Determination of Sulfites in Dried Fruits by Paper Spray Ionization Tandem Mass Spectrometry.

Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits.

Discrimination of untreated and sodium sulphite treated bean sprouts by Fourier transform infrared spectroscopy and chemometrics.

Sprouts of black beans (Phaseolus vulgaris L.), soybeans (Glycine max L.) and mung beans (Vigna radiata L.) are widely consumed foods containing abundant nutrients with biological activities. They are commonly treated with sulphites for the preservation and extension of shelf-life. However, our previous investigation found that immersing the bean sprouts in sulphite might convert the active components into sulphur-containing derivatives, which can affect both the quality and safety of the sprouts. This study explores the use of FTIR in conjunction with chemometric techniques to differentiate between non-immersed (NI) and sodium sulphite immersed (SI) black bean, soybean and mung bean sprouts. A total of 168 batches of raw spectra were obtained from NI and SI-bean sprouts using FTIR spectroscopy. Four pre-processing techniques, three modelling assessment techniques and four model evaluation indices were examined for differences in performance. The results show that the multiplicative scatter correction is the most effective pre-processing method. Among the models, the accuracy rate of the three models was as follows: radial basis function neural network (95%) > convolutional neural network (91%) > random forest (82%). The overall findings indicate that FTIR spectroscopy, in conjunction with appropriate chemometric approaches, has a high potential for rapidly determining the difference between NI and SI-bean sprouts.

Metals/bisulfite system involved generation of 24-sulfonic-25-ene ginsenoside Rg1, a potential quality control marker for sulfur-fumigated ginseng.

Ginseng is a most popular health-promoting food with ginsenosides as its main bioactive ingredients. Illegal sulfur-fumigation causes ginsenosides convert to toxic sulfur-containing derivatives, and reduced the efficacy/safety of ginseng. 24-sulfo-25-ene ginsenoside Rg1 (25-ene SRg1), one of the sulfur-containing derivatives, is a potential quality control marker of fumigated ginseng, but with low accessibility owing to its unknown generation mechanism. In this study, metals/bisulfite system involved generation mechanism was investigated and verified. The generation of 25-ene SRg1 in sulfur-fumigated ginseng is that SO2, formed during sulfur-fumigation, reacted with water and ionized into HSO3-. On the one hand, under the metals/bisulfite system, HSO3- generates HSO5- and free radicals which converted ginsenoside Rg1 to 24,25-epoxide Rg1; on the other hand, as a nucleophilic group, HSO3- reacted with 24,25-epoxide Rg1 and further dehydrated to 25-ene SRg1. This study provided a technical support for the promotion of 25-ene SRg1 as the characteristic quality control marker of sulfur-fumigated ginseng.

A highly sensitive ratiometric fluorescent probe for detecting HSO3-/SO32- and viscosity change based on FRET/TICT mechanism.

BACKGROUND: Sulfur dioxide (SO2) is a significant gas signaling molecule in organisms, and viscosity is a crucial parameter of the cellular microenvironment. They are both involved in regulating many physiological processes in the human body. However, abnormalities in SO2 and viscosity levels are associated with various diseases, such as cardiovascular disease, lung cancer, respiratory diseases, neurological disorders, diabetes and Alzheimer's disease. Hence, it is essential to explore novel and efficient fluorescent probes for simultaneously monitoring SO2 and viscosity in organisms. RESULTS: We selected quinolinium salt with good stability, high fluorescence intensity, good solubility and low cytotoxicity as the fluorophore and developed a highly sensitive ratiometric probe QQD to identify SO2 and viscosity changes based on Förster resonance energy transfer/twisted intramolecular charge transfer (FRET/TICT) mechanism. Excitingly, compared with other probes for SO2 detection, QQD not only identified HSO3-/SO32- with a large Stokes shift (218 nm), low detection limit (1.87 µM), good selectivity, high energy transfer efficiency (92 %) and wide recognition range (1.87-200 µM), but also identified viscosity with a 26-fold fluorescence enhancement and good linearity. Crucially, QQD was applied to detect HSO3-/SO32- and viscosity in actual water and food samples. In addition, QQD had low toxicity and good photostability for imaging HSO3-/SO32- and viscosity in cells. These results confirmed the feasibility and reliability of QQD for HSO3-/SO32- and viscosity imaging and environmental detection. SIGNIFICANCE: We reported a unique ratiometric probe QQD for detecting HSO3-/SO32- and viscosity based on the quinolinium skeleton. In addition to detecting HSO3-/SO32- and viscosity change in actual water and food samples, QQD could also monitor the variations of HSO3-/SO32- and viscosity in cells, which provided an experimental basis for further exploration of the role of SO2 derivatives and viscosity in biological systems.

