Kombucha is a traditional remedy prepared at home by

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1 FOOD COMPOSITION AND ADDITIVES Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, Determintion of Ethnol in Kombuch, Juices, nd Alcohol-Free Beer by Enzytec TM Liquid Ethnol: Single-Lbortory Vlidtion, First Action Mrkus Lcorn nd Thoms Hektor R-Biophrm AG, An der neuen Bergstrsse 17, D Drmstdt, Germny Enzytec TM Liquid Ethnol is n enzymtic test for the determintion of ethnol in kombuch, juices, nd lcohol-free beer. The kit contins two components in redy-to-use formt. Quntifiction is bsed on the ctlytic ctivity of lcohol dehydrogense, which oxidizes ethnol to cetldehyde nd converts NAD + to NADH. Mesurement is performed in 3 ml cuvettes t 340 nm within 20 min. Smples with lcohol contents round 0.5% lcohol by volume need to be diluted 1:20 or 1:50 with wter before mesurement. Acetldehyde interferes t concentrtions higher thn 3000 mg/l, wheres sulfite interferes t concentrtions higher thn 300 mg/l. The liner mesurement rnge is from 0.03 up to 0.5 g/l ethnol, wheres LOD nd LOQ re 1.9 nd 3.3 mg/l ethnol, respectively. Kombuch with concentrtions between 2.85 nd 5.82 g/l showed reltive repetbility stndrd devition round 1%, wheres juices were below 2%. Results from reproducibility experiment reveled tht t concentrtion of 0.1 g/l, the RSD R ws t 2.5%, wheres t higher concentrtions between 0.2 nd 0.3 g/l, coefficients round 1% were obtined. Trueness ws checked by using Cerillint queous ethnol solutions nd beer with concentrtion of 0.4 nd 4 g/l (BCR-651 nd BCR-652). Spiking of kombuch nd juice smples resulted in recoveries between 95% nd 104%. Acceptble stbility ws found for the whole test kit under ccelerted conditions t 37 C for 2 weeks. The kit is lso not susceptible to short freezing thwing cycles nd hrsh trnsport conditions. Received November 17, Accepted by SG Jnury 5, This method ws pproved by the Expert Review Pnel for Kombuch in Ethnol s First Action. The Expert Review Pnel for Ethnol in Kombuch invites method users to provide feedbck on the First Action methods. Feedbck from method users will help verify tht the methods re fit-for-purpose nd re criticl for gining globl recognition nd cceptnce of the methods. Comments cn be sent directly to the corresponding uthor or methodfeedbck@oc.org. Supplementl informtion is vilble on the J. AOAC Int. website, Corresponding uthor s emil: m.lcorn@r-biophrm.de DOI: Kombuch is trditionl remedy prepred t home by fermenting sweetened blck te with symbiotic culture of yests nd bcteri. The fermented te is produced by the ction of floting microbil mt/colony consisting of erobic bcteri nd yests. The colony s ppernce often resembles surfce mold or mushroom but is ctully floting cellulose mt produced during microbil growth. The kombuch culture hs been shown to be symbiosis of Acetobcter nd vriety of yests. The kombuch recipe my vry; however, it is commonly mde by infusing blck te leves into freshly boiled wter, sweetened with g/l (5 15%) sucrose, for bout 10 min. After removing the te leves, the te is llowed to cool to room temperture nd the microbil mt/colony from previous btch is dded to the sweetened te with bout 100 ml of kombuch from previous fermenttion. It is then covered with clen cotton cloth nd incubted t room temperture for bout 7 10 dys. Crbon conversion of sucrose begins by the yests breking down the sugr into glucose nd fructose. Glucose is primrily used up by the yests to yield ethnol nd crbon dioxide. The ethnol is then oxidized by the bcteri to cetldehyde nd then to cetic cid. As result, the lcohol content of kombuch is thought to never exceed 10 g/l, nd the cetic cid concentrtions my rise to levels s high s 10 g/l. The finl product is slightly crbonted beverge composed of fructose, ethnol, orgnic cids, vitmins, minerls, nd te components, resembling the tste of cider (1). Ethnol is ubiquitous in its nturl occurrence due to the ctivity of yests in sugr-contining food. Its quntittion is not only importnt in the mnufcture of wines, beers, nd spirits, but lso for low-lcohol nd nonlcoholic beverges, juices, nd rnge of other foodstuffs, including jm, honey, vinegr, nd diry products. Becuse of its ubiquitous occurrence, determintion of ethnol in lbortories requires specil ttention to ll kinds of cross contmintion. Within the Compendium of Interntionl Anlysis of Methods of the Orgnistion Interntionl de l Vigne et du Vin, there re some fully vlidted type I methods for quntifiction of ethnol in wine (2). Besides these worldwide ccepted methods, there is no fully vlidted method to quntify ethnol in kombuch by simple nd robust procedure. In 2016, AOAC initited cll for methods bsed on the Stndrd Method Performnce Requirements (SMPR ) for determintion of ethnol content in kombuch. This study investigted the test kit Enzytec TM Liquid Ethnol (E 8340; R-Biophrm AG, Drmstdt, Germny) to be pplicble for the quntifiction of ethnol not only in kombuch, but lso in fruit juice, vegetble juice, nd lcohol-free beer.

