ALCOHOL CONCENTRATION IN MEAD DURING FERMENTATION
WITH SACCHAROMYCES CEREVISIAE
A. C. Şarba1*, A.Timar2, L. Al. Mărghitaş1
1
University of Agricultural Sciences and Veterinary Medicine,
3-5, Mănăştur, Cluj-Napoca,CJ- 400372, Romania
2
University of Oradea, Environmental Protection Faculty,
Gen. Magheru 26, Oradea, BH- 410058, Romania,
E-mail: andreea.sarba@yahoo.com
Abstract. The alcohol content in mead varies between 8% and 18% vol and it can range drastically from one batch
to other depending by the quality of honey. This is the main reason why it is of extreme importance to take account the
characteristics of the honey, and supplements used in the fermentation environment. Sugar content represents the potential of
mead in alcohol content. Sugar profile of the honey was determinated following a HPLC-RID method. pH was determinated
by automatic titration. Alcohol content was determinated according to official methods. Alcohol content curve reflects the
fermentation process stages. However it is of extreme importance to take into account the characteristics of the honey.
Keywords:honey, honey fermentation, mead, alcohol content.
AIMS
The aim of this study was to determinate the ethyl alcohol content curve during the
honey fermentation process in order to obtain honey wine known as mead. The fermentation
rate depends on honey characteristics, yeast strain, yeast nutrition and pH. There is a lack of
scientific information about honey-must fermentations but it is accepted by mead makers that
mead quality improvement includes the development of the proper additive formulation and
optimization of fermentation conditions. Nevertheless, research is still needed on the
physiology and metabolism of Saccharomyces cerevisiae under the particular harsh honeymusts environment (Ref. 1). Our purpose was to determinate the limits of the yeast strain
fermentation conditions regarding pH and ethyl alcohol levels.
BACKGROUND
The melliferous resources must be rationally used in order to efficiency the bee forage
of stationary and pastoral apiaries. A correct distribution of these require an assessment of the
honey yield, to ensure an optimum load of bee colonies/unit area of foraging and for a correct
*
For correspondence.
knowledge of the melliferous resources. Exploitation of natural resources available locally in
rural areas involve the mobilisation and the promotion of traditional activities such as
beekeeping, by the decisions on the development and implementation of strategies for
sustainable development of rural communities (Ref. 2). Mead represents an alternative to
develop new products from honey, optimizing to it’s full economical and nutritional potential.
The high content in glucose and fructose, which are easily assimilated, confers to honey
excellent energy and nutritional values. As fructose is metabolised without needing insulin,
honey is a product that is recommended for diabetics. Moreover, honey protects the digestive
tract and favours the absorption of iron in the intestine. Honey has successfully replaced sugar
substitutes. It has been proven that honey helps us assimilate calcium and magnesium, which
are essential elements in bone structure formation. Some studies have signalled an antianemic
effect and an increase of haemoglobin in blood, an improvement of health in general, an
increased appetite and increasing weight (Ref. 3). These are the main reasons why honey wine
can be considered a functional food. Also, it is very important that the main material is
according to the legal environment and quality parameters agreed by national and
international legislation. Trasability must be able to be made anytime. That’s why the
collecting process is an important step in mead making technology. Maintaining the
contamination level within accessible, minimum limits lead to a linear and constant process
when it comes to ensuring quality and food safety. Thus, the harvesting, collecting,
manufacturing and storing of honey are a field in a continuous evolution and adjustment to
the European legislation, industry needs and the harsh market competition (Ref. 4). Connected
to this we have developed an complex technology to produce a fermented product of good
quality including a sterilizing feature in the technologic diagram. This process does not affect
the physical and chemical properties of honey (Ref. 5-6).
EXPERIMENTAL
The fermented product was obtained from a fluid multi floral honey sample obtained from a
local beekeeper. Characteristics and quality were confirmed in accordance with the limits
established by the Council Directive 2001/110/EC (Ref. 7). All honeys share certain general
characteristics, including a moisture content below 20%, a sugar content of 70-80%, an ash
content ranging from 0,1% to 0,2%, and a pH between 3,8 and 4.7 (Ref. 8-10).