A ratiometric fluorescence probe for bisulfite detection in live cells and meat samples.

The development of reliable probe technology for the detection of bisulfite (HSO3-) in situ in food and biological samples is contributing significantly to food quality and safety assurance as well as community health. In this work, a responsive probe, EHDI, is developed for ratiometric fluorescence detection of HSO3- in aqueous solution, meat samples, and living cells. The probe is designed based on the HSO3- triggered 1,4-addition of electron deficit C = C bond of EHDI. As a result of this specific 1,4-addition, the π-conjugation system was destructed, resulting in blue shifts of the emission from 687 to 440 nm and absorption from 577 to 355 nm. The probe has good water solubility, high sensitivity and selectivity, allowing it to be used for imaging of HSO3- internalization and production endogenously. The capability of probe EHDI for HSO3- was then validated by traditional HPLC technology, enabling accurately detect HSO3- in beef samples. The successful development of this probe thus offers a new tool for investigating HSO3- in situ in food and biological conditions.

Taming Janus-Faced Quinoline-Derived Fluorescent Probes for Dual-Channel Distinguishable Visualization of HSO3- and HClO in Dried Foods and Living Cells.

With the booming development of food manufacturing, developing ideal analytical tools to precisely quantify food additives is highly sought after in the food science field. Herein, a new series of quinoline-derived multifunctional fluorescent probes has been synthesized. Bearing double reactive sites, these compounds display fluorescence response toward both bisulfite (HSO3-) and hypochlorous acid (HClO). Among these compact structures, compound ethyl-2-cyano-3-(6-(methylthio)quinolin-2-yl)acrylate (QTE) was screened out. Probe QTE not only shows ratiometric variation toward HSO3- with little cross talk but also performs turn-off signal toward HClO. In addition, probe QTE has been utilized for bioimaging of HClO in living cells. Furthermore, the HSO3- content in dried food samples has been appraised by QTE with satisfactory results. Meanwhile, relying on the apparent chromaticity change, a flexible dark-box device has been elaborated for chromatic analysis, promoting visualization of HSO3- in the field.

Sulfites detection by surface-enhanced Raman spectroscopy: A feasibility study.

The exhaustive control required for the correct wine production needs of many chemical analysis throughout the process. The most extended investigations for wine production control are focused on the quantification of total and free SO2. Most methods described in the literature have an adequate detection limit, but they usually lack reproducibility and require a previous sample treatment for the extraction of the SO2 from the wine-matrix. In this context, Surface-Enhanced Raman Spectroscopy (SERS) can be a promising technique for free SO2 determination without the need for any sample pre-processing. This work describes a proof of concept of a new methodology based on SERS and supported by Density Functional Theory (DFT) calculations to identify the active vibrational modes of the key molecules that contribute to the concentration of free SO2 in solution. Theoretical predictions and experimental outcomes are brought together to chemometrics to get a simple and real-time free SO2 monitoring. This general procedure could pave the way towards an implementation of a portable SERS detection module for in-field measurements.

Multi-well plate as headspaces for paper-based colorimetric detection of sulfur dioxide gas: An alternative method of sulfite titration for determination of formaldehyde.

