Virus Inactivation by Grapes and Wines

Transcription

1 APPLIZD AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1976, p Vol. 32, No. 6 Copyright C) 1976 Americn Society for Microiology Printed in U.S.A. Virus Inctivtion y Grpes nd Wines JACK KONOWALCHUK* AND JOAN I. SPEIRS Bureu of Microil Hzrds, Helth nd Welfre Cnd, Ottw KiA OL2, Cnd Received for puliction 7 July 1976 Infusions nd extrcts of different grpes inctivted poliovirus; gents responsile for this property resided in the skin of the grpe. Commercil grpe juice t oth nturl nd neutrl ph inctivted vrious enteric viruses nd herpes simplex virus; 1,000-fold reduction in poliovirus infectivity occurred fter incution with grpe juice, ph 7.0, for 24 h t 4 C. A vriety of wines were ntivirl ut to lesser extent thn grpe juice; red wines were more ntivirl thn white. Antivirl ctivity ws demonstrle in frctions of grpe juice vrying in moleculr weight from <1,000 to >30,000 s determined y memrne filtrtion. Some restortion of poliovirus infectivity from virus-grpe juice complexes ws chieved with 1% geltin, 0.1% Tween 80, 0.5% polyvinyl pyrrolidone, nd 0.5% polyethylene glycol. Mny reports hve descried the helth enefits tht cn e derived from the moderte nizing n entire wshed smple of fruit with dischsed. Grpe extrcts were prepred y homoge- consumption of wine (8, 9, 13). Some hve referred to the disinfectnt properties of wine, s trifuged t low speed to remove pulp, nd the supertilled wter, 1:1 (wt/vol). The homogente ws cen- noted when Egyptin wrriors mixed wine with ntnt fluid ws centrifuged gin t 15,000 x g for wter whenever the troops invded foreign 30 min to remove prticultes nd cteri. The cler liquid ws clled the extrct. Pulp nd skin lnds; s lte s the nineteenth century, wine extrcts were prepred from hlf of purchsed ws recommended to e mixed with impure smple y seprting pulp nd skins nd then processing ccording to the procedure outlined for grpe wter tht could cuse choler (8). Although the ctericidl properties of wine hve een extrct. rodly defined for some time (10, 12), its effect Infusions of Toky nd Riier grpes were prepred y plcing, stems up, eight unlemished on niml viruses hs, to our knowledge, not een descried. This report descries some of grpes (out 1 inch [2.5 cm] in dimeter) in Pyrex the ntivirl properties of grpes nd their dish. Twenty-five milliliters of distilled wter ws dded to immerse the ottom hlf of ech grpe. products. After incution t 4 C for 24 h, the fluid ws removed nd centrifuged for 30 min t 15,000 x g. The MATERIALS AND METHODS cler liquid ws clled the infusion. An infusion of Viruses nd cells. Coxsckievirus type B5 nd risins ws prepred y soking 10 g of Sultn poliovirus type 1 (Sin) were grown nd ssyed in seedless risins in 10 ml of distilled wter t 4 C for HEp-2 cells; echovirus type 7 nd herpes simplex 24 h. The fluid ws removed nd centrifuged s virus type 1 were grown nd ssyed in Vero cells; ove. nd reovirus type 1 ws grown nd ssyed in primry Africn green monkey kidney cells. The juice, three Cndin wines, red, white, nd rose, Commercil (Welch) unsweetened Concord grpe sources of viruses nd cells hve een descried three French reds, one Itlin red, nd three Germn whites were tested. The ph vried s follows: elsewhere (6). Virus stocks were stored in seled mpoules t -70 C. HEp-2 nd Vero cells were grown grpe extrcts, 3.3 to 3.9; infusions, 4.4 to 5.9; wines, in medium 199 contining 10% fetl ovine serum; 3.3 to 3.8. Grpe juice ws recorded t ph 3.3. When monkey kidney cells were grown in medium 199 indicted, the ph ws djusted to 7.0 with NOH. contining 2% fetl