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STATUS REPORT: AGRICULTURAL RESEARCH PROJECTS

(Please fill one form per project)

1. Project Details

Title: Alternative use of wine grapes: Utilisation of non-Saccharomyces yeast for the production of Balsamic-styled vinegar.

Project ID:

21.1.1/12/PRS – 02/INF

Report Number

Final Commencement Date:

3 1 0 8 2 0 1 2

d d m m y y y y

Conclusion Date:

3 0 0 9 2 0 1 5

d d m m y y y y

Extension for final report granted (15/12/2015)

Research Institute/Agency:

ARC Infruitec-Nietvoorbij Lead Researcher/ Project Manager:

Dr Neil Jolly

E-mail: jollyn@arc.agric.za

Fax: 021 809 3002

Tel: 021 809 3060

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2. Project focus and outputs

Research Objectives:

An alternative outlet for wine grape producers by developing technology for the production of Balsamic-styled vinegar to overcome problems experienced by the wine industry such as global recession, over-production and anti-alcohol groups.

Research Outputs/Deliverables: Please make a comparison of planned activities and actual work accomplished during the reporting period (please use tables if possible). Any major deviations from the planned work schedule should be delineated and discussed, corrective actions taken or planned.

As agreed the project ran according to the objectives as shown in the comprehensive proposal (and below) and not as shown in the Service Level Agreement. Milestones, due date and progress made on the project.

Milestone

ARC

Financial

year and

due date

Output

2013/14

1. Preliminary literature survey July 2013 Scientific articles obtained. Aspects of acetic acid bacteria and non-Saccharomyces yeast formed part of two National Diploma ‘Work Integrated Learning’ student project reports, and currently an MTech project.

2. Sourcing of acetic acid bacteria (purchases and isolations)

September 2013

Acetic acid bacteria isolated and reference strains purchased from international culture collections, or acquired from the collaborator.

3. Visit by collaborator September 2013

Collaborator from Spain visited in February 2015.

4. Initial screening of non-Saccharomyces yeasts (VA formation)

October 2013

Screening completed and five non-Saccharomyces yeasts selected.

5. Screening of acetic acid bacteria November 2013

Acetic acid bacteria screened in ethanol and sugar environment and 20 strains selected.

6. Laboratory-scale trials (yeast and bacteria combinations)

January 2014

Six laboratory-scale-trials carried out with various yeast and bacteria combinations.

7. Preliminary sensory evaluations February 2014

Tasting sheet formulated. Core panel of tasters convened. Twenty-four commercial vinegars and laboratory-scale vinegars evaluated.

2014/15

8.. Fermentation trials in fresh juice May 2014 Laboratory-scale trials continued in 2014 and 2015 grape juice.

9. Chemical and sensory evaluations September 2014

Commercial and laboratory-scale vinegar continually evaluated.

10. Continuation of laboratory-scale trials December 2014

Laboratory-scale trials completed.

11. Refinements in fermentation process December 2014

Methodology standardised to 6% inoculation strategy.

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2015/16

12. Small-scale production trials May 2015 Pilot-scale production trial commenced in standard grape juice and cooked grape juice.

13. Chemical and sensory evaluation of products

August 2015 Laboratory-scale and pilot-scale vinegar evaluation completed.

14. Final report September 2015

Completed and submitted to DAFF and Winetech. Extension for final report granted (15/12/2015)

Results obtained: Please provide overview of the technical progress during the reporting period and its main results

INTRODUCTION This project started on 1 September 2012 after the service level agreement was signed.

Economic factors and global trends has led to decreased wine consumption and, in some instances, less exports of local wines. This has adversely affected the South African grape producer. In addition, the wine industry is under pressure from anti-alcohol legislation and a perception of a lack in transformation and empowerment in certain sectors of society. Global warming will also lead to some viticultural areas being unable to produce premium quality wines. Over-production is also a constant threat. With aforementioned in mind it is critical that an alternative use is found for wine grapes and that entrepreneurial opportunities are identified. Production of Balsamic-styled vinegar can serve both purposes. Grapes can be channelled to an alternative high-priced product, while necessitating low technological inputs, making it easy for a low capital start-up business.

Traditional Balsamic Vinegar (Aceto Balsamico Tradizionale) is an international regulated name (Italy’s Denominazione di Origine Protetta and the European Union's Protected Designation of Origin) and can only be produced in the Modena and Reggio-Emilia regions in Italy. It differs from normal wine vinegar in that it is a primary product from grape juice and not a by-product or downstream product of wine. Traditionally Balsamic vinegar is made from cooked Trebbiano and/or Lambrusco grape juice by a natural fermentation and acetification process in a solera styled system of barrels. Barrels are traditionally made from Cherry, Oak, Mulberry, Ash and Juniper wood. The production and maturation process can take up to 12 years to complete and are subject to legislative supervision and approval.

Balsamic Vinegar of Modena (Aceto Balsamico di Modena) is a more modern product made on industrial scale that ranges in quality and price, but must be produced in Modena, Italy. This method makes use of a wine vinegar base, with added colourant (caramel), and concentrated grape juice.

A third category is ”Condiments” (Condimento) which can be mixtures of the above two types, made in the traditional way but without legislative supervision and approval, aged for a shorter period of time, or made by producers located outside of the Modena and Reggio Emilia regions.

Traditional Balsamic vinegar production utilises yeasts (specifically non-Saccharomyces yeast) in conjunction with acetic acid bacteria for production and should not be a wine vinegar sweetened with grape juice. The initial yeast fermentation is critical to the formation of flavour compounds and complex volatile acids other than acetic acid, before the acetic acid bacteria complete the production process. Research has shown that a succession of various microorganism species during production is desirable. Balsamic Vinegars of Modena available in South African supermarkets all originate in Italy. These vinegars are either imported already bottled or are bottled locally under the specific supermarket or other local brand.

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The nature of vinegar is such that a wine cellar cannot be used to make vinegar as acetic acid bacteria are wine spoilage organisms. Therefore a separate facility is required. However, basic equipment (e.g. plastic food grade buckets, or old wine barrels) could be used for production without the use of cooling. This makes it an ideal venture for a small business with minimal capital outlay.

