively describe the characteristics of the unpleasant odor of scallops. According to the sensory analysis results (Figure 2), the unpleasant odor score of samples increases rapidly with the decline in quality, while the earthy-musty score increases slightly, and the sulfur score shows little change. Te stench score of samples in poor condition decreases slightly after being cooked, and the  Aquaculture Research earthy-musty and sulfur scores increase signifcantly. After the scallops in excellent condition are cooked, the unpleasant odor characteristics are generally maintained at a low level, the stench and earthy-musty scores are decreased, and the sulfur score is increased. In general, the sensory evaluation results for samples in diferent conditions are comparable to a certain extent. Te quantitative results for diferent descriptors are somewhat inconsistent, so they were qualifed by the correlation analysis with the results of the volatile component analysis.

Analysis and Comparison of Volatile Components for Yesso Scallops in Diferent Conditions.
Te volatile components of scallop samples for the four diferent conditions were detected, and the results are shown in Figure 3 and Table 2. Seventy-eight volatile compounds were identifed from scallop samples in the four states, including 10 sulfur compounds labeled A1-A10, 17 hydrocarbons labeled B1-B17, fve amines labeled C1-C5, 15 heterocyclic compounds labeled D1-D15, 14 aromatic compounds labeled E1-E14, seven alcohols labeled F1-F7, three ketones labeled G1-G3, and seven aldehydes labeled H1-H7.
Te relative contents of sulfur compounds increase as a whole after cooking. High concentrations of sulfur compounds generally generate a pungent smell, but some sulfur compounds provide good favor characteristics at low concentrations [17], such as a pleasant crab-like favor. Sulfur compounds participate in the formation of the favor of scallops after cooking. Nitrogen compounds are important favor compounds in aquatic products, such as trimethylamine, which is an important fshy substance in aquatic products, and one of the indicators for evaluating the freshness of aquatic products [18]. It is generally believed that alcohols and alkanes have high odor thresholds and make little contribution to the formation of scallop favor, but alkenes can act as precursors of aldehydes, ketones, and other compounds and are potential favor compounds [19]. Te low threshold value of aldehydes has an important infuence on the odor characteristics of the scallop. Te heterocyclic compounds of cooked scallop samples increase as a whole. Specifcally, some pyrazine and furan compounds with barbecue-like favors participate in the formation of cooked scallop aroma [20].

Correlation Analysis between Sensory Evaluation and
Volatile Component Results. Te results of sensory quantitative description analysis and volatile component quantitative analysis were analyzed by partial least square regression. First, all 78 volatile components were analyzed and six PLSR models were established. Te R 2 values of all six models were greater than 0.7907 (stench), with a maximum value of 0.9621 (cooked meat). PLSR models of pleasant odors were signifcantly higher than unpleasant odors. Tese models can be used for correlation ranking of volatile components, not be used for predicting sensory characteristics.
According to the PLRS standardization coefcient and load result analysis (as shown in Figures 4 and 5), the 16 most relevant volatile components were selected for each descriptor, without considering the interaction between volatile components and only analyzing the role of individual volatile components in the formation of odor characteristics. Furthermore, with the 16 most relevant volatile components selected as the independent variable, six PLSR models were established. Te R 2 value of all 6 models were greater than 0.9508 (sulfur favor), indicating that the model can well refect the correlation between sensory characteristics and volatile odor components.
At the same time, considering the accuracy and difculty of quantifying GC-O results, a new standard star-marking method was used to further evaluate the role of each volatile component in the formation of odor characteristics. Based on the 16 volatile components, most related to each descriptor obtained by partial least squares regression and combined with the correlation with GC-O odor description and the size of the olfactory threshold, each volatile component was star rated. Te number of asterisks obtained is used to grade each volatile component in degrees. Te specifc method is shown in Tables 3 and 4.
Among the most relevant volatile components for "cooked meat," there are no 4-star components, three 3-star components (2-ethylfuran, 2,3,5-trimethylpyrazine, and 2-pentyl furan), three 2-star components (2,5-dimethylpyrazine,      Aquaculture Research basis for odor characteristic formation and change in scallops. Odor precursors in meat products generally include certain polypeptides, free amino acids, lipids, free fatty acids, reducing sugars, and thiamine [21]. Of these, there are a wide variety of free amino acids and free fatty acids, and these substances are a particular focus of meat favor research. HPLC chromatograms of the free amino acids are shown in Figure 6; GC-MS total ion current diagrams for the free fatty acids are shown in Figure 7. Te results for odor precursors under diferent conditions are shown in Table 5. Generally, with the decline of scallop quality caused by environmental stress, the content of free amino acids for scallops in the poor fresh state is signifcantly higher than that of the excellent state, which indicates that proteins or polypeptides have been decomposed to a certain extent under the action of microorganisms and enzymes during environmental stress, leading to the content of free amino acids increasing. Tis provides numerous favor precursors  for the formation of many volatile components. Te content of free amino acids in cooked scallop is signifcantly lower than that in live scallop meat, indicating that free amino acids react to form other small-molecular compounds during the cooking process, thus forming the odor characteristics of cooked scallop. Free fatty acids also play an important role in the formation of scallop odor characteristics. It can be seen from Table 5 that, on the whole, the content of free fatty acids in the meat of live scallops in poor condition is signifcantly lower than that in excellent condition, and some free fatty acids are not even detected, which is related to fatty acid oxidation by microbial and enzymatic action during environmental stress. Te content of free fatty acids in cooked scallops increases as a whole, indicating that some of the fat is decomposed to a certain extent during the cooking process and more free fatty acids are produced.

