Polyphenol Oxidase Inhibitor from Blue Mussel ( Mytilus edulis ) Extract

Polyphenol Oxidase Inhibitor from Blue Mussel (Mytilus edulis) Extract

Enzymatic browning remains a problem for the fruit and vegetable industry, especially new emerging markets like pre-cuts. A crude inhibitor from blue mussel (Mytilus edulis) showed broad inhibition for apple (58%), mushroom (32%), and potato (44%) polyphenol oxidase (PPO) and was further characterized. Inhibition increased as the concentration of inhibitor increased in the reaction mixture eventually leveling off at a maximum inhibition of 92% for apple PPO. The inhibitor was capable of bleaching the brown color formed in the reaction mixture with apple PPO. Identification of the inhibitor by mass spectrometry and high-performance liquid chromatography revealed it to be hypotaurine (C2 H7 NO2 S). Hypotaurine and other sulfinic acid analogs (methane and benzene sulfinic acids) showed very good inhibition for apple PPO at various concentrations with the highest inhibition occurring at 500 μM for hypotaurine (89%), methane sulfinic acid (100%), and benzene sulfinic acid (100%).

Abstract:

Keywords: apple polyphenol oxidase, blue mussel (Mytilus edulis), browning inhibitor, hypotaurine, sulfinic acids

An inhibitor found in the expressed liquid from blue mussel shows very good inhibition on enzymatic browning. Since this enzyme is responsible for losses to the fruit and vegetable industry, natural inhibitors that prevent browning would be valuable. Finding alternative chemistries that inhibit browning and understanding their mode of action would be beneficial to the fruit and vegetable industries and their segments such as pre-cuts, juices, and so on. Inhibitors from products ingested by consumers are more acceptable as natural ingredients.

Practical Application:

Introduction Enzymatic browning remains a major problem in fruits and vegetables with an estimated 50% loss resulting from this postharvest reaction (Martinez and Whitaker 1995). In addition, new market segments for fruits and vegetables such as minimally processed pre-cuts are generally more susceptible to browning reactions (Gorny and others 1998; Paola and others 1999; Soliva-Fortuny and Mart´ın-Belloso 2003). Apple (Rojas-Gra¨u and others 2008), lettuce (Altunkaya and Goekmen 2008), pear (Sapers and Miller 1998), banana (Apintanapong and others 2007), potato (Cantos and others 2002), mushroom (Gerald and others 1994), and other pre-cuts all describe browning as one of the limitations for extending shelf life. Polyphenol oxidases (EC 1.10.3.1, [PPO]) found in fruits, vegetables, fungi, seafood, and mammals have been extensively studied

MS 20121357 Submitted 10/2/2012, Accepted 1/2/2013. Authors Schulbach, Yagiz, and Marshall are with Inst. of Food and Agricultural Sciences, Food Science and Human Nutrition Dept., Food and Environmental Toxicology Laboratory, SW 23rd Drive, Univ. of Florida, P.O. Box 110720, Gainesville, FL 32611–0720, U.S.A. Author Johnson is with the Mass Spectrometry Services, Chemistry Dept., Univ. of Florida, P.O. Box 117200, Gainesville, FL 32611–7200, U.S.A. Author Simonne is with the Inst. of Food and Agricultural Sciences, Family Youth and Community Sciences, Food and Environmental Toxicology Laboratory, SW 23rd Drive, Univ. of Florida, P.O. Box 110720, Gainesville, FL 32611–0720, U.S.A. Author Kim is with the Dept. of Food Science and Technology, Mokpo Natl. Univ., 1666 Youngsanro, Chungkye-myun, Muan-gun, Jeonnam 534–729, Republic of Korea. Author Jeong is with the Dept. of Food Science and Nutrition, Dankook Univ., 152 Jukjeon-Ro, Suji-Gu, Yongin-Si, Gyeonggi-Do, 448–701, Republic of Korea. Direct inquiries to author Marshall (E-mail: [email protected]).