In this project yeast choice and acetic acid bacteria selections needed to be investigated, while a small-scale production process had to be devised. The ARC Infruitec-Nietvoorbij has an extensive non-Saccharomyces yeast collection that could be screened for yeasts with desirable characteristics. The project team included a Spanish collaborator that has done research on acetic acid bacteria as part of a European Balsamic Vinegar project. The Spanish collaborator could supply acetic acid reference bacteria and research expertise. The project comprised of various sections, some that ran concurrently. These were: 1) Reference vinegars: procurement, training of judges, chemical and sensory analyses; 2) Selection and evaluation of non-Saccharomyces yeast; 3) Isolation, evaluation, selection and identification of acetic acid bacteria; 4) Laboratory-scale production trials in normal (single strength) grape juice 5) Pilot-scale production trial in normal grape juice; 6) Pilot-scale Balsamic-style vinegar production in cooked (concentrated) juice; 7) Evaluation of final products, shelf-life evaluation; 8) Other products: Port wine style vinegar. REFERENCE VINEGARS Twenty-five vinegars were sourced locally and internationally (Fig. 1 and Table 1). These included 15 Balsamic Vinegars of Modena (Italy), two condiments (Italy), four Balsamic-styled vinegars (two from Italy, one from Spain and one locally produced vinegar), two Traditional Balsamic Vinegars (Italy), and a locally produced hobbyist product. A South African wine vinegar was used as a sensory and analytical standard. Each vinegar was assigned a code-number for easy reference. The price ranged from R20 to just over R4000 per 250 mL. The 25 vinegars were used for chemical analyses (testing of analytical equipment; standardisation of analysation methods; determination of a vinegar chemical profile) and sensory analyses (development of a sensory evaluation sheet; training of a sensory evaluation panel). Due to a very limited sample volume, the locally produced hobbyist product was eliminated from further analyses.

Fig. 1. A selection of commercial reference vinegars used in the project.

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Table 1. Details of reference vinegars used in this research project.

Code

no

Product Type1 Origin Pack-

aging

size

(ml)

Price/

250mL (R)2

Analytical and sensory standard vinegar 1 Wellington's Red Wine Vinegar Wine vinegar South Africa 500 14.00

Balsamic Vinegar of Modena and Balsamic-styled vinegars 2 Wellington's Imported Balsamic Vinegar BvM Modena, Italy 500 20.25 3 Staffords Organic Balsamic Vinegar BvM Modena, Italy 250 39.99 4 Ortalli Balsamic Vinegar of Modena (500mL) BvM Modena, Italy 500 18.75 5 Ortalli Balsamic Vinegar of Modena (250mL) BvM Modena, Italy 250 56.99 6 Willow Creek Cabernet Sauvignon Estate Balsamic Vinegar Bsv South Africa 500 39.00 7 De Nigris Aceto Balsamico BvM Modena, Italy 250 179.99 8 Ortalli Balsamic Vinegar of Modena - Aged BvM Modena, Italy 250 79.99 9 Serena Balsamic Vinegar of Modena BvM Modena, Italy 500 13.40 10 PnP Finest Balsamic Vinegar of Modena BvM Modena, Italy 250 99.99 12 Agredolç Bsv Catalonia, Spain 250 90.05 15 Staffords White Balsamic Vinegar Bsv Italy 250 24.99 14 Davies hobbyist product Bsv South Africa 100 -3 16 De Nigris Aceto Balsamico di Modena IGP (250mL) BvM IGP Italy 250 46.99 17 Gourmet Balsamic Vinegar (Hillcrest Berry Orchards) BvM Italy 500 24.00 18 Woolworths BoM PGI Imported BvM IGP Italy 250 44.95 19 Woolworths BoM PGI Organic Imported BvM IGP Italy 250 59.95 20 Aceto Balsamico di Modena PGI BvM IGP Italy 250 144.95 21 Olyfberg Balsamic vinegar BvM Italy (bottled in SA) 500 35.00 22 De Nigris Balsamic Vinegar of Modena Platimum Eagle IGP BvM IGP Italy 250 316.90

Traditional Balsamic, Condiments, Reserve vinegars 11 De Nigris I Fruttati Cooked Grape Must (La Saba) Condiment Afragola-Napoli, Italy 250 84.99 13 Acetaia Dodi Riserva di Famiglia Reserve Emelia-Romagna, Italy 100 758.03 23 Acetaia di Giorgio Aceto Balsamico Tradizionale Di Modena

D.O.P. Capsula Bianca (white cap) (over 12 years old) TBV Modena, Italy 100 1676.83

24 Acetaia di Giorgio Aceto Balsamico Tradizionale Di Modena D.O.P. Extravecchio Capsula Oro “Carlotta 1986” Extra aged (over 25 years)

TBV Modena, Italy 100 4098.88

25 Acetaia Leonardi Condimento Riserva Ciliegio Condiment Modena, Italy 100 1448.45 1BvM = Balsamic Vinegar of Modena; BvM IGP = Balsamic vinegar of Modena with Protected Geographic Identification (Indicazione Geografica Protetta); Bsv = Balsamic styled vinegar; TBV = Traditional Balsamic Vinegar. 2Price standardised to 250 mL for comparison between products. 3Not for sale.

Chemical analyses

The chemical analyses of the vinegars was one of the most challenging aspects of the project. The instrumentation and methodology available to the project team was designed with wine as the matrix. Due to ease of use, low sample volume (1 mL) and low cost of analyses, the OenoFOSS (FOSS, Denmark) instrument was evaluated for suitability for analysing vinegar. The existing wine calibrations were not suitable so a new set of calibration values specific for vinegar were created. An acceptable calibration curve for volatile acidity could initially be set up, but not for pH, alcohol and total acidity. However, the volatile acidity results could not be verified and the use of the OenoFOSS as an analytical instrument for vinegar was discarded. Other analytical methods were subsequently used. The three methods used to measure sugar levels i.e. Rebelein, Densitometer (DMA 35, Anton Paar, Austria) and enzymatic (CAF, Stellenbosch University, Stellenbosch) all gave varying results. Considering

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that all three methods were designed for grape juice and wine the variations could be expected. However, the order of magnitude between the methods was similar. As the enzymatic method is automated, these values could be accepted as the most accurate. Analyses for pH, total acidity and alcohol were done using a pH meter (Martini Instruments), mini-titrator (Hanna Instruments) and Alcolyser Wine M (Anton Paar), respectively. Acetic acid was also determined enzymatically (CAF, Stellenbosch University, Stellenbosch). Volatile acidity was done by the distillation method (Koelenhof Wynkelder Laboratorium, Stellenbosch). For all analyses a standard dilution of 1:25 was used. The total acidity of all the commercial samples ranged from about 60 to 80 g/L for both Balsamic Vinegar of Modena as well as Traditional, Reserve and Condiment vinegars (Table 2). Traditional, Reserve and Condiment vinegars generally had higher sugar levels than Balsamic Vinegars of Modena. Analytically, there appeared to be no “ideal” acid/sugar ratio that was characteristic to these vinegars (data not shown).