This work describes the analysis of formaldehyde using a 96-well microplate as multiple headspaces for the separation of sulfur dioxide gas generated from the sulfite remaining after its reaction with the formaldehyde in the sample. The quantitation of the gas is by colorimetric detection of an indicator paper placed over the microplate. The samples are aqueous extracts of various foods that are possibly adulterated with formaldehyde. A known excess amount of sulfite is added to the extract solution aliquoted in the well. The remaining sulfite is acidified with hydrochloric acid to generate sulfur dioxide gas which diffuses through the headspace above the solution to be absorbed at the moist strip of the indicator paper placed over the mouth of the wells. Anthocyanins extracted from the butterfly pea flower is used as the pH indicator giving a color change from the increase of hydrogen ions by hydrolysis of the absorbed sulfur dioxide gas. The exposed paper strip is scanned, and the digital images of the colored region analyzed using ImageJ software. The optimized method has a linear range of 200-1000 mg L-1 formaldehyde with limit of detection ((2.57*SD of intercept)/(slope of calibration line)) of the aqueous extract of 40 mg L-1 and coefficient of determination (r2) > 0.9979. Samples of fresh produce, such as seafood, meat, and vegetables, and various processed food were analyzed for their possible formaldehyde content. The results obtained from the headspace paper-based colorimetric detection are not statistically different from the values obtained from the titration method by paired t-tests.

A fully integrated 3D printed platform for sulfite determination in beverages via gas diffusion membrane extraction and digital video treatment.

In this study, we have described a miniaturized, simple, and low-cost device for sulfite determination in beverages by coupling Gas Diffusion Microextraction to paper-based analytical devices. The color change of an acid-base indicator - promoted by the generated gaseous SO2 - impregnated onto the paper surface was monitored in the function of time by video recording using a smartphone. The analytical information was related to the Hue, Saturation, Value (HSV) color space extracted from the video file. The complete analytical platform was built using a 3D printer, allowing the easy fabrication of a low-cost tailored device. Under optimized conditions, a linear relation from 5 to 90 mg L-1 was obtained using 30 µL of the reagent, 1 mL of sample, and 10 min of analysis. The relative standard deviation and the limit of detection were 2.2 % and 1.6 mg L-1, respectively. The method was successfully employed in several beverages, such as juices, soda, and coconut water.

Baseline composition and microbial quality assessment of raw milk from small ruminants and Maghrebi camels in the oasis area of Tunisia.

The milk quality and characteristics of the local Gharbi sheep and autochthonous goat population were studied and compared to those of the local Maghrebi camel. Milk samples from 378 lactating animals raised in the Tunisian oasis region were obtained and processed for various physicochemical compositions (pH, density, acidity, dry matter, fat, protein, lactose, casein, ash, and casein-protein ratio), mineral concentrations (Ca, P, Na, and K), and bacteriological properties (total mesophilic aerobic bacteria (TMAB), total coliform count (TCC), lactic acid bacteria (LAB), sulfite-reducing Clostridium (CSR), yeast and molds (Y/M), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Salmonella) using standard methods. Milk from sheep breeds had a higher average of all physical parameters (pH, density, and dornic acidity) than milk from goat species. The sheep population produced milk with a similar pH to the camel population, but with a higher density and acidity content. The pH and acidity were higher in Neggas than in goat species, while density was similar in both. For chemical composition, the results showed significant heterogeneity in milk content across all species. Except for the casein-protein ratio, which favors goat species, the analysis indicates that sheep species were superior to populations of goats and camels in all chemical compositions. The present results showed considerable variation in the mineral content of milk from different species. The levels of calcium and phosphorus are higher in sheep than in goat and camel milk. Compared to small ruminants, milk from camels is the richest in Na and K. Additionally, more calcium is present in the milk of camels than that of goats. Goat milk, the lowest in Ca and Na, contains more P than camel milk and more K than sheep's milk on average. The poorest microbial quality was that of camel milk for all bacterial counts. Based on TMAB, TCC, and E. coli counts, the microbiological quality of goat milk was higher than that of ovine milk, while ovine milk had better quality based on LAB, Y/M, and S. aureus values. For Escherichia coli and Staphylococcus aureus, there were no significant variations between the species studied. Results showed that all milk samples studied were completely free of two dangerous pathogens, Salmonella and sulfite-reducing Clostridium. The bacteriological quality of small ruminant's milk was acceptable and met the regulatory limits set by Tunisian dairy legislation. Regarding camel milk, the microbial analysis revealed poor quality that exceeds standard criteria.

