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International Journal of Trend in Scientific Research and Development (IJTSRD)
Volume: 3 | Issue: 2 | Jan-Feb 2019 Available Online: www.ijtsrd.com e-ISSN: 2456 - 6470
@ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 827
Novel Sources of Saccharomyces Species as
Leavening Agent in Bread Making
Umeh, S. O1., Okpalla, J.2, Okafor, J. N. C.1
1Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Nigeria
2Department of Microbiology, Chukwuemeka Odimegwu Ojukwu University Uli, Anambra, Nigeria
ABSTRACT
Mega biodiversity of Nigerian’s flora and fauna which include microorganism could be conserved and served as alternative
source forbaker’s yeast, the leavening agent in bread making. This study was conducted in attempt to exploit the potential of
Saccharomyces cerevisiae strains isolated from two local beverages (“burukutu” and “fura”) andatropicalfruit(shaddock)as a
leavening agent in bread making. The enrichment was carried out by fermenting the samples (burukutu, fura and Shaddock
juice) in medium containing sterilized grape must at 30°C for 3 days followed by isolation of tentative yeastsat30°C for3 days.
The samples tested showed the presence of yeasts and thirty (30) isolates from the three samples was selected for
identification of S. cerevisiae strains through biochemical andphysiologicaltests. Ofthethirty yeaststrainsidentified,12 strains
were chosen as baker’s yeast due to their potential to tolerate ethanol concentration of up to 13 % (v/v), grow at temperature
of 30 - 45°C and low or no hydrogen sulphide production. They are able to flocculate intensively, grow at high stress levels and
even showed better fermentative performance compared to commercial baker’s yeast(CY).These attributesthusindicatethat
the local beverages and fruits could be potential sources of indigenous S. cerevisiae strains for leaveningagentin breadmaking
and for other confectionary industries.
Keywords: Grape fruits, Burukutu, Fura, Shaddock, Bread making, Saccharomyces cerevisiae, leavening agent
INTRODUCTION
As a predominant species it is well known that
Saccharomyces cerevisiae plays an important role as a
leavening agent in bread making. The leavening step is
essential in the fermentationofdough.Itdoesnotonlyinduce
and increase the volume of dough through gas incorporation
buthelps creating the desiredflavorandtexture(Fleuryetal.,
2002). PreviousresearcherhavearguedthatS.cerevisiaedoes
notlive in other environmentexcept nature,butcanbefound
only in vinery environmentas a wild or domesticatedspecies
(Martini et al., 1996). In many instances, the strains are
clearly specializedfor use in the laboratory (Mortimer and
Johnston, 1986) and in the production of wine (Kunkee and
Bisson, 1993), beer (Hammond, 1993) and bread (Rose and
Vijayalakshmi, 1993). This has supported the common view
that S. cerevisiae is a domesticated species that has
continuously evolved in association with the production of
alcoholicbeverages(Mortimer,2000;Martini,1993;Naumov,
1996). Nowadays there are controversial arguments
regarding the nature of S. cerevisiae. Kurtzman and Fell
(1998) reported thatfruits, vegetables, drink andagriculture
products are among important microhabitats for yeast
species in thewild. Adequate work has been reported to look
at the potential of indigenous S. cerevisiae as leavening agent
during bread making from local sources.
The leavening agents (yeasts) currently used by bakery
industries in Nigeria are mostly imported fromEurope and
America. Thus, in this work the presence of yeasts from
Nigerian local beverages and fruits are exploited, thus S.
cerevisiae strains isolatedfrom them was characterizedtobe
used as baker’s yeast as leavening agent in bread making.
“Burukutu” and “Fura” are two cheap beverage drinks that
are produced by rural women using their local technology.