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Table 2. Chemical analyses of commercial wine, Balsamic, Balsamic-styled and hobbyist Balsamic-styled vinegars.

1Not determined.

Code

no.

Sample name pH Total

acidity

(g/L)

Sugar Acetic

acid

(g/L)

Volatile

acidity

(g/L) Sugar

(°B)

Rebe-

lein

method

(g/L)

Total

sugar

(F + G +

S)

(g/L)

Analytical and sensory standard vinegar

1 Wellingtons red wine vinegar 3.22 120.5 5.0 13.8 1.48 70.70 58.32

Balsamic Vinegar of Modena and Balsamic-styled vinegars

2 Wellingtons imported Balsamic Vinegar 3.36 82.8 37.5 346.3 238.61 59.48 48.00

3 Staffords Organic Balsamic Vinegar 3.47 74.0 37.5 275.0 243.39 54.03 42.00

4 Ortalli Balsamic Vinegar of Modena (500mL) 3.51 78.3 32.5 305.0 202.05 25.35 19.50

5 Ortalli Balsamic vinegar of Modena (250mL) 3.42 74.0 57.5 497.5 246.40 53.68 43.75

6 Willow Creek Cabernet Sauvignon 3.44 56.8 32.5 270.0 236.28 55.13 42.00

7 De Nigris Aceto Balsamico di Modena I.G.P 3.48 56.0 70.0 627.5 288.89 51.45 41.25

8 Ortalli Balsamic Vinegar of Modena - Aged 3.46 76.5 55.0 497.5 230.73 65.63 47.75

9 Serena Balsamic vinegar of Modena 3.44 71.3 25.0 193.8 138.09 59.33 50.25

10 PNP finest Balsamic Vinegar 3.58 65.0 77.5 635.0 313.47 48.40 40.00

12 Agredolç 3.39 78.0 17.5 96.3 76.53 66.98 53.00

14 Davies hobbyist product 2.94 117 65.9 708.8 -1 -1 64.5

15 Staffords white Balsamic Vinegar 3.39 61.3 25.0 202.5 159.77 55.20 42.00

16 De Nigris Aceto Balsamico di Modena IGP 3.27 75.0 25.0 145.0 119.18 56.08 45.00

17 Gourmet Balsamic vinegar (Hillcrest Berry Orchards) 3.46 69.3 40.0 353.8 251.05 62.25 50.25

18 Woolworths Balsamic Vinegar of Modena PGI Imported 3.63 75.0 57.5 437.5 239.94 60.63 45.25

19 Woolworths Balsamic Vinegar of Modena PGI Organic Imported 3.50 68.0 52.5 403.8 220.12 58.93 47.50

20 Aceto Balsamico di Modena PGI 3.21 64.5 80.0 640.0 330.62 43.68 25.75

21 Olyfberg Balsamic Vinegar 3.42 69.0 45.0 381.3 208.22 65.05 52.50

22 De Nigris Balsamic Vinegar of Modena Platimum Eagle 3.44 57.5 70.0 625.0 313.86 45.73 30.00

Traditional Balsamic, Condiments, Reserve vinegars

11 De Nigris i Fruttati (La Saba) 3.78 52.8 82.5 715.0 378.58 9.50 6.25

13 Acetaia Dodi Riserva di Famiglia 3.77 50.5 90.0 715.0 371.85 31.70 26.00

23 Acetaia di Giorgio Aceto Balsamico Tradizionale 3.15 83.5 82.5 650.0 306.24 49.18 41.25

24 Acetaia di Giorgio “Carlotta 1986” 3.09 79.0 100.0 777.5 346.78 34.50 25.25

25 Acetaia Leonardi Condimento Riserva Ciliegio (cherry) 3.32 53.5 90.0 852.5 376.52 27.18 17.00

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Sensory analyses

A tasting sheet for the sensory evaluation of Balsamic vinegar was developed while a core group of staff was trained for the evaluation of vinegar. The first score sheet (data not shown) was found to be unsatisfactory and needed to be improved. The improved score sheet (Fig. 2) included eight descriptors (General appearance and colour; Aroma, Viscosity, Initial taste, Middle taste, Aftertaste, Acidity/sweetness balance, Overall quality). For each descriptor a rating from 1 to 5 was given (Unacceptable, Bad, Average, Good, and Excellent). The exception was Acidity/sweetness balance where a different scale was used i.e. Too acidic, Slightly too acidic, Good balance, Slightly too sweet and Too sweet. A good balance equated to the highest score of 5 and the other criteria on both acidity and sweetness sides scored 2.5 or 1. The sensory evaluation results of the commercial vinegars are shown in Fig. 3. Overall the vinegars showed a wide range of sensory profiles. It was especially on Acid/sweetness balance and Viscosity that the biggest differences occurred. The Traditional, Reserve and Condiment vinegars rated the highest in all categories evaluated. The Spanish Agredolç and South African Willow Creek vinegars were judged not to be true Balsamic-style vinegars, but were more akin to wine vinegars. None-the-less these two vinegars were kept in the line-up of reference vinegars.

EVALUATOR: DATE: Tasting sheet 5

SAMPLE:

1. General appearance and colour: 2. Aroma: 3. Viscosity 4. Initial taste

Unacceptable Unacceptable Unacceptable Unacceptable

Bad Bad Bad Bad

Average Average Average Average

Good Good Good Good

Excellent Excellent Excellent Excellent

5. Middle Taste 6. After Taste 7. Acidity/sweetness balance 8. Overall quality

Unacceptable Unacceptable Too acidic Unacceptable

Bad Bad Slightly too acidic Bad

Average Average Good balance Average

Good Good Slightly too sweet Good

Excellent Excellent Too sweet Excellent

Comments

BALSAMIC VINEGAR TASTE PANEL EVALUATION

Fig. 2. Tasting sheet used for sensory evaluation of Balsamic and Balsamic styled vinegars (© ARC Infruitec-Nietvoorbij). Further analyses of the chemical and sensory data showed that there was no relationship between the overall quality and total acidity (Fig. 4A), however, a direct relationship was apparent between total sugar and quality (Fig. 4B). The higher the sugar, the higher the perceived quality. The ratio between sugar and acidity, or sugar, acidity and pH, could also be linked to high quality (Fig. 4C and D).

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Fig. 3. Sensory profile and quality of commercial Balsamic and a Balsamic-styled vinegars compared to a red wine standard vinegar (red dashed line).