ovine serum. Stock cultures Dilutions were mde in wter. were grown in Roux ottles s monolyers t 36 C. Virus inctivtion with grpe products. Unless For virus ssy, monolyers were prepred from otherwise indicted, virus inoculum of out 8,000 stock cultures in plstic dishes (60 y 15 mm) 24 h plque-forming units (PFU) in 0.05 ml ws dded to efore use. duplicte or triplicte screw-cpped ottles contining 2 ml of wter (virus control) or 2 ml of grpe Source nd preprtion of grpes nd grpe products. Grpes, risins, nd grpe juice were purchsed from locl grocery stores; wines were pur- nturl ph or ph 7.0. Supensions were incuted t extrct, infusion, juice or wines, or dilutions t the chsed from locl outlets of the Ontrio Liquor Control Bord. Vitis vinifer tle grpes (green seeding ech suspension 1:100 to eliminte cell toxicity. 40C for vrious periods. Virus ws ssyed y dilutless, Toky [red], Riier [lue] nd lue seedless) Triplicte liquots of 2.0 ml were mixed with 0.5 ml nd Vitis lrusc (Concord [lue]) grpes were pur- of 1:1 mixtures of lox medium 199 nd fetl ovine 757

2 758 KONOWALCHUK AND SPEIRS serum efore pipetting to pproprite monolyers. Cultures were shken for 2 h t mient temperture (out 22 C) to llow dsorption of virus. Smples were poured off, n gr overly ws pplied (5), nd the cultures were incuted t 36 C in humidified 5% CO2 tmosphere. Enterovirus plques were counted fter 3 dys, herpes simplex virus fter 5 dys, nd reovirus fter 8 dys. Plque counts for ech smple (counts on three monolyers) were verged. The counts otined for duplicte or triplicte smples were verged nd expressed s percentge of the vlue otined for the controls. The percent error (stndrd devition divided y the men nd multiplied y 100) of the ssy system vried from 2 to 25%, with men of 9%. Differences of >25% (the mximum experimentl error) were considered significnt. Frctiontion of grpe juice nd wine. Twenty milliliters of Concord grpe juice or Cndin red wine, oth djusted to ph 7.0 (dsorption to memrnes occurred t the nturl ph), ws frctionted y filtrtion, in series, through Diflo memrnes PM30 nd UM10 (Amicon Corp., Lexington, Mss.); grpe juice ws further filtered through UM2 memrne. These memrnes re designed to restrict pssge of mcromolecules in excess of moleculr weight 30,000, 10,000, nd 1,000, respectively. Memrnes, 76 mm in dimeter, were fitted in mgneticlly stirred ultrfiltrtion cell (Diflo model 402). Filtrtion pressure ws 40 l/in2. To ssist in the size seprtion of prticles, ech filtrtion ws concluded with discontinuous difiltrtion with 10 ml of wter, three or four times. Ten milliliters of filtrte from ech memrne ws retined for ssy of ntivirl ctivity; the reminder ws filtered through the next grde of memrne. To otin retenttes, memrnes were removed from the filtrtion cell, plced with 20 ml of wter in fltottomed crystllizing dish, nd gently scrped with stirring mgnetic r for 5 min. Antivirl ctivity of retenttes nd filtrtes ws compred with tht of n pproprite dilution of strting mteril (control). Complex dissocition. The possiility of rectivtion of poliovirus from its complex with grpe juice ws studied y dding queous solutions of 2% geltin (Difco), 0.2% Tween 80 (Sigm Chemicl Co.), 1% polyvinyl pyrrolidone (British Drug House Ltd.), nd 1% polyethylene glycol 20,000 (Fisher Scientific Co.) (2, 7). One-milliliter volumes ofthe gents were mixed for 1 h with 1-ml portions of poliovirus-grpe juice complexes. RESULTS Poliovirus 1 ws inctivted fter 24-h incution with queous extrcts of Toky, Riier, or green seedless grpes, djusted to ph 7.0, nd with infusion prepred from the pprently intct surfces oftoky nd Riier grpes t the nturl ph or ph 7.0 (Tles 1 nd 2). Exmintion of extrcts prepred from the skin nd pulp of Concord nd lue seedless grpes indicted tht little ctivity ws present in the pulp (Tle 3). TABLE 1. Recovery ofpoliovirus from grpe extrcts (ph 7.0) fter incution t 4 C for 24 h Type of grpe extrct APPL. ENVIRON. MICROBIOL. Dilution of extrct in wter Undiluted 1:20 PFU/ % of in- PFU/mon- % of inmono- put vi- olyer put vilyer rus olyer rus Toky... 0 <1 1(1-1) 1 Riier... 