Analysis and Comparison of Aroma Compounds of Brown Sugar in Guangdong, Guangxi and Yunnan Using GC-O-MS

Guangdong, Guangxi and Yunnan are the three provinces in China that yield the most brown sugar, a brown-red colored solid or powdered sugar product made from sugar cane. In the present study, the differences between odor compounds of brown sugar from Guangdong, Guangxi, and Yunnan provinces in China were compared and analyzed by gas chromatography-olfactometry-mass spectrometry (GC-O-MS). A total of 80 odor compounds, including 5 alcohols, 9 aldehydes, 8 phenols, 21 acids, 14 ketones, 5 esters, 12 pyrazines, and 6 other compounds, were detected. The fingerprint analysis of the brown sugar odor compounds showed 90% similarity, indicating a close relationship among the odor properties of brown sugar in each province. Moreover, the orthogonal partial least squares discriminant analysis (OPLS-DA) was performed to identify the compounds contributing to the volatile classification of the brown sugar from three provinces, which confirmed that OPLS-DA could be a potential tool to distinguish the brown sugar of three origins.


Introduction
Brown sugar, a traditional sweetener with a distinctive flavor, is mainly made from sugarcane through extraction, clarification, and boiling [1]. It is also called non-centrifugal cane sugar (NCS), which does not separate molasses, so it retains the original flavor and nutrients of sugarcane. Brown sugar is rich in flavonoids and phenols that may act as antioxidants and, therefore, exert benefits on organisms [2][3][4]. Furthermore, it exerts immunomodulatory, cytoprotective, anti-carcinogenic, and anti-cancer properties [5].
A study on the physicochemical properties and storage stability of brown sugar revealed darker color, increased water content and water activity, but decreased glucose and fructose contents due to the Maillard reaction [6]. Similarly, a study on the odor components of brown sugar revealed that acetaldehyde, 2-methylbutyraldehyde, 3-methylbutyraldehyde, 2,6-dimethylpyrazine, nonanal, 2,6-diethylpyrazine, 2,3,5-trimethy lpyrazine, furfural, 2,3-dimethylpyrazine, decanal, and 2-acetylpyrrole were the primary components based on their relative concentration [7]. Juliana et al. [8] extracted a total of six odor compounds from brown sugar beverages through simultaneous steam distillationsolvent extraction using a mixture of diethyl ether-pentane (1:1, w/w) as the solvent. Of the six components, 2-methylpyrazine was the key aroma compound in this beverage. Our previous research has proved that heating of syrup was the primary production step affecting the brown sugar flavor because of the production of a large number of pyrazine compounds [9]. 1 2        Guan gxi1 Guan gxi2 Guan gxi3 Guan gxi4 Guan gxi5 Guan gxi6 Yun-nan1 Yun-nan2 Yun-nan3 Yun-nan4 Yun-nan5

Analysis of Key Aroma Compounds in Brown Sugar Samples
A total of 46 aroma-active compounds were identified in 18 brown sugar samples by olfactometry, including 4 alcohols, 4 aldehydes, 3 phenols, 15 acids, 11 ketones, 7 pyrazines, and 2 other compounds. According to the odor properties of the aroma active compounds, these compounds can be classified into nine types: sweet/caramel, fruity, green/grassy, sour, sweaty/cheese, nutty, roasted, fatty and potato, which indicated that the aroma profile of brown sugar was the result of the synergistic effect of various odors.
In fact, it is the OAV of the aroma compound, and not its amount, that determines the contribution of the aroma compound. Aroma activity is generally defined as compounds with OAVs greater than 1 [14]. Therefore, the calculation of OAV was carried out for aroma compounds that can be sniffed (Table 2). Among the 18 brown sugar samples, 26 compounds with OAV >1 were considered as the key aroma active compounds of the brown sugar samples in this study and contributed to the overall flavor.
Acids: A total of 21 kinds of acid compounds were detected in 18 brown sugars, among which the OAV of 11 kinds of acid compounds was greater than 1. Acetic acid, one of the most abundant compounds in brown sugar, had the highest OAV and contributed sour aroma to the samples. 2-Methylbutanoic acid and 3-methylbutanoic acid exhibited a sour aroma and had been reported to be the key aroma components in Japanese sweet rice wine, which played an important role in the overall flavor of sweet rice wine [28]. Benzoic acid, however, has an unpleasant urine-like odor, which may be caused by phenylalanine under the action of phenylalanine ammonia-lyase in plants [29].