A B

C D

Fig. 4. Relationship between overall quality as determined by a sensory panel and chemical analytical measurements. The standard wine vinegar is shown as a red point.

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ACETIC ACID BACTERIA: ISOLATION, IDENTIFICATION AND EVALUATION Isolation and identification

Acetic acid bacteria cultures are not commercially available, therefore the project team had to isolate bacteria locally. A targeted approach was used with growth media selective for acetic acid bacteria. The source material included, healthy and rotten Kei Apple fruit (Dovyalis caffra) from trees on the ARC Infruitec-Nietvoorbij premises, grape pomace from the Nietvoorbij Research Cellar waste heap, healthy and rotten Sauvignon blanc grapes from the ARC Robertson Research Farm and a locally-made hobbyist vinegar. All samples were aseptically collected. A total of 81 presumptuously identified acetic acid bacteria were isolated (Gram negative, catalase positive). Identification of the presumptuously identified bacteria were done by a PCR technique (16s rDNA and restriction by two enzymes, AluI, TaqI) supplied by the Spanish collaborator. Twenty-eight references strains were acquired, 16 purchased form BCCM/LMG, Belgium and 12 donated by the Spanish collaborator (Prof. A. Mas, University Rovira i Virgili, Spain). An example of the profiles generated are given in Fig. 5. The majority of isolates appeared to be Acetobacter pasteurianus, with only a few A. aceti, Gluconacetobacter. hansenii and Gluconobacter oxydans. No isolates were identified as Glucoacetobacter europaeus. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fig. 5. A selection of profiles of acetic acid bacteria isolates and reference strains generated by ITS-PCR digested by TaqI restriction enzyme.

Evaluation of acetic acid bacteria

One acetic acid bacteria isolate was evaluated under three different aeration strategies (stirred Erlenmeyer, stationary Erlenmeyer, Thompson flat-bed flask) in 6.64% base wine to determine the most appropriate strategy for use in laboratory-scale evaluation of the other isolates (Fig. 6). Monitoring ethanol depletion showed that the best results were obtained in the Thompson flat-bed flasks. However, the results in the stationary Erlenmeyer flasks, although slower, were a close second-best and finished at the same time as the Thompson flasks (Fig. 7). Both aforementioned finished under 30 days compared to the stirred Erlenmeyer that was still unfinished at 50 days. However, due to limited availability of the Thompson flasks and considering that the duration was similar, stationary Erlenmeyer flasks were used for all subsequent evaluations and trials.

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A B

Fig. 6. Thompson flat-bed flasks, Erlenmeyer flasks (A) and Erlenmeyer flasks on stirrers (B) to compare growth of selected acetic acid bacteria strains.

Fig. 7. Comparison of growth kinetics of acetic acid bacteria strain 179/12 in stirred and stationary Erlenmeyer flasks, and flat-bed Thompson flasks.

The initial raw material for Balsamic vinegar production is cooked (concentrated) grape juice (high sugar environment), but alcohol can also be present due to alcoholic fermentation by yeast. The bacteria are therefore required to grow in both environments, with some playing a bigger role in as “starters” and others as “finishers”. The screening of the acetic acid bacteria was therefore conducted separately in an alcoholic (diluted wine, ca. 6% ethanol) and high sugar environment (standard grape juice 213 g/L sugar, pH 3.54, 5.2 g/L total acidity), respectively to find the most suitable isolate(s) for each matrix. Alcohol levels and pH were monitored in the wine and grape juice, respectively. A graphical representation of a sub-set of data for wine and grape juice is shown in Figs 8 and 9, respectively

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Fig. 8. Depletion of alcohol in 5.76% (v/v) wine by various acetic acid bacteria isolates over a period of 30 days.

Fig. 9 Change in pH due to growth of acetic acid bacteria in grape juice over a period of 32 days.

After the first screening, 11 isolates showing the best growth in alcohol and 17 showing the best growth in grape juice were evaluated in a second round of screening (Figs 10 and 11). This data was used to make a final selection of 20 acetic acid bacteria isolates for vinegar production trials (Table 3). A sub-selection of bacteria was also was also evaluated in different concentrations of grape juice and cooked grape juice (24 – 40°B). Monitoring of the pH showed that the ability of the bacteria to grow became less as the sugar concentration increased (Fig. 12). However, growth was still recorded, albeit slowly, in the highest sugar concentration of 40°B.

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Fig. 10. Depletion of alcohol in 6 % (v/v) wine by various acetic acid bacteria isolates over a period of 42 days.

Fig. 11. Change in pH during growth of acetic acid bacteria isolates in a grape juice (213 g/L sugar pH 3.54, 5.2 g/L total acidity) over a period of 28 days.

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Table 3. Source and identity of selected acetic acid bacteria used for vinegar production. Acetic acid

bacteria isolate

code

Source

Identity

179/12 Healthy Chardonnay grapes from Robertson Acetobacter pasteurianus

87/30 Genebank Acetobacter aceti

126/34 Jaboticaba fruit (Plinia cauliflora) Gluconacetobacter sp.

126/34a Genebank1 No identity / inconclusive2

126/34b Genebank No identity / inconclusive2

172/36 Genebank Acetobacter tropicalis

172/36a Genebank Acetobacter tropicalis

172/36b Genebank Acetobacter tropicalis

172/43 Genebank No identity / inconclusive2

172/43a Genebank No identity / inconclusive2

172/43b Genebank No identity / inconclusive2

179/19 Nietvoorbij Shiraz juice Gluconobacter sphaericus

179/19a Nietvoorbij Shiraz juice Gluconobacter sphaericus

179/19b Nietvoorbij Shiraz juice No identity / inconclusive2

179/48 Nietvoorbij Cellar Pomace Acetobacter pasteurianus

179/59 Nietvoorbij Cellar Pomace Acetobacter pasteurianus

179/64 Nietvoorbij Cellar Pomace Acetobacter pasteurianus

179/65 Rotten Kei Apple (Dovyalis caffra) No identity / inconclusive2

179/68 Davies hobbyist product No identity / inconclusive2

179/68a Davies hobbyist product Acetobacter pasteurianus

179/68b Davies hobbyist product Gluconobacter sphaericus 1ARC Infruitec-Nietvoorbij Oenological Genebank. 2Does not match current reference species.

Fig. 12. Evaluation of selected bacteria in cooked grape juices from 24 to 40°B. Final pH after eight days.

