Volatile Sulfur-Containing Compounds from Methionine Metabolism in

Chapter 12

Volatile Sulfur-Containing Compounds from Methionine Metabolism in Genetically Modified Lactobacillus helveticus CNRZ32 Strains Downloaded by UNIV OF GUELPH LIBRARY on May 10, 2012 | http://pubs.acs.org Publication Date: August 9, 2007 | doi: 10.1021/bk-2007-0971.ch012

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Scott A. Rankin , Dattatreya S. Banavara , Ed S. Mooberry , James L. Steele , Jeffery R. Broadbent , and Joanne E. Hughes 1

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Departments of Food Sciences and Biochemistry, University of Wisconsin, Madison, WI53706 Departments of Nutrition and Food Science and BioIogy, Utah State University, Logan, UT 84322-8700 3

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Cystathionine-β-lyase (CBL) catalyzes the conversion of methionine to methanethiol, dimethyl disulfide and dimethyl trisulfide. These three compounds are collectively referred to as volatile sulfur compounds (VSC). Two strains of Lactobacillus helveticus CNRZ32 were evaluated for VSC production; wild-type and a genetically modified variant that produced elevated levels CBL. Whole cell suspensions and cell free extracts were incubated in buffer (pH 6.0) with methionine substrate; furfuryl alcohol was added as an internal standard. Results demonstrated that the modified strain produced ~2-fold higher levels of VSC, primarily dimethyl disulfide. In parallel, NMR was used to substantiate a lyasemediated pathway by tracking metabolites of uniformly labeled C methionine (17.5mM). 13

© 2007 American Chemical Society

In Flavor of Dairy Products; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Downloaded by UNIV OF GUELPH LIBRARY on May 10, 2012 | http://pubs.acs.org Publication Date: August 9, 2007 | doi: 10.1021/bk-2007-0971.ch012

206 Flavor development in cheese is attributed mainly to the metabolic activities of starter and non-starter lactic acid bacteria. Catabolism of free amino acids yields desirable and undesirable flavor compounds (7). One class of flavor compounds, volatile sulfur compounds (VSC), can constitute a major portion of the odor active volatiles in ripened cheeses (2). During cheese ripening, microbial proteases, rennet and other enzymes, metabolize casein into smaller peptides. These peptides further yield a pool of amino acids by the action of peptidase enzymes. Methionine and cysteine are believed to be present in cheese at levels up to 0.02%. Methionine has been identified as a main precursor of VSC in certain cheeses (3). The catabolism of methionine to V S C s has been studied extensively in a variety of lactic acid bacteria (4, 5) and yeasts (6). Methionine conversion is believed to take place by at least two major pathways, one initiated by aminotransferases (ATases) and another initiated by lyases such as methionine-y-lyase and cystathionine-p-lyase (CBL) (7). The mechanisms, pathways and intermediate compounds formed are very different for these two enzymes. The ATase pathway has been shown to play a major role in converting methionine to V S C s in lactococcal strains (8). Lyases and ATases share a common co-factor, pyridoxal phosphate. However, VSC generation by the ATase-dependent pathway is also contingent on the presence of a-keto acids. In cheese, multiple pathways of VSC generation might exist based on factors such as bacterial strain, pH, and substrate/co-factor/enzyme availability. In this study, Lactobacillus helveticus CNRZ32 modified to overexpress the lyase enzyme CBL was studied for its ability to generate increased amounts of VSCs.

