Indicate (X) client(s) to whom this final report is submitted. Replace any of these with other relevant clients if required. FINAL REPORT FOR 2011/12

Transcription

1 Finl report 1 CFPA Cnning Fruit Producers Assoc. Sumit to: Wiehhn Victor Tel: +27 (0) inmk@mwe.co.z SAAPPA / SASPA / SAT Fruitgro Science Sumit to: Louise Lieenerg Tel: +27 (0) /1 louise@fruitgro.co.z DFTS Dried Fruit Technicl Services Sumit to: Dppie Smit Tel: +27 (0) dppies@dtd.co.z Winetech Sumit to: Anel Andrg Tel: +27 (0) ndrg@winetech.co.z X Indicte (X) client(s) to whom this finl report is sumitted. Replce ny of these with other relevnt clients if required. FINAL REPORT FOR 2011/12 PROGRAMME & PROJECT LEADER INFORMATION Progrmme leder Project leder Title, initils, surnme Dr F. Vries Mr H.W. du Plessis Present position Acting Progrmme Mnger Microiologist Address ARC Infruitec-Nietvoorij Privte Bg X5026 Stellenosch, 7599 ARC Infruitec-Nietvoorij Privte Bg X5026 Stellenosch, 7599 Tel. / Cell no Fx E-mil vriesf@rc.gric.z dplessishe@rc.gric.z PROJECT INFORMATION Project numer Project title Project Keywords Industry progrmme WW10-22 Effect of non-scchromyces yest on mlolctic fermenttion Lctic cid cteri, yest nd cteri interctions, wine qulity CFPA Deciduous DFTS Winetech Microiology Other Fruit kind(s) Wine grpes Strt dte (dd/mm/yyyy) 01/04/2009 End dte (dd/mm/yyyy) 31/03/2012

2 Finl report 2 FINAL REPORT 1. Executive summry This project showed tht there is lot of vrition mong non-scchromyces yests in terms of their fermenttion ilities, s well s their impct on MLF. This vried etween species, ut lso etween strins within the sme species. During the 2010-hrvest, three non-scchromyces strins, i.e. Kloecker picult, Schizoscchromyces pome nd Torulspor delrueckii were used in the production of Chenin lnc nd Pinotge wines. Non-Scchromyces fermented wines completed MLF fster thn wines fermented with Scchromyces cerevisie only. Sensory dt showed tht Chenin lnc wines not hving undergone MLF were preferred nd scored higher for rom intensity nd overll qulity. Pinotge wines not hving undergone MLF scored higher for rom intensity nd fruity rom, ut Pinotge wines hving undergone MLF hd more ody nd scored higher for overll qulity. During the 2011-hrvest, two commercil T. delrueckii strins were evluted in Chenin lnc nd Pinotge. Mlolctic fermenttion ws completed fter three dys in ll of the Pinotge wines. Due to sluggish lcoholic fermenttions of the non- Scchromyces yests, the Chenin lnc wines underwent spontneous MLF. These results showed tht non-scchromyces yests hve positive effect on MLF, ut it my increse the occurrence of spontneous MLF, especilly in sluggish fermenttions. Pinotge wines produced with non-scchromyces yests were considered to e etter thn wines produced with Scchromyces yest only. Depending on species nd strin selection, non- Scchromyces yests hve positive effect MLF nd wine qulity. 2. Prolem identifiction nd ojectives Successful induction nd completion of mlolctic fermenttion (MLF) is n ongoing prolem worldwide, despite the considerle ody of knowledge concerning wine properties, fctors ffecting lctic cid cteri (LAB) nd developments in strin selection nd strter cultures. The dvent of the use of non-scchromyces yests in wine production constitutes nother unknown fctor for mlolctic fermenttion. To dte the interction etween wild nd industril yests nd lctic cid cteri hs received little ttention. The ojective of this project is therefore to investigte the interction etween non-scchromyces yests nd LAB, nd susequent effects on MLF nd wine qulity. 3. Workpln (mterils & methods) Screening of non-scchromyces yests nd O. oeni This procedure is sed on the screening technique reported y Krieger nd Arnink (2003). Frozen Chrdonny juice from 2009 ws fermented with different commercil Scchromyces cerevisie, selective non-scchromyces yest nd LAB strins listed in Tle 1. At the end of AF, filter disk ws impregnted with the wine fermented with the selected yests strins nd plced on MRS grpe juice gr (MRS supplemented with 25% ( v / v) grpe juice), on which specific commercil LAB strins i.e. Viniflor oenos, Viniflor CH16, Enoferm lph nd Lctoenos SB3 ws lwned. MRS pltes were incuted t 30 C until growth ws oserved.

3 Finl report 3 Tle 1. Yests nd cteri strins used in this study. Species Strin Source Scchromyces cerevisie N 96* Anchor Yest Scchromyces cerevisie VIN 13* Anchor Yest Scchromyces cerevisie NT 202 Anchor Yest Scchromyces cerevisie NT 112 Anchor Yest Scchromyces cerevisie QA23 Lllemnd Inc. Scchromyces cerevisie BM45 Lllemnd Inc. Scchromyces cerevisie BM4X4 Lllemnd Inc. Scchromyces cerevisie EC1118 Lllemnd Inc. Scchromyces cerevisie FX10 Lffort Cndid pulcherrim Y0838 ARC Infruitec-Nietvoorij genenk Cndid pulcherrim Y0839 ARC Infruitec-Nietvoorij genenk Cndid pulcherrim Y0842 ARC Infruitec-Nietvoorij genenk Cndid pulcherrim Y0857 ARC Infruitec-Nietvoorij genenk Cndid pulcherrim Y0861 ARC Infruitec-Nietvoorij genenk Cndid zemplinin Y0841 ARC Infruitec-Nietvoorij genenk Cndid zemplinin Y0844 ARC Infruitec-Nietvoorij genenk Cndid zemplinin Y0849 ARC Infruitec-Nietvoorij genenk Cndid zemplinin Y0872 ARC Infruitec-Nietvoorij genenk Cndid zemplinin Y0879 ARC Infruitec-Nietvoorij genenk Kloecker picult Y0840 ARC Infruitec-Nietvoorij genenk Kloecker picult Y0845 ARC Infruitec-Nietvoorij genenk Kloecker picult Y0846 ARC Infruitec-Nietvoorij genenk Kloecker picult Y0858 ARC Infruitec-Nietvoorij genenk Kloecker picult Y0860 ARC Infruitec-Nietvoorij genenk Schizoscchromyces pome Y0197 ARC Infruitec-Nietvoorij genenk Torulspor delrueckii Y0163 ARC Infruitec-Nietvoorij genenk Torulspor delrueckii M2/1 ARC Infruitec-Nietvoorij culture collection Torulspor delrueckii M2/15 ARC Infruitec-Nietvoorij culture collection Torulspor delrueckii M2/27 ARC Infruitec-Nietvoorij culture collection Torulspor delrueckii Hrmony Chr. Hnsen A/S Torulspor delrueckii Level 2 Td Lllemnd Inc. Torulspor delrueckii 301 ARC Infruitec-Nietvoorij culture collection Torulspor delrueckii 654 ARC Infruitec-Nietvoorij culture collection Oenococcus oeni** Viniflor oenos Chr. Hnsen A/S Oenococcus oeni Viniflor CH16 Chr Hnsen A/S Oenococcus oeni Enoferm lph Lllemnd Inc Oenococcus oeni Lctoenos SB3 Lffort *Reference yests. **Commercil lctic cid cteri strins.

4 Finl report 4 Evlution of commercil MLF strins A synthetic (chemiclly defined) medium used y Costello et l. (2003) ws used to study Scchromyces yest nd LAB comptiility. Five hundred millilitres liquots of the synthetic juice (SJ) were fermented to dryness y S. cerevisie strins NT 202, NT 112, EC 1118 nd FX10. After lcoholic fermenttion (AF), 100 ml liquots were divided into sterile 250 ml ottles nd inoculted with commercil MLF strter cultures (Tle 1). Both, AF nd MLF were conducted t c. 22 C. All commercil cultures were used s prescried y the suppliers. Progress of MLF in the synthetic wine (SW) ws monitored using pper chromtogrphy (Kunkee 1968). Lortory-scle wine production trils The SJ medium of Costello et l. (2003) ws used to study Scchromyces yest nd LAB comptiility. The following components were excluded from the SJ: D-erythritol, L- rhmnose, D-ritol, D-mnnose, Zncl 2, Co(NO 3) 2.6H 2O, N 2MoO 4.2H 2O, denine sulphte, gunine, urcil, xnthine, cytosine, choline chloride nd colmine. Yest strins used in this study were grown in Yest Peptone Dextrose (YPD) roth t 30 C prior to inocultion. Two hundred nd fifty millilitres of the SJ were inoculted with pure cultures of the different non-scchromyces strins nd fermented to dryness or for 33 dys. Alcoholic fermenttion (AF) ws crried out in duplicte. After AF, the SW ws sterile filtered through 0.22 µm filter nd 100 ml ws liquoted into sterilised 250 ml ottles for the three different tretments. The three tretments were: (1) ddition of LAB, (2) ddition of nutrients nd LAB, nd (3) the reduction of inhiitory compounds in SW, prior to the ddition of LAB. Tretments 1 nd 2 were performed in duplicte. Oenococcus oeni strin, V. oenos ws used to induce MLF. V. oenos ws inoculted s prescried y supplier. The ethnol concentrtion of the SW ws djusted to 10% ( v / v) prior to ddition of V. oenos. The screening of non- Scchromyces yests in SJ ws conducted t ±21 C. The Ripper method ws used to determine free nd totl sulphur dioxide (SO 2) nd lcohol concentrtion ws determined y Koelenhof Wine Cellr Lortory. Smll-scle wine production trils of 2010 (1 st vintge) Grpes were otined from the Nietvoorij Reserch Frm nd the nlysis of the juice fter crushing nd pressing re listed in Tle 2. Kloecker picult (Y0858), Schizoscchromyces pome (Y0197) nd Torulspor delrueckii (M2/15) were used in smll-scle (20 L) Chenin lnc (14 C) nd Pinotge (23 C) wine production trils during the 2010-hrvest. S. cerevisie strin, N 96 ws used s the reference yest. V. oenos inoculted t the end of AF to induce MLF. Two tretments were pplied i.e. tretment 1: non-scchromyces yest without undergoing MLF nd tretment 2: non-scchromyces yest with MLF. All fermenttions were conducted in duplicte. Juice smples were nlysed (ph, totl cidity, sugr, free nd totl SO 2) in the Nietvoorij cellr lortory using stndrd techniques (Ilnd et l., 2000). The progress of MLF ws monitored using pper chromtogrphy nd Wine Scn nlyses (IWBT, Stellenosch University). Tle 2. Chemicl nlyses of Chenin lnc nd Pinotge juice of the 2010-hrvest. Cultivr Sugr ( B) ph Totl cid Free SO 2 (mg/l) Totl SO 2 (mg/l) Chenin lnc Pinotge

5 Finl report 5 Sensory evlution nd chemicl nlyses of 2010 wines The wines were sensorilly evluted y descriptive nlyses during the nnul experimentl wine evlutions t Nietvoorij. The pnels consisted of commercil winemkers (7) nd Nietvoorij stff (2). A ten centimetre unstructured line scle ws used nd the judges were sked to score wines for the different rom nd tste descriptors, s well s overll qulity. The wines were coded nd presented in rndom order. The concentrtions of the voltile compounds in the wines were mesured using Gs chromtogrphy with flme ioniztion detector (GC-FID) (Anlyticl Lortory, Distell, Stellenosch). Smll-scle wine production trils of 2011 (2 nd vintge) Torulspor delrueckii strins isolted from commercil yest cultures, i.e. Hrmony (Chr. Hnsen) nd Level 2 Td (Lllemnd), were used in smll-scle (20 L) Chenin lnc (14 C) nd Pinotge (23 C) wine production trils during the 2011-hrvest. VIN 13 ws used s the reference yest. Two tretments were pplied, i.e. tretment 1: yest without undergoing MLF nd tretment 2: yest with MLF. V. oenos ws inoculted t the end of the lcoholic fermenttion (AF) in the Pinotge wines. All fermenttions were conducted in duplicte. Juice smples were nlysed (ph, totl cidity, sugr, free nd totl SO 2) in the Nietvoorij Cellr Lortory using stndrd techniques. The totl nitrogen content of the juice ws determined y Vinl (Pty) Ltd. The progress of MLF ws monitored using pper chromtogrphy nd WineScn nlyses (IWBT). Tle 3. Chemicl nlyses of Chenin lnc nd Pinotge juice of the 2011-hrvest. Cultivr Sugr (ºB) ph Totl cidity Free SO 2 (mg/l) Totl SO 2 (mg/l) YAN (mg/l) FAN (mg/l) NH 4 (mg/l) Chenin lnc Pinotge Sensory evlution nd chemicl nlyses of 2011 wines The wines were sensorilly evluted y descriptive nlyses during the nnul experimentl wine evlutions t Nietvoorij. The pnels consisted of commercil winemkers (2) nd Nietvoorij stff (10). ). A ten centimetre unstructured line scle ws used nd the judges were sked to score wines for the different rom nd tste descriptors, s well s overll qulity. The wines were coded nd presented in rndom order. The concentrtions of the voltile compounds in the wines were mesured using Gs chromtogrphy with flme ioniztion detector (GC-FID) (Anlyticl Lortory, Distell).

6 Finl report 6 4. Results nd discussion Milestone 1. Literture study Achievement Completed. 2. Screening of non-scchromyces yests nd O. oeni Completed. Plte ssy needs optimiztion. 3. Evlution of commercil LAB strins Completed. E. lph nd CH16 fster nd less sensitive MLF cultures thn V. oenos nd Lctoenos. 4. Lortory-scle wine production trils Completed. Cndid zemplinin isoltes lest inhiitory effect on MLF. Torulspor delrueckii isoltes showed good fermenttion ilities. 5. Smll-scle wine production trils of 2010 (1 st vintge) Completed. 14 Chenin lnc nd 14 Pinotge wines produced with three different 6. Chemicl nd sensory nlyses of 2010 vintge wines 7. Smll-scle wine production trils of 2011 (2 nd vintge) 8. Chemicl nd sensory nlyses of wines 2011 vintge wines non-scchromyces yests. Completed. Non-Scchromyces wines completed MLF fster, nd were preferred to reference Scchromyces wines. Completed. 12 Chenin lnc nd 12 Pintotge wines produced with two commercil T. delrueckii yests. Completed. T. delrueckii wines hving undergone MLF preferred to Scchromyces reference wines. Milestone 1: Literture study A literture serch ws performed to find relevnt nd relted informtion out the effect non-scchromyces yests hve on the growth of LAB. Non-Scchromyces yests cn produce compounds such s cetldehyde, cetic cid, esters, glycerol, sulphur dioxide, hydrogen sulphide nd other y-products (Romno et l., 1997, 2003; Cini et l., 2010), which my e inhiiting or stimulting to LAB. Non-Scchromyces yests cn secrete pectinses, proteses nd glycosidses (Fernández et l., 2000; Cini et l., 2010). Enzymtic hydrolysis of the proteins into smller more solule nitrogen-contining molecules (peptides nd mino cids) could stimulte LAB growth. Terpenes nd other voltile compounds such s stright-chin nd cyclic lcohols, to nme just few, help improve wine rom. These voltile odorous compounds re present in free form in the grpes or re minly ind to sugr to form odourless non-voltile glycosidic complexes. β-glucosidse cleves off rom precursors ound to the sugr molecules contriuting to wine rom. These sugr molecules cn e utilised y LAB. Wine mcro-molecules such s polyscchrides nd mnnoproteins cn lso ffect LAB growth nd Diez et l. (2012) found tht mnnoproteins could stimulte LAB growth. Some non-scchromyces yest such s Hnsenul noml (Pichi noml), Schizoscchromyces pome, Zygoscchromyces ilii nd Isstchenki orientlis cn degrde mlic cid (Symn & Viljoen-Bloom 2006; Seo et l., 2007; Cini et l., 2010), which will ffect LAB growth nd MLF. During the eginning of fermenttion, these yests my deplete the nutrients found in wine. These deficiencies, comined with toxic metolites, cn inhiit the growth of LAB (Riéreu-Gyon et l., 2006; Costello et l., 2003). It hs lso een reported tht the growth of ntive yest species, such s Scchromycodes ludwigii, Cndid pulcherrim nd Pichi spp., is ntgonistic to the growth of LAB (Fornchon, 1968). However, in the lst mentioned study only six non-scchromyces yest strins were

7 Finl report 7 used nd their effect on one Lctocillus revis strin, one Lctocillus hilgrdii strin nd two Leuconostoc mesenteroides strins were investigted. Mendoz & Frís (2010) evluted the use of S. cerevisie nd K. picult in mixed fermenttions with O. oeni. They found tht the yest/lab interction did not ffect MLF nd tht sequentil inocultion of O. oeni llowed etter control on the sensory qulity of the wine. Wine yest my exert rnge of effects upon LAB, including inhiition, stimultion, or neutrl effect. The interctions etween Scchromyces cerevisie nd O. oeni hve een studied in greter detil, nd the sme rules should pply to non-scchromyces yests. It is cler, however, tht the interction etween Scchromyces/non-Scchromyces/LAB is complex nd tht there re mny fctors tht ply role. Milestone 2: Screening of non-scchromyces yests nd O. oeni A simple screening technique to determine yest nd LAB comptiility ws evluted. If the yest nd LAB strin ws not comptile cler zone of no growth would e visile round the filter disk. If the yest nd LAB ws comptile no zone would e visile round the filter disk. The results of the plte screening technique were inconclusive ecuse ll yest nd LAB comintion ppered to e comptile, including Scchromyces reference strins previously reported to e incomptile (S. Krieger-Weer, personl communiction). Milestone 3: Evlution of commercil LAB strins The im of this experiment ws to determine the effect of S. cerevisie strins, NT 202, NT 112, EC 1118 nd FX10, hve on sequentil MLF in SW. These commercil yests were used ecuse it is known wht effects they hve on LAB nd MLF in wine. Mlolctic fermenttion took longer to complete in the SW fermented with NT 112, thn with the other yests (Tle 4). Mlolctic fermenttion completed the fstest in SW fermented with NT 202. Enoferm lph completed MLF the fstest nd ws comptile with ll the yest strins. This tril showed tht LAB strins, E. lph nd CH16 were less ffected y yest selection thn V. oenos nd Lctoenos nd tht the wrong yest nd LAB selection could dely MLF. Tle 4. Durtion of sequentil mlolctic fermenttion (MLF) in synthetic wine fermented with different commercil yest strins. Tretment MLF (dys) NT Viniflor CH16 7 NT Viniflor oenos 20 NT Enoferm lph 7 NT Lctoenos SB3 20 NT Viniflor CH16 25 NT Viniflor oenos 32 NT Enoferm lph 25 NT Lctoenos SB3 41 EC Viniflor CH16 41 EC Viniflor oenos 20 EC Enoferm lph 20 EC Lctoenos SB3 20 FX10 + Viniflor CH16 20 FX10 + Viniflor oenos 25 FX10 + Enoferm lph 20 FX10 + Lctoenos SB3 25

8 Finl report 8 Milestone 4: Lortory-scle wine production trils The im of this tril ws to determine the effect non-scchromyces yests hve on O. oeni strin, V. oenos nd MLF in synthetic grpe juice. This medium ws chosen ecuse it could help to determine if the different yest strins stimulted or inhiited LAB growth nd MLF. If yest inhiited LAB growth nd MLF, it could e determined whether inhiition ws due to nutrient depletion or yest derived compounds. Progress of AF (mesured in CO 2 weight loss) of the different non-scchromyces strins in SJ, re shown in Fig. 1. Some non-scchromyces yest ppered to e strong fermenters, wheres others ppered to e wek. However, none of the non-scchromyces yests fermented to dryness nd few of the SW smples contined high residul sugr concentrtions t the end of AF (Tle 5). There were cler differences in the ility of the different non-scchromyces yests to produce SO 2, with Kloecker picult Y0840 nd Torulspor delrueckii M2/15 producing the highest totl SO 2 levels. There ws less vrition in the lcohol levels for the Torulspor delrueckii strins thn for the other non- Scchromyces strins, with the Cndid strins showing the most vrition nd the highest residul sugr vlues. Mny of the non-scchromyces yests produced reltively high lcohol concentrtions CO 2 mss loss (g) Time (dys) Y0163 Y0841 Y0858 M2/15 M2/1 Y0838 Y0857 Y0839 M2/27 Y0840 Y0849 Y0872 Hrmony Y0846 Y0860 N Y0861 Y0844 Y0845 Y0842 Y0879 Y0197 Figure 1. Progress of lcoholic fermenttion in synthetic grpe juice fermented with different non-scchromyces yests. The chemicl nlyses nd durtion of MLF of the SW re listed in Tles 5 nd 6, respectively. The different non-scchromyces strins hd definite effect on the progress of MLF (Tle 6). In this synthetic medium, most of the non-scchromyces strins hd n inhiitory effect on MLF. Only few non-scchromyces strins completed MLF for ll three tretments, with Cndid zemplinin Y0844 performing the est. The only other non- Scchromyces strins where MLF ws completed for ll three tretments were two Cndid zemplinin strins, Y0849 nd Y0872, nd Kloecker picult strin, Y0860. All three tretments of the S. cerevisie reference yest, N 96 completed MLF. It is cler from tretments 2 nd 3 tht the inhiition of MLF y some strins ws due to nutrient depletion or production of inhiitory components/metolites.

9 Finl report 9 Tle 5. Chemicl nlyses of the synthetic wine t the end of lcoholic fermenttion. Species Strin Free SO 2 (mg/l) Totl SO 2 (mg/l) Alcohol (% v / v) Residul sugr Scchromyces cerevisie N Cndid pulcherrim Y Cndid pulcherrim Y Cndid pulcherrim Y Cndid pulcherrim Y Cndid pulcherrim Y Cndid zemplinin Y Cndid zemplinin Y Cndid zemplinin Y Cndid zemplinin Y Cndid zemplinin Y Kloecker picult Y Kloecker picult Y Kloecker picult Y Kloecker picult Y Kloecker picult Y Schizoscchromyces pome Y Torulspor delrueckii Y Torulspor delrueckii M2/ Torulspor delrueckii M2/ Torulspor delrueckii M2/ Torulspor delrueckii Hrmony Torulspor delrueckii Torulspor delrueckii Averge vlues of the duplicte smples. Reference yest. Milestone 5: Smll-scle wine production trils of 2011 (1 st vintge) Three non-scchromyces nd one Scchromyces reference strin were used to produce Chenin lnc nd Pinotge wines. Chenin lnc nd Pinotge wine nlyses t the end of AF re listed in Tles 7 nd 8. All the Chenin lnc wines fermented to dryness (Tle 7), ut the Pinotge wines still contined some residul sugr (Tle 8) t the end of AF. In oth cultivrs, the indigenous Scchromyces popultion completed the AF (dt not shown). The wines fermented with Sc. pome ll underwent prtil MLF t the end of AF (Tles 7 nd 8).

10 Finl report 10 Tle 6. Durtion of sequentil mlolctic fermenttion (MLF) in synthetic wine fermented y different yest strins. Species Strin MLF durtion (dys) Tretment 1 Tretment 2 Tretment 3 c Scchromyces cerevisie d N Cndid pulcherrim Y0838 >68 39 >68 Cndid pulcherrim Y0839 (42 - >68) (22 - >68) >68 Cndid pulcherrim Y0842 (8 - >68) >68 6 Cndid pulcherrim Y0857 > >68 >68 Cndid pulcherrim Y > Cndid zemplinin Y0841 (42 - >68) (17 - >68) >68 Cndid zemplinin Y Cndid zemplinin Y Cndid zemplinin Y Cndid zemplinin Y >68 Kloecker picult Y0840 (42 - >68) (42 - >68) 50 Kloecker picult Y0845 MLF completed prior to inocultion Kloecker picult Y0846 (42 - >68) > Kloecker picult Y0858 >68 >68 Not tested Kloecker picult Y Torulspor delrueckii M2/15 >68 >68 >68 Torulspor delrueckii M2/1 42 (8 - >68) >68 Torulspor delrueckii M2/27 >68 >68 >68 Torulspor delrueckii Y0163 >68 >68 >68 Torulspor delrueckii Hrmony (65 - >68) (58 - >68) >68 Torulspor delrueckii 301 (35 - >68) 40 >68 Torulspor delrueckii >68 Schizoscchromyces pome Y0197 MLF completed prior to inocultion Tretment 1: ddition of Viniflor oenos (commercil MLF culture). Tretment 2: nutrient supplementtion nd ddition of V. oenos. c Tretment 3: detoxifiction of the fermented synthetic grpe juice prior to ddition of V. oenos. d Reference yest. Tle 7. Chemicl nlyses of the 2010 Chenin lnc wines t the end of lcoholic fermenttion. Tretment ph Voltile cidity Totl cidity Mlic cid Lctic cid Alcohol (% v / v) Glycerol Residul sugr N96 + MLF < Y < Y < M2/ < Y MLF < Y MLF < M2/15 + MLF < Vlues in tle re verges of the duplictes smples. Reference yest.

11 Finl report 11 Tle 8. Chemicl nlyses of 2010 Pinotge wines t the end of lcoholic fermenttion. Tretment ph Voltile cidity Totl cidy Mlic cid Lctic cid Alcohol (% v / v) Glycerol Residul sugr N96 + MLF Y Y M2/ < Y MLF Y MLF < M2/15 + MLF Vlues in tle re verges of the duplictes smples. Reference yest. All the tretments completed MLF successfully, with the Sc. pome wines finishing MLF the quickest nd the reference wines (N 96) tking the longest to complete (Tles 9 nd 10). In the Chenin lnc wines, higher LAB numers resulted in fster MLF. The LAB numers in the Pinotge wines showed more vrition, ut there is no cler correltion etween LAB numers nd durtion of MLF. This tril showed tht non-scchromyces fermented wines completed MLF fster thn wines fermented with N 96 (Scchromyces reference yest). The non-scchromyces yests did not inhiit MLF s expected from dt of the SW tril. Mlolctic fermenttion ctully completed quickly nd without ny prolems, indicting tht the compounds tht were excluded from the SJ medium were proly essentil to LAB growth, which explins the slow MLF progress in the SW. Tle 9. Vile lctic cid cteri (LAB) count, durtion of mlolctic fermenttion nd chemicl nlyses of 2010 Chenin lnc wines fermented with different yests. Tretment LAB count (cfu/ml) MLF (dys) ph Voltile cidity Totl cidity Alcohol (% v / v) Glycerol N 96 + MLF 1.5E Y MLF 1.8E Y MLF 1.9E M2/15 + MLF 1.7E Vlues in tle re verges of the duplictes smples. Reference yest. Tle 10. Vile lctic cid cteri (LAB) count, durtion of mlolctic fermenttion nd chemicl nlyses of Pinotge wines fermented with different yests. Tretment LAB count (cfu/ml) MLF (dys) ph Voltile cidity Totl cidity Alcohol (% v / v) Glycerol N 96 + MLF 3.7E Y MLF 1.8E Y MLF 1.8E M2/15 + MLF 2.5E Vlues in tle re verges of the duplictes smples. Reference yest.

12 Finl report 12 Milestone 6: Chemicl nd sensory nlyses of 2010 wines The K. picult tretment without MLF scored the highest for most of the descriptors during the sensory evlution of the Chenin lnc wines nd the S. pome with MLF tretment scored the lowest (Fig. 2). The Chenin lnc wines hving undergone MLF scored lower for rom intensity nd overll qulity thn the tretments tht did not undergo MLF. However, the wines tht underwent MLF hd more ody thn the wines tht did not undergo MLF. Descriptive sensory nlyses of the Pinotge wines indicted tht there ws no or little difference etween the tretments (Fig. 3). The T. delrueckii tretment without MLF scored the highest for most of the descriptors nd N 96 tretment scored the lowest. Pinotge wines hving undergone MLF were less fruity thn wines not hving undergone MLF, ut scored higher for vegettive rom, ody nd overll qulity. The sensory dt showed tht MLF negted the romtic contriution of non-scchromyces yests in Chenin lnc wines, ut enhnced the mouth-feel nd generl qulity of Pinotge wines produced with non- Scchromyces yests. Gs chromtogrphic nlysis of the Chenin lnc wines reveled tht the K. picult tretments, with nd without MLF, hd noticele higher ethyl cette levels thn the other tretments (Tle 11). The K. picult tretment with no MLF lso contined slightly higher levels of the romtic esters, iso-myl cette nd hexyl cette. The concentrtions of the other esters did not differ or differed only slightly nd were therefore not included. None of the tretments contined high methnol levels (Tle 12). The Sc. pome tretments contined slightly lower levels of most of the higher lcohols thn the other tretments, except for iso-utnol, which ws slightly higher thn the other tretments. Wines fermented with N 96 contined higher voltile cidity levels thn wines produced with the non- Scchromyces yests. The concentrtion of hexnoic nd octnoic cid ws lso higher in the N 96 wines (Tle 13). Figure 2. Descriptive sensory nlyses of Chenin lnc wines of 2010.

13 Finl report 13 Figure 3. Descriptive sensory nlyses of Pinotge wines of Tle 11. Ester concentrtions (mg/l) in Chenin lnc wines of Tretment Ethyl cette iso- Amyl cette Hexyl cette Ethyl lctte Ethyl cprylte Di-ethyl succinte N 96 + MLF K c Y Sp d Y Td e M2/ K Y MLF Sp Y MLF Td M2/15 + MLF Vlues in tle re verges of two replictes. Reference yest, c Kloecker picult, d Schizoscchromyces pome nd e Torulspor delrueckii. Tle 12. Higher lcohol concentrtions (mg/l) in Chenin lnc wines of Tretment Methnol n- iso- n- iso-amyl 2-Phenyl Propnol Butnol Butnol lcohol ethnol N 96 + MLF K c Y Sp d Y Td e M2/ K Y MLF Sp Y MLF Td M2/15 + MLF Vlues in tle re verges of two replictes. Reference yest, c Kloecker picult, d Schizoscchromyces pome nd e Torulspor delrueckii.

14 Finl report 14 Tle 13. Voltile cid concentrtions (mg/l) in Chenin lnc wines of Tretment Acetic Propionic Hexnoic Octnoic Decnoic n-butyric cid cid cid cid cid cid N 96 + MLF K c Y Sp d Y Td e M2/ K Y MLF Sp Y MLF Td M2/15 + MLF Vlues in tle re verges of two replictes. Reference yest, c Kloecker picult, d Schizoscchromyces pome nd e Torulspor delrueckii. The GC dt of the Pinotge wines confirmed the sensory evlution results, with only few compounds differing for the vrious tretments. These compounds re listed in Tles 14 nd 15. Compounds not included in Tles 14 nd 15 did not differ much. As oserved in the Chenin lnc wines, the K. picult tretments contined higher ethyl cette levels. As expected, ethyl lctte nd cetic cid levels were noticely higher in the wines hving undergone MLF (Tle 14). N 96 wines contined higher iso-vleric cid levels thn the other wines. There were cler differences in the levels of higher lcohols for the vrious tretments. The N 96 wines contined the lowest methnol nd the highest propnol levels. The K. picult tretments lso contined higher iso-utnol levels thn the other tretments. Tle 14. Concentrtions of voltile compounds (mg/l) in Pinotge wines of Tretment Ethyl cette iso- Amyl cette Ethyl lctte Acetoin Acetic cid iso- Vleric cid N 96 + MLF K c Y Sp d Y Td e M2/ K Y MLF Sp Y MLF Td M2/15 + MLF Vlues in tle re verges of two replictes. Reference yest, c Kloecker picult, d Schizoscchromyces pome nd e Torulspor delrueckii. Tle 15. Higher lcohol concentrtions (mg/l) in Pinotge wines of Tretment Methnol n-propnol iso- iso-amyl 2-Phenyl Butnol lcohol ethnol N 96 + MLF K c Y Sp d Y Td e M2/ K Y MLF Sp Y MLF Td M2/15 + MLF Vlues in tle re verges of two replictes. Reference yest, c Kloecker picult, d Schizoscchromyces pome nd e Torulspor delrueckii.

15 Finl report 15 Milestone 7: Smll-scle wine production trils of 2011 (2 nd vintge) Purified T. delrueckii isoltes from commercil cultures, Hrmony nd Level 2 Td, were evluted in Chenin lnc nd Pinotge during the 2011-hrvest. S. cerevisie, VIN 13 ws used s reference yest. The nitrogen level of the Chenin lnc ws lower thn tht of the Pinotge juice (Tle 3). The initil SO 2 concentrtion ws very low for oth cultivrs nd no SO 2 ws dded. All the tretments of the Pinotge wines fermented to dryness nd the nlyses of the wines fter AF re listed in Tle 16. The nlyses t the end of AF were very similr, with one or two tretments showing slight vrition. Mlolctic fermenttion completed quickly nd ll the tretments were finished fter three dys (Tle 17). The chemicl nlyses of the wines fter MLF were lso very similr. Tle 16. Chemicl nlyses of 2011 Pinotge wines t the end of lcoholic fermenttion. Tretment ph Voltile cidity Totl cidity Mlic cid Glucose Fructose Alcohol (% v / v) Glycerol VIN Hrmony c Level 2 Td * Vlues in tle re verges of two replictes. Reference yest c Isolte from commercil Torulspor delrueckii culture of Chr. Hnsen A/S. *Isolte from commercil Torulspor delrueckii culture of Lllemnd Inc. Tle 17. Durtion of mlolctic fermenttion (MLF) nd chemicl nlyses of 2011 Pinotge wines fermented with different yests. Tretment Durtion of MLF (dys) ph Voltile cidity Totl cidity Alcohol (% v / v) Glycerol VIN Hrmony c Level 2 Td * Vlues in tle re verges of two replictes. Reference yest *Isolte from commercil Torulspor delrueckii culture of Lllemnd Inc. Chenin lnc juice inoculted with the different T. delrueckii strins struggled with AF t 14ºC nd only the VIN 13 tretment fermented to dryness. As result the temperture ws incresed to 20ºC. The fermenttions remined sluggish nd on completion still contined some residul sugr, most of which ws fructose (Tle 18). The AF of the T. delrueckii tretments took etween dys to complete (glucose levels) nd during this time spontneous MLF occurred in most of the smples, including the tretments tht were not supposed to undergo MLF. Level 2 Td wines completed MLF fster thn the other tretments, showing tht there re some strin differences. The only tretments tht did not undergo MLF were the VIN 13 tretments (reference strin). After 80 dys, MLF did not even strt in the VIN 13 tretment tht ws inoculted for MLF (Tle 19). Two different commercil MLF strters, V. oenos nd Enoferm lph, were used to induce MLF, without success. This tril confirmed tht non-scchromyces yests hve positive effect on the occurrence of MLF.

16 Finl report 16 Tle 18. Chemicl nlyses of 2011 Chenin lnc wines t the end of lcoholic fermenttion. Tretment ph Voltile cidity Mlic cid Glucose Fructose Alcohol (% v / v) VIN < Hrmony c Level 2 Td * < VIN 13 + MLF d < Hrmony + MLF d Level 2 Td + MLF d < Vlues in tle re verges of two replictes. Reference yest c Isolte from commercil Torulspor delrueckii culture of Chr. Hnsen A/S. *Isolte from commercil Torulspor delrueckii culture of Lllemnd Inc. d Spontneous MLF Tle 19. Durtion of spontneous mlolctic fermenttion (MLF) in Chenin lnc wines of 2011 fermented with different yests. Tretment Durtion of MLF (dys) VIN 13 >80 Hrmony c 55 d Level 2 Td * 53 d Vlues in tle re verges of four replictes. Reference yest c Isolte from commercil Torulspor delrueckii culture of Chr. Hnsen A/S. *Isolte from commercil Torulspor delrueckii culture of Lllemnd Inc. d Spontneous MLF Milestone 8: Sensory evlution nd chemicl nlyses of 2011 wines Results of sensory evlution of the Pinotge wines re shown in Figure 4. Pinotge wines hving undergone MLF scored higher thn the wines not hving undergone MLF. The wines produced with T. delrueckii yests scored higher thn the wines fermented with the Scchromyces yest, VIN 13. Due to the sluggish fermenttion of the Chenin lnc wines nd the difference in residul sugrs, it ws decided to only evlute the rom nd not the tste of the wine. The reference wine, VIN 13 scored the highest for generl rom intensity, tree fruit nd tropicl fruit, wheres the T. delrueckii wines were considered to hve more dried fruit chrcter (Fig. 5). The T. delrueckii wines lso scored higher for other rom, which referred to sweet ssocited roms or in some cses off-flvours. The voltile chemicl composition of the Chenin lnc wines ws not determined.

17 Finl report 17 Figure 4. Descriptive sensory nlyses of Pinotge wines of Figure 5. Descriptive rom nlyses of Chenin lnc wines of 2011.

18 Finl report 18 Results of the GC nlyses of the Pinotge wines re listed in Tles 20 to 22. Except for hexyl cette, Pinotge wines hving undergone MLF contined higher esters levels thn the wines not hving undergone MLF (Tle 20). Tle 20. Concentrtions of esters (mg/l) in Pinotge wines of Tretment Ethyl iso-amyl Ethyl Hexyl 2-Phenylethyl cette cette cprote cette cette VIN Hrmony c Level 2 Td* VIN 13 + MLF Hrmony + MLF Level 2 Td + MLF Vlues in tle re verges of two replictes. Reference yest c Isolte from commercil Torulspor delrueckii culture of Chr. Hnsen A/S. *Isolte from commercil Torulspor delrueckii culture of Lllemnd Inc. Methnol, propnol nd iso-utnol levels re higher in Pinotge wines hving undergone MLF (Tle 21). The different yest strins lso produced vrying levels of higher lcohols. The levels of propnol nd iso-myl lcohol were higher in VIN 13 wines thn the T. delrueckii wines. Tle 21. Higher lcohol concentrtions (mg/l) in Pinotge wines of Tretment Methnol n-propnol iso- iso-amyl Butnol lcohol 2-Phenyl ethnol VIN Hrmony c Level 2 Td * VIN 13 + MLF Hrmony + MLF Level 2 Td + MLF Vlues in tle re verges of two replictes. Reference yest c Isolte from commercil Torulspor delrueckii culture of Chr. Hnsen A/S. *Isolte from commercil Torulspor delrueckii culture of Lllemnd Inc. Pinotge wines hving undergone MLF contined higher cetic cid nd iso-vleric cid levels thn wines not hving undergone MLF (Tle 22). None of the yests produced high cetic cid levels. The different yest strins produced vrying levels of voltile cids. VIN 13 produced the higher levels of propionic cid thn the T. delrueckii yests.