0 <1 0 <1 Green seedless 0. <1 18 (16-20) 22 Averge of two smples, rnge in prentheses. Input virus, 83 (79-87). TABLE 2. Recovery ofpoliovirus from grpe infusions fter incution t 4 C for 24 h Type of Nturl ph ph 7.0 grpe in- PFU/mon- % of in- PFU/mon- % of infusion olyer put virus olyer put virus Toky.. 50 (48-51) (12-16) 17 Riier.. 21 (19-22) (16-18) 21 ph 5.2 for the Toky infusion, ph 5.9 for the Riier infusion. Averge of two smples, rnge in prentheses. Input virus, 82 (79-85). The effect of grpe juice on severl viruses fter 1-h incution is shown in Tle 4. There ws less inctivtion of coxsckievirus B5 nd echovirus 7 thn of poliovirus 1 nd herpes simplex virus 1. Incution of high inoculum of poliovirus for 24 h with undiluted grpe juice t ph 7.0 resulted in 1,000-fold reduction; e.g., in three seprte experiments, n input of 3.6 (3.0 to 4.0) x 106 PFU in 1 ml of grpe juice ws reduced to 4.1 (3.8 to 4.4) x 103 PFU. An infusion of seedless risins, t oth the nturl ph nd ph 7.0, inctivted severl viruses (Tle 5). The effect of three Cndin wines on severl viruses fter 1-h incution is shown in Tle 6. Coxsckievirus ppered not to e inctivted. Echovirus ws ffected less thn herpes simplex virus, poliovirus, or reovirus; inctivtion occurred t oth the nturl wine ph nd t ph 7.0 with ll three wines. Wines diluted 1:100 showed no ctivity except for red wine, ph 7.0, with poliovirus nd herpes simplex virus. The effect of severl imported wines on poliovirus fter 24-h incution is shown in Tle 7. In ll instnces, red wines inctivted virus etter thn white wines. With few exceptions, t oth the nturl ph nd ph 7.0, red wines diluted 1:1,000 inctivted virus, ut white wines diluted 1:100 showed no ctivity. Filtrtion of grpe juice through series of three Amicon memrnes, PM30, UM10, nd

3 VOL. 32, 1976 TABLE 3. VIRUS INACTIVATION BY GRAPES AND WINES 759 Recovery ofpoliovirus from extrcts ofskin, pulp, or whole lue grpes tph 7.0 fter incution t 4C for 24 h Dilution of extrct in wter Type of lue Type of ex- Undiluted grpe trct PFU/mono- % of input PFU/mono- % of input PFU/mono- % of input lyer virus lyer virus lyer virus Concord Whole 0 < Skin 0 < Pulp Seedless Whole 0 <1 0 <1 0 <1 Skin 0 <1 0 <1 0 <1 Pulp Single smples. Input virus, 85. i Seeded. TABLE 4. Recovery of viruses from grpe juice, fter incution t 4 C for 1 h, s percentge of input virus Dilution of GjieH grpe Virus Juice in wter Coxsckie Echo Herpes simplex Polio 3.3 Undiluted 3 (2_3) <1C < ' 20 (18-21) 4 (3-5) < (36-44) 10 (9-10) <1 7.0 Undiluted <1 <1 <1 < (97-102) 22 (21-23) <1 < (95-100) 56 (52-59) <1 10 (8-11) The low ph is the nturl ph. Averge of two smples, rnge in prentheses. c No plques in two smples. TABLE 5. Recovery of viruses from risin infusion, fter incution t4 C fori h, s percentge ofinput virus Infusion ph Virus Echo Herpes simplex Polio 4.4, nturl 7.0, djusted 56 (51-60) 67 (63-69) <1 8 (4-11) 45 (39_49) 49 (43-54) Averge of three smples, rnge in prentheses. No plques in three smples. UM2, reduced the ctivity ginst poliovirus out 10,000 fold; 1,000-fold reduction occurred fter filtrtion through the PM30 