2 2 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, 2018 The underlying enzymtic rection of this kit requires one enzyme nd one coenzyme only. Ethnol is oxidized by the ctlytic ctivity of lcohol dehydrogense (ADH) in the presence of nicotinmide-denine dinucleotide (NAD + ) to cetldehyde. Becuse the equilibrium of this rection lies in fvor of ethnol nd NAD +, specil experimentl conditions were set up to ensure quntittive rection to cetldehyde. In consequence, the test kit only contins two redy-to-use components, which is the bsis for robust nd precise simple quntifiction of ethnol. AOAC Officil Method Ethnol in Kombuch, Juices, nd Alcohol-Free Beer ENZYTEC TM Liquid Ethnol First Action 2017 The Enzytec Liquid Ethnol quntifies ethnol in diluted or undiluted smples between 30 nd 300 mg/l ethnol with high precision (CV 2%). The liner rnge of the system is between 3.3 nd 500 mg/l. The LOD is t 1.9 mg/l. All chemiclly relted primry, secondry, nd tertiry lcohols show sidechin ctivity with the exception of methnol. Acetldehyde interferes t concentrtions higher thn 3000 mg/l, wheres sulfite interferes t concentrtions higher thn 300 mg/l. Cution: see Mteril Sfety Dt Sheet A. Principle The enzymtic rection requires one enzyme nd one coenzyme only. As cn be seen from (I), ethnol is oxidized by the ctlytic ctivity of ADH in the presence of NAD + to cetldehyde nd NADH/H +. NADH formed is stoichiometric with mount of ethnol originlly present. NADH produced is mesured t 340 nm with spectrophotometer. ( ) ( ADH) + + I Ethnol + NAD Acetldehyde + NADH + H Becuse the equilibrium of this rection lies in fvor of ethnol nd NAD +, specil experimentl conditions were set up to ensure quntittive rection to cetldehyde. In consequence, the test kit only contins two redy-to-use components, which is the bsis for robust nd precise simple quntifiction of ethnol. B. Apprtus Apprtus specified hs been tested. Equivlent pprtus my be used. () Rection tube (2 ml); reclosble. (b) Disposble plstic cuvettes (1 cm light pth). (c) Micro-pipettors ( μl nd μl). (d) Multipette (to dispense 2 ml liquots of regent 1). (e) Spectrophotometer set t 340 nm. (f) Vortex mixer. (g) Ultrsound for degssing. (h) Centrifuge (min g). C. Regents Items () to (c) re vilble s test kit (ENZYTEC Liquid Ethnol, E 8340; R-Biophrm Drmstdt, Germny). All regents re stble s indicted on the lbel t 2 8 C (36 46 F). () The test kit consists of regent 1 (2 x) nd regent 2 (2 x). (b) Regent ml (contining buffer; redy to use). (c) Regent ml (contining NAD + nd ADH; redy to use). D. Stndrd Reference Mteril Trcebility ws estblished by using certified reference mterils (AQ01-015, AQ02-015, nd AQ03-015) from ACQ Science (Rottenburg-Hilfingen, Germny) with ethnol concentrtions from 0.1, 0.2, nd 0.3 g/l. E. Stndrd nd Spike Solution For spiking experiments, EMSURE Ethnol (bsolute for nlysis; Merck, ) ws used. The mteril is free of wter. Becuse ethnol is voltile, it ws decided to use 20% lcohol by volume (ABV) ethnolic solution for spiking smples. For preprtion, 100 ml volumetric flsk ws filled with bout 50 ml distilled wter. Twenty milliliters of bsolute ethnol ws pipetted into the flsk, mixed, nd filled with wter up to 100 ml. F. Generl Preprtion () Both components of the test kits re redy to use but should be wrmed up to room temperture before use. (b) Mke sure tht the lbortory environment, especilly the ir, is not contminted with ethnol, e.g., cused by use of disinfection or clening regents. Otherwise, ll results will be bised due to the high sensitivity of the test kit towrd ethnol. (c) This test should only be crried out by trined lbortory employees. (d) The instructions for use must be strictly followed. (e) No qulity gurntee is ccepted fter expirtion of the kit (see expirtion lbel). (f) Do not interchnge individul regents between kits of different lot numbers. (g) Follow recommendtions of the mnufcturer of the spectrophotometer for proper wrm-up nd mintennce. G. Smple Preprtion () Generl recommendtion. (1) Ethnol is very voltile; therefore, when diluting smple solutions, lwys pipette beneth the surfce of the diluent; when filtering smple solution, the filtrte shll not drop but rinse down the wll of the vil; close vil tightly before centrifugtion. (2) Use cler, slightly colored, nd ph-neutrl liquid smples directly, or fter dilution into the relevnt mesurement rnge of mg/l ethnol; slightly cidic or lkline smple my be used directly fter dilution; check strong cidic smple solution for recovery by spiking even fter dilution in cse of ny doubt. (3) Degs smples contining crbon dioxide by short burst of ultrsound t 0 C (ultrsonic device filled with ice cubes nd distilled wter). (b) Smple preprtion. (1) Cler kombuch, lcohol-free beer, nd juices: dilute with wter. (2) Turbid kombuch, lcohol-free beer, nd juices: centrifuge before dilution nd dilute superntnt with wter.