 R  C 2013 Institute of Food Technologists doi: 10.1111/1750-3841.12059

Further reproduction without permission is prohibited

and are primarily responsible for enzymatic browning (Yoruk and Marshall 2003; Mayer 2006). PPO is a copper-containing enzyme that catalyzes o-hydroxylation of monophenols and/or the oxidation of o-diphenols to quinones in the presence of oxygen. Thus, under conditions of injury, whether natural or during processing, the breakdown of intracellular barriers can allow the reactants to interact (Toivonen 2004; Mishra and others 2012) causing discoloration. This is especially important to sectors such as pre-cuts, which removes intracellular barriers as well as exposes more surface area to oxygen. To prevent discoloration in fruits, vegetables, seafood, and so on, various inhibitors of enzymatic browning are used. There are a number of inhibitor classes including reducing agents, chelators, complexing agents, acidulants, enzyme inhibitors, and so on. (Chang 2009; Oms-Oliu and others 2010). Another type of inhibitor may be found in those biological systems that require PPO for physiological functions. Insects and crustaceans use PPO to harden their cuticle (Terwilliger 1999) while mollusks use PPO for adhesion to surfaces (Silverman and Roberto 2007). Inhibitors of PPO have been isolated from insects and demonstrated to inhibit insect PPO (Tsukamoto and others 1992; Sugumaran and Nellaiappan 2000) and PPO in fruits and vegetables (Yoruk and others 2003; Grotheer and others 2012). Consumer acceptance for pre-cuts containing PPO inhibitors from insects would be hard to endorse while inhibitors from crustaceans and mollusks would not be a concern since these are consumed regularly. However, little information exists on inhibitors from crustaceans and mollusks. It is hypothesized that inhibitors from mollusks exist to control its natural PPO and that these may offer alternative enzymatic browning control of PPO in fruits and

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Kurt F. Schulbach, Jodie V. Johnson, Amarat H. Simonne, Jeong-Mok Kim, Yoonhwa Jeong, Yavuz Yagiz, and Maurice R. Marshall

Browning inhibitor from mussel . . .

C: Food Chemistry

vegetables. The objective was to isolate and identify inhibitor(s) for 1 min with 400 mL acetone (–20 ◦ C). The sample was then from blue mussel (Mytilus edulis) that inhibit apple PPO. filtered through Whatman No. 1 filter paper (Fisher Scientific) and the residue after drying at room temperature (approximately Materials and Methods 15 min) was placed back in the blender and blended again with 200 mL of the cold acetone. This was repeated 2 additional times. Materials The residue after the 4th extraction was left overnight under vacFrozen blue mussels (Mytilus edulis) (from Canada or China) uum to dry and then placed in vacuum storage bags at –20 ◦ C were acquired through a local seafood market. Red delicious until needed. apples, Russet potatoes, and mushrooms were purchased at a PPO extraction was performed on the stored residues. Onelocal supermarket (Gainesville, Fla., U.S.A.). Catechol (Certi- gram residue was mixed with 50-mL 0.1M phosphate buffer fied), acetone (Certified), and HPLC grade acetonitrile, methanol, pH 7.2, with stirring for 30 min at 4 ◦ C and then centrifuging 1-propanol, and phosphoric acid were purchased from Fisher at 12000 g for 30 min in a Beckman Coulter Optima Centrifuge Scientific (Pittsburgh, Pa., U.S.A.). Hypotaurine, sodium (Beckman Instruments Inc., Fullerton, Calif., U.S.A.). The supermethanesulfinate, and benzene sulfinic acid were purchased from natant was filtered through Pyrex glass wool (Fisher Scientific) and Sigma-Aldrich Co. (St. Louis, Mo., U.S.A.). stored in microcentrifuge tubes at –20 ◦ C until needed (Yoruk and others 2003; Grotheer and others 2012).

