Production Of Volatile Sulfur Compounds Research Articles

Evaluation of a quantitative screening method for hydrogen sulfide production by cheese-ripening microorganisms: The first step towards l-cysteine catabolism

A practical adaptation of the methylene blue reaction for hydrogen sulfide quantification was developed to perform microbial selection. Closed plate flasks containing a zinc-agar layer above the liquid microbial culture are proposed as a trap system where the H 2S can be retained and then quantified by the methylene blue reaction. Using this quantitative method, the ability to produce H 2S was studied in several cheese-ripening microorganisms. Our aim was to select strains that produce the highest quantities of H 2S as the main product of l-cysteine catabolism. Thirty seven yeast and bacteria strains were cultivated with or without l-cysteine. The separation between the growth medium and the H 2S trapping layer displayed good performance: all the studied strains grew efficiently and only negligible loss of H 2S was observed during culturing. The strains displayed large differences in their H 2S production capabilities: yeast strains were greater producers of H 2S than bacteria with production strain-related in both cases. Furthermore, the relationship between H 2S production and l-cysteine consumption was analyzed, which made it possible for us to select microorganisms with high capacity in l-cysteine degradation. The production of volatile sulfur compounds was also studied and the possible effect of culture pH and metabolic differences between strains are discussed.

Short Communication: Empirical and Mechanistic Evidence for the Role of Pyridoxal-5′-Phosphate in the Generation of Methanethiol from Methionine

The catabolism of the sulfur-containing AA Met to form flavor-active volatile sulfur compounds (VSC) is an important mechanism in flavor development during cheese maturation. Numerous enzymes catalyzing AA catabolism require the presence of the cofactor pyri-doxal-5′-phosphate (PLP). In fact, reports have shown that some of these reactions can be catalyzed by PLP alone, albeit at a reduced rate. We hypothesized that, as a specific application in cheese flavor reactions, PLP can react directly with free Met to generate a specific VSC, methanethiol (MTH). In this study, the ability of PLP to catalyze MTH generation from Met was examined under “cheeselike” conditions of salt and pH. Methionine and varying concentrations of PLP were incubated in a buffer (pH 5.2 + 4.0% NaCl) analogous to the aqueous phase of aged Cheddar cheese. Samples were analyzed using headspace solid-phase microextraction, and relative concentrations of VSC were determined by gas chromatography–mass spectrometry. Results showed MTH, dimethyl disulfide, and dimethyl trisulfide production when Met and PLP were incubated together at 7°C (cheese-aging temperature). These results indicate that the production of VSC from Met can occur nonenzymatically as catalyzed by free PLP.

Control of soilborne potato diseases using Brassica green manures

Brassica crops used in crop rotations and as green manures have been associated with reductions in soilborne pests and pathogens. These reductions have been attributed to the production of volatile sulfur compounds through a process known as biofumigation, and to changes in soil microbial community structure. In this study, selected Brassica crops, including canola, rapeseed, radish, turnip, yellow mustard, and Indian mustard, were evaluated for control of various soilborne potato pathogens and diseases in culture, in greenhouse trials, and in field trials on commercial potato farms. In in vitro assays, volatiles released from chopped leaf material of Brassica crops and barley inhibited growth of a variety of soilborne pathogens of potato, including Rhizoctonia solani, Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, and Fusarium sambucinam, with Indian mustard resulting in nearly complete inhibition (80–100%). All Brassica crops and barley reduced inoculum levels of R. solani (20–56% reduction) in greenhouse tests, and radish, rapeseed, and Indian mustard reduced subsequent potato seedling disease by 40–83%. In an on-farm field trial at a site with a substantial powdery scab problem, Indian mustard, rapeseed, canola, and ryegrass grown as green manure rotation crops reduced powdery scab in the subsequent potato crop by 15–40%, and canola and rapeseed reduced black scurf by 70–80% relative to a standard oats rotation. At another field site where common scab was the primary disease problem, an Indian mustard green manure reduced common scab by 25%, and rapeseed, yellow mustard, and ‘Lemtal’ ryegrass also reduced black scurf relative to a standard ryegrass rotation. Disease reductions were not always associated with higher glucosinolate-producing crops, and were also observed with non- Brassica crops (barley and ryegrass), indicating other mechanisms and interactions are important, particularly for control of R. solani. Overall, Indian mustard was most effective for reducing powdery scab and common scab diseases, whereas rapeseed and canola were most effective in reducing Rhizoctonia diseases. These results indicate that Brassica crops have potential for use as green manures for the control of multiple soilborne disease problems.