3 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, (3) Smples with % ABV (0.6 up to 6 g/l ethnol) should be diluted with wter, e.g., 100 μl smple is pipetted into 1900 μl distilled wter. (4) Smples with % ABV (3 up to 30 g/l ethnol) should be diluted , e.g., 100 μl smple is pipetted into 9.90 ml distilled wter. (5) Other dilutions s, e.g., 1:50 or 1:10, re possible if the ethnol concentrtion of the diluted smples lies within the mesurement rnge (0.03 up to 0.3 g/l). (6) Dilution of ethnol-contining smples with wter is very susceptible to pipetted volumes used for dilution. Therefore, pipette t minimum 100 μl ethnol-contining smple into the respected volume of wter; lower volumes, e.g., 20 μl, will result in higher CVs. (7) Use diluted smple solutions within 3 dys for ethnol mesurement (storge temperture 4 C). H. Determintion () Plce cuvette for one blnk (RB) nd ech smple/ control into rck nd pipette 2000 μl regent 1 (R1) into ech cuvette. (b) Add 100 μl ech smple or control into designted cuvette nd 100 μl distilled wter into the designted cuvette (blnk). (c) Mix with plstic sptul or nother pproprite device. (d) Incubte for 3 min t C. (e) Red nd document bsorbnce A1 in spectrophotometer set t 340 nm for ech cuvette. (f) Add 500 μl regent 2 (R2) in ech cuvette nd mix well. (g) Incubte for 15 min t C. (h) Red nd document bsorbnce A2 in spectrophotometer set t 340 nm for ech cuvette. I. SLV Prmeters The mnufcturer s in-house vlidtion scheme followed the AOAC Appendix K recommendtions s described in the SMPR for ethnol quntifiction in kombuch nd long-lsting prcticl experiences of the method developer for redy-to-use enzymtic test kits. () Linerity. Linerity check over rnge of up to 0.7 g/l ethnol (in wter) in three different runs with one replicte in ech run. Additionlly, clcultion of residul plot to chrcterize rnge of linerity. (b) LOD nd LOQ. According to DIN 32645: (bsed on DIN ISO : ) with concentrtions rnging from 2.5 up to 45 mg/l ethnol (in wter) nlyzed in three independent runs (n = 1). (c) Selectivity. Experiment using methnol, 1-propnol, 2-propnol, n-butnol, isobutnol, 1-pentnol, 2-pentnol, 3-pentnol, nd 1-hexnol t concentrtion of 0.2 g/l in comprison with 0.2 g/l ethnol. All solutions were prepred in distilled wter. (d) Precision profile. Dt were clculted from the linerity dt set (queous ethnol clibrtors). The clculted RSD derived from replictes from three independent runs. (e) Repetbility. Four nonspiked kombuch smples (degssed, centrifuged, diluted 1:50) were used over period of 3 dys by two persons on two occsions per dy with two technicl replictes per extrct nd dy. Additionlly, four nonspiked kombuch smples (degssed, centrifuged, diluted 1:50) were used in n = 6 t ech occsion over period of 2 dys on three occsions in totl with one technicl replicte per extrct. Four different nonspiked fruit juices were degssed, centrifuged, nd tested in n = 6 t ech occsion over period of 2 dys on three occsions in totl with one technicl replicte per extrct. Three different nonspiked vegetble juices were degssed, centrifuged, nd tested in n = 6 t ech occsion over period of 2 dys on three occsions in totl with one technicl replicte per extrct. (f) Inter-lot precision. Aqueous ethnol solutions with 0.03 (solution A), 0.15 (solution B), nd 0.30 g/l (solution C) were nlyzed with six replictes on 1 dy by one person in three different test kit lots. (g) Lbortory-internl reproducibility. Mesurement of certified reference mteril (queous ethnol solutions from ACQ Science GmbH with ethnol concentrtions of 0.1, 0.2, nd 0.3 g/l); ech mteril ws used directly for mesurement nd nlyzed in four different lots by four different persons with two replictes per mesurement. From these vlues s r, RSD r, s R, nd RSD R were clculted. (h) Trueness. Mesurement of certified reference mterils (queous ethnol solutions from Cerillint with ethnol concentrtions from 0.8 up to 4 g/l nd BCR beer with concentrtions of 0.05 nd 0.5% ABV). (i) Recovery. Four different kombuch smples used for repetbility chrcteriztion were spiked to their endogenous ethnol concentrtions (between 2.7 nd 5.7 g/l); smples were degssed, centrifuged, nd diluted in n = 6 t ech occsion by two persons over period of 2 dys on three occsions in totl with one technicl replicte per extrct per person. For ll juices, smples were individully spiked to rech the upper mesurement rnge of 0.3 g/l. Smples were degssed, centrifuged, nd tested in n = 6 t ech occsion over period of 2 dys on three occsions in totl with one technicl replicte per extrct. (j) Interferences. Sulfite nd cetldehyde were tested in presence of 0.15 g/l ethnol (n = 1) with concentrtions of 0.06 up to 30 nd 1.5 up to 30 g/l, respectively. (k) Robustness. Incubtion temperture ws vried between 18 nd 37 C. Incubtion time before mesuring A2 t 340 nm ws vried between 5 nd 20 min. An lcohol-free beer smple nd kombuch smple with ethnol concentrtions of 3.5 nd 2.0 g/l were checked for dilutbility. (l) Stbility. See supplementl mteril. J. Clcultions () Clculte ΔA for every smple or control: ΔA = (A 2 df A 1 ) smple or control (A 2 df A 1 ) RB where df is dilution fctor clculted s follows: df = (smple volume + R1) / (smple volume + R1 + R2) = (b) Clculte concentrtions for every smple or control: c = (V MW ΔA) / (ε d v 1000) where V = finl volume; MW = moleculr weight of ethnol; ε = bsorption coefficient of NADH t 340 nm; d = light pth within