Preparation of mussel inhibitor At purchase, the frozen mussels were packaged in sealed plastic bags (approximately 900 g) and transported on ice to the lab. The package was thawed under running water and the juice surrounding the mussels was removed and collected. The mussels were shucked, the contents pressed through EMD Chemical Inc. 3P Miracloth (Fisher Scientific, Pittsburgh, Pa., U.S.A.), combined with the juice and filtered through a Whatman GF/B glass microfiber filter (Fisher Scientific) under vacuum. The juice mixture was filtered again through a 0.45-μm pore diameter Whatman nylon filter (Fisher Scientific) under vacuum, and was dialyzed using a Spectra/Por CE 500 Da MWCO dialysis tubing (Spectrum Lab Inc., Rancho Dominguez, Calif., U.S.A.) with 3 changes of deionized water at 4 ◦ C for 24 h. This dialysate (crude inhibitor) was used for determining initial inhibition with apple PPO. Once inhibition was established, the dialysate was then filtered through a 2nd 0.45-μm pore diameter Whatman nylon filter. Thirty milliliter of the final filtrate was applied to a column (Sephadex G-25–80, 2.5 cm D × 38 cm L, Sigma-Aldrich Co.). The column was eluted with 0.02 M acetate buffer, pH 5.5 at a flow rate of 0.3 mL/min, resulting in 2 fractions A and B (Figure 1) showing inhibition. Preparation of plant PPO Apple, potato, and mushroom were cut into small pieces and approximately 200 g each were homogenized in a prechilled blender (Waring Products Inc, Torrington, Conn., U.S.A.) on high setting

Figure 1–Elution of the juice from blue mussel after filtration and dialysis on a Sephadex G-25 column (2.5 cm D × 38 cm L) (“Materials and Methods”). Ten to 30 mL of sample was applied and the flow rate was 0.3 mL/min. Fractions were collected every 15 min beginning at 72 min. Percent inhibition was determined using the assay described in “Materials and Methods.”

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Polyphenol oxidase assay PPO activity was determined in the presence or absence of mussel inhibitor using a spectrophotometric assay at 420 nm and 25 ◦ C (Yoruk and others 2003; Grotheer and others 2012). Rates were determined over 2 min using the greatest linear initial reaction rate under the conditions of the reaction. The reaction mixture consisted of 2.45 mL of 0.1 M sodium acetate-acetic acid buffer, pH 5.5, 0.3 mL of 0.5 M catechol (Sigma Chemical Co., St. Louis, Mo., U.S.A.), 0.05 mL of PPO, and 0 to 0.2 mL of inhibitor (fractions A or B) or buffer (control). Percent inhibition was calculated by comparing the rate with inhibitor to the rate of the control in the standard reaction mixture. High-performance liquid chromatography (HPLC) All HPLC methods with the exception of those run for mass spectral analysis (Univ. of Florida Mass Spectrometry Services, Gainesville, Fla., U.S.A.) were performed on a Perkin Elmer HPLC system consisting of a series 200 autosampler, series 200 LC pump and series 235C diode array detector. Fraction B from the G-25 column was concentrated using a Labconco model 5 freeze-dryer equipment (Labconco Co. Kansas City, Mo., U.S.A.) and reconstituted with 8 mL of methanol:water (75:25 v/v). Approximately, 50 to 70 replicate 100 μL injections of this were run on the HPLC system (A; Figure 3). The conditions were column: Agilent Zorbax SB-C18 (5 μm, 4.6 × 250 mm, Agilent Technologies, Santa Clara, Calif., U.S.A.); mobile phase: solvent A, 0.1 M phosphoric acid, pH 2.5, solvent B, 0.1 M phosphoric acid, pH 2.5 in 50% acetonitrile; gradient elution: 100% A to 100% B over 6 min, then hold for 2 min, 100% B to 100% A over 4 min, then hold for 8 min for equilibration; flow rate: 0.8 mL/min; detection: 215 nm. Inhibitor was collected in the fractions between 3.5 and 3.8 min (Figure 3A). Approximately, 5 to 7 mL of the collected sample was dialyzed as described previously using only 1 change of deionized water. This was then freeze dried as previously described and brought up to 1 to 2 mL with methanol:water (75:25 v/v). This fraction was then applied to a C-18 column (Alltech Econosil, 5 μm, 4.6 × 250 mm, Fisher Scientific) with the following conditions (B; Figure 3): solvent A, 1% propanol in acetonitrile, solvent B, 1% propanol in water; gradient elution: 100% A, then inject (50 μL) sample and hold for 2 min, ramp to 70% B over 12 min, ramp to 100% A over 6 min, then hold for 10 min for equilibration; flow rate: 0.8 mL/min; detection: 215 nm. Inhibitor was collected in the fractions representing the center of the peak (approximately 8.3 to 8.5 min) (Figure 3B) until the 1