These beverages are produced and consumed all year round
and are always available. They are produced from local
sorghum and millet (Sorghum beer and Millet meal)
respectively. They cannot be preserved for a long time even
in the refrigerator due to their perishable nature. On the
other hand, shaddock fruits are wild fruits from wild grape
that has no recognizable use attributed to it. It ripes and
wastes in the forests. Due to the report of Kurtzman and Fell
(1998) thatfruits, vegetables, drink and agricultureproducts
areamong important microhabitats for yeast species in the
wild, the shaddock fruits were harvested, peeled and their
juice extracted and fermented to check if they contain wild
yeasts specifically Saccharomyces cerevisiae. Having isolated
the yeasts from these local sources, they were enriched and
tested for their ability to serve as baker’s yeast. The isolated
yeasts were compared with the attributes of commercial
baker’s yeast and found to be very close and even batterthan
the imported yeasts. They are fresh and directly isolated but
the imported yeasts had stayed for so long before reaching
the bakers. This work has recommended the isolation and
enrichment of yeasts from indigenous products to beused in
our baking industries as this will encourage our local
production of bread and other confectionaries.
MATERIALS AND METHODS
SAMPLE COLLECTION
Millet meal (Fura), Sorghum beer (Burukutu), Commercial
bakers’ yeast (Saccharomyces cerevisiae), flour and other
baking ingredients were purchased from Eke-Awka market,
Anambra State using sterile plastic containers and were
transported to the laboratory. Shaddock fruits were
harvested from a farm at Umuzocha Village Awka and
identified by a botanist in theDepartmentof Botany,Nnamdi
Azikiwe University Awka, Anambra State, Nigeria. Other
chemicals and reagents used were obtained from the
Department of Applied Microbiology and Brewing of the
same institution and are of analytical grade.
International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470
@ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 828
PREPERATION OF SAMPLES
The shaddock fruits were washed with water containing
sanitizers and peeled. The juice was aseptically extracted
using a hand juice extractor, filtered with muslin cloth and
the filtrate collected in a sterile plastic container.
ISOLATION OF THE YEASTS
The enrichment procedure to detect and isolate fermenting
yeast species from the collected samples was carried out by
adding 1 ml of each sample into high-sugar medium (grape
must, pH 3.2, with sugar added to a final concentration of
27%, w/v). All the micro-fermentations were carried out at
27°C in 100ml Erlenmeyer flasks containing 50 ml sterilized
grape must (Thais et al., 2006) and incubated for 3 days.
After incubation 1ml of each of the enriched samples was
serially diluted in triplicates rangingfrom101 to104 and 1ml
of each dilution was inoculated onto Sabouraud dextrose
agar (SDA) containing chloramphenicol (to avoid bacterial
growth) by a pour plate method and subsequentlyincubated
for 3 days. The colonies were then counted and selected
according totheirmorphologicalcharacteristicsasdescribed
by Martini et al. (1996). Tentative yeast isolates from the
SDA plates of 102and 104 dilutions were then sub- cultured
onto Yeast Peptone Dextrose (YPD) medium (10 g /L Yeast
Extract, 20 g/L Peptone, 20 g/L dextrose and 20g/L agar)
and incubated at 30°C for 3 days. Representative colonies
were picked from the plates and their pure cultures
preserved in slants and stored in the refrigerator at 40C.The
yeast isolates were coded as follows;
SY (shaddock yeast)
BY (burukutu yeast)
FY (fura yeast) and the commercial baker’s yeasts were
designated as CY.
MICROSCOPIC OBSERVATION OF THE YEASTS
MORPHOLOGY
A loop full of each colony of the isolates was supplemented
in a drop of sterile distilled water placed on glass slide and
smeared until the smear dry off. The smear was then stained
with dilute methylene blue dye, air dried and observed
under light microscope at 100 X magnification according to
Thais et al., 2006.
ETHANOL TOLERANCE OF THE ISOLATES
The ability of the isolates to grow in higher ethanol
concentrations was testedbygrowingthemin Yeast peptone
glucose(YPG) broth containing threedifferentconcentration
of ethanol (Thais et al., 2006). A loop full of each isolate was
inoculated into a freshly prepared YPG broth containing
different concentrationsofethanol10%,13% and15%(v/v)
respectively and observed after 72 hours.