4 4 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, 2018 cuvette; nd v = test portion volume explin the ethnol-specific formul: OD residul or Ethnol, g/l = (2.6 ml g mole 1 ΔA)/ (6.3 L mmole 1 cm 1 1 cm 0.1 ml 1000) Ethnol, g/l = ΔA If smple ws diluted before mesurement, this result hs to be multiplied with the dilution fctor F nd converted to % ABV using the formul: Results nd Discussion Linerity % ABV = Ethnol [g/l] / The rnge of concentrtions tested for linerity behvior of the enzymtic system is from up to 0.7 g/l ethnol. This set of dilutions ws tested three times independently, nd results re depicted in Figure 1. It is lredy visible by eye tht t concentrtions higher thn 0.5 g/l ethnol, the linerity of the system is no longer given. A correltion fctor r 2 is not given in Figure 1 becuse this fctor lone does not result in ny relevnt informtion bout sufficient linerity. Insted, residuls of ΔOD vlues were clculted nd presented in Figure 2. This plot clerly reveled tht the linerity ends between 0.4 nd 0.5 g/l ethnol. The lower point of quntifiction will be chrcterized in the LOD nd LOQ section. LOD nd LOQ The enzymtic system Enzytec Liquid Ethnol is very sensitive. Therefore, it is not remrkble tht nerly ll mtrices checked to be ethnol-free contined low but detectble or even quntifible mounts of ethnol. As consequence, only clibrtion dt cn be used to estimte LOD nd LOQ. This procedure is lid down in DIN 32645: (bsed on DIN OD Figure 1. Linerity check over rnge of up to 0.7 g/l ethnol in three different runs with one replicte in ech run. ΔOD vlues fter subtrction of blnk vlues re shown. g/l ethnol g/l ethnol Figure 2. Residul plot of dt presented in Figure 1. Rnge of concentrtion is from up to 0.7 g/l. For ech concentrtion, three independent mesurements were vilble. OD mg/l ethnol Figure 3. Anlysis of clibrtors with concentrtions ner to zero concentrtion. A rnge from 2.5 up to 45 mg/l ws covered nd nlyzed in three independent runs (n = 1). Clibrtion function is described by ΔOD = mg/l ethnol ISO : ) nd required clibrtion dt ner to zero concentrtion. Therefore, clibrtors between 2.5 nd 45 mg/l ethnol were prepred nd nlyzed in three independent runs (Figure 3). From this figure, the liner clibrtion function ws clculted to ΔOD = mg/l ethnol For the following clcultion, the slope of this line (b = ) is used (Figure 3). According to DIN 32645: , quick estimte of LOD nd LOQ is possible. In principle, the residul stndrd devition s y,x of the mesurement signl is converted to stndrd devition s x0 of the concentrtion by dividing s y,x with the slope b of the grph, which is given bove. This stndrd devition of the procedure s x0 is multiplied with severl fctors to estimte LOD nd LOQ. In this cse, LOD is t 1.85 mg/l (0.0002% ABV), wheres LOQ is t 3.26 mg/l (0.0004% ABV). The estimtes from two dditionl lots reveled LODs t 0.89 nd 1.43 mg/l, wheres LOQs were estimted t 1.60 nd 2.54 mg/l (rw dt nd clcultion not shown). These three lots were lso used to chrcterize stbility of the system. An ccelerted stbility study ws performed over period of

5 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, dys t 37 C. At the end of this period, LOD nd LOQ were estimted gin nd reveled LOD vlues of 0.53, 1.33, nd 1.25 mg/l, wheres LOQ vlues were clculted to 0.96, 2.36, nd 2.22 mg/l. Selectivity Relevnt chemiclly relted lcohols tht were reported to exist in beverges nd juices were tested in the enzymtic system t concentrtion of 0.2 g/l (Tble 1). Methnol is not recting in the enzymtic system. In contrst, 1-propnol, 2-propnol, n-butnol, 1-pentnol, nd 1-hexnol showed significnt or even comprble results to ethnol. This ws expected becuse ny lcohol dehydrogense exerts these kinds of side-chin ctivities. Theoreticlly, these lcohols could led to creep rections. But the published dt on these lcohols revel tht in the best cse, fctor of 1000 exists between the concentrtion of ethnol nd the chemiclly relted side-chin ctive lcohols, so the side-chin ctivity should not led to mesurble errors. Precision Profiles The chrcteriztion of precision over the whole mesurement rnge is depicted in Figure 4. These dt were clculted from the linerity dt set (queous ethnol clibrtors). The clculted RSD ws derived from three independent replictes from three independent runs. Repetbility For chrcteriztion of repetbility, diluted kombuch smples with endogenous ethnol concentrtions between 2.73 nd 5.65 g/l were nlyzed by two persons over period of 3 dys with two testing series per dy; RSDs were somewht higher (Tble 