potato (86%) PPO using a large MW inhibitor from tobacco horn worm. Since, Fraction B showed broader inhibition for various PPOs, it was further studied and identified in this paper, using apple PPO for testing inhibition. Based on previous works and its elution on the gel permeation column, Fraction B was thought initially to be a small protein or peptide. The peptide/small protein nature of the inhibitor seemed to be confirmed as purification of the HPLC fractions was initiated and the inhibitor activity was retained in 500 Da MWCO dialysis tubing but lost using 1000 Da MWCO dialysis tubing. However, when the sample retained after dialysis was sent to the Univ. of Florida Interdisciplinary Center for Biotechnology Research, Protein Core Facility, (Gainesville, Fla., U.S.A.) for analysis and sequencing, only 2 major peaks were identified histidine and an unknown peak (inhibitor). It was quickly recognized from these data that the inhibitor was not a small protein/peptide.

40 30 25 20 15 10 5 0

% Inhibion

Results and Discussion

50 45 40 35 30 25 20 15 10 5 0

Inhibitor

Frac A

Frac B

Potato

Inhibitor

100 90 80 70 60 50 40 30 20 10 0

Frac A

Frac B

Apple

% Inhibion

Purification and characterization of inhibitor from blue mussel The crude mussel juice extract after initial dialysis inhibited apple PPO by 84 ± 3% (control rateavg , 0.27 ± 0.02 A420 nm /min; 3 extractions with 2 replicates, n = 6). Inhibition for over 25 samples varied from 65% to 84% for most batches of crude juice after initial dialysis with the exception of 1 batch which showed only 45% inhibition. The dialyzed juice was then placed on a G-25 Sephadex column and the following elution profile was observed (Figure 1). Two fractions, A and B, showed inhibition for apple PPO; inhibiting the enzyme by 80% and 62%, respectively. Based on the elution profile, inhibitors from mussel showed a large molecule (Fraction A) eluting close to the void volume and a small molecule eluting late in the profile. Previous studies from insects showed PPO inhibitors were either peptides or proteins based on molecular mass estimates from 3000 to 3800 Da from housefly (Tsukamoto and others 1992), to approximately 30000 to 50000 Da for housefly (Yoruk and others 2003), to >100000 Da for German cockroach (Grotheer and others 2012), to 380000 Da for tobacco horn worm (Sugumaran and Nellaiappan 2000). Both fractions were tested for PPO inhibition using apple, mushroom, and potato PPO (Figure 2). The inhibitor activity prior to the G-25 column was 84 ± 1.6% for apple, 19 ± 0% for mushroom, and 18 ± 1% for potato. Fraction A from the G-25 column showed 81 ± 0%, 0%, and 1.8 ± 0.8% inhibition for apple, mushroom, and potato PPO, respectively while Fraction B inhibited PPO by 58 ± 1% (apple), 32 ± 4% (mushroom), and 44 ± 1% (potato). Grotheer and others (2012) showed similar inhibition for potato (20%) and apple (72%) PPO for an inhibitor from German cockroach while calculating an estimate from their graphical data, Sugumaran and Nellaiappan (2000) showed higher inhibition for apple (99%) and

Mushroom

35

% Inhibion

to 2 mL sample was completely injected. This was dried under N2 gas and reconstituted with buffer (0.1 M sodium acetate-acetic acid buffer, pH 5.5) for assaying or further characterization. Mass spectral analyses of the inhibitor were performed by the Mass Spectrometry Services, Dept. of Chemistry, Univ. of Florida, Gainesville, Fla., U.S.A. HPLC/UV/MS analyses were performed with an Agilent (Agilent Technologies, Santa Clara, Calif., U.S.A.) 1100 series binary pump, Phenomenex (Torrace, Calif., U.S.A.) ˚ (2 × 150 mm; 4 μm) plus C18 Synergi 4μ Hydro-RP 80A guard column (2 × 4 mm), Agilent 1100 G1314A variable wavelength detector (254 or 215 nm monitored) and Thermofinnigan (Thermo Scientific, West Palm Beach, Fla., U.S.A.) LCQ quadruple ion trap mass spectrometer operated with an electrospray ionization source. Several different gradient programs were evaluated before adopting a normal phase gradient of 1% isopropanol in water (phase A) and 1% isopropanol in acetonitrile (phase B) coupled with a C18 column. The normal phase gradient was: 100% B for 0 to 2 min, linear ramp to 5% B over 45 min; hold at 5% B for 13 min; linear gradient to 100% B over 30 min and hold for 30 min at 100% B. A number of different MSn methods were used; typically, collision-induced dissociation (CID) MS/MS data were obtained at 37.5% CID energy and q-CID of 0.25 or at 45% CID energy and q-CID of 0.3. High-resolution mass spectrometry (HRMS) analyses were performed on an Agilent (Santa Clara, Calif., U.S.A.) 6210 time-offlight mass spectrometer (TOFM) operated with an electrospray ionization source. Samples were introduced via flow injection with an Agilent 1200 series binary pump.

Inhibitor

Frac A

Frac B

Figure 2–Inhibition by mussel juice precolumn (Inhibitor), and Fractions A and B from the G-25 Sephadex column on various plant PPO using the standard assay method described (“Materials and Methods”). Bar graph represents average percent inhibition ± standard deviation, n = 2; and is representative of 3 total trials. Control PPO activity was 0.17 ± 0, 0.29 ± 0.01, and 0.25 ± 0 A420 nm /min for apple, mushroom, and potato, respectively.

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Browning inhibitor from mussel . . .

Browning inhibitor from mussel . . . the peak was collected. Inhibition with solvent removed was between 74% and 90%. The fraction collected was further evaluated for inhibition and identification using LC-MS. The inhibitor from the 2nd chromatographic run was evaluated for percent inhibition of apple PPO compared with concentration (Figure 4A). From the figure, inhibition was fairly linear as inhibitor concentration in the reaction mixture (3 mL total) was increased to 150 μL and then gradually leveled off as concentration further increased. It should be noted that the maximum

Figure 3–HPLC chromatograms of the inhibitor (arrow) under 2 different chromatographic conditions as described in “Materials and Methods.”

950 900 850

A

800 750 700 650 600 550 500 450 400 350 300 250 200 150 100

Response [mV]

C: Food Chemistry

The 1st chromatographic run showed the inhibitor eluting between 3.3 and 4.3 min (Figure 3A) and it was not completely separated from other components from the G-25 separation. The center of the peak was collected for further HPLC separation and inhibition with solvent removed was around 80% to 96%. This fraction was then purified using a 2nd chromatography separation (Figure 3B). This chromatogram shows the inhibitor to be separated from other components of the 1st chromatographic run. The inhibitor eluted between 8.3 and 8.8 min and again, the center of

50 0

2

4

6

8

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14

16

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850 800

B

750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 0

1

2

3

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6

7

Retenon me (min)

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8

9

10

11

12

13

Browning inhibitor from mussel . . .

Figure 4–Inhibition of apple PPO as a function of inhibitor concentration (A). Bleaching of the browning color by the inhibitor (B). Standard assay was run as described in “Materials and Methods” for both A and B. Various amounts of inhibitor were added in the standard reaction mixture (3 mL). Values are averages ± standard deviation, n = 2 and are representative of 3 total trials for A and B. For B, the standard reaction mixture without inhibitor was run for 4 min, and 300 μL of inhibitor was added (arrows) and absorbance at 420 nm continued to be monitored.

Table 1–Data for flow injection/ESI-TOF-HRMS of the blue mussel inhibitor. ESI polarity Ion (+)ESI-MS (–)ESI-MS

Theoretical Detected Delta m/z m/z (ppm)

[M+H]+ [M–H+2Na]+ [M–H]– [(M–H)+(M–H+Na)]–

110.027 153.9909 108.0125 239.0142

−3.64 −3.25 −2.78 −0.84

110.0266 153.9904 108.0122 239.014

Table 2–ESI-MSn collision-induced dissociation (CID) of major molecular-type-ions. All major ions in the product spectra are shown with their percent relative abundances in parentheses. Inhibitor (+)ESI-MSn:

m/z 110 [M+H]+ m/z 122 = [(M–H+Li)+Li]+ m/z 154 = [(M–H+Na)+Na]+ m/z 108 [M–H]–

(–)ESI-MSn: Hypotaurine Std (+)ESI-MSn m/z 110 [M+H]+ (–)ESI-MSn m/z 108 [M–H]–

110 → 92 (100) → 122 → 79 (100)

30 (100)

154 → 111 (100) 108 → 65 (100), 64 (51) 110 → 92 (100) → 30 (100) 108 → 65 (100), 64 (34)