TEMPERATURE TOLERANCE OF THE ISOLATED YEASTS
The ability of the yeast isolates to grow at higher
temperatures was verified by plating the yeast isolates onto
YPG medium and incubated at three different temperatures
(Thais et al., 2006). A loop full of each isolate was streaked
on a freshly prepared and dried YPG medium and incubated
at three different temperatures of 27oC, 37oC and 45ºC and
observed for 72 hours.
HYDROGEN SULFIDE PRODUCTION TEST
The ability of the yeast to produce hydrogen sulphide (H2S)
was examined by growing the yeast isolates on lead acetate
medium (40 g/L glucose, 5 g/L yeast extract, 3 g/L peptone,
0.2 g/L ammonium sulfate, 1 g/L lead acetate and 20 g/L
agar) and incubated 30ºC for 10 days (Ono et al., 1991).
FLOCCULATION ABILITY OF THE ISOLATES
In this test, isolates were inoculated in 10 ml of freshly
prepared YPG broth and incubated at 30ºC for 3 days (Thais
et al., 2006). A loop full of each isolates was inoculated intoa
freshly prepared YPG broth and observed for 72 hours after
which the tubes were agitated to observe flocculation
formation.
STRESS EXCLUSION TEST
Stress exclusion test was conducted as described by Thais et
al., (2006). The ability of the isolates to grow at different
stress conditions was done for 15 days’ incubation onto
different media. This was done by first growing the yeast
isolates onto Yeast Peptone Glucose (YPG) (10 g/L yeast
extract, 20 g/L peptone, 20 g/L glucose and 20 g/L agar)
medium at 30ºC for 3 days, then on YPG medium containing
8% (v/v) ethanol, YPG with 20% (w/v) glucose and then in
YPG medium supplemented with 2% (w/v) sucrose and 8%
(v/v) ethanol and incubated under the same conditions.
FERMENTATIVE CAPACITY OF THE ISOLATED YEAST
STRAINS
In this test, the fermentative capacity media was prepared
and the test was conducted as described by Atlas and Parks
(1996). Prior to yeast cells growth into a freshly prepared
Yeast fermentation broth (YFB) containing Peptone 7.5 g/L,
yeast extract 4.5g/L; 1ml of 1.6%(w/v)bromothymoblue as
an indicator, 6% (w/v) each of glucose, sucrose, fructose,
galactose, raffinose, lactose and maltose were autoclaved
separately. The yeast cells were grown at 30oC for 3 days.
The YFB was added with respective sugar, and then yeast
cells were examined on the fermentative ability of the
different carbon sources. The Durham tubes were also
placed into the media to trap the carbon (iv) oxide released
and the indicator to determine acid production by a colour
change (green to yellow) if the yeast cells have the ability to
ferment the respective sugar.
RESULTS
The following results were obtained from the study. Table 1
showed the effect of ethanol concentrations, different
temperatures, flocculation ability and Hydrogen sulphide
production of the isolated yeasts revealing their potential as
leavening agent. Table 2 presents theabilityof theisolates to
grow on different media and Table 3 showed the
fermentative capacity of the yeast on different carbon
sources.