The
individual glucidic spectrum for the multi floral honey was determinate with HPLC according
to the following method HPLC-RID method on a Shimadzu instrument with refractive index
detector, amino silica gel modified column Alltima Amino (100 A, 5μm, 250mm×4.6 mm),
using a mixture of acetilonitrile and water as mobile phase (75:25 v/v). Injection volume was
20μL, column pressure 6.3 MPa, and 1mL/min the flow rate of the mobile phase. Sugar
spectrum for glucose, fructose, sucrose, turanose, maltose, trehalose, isomaltose and erlose
was injected separately and in mixture in the range of 0-50% concentration, depending on the
amount present in honey, for calibration curve and linearity range test. Linear regression
factor for calibration curves has been greater than 0,9982 for all of sugars. The honey must
was prepared by diluting 250 g of honey in 750 ml of spring water in glass bottles with
fermentation tubes. Yeast from the Saccharomyces cerevisiae strain, used in wine and beer
production, has also been used as a starter in mead production (Ref. 11). Selected yeast
Saccharomyces cerevisiae was used to inoculate the honey must (0,2g/l) under sanitary
conditions. The yeast was added to 100 ml of must in a 150 ml Erlenmeyer flask and plugged
with sterile cotton and incubated at 28ºC to allow the culture to grow. In some studies, in
order to optimise honey must preparation for mead production the must has been
supplemented with potassium tartrate, malic acid and diammonium phosphate and were be
able to reduce the fermentation time to 11 days (Ref. 12). In order for the fermentation to have
place, after 5 hours the yeast was poured in the honey must along with a fermentation starter
mix 0,26 g/l (ammonium sulphate 61,8%, ammonium phosphate dibasic 33%, potassium
bicarbonate 5%, vitamin B1 0,2%). This mix is used to supply the yeast and enrich the honey
must for a healthy growth and fermentation of the yeast. pH was determinate for the honey
must and during the fermentation process. Alcohol content was determinate during the
fermentation process by using the SR 184-2:2010 standard method for ethyl alcohol and
alcoholic drinks (determination of alcoholic concentration) with Kjeldal steam distillation
unit.
RESULTS AND DISCUSSIONS
The principle of alcoholic fermentation is to convert sugars into carbon dioxide and
ethyl alcohol. Preparation of this alcoholic beverage is dependent of the raw material. In
almost all honey types, fructose and glucose predominate. These two sugars account for
nearly 85-95% of the honey carbohydrates (Ref. 13) which are fermented into ethyl alcohol.
The glucidic spectrum also showed quantities of turanose, maltose, trehalose, isomaltose,
melesitose. After fermentation the quantities of sugars are quantified in small amounts in the
final product. The most important decrease in content is identified to be for fructose, being the
main sugar and the precursor for the fermentation process.
Table 1. Content of 7 different and total sugar in honey, honey must and fermented final
product
Monitoring pH level during the fermentation represents the indicator of fermentation
process. The initial pH (3,82) decreased during fermentation process down to (3,31). pH
values and alcoholic fermentation are strongly connected as long as yeasts can’t ferment at
low pH.
Table 2. Alcohol content and pH level
CONCLUSIONS
Low pH, among other factors, has been pointed out as one cause of sluggish or
premature fermentation arrest in alcoholic beverage (Ref. 14). Monitoring pH after honey
must preparation and during fermentation it is an important issue to prevent premature
fermentation arrest and incomplete sugar breakdown. As expected, the pH of the fermenting
honey must decreased as the fermentation process occurred due to the changes in the organic
acids spectrum (Ref. 15). pH level fall constantly along with ethyl alcohol grow levels.
Fig. 1. Alcohol content during fermentation
Fig. 2. pH levels during fermentation
In the figures it can be observed that honey must became more acidic as the yeast
starts to metabolize sugars.
It has occurred an important decrease in sugar content during fermentation, especially
fructose and glucose. Sucrose and erlose were completely consumed by yeasts during the
fermentation. Also it was prove that the yeast strain used, Saccharomyces cerevisiae, is
suitable to ferment an acidic environment such as honey must because can adapted but only to
a point. In our experiment the extreme pH drop was 3.1. At this point the yeast is inhibited.
Yeast can also be inhibited by the level of ethanol. The yeast used in this study,
Saccharomyces cerevisiae, proved to have high alcohol tolerance by fermenting the honey
must up to 12,96% (v/v).
Even if fermentation rate depends on the honey variety and it’s characteristics, through
proper selection of yeast strain and fermentation conditions, such as mixing during
fermentation, yeast nutrition and pH’s control, it may be possible to dramatically increase
fermentation rate (Ref. 16).
Acknowledgements
This paper was published under the frame of European Social Found, Human
Resources
Development
Operational
Programme
2007-2013,
project
no. POSDRU/159/1.5/S/132765
REFERENCES
1. A. MENDES-FERREIRA, F. COSME, C. BARBOSA, V. FALCO, A. INÊS, A.
MENDES-FAIA: Optimization of honey-must preparation and alcoholic fermentation
by Saccharomyces cerevisiae for mead production, International Journal of Food
Microbiology, 144, 193-198, (2010).
2. D. JITARIU, M. POPESCU, I. MOISE, C. URDEA. Rural sustainable development
opportunity by exploiting the melliferous resources – case study: the Ciucurova
village, Tulcea County, Romania. Journal of Environmental Protection and Ecology
15, No 3, 1074-1085, (2014).
3. M. POPA, R. AXINTE, S. VARVARA. Considerations regarding the quality of honey
on heating and storage – changes in hydroxymethylfurfuraldehyde content of the
honey from Transylvania (Romania), Journal of Environmental Protection and
Ecology 11, No. 2, 555-561, (2010).