memrne; ntivirl ctivity ws found in ll frctions tht rnged in moleculr weight from <1,000 to >30,000 (Tle 8). Filtrtion of red wine, in series, through PM30 nd UM10 memrne reduced the ctivity ginst poliovirus out 10-fold (Tle 9); ntivirl ctivity ws found in ll frctions tht rnged in moleculr weight from <10,000 to >30,000. Results of dding geltin for 1 h to 2-, 5-, or 24-h poliovirus-grpe juice complexes re shown in Fig. 1. In controls without geltin, inctivtion of poliovirus incresed with time nd ws more rpid t ph 7.0 thn t ph 3.3; it ws lso more rpid with undiluted thn diluted grpe juice. The restortion of virus ctivity with geltin ws inversely proportionl to the degree of inctivtion. The effects of geltin, Tween 80, polyvinyl pyrrolidone, nd polyethylene glycol in 24-h poliovirus-grpe juice complex re shown in Tle 10. The lst mteril ws the most effective dissociting gent; ll gents were more effective in dissociting complexes tht were formed y diluted rther thn undiluted grpe juice nd t ph 3.3 rther thn ph 7.0. The effect of polyethylene glycol in 1- to 28- dy poliovirus-grpe juice complexes is shown in Tle 11. No virus recovery (<1%) ws otined from smples without polyethylene glycol; complex dissocition with polyethylene glycol decresed s the time of complex formtion incresed. DISCUSSION The nticteril property of wines hs een ttriuted to phenolic sustnces (10). It hs een suggested tht ll phenols my show ctericidl effects, nd this property is proly relted to their protein-inding cpcity (13). The effect on viruses my e somewht the sme, cusing loss of infectivity (t lest tem-

4 760 KONOWALCHUK AND SPEIRS APPL. ENVIRON. MICROBIOL. TABLE 6. Recovery of viruses from three Cndin wines, fter incution t 4 C for 1 h, s percentge of input virus Virus Wine type phdilution of wine Wine type Wine ph' in wter Coxsckie Echo Herpes Polio Reo Red 3.5 Undiluted <lc Red Red Red 7.0 Undiluted <1 <1 <1 Red < Red White 3.5 Undiluted <1 White White White 7.0 Undiluted White White Ros6 3.5 Undiluted <1 Ros Rose Ros6 7.0 Undiluted Rose Rose The low ph is the nturl wine ph. Single smples. c No plques. TABLE 7. Recovery ofpoliovirus from severl imported wines, fter incution t 4 C for 24 h, s percentge of input virus Dilution of wine in wter Wine type Wine ph Undiluted 10-' Red 1, French 3.8 <l Red 1, French 7.0 <1 < Red 2, French 3.6 < Red 2, French 7.0 <1 < Red 3, French 3.3 <1 <1 < Red 3, French 7.0 <1 < Red 4, Itlin 3.4 <1 < Red 4, Itlin 7.0 <1 < White 1, Germn White 1, Germn White 2, Germn White 2, Germn White 3, Germn White 3, Germn The low ph is the nturl wine ph. No plques. Single smples. porrily). In n erlier report (6), we showed tht extrcts of strwerries nd severl other fruits nd vrious compounds of plnt origin, e.g., tnnic (gllotnnic) cid, gllic cid, chlorogenic cid, cynin chloride, mlvin chloride, L-scoric cid, nd vnillin, were ll ntivirl, s were grpes nd grpe products s shown in this report. As with strwerry extrct, components of grpe juice, when seprted y memrne filtrtion, showed ntivirl ctivity in wide rnge of moleculr weights, i.e., <1,000 to >30,000. The comprtively lrge proportion of ctive components of grpes nd wines retined y the PM30 memrne, moleculr weight >30,000, my e due in prt to concentrtion polriztion, i.e., incresed concentrtion of rejected mcrosolutes t the memrne surfce, incresing memrne re-