19 Finl report 19 Tle 22. Voltile cid concentrtions (mg/l) in Pinotge wines of Tretment Acetic cid Propionic cid iso- Vleric cid Hexnoic cid Octnoic cid Decnoic cid VIN Hrmony c Level 2 Td * VIN 13 + MLF Hrmony + MLF Level 2 Td + MLF Vlues in tle re verges of two replictes. Reference yest c Isolte from commercil Torulspor delrueckii culture of Chr. Hnsen A/S. *Isolte from commercil Torulspor delrueckii culture of Lllemnd Inc. Discussion Results showed tht there is lot of vrition mong non-scchromyces yests in terms of their fermenttion ilities, s well s their impct on MLF. This vried etween species, ut lso etween strins within the sme species. Some of the non-scchromyces yest strin showed lot of potentil nd will e investigted further, to improve on ville commercil yests. Non-Scchromyces yests hve positive effect on MLF, ut it my increse the occurrence of spontneous MLF, especilly in sluggish fermenttions. Non-Scchromyces wines were considered to e etter nd more complex thn wines fermented with Scchromyces yests only, nd this ws shown with the non-scchromyces isoltes nd commercil strins. Wines produced with non-scchromyces yests, which underwent MLF scored higher for overll qulity nd were generlly etter thn wines not hving undergone MLF. Depending on strin selection, non-scchromyces yests hve positive effect on MLF nd wine qulity. This knowledge should help the industry to llevite some of the MLF prolems nd improve the qulity of South Africn wines. Literture cited Cini, M., Comitini, F., Mnnzzu, I. & Domizio, P., Controlled mixed culture fermenttion: new perspective on the use of non-scchromyces yests in winemking. FEMS Yest Res 10, Costello, P.J., Henschke, P.A. & Mrkides, A.J., Stndrdised methodology for testing mlolctic cteri nd wine yest comptiility. Austrlin Journl of Grpe nd Wine Reserch 9, Diez, L., Gudlupe, Z., Ayestrán, B., & Ruiz-Lrre, F., Effect of yest mnnoproteins nd grpe polyscchrides on the growth of wine lctic cid nd cetic cid cteri. Journl of Agriculturl nd Food Chemistry, 58, Fernández, M., Úed, J.F. & Briones, A.I., Typing of non-scchromyces yests with enzymtic ctivities of interest in wine-mking. Interntionl Journl of Food Microiology 59, Fornchon, J.C.M., Influence of different yests on the growth of lctic cid cteri in wine. Journl of the Science of Food nd Agriculture 19, Ilnd, P.G., Ewrt, A., Sitters, J., Mrkides, A. & Bruer, N., Techniques for chemicl nlysis nd qulity monitoring during winemking. Ptrick Ilnd Promotions, Cmpelltown, Austrli. Krieger, S. & Arnink, K., Mlolctic fermenttion- A review of recent reserch on timing of inocultion nd possile yest-cteri comintions. Proceedings of the 32nd Annul New York Wine Industry Workshop, Genev, NY, 2-4 April pp

20 Finl report 20 Kunkee, R.E., A simplified chromtogrphic procedure for detection of mlolctic fermenttion. Wines nd Vines 49, Mendoz, L. & Frís, M.E., Improvement of wine orgnoleptic chrcteristics y non- Scchromyces yests. Current Reserch, Technology nd Eduction Topics in Applied Microiology nd Microil Biotechnology 2, Riéreu-Gyon, P., Duourdieu, D., Donéche, B. nd Lonvud, A., The Microiology of Wine nd Vinifictions. Hndook of Enology. John Wiley & Sons Ltd. Chichester, Englnd. Romno, P., Fiore, C., Prggio, M., Cruso, M. & Cpece, A., Function of yest species nd strins in wine flvour. Interntionl Journl of Food Microiology 86, Romno, P., Suzzi, G., Domizio, P. & Ftichenti, F., Secondry products formtion s tool for discriminting non-scchromyces wine strins. Antonie vn Leeuwenhoek 71, Symn, M. & Viljoen-Bloom, M., The iochemistry of mlic cid metolism y wine yests - review. South Africn Journl of Enology nd Viticulture 27, Seo, S-H., Rhee, C-H. & Prk, H-D., Degrdtion of mlic cid y Isstchenki orientlis KMBL 5774, n cidophilic yest strin isolted from Koren grpe wine pomce. The Journl of Microiology 45, Accumulted outputs List ALL the outputs from the strt of the project. The yer of ech output must lso e indicted. Technology development, products nd ptents Indicte the commercil potentil of this project (intellectul property rights or commercil product(s)). Severl non-scchromyces yests were evluted nd some hve the potentil to e commercilised. Humn resources development/trining Indicte the numer nd level (e.g. MSc, PhD, post doc) of students/support personnel tht were trined s well s their cost to industry through this project. Add in more lines if necessry. Student level (BSc, MSc, PhD, Post doc) Cost to project (R) 1. Mr H.W. du Plessis (PhD) R103, Mr J.W. Hoff (MSc) R44, Ms V.M. Bred (MTech) R29, Ms S. Podgorski (ND Biotechnology) R6,000 Pulictions (populr, press releses, semi-scientific, scientific) Heinrich du Plessis & Neil Jolly. Impct of non-scchromyces yests on mlolctic fermenttion nd flvour in Chenin lnc wines (Populr sumitted to Winelnds). Heinrich du Plessis & Neil Jolly. Impct of non-scchromyces yests on mlolctic fermenttion nd flvour in Pinotge wines (Populr sumitted to Winelnds). Heinrich du Plessis & Neil Jolly. Wines produced using Torulspor delrueckii yests (Populr to e sumitted to Winelnds). H.W. du Plessis, N.S. Ntushelo nd N.P. Jolly. Impct of non-scchromyces yests on mlolctic fermenttion nd flvour in Chenin lnc nd Pinotge wines. (Scientific to e sumitted to South Africn Journl of Enology nd Viticulture).

21 Finl report 21 Presenttions/ppers delivered Du Plessis, H.W. & Jolly, N.P Effect of non-scchromyces yest on mlolctic fermenttion. 32 nd Conference of the Society of Enology nd Viticulture, Somerset West, South Afric. Poster Du Plessis, H.W., vn Bred, V.M., Hoff, J.W., Ohlson, S. & Jolly, N.P., Effect of non- Scchromyces yests on mlolctic fermenttion nd wine flvour. South Africn Society for Microiology Conference Novemer Poster Du Plessis, H.W Other. Nie-Scchromyces gis en melksuurkterieë interksies in wyn. Rdio Elsenurg. 19 Octoer Rdio tlk

22 Finl report Totl cost summry of project Yer CFPA Deciduous DFTS Winetech THRIP Other TOTAL Totl cost in rel terms for yer /10 146, , ,514 Totl cost in rel terms for yer /11 161, , ,573 Totl cost in rel terms for yer /12 211, , , ,033 Totl cost in rel terms for yer 4 Totl cost in rel terms for yer 5 TOTAL 519, , ,183 1,276,120 Agriculturl Reserch Council, 2014 The content of this document my constitute vlule Intellectul Property nd is confidentil. It my not e red, copied, disclosed or used in ny other mnner y ny person other thn the ddressee(s) nd specificlly not disclosed to nother prty sumitting proposl herein. Unuthorised use, disclosure or copying is strictly prohiited nd unlwful.