Reagent-less and sub-minute quantification of sulfite in food samples using substrate-integrated hollow waveguide gas sensors coupled to deep-UV LED.

The increasing consumption of processed foods demands the usage of chemical preservatives to ensure freshness and extended shelf life. For this purpose, sodium sulfite and its derivatives have been widely used in a variety of food products to inhibit microbial spoilage and for mitigating oxidative decay. However, the excessive consumption of sulfite may cause health problems, thus requiring rapid and accurate analytical methods for the rapid identification of threshold levels. Conventionally, sulfite is volatilized from food samples by acidification followed by trapping of the gaseous SO2 and determination using a suitable analytical technique. Herein, we propose a yet unprecedented reagent-less approach via direct absorbance measurements of gaseous SO2 at 280 nm after sample acidification. The detection system combines a deep-UV LED and a SiC photodiode with a substrate-integrated hollow waveguide (iHWG) gas cell. Absorbance measurements were performed using a log-ratio amplifier circuitry, resulting in noise levels <0.7 mAU. This innovative concept enabled the determination of sulfite in beverages in the range of 25-1000 mg L-1 with suitable linearity (r2 > 0.99) and an analysis time <30 s. The limit of detection (LOD) was calculated at 14.3 mg L-1 (3σ) with an iHWG providing an optical path length of 75 mm. As a proof of concept, this innovative analytical platform was employed for sulfite quantification in concentrated grape juice, coconut water and beer, with suitable accuracy in terms of recovery (83-117%) and favorable comparison with the official Monier-Williams method. Given the inherent modularity and adaptability of the device concept, we anticipate the application of the proposed analytical platform for the in-situ studies addressing sulfite and other volatilized preservatives in a wide variety of food products with tailorable detectability.

Methyl Salicylate, an Odor-Active Compound in Bordeaux Red Wines Produced without Sulfites Addition.

Red wines produced without the addition of any SO2 are currently the source of a new consumer trend. The first characterization approaches regarding these specific wines were devoted to sensory studies that highlighted differences according to the use of SO2 during winemaking. The goal of this paper is to extend our knowledge of such aromatic specificities. Examining experimental wines produced with and without the addition of SO2, aroma fractionation, gas chromatography coupled with olfactometry, and mass spectrometry were applied to identify compounds at the origin of the specificities of these wines. Thus, we identified methyl salicylate as being impacted by the use of SO2 during the winemaking process. Studying a large number of commercial Bordeaux red wines, methyl salicylate was significantly quantified at a higher content in wines without added SO2. A sensory approach revealed a significant impact of methyl salicylate on wines without the sulfite aroma, particularly concerning fruity aromas and wine freshness.

Desulfoluna limicola sp. nov., a sulfate-reducing bacterium isolated from sediment of a brackish lake.

A novel sulfate-reducing bacterium, strain ASN36T, was isolated from sediment of a brackish lake in Japan. Cells of strain ASN36T were not motile and rod-shaped, with length of 2.0-4.9 µm and width of 0.6-0.9 µm. Growth was observed at 5-35 °C with an optimum growth temperature of 25-30 °C. The pH range for growth was 6.6-8.8 with an optimum pH of 7.3. Major fatty acids were C16:1 ω7c and C16:0. Under sulfate-reducing conditions, strain ASN36T utilized lactate, malate, pyruvate, butyrate, ethanol, butanol, glycerol, yeast extract and H2/CO2 as growth substrate. Fermentative growth occurred on malate and pyruvate. The novel isolate used sulfate, sulfite and thiosulfate as electron acceptors. The genome of strain ASN36T is composed of a chromosome with length of 6.3 Mbp and G + C content of 55.1 mol%. Analyses of the 16S rRNA gene indicated that strain ASN36T is related to Desulfoluna species. Overall genome relatedness indices indicated that strain ASN36T does not belong to any existing species. In contrast to the closest relatives, strain ASN36T lacks genes for reductive dehalogenase required for organohalide respiration and does not use halogenated aromatics as electron acceptors. On the basis of its genomic and phenotypic properties, strain ASN36T (= DSM 111985 T = JCM 39257 T) is proposed as the type strain of a new species, Desulfoluna limicola sp. nov.