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NON-SACCHAROMYCES YEAST: SELECTION AND EVALUATION The research group had over 3000 non-Saccharomyces yeast isolates in cryo-preservation (-80°C) in the Oenological Culture Collection (ARC Infruitec-Nietvoorbij) at their disposal. The majority of the yeasts are unidentified and uncharacterised. The isolates emanate from a wide variety of source material (grapes and other fruits) and geographical areas. From information available in the culture collection database, a preliminary selection of 1557 yeast isolates was made for screening purposes. The first step entailed screening for the formation of acidity on CaCO3 agar plates. Thereafter, a smaller selection of yeasts were screened for aroma in grape juice to eliminate potential off-odour forming yeasts (data not shown). Twenty yeasts were found to produce off-odours, 24 were neutral and 49 produced pleasant fruity aromas. Residual sugar and alcohol levels were also measured. All this data was used to further refine the yeast selection to 20 isolates, 19 of which could be identified by ITS-PCR. These isolates were subsequently investigated further for fermentation kinetics, aroma production, volatile acidity production and ethanol production (Figs 13 and 14). As all the preliminary screenings were done in standard grape juice, the final yeast selection was also evaluated in cooked juice (Fig. 15).

Fig. 13. Fermentation kinetics of 20 selected non-Saccharomyces yeasts in standard grape juice.

A review of all the data resulted in a final selection of five non-Saccharomyces yeast i.e. Zygosaccharomyces bailii strain C48V19, Hanseniaspora guilliermondii strain C2-15, Candida pulcherrima (Metschnikowia pulcherrima) strain C1-15, Kloeckera apiculata (Hanseniaspora uvarum) strain 45/69 and Candida zemplinina (Starmerella bacillaris) strain C2/19 or 74/56. The selected yeasts showed good fermentation kinetics, aroma production, low volatile acidity and maximal ethanol production of approximately 6% (the optimal ethanol level where acetic acid bacteria can grow). The yeasts could also grow in a 38°B (388.4 g/L) cooked grape juice.

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Fig. 14. Ethanol and volatile acidity production, and aroma intensity after fermentation of standard grape juice by 20 selected non-Saccharomyces yeasts.

Fig. 15. Fermentation kinetics of eight non-Saccharomyces yeasts in cooked grape juice (388.4 g/L sugar) over a period of 87 days.

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PREPARATION OF BASE MATERIAL: COOKING OF JUICE Preparation of juice by boiling to increase sugar content (as would be done for Traditional Balsamic Vinegar production) was carried out. Dependant on the volume required, either a normal pot and hot plate combination, or steam pot was used (Fig. 16). The base juice was brought to boiling point and allowed to simmer until concentrated. The initial sugar of the grape juice of e.g. 24.6°B could thereby be increased to 33-40°B within 45 – 60 minutes (Fig. 17). At this stage there was a large reduction of naturally occurring yeast and bacteria, but the grape juice was not sterile (data not shown).

A

B

Fig. 16. Cooking of grape juice in (A) normal pot and for pilot-scale in (B) a steam pot (Pilot Plant, Post-Harvest and wine Technology Division, ARC Infruitec-Nietvoorbij).

Fig. 17. Increase in concentration of sugar during boiling of grape juice over a period of 45 minutes.

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LABORATORY-SCALE TRIALS: COMBINED FERMENTATION AND

ACETIFICATION Six laboratory-scale trials were carried out. Each trial tested various inoculation strategies, bacteria and yeast combinations and volumes (Table 4). There were two basic strategies for inoculation, 1) a co-inoculation of the yeast and bacteria on day zero, 2) a sequential inoculation where the yeast was inoculated on day zero, and the acetic acid bacteria were inoculated when the alcohol levels reached 6%. Yeast only, bacteria only and uninoculated fermentations were done, where appropriate, for references purposes. The base material was either sterile standard grape juice or cooked juice (Table 5). In all the vinegar production trials, three repetitions were carried out and reported data are the calculated averages.

Table 4. Non-Saccharomyces yeasts and acetic acid bacteria used in laboratory (Trials 1-6) and pilot-scale (Trails 7-8) vinegar production trials.

Yeast / Bacteria strain Trial number / Base material / Volume of trial (L)1 1

Std juice (1L)

2 Std

juice (1L)

3 Std

juice (1L)

4 Std

juice (1l)

5 Std

juice (1L)

6 Cooked

juice (1L)

7 Std

juice (“May”) (3.2L)

8 Cooked

juice (“Charlotte”)

(3.2L)

Non-Saccharomyces yeasts 74/56 Candida zemplinina √ √ √ C2-19 Candida zemplinina √ √ C48 V19 Zygosaccharomyces bailii √ √ √ √ √ C2-15 Hanseniaspora guilliermondii √ √ √ √ C1-15 Candida pulcherrima √ √ √ √ 45/69 Kloeckera apiculata √ √ √ √ √ √ Acetic acid bacteria strain no. 179/12 √ √ 87/30 √ √ √ √ √ √ 126/34 √ √ √ √ √ √ 126/34a √ √ √ √ 126/34b √ √ √ √ 172/36 √ √ √ √ 172/36a √ √ √ √ 172/36b √ √ √ √ 172/43 √ √ √ √ 172/43a √ √ √ √ 172/43b √ √ √ √ 179/19 √ √ √ √ 179/19a √ √ √ √ 179/19b √ √ √ √ 179/48 √ √ √ √ 179/59 √ √ √ √ 179/64 √ √ √ √ 179/65 √ √ √ √ 179/68 √ √ √ √ 179/68a √ √ √ √ 179/68b √ √ √ √

1Std = standard single strength grape juices. Trials 7 and 8 were named “May” and “Charlotte” as per Italian tradition.

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Table 5. Chemical analyses of standard grape juice used in laboratory-scale and pilot-scale vinegar production trials. Trial Grape juice Analyses1

Sugar Alcohol

(%v/v)

pH

Total acidity

(g/L)

Total

soluble

Solids

(°B)

Calculated2

(g/L)

Rebelein

method

(g/L)

1 Chenin blanc (autoclaved x1) 20.5 205 Na2 0.20 3.53 5.4

2 Chenin blanc (autoclaved x1) 22.0 220 Na2 0 3.29 5.34

3 Chenin blanc (autoclaved x1) 23.4 234 253 0.11 3.25 6.30 4 Chenin blanc (autoclaved x1) 22.3 223 266 0.08 3.25 6.65 5 Chenin blanc (autoclaved x1 ) 24.0 240 240 0.66 3.36 6.58 6 Chenin blanc (cooked) 39.5 395 428 0 3.32 9.62 7 Chenin blanc (autoclaved 5X) 24.0 240 Na2 0.21 3.14 9.05 8 Chenin blanc (cooked) 37.7 377 Na2 0.6 3.24 12.3 9 Nietvoorbij vintage barrel sample 2.7 27 39 6 2.09 2.3

1Analyses for Trial 1 done on the OenoFOSS while the densitometer, alcolyser, pH meter and auto-titrator were used for trials 2 to 9. 2Calculated at 1°B = 10g/L sugar. 2Not analysed.

Fermentation and acetification kinetics of laboratory-scale vinegar production trials

Trial 1 In the first trial the non-Saccharomyces yeast K. apiculata strain 45/69 was combined with acetic acid bacteria A. pasteurianus strain 179/12 in various combinations (yeast only; simultaneous co-inoculation of yeast and bacteria; sequential co-inoculation with yeast and bacteria at 6% ethanol). The main aim of this trial was to evaluate the accuracy and reliability of the OenoFOSS instrument for monitoring alcoholic and acetification fermentations. This trial was conducted concurrent with the analyses of the commercial reference vinegars. Analytical results of the grape juice and the initial alcoholic fermentation could be used, but as was the case with the reference vinegars, the OenoFOSS was unable to perform measurements once high levels of acetic acid became part of the matrix (data not shown). This trial was terminated after 37 days. Trial 2 The second trial was initiated with the same microorganisms as in Trial 1 (K. apiculata strain 45/69 and A. pasteurianus strain 179/12) and the densitometer, alcolyser, pH meter and auto-titrator instruments were used for analyses. Better results were obtained (Fig. 18) and an interesting trend was noted on the densitometer data. This instrument was capable of measuring the declining sugar concentration during the alcoholic fermentation, but the when acetification commenced the measured values increased. This was subsequently a very useful tool to quickly monitor the progress of the vinegar production. The alcolyser also gave good results showing ethanol production during the alcoholic phase and then depletion as the ethanol was utilised by the acetic acid bacteria. The trial was allowed to run for 98 days. The 6% production strategy showed a better acidity development than the 0% method. This was also reflected in the ethanol monitoring which showed that ethanol was not completely utilised in the 0% strategy.

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Fig. 18. Changes in alcohol, sugar, pH and total acidity during laboratory-scale vinegar production (Trial 2). K. apiculata strain 45/69 with acetic acid bacteria 179/12 (start juice analyses: 22.0°B, 0% alcohol, pH 3.29, 5.34 g/L total acidity).

Trials 3 and 4 The third and fourth trials compared two non-Saccharomyces yeasts, C. zemplinina strain 74/56 and Z. bailii strain C48V19, respectively, combined with the same two acetic acid bacteria isolates (strains 87/30 and 126/34) (Figs 19 and 20). For the C. zemplinina yeast/acetic acid bacteria combination the 0 and 6% strategies differed in alcohol and acidity formation (Fig. 19). The 0% strategy gave the best acid formation. In contrast the Z. bailii/acetic acid bacteria combination gave similar result for both 0 and 6% strategies, with both forming high acidity levels (Fig. 20). The trial was terminated after 357 days.

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Fig. 19. Alcohol, sugar, pH and total acidity progress during laboratory-scale vinegar production (Trial 3). C. zemplinina strain 74/56 with acetic acid bacteria 87/30 and 126/34 (start juice analyses: 23.4°B/253 g/L sugar, 0.11% alcohol, pH 3.25, 6.3 g/L total acidity).

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Fig. 20. Alcohol, sugar, pH and total acidity progress during laboratory-scale vinegar production (Trial 4: Z. bailii strain C48V19 with acetic acid bacteria87/30 and 126/34 22.3; start juice analyses: 23.3°B/266 g/L sugar, 0.08% alcohol, pH3.25, 6.65 g/L total acidity).

Trial 5 Trial 5 was the first trial that explored the multi-culture approach with five non-Saccharomyces yeasts and twenty acetic acid bacteria isolates. This trial, done in an autoclaved juice (24.0°B), showed good ethanol production for the 6% strategy followed by a good acidity production (Fig. 21). The 0% strategy only showed a marginal ethanol production with moderate acidity production. It was apparent that the 6% strategy was leading to a faster and higher acidity production. The trial was terminated after 235 days.

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Fig. 21 Alcohol, sugar, pH and total acidity progress during laboratory-scale vinegar production (Trial 5: five non-Saccharomyces yeast and 20 acetic acid bacteria strains; start juice analyses: 24.0°B sugar, pH3.36, 6.58 g/L total acidity).

Trials 6 Trial 6 was identical to Trial 5, except that a cooked juice of 39.5°B was used. The results indicated that although alcohol was being formed by the yeasts, no notable acidity production could be observed (Fig. 22). This was an unexpected result as the data from the initial acetic acid bacteria evaluations showed the selected bacteria were able to grow in high balling grape juice (Fig. 12). The trial was terminated prematurely after 53 days in the belief that all the acetic acid bacteria had died. It was only later concluded that the trial should have been left to continue as the acetification would probably have started in due course.

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Fig. 22. Alcohol, sugar, pH and total acidity progress in cooked grape juice during laboratory-scale vinegar production (Trial 6: five non-Saccharomyces yeast and 20 acetic acid bacteria strains; start juice analyses: 39.5°B sugar, pH3.32, 9.62 g/L total acidity).

Sensory analyses of laboratory-scale vinegars

Vinegar trials 2, 3, 4 and 5 The vinegar products of Trials 2, 3, 4 and 5 were sensorially evaluated by a panel comprising of students and staff members. Initially the appearance of the vinegar products were rated as unacceptable due to insufficient colour. This was a result of limited or no caramelisation occurring during the juice preparation process, and no ageing of the product. During preparation of the base juice for subsequent trials the colour was improved by repeated autoclaving and/or cooking to give a deeper initial colour. If required, food colourant was also added to deepen the vinegar colour before evaluation. The vinegars were also sweetened with cooked grape juice (ca. 37°B in a 1:1 ratio) to follow the “Balsamic Vinegar of Modena” production practise. The sensory profiles are given in Fig. 23. Overall the 6% inoculation strategy was preferred above the 0% inoculation strategy. One of the vinegars was also sweetened to a higher degree (1:2), with the result that the sensory profile was marginally improved (Fig. 24).

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Fig. 23. Sensory profile of Balsamic-style vinegars produced in Trials 2-5 (Trial 5 was not evaluated for acidity/sweetness balance).