2). But becuse different persons were involved on different dys, the results re still excellent. For this experiment, only one dilution ws prepred from ech smple to exclude effects due to vrition of dilution. If dilution is included into the uncertinty budget, the results re still cceptble (Tble 3). At this time, only one nlyst performed the experiments, becuse Tble 1. Selectivity experiment using methnol, 1-propnol, 2-propnol, n-butnol, isobutnol, 1-pentnol, 2-pentnol, 3-pentnol, nd 1-hexnol t concentrtion of 0.2 g/l in comprison with 0.2 g/l ethnol. All solutions were prepred in distilled wter Substnce Trget vlue, g/l Concn, g/l Rec., % Ethnol Methnol Propnol Propnol n-butnol Isobutnol Pentnol Pentnol Pentnol Hexnol RSD OD (%) g/l ethnol Figure 4. Precision profile of dt used to chrcterize linerity (see Linerity section; Figure 1). it ws shown in Tble 2 tht the person hs negligible influence on vrition of results. It will be presented in the Robustness section tht it is bsolutely necessry to hve smple intke of 100 μl kombuch for dilution. Otherwise, RSDs will rise up to 4% if, e.g., only 20 μl kombuch is used for dilution before mesurement in the enzymtic system (results not shown). When undiluted fruit juice smples (pple, crnberry, multivitmin, ornge) with endogenous ethnol concentrtions between nd g/l were nlyzed by one person over period of 2 dys with three tests in totl, RSDs were below 2%, with the exception of crnberry juice, in which, due to the low ethnol concentrtions, the RSD ws round 3% (Tble 4). When undiluted vegetble juice smples (tomto, tomtovegetble, crrot) with endogenous ethnol concentrtions between nd g/l were nlyzed by one person over period of 2 dys with three tests in totl, RSDs were below 1%, with the exception of tomto-vegetble juice, in which, due to the very low ethnol concentrtions, the RSD ws round 2.3% (Tble 5). Inter-Lot Precision Becuse the enzymtic test kit is proprietry redy-to-use test kit, chrcteriztion of inter-lot vrition is necessry to show tht ll lots produced under routine conditions in the production fcility t R-Biophrm re comprble with respect to vrition. Results re shown in Tble 6 nd show tht ll three lots were comprble over the mesurement rnge. Lbortory-Internl Reproducibility (Intermedite Precision) Tble 7 contins dt from R-Biophrm s QC deprtment, where rel-time stbility tests re performed. For QC tests, certified reference mterils from ACQ re used becuse they hve optiml volumes of 1.5 ml, which mkes it possible to use new vil for ech testing. As cn be seen from Tble 7, four different lots were tested by four technicins on different dys in duplicte. Using the AOAC excel work sheet for collbortive tests, results from Tble 7 were tken to clculte s r, RSD r, s R, nd RSD R (Tble 8). Reproducibility

6 6 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, 2018 Tble 2. Repetbility Kombuch (nonspiked). Smple ws degssed, centrifuged, diluted (1:50), nd used over period of 3 dys by two persons on two occsions per dy. Two technicl replictes per extrct nd dy. Result in g/l ethnol Dy 1, test 1 Dy 1, test 2 Dy 2, test 1 Dy 2, test 2 Dy 3, test 1 Dy 3, test 2 Kombuch 1 Kombuch 2 Kombuch 3 Kombuch 4 Person 1 Person 2 Person 1 Person 2 Person 1 Person 2 Person 1 Person Men, g/l SD, g/l RSD, % reltive stndrd devitions for concentrtions between 0.1 nd 0.3 g/l were between 0.91 nd 2.53%, which is quite excellent. Tble 3. Repetbility including dilution Kombuch (nonspiked). Smple ws degssed, centrifuged, nd diluted (1:50) in n = 6 t ech occsion over period of 2 dys on three occsions in totl. One technicl replicte per extrct. Result in g/l ethnol; 100 μl kombuch ws pipetted into 4.90 ml wter Kombuch 1 Kombuch 2 Kombuch 3 Kombuch 4 Dy 1, test Dy 1, test Dy 2, test Men, g/l SD, g/l RSD, % Trueness Severl certified reference mterils (CRM) re vilble, lthough most of them re queous ethnol solutions. Only two beers re vilble. In consequence, most of the dt presented in this chpter re results for queous ethnol solutions. In the opinion of the method developer, this is not generl problem becuse ll smples with ethnol concentrtions round the threshold of 0.5% ABV (3.945 g/l) need to be diluted by fctor of 20, which minimizes the influence of mtrix effects. The Recovery section will del with possible mtrix effects. Tble 9 shows the results for CRMs from Cerillint nd two BCR beers Tble 4. Repetbility including dilution Four different fruit juices (nonspiked) were degssed, centrifuged, nd tested in n = 6 t ech occsion over period of 2 dys on three occsions in totl. One technicl replicte per extrct Apple Crnberry Multivitmin Ornge Men, g/l SD, g/l RSD, % Apple, ornge, pinepple, cerol, pssion fruit, mngo, lemon, bnn, nectrine. Tble 5. Repetbility including dilution Three different vegetble juices (nonspiked) were degssed, centrifuged, nd tested in n = 6 t ech occsion over period of 2 dys on three occsions in totl. One technicl replicte per extrct. Result in g/l ethnol Tomto Tomto-vegetble Crrot Men, g/l SD, g/l RSD, %