5 VOL. 32, 1976 TABLE 8. Smple VIRUS INACTIVATION BY GRAPES AND WINES 761 Recovery ofpoliovirus from filtrtion frctions ofgrpejuice (ph 7.0), fter incution t 4 C for24 h, s percentge of input virus Dilution in wter Undiluted Control <l <1 19 (17-20)c 60 (57-63) PM30 retentte <1 <1 13 (12-14) 102 ( ) PM30 filtrte 5 (4-6) 59 (58-60) 104 ( ) 95 (92-98) UM10 retentte 5 (3-6) 44 (41-46) 94 (91-96) 98 (96-100) UM10 filtrte 5 (5-5) 67 (66-67) 89 (88-90) UM2 retentte 3 (2-4) 40 (38-41) 90 (87-92) 97 (94-99) UM2 filtrte 53 (50-55) 99 (97-101) 101 (99-102) Unfiltered grpe juice, ph 7.0. No plques in two smples. c Averge of two smples, rnge in prentheses. TABLE 9. Recovery ofpoliovirus from filtrtion frctions ofred wine (ph 7.0), fter incution t 4 C for 24 h, s percentge of input virus Dilution in wter Smple 1/4 1/8 1/16 1/32 1/64 1/256 Control <1 16 (14-17)C 39 (37-41) 92 (91-93) PM30 retentte 8 (7-9) 60 (56-64) 90 (87-93) 97 (95-99) PM30 filtrte 11 (10-11) 41 (39-42) 94 (92-95) 102 ( ) UM10 retentte 38 (35-40) 78 (75-81) 101 (99-102) UM10 filtrte 57 (54-60) 102 (98-105) In C- ui cc In d Unfiltered wine, ph 7.0. No plques in two smples. c Averge of two smples, rnge in prentheses WITHOUT GELATIN HOURS OF INCUBATION FIG. 1. Recovery of poliovirus from grpe juice. Left to right: ph 7.0 undiluted (X) nd diluted 1:100 (0); ph 3.3 undiluted (U) nd diluted 1:100 (O), fter incution for 2, 5, or 24 h followed y n dditionl 1-h incution with or without 1 %geltin. Duplicte smples were verged. tentivity. (This prolem ws minimized y constnt stirring during filtrtion followed y repeted wshing of the residue.) More likely, however, ctive components were nturlly polymerized phenols, found undntly in grpes nd wines (13). Grpe pulp, in contrst to the skin, ws not ntivirl. Reports indicte tht phenols re minly locted in the skin nd seeds of grpes nd other fruits (13). In our studies, poliovirus ws reduced to out 1/10 of its ctivity t ph 7.0 in 24 h y 10-4 dilution of grpe juice (Tle 8) or 10-2 dilution of red wine or undiluted white wine (Tle 7). The phenolic content of these products hs een clculted y mny workers (13). Singleton nd Ough (14) estimted tht the phenolic content, in tnnic cid equivlents, of 30 white wines verged 263 mg/liter; tht of 24 red wines verged 2,171 mg/liter. Noyes et l. (11) found n verge of 3,310 mg/liter in Concord grpe juice. The lower phenolic content of white wine is proly the result of the method of preprtion, since most white wines re produced from juice only nd red wines re produced y fermenttion in the presence of grpe solids. The totl phenolic mesurements of white grpes, including skins, seeds, nd pulp, compre fvorly with those found in red grpes (13). However, due to such fctors s virus type, phenol type (6), nd ph, one should not expect simple reltion etween virus inctivtion nd totl phenol content of fruit. Severl studies hve shown plnt extrcts, tnnins, nd relted compounds to e ntivirl

6 762 KONOWALCHUK AND SPEIRS TABLE 10. Recovery ofpoliovirus from grpejuice, s percentge ofinput virus, fter incution t 4C for 24 h followed y n dditionl 1-h incution in complex-dissociting gents Grpe juice Dilution of grpe juce Agent APPL. ENVIRON. MICROBIOL. ph in wter None Geltin (1%) Tween 80 (0.1%) Polyidnyl pyr- Polyethylene 3.3 Undiluted <1 <1 7 (5-8)C <1 63 (60-65) <1 52 (48-55) 63 (60-65) 71 (69-73) 85 (82-87) 7.0 Undiluted <1 <1 <1 <1 20 (18-21) <1 3 (2-4) 47 (44-49) 66 (64-68) 68 (64-71) The low ph is the nturl ph. No plques in three smples. C Averge of three smples, rnges in prentheses. TABLE 11. Recovery ofpoliovirus from grpe juice, s percentge of input virus, fter incution t 4 C for 1 to 28 dys followed y n dditionl 1-h incution in 0.5 % polyethylene glycol Grpe Dilution of grpe Dys of incution juice ph juice in wter Undiluted 64 (62-65) 30 (28-31) 11 (9-12) 8 (6-9) (83-87) 78 (75-80) 77 (74-80) 65 (63-67) 7.0 Undiluted 20 (19-20) <1C <1 < (64-70) 10 (9-11) 4 (3-4) <1 The low ph is the nturl ph. Averge of two smples, rnge in prentheses. c No plques in two smples. to influenz viruses, myxoviruses sugroup 2, Semliki forest, West Nile, herpes simplex, nd vccini viruses (1, 3, 4, 7), nd tocco mosic nd tocco necrosis viruses (15). In generl, inctivted virus could e restored to infectivity y dilution, y ddition of geltin in the cse of niml viruses, or y lumin or nicotine sulfte in the cse of plnt viruses. The interction etween tnnins nd viruses (or proteins in generl) is considered to e due to hydrogen onds nd is proly reversile (15, 16). However, Goldstein nd Swin (2) noted tht the enzyme 13-glucosidse formed more stle complex with the condensed tnnin from wttle thn with tnnic cid; in this cse onds other thn hydrogen my hve een formed. Previously (6) we found tht 24-h complex of poliovirus nd strwerry extrct, ph 7.0, ws not detectly dissocited y ddition of geltin. In the present study, poliovirus nd grpe juice showed similr stility, nd this ws influenced y ph, durtion of incution, nd concentrtion of the ctive components. Although some methods my not detect virus complexes from fruit, it ppers likely tht such complexes, when ingested, my dissocite in the humn gut nd my e cple of inititing disese. In view ofthis, it ppers pproprite tht reserchers devising new methods for virus recovery incorporte some mens of dissociting complexed virus. This would help to give more relistic ssessment of the totl virus present. LITERATURE CITED 1. Crson, R. S., nd A. W. Frisch The inctivtion of influenz viruses y tnnic cid nd relted compounds. J. Bcteriol. 66: Goldstein, J. L., nd T. Swin The inhiition of enzymes y tnnins. Phytochemistry 4: Herrmnn, E. C., Jr., nd L. S. Kucer Antivirl sustnces in plnts of the mint fmily (Lite). II. Nontnnin polyphenol of Meliss officinlis. Proc. Soc. Exp. Biol. Med. 124: Herrmnn, E. C., Jr., nd L. S. Kucer Antivirl sustnces in plnts of the mint fmily (Lite). III. Peppermint (Menth piperit) nd other mint plnts. Proc. Soc. Exp. Biol. Med. 124: Konowlchuk, J., nd J. I. Speirs Enterovirus recovery with vegetle floc. Appl. Microiol. 26: Konowlchuk, J., nd J. I. Speirs Antivirl ctivity of fruit extrcts. J. Food Sci. 41: Kucer, L. S., nd E. C. Herrmnn, Jr Antivirl sustnces in plnts of the mint fmily (Lite). I. Tnnin of Meliss offcinlis. Proc. Soc. Exp. Biol. Med. 124: Lichine, A Alexis Lichine's new encyclopedi of wines nd spirits. Alfred A. Knopf, Inc., New York. 9. Luci, S. P A history of wine s therpy. J. B. Lippincott Co., Montrel. 10. Msquelier, J The ctericidl ction of certin phenolics of grpes nd wine, p In J. W. Firirn (ed.), The phrmcology of plnt phenolics. Acdemic Press Inc., New York.

7 VOL. 32, 1976 VIRUS INACTIVATION BY GRAPES AND WINES Noyes, H. A., H. T. King, nd J. H. Mrtsoff Vritions in the Concord grpe during ripening. J. Assoc. Off. Agric. Chem. 6: Powers, J. J Action of nthocynin nd relted compounds on cteril cells, p In N. Molin (ed.), Microil inhiitors in food. Almqvist nd Wiksell, Stockholm. 13. Singleton, V. L., nd P. Esu Phenolic sustnces in grpes nd wine, nd their significnce. In Advnces in food reserch, Suppl. 1. Acdemic Press Inc., New York. 14. Singleton, V. L., nd C. S. Ough Complexity of flvor nd lending of wines. J. Food Sci. 27: Thresh, J. M Some effects of tnnic cid nd of lef extrcts which contin tnnins on the infectivity of tocco mosic nd tocco necrosis viruses. Ann. Appl. Biol. 44: White, T Tnnins-their occurrence nd significnce. J. Sci. Food Agric. 8: