mead production: selection and characterization assays of

A.P. Pereira1, T Dias 1, J. Andrade1, E. Ramalhosa 1, A. Mendes-Ferreira 2, A. Mendes-Faia 2 , and M.L. Estevinho 1

Mead production: selection and characterization assays of Saccharomyces cerevisiae strains

1 CIMO- Centro de Investigação de Montanha/Escola Superior Agrária de Bragança, Quinta de Sta. Apolónia, Apartado 1172, 5301-855 Bragança, Portugal2 Institute for Biotechnology and Bioengineering-Centro de Genética e Biotecnologia-IBB/ CGB -Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

3

Mead is a traditional alcoholic drink which results from the fermentation of diluted honey. Yeasts used in mead production are, usually, wine Saccharomyces cerevisiae strains.Most of these yeasts are not adapted to the conditions of mead production namely, high sugar levels, low pH values and reduced nitrogen concentrations. The inability of yeaststrains to respond and adapt to unfavorable stressful growth conditions, leads to several problems, such as lack of uniformity of the final product, delays and “pouts”fermentations, as well as the production of off-flavors by the yeasts. Therefore, it is necessary to find yeast strains more suitable for mead production.

BACKGROUND

AIMS

METHODS

The main objectives of this work were to select, from honey, the most appropriate indigenous yeasts for of mead production and to optimize fermentation conditions.

YEAST ISOLATION YEAST IDENTIFICATION CHARACTERIZATION OF TWO HONEY SAMPLES USED INMEAD PRODUCTION

Stress resistance to ethanol, sulfur dioxide and osmoticstress were evaluated in seven Saccharomyces cerevisiaestrains (five isolated from honey, a commercial wine strain and

MEAD PRODUCTION a laboratory strain), using YPD medium.

For mead production, two S. cerevisiae yeasts strains isolatedfrom honey, randomly selected, and the commercial winestrain were used. The fermentation media obtained from twodifferent honeys (a dark and a light honeys) were enrichedwith two different supplements, a commercial one or onedeveloped by the research team.

Honey samples

Rhodotorula mucilaginosa

17%

Saccharomyces cerevisiae

8%

Zygosaccharomyces rouxii

8%

Pichia membranaefaciens

4%

Zygosaccharomyces mellis13% Candida sorbosivorans

8%

RESULTSYEAST SPECIES ISOLATED FROM HONEY SAMPLES YEAST TOLERANCE TO ETHANOL, SO2 AND OSMOTIC STRESS

Strain

μ (h-1)

ControlSO2 10% (v/v)

ethanol20% glucose +20% fructose100 mg/L 250mg/L

1 (honey) 0.18 0.18 0.18 0.10 0.18

2 (honey) 0.16 0.17 0.17 0.11 0.20

3 (reference) 0.18 0.17 0.17 0.10 0.19

4 (honey) 0.18 0.18 0.18 0.10 0.201000

m1

TAB

Z.ro

uxii

ESA5

1

5B3

M1

M70

M12

5

7ER

ESA1

7

ESA1

1

1Lisa

2Lisa

1694

-P01

1

1694

-P01

5

m2

10001000

m1

TAB

Z.ro

uxii

ESA5

1

5B3

M1

M70

M12

5

7ER

ESA1

7

ESA1

1

1Lisa

2Lisa

1694

-P01

1

1694

-P01

5

m2

Trichosporon mucoides4%

Candida parapsilosis13%

Candida magnoliae25%

Size of the PCR-amplified rDNA region of some yeast strains

5 (honey) 0.17 0.18 0.18 0.09 0.19

6 (honey) 0.17 0.17 0.17 0.10 0.19

7 (commercial) 0.17 0.18 0.17 0.10 0.19

μ -Specific growth rates of the strains in absence and presence of SO2 (100 and 250 mg/L), ethanol (10%) and sugars (20% glucose + 20% fructose

CHARACTERIZATION OF HONEYS FOR MEAD PRODUCTIONGROWTH AND FERMENTATION KINETICS

1000

750

500

250

500

400

300

200

100

1000

750

500

250

500

400

300

200

100

1000

750

500

250

500

400

300

200

100

4

5

6

80

100

120

Ln (g/L)

Dark honey enriched with supplement 1

4

5

6

80

100

120

Ln (g/L)

Dark honey enriched with supplement 2

Pollen

Erica (61.91%)Castanea (14.28%)

Lavandula (14.28%)Rubus (9.53%)

Lavandula (52.0%) Trifolium (24.0%)Rubus (16.0%)Others (8.0%)

Moisture (%) 16.80 16.20

pH 4.90 3.84

Acidity (meq.Ac/Kg) 30.00 23.00

Diastase Index 14.60 8.65

HMF (mg/Kg) 3.59 16.02

Electrical Conductance (mS/cm) 0.77 0.32

Total Ashes (%) 0.55 0.17

Reducing Sugars (%) 71 43 68 03

0

1

2

3

0

20

40

60

0 24 48 72 96 120 144 168 192 216

(OD x 10)

[ ] (g

Time (hours)

0

1

2

3

0

20

40

60

0 24 48 72 96 120 144 168 192 216

(OD x 10)

[ ] (g

Time (hours)

2

3

4

5

6

40

60

80

100

120

Ln (OD x 10)

[ ] (g

/L)

Light honey enriched with supplement 1

2

3

4

5

6

40

60

80

100

120

Ln (OD x 10)

[ ] (g

/L)

Light honey enriched with supplement 2

GlucoseFructoseEthanolGlicerolAcetic acidBiomass

This work was supported by FCT project PTDC/AGR-ALI/68284/2006

Reducing Sugars (%) 71.43 68.03

CONCLUSIONSThe results obtained with the three of S. cerevisiae indicate that the success of mead production greatly depends on the fermentation medium composition, namely on the type ofhoney, as well as on the formulation of the supplements used.

0

1

2

0

20

40

0 24 48 72 96 120 144 168 192 216

)

Time (hours)

0

1

2

0

20

40

0 24 48 72 96 120 144 168 192 216

)

Time (hours)