Experimental Chemicals The compounds L-methionine, a-keto-4-(methylthio)-butanoic acid (KMBA), 2-hydroxy-4-(methylthio)-butanoic acid (HMBA), 2-ketobutyric acid (KBA), NADH, pyridoxal phosphate, a-ketoglutarate (ct-KG), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), methional and furfuryl alcohol were purchased from Sigma-Aldrich (St Louis, MO). (U) C Methionine was obtained from Cambridge Isotope Laboratories (Andover, MA). 13

C B L Overexpression Recent analysis of a draft quality (4X) genome sequence for Lb. helveticus CNRZ32 identified a gene cluster encoding 3 enzymes involved in Cys


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biosynthesis: O-acetyiserine lyase (cysK), CBL, and serine acetyltransferase (cysE). To further characterize the role of CBL in VSC generation, the Lb. helveticus CBL gene was cloned into the expression vector pTRK687 (9) then transformed by electroporation into Lb. helveticus CNRZ32 as described previously (10) . Two independent clones containing the recombinant plasmid (pTRK687:cW) were collected, and the integrity of the plasmid construct was confirmed by DNA sequence analysis. Lb. helveticus CNRZ32 wild type cells were also transformed with unmodified pTRK687 and designated LH32. Stock cultures of LH32 and each of the genetically modified variants, designated LH32cW.7 and LH32cW.2, were stored at -80°C in nonfat dry milk with 11% glycerol until needed.

Organisms and Cell growth Working cultures of Lb. helveticus LH32, LH32cbl.l and LH32cW.2 were prepared from frozen stocks by transfer into in 5.5% MRS broth media (Difco, Detroit, MI) that contained chloramphenicol (5\ig/m\) with incubation at 42±1°C for 20 hours. Cells were grown in triplicate (45ml media, 90-100^1 inocula at comparable OD o). Growth of bacteria was monitored hourly by OD o and pH of media. 60

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Cell harvest and preparation Cells were harvested in late log phase to early stationary phase (at comparable growth periods), washed twice with potassium phosphate monobasic-disodium phosphate buffer solution (pH 6.0) and pelleted by centrifiigation (6,500 x g, 15 min). The pH of the remaining cell free media was noted. The pelleted cells were washed twice by vortexing and centrifiiging with a 0.05 M potassium phosphate buffer (adjusted to pH 6.0 with 0.1 N NaOH) at 4°C. Buffer (24 ml) was added to the washed cell pellet with 5 ml of methionine (100 mM) substrate, 100 jllI of pyridoxal phosphate (1 mM) co-factor, and 150 ju.1 of furfuryl alcohol (10 fil/ml, internal standard) in a 4 0 ml glass vial fitted with teflon lined septum cap. After vortexing, 2 ml were removed and analyzed for cell concentration spectrophotometrically at 600 nm. The final OD oo of the cell was adjusted to 2.0. Two negative controls were prepared for each sample, one by adding substrate and cofactor to the cells autoclaved for 20 min and the other without any substrate with the native cell suspension. Each of the experimental treatments was prepared in duplicate with two controls. 6


208 Cell extracts


Cell extracts were prepared by sonication (Branson 1510, Danbury, CT) of 20 ml cell suspensions under ice at 4°C. A preliminary analysis of cell lysis showed that maximum extracellular protein was obtained in cell free extracts under sonication for 25 min with the sonicator used in the study. Cell debris was removed by centrifugation (14,000 x g, 30 min, 4°C) to yield a crude cell free extract. Protein analysis of the samples was done using a BCA protein assay kit (Pierce Chemicals, Rockford, IL).

SPME-GC-MS

The formation of methionine-derived volatiles was studied using solid phase microextraction (SMPE) with GC-MS analysis. Approximately 30 min prior to each assay, samples incubated for each designated time period were removed, shaken well and allowed to equilibrate for 30 min. in a multiblok heater at 30°C. A SPME fiber coated with carboxen/PDMS (85 |im) was used for adsorbing the volatiles by placing it in the headspace of the equilibrated (static headspace) sample for 10 min. The volatiles absorbed onto the fiber were desorbed into the GC inlet at 280°C. The fiber was held in the injection port over the complete program cycle (17 min) to ensure complete desorption. The GC temperature program was as follows: 35°C initial temp, 2 min hold time, 5°C per min to 70°C, 20°C per min to 200°C, hold for 5min. The quantitative assessments of VSC were done in the selected ion monitoring mode (m/z 48 for MTL, m/z 94 for DMDS and m/z 126 for DMTS). Total mM of VSC was calculated using standard curves. The variation in adsorption efficiencies was corrected by the use of as internal standard (fiirfuryl alcohol at a concentration noted above).