MS/MS to produce m/z 92 via loss of H2 O. The m/z 108 [M–H]− ion underwent CID to product m/z 65 and m/z 64 via loss of 43 and 44 u, respectively. Subsequently, a more purified inhibitor fraction was analyzed with a “normal” phase gradient C18 HPLC/215 nm UV/ESI-MS described in the “Material and Methods” section. The MW 109 inhibitor eluted at RT 16.51 min with a strong 215 nm UV peak and produced abundant (+)ESI-MS m/z 110 [M+H]+ and m/z 151 [M+H+Acetonitrile]+ ions and (– )ESI-MS m/z 108 [M–H]− ions. Postcolumn addition of dilute solutions of lithium acetate and sodium acetate into the column effluent prior to the ESI source resulted in the formation of m/z 122 [(M–H+Li)+Li]+, m/z 138 [M–H+Li+Na]+ and m/z 154 [(M–H+Na)+Na]+ ions and a number of self-adduct ions involving the neutral 115 u (M–H+Li) and 131 u [M–H+Na]+. The Li-containing ions confirmed the m/z 154 [(M–H+Na)+Na]+ ion and the m/z 110 [M+H]+ ion. The CID of m/z 122 [(M– H+Li)+Li]+ and m/z 154 [(M–H+Na)+Na]+ ions resulted in m/z 79 and m/z 111 product ions, respectively, due to loss of 43 u. Flow injection/ESI-TOF-HRMS of a more purified inhibitor fraction resulted in a molecular formula of C2.H7.N1.O2.S1 based upon the following data (Table 1). Once hypotaurine was determined to be the most likely candidate for the MW 109 inhibitor, reverse phase HPLC/UV/ESIMSn analyses showed it behaved just as the inhibitor did. Hypotaurine produced an m/z 110 [M+H]+ ion which underwent CID to produce m/z 92 which was further dissociated to m/z 30. Hypotaurine produced an m/z 108 [M–H]– ion which underwent CID to produce m/z 65 and 64 product ions. Table 2 and Figure 5 summarize the ESI-MSn dissociations observed for the inhibitor and the hypotaurine standard. Final confirmation of hypotaurine was by spiking pure hypotaurine in the extracts and running the chromatography as in Figure 3. Pure hypotaurine eluted at the same retention times as the inhibitor; for the 1st chromatography at 3.3 to 4 min and the 2nd chromatography at 8.4 to 8.6 min. Hypotaurine and taurine are found in many animal tissues including marine species (Roe and Weston 1965). They are thought to function metabolically as antioxidants (Aruoma and others 1988), osmolytes (Yin and others 2000; Rosenberg and others Vol. 00, Nr. 0, 2013 r Journal of Food Science C5

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inhibition was at 92% and never reached 100%. This relationship is similar to other reports for inhibitors of PPO (Ding and others 2002; Yoruk and others 2003; Arias and others 2011). The inhibitor when added after color formation seemed to bleach the color in the reaction mixture (Figure 4B). The initial lowering of absorbance may be due to dilution (∼10%); however, the absorbance at 420 nm continued to decline over time. Further additions of inhibitor did not appreciably decrease the absorbance. Absorbance changed from 0.8 to below 0.3 with the addition of inhibitor confirming its bleaching characteristic and its ability to react with the product. This was confirmed by LC-MS with a molecular weight of 218 and the formula of C8 H12 N1 O4 S1 (data not shown). This was also seen for inhibitors of PPO such as sodium chlorite (Lu and others 2006), kojic acid (Chen and others 1991), and sulfite (McEvily and others 1992). The inhibitor collected from the 2nd HPLC run was presented to the Univ. of Florida Mass Spectrometry Services, Dept. of Chemistry, Gainesville, Fla., U.S.A.. With reverse phase gradient C18 HPLC/254 nm UV/ESI-MS, a number of compounds were detected. The inhibitor compound of interest eluted essentially in the void volume at 100% aqueous mobile phase, which resulted in poor sensitivity and difficulty in interpreting the MS data as a number of compounds eluted in the void volume. The MW 109 inhibitor produced m/z 110 [M+H]+ and m/z 154 [(M–H+Na)+Na]+ ions and little or no m/z 132 [M+Na]+ ions with (+)ESI-MS and m/z 108 [M–H]− ions with (–)ESI-MS. The m/z 154 [(M–H+Na)+Na]+ ion was indicative of the presence of an acidic proton. The m/z 110 [M+H]+ underwent collision-induced dissociation (CID)

Browning inhibitor from mussel . . .