Preliminary Analysis of the Formation and Change Mechanisms of the Unpleasant Odor Characteristics of Yesso
Scallops. Te reason for the formation of unpleasant odor characteristics in Yesso scallops is that some bad volatile components are produced directly during long periods of environmental stress, or the relevant odor precursors are produced and then further produce bad volatile components. Without considering the interaction between volatile components, the formation mechanism of unpleasant odor characteristics in scallops can be preliminarily obtained by analyzing the volatile components and favor precursors with scores above 3-stars for these experimental conditions. Te unpleasant odor characteristics are mainly related to trimethylamine, some small-molecule aldehydes and ketones, and sulfur compounds. Trimethylamine is an important source of the fshy smell of scallops, and its production mechanism has been thoroughly studied. It is formed by the reduction of trimethylamine oxide under the action of reductase or microorganisms under environmental stress [22]. Te aldehydes octanal, nonanal, and sunfower aldehydes, as well as 3-octanone, are involved in the formation of unpleasant odor characteristics upon deterioration of the quality of scallops. Tese small-molecule aldehydes or ketones are generally considered to be the products of the oxidative decomposition of unsaturated fatty acids. For example, Wang et al. reported that the smallmolecule aldehydes are related to linoleic acid and some of the volatile components of abalone ω-3. And the unpleasant odor characteristics are related to the oxidation of fatty acids, which is consistent with the detection of free fatty acids in this study [23]. 2-Methylbutyraldehyde is also an important aldehyde involved in the formation of bad odor characteristics in scallops, and some studies have indicated that 2methylbutyraldehyde in meat is produced by Strecker degradation of free amino acids such as leucine and isoleucine [24]. Sulfur compounds are another source of the  amino acids in fsh muscle, which is consistent with the detection of free amino acids in this study [25].
In addition, among the volatile components with scores below 3-stars, dimethyl sulfde is also considered to be an important source of unpleasant odor characteristics in aquatic products. Some studies have indicated that methionine is converted into dimethyl sulfde upon cooking through a series of reactions including Strecker degradation, thermal degradation, and oxidation [26].

Preliminary Analysis of the Formation and Change
Mechanisms of the Pleasant Odor Characteristics of Yesso Scallops. Tere are relatively few volatile components for experimental conditions related to pleasant odor characteristics. Analysis of these volatile components and favor precursors shows that some furans, pyrazines, and sulfur compounds participate in the formation of pleasant odor characteristics during the cooking process. Among them, 2ethylfuran, 2-pentylfuran, and 2,3,5-trimethylpyrazine are considered to be derived from amino acids. Reducing sugar and other substances are produced by the Maillard reaction [3,27]; Furans are found in cooked or sterilized crustacean meat and are thermal-degradation products of lipids or thiamine [28]. Te sulfur compound dimethyl trisulfde is considered to be produced by thermal degradation of some sulfur-containing amino acids [21].
In addition, among the volatile components with scores below 3-stars, 2,5-dimethylpyrazine is also an important odor substance of cooked meat and a product of the Maillard reaction [3]; 1-Penten-3-ol is considered to make a certain contribution to meat favor. Sun et al. found that 1-octen-3-ol can be produced by linoleic acid peroxide degradation and arachidonic acid degradation by lipoxygenase through GC-O-MS [29].
Te formation and change mechanisms of odor characteristics of Yesso scallops discussed in detail above are shown in Figure 8.

Additional Analysis of the Sulfur Compounds in
Scallops. According to the results of this study, sulfur compounds have a very important infuence on the formation of odor characteristics in scallops. It is generally believed that the sulfur compounds in marine products have a very low odor threshold, and with the increase of concentration, the aroma will transition from "fresh sea water" to a "rotten stink" sulfur-like odor [25]. Te sulfur compounds screened in this study mainly include mercaptans, thioethers, thiazoles, and other heterocyclic compounds. Similarly, the efects of these sulfur compounds on the formation of odor characteristics in scallops are complex. With changing concentration, their odor characteristics will also change, and the conclusions mentioned above need to be verifed with further research. In addition, scallops are flter feeders and feed on algae in the ocean. Sulfur compounds are important contributors to the characteristic odor of algae, and their feeding and growth environment may also be one of the sources of sulfur compounds in scallops [30]. Propyl dimethyl sulfonate is degraded to dimethyl sulfde under the action of microorganisms [31].

Conclusions
In this study, the pleasant and unpleasant odor characteristics of scallops were analyzed separately. Although their correlation was not taken into account, the quantifcation of sensory descriptors was found to improve the accuracy of the quantitative description analysis. Te sensory results were used for regression analysis with the GC-MS measurement results, and the 16 most relevant volatile components for each odor characteristic were identifed. Considering the accuracy and difculty of quantifying GC-O olfactory results, this study limited the GC-O olfactory range to six sensory descriptors and combined olfactory results with olfactory thresholds to establish a new standard star-mark grading method to further evaluate the role of the most relevant 16 volatile components in the formation of odor characteristics, dividing the volatile components into four grades.
On the basis of the abovementioned research, two types of possible favor precursors were selected for analysis, and combined with the literature, the possible favor precursors were identifed, completing a preliminary analysis of the formation and change mechanisms of odor characteristics.
Tis study did not consider the synergistic efect between volatile components and only studied the formation of odor characteristics by individual volatile components. Terefore, in our further research, favor loss and recombination tests will be further carried out on the basis of the results of this study. In addition, the determination range of favor precursors will be expanded, including lipids, reducing sugars, and other possible favor precursors, and removal analysis of favor precursors will also be performed in order to better reveal the formation and change mechanisms of odor characteristics, and even provide a reference for the development of scallop favor essence.

Data Availability
Te data that supports the fndings of this study are available in the tables of this article.