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C Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) has been used in the field of biochemistry and medicine for varied purposes. Nuclei with odd number of protons and neutrons (e.g. H , C , N , F, P) exhibit a mechanical spin phenomenon. When placed in a static magnetic field, such atoms are driven to a spin balanced state known as polarization. An exciting radiofrequency applied in definite pulses causes perturbation (resonance) and relaxation of the spin balanced state and magnetic resonance signal. Different nuclei resonate at differentfrequenciesand NMR signal of a given nuclei is referred to as chemical shift. The scale of chemical shifts varies for different nuclei. Chemical shifts of nuclei attached to different atoms also vary within the scale. Analysis of molecular transitions and changes can be tracked by analyzing NMR spectra of !

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samples over time. The use of C enriched molecules increases the specificity and sensitivity of the method. In this study, (U) C Met was used as the substrate in whole and cell free extracts. A l l C NMR spectra were obtained with a Bruker model DMX400 wide-bore NMR spectrometer operating at a carbon NMR frequency of 100.6 MHz with a broadband 5-mm-diameter NMR probe at a temperature of 30°C. The C NMR spectra were obtained with power-gated proton decoupling as directed by a Bruker pulse program zgpg30 by using the following parameters: C spectral window, 225 ppm; 90-degree pulse width, 10 ms; 1 s relaxation delay; 2048 scans per spectrum. The catabolic process was monitored at 2 hr intervals over time. ,3

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Results and Discussion Bacterial Growth Growth of both CBL expression clones significantly differed from the wildtype strain relative to the onset of exponential growth phase. Fig. 1 represents growth curves for the bacteria as determined by pH and OD oo. For analyzing VSC production capability, bacteria were harvested at comparable growth periods (18 hr for CBL expression clones, 16 hr for wild-type). 6

Figure J. Growth curve for LH32, LH32cbl 1 and LH32cb\.2 Strains.


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Production of VSC In the control samples (autoclaved cells with Met and whole cells without added Met), there were no detectable levels of VSC. There were no differences in VSC concentration trends between studies done using whole cells or cell free extracts. Three VSCs, methanethiol (MTL), dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) were found in the headspace of whole cell media of the G M and native treatments. The concentrations of MTL and DMTS found in all the samples were relatively low as compared to DMDS. This is probably due to the fact that MTL readily converts to DMDS in aqueous media via the formation of free radicals especially in presence of oxygen; DMDS is relatively more stable. Among the V S C s found in incubated samples, DMTS formation requires three molecules of MTL while DMDS involves two. The conversion of MTL into DMDS and DMTS may also vary with water activity and pH. Previous research has reported total VSCs as a measure of methionine conversion since total VSC concentration is thought to be related by reactions involving the same pool of MTL. The mechanism of interconversion of MTL to DMDS and DMTS is given in the series of reactions below.

CH S" + H

CH SH

+

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3

CH S'

-•

CH S* + e

CH S* + CH S*

•>

CH3S-SCH3 (predom.)

C H 3 S - S C H 3 + e-

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CH S-S" + C H

CH3S-S- + CH S*

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CH3S-S-SCH3

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Production of V S C s by .H32, LU32cbl.l and LH32M.2 was repetitive under like conditions. Both CBL clones (LH32cW.7 and LH32cM2) produced V S C s at higher concentrations (at least 2 times) as compared to the native strain, LH32 (Fig. 2). VSC production had no correlation to cell autolysis. Because of the reactive nature of S-compounds and their susceptibility to interconversion as influenced by such factors as redox potential, pH, etc., the ratio of V S C s may vary while the total pool remains constant. However, in the controlled conditions of this study, there were no significant differences in the relative concentrations of MTL, DMDS and DMTS, but, as was predicted from



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Figure 2. Production of VSC by whole cells of Lb. helveticus LH32, LH32cb\. 1 and LH32cbl.2. (Error bars represent one standard deviation, n = 2).

the relative stabilities, DMDS was the predominant VSC by a factor of approximately ten.