Fingerprint Analysis of Sugar Products from Three Different Regions
A food fingerprint can be defined as molecular markers that indicate a characteristic state or condition of food, thus enabling more accurate product identification [30]. Each sample is regarded as a multidimensional space vector. If two samples are more similar, their space will be closer, and the angle between the two samples' space vectors will be smaller, which leads the cosine of the angle between the two vectors to move closer to 1. Therefore, the similarity of samples can be expressed by the cosine of the included angle. On the contrary, if the difference between the two samples is greater, the cosine of the included angle becomes smaller. In this study, the samples were determined by GC-O-MS, and the odor-active compounds were selected for fingerprint and similarity evaluation.
It is worth mentioning that the similarity of samples becomes higher when the similarity or the cosine of the angle is above 90%. As depicted in Table 3 and Figure 2, of the six samples in Guangdong, except for Guangdong3, the similarity and cosine of the included angle of the other five samples were above 90%. This indicated that the odor properties of Guangdong3 were quite different than the other five samples, which might have happened due to different processing technology.  The cosine of the included angle of six samples in Guangxi was above 90%, and the similarity of Guangxi3 was just less than 90% (89.80%). This result indicated that the odor properties of these six samples in Guangxi were similar, without much difference Of the six samples in Yunnan, only Yunnan2 had similarity and cosine of included angle lower than 90%, while the other five samples had similarity and cosine of included angle higher than 90%. This result indicated that the odor attributes of the other five samples were similar, but Yunnan2 had significant differences with them.

Verification of Fingerprint
In order to verify whether the fingerprint method is suitable for the analysis of brown sugar, the verification was carried out. Fingerprint verification includes three parts: stability experiment, precision experiment, and repeatability experiment. Following the sample preparation described in Section 2.4, a brown sugar sample was selected and analyzed by GC-MS after 0, 2, 4, 8, 16, and 24 h. Furthermore, the relative standard deviations (RSD) of the relative retention times (RT) and relative peak areas of the odor-active compounds were calculated. The results showed that the RSD of the relative RT of the odor-active compounds was less than 0.3%, and the RSD of the relative peak areas was less than 5%, indicating that the samples were stable within 24 h and met the requirements of the fingerprint method.
A brown sugar sample was extracted and concentrated with the organic solvent, and then the concentration was injected six times consecutively to calculate the RSD of relative RT and relative peak area of the odor-active compounds. These results showed that the RSD of the relative RT of the odor active compounds was less than 0.5%, and the RSD of the relative peak area was less than 6%, indicating that the precision of the instrument was good and met the requirements of the fingerprint method.
Five brown sugar samples were extracted and analyzed for their odor compounds, followed by the RSD of relative RT and relative peak area of the odor active compounds analysis. The results showed that the RSD of relative RT was less than 0.3%, and the RSD of the relative peak area was less than 7%, indicating that they had good repeatability and met the requirements of the fingerprint method.

Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)
The fingerprinting analysis of samples from the three origins of Guangdong, Guangxi, and Yunnan revealed that the majority of samples within each province had similar odor types. In addition, a supervised OPLS-DA multivariate statistical analysis method was used to establish a statistical model in order to distinguish odor compounds between Guangdong and Guangxi, Guangdong and Yunnan, and Guangxi and Yunnan.
By conducting OPLS-DA analysis on the brown sugar, a variable importance of projection diagram (VIP) of the model was obtained. A VIP is a vector that summarizes the contribution of a variable to the explanation of the model. Variables with a VIP >1 are generally considered to contribute to the explanation of the model [31,32]. The samples were assessed as independent variables, and the OPLS-DA model was fitted automatically.
The OPLS-DA and VIP results ( Figure 3) indicate that the brown sugars from Guangdong and Guangxi were well separated. The brown sugar from Guangdong and Guangxi showed the greatest degree of separation and low intra-group differences, facilitating an accurate exploration of the differences in composition. VIP diagram elucidated that 4-hydroxybenzaldehyde, 3,5-dimethoxy-4-hydroxybenzaldehyde, n-hexadecanoic acid, butanoic acid, acetic acid, 2-methoxy-4-acetylphenol, 2-acetylpyrrole, pentadecanoic acid, furfuryl alcohol, 4-hydroxyacetophenone, etc., were the main contributors to the distinction between Guangdong and Guangxi samples. These compounds were basically aldehydes, acids, ketones, and phenols. Among these, 3,5-dimethoxy-4-hydroxybenzaldehyde and 4-hydroxybenzaldehyde played an important role in classifying Guangdong and Guangxi. 4-Hydroxybenzaldehyde and 3,5-dimethoxy-4-hydroxybenzaldehyde presented a pleasant nutty and creamy odor. Previously, 4-hydroxybenzaldehyde and 3,5-dimethoxy-4-hydroxybenzaldehyde were identified as the major volatile constituents in brown sugars [33]. Acetic acid is also one of the key compounds that can distinguish brown sugar from two provinces. Acetate is a well-known product of the thermal degradation of saccharides, and it is primarily formed during the early stage of the Maillard reaction, under neutral and alkaline conditions. Acetic acid is formed exclusively by hydrolytic cleavage of β-dicarbonyl in hexose-based systems [34]. As shown in Figure 4, OPLS-DA analysis and VIP results indicate that the brown sugars from Guangdong and Yunnan are distinguishable. The principal compounds contributing to this distinction include n-hexadecanoic acid, acetic acid, dibutylphthalate, 2acetylpyrrole, 2,5-dimethylpyrazine, and 2-methylpyrazine. Of the compounds with VIP greater than 1, pyrazine compounds appeared, which indicated that pyrazine compounds played a significant role in distinguishing brown sugar between Guangdong and Yunnan. The average content of pyrazines in Guangdong and Yunnan was 2897.28 ng/g and 1441.20 ng/g, respectively, and the pyrazine contents in Guangdong samples were higher than in Yunnan. These compounds could impart a popcorn, nutty, and roasted aroma to brown sugar.  As shown in Figure 4, OPLS-DA analysis and VIP results indicate that the brown sugars from Guangdong and Yunnan are distinguishable. The principal compounds contributing to this distinction include n-hexadecanoic acid, acetic acid, dibutylphthalate, 2-acetylpyrrole, 2,5-dimethylpyrazine, and 2-methylpyrazine. Of the compounds with VIP greater than 1, pyrazine compounds appeared, which indicated that pyrazine compounds played a significant role in distinguishing brown sugar between Guangdong and Yunnan. The average content of pyrazines in Guangdong and Yunnan was 2897.28 ng/g and 1441.20 ng/g, respectively, and the pyrazine contents in Guangdong samples were higher than in Yunnan. These compounds could impart a popcorn, nutty, and roasted aroma to brown sugar. As shown in Figure 4, OPLS-DA analysis and VIP results indicate that the brown sugars from Guangdong and Yunnan are distinguishable. The principal compounds contributing to this distinction include n-hexadecanoic acid, acetic acid, dibutylphthalate, 2acetylpyrrole, 2,5-dimethylpyrazine, and 2-methylpyrazine. Of the compounds with VIP greater than 1, pyrazine compounds appeared, which indicated that pyrazine compounds played a significant role in distinguishing brown sugar between Guangdong and Yunnan. The average content of pyrazines in Guangdong and Yunnan was 2897.28 ng/g and 1441.20 ng/g, respectively, and the pyrazine contents in Guangdong samples were higher than in Yunnan. These compounds could impart a popcorn, nutty, and roasted aroma to brown sugar.  Based on the VIP diagram and OPLS-DA analysis of brown sugar between Guangxi and Yunnan ( Figure 5), they were well separated. A number of compounds contributed to the differentiation between the two provinces, including 4-hydroxybenzaldehyde, 3,5-dimethoxy-4-hydroxybenzaldehyde, n-hexadecanoic acid, acetic acid, butanoic acid, and 4hydroxyacetophenone. Of these volatile compounds, the contribution of 4-hydroxybenzalde hyde was the greatest. The average content of 4-hydroxybenzaldehyde in Guangxi was 2728.55 ng/g, while the samples from Guangxi had no odor compounds. The average contents of 3,5-dimethoxy-4-hydroxybenzaldehyde in Guangxi and Yunnan were 926.34 ng/g and 2967.95 ng/g and the contents in Yunnan were significantly higher than in Guangxi. Perhaps these compounds play an important role in distinguishing the sugars from Guangxi and Yunnan.
Based on the VIP diagram and OPLS-DA analysis of brown sugar between Guangxi and Yunnan ( Figure 5), they were well separated. A number of compounds contributed to the differentiation between the two provinces, including 4-hydroxybenzaldehyde, 3,5dimethoxy-4-hydroxybenzaldehyde, n-hexadecanoic acid, acetic acid, butanoic acid, and 4-hydroxyacetophenone. Of these volatile compounds, the contribution of 4-hydroxybenzaldehyde was the greatest. The average content of 4-hydroxybenzaldehyde in Guangxi was 2728.55 ng/g, while the samples from Guangxi had no odor compounds. The average contents of 3,5-dimethoxy-4-hydroxybenzaldehyde in Guangxi and Yunnan were 926.34 ng/g and 2967.95 ng/g and the contents in Yunnan were significantly higher than in Guangxi. Perhaps these compounds play an important role in distinguishing the sugars from Guangxi and Yunnan.