Generation Pattern of Sulfur Containing Gases from Anaerobically Digested Sludge Cakes

Eleven dewatered sludge cakes collected from anaerobic digesters at different treatment plants were evaluated for the amount, type, and pattern of odorous gas production. All but one of the sludge cakes were from mesophilic anaerobic digesters. One was from a thermophilic digester. The pattern and quantities of sulfur gases were found to be unique for each of the samples with regard to the products produced, magnitude, and subsequent decline. The main odor-causing chemicals were volatile sulfur compounds, which included hydrogen sulfide, methanethiol, and dimethyl sulfide. Volatile sulfur compound production peaked in 3 to 8 days and then declined. The decline was a result of conversion of organic sulfur compounds to sulfide. In one side-by-side test, a high-solids centrifuge cake generated more odorous compounds than the low-solids centrifuge cake. The data show that anaerobic digestion does not eliminate the odor potential of anaerobically digested dewatered cakes.

YtjE from Lactococcus lactis IL1403 Is a C-S Lyase with α,γ-Elimination Activity toward Methionine

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.

Composition of the bacterial flora in tonsilloliths

Tonsilloliths are a potential cause of oral malodor. In this study, microbial profiles and composition of tonsilloliths were determined using culture-independent molecular methods and scanning electron microscopy. 16S ribosomal RNA bacterial genes (16S rDNAs) isolated from tonsilloliths of 6 individuals were amplified by PCR and cloned into Escherichia coli. Partial 16S rDNA sequences of approximately 600 bases of cloned inserts were used to determine species identity by comparison with sequences of known species. Characteristics of bacteria on the surface and inside the tonsillolith were analyzed using scanning electron microscopy. Anaerobic bacteria detected in tonsilloliths belonged to the genera Eubacterium, Fusobacterium, Megasphaera, Porphyromonas, Prevotella, Selenomonas and Tannerella, all of which appear to be associated with production of volatile sulfur compounds. Electron microscopy revealed cocci and rods on the surface and rods predominating inside the tonsilloliths. These results support the tonsillolith as an origin of oral malodor.

REDUCTION OF IRRADIATION-INDUCED QUALITY CHANGES BY ROSEMARY EXTRACT IN READY-TO-EAT TURKEY MEAT PRODUCT

Ionizing radiation can effectively inactivate food borne pathogens in meat and meat products. However, at high doses, it may induce an undesirable color change and an off-odor which is partially due to production of volatile sulfur compounds. This study was conducted to investigate the effects of rosemary extract applied either in formulation or as a post manufacture dip on irradiation-induced volatile sulfur compounds as well as changes in color and lipid oxidation in turkey bologna. Turkey bologna was prepared from ground turkey emulsions with or without rosemary extraction at a final concentration of 0.075%. After cooking, bologna was sliced, sealed in gas impermeable bags, exposed to 0, 1.5 and 3.0 kGy gamma rays, and then stored at 5°C for up to 8 weeks. In the second experiment, slices of turkey bologna were dipped in water or 0.75% of rosemary extract for 2 min followed by irradiation at 3.0 kGy. Results showed that rosemary extract applied in formulation inhibited lipid oxidation in both irradiated and nonirradiated samples. Irradiation increased redness and lightness while reduced yellowness of samples. Rosemary extract was able to inhibit the irradiation-induced color changes. Irradiation induced production of volatile sulfur compounds, such as methanethiol and dimethyl disulfide. Rosemary extract applied either in formulation or as a dip, however, did not significantly reduce the formation of the volatile sulfur compounds.

Volatile Sulfur Compounds Produced by Helicobacter pylori

To assess the volatile sulfur compounds produced by three strains of Helicobacter pylori in broth cultures mixed with sulfur-containing amino acids. Halitosis has been reported in H. pylori-positive patients, and volatile sulfur compounds such as hydrogen sulfide and methyl mercaptan are known to be responsible for inducing oral malodor. Whether H. pylori produces these volatile sulfur compounds has yet to be established. Three strains of H. pylori (ATCC 43504, SS 1, DSM 4867) were cultured with 5 mM cysteine and methionine. After 72 hours of incubation, the headspace air was aspirated and injected directly into a gas chromatograph. The concentrations of hydrogen sulfide and methyl mercaptan were analyzed and compared between experimental and control cultures In broth containing 5 mM cysteine, hydrogen sulfide was increased by ATCC 43504 (P < 0.01) and SS 1 (P < 0.05), while methyl mercaptan was elevated only by SS 1 (P < 0.05). In broth containing 5 mM methionine, methyl mercaptan increases were significant for SS 1 (P < 0.05) and DSM 4867 (P < 0.05). In broth containing 5 mM cysteine and 5 mM methionine, the concentration of hydrogen sulfide was higher than in controls for all three strains (P < 0.01); that of methyl mercaptan was higher only for SS 1 (P < 0.01). Cysteine addition to cultures containing methionine increased hydrogen sulfide and methyl mercaptan for ATCC 43504 (P < 0.05) and SS 1 (P < 0.05). Conversely, addition of methionine to cultures containing cysteine increased methyl mercaptan only for DSM 4867 (P < 0.01). The production of volatile sulfur compounds by H. pylori is not only very complicated but also strain-specific. Nevertheless, H. pylori was shown to produce hydrogen sulfide and methyl mercaptan, which suggests that this microorganism can contribute to the development of halitosis.