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C Nuclear Magnetic Resonance

Gao et al (8) demonstrated that L. lactis strains follow an ATase mediated pathway to produce VSC from methionine. The presence of a-ketoglutarate (aKG) is found to be important for this reaction. In this study with Lb. helveticus, both wild-type and CBL expression mutants evaluated with NMR analysis did not show any ATase-related pathway intermediates (KMBA or HMBA, Fig. 3). The chemical shifts for DMDS and DMTS were observed with no shifts for KMBA or HMBA. Lyases are known to hydrolyze methionine yielding MTL, ammonia and KBA. In this study, the chemical shifts for K B A were also not found. There are several reasons for this observation. First, methionine catabolism was slower in Lb. helveticus as compared to L. lactis studies making the limit of detection a possible issue. Also, once methionine is catabolised by ATase to K M B A or HMBA, conversion to VSCs proceeds slowly, either by redox-driven chemical degradation or via low-activity enzymes and hence KMBA or HMBA may



212 accumulate in the system where they can be more readily detected. Though there was much higher VSC production with the CBL overexpression variants, chemical shifts of ATase-derived intermediates were not observed. Addition of a-KG to cell suspensions, decreased VSC production significantly. It is possible that VSC production predominantly proceeds via a lyase pathway and upon addition of a-KG, the substrate is utilized by both ATase and lyase increasing the intermediates and decreasing VSC. Also, NMR studies did not show any chemical shifts for KMBA, HMBA or K B A under these conditions for lactobacilli even with the addition of a-KG. However, in NMR studies with L. lactis, K M B A was previously observed (8) as well as denoted in our repeated study with L. lactis (Fig. 3).

203

140

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100

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Figure 3. NMR signal of C methionine metabolized by Lb. helveticus CNRZ32 (A) and Lc. lactis ssp. cremoris (B). In B, peaks at 168 and 203 ppm denote the first and second carbon nuclei, respectfully, of KMBA absent in the lactobacillus signal (A).

References 1. 2. 3. 4.

Curtin, A.C.; McSweeney P.L.H. J. Dairy. Res. 2003, 70, 249-252. Singh, T.K.; Drake, M.A.; Cadwallader, K.R. Comp. Rev. in Food Sci. and Food Safety 2003, 2, 139-162. Seefeldt, K.E.; Weimer, B.C.. J.Dairy Sci. 2000, 83,2740-2746. Helinck, S.; Le Bars, D.; Moreau, D.; Yvon, M . Appl. Environ. Microbiol. 2004, 70(7;, 3855-3861.


213 Pripis-Nicolau, L.; De Revel, G ; Bertrand, A.; Lonvaud-Funel, A. J. Appl. Microbiol. 2004, 96(5), 1176-1184. 6. Dias. B.; Weimer, B. Appl Environ. Microbiol 1998, 64(9), 3320-3326. 7. Tamman. J.D.; William, A.G.; Nobel, J.; Lloyd, D. Lett .Appl Microbiol 2000, 30, 370. 8. Gao. S.; Mooberry, E.S.; Steele, J.L. Appl Environ.Microbiol.1998, 64, 4670-4675. 9. Sturino, J.M.; Klaenhammer, T.R. Appl Environ Microbiol 2004, 70, 17351743. 10. Bhowmik, T.; Steele, J.L. J. Gen Microbiol. 1993,139, 1433-1439. Downloaded by UNIV OF GUELPH LIBRARY on May 10, 2012 | http://pubs.acs.org Publication Date: August 9, 2007 | doi: 10.1021/bk-2007-0971.ch012

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Volatile Sulfur-Containing Compounds from Methionine Metabolism in

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