Materials
Eighteen brown sugar samples from Guangdong, Guangxi and Yunnan were provided by COFCO. These samples were stored in a refrigerator at −80 °C before analysis.

Extraction of Odor Compounds from Sugars
The odor compounds in brown sugar were extracted by a liquid-liquid extraction (LLE) method according to Chen, et al. [33]. In brief, 50.00 g of brown sugar was placed in a triangular flask, 50 mL of distilled water was added to dissolve the brown sugar, then, 50 mL of ether, 50 mL of dichloromethane and 5 μL of internal standard 2-methyl-3-heptanone (81.6 mg/mL) were added, and the mixture was magnetically stirred at 1000 rpm for 10 min. After centrifugation (Hitachi, Japan) for 30 min at 10,000 rpm, the extract containing the volatile aroma compounds was separated by a funnel. Subsequently, 150.0 g anhydrous sodium sulfate was added to the extract and put into a refrigerator at 4 °C to

Materials
Eighteen brown sugar samples from Guangdong, Guangxi and Yunnan were provided by COFCO. These samples were stored in a refrigerator at −80 • C before analysis.

Extraction of Odor Compounds from Sugars
The odor compounds in brown sugar were extracted by a liquid-liquid extraction (LLE) method according to Chen et al. [33]. In brief, 50.00 g of brown sugar was placed in a triangular flask, 50 mL of distilled water was added to dissolve the brown sugar, then, 50 mL of ether, 50 mL of dichloromethane and 5 µL of internal standard 2-methyl-3-heptanone (81.6 mg/mL) were added, and the mixture was magnetically stirred at 1000 rpm for 10 min. After centrifugation (Hitachi, Japan) for 30 min at 10,000 rpm, the extract containing the volatile aroma compounds was separated by a funnel. Subsequently, 150.0 g anhydrous sodium sulfate was added to the extract and put into a refrigerator at 4 • C to remove water for 12 h, and filtered with a filter paper. A gentle nitrogen stream was used to concentrate the volume into 100 µL, and the odor compounds were extracted and stored at −80 • C for further analysis.