Fig. 24. Comparison of Trial 5’s vinegar sweetened in 1:1 and 1:2 ratio with cooked grape juice (ca. 37°B). Acidity/Sweetness balance was not evaluated for Trial 5. Blended vinegars and shelf life trial Aliquots of the three individual repetitions per trial were blended and sweetened in a 1:1 ratio with cooked grape juice. It can be seen from Fig. 25 that a wide range of profiles and quality could be achieved. This is normal for a product that is based on a biological process. After filtration (0.45 micron filter) the blended vinegars were bottled and allowed to age for three months (Fig.

26

25). The evaluation after three months showed that one product had increased in quality while two had remained stable (Fig. 26). Unfortunately, five products had deteriorated in quality due to the added sweetener being further metabolised. This was an indication that the initial vinegar had not reached a natural stable state, and that the bottling process was not sterile enough. This aspect will need further attention in a follow-up project.

A B

Fig. 25. Sensory profile of vinegar blends composed of the three repetitions per trial, at time zero (A) and after three months (B).

Fig. 26. Shelf-life evaluation: comparison of vinegar blends over a three month period.

OTHER PRODUCT: PORT-STYLED WINE VINEGAR TRIAL A characteristic sensory attribute of Balsamic vinegar is the “matured/aged” aroma and flavour typically found. This is developed during the lengthy production period (over 12 years). In an attempt to hasten this process, a Port-style of wine (Table 5) was used as a base material for laboratory-scale vinegar production.

27

The Port-style wine already had certain aged aromas. After dilution to 6% ethanol, two inoculation strategies were followed i.e. five non-Saccharomyces yeast and 20 acetic acid bacteria consortium; and 20 acetic acid bacteria-only (Trial 9). Fermentation and acetification kinetics of the Port-style wine vinegar production

The expected patterns of alcohol depletion and acid formation were observed for both strategies (Fig. 27). A subsequent decline in total acidity was noted in the ‘acetic acid bacteria-only”. The reasons for this cannot currently be explained.

Fig. 27. Alcohol, sugar, pH and total acidity progress during a pilot-scale vinegar production in a diluted port (Trial 9: five non-Saccharomyces yeasts and 20 acetic acid bacteria strains; start wine analyses: 2.7°B sugar, pH 2.09, 2.3 g/L total acidity; 6% alcohol).

Sensory evaluation of Port-style wine vinegars

A sensory evaluation of the port-style wine vinegar was conducted at 6 months and at 11 months production time. For both time intervals the vinegar was evaluated as an unsweetened and sweetened (with cooked juice) product with a wine or Balsamic-styled vinegar as a reference. The 6 month evaluation showed no differences in the sensory profiles between the two strategies (Fig. 28A). However, sweetening improved all the sensory attributes measured (Fig. 28B). At 11 months the yeast-bacteria consortium product was marginally better, with a marked improvement when the product was sweetened (Fig. 28C and D). The use of Port-styled wine should be further investigated with a higher degree of sweetening.

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A B

C D

Fig. 28. Sensory profile of vinegars made from a Port-style wine after six (A, B) and eleven months (C, D) (unsweetened and sweetened).

PILOT-SCALE TRIALS: COMBINED ACETIFICATION AND FERMENTATION Two pilot-scale trials (Trial 7 and 8) were conducted using the accumulated data of the laboratory-scale trials as guidelines. The multi-culture yeast and bacterium consortia approach was used (Table 4) and a standard and cooked juice was used as the base material, respectively (Table 5). The 6% inoculation strategy was followed. In line with Italian tradition, the two pilot-scale trials were given lady’s names i.e. “May” and “Charlotte”. Trial 7: Fermentation and acetification kinetics, and sensory evaluation

Vinegar Trial 7 (“May”) in the standard grape juice showed good alcohol development, followed by depletion, with concomitant increase in acidity and decrease in pH (Fig. 29). After 111 days, as per Balsamic Vinegar production methods, half the vinegar was racked off into a second container (“May 2”) and fresh grape juice was added to the initial “May”, i.e. ”May 1”. This can be seen in Fig. 29 as the increase in sugar and decrease (dilution) of acidity. Further monitoring of the acidity will determine whether a re-inoculation of microbiota will be required, or if the original consortium will be able to continue the acetification process. The two products also underwent a sensory evaluation at six months. “May 2” showed a good aroma, but as it was not sweetened, the other sensory attributes scored low (Fig. 30). “May 1” with the added grape juice showed a good aroma profile. The exception was ‘appearance and colour’ and ‘viscosity’. However, these two attributes can be corrected in the final product by addition of grape juice concentrate and caramel colourant as is done for Balsamic Vinegar of Modena production. The trial is currently still running and can receive further attention in a follow up project.

29

Fig. 29. Alcohol, sugar, pH and total acidity progress during a pilot-scale vinegar production (Trial 7 [May]: five non-Saccharomyces yeasts and 20 acetic acid bacteria strains; start juice analyses: 24.0°B sugar, pH3.14, 9.05 g/L total acidity).

Fig. 30. Sensory profile of two stages of a Balsamic-styled vinegar production following the Traditional Balsamic Vinegar production method (Trial 7 [May]).

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Trial 8: Fermentation and acetification kinetics

Vinegar Trial 8 (”Charlotte”) is a true Traditional Balsamic Vinegar production trial in cooked grape juice. The expected alcohol development was observed (Fig. 31) and the acetic acid bacteria were inoculated at 6% ethanol, 38 days after the yeast inoculation. The start of acetification has just begun at 140 days, albeit slowly. The trial is currently still in progress and further monitoring can be done under a new follow up project. Further inoculation of acetic acid bacteria can also be considered if the acetification lags.

Fig. 31. Alcohol, sugar, pH and total acidity progress during a pilot-scale vinegar production (Trial 8 [Charlotte]: five non-Saccharomyces yeasts and 20 acetic acid bacteria strains; start juice analyses: 37.7.0°B sugar, pH3.24, 12.3 g/L total acidity).

INTERESTED PARTIES Although not an aim of this project, some potential entrepreneurs have shown interest in starting Balsamic-styled vinegar production. The one candidate already produces vinegar from other fruits (not grapes). The other candidates have culinary and wine production backgrounds. SUMMARY AND CLOSING COMMENTS The project team was successful in screening and evaluating non-Saccharomyces yeast and acetic acid bacteria strains. A final selection of five non-Saccharomyces yeasts and 20 acetic acid bacteria can be successfully used in a multi-culture approach for Balsamic-styled Vinegar production. Of the two strategies investigated i.e. 0 and 6%, the 6% strategy gave the best results. Two base materials could be used, i.e. a single strength grape juice, or a cooked (concentrated) grape juice. The single strength grape juice underwent alcoholic and acetification quickly and could then be sweetened and coloured to produce an acceptable Balsamic-styled vinegar. However, further ageing would be required to improve the product. This is in line with Balsamic Vinegar of Modena production methods that need a minimum of two months ageing.