7 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, Tble 6. Inter-lot precision using queous ethnol solutions with 0.03 (solution A), 0.15 (solution B), nd 0.30 g/l (solution C). Six replictes were nlyzed on one dy by one person in three lots (BCR-651 nd BCR-652). Compred with the certified vlue, ll results were excellent nd only differed in one cse by 4% (beer with 0.051% ABV, corresponding to 0.4 g/l ethnol) Recovery Lot 1 g/l Lot 2 g/l Solution A Lot 3 g/l Men SD RSD, % Solution B Men SD RSD, % Solution C Men SD RSD, % All kombuch smples from the mrket showed ethnol concentrtions between 2.85 nd 5.82 g/l (see top of Tble 10). It ws decided to spike these smples s high s their endogenous ethnol concentrtion. The four different kombuch smples were nlyzed by two persons on three occsions over period Tble 8. Clcultion of s r, RSD r, s R, nd RSD R for three certified reference mterils (rw dt in Tble 7) from QC dt Reference 1 Reference 2 Reference g/l 0.2 g/l 0.3 g/l Totl number p Totl number of replictes Overll men of ll dt (grnd men) Repetbility stndrd devition Reproducibility stndrd devition Repetbility reltive stndrd devition Reproducibility reltive stndrd devition Sum(n(L)) XBARBAR g/l g/l g/l s(r) g/l g/l g/l s(r) g/l g/l g/l RSD(r) 0.58% 0.71% 0.72% RSD(R) 2.53% 0.91% 1.65% of 2 dys s 6-fold repliction. Every smple of these six replictes ws diluted nd not stored for the next occsion. The concentrtion of the spiked smples ws clculted by subtrction of the endogenous ethnol concentrtion from the nlyticl result. From this, the recovery ws clculted. Results of this recovery experiment re depicted in Tble 10 nd reveled very high precision (RSDs smller thn 1.6%). This high precision ws only chievble if 100 μl kombuch ws used for dilution. If only 20 μl ws used, RSD ws less thn 4% (results not Tble 7. Mesurement of certified reference mteril (queous ethnol solutions from ACQ Science GmbH). Ech mteril ws used directly for mesurement nd nlyzed in four different lots by four different persons with two replictes per mesurement ACQ reference 1 Certified: 0.1 g/l ACQ reference 2 Certified: 0.2 g/l ACQ reference 3 Certified: 0.3 g/l Rep. 1 Rep. 2 Rep. 1 Rep. 2 Rep. 1 Rep. 2 Lot Dte Person g/l ethnol g/l ethnol g/l ethnol A B A B A B C D D D D D D Rep. = Replicte.

8 8 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, 2018 Tble 9. Mesurement of certified reference mteril (queous ethnol solutions from Cerillint nd BCR beer). Ech mteril ws diluted before mesurement nd nlyzed Smple Dilution Certified vlue Mesured Rec. g/l ethnol g/l ethnol % Cerillint 800 mg/l 1: Cerillint 800 mg/l 1: Cerillint 1000 mg/l 1: Cerillint 1000 mg/l 1: Cerillint 1500 mg/l 1: Cerillint 1500 mg/l 1: Cerillint 2000 mg/l 1: Cerillint 2000 mg/l 1: Cerillint 4000 mg/l 1: Cerillint 4000 mg/l 1: BCR % 1: BCR % 1: BCR % 1: BCR % 1: shown). Tble 11 contins recovery dt converted to percentge of recovery. Men recovery vried from 96 up to 102%, wheres the rnge of individul recoveries ws between 95 nd 104%. Fruit juice smples from the mrket showed ethnol concentrtions between nd g/l (see top of Tble 12). It ws decided to spike these smples t bout 0.3 g/l in totl becuse the liner rnge of the clibrtion is limited, nd the spiked smples should be treted like unspiked smples (undiluted). The three different juice smples were nlyzed by one person on three occsions over period of 2 dys s 6-fold repliction. The concentrtion of the spiked smples ws clculted by subtrction of the endogenous ethnol concentrtion from the nlyticl result. From this, the recovery ws clculted. Results of this recovery experiment re depicted in Tble 12 nd reveled very high precision (RSDs smller thn 2%). Tble 12 lso contins recovery dt converted to percentge of recovery. Men recovery vried from 95 up to 97%, wheres the rnge of individul recoveries ws between 90 nd 101%. Vegetble juice smples (tomto, tomto-vegetble, nd crrot) from the mrket reveled ethnol concentrtions between nd g/l (see top of Tble 13). It ws decided to spike these smples t bout 0.3 g/l in totl becuse the liner rnge of the clibrtion is limited, nd the spiked smples should be treted like unspiked smples (undiluted). Tble 10. Recovery Kombuch. Four different kombuch smples with endogenous ethnol concentrtions between 2.85 nd 5.82 g/l were spiked with ethnol between 2.70 nd 5.70 g/l (see top of tble). Smples were degssed, centrifuged, nd diluted in n = 6 t ech occsion by two persons over period of 2 dys on three occsions in totl. One technicl replicte per extrct per person. Result in g/l ethnol. The concentrtion of the spiked smples is clculted by subtrction of the endogenous ethnol concentrtion from the nlyticl result; 100 μl kombuch ws pipetted into 4.90 ml wter Blnk Spike Kombuch 1 Kombuch 2 Kombuch 3 Kombuch Person 1 Person 2 Person 1 Person 2 Person 1 Person 2 Person 1 Person 2 Dy 1, test Dy 1, test b Dy 2, test Men, g/l SD, g/l RSD, % b See Tble 3. Outlying vlue.