L-methionine degradation pathway in Kluyveromyces lactis: identification and functional analysis of the genes encoding L-methionine aminotransferase.

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of L-methionine. L-methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, KlARO8.1 and KlARO8.2, were found to be responsible for L-methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.

Influence of anxiety on the production of oral volatile sulfur compounds

Since many patients complain about halitosis without there being any clinical evidence of its cause, psychological symptoms have been pointed out as halitosis-inducing factors. The aim of this study was to evaluate the influence of anxiety on the production of volatile sulfur compounds (VSC). Seventeen undergraduate men in good oral and general health participated in this study, after approval by the ethics committee. The volunteers were requested to refrain from toothbrushing, using mouth rinse and eating on the experimental day. Before presenting the anxiogenic condition, the volunteer was asked to fill out the Beck Anxiety Inventory questionnaire, to check whether he had been exposed to stressors during the previous week. The Video-Recorded Stroop Color-Word Test (VRSCWT) was used to elicit anxiety. The VSC (halimeter), blood pressure, heart rate and salivary flow measurements were taken before and after the VRSCWT. The volunteers presented a minimal or slight level of anxiety before the test. There was an increase in the oral concentration of VSC, Systolic Blood Pressure and of heart rate ( p < 0.05) after the VRSCWT, and no changes in the salivary flow. The results of the present study showed that the anxiogenic condition (VRSCWT) induced increases in VSC concentration, which might contribute to halitosis.

Evidence for Distinct l -Methionine Catabolic Pathways in the Yeast Geotrichum candidum and the Bacterium Brevibacterium linens

Tracing experiments were carried out to identify volatile and nonvolatile L-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the L-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and alpha-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of L-methionine catabolism. However, the L-methionine degradation pathways differ in the first steps of L-methionine degradation. L-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens.

Inhibitory effect of Weissella cibaria isolates on the production of volatile sulphur compounds

The objective of this study was to characterize the inhibitory effects of Weissella cibaria isolates on volatile sulphur compounds (VSC) production both in vitro and in vivo. We isolated and identified three hydrogen peroxide-generating lactobacilli from children's saliva, and assessed their inhibitory effects on VSC production and Fusobacterium nucleatum proliferation. Clinical studies were conducted with 46 subjects in order to measure the VSC of their mouth air. These lactobacilli were identified as W. cibaria. These isolates inhibited the production of VSC by F. nucleatum (p<0.05). The concentration of F. nucleatum was decreased by 5-log cycles as a result of exposure to the W. cibaria strains (p<0.05), whereas the catalase-treated W. cibaria cultures exerted no evident inhibitory effects on F. nucleatum replication. In the clinical studies, gargling with one isolate resulted in a significant reduction in the levels of H2S and CH3SH by approximately 48.2% (p<0.01) and 59.4% (p<0.05), respectively. These results indicate that W. cibaria isolates possess the ability to inhibit VSC production under both in vitro and in vivo conditions, demonstrating that they bear the potential for development into novel probiotics for use in the oral cavity.

Identification of volatile sulfur compounds produced by Shigella sonnei using gas chromatography–olfactometry

This study investigates volatile sulfur compound production by Shigella sonnei cultured on tryptic soy agar (TSA) and TSA supplemented with 80 μg/ml of the antibiotic rifampicin (TSA-R). Headspace volatiles were trapped and concentrated using solid phase microextraction and identified by gas chromatography–olfactometry (GC–O) and gas chromatography–mass spectroscopy (GC–MS). Results identified two aroma active compounds: methanethiol (MT) and dimethyl sulfide (DMS). Potential pathways of MT and DMS formation were identified as the breakdown of l-methionine by l-methionine-α-deamino-γ-mercaptomethane-lyase (METase) and METase-mediated metabolism of methylated l-methionine, respectively. DMSO, used in TSA-R preparation, may have contributed to an increase in malodor production due to the action of DMSO reductase.

Involvement of a Branched-Chain Aminotransferase in Production of Volatile Sulfur Compounds in Yarrowia lipolytica

The enzymatic degradation of L-methionine and the subsequent formation of volatile sulfur compounds (VSCs) are essential for the development of the typical flavor in cheese. In the yeast Yarrowia lipolytica, the degradation of L-methionine was accompanied by the formation of the transamination product 4-methylthio-2-oxobutyric acid. A branched-chain aminotransferase gene (YlBCA1) of Y. lipolytica was amplified, and the L-methionine-degrading activity and the aminotransferase activity were measured in a genetically modified strain and compared to those of the parental strain. Our work shows that L-methionine degradation via transamination is involved in formation of VSCs in Y. lipolytica.

Short‐term and long‐term effects of increasing temperatures on the stability and the production of volatile sulfur compounds in full‐scale thermophilic anaerobic digesters

This study compares the effect of a rapid increase of the digester temperature (from 54 degrees C to 58 degrees C in 2 weeks) with a slow increase (from 53.9 degrees C to 57.2 degrees C at a rate of 0.55 degrees C per month) on full-scale thermophilic anaerobic digestion at Hyperion Treatment Plant. The short-term test demonstrated that rapidly increasing the digester temperature caused elevated production of volatile sulfur compounds, most notably methyl mercaptan, but volatile solids destruction and methane production were not significantly affected. The increase of the volatile fatty acid to alkalinity ratio from 0.1 to over 0.3 indicated a transient change in digester biochemistry, which was reversed by lowering the temperature. In the long term-test, a slow increase of digester temperature, the production of hydrogen sulfide increased above temperatures of 56.1 degrees C, but was controlled by increased injection of ferrous chloride. Methyl mercaptan was detected in trace amounts at the highest temperature tested (57.2 degrees C). This test showed insignificant effects on other digestion parameters, although some temperature-independent changes were observed that could have been seasonal effects over the year that the long-term test lasted. Thus a slow temperature increase was preferable. This observation contrasts with previous results showing the desirability of a rapid temperature rise to first establish a thermophilic culture when converting from mesophilic operation. Further research is warranted on temperature limits and process changes to optimize thermophilic anaerobic digestion.

Importance of curd-neutralising yeasts on the aromatic potential of Brevibacterium linens during cheese ripening

Three cheese-ripening yeasts Kluyveromyces lactis , Geotrichum candidum and Debaryomyces hansenii were grown in a cheese slurry either individually or in association with the cheese-ripening bacterium Brevibacterium linens . Alcohols, aldehydes and esters were produced by the yeasts, and appeared in the early stages of ripening, whereas volatile sulphur compounds (VSCs) were produced late during the ripening process. The production of VSCs was greatly improved when the yeasts D. hansenii and K. lactis were associated with B. linens . In contrast to G. candidum , these yeasts efficiently neutralised the cheese slurry, which promoted the growth of B. linens . Our results gave evidence of metabolic synergies between the yeasts and B. linens . For instance, generation of S -methyl thioacetate was improved when a yeast that was able to produce esters and thus to accumulate acyl CoA was associated with a bacterium that was able to form large amounts of methanethiol.

O9 Creation of oral care flavours to deliver breath freshening benefits – an in vitro method

Flavours conventionally help to deliver breath freshening in oral care products via sensory masking. The aim of this study was to determine whether flavours could additionally inhibit formation of malodorous compounds by bacteria. Oral malodour is in large part related to the production of volatile sulphur compounds (VSCs) by Gram‐negative organisms. We therefore developed a quantitative in vitro assay for hydrogen sulphide production by Klebsiella pneumoniae. The entire Quest dental flavour palette was screened for inhibition of H2S production. A database of flavour ingredient effects was created, and used to create VSC‐inhibitory flavours. These flavours were assessed in the in vitro assay to confirm that the compounded flavour performed as expected. The best performing flavours developed in this way were selected for testing in the Quest Breath Freshness Panel (see poster, P21). Rules based on the performance of dental flavour ingredients, singly or in combination, were also used to develop a patent position. Flavours formulated using these rules (termed Q‐fresh flavours) have been incorporated into a range of oral care products including toothpastes, mouthwashes, chewing gums and breath films. These products have been tested in the Quest Breath freshness panel, and have been shown to deliver significant breath freshening benefits. An in vitro VSC‐inhibition method has allowed reliable development of flavours with breath‐freshening benefits.