C: Food Chemistry

2006), involved in immunomodulation, bile salt formation, and neuroprotection (Bouckenooghe and others 2006), and reduce H2 S toxicity (Rosenberg and others 2006; Ortega and others 2008) in marine organisms found near hydrothermal vents and cold seeps. Levels of taurine (as mg/100 mL) are found in lamb (47.3), pork (49.6), chicken (33.7), cod (31.4), oysters (69.8), and clams (240) (Roe and Weston 1965). Taurine is synthesized from hypotaurine (Bouckenooghe and others 2006; Ortega and others 2008), and it is feasible for hypotaurine and taurine to be found in the expressed liquid of the blue mussel. Pure hypotaurine and other sulfinic acid analogs were tested for inhibition using apple PPO (Table 3). Table 3 shows that all sulfinic acids tested showed inhibition towards apple PPO. At the low concentration (67.5 μM), inhibition was around 20% to 30%, and as the concentration increased, inhibition increased to a maximum of 100%. The lowest inhibition at the highest concentration was hypotaurine at 89% inhibition compared to methane and benzene sulfinic acids at 100%. Taurine, a sulfonic acid and product of the intermediate hypotaurine did not inhibit apple PPO: the activity (n = 3) for control was 0.24 ± 0.02 A420 nm /min while the activity at 67.5, 250, and 500 μM was 0.23 ± 0.04, 0.25 ± 0.01, and 0.23 ± 0.01 A420 nm /min, respectively. There is very little information on the use of hypotaurine in the prevention of browning in fruits and vegetables. However, hypotaurine has been used as an antioxidant and bleaching ingredient in cosmetics (Kruse and others 2002) while other sulfur compounds like cysteine and glu-

+

OH2

H2N

Table 3–Percent inhibition of apple PPO using hypotaurine and other sulfinic acid analogs. Concentration [μM]

Methane sulfinic acid Benzene sulfinic acid

Inhibitors of enzymatic browning were found in the expressed liquid of a commercial blue mussel product. One showing broader inhibition toward apple, potato, and mushroom PPO was further identified as hypotaurine by mass spectrometry and HPLC. Hypotaurine and other sulfinic acid analogs showed excellent inhibition (ranging from 89% to 100%) of apple PPO. The effectiveness of other sulfinic acids in inhibiting browning may warrant further

OH

H2N S

O

O

Hypotaurine Molecular Formula = C2H7NO2S Monoisotopic Mass = 109.0197 u O

-

(-)ESI

O

m/z 108 [M-H]CID

+

O

S

Li+

H

-

O S

S

Na+

(+)ESI

m/z 92

O

m/z 65

CID

m/z 30

O

S

CID

O

m/z 64

+

CH2

Li

H2N S

O

O

Li

Na

H2N

+

m/z 122 [(M-H+Li)+Li]+ CID

S

O

O

Na

S

O

O

Li

CID

-43u, C2H5N1

m/z 79

+

+

m/z 154 [(M-H+Na)+Na]+

Na

Li H

H

S

O

O

Na

+

m/z 111

Figure 5–Summary scheme of the ESI-MSn collision-induced dissociation (CID) of the molecular-type ions for the inhibitor and hypotaurine.

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4a 15 8 1 1 0 16 1 0

Conclusion

H2N

m/z 110 [M+H]+

± ± ± ± ± ± ± ± ±

tathione have been used to inhibit enzymatic browning (Kuijpers and others 2012).

(+)ESI

H2N

39 80 90 34 97 100 21 98 100

Data are averages ± standard deviations, n = 6.

S

H2N

67.5 250 500 67.5 250 500 67.5 250 500

Hypotaurine

a

Average% Inhibition

evaluation. Hypotaurine is a natural compound found in many foods and may offer a benefit in preventing browning in fruits and vegetables, especially pre-cuts and juices.

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Vol. 00, Nr. 0, 2013 r Journal of Food Science C7

C: Food Chemistry

Browning inhibitor from mussel . . .