9 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, Tble 11. Recovery Kombuch. Results re expressed s percentge recovery clculted from vlues in Tble 10. The concentrtion of the spiked smples is clculted by subtrction of the endogenous ethnol concentrtion from the nlyticl result; from this, the recovery ws clculted Kombuch 1 Kombuch 2 Kombuch 3 Kombuch 4 Person 1 Person 2 Person 1 Person 2 Person 1 Person 2 Person 1 Person 2 Dy 1, test Dy 1, test Dy 2, test Men, rec. % SD, rec. % Outlying vlue; see Tble 10. The concentrtion of the spiked smples ws clculted by subtrction of the endogenous ethnol concentrtion from the nlyticl result. From this, the recovery ws clculted. Results of this recovery experiment re depicted in Tble 13 nd reveled very high precision (RSDs smller thn 1.6%). Tble 13 lso contins recovery dt converted to percentge of recovery. Men recovery vried from 96 up to 97%, wheres the rnge of individul recoveries ws between 93 nd 99%. Interferences The liphtic lcohols 1-propnol, 2-propnol, nd n-butnol tht showed the highest side-chin ctivities (see Selectivity section) were gin tested t different levels between nd 0.15 g/l in presence of 0.15 g/l ethnol. As cn be seen in Figure 5, the three tested higher lcohols exert significnt positive interferences to the ethnol quntifiction. As expected from selectivity experiments, the grph is quite liner. Nevertheless, the reltionship between ethnol concentrtions nd concentrtion of these interfering lcohols will lwys be greter thn fctor 1000 under prcticl conditions. In the present cse, this would be, e.g., 1-propnol concentrtion of g/l. This will never influence the ethnol determintion significntly. Results for other possible interfernts re shown in Tble 14. Sugrs nd short-chin orgnic cids exert no interfering effect (results not shown). Up to 300 mg/l, sulfite does not interfere with the ethnol mesurement. Higher vlues re not prcticl becuse even in wine the thresholds re between 200 nd 300 mg/l. Kombuch, lcohol-free beer, nd juices re normlly not conserved by sulfite. Acetldehyde does not interfere up to 3000 mg/l. Becuse cetldehyde is only fermenttion byproduct nd normlly present t very low concentrtions in finl products, it is ssumed tht this interference hs no influence on ethnol quntifiction under prcticl conditions. Robustness Enzymtic systems re sometimes susceptible to vritions in incubtion times nd incubtion tempertures. Therefore, it ws checked if the ssy still produces true results if the temperture is lowered to 18 C or incresed up to 37 C. Incubtion time before mesuring A2 t 340 nm ws vried between 5 nd 20 min. There is no influence when vrying the incubtion temperture or incubtion time (results not shown). Due to the smll mesurement rnge between 30 nd 300 mg/l, it will often hppen in prxis tht smple shows ethnol concentrtion higher thn 300 mg/l. Therefore, n lcohol-free beer smple nd kombuch smple with ethnol concentrtions of 3.5 nd 2.0 g/l were checked for dilutbility. As cn be clerly seen in Figure 6, both smples re dilutble over the whole rnge. Discussion The test kit Enzytec Liquid Ethnol investigted in this vlidtion study ws proven to be pplicble for the quntifiction of ethnol in kombuch, fruit juice, vegetble juice, nd lcohol-free beer. It consists of two components, in which one

10 10 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, 2018 Tble 12. Recovery Fruit juice. Three different juice smples with endogenous ethnol concentrtions between nd g/l were spiked with ethnol between nd g/l (see top of tble). Smples were degssed, centrifuged, nd tested in n = 6 t ech occsion over period of 2 dys on three occsions in totl. One technicl replicte per extrct. The concentrtion of the spiked smples is clculted by subtrction of the endogenous ethnol concentrtion from the nlyticl result nd is given in g/l ethnol; from this, the recovery ws clculted nd is presented in prentheses Apple Crnberry Multivitmin Blnk Spike Dy 1, test (97%) (94%) (95%) (97%) (93%) ( ) b (97%) (95%) (98%) (101%) (95%) (95%) (100%) (96%) (96%) (98%) (98%) (95%) Dy 1, test (95%) (95%) (95%) (96%) (94%) (94%) (96%) (94%) (98%) (98%) (95%) (93%) (96%) (95%) (95%) (96%) (96%) (95%) Dy 2, test (100%) (90%) ( ) b (97%) (95%) (91%) (93%) (93%) (96%) (100%) (96%) (96%) (98%) (95%) (95%) (96%) (96%) (97%) Men, g/l (97%) (95%) (95%) SD, g/l (1.9%) (1.7%) (1.6%) RSD, % b See Tble 4. Outlier. Tble 13. Recovery Vegetble juice. Three different juice smples with endogenous ethnol concentrtions between nd g/l were spiked with ethnol between nd g/l (see top of tble). Smples were degssed, centrifuged, nd tested in n = 6 t ech occsion over period of 2 dys on three occsions in totl. One technicl replicte per extrct. The