Desulfofustis limnaeus sp. nov., a freshwater sulfate-reducing bacterium isolated from marsh soil.

A novel sulfate-reducing bacterium, strain PPLLT, was isolated from marsh soil. Cells of strain PPLLT were rod-shaped with length of 1.5 µm and width of 0.7 µm. Growth was observed at 22-37 °C (optimum 35 °C) and pH 6.8-8.4 (optimum 7.3). Lactate, succinate, fumarate, formate and malate were utilized as electron donors for sulfate reduction. Fermentative growth was not observed on tested organic acids. Besides sulfate, sulfite, thiosulfate and elemental sulfur were utilized as electron acceptors. Hydrogen is used only in the presence acetate or yeast extract. The major fatty acid was C16:0. The complete genome of strain PPLLT was composed of a circular chromosome with length of 4.2 Mbp and G + C content of 57.7 mol%. Sequence analysis of the 16S rRNA gene showed that strain PPLLT was affiliated with the genus Desulfofustis in the family Desulfocapsaceae. On the basis of differences in the phylogenetic and phenotypic properties between the strain and the type strain of the genus Desulfofustis, strain PPLLT (DSM 110475T = JCM 39161T) is proposed as the type strain of a new species, with name of Desulfofustis limnaeus sp. nov.

Simple and sensitive determination of sulfites in Chinese herbal teas by ultrahigh-performance liquid chromatography tandem mass spectrometry.

Sulfites are used widely in food and beverage production to prevent browning or oxidation. However, the overingestion of sulfites is harmful to human health and may cause medical complications. Chinese herbal teas have been widely consumed for centuries. However, sulfite levels in Chinese herbal teas are rarely investigated and reported. Here, we present a simple, sensitive, and quantitative method to determine sulfites in Chinese herbal teas using ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) coupled with dispersive solid phase extraction. The method utilized a SeQuant ZIC-HILIC column for separation, and the optimal gradient eluents consisted of acetonitrile and aqueous solution with 0.1% acetic acid and 10 mM ammonium acetate. Porous chitosan/partially reduced graphene oxide/diatomite (CS/prGO/DM) composites were used as efficient dispersive solid phase extraction adsorbents for sample preparation. Several parameters were investigated during the extraction process, including sample-to-extraction solvent volume ratios, the extraction procedure and dosage of the adsorbent. Under the optimum conditions, the developed method gave a good determination coefficient (r2 > 0.99), low detection limits (0.51-12.1 µg kg-1) and high recoveries in the range of 83.8-102.7% at different spiked levels. The method has the great advantages of being time saving, good reproducibility and much lower detection limits when compared to titration methods. The method was further applied to analyze real herbal tea samples collected from the local market, demonstrating that our developed method is robust and useful for determining sulfites in practical application.

Amperometric Sensor for Selective On-Site Analysis of Free Sulfite in Wines.

Accurate and rapid on-site analysis of free SO2 content is crucial in the process of winemaking from a producer and consumer perspective. Herein, we present an amperometric sensor based on commercially available screen-printed electrodes coupled with an electrochemical oxygen filter. The developed amperometric method gave a linear response in a concentration range up to 200 mg L-1 with a limit of quantification of 7.5 mg L-1. The applicability of the developed sensor was successfully tested on 27 white and red wine samples and compared to the Ripper method (iodometry) that is a standard procedure for free SO2 determination. The sensor exhibits similar precision and accuracy but shows no interference from oxidizable species such as ascorbic acid, which is a major advantage over iodometric titration. The performance of the sensor was in addition positively evaluated during on-site analysis in a winery.

A mitochondria-targeted near-infrared fluorescent probe for detection and imaging of HSO3- in living cells.