GC-O-MS
Three well-trained panelists conducted a GC-O analysis of the concentrated distillate. The panelists were recruited from Beijing Technology and Business University's Molecular Sensory Laboratory. To identify and describe the aroma characteristics of the reference compounds, they smelled several concentrations of reference compounds in model solutions 2 h per day before analysis. The training lasted for one month. For the GC-O analysis, wet gas was delivered to the nose using a blank capillary column to improve the sensitivity of the panelists. The aroma perceptions, intensity, and RT were recorded by the panelists. If two or more panelists detected the aroma, an aroma-active compound was identified [35].
To determine the volatile aroma profile of sugars, an Agilent 7890A gas chromatograph (GC) coupled with an Agilent 5977B mass spectrometer (MS) and a sniffing port (Gerstel, Germany) was used. The aroma extract (1 µL) was injected into a DB-Wax column (60 m × 0.25 mm i.d., film thickness 0.25 µm, Agilent J&W) through splitless mode, and the flow rate of the helium carrier gas was maintained at 1.7 mL/min. The oven temperature was initially programmed at 40 • C, further raised to 100 • C at a rate of 4 • C/min, following a gradual increase up to 200 • C at a rate of 3 • C/min for 5 min, and after achieving an ultimate temperature of 230 • C at a rate of 3 • C/min, it was maintained for 10 min. The interface and ion source were set at 250 • C and 230 • C, respectively, while the electron-impact ionization was set at 70 eV, the acquisition range (m/z) at 35-350 amu, and the scan rate at 1.77 scans/s. The transmission line temperature of the olfactory detection port (ODP) was maintained at 235 • C.

Qualitative Analysis
The ionization of a molecule in a vacuum produces a characteristic group of ions of different masses. The plot of relative abundance versus mass of these ions constitutes a mass spectrum. The spectrum can be used to identify the molecule. The unknowns were identified by comparing the fragments with the National Institute of Standards and Technology (NIST) MS Spectral Library (Version 2020), by comparing the odor percepts with the database (http://www.thegoodscentscompany.com) and by calculating the linear retention indices (LRIs) using a homologous series of n-alkanes (C 7 -C 30 ). The use of multiple methods can increase the accuracy of qualitative results. Using the internal standard area, the resulting peaks were calibrated, and the aroma compound contents were expressed as nanograms per gram of sample [10].

Odor Activity Value (OAV)
In order to evaluate the contribution of each odorant to the overall aroma of brown sugar, the OAV (ratio of concentration to its odor threshold) was calculated [36]. These threshold values were derived from the literature in water [37].

Statistical Analysis
All experiments in this study were conducted in triplicates, and the data were expressed as mean ± standard deviation. The bar graph was drawn by OriginPro 2022 (OriginLab Corp., Northampton, MA, USA), the OPLS-DA analysis was conducted by SIMCA 14.1 (MKS Instruments, Andover, MA, USA), and the tables were organized by Microsoft Excel 2021 (Microsoft Corp., Redmond, WA, USA).

Conclusions
In summary, a total of 80 odor compounds, including 5 alcohols, 9 aldehydes, 8 phenols, 21 acids, 14 ketones, 5 esters, 12 pyrazines, and 6 other compounds, were detected in 18 brown sugar samples from three different provinces. The fingerprint analysis showed 90% similarity, indicating a close relationship among the odor components of brown sugars from each province without much difference. Further, the stability, accuracy, and repeatability of the fingerprint method were verified, and speculated that the method could meet the requirements of the fingerprint. In the future, fingerprint might have wider applica-tions due to its characteristic of distinguishing geographical origin and food adulteration. Additionally, the OPLS-DA was employed to identify the tracing of brown sugar and to identify the compounds contributing to brown sugars' volatile classification. The results demonstrated that 4-hydroxybenzaldehyde, 3,5-dimethoxy-4-hydroxybenzaldehyde, nhexadecanoic acid, and acetic acid were the essential components in distinguishing the sugars from Guangdong, Guangxi, and Yunnan, validating the efficiency of OPLS-DA. Acknowledgments: The present work was supported by China Oil and Food Import and Export Corporation (COFCO). No project number.

Conflicts of Interest:
The authors declare no conflict of interest.