concentrtion of the spiked smples is clculted by subtrction of the endogenous ethnol concentrtion from the nlyticl result nd is given in g/l ethnol; from this, the recovery ws clculted nd is presented in prentheses Tomto Tomto-vegetble Crrot Blnk Spike Dy 1, test (95%) (96%) (97%) (96%) (95%) (99%) (95%) (96%) (97%) (97%) (95%) (99%) (96%) (95%) (98%) (96%) (96%) (98%) Dy 1, test (98%) (96%) (96%) (96%) (95%) (96%) (96%) (95%) (93%) (98%) (95%) (98%) (97%) (95%) (97%) (97%) (95%) (98%) Dy 2, test (95%) (95%) (96%) (96%) (97%) (97%) (97%) (97%) (98%) (96%) (96%) (98%) (97%) (97%) (99%) (97%) (96%) (96%) Men, g/l (96%) (96%) (97%) SD, g/l (0.9%) (0.7%) (1.5%) RSD, % See Tble 5. component contins buffer nd the second component lcohol dehydrogense nd NAD. For smple preprtion, kombuch, juices, nd beer were centrifuged nd diluted if necessry. Mesurement is monitored t 340 nm nd is finished within 20 min. Becuse only few steps re necessry to finish smple preprtion nd mesurement, the ssy is especilly useful for technicins with bse-level lbortory experience. The in-house vlidtion included linerity study, estimtion of LOD nd LOQ, selectivity nd inferences, different types of precision, chrcteriztion of trueness, recovery, lot-to-lot comprbility, stbility testing, nd ruggedness testing. Experiments showed excellent linerity nd high precision tht is minly driven by precision of pipettes nd not test kit components. The ssy fulfills ll requirements listed in the AOAC SMPR Due to the low LOQ of 3.3 mg/l ethnol (0.0004% ABV), kombuch nd lcohol-free beer need to be diluted before mesurement, typiclly round 1:20 or 1:50. Therefore, possible mtrix interferences due to, e.g., color or ph re often recovery (%) propnol 2-propnol n-butnol g/l interfernt Figure 5. Interference of other lcohols. Addition of different mounts of 1-propnol, 2-propnol, nd n-butnol (0.015 up to 0.15 g/l) to 0.15 g/l ethnol (n = 1).

11 Lcorn & Hektor: Journl of AOAC Interntionl Vol. 101, No. x, Tble 14. Interfernts sulfite nd cetldehyde t different concentrtions in presence of 0.15 g/l ethnol (n = 1) Probe Interfernt, g/l Mesured in g/l Rec., % Ethnol 0.15 g/l Ethnol 0.15 g/l Ethnol 0.15 g/l + sulfite Ethnol 0.15 g/l + cetldehyde b = Not pplicble. Significnt interferences. g/l ethnol b b 445 b 3 b b 151 b 1.5 b b 125 b 0.6 b b 111 b b b 77 b kombuch beer OD Figure 6. Dilutbility of kombuch nd lcohol-free beer over the whole mesurement rnge. Smples were diluted 1:4 up to 1:150 (kombuch) nd 1:6 up to 1:300 (beer) with wter. not relevnt. In the cse of juices, ethnol concentrtions were low, nd no dilution ws necessry. Nevertheless, precision nd recovery were not ffected. The enzyme contined in the test kit will lso convert other primry liphtic lcohol s, e.g., 1-propnol, n-butnol, nd 1-pentnol, but not methnol. These primry lcohols re only contined t very low levels fter lcoholic fermenttion. Typiclly, concentrtions of higher lcohols re 1000 times lower thn ethnol. Therefore, this side-chin ctivity is not relevnt under prcticl conditions for mesurement of ethnol in kombuch, juices, nd lcoholfree beer. Acetldehyde interferes t concentrtions higher thn 3000 mg/l, wheres sulfite interferes t concentrtions higher thn 300 mg/l. Under prcticl conditions, this is lso not relevnt becuse kombuch, juices, nd beer re not treted with sulfite, nd cetldehyde is never present t 3 g/l fter norml fermenttion process. Trueness ws nlyzed using queous ethnol stndrd reference solutions nd certified reference mteril (lcohol-free beer). A thorough robustness testing included the nlysis of incubtion temperture (18, 25, nd 37 C) nd incubtion time (5, 10, 15, nd 20 min). No prmeter ws found to influence the result in wy tht could be criticl under prcticl conditions. Kombuch nd beer smples re dilutble. Cre should be tken when lcoholcontining smple re diluted, becuse the volume of smple used for dilution is criticl. It is strongly recommended to use 100 μl t minimum for dilution. At lower volumes (e.g., 20 μl), results will turn out to be less precise. The test kit is stble for 2 weeks t 37 C nd until now rel time stbility of 12 months is covered. Conclusions In summry, the dt of the in-house vlidtion study proved tht the performnce clims for kombuch re fulfilled nd re in ccordnce with AOAC SMPR Acknowledgments We would like to thnk Michel Noll, R-Biophrm Ag (Drmstdt, Germny), for performnce of ll lbortory work nd Ptrici Meinhrdt R-Biophrm Inc. (Wshington, MO), for proofreding nd ll her prcticl experience. References (1) Greenwlt, C.J., Steinkrus, K.H., & Ledford, R.A. (2000) J. Food Prot. 63, (2) Orgnistion Interntionl de l Vigne et du Vin (OIV), Compendium of Interntionl Methods of Anlysis, OIV-MA-AS312-01A: R2009