P33 In vitro perfusion biofilm model for the growth of oral microbes associated with oral malodour

Objectives Oral malodour is mainly due to the generation of volatile sulphur compounds (VSC) by oral anaerobes present in the tongue biofilm. The aim of this study was to evaluate the use of a biofilm system to model the ecology and VSC physiology of tongue biofilm microcosms. Of particular interest was the use of the model to maintain a high diversity of bacterial groups and study their response to environmental changes including exposure to VSC substrates and inhibitors.Methods Biofilms were grown using a Sorbarod perfusion model modified by supply of anaerobic gas to grow oral anaerobes. Sorbarods were inoculated using tongue scrape suspension containing 3 × 108 cfu ml−1 and pre‐incubated anaerobically for 24 h at 37°C to promote cell adhesion. After 24 h, the biofilm system was perfused at 12 ml h−1 with BHI (0.74 g L−1) supplemented with haemin, cysteine and/or DTT. Biofilms and eluates (released cells) were sampled up to 96 h and numbers of broad bacterial groups enumerated by selective media. VSC production was measured using a HalimeterTM.Results Repeated analysis of biofilms showed that a quasi‐steady state was achieved between 48 and 96 h. The mean specific growth rate was 0.018 h−1. Ecological analysis showed all broad groups were maintained at a constant proportion reflecting the original inoculum for both biofilms and eluates. VSC specific activity was upregulated when biofilms were perfused with appropriate VSC‐substrates (cysteine, glutathione and methionine). In situ exposure of the biofilms to pulses of putative inhibitors (Zn, H2O2 and chlorhexidine) at a range of concentrations typically used in vivo gave dose‐dependent inhibition of VSC production (for Zn and H2O2) and reduced viable numbers by one to two log‐fold (H2O2 and chlorhexidine).Conclusions The modified Sorbarod perfusion model is suitable for maintaining high diversity biofilms representative of the tongue flora in terms of both ecological behaviour and response towards VSC substrates and inhibitors.

P30 Relationship of oral malodor with tongue microbiota analyzed with real‐time PCR

Oral malodor is thought to mainly originate from the bacterial population present on the surfaces of tongue. However, the relationship between oral malodor and tongue microbiota has been little studied. In the present study, we analyzed tongue microbiota with real‐time PCR and examined an association of oral malodor with tongue microbiota. The subject population consisted of 37 patients who complained of oral malodor. Oral malodor was assessed by an organoleptic test (OLT) and volatile sulfur compound (VSC) levels by gas chromatography. Real‐time PCR was carried out in tongue biofilm samples using a LightCyclerTM system as previously described (Kuboniwa et al. 2002) and the bacterial proportion in clinical sample was used as a quantitative parameter. The correlation coefficient between the proportion of total five Gram‐negative anaerobes (P. gingivalis, T. forsythensis, P. intermedia, P. nigrescens,and T. denticola) and total VSC level (r = 0.84) was higher than that between the bacterial proportion and OLT score (r = 0.24). In these five Gram‐negative anaerobes, the proportions of P. intermedia, P. nigrescens and T. denticola were correlated with the concentration of hydrogen sulfide (r = 0.79, 0.73 and 0.50, respectively). The proportions of P. gingivalis, P. intermedia and P. nigrescens were also correlated with the concentration of methyl mercaptan (r = 0.91, 0.40 and 0.71, respectively). When a linear regression analysis was conducted using the proportion of each of five Gram‐negative anaerobes as independent variable, the explanatory power of these independent variables showed 77% for total VSC level and 10% for OLT score. These results suggest that Gram‐negative anaerobes in tongue microbiota may contribute VSC production and may be also related to oral malodor.

Development and validation of a plate technique for screening of microorganisms that produce volatile sulfur compounds

Volatile sulfur compounds (VSCs) are of major importance for flavor development in foodstuffs such as cheeses. Such compounds originate from the amino acid l-methionine, which can be degraded to methanethiol (MTL), a common precursor to a variety of other VSCs. A plate assay based on double-layer petri dishes containing 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB), a chemical used for the estimation of free thiols, in the upper layer provides an easy and reliable detection method for thiol-producing, cheese-ripening microorganisms. MTL production was quantitated by measuring the yellow–orange color intensity resulting from reaction with DTNB. Using this method, 18 Geotrichum candidum strains isolated from cheeses were compared, and the color intensity was found to be correlated with the production of microbial VSCs as measured by gas chromatographic analysis.