4. M. POPA, M. VICA, R. AXINTE, M. GLEVITZKY, S. VARVARA. Correlations on
the microbiological and physicochemical characteristics of different types of honey,
Journal of Environmental Protection and Ecology 10, No. 4, 1113-1121, (2009).
5. A. C. ŞARBA, L. Al. MĂRGHITAŞ, A. TIMAR. Honey sterilization and mead
production, Bulletin USAMV Animal Science and Biotechnologies, 71(2), 313-314,
(2014).
6. A. C. ŞARBA, L. Al. MĂRGHITAŞ, A. TIMAR: UV sterilization of honey,
University of Oradea Anals, Ecotoxicology, Zootechnie and Food Industry, vol.
XIII/B, pp. 241-244, (2014).
7. COUNCIL DIRECTIVE 2001/110/EC of 20 December 2001 relating the honey. Off.
J. Eur. Union 2002, L10, 47-52.
8. T. NAGAI, R. INOUE, N. KANAMORI, N. SUZUKI, T. NAGASHIMA:
Characterization of honey from different floral sources. It’s functional properties and
effects of honey species on storage of meat, Food Chemistry, 97, 256-262, (2006).
9. S. OUCHEMOUKH, H. LOUAILECHE, P. SCHWEITZER: Physicochemical
characteristics and pollen spectrum of some Algerian honeys, Food Control, 18, 52-58,
(2007).
10. L.R. SILVA, R. VEIDEIRA, A.P. MONTEIRO, P. VALENTÃO, P.B. ANDRADE:
Honey from Luso region (Portugal): Physicochemical characteristics and mineral
contents, Microchemical Journal 93, 73-77, (2009).
11. A. ROLAND, G.C.J. VAN MUISWINKEL, C. LASANTA, V. PALACIOS, I.
CARO: Influence of pollen addition on mead elaboration: Physicochemical sensory
characteristics, Food Chemistry 126, 574-582, (2011).
12. A. MENDES-FERREIRA, C. BARBOSA, V. FALCO, C. LEÃO, A. MENDESFAIA: The production of hydrogen sulphide and other aroma compounda by wine
strains of Saccharomyces cerevisiae in synthetic media with different nitrogen
concentrations, Journal of Industrial Microbiology and Biotechnology 36, 571-583,
(2009).
13. M.S. FINOLA, M.C. LASAGNO, J.M. MARIOLI: Microbiological and chemical
characterization of honeys from central Argentina, Food Chemistry 100, 1649-1653,
(2007).
14. L.F. BISSON: Stuck and sluggish fermentations. American Journal of Enology and
Viticulture 50, 107-119, (1999).
15. G.I. DEZMIREAN, L.AL. MĂRGHITAŞ, O. BOBIŞ, D.S. DEZMIREAN, V.
BONTA, S. ERLER: Botanical origin causes changes in nutritional profile and
antioxidant activity of fermented products obtained from honey, Journal of
Agricultural and Food Chemistry, 60, 8028-8025, (2012).
16. E. RAMALHOSA, T. GOMES, A.P. PEREIRA, T. DIAS, L.M. ESTEVINHO: Mead
production: tradition versus modernity, Advances in food and nutrition research,
63:101-18, (2011).
Table 1. Content of 7 different and total sugar in honey, honey must and fermented final product
Fructose Glucose Sucrose Turanose Maltose Trehalose Isomaltose
Erlose
H
43,25
27,97
0,15
2,53
1,67
0,82
0,79
1,3
HM
11,506
7,793
0,044
0,681
0,476
0,199
0,201
0,454
FP-M 0,728
0,063
0,597
0,3
0,208
0,217
H – honey; HM – honey must; FP-M – final product - mead.
Table 2. Alcohol content and pH level
DOF
1
2
3
5
7
10
11
14
Alcohol content
ρ
c % (v/v)
0,9997
0,21
0,9978
1,48
0,9958
2,88
0,9938
4,24
0,9914
6,12
0,9888
8,16
0,9878
8,96
0,9866
9,94
pH
DOF
3,82
3,80
17
21
25
29
32
35
37
40
3,77
3,57
3,53
Alcohol content
ρ
c % (v/v)
0,9856
10,78
0,9851
11,17
0,9849
11,39
0,9846
11,64
0,9843
11,92
0,9839
12,25
0,9834
12,69
0,99831
12,96
pH
3,47
3,42
3,38
3,31
DOF – Day of fermentation; ρ – relative density; c% (v/v) – alcohol content at 20°C.
Figure 1. Alcohol content during fermentation
Alcohol content during fermentation
Day of fermentation
45
40
35
30
25
20
15
10
5
0
0,21 1,48 2,88 4,24 6,12 8,16 8,96 9,94 10,78 11,17 11,39 11,64 11,92 12,25 12,69 12,96
alcohol content
Figure 2. pH levels during fermentation
pH levels during fermentation
3.9
3.8
3.7
PH LEVEL
3.6
3.5
3.4
3.3
3.2
3.1
3
1
2
5
10
14
ph
21
29
35
40