Sulfur dioxide, an essential gas signaling molecule mainly produced in mitochondria, plays important roles in many physiological and pathological processes. Herein, a near-infrared fluorescent probe, A1, with good mitochondria targeting ability was developed for colorimetric and fluorescence detection of HSO3-. Probe A1 has a conjugated cyanine structure that can selectively react with HSO3- through the nucleophilic addition. The reaction with HSO3- destroys the conjugated structure of probe A1, resulting in fluorescence quenching, and accompaniedby color change of probe A1 solution from purple-red to colorless. Probe A1 showed high selectivity and good sensitivity to HSO3- in PBS. And the limit of detection was calculated to be 1.28 and 0.037 µM for colorimetry and fluorescence spectrophotometry respectively. In addition, probe A1 mainly entered the mitochondria in living cells, and was successfully used for imaging the exogenous/endogenous HSO3- in cells. These results suggest the potential applications of probe A1 in biological systems.

Disposable Microchamber with a Microfluidic Paper-Based Lid for Generation and Membrane Separation of SO2 Gas Employing an In Situ Electrochemical Gas Sensor for Quantifying Sulfite in Wine.

This work presents a fully disposable microchamber for gas generation of a sample solution. The microchamber consists of a cylindrical well-reactor and a paper-based microfluidic lid (µFluidic lid), which also serves as the reagent loading and dispensing unit. The base of the reactor consists of a hydrophobic membrane covering an in-house graphene electrochemical gas sensor. Fabrication of the gas sensor and the three-layer µFluidic lid is described. The µFluidic lid is designed to provide a steady addition of the acid reagent into the sample solution instead of liquid drops from a disposable syringe. There are three steps in the procedure: (i) acidification of the sample in the reactor to generate SO2 gas by the slow dispensing of the acid reagent from the µFluidic lid, (ii) diffusion of the liberated SO2 gas through the hydrophobic membrane at the base of the reactor, and (iii) in situ detection of SO2 by cathodic reduction at the graphene electrode. The device was demonstrated for quantitation of the sulfite preservative in wine without heating or stirring. The selectivity of the analysis is ensured by the combination of the gas-diffusion membrane and the selectivity of the electrochemical sensor. The linear working range is 2-60 mg L-1 SO2, with a limit of detection (3SD of intercept/slope) of 1.5 mg L-1 SO2. This in situ method has the shortest analysis time (8 min per sample) among all voltammetric methods that detect SO2(g) via membrane gas diffusion.

A quinoline-based probe for the ratiometric fluorescent detection of sulfite in lysosomes of living cells.

A lysosome-targeting ratiometric fluorescent probe was synthesized for detecting sulfite based on sulfite-triggered nucleophilic addition reaction. Due to the specific reaction, the fluorescence intensity ratio (I530/I390) of the probe in an almost aqueous solution (0.5% DMSO) changed significantly after the addition of HSO3-, corresponding to the change in the fluorescence color of the solution from green to blue. The recognition was conducted using high-resolution mass spectrometry, proton nuclear magnetic resonance, and density functional theory calculations. The fluorescent probe could be utilized to quantitatively monitor HSO3- in lysosomes of living C6 glioma cells and real-water samples.

Induced sulfur metabolism by sulfur dioxide maintains postharvest quality of 'Thompson Seedless' grape through increasing sulfite content.

BACKGROUND: The commercial preservation of table grapes largely depends on the application of sulfur dioxide (SO2 ). However, little is known about whether SO2 participates in sulfur metabolism to improve the postharvest quality of table grapes. In this study, the contents of sulfur-containing compounds, activities of enzymes, and expression of genes involved in sulfur metabolism in table grapes (Vitis vinifera cv. Thompson Seedless) were evaluated. RESULTS: The results indicated that SO2 treatment maintained the postharvest quality of table grapes. The sulfite content in rachises and berries, but not the sulfate content, increased in response to SO2 treatment. SO2 caused high activities of sulfite reductase, O-acetylserine (thiol)-lyase, and γ-glutamylcysteine synthetase, thereby increasing the contents of cysteine, hydrogen sulfide, and glutathione in the rachises and berries. The expression of VvSURTL, VvATPS1, VvATPS2, and VvAPR3 decreased in response to SO2 treatment; however, the transcript levels of VvSiR1 and VvOASTL exhibited the opposite tendency. CONCLUSION: These findings indicated that the sulfite converted from SO2 participated in sulfur metabolism and maintained the postharvest quality of table grapes by modulating the contents of metabolites, activities of enzymes, and expression of genes related to sulfur metabolism. © 2021 Society of Chemical Industry.