31

The use of cooked juice for vinegar production is a much slower process and will require a lengthy production and maturation time. The use of an already aged product as a base material (i.e. “Port styled wine”) can be considered as a faster alternative process. Thickening and sweetening will have to be used here as well. Currently a student is conducting extensive trials as part of an MTech project (Cape Peninsula University of Technology). These trials are being conducted at ARC Infruitec-Nietvoorbij and cover both 0 and 6% inoculation strategies, at three different temperature conditions (low, high and fluctuating). Key aims of the project are the uses of process modelling to measure substrate and product outcomes, while investigating to what extent the various yeast and bacteria in the consortia are actually participating in the alcoholic fermentation and acetification process. The culturing of the microorganisms once acetification has started, is proving to be particularly challenging, due to the microorganisms having entered a ‘viable but not culturable’ state. MOTIVATION FOR A NEW PROJECT This current three year DAFF funded and Winetech evaluated project reached its original objectives. A consortia of yeast and bacteria are available for use for producing Balsamic styled vinegar from either standard grape juice or cooked grape juice. However, the data generated has led to new research questions that can be covered in a new two-three year project. This application is for the first year to address some of the new goals and will also allow the student to complete her MTech degree. One of the initial questions for the new project regards the microbiota used. Chemical monitoring of the combined fermentation/acetifications show the required development of ethanol and total acidity. However, it is not known if all the yeast and bacteria are metabolically active and to what degree they contribute to the final product. For a better understanding of the process and to avoid any potential economic losses by the use of unnecessary starter cultures, there is also a need to investigate the population dynamics occurring during production and maturation. This will allow tweaking the process and allow an improvement of quality. The new project will allow the aforementioned voids to be covered by investigating the science underlying the production processes.

ACKNOWLEDGEMENTS • The Department of Agriculture, Forestry and Fisheries (DAFF) and the Agricultural Research

Council (ARC Infruitec-Nietvoorbij) for funding; • The Agricultural Research Council (ARC Infruitec-Nietvoorbij) for infrastructure. • Winetech for administrative and scientific evaluation. In particular Mr Jan Booysen who was key

to initially obtaining funding from DAFF; • The CPUT students (National Diploma, BTech, MTech) and NRF-DST interns that participated at

various stages on the project; • ARC colleagues for administrative and research support; • The members of the sensory panels for critical evaluation of the vienagrs; • Prof. Albert Mas, University of Rovira i Virgili, Spain for acetic acid bacteria reference strains,

sensory training and expert advice.

3. Scope and geographical coverage of the project activities (Please indicate with a cross, X)

National

X Regional Stellenbosch, Western Cape

Please Specify:

32

Provincial

Please Specify:

Other

Please specify:

4. Current State of project (Please indicate with a cross, X)

Starting

Moderately advanced

Nearing Completion

Completed and published

X Completed and awaiting publication

Are the deliverables on schedule according to the SLA?: Yes Please highlight the list of deliverables that has been completed for the period of reporting.

If not, what is the reason: Remark on the nature of the constraint, whether is it surmountable and what is/are the envisaged change(s) to the project protocol:

5. Amounts (in Rands) spent on the project since its inception.

Total requested Actual expenditure Roll over

R538 033 (year 1)

R538 033 (year 2)

R538 033 (year 3)

R83 031

R880 370

R484 589

R455 022

R221 656

R0.00

33

5.1 Budget breakdown for the period of reporting

Category Budget Actual cost Total % spent Remaining

Labour 285 014.65 304 823.47 100%

Overheads 93 408.00 85 059.23 91%

Total labour + overheads 378 422.65 139 893.82

Travel 32 890.00 22 797.18 69%

Durable equipment - -

Consumables 73 276.00 71 908.76 98%

Total 106 166.00 94 705.95

GRAND TOTAL 484 588.65 484 588.65 100% 0

* Values in Table 5.1 as supplied by the Financial Office of ARC Infruitec-Nietvoorbij.

6. Partners/collaborators or the role played by the other

organisations/institutions (local and/ or foreign) in support of this

project. Indicate the kind of support (i.e. financial or technical expertise) received or to be received for this project.

6.1 Local Organisation/Institutes Type of support:

Expertise Financial (Rands)

Universities/Technikons/Colleges

Research Institutes (incl. non-profit)

Science Councils (e.g. HSRC, CSIR, etc.)

Private Companies/Business

Others (please specify):

Winetech (Wine industry funding body)

Submit annual

progress report to

Winetech.

0

6.2 Foreign/multilateral organisations/institutes

Type of support:

Expertise Financial (Rands)

Development agencies (i.e.DFID, GTZ, etc)

Others (please specify): University of Rovira i Virgili, Tarragona, Spain

Expertise and reference acetic acid bacteria.

0

7. The NUMBER of personnel in your Institute/division directly involved in this

project according to the categories below.

Personnel currently involved in R&D

Qualifications and Personnel

Categories

African Indian Coloured White South

African

TOTAL

M F M F M F F M Y *N M F

7.1 Researchers

34

Doctoral degree 1 1 1

Masters degree (MSc & MTech) 1 1 2 1 1

Honours degree or equivalent

Bachelors degree or equivalent 1 1 1

National Higher Diploma

Non-degree/non-formal qualifications

7.2 Technicians directly supporting this project All degree qualifications 2 1 1 4 0 4

Technical qualifications (Higher

Diplomas)

1 1 2 1 1

Other qualifications (incl. Non-formal) 1 1 1

Total 11 0 4 7

8. The NUMBER OF STAFF UNDER THE DIFFERENT AGE GROUPS that are

engaged in R&D activities for this project. Please follow the categories below.

R&D Staff Categories Age Categories

< 25 25-34 35-44 45-54 55-64 > 64 TOTAL

8.1 South African R&D Staff

Researchers 3 1 4

Technicians directly supporting R&D

Other staff directly supporting R&D

8.2 Non-South African R&D Staff Researchers

Technicians directly supporting R&D 5 1 1 7

Other staff directly supporting R&D

* This includes personnel with visa status and/or permanent and temporary work permits.

THANK YOU FOR YOUR COORPERATION