International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470
@ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 829
Table 1: Effect of ethanol concentrations, temperatures, flocculation and hydrogen sulfide production of
Saccharomyces cerevisiae strains
Yeast
Strains
Ethanol Concentration
(V/V)
Temperature °C
Flocculation
Hydrogen Sulfide
Production
Potential To Be
Employed As Leavening
Agent10% 13% 15% 25 30 37 45
BY2 +++ ++ - +++ +++ +++ +++ +++ - Yes
BY3 +++ ++ - +++ +++ +++ +++ +++ - Yes
BY6 +++ ++ - +++ +++ +++ + +++ + Yes
BY8 +++ ++ - +++ +++ +++ + +++ + Yes
FY4 + + - +++ +++ ++ - +++ + Yes
FY7 ++ + - +++ +++ ++ - +++ + Yes
FY8 ++ + - +++ +++ ++ - +++ + Yes
FY9 +++ ++ - +++ +++ ++ +++ +++ - Yes
SY1 +++ ++ - +++ +++ ++ ++ +++ - Yes
SY3 +++ +++ - +++ +++ +++ - ++ + Yes
SY6 +++ +++ - +++ +++ +++ - ++ + Yes
SY10 +++ +++ - +++ +++ +++ +++ ++ - Yes
CY +++ ++ - +++ +++ +++ + ++ - Yes
Key: Intensive response (+++);moderate response (++);low response (+);no response (-);CY- Saccharomyces cerevisiae
commercial as control YPG – yeast peptone glucose medium YPS – yeast peptone sucrose medium Yes - potentially use as
leavening agent No – could not being use as leavening agent
Table 2: Stress exclusion tests on isolated yeasts for temperature and cell osmotic pressure in high concentration
of ethanol and sugar
Yeast
strains
Source
Growth on different Media Yeast strains
YPG
Temperature
37°C
Ethanol
(8%, v/v)
YPG
(20%, v/v)
YPS (20%,
w/v) + ETOH
(8%,v/v)
Potentially
use as
leavening agent
BY2 Burukutu +++ +++ +++ +++ ++ Yes
BY3 Burukutu +++ +++ +++ +++ +++ Yes
BY6 Burukuru +++ +++ +++ +++ +++ Yes
BY8 Burukutu +++ +++ +++ +++ ++ Yes
FY4 Fura +++ +++ +++ +++ +++ Yes
FY7 Fura +++ +++ +++ +++ ++ Yes
FY8 Fura +++ +++ +++ +++ ++ Yes
FY9 Fura +++ +++ +++ +++ +++ yes
SY1 Shaddock +++ +++ +++ +++ ++ Yes
SY3 Shaddock +++ +++ +++ +++ +++ Yes
SY6 Shaddock +++ +++ +++ +++ ++ Yes
SY10 Shaddock +++ +++ +++ +++ +++ Yes
CY Commercial yeast +++ +++ +++ +++ +++ yes
KEY: Intensive response (+++);moderate response (++);low response (+);no response (-);CY- Saccharomyces cerevisiae
commercial as control YPG – yeast peptone glucose medium YPS – yeast peptone sucrose medium Yes - potentially use as
leavening agent No – could not being use as leavening agent
Table 3 Fermentative capacity of the isolated yeasts
Yeasts
strains
Car
Glucose
bon
sources
Maltose
and carbon dioxide
relea Fructose
Sucrose
se in each sugar
fermentationa Lactose
Galactose Raffinose
Yeast strains identified as
Saccharomyces cerevisiae
BY2 + + + + - + + Yes
BY3 + + + + - + + Yes
BY6 + + + + - + *+ Yes
BY8 + + + + - + + Yes
FY4 + + + + - + *+ Yes
FY7 + + + + - + + Yes
FY8 + + + + - + + Yes
FY9 + + + + - + + Yes
SY1 + + + + - + + Yes
SY3 + + + + - + + Yes
SY6 + + + + - + *+ Yes
SY10 + + + + - + + Yes
C.Y
cerevisiae
+ + + + - + + Yes
Key: aAssimilate carbon and simultaneously release of carbon dioxide indicated (+); did not assimilate carbon source andnot
release carbon dioxide indicated as (-) Acidic fermentation (Broth turn into yellow), *Alkaline fermentation (Broth turn into
blue), Yes - potentially use as leavening agent , No – could not being use as leavening agent
International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470
@ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 830
DISCUSSIONS
Baker’s yeast is one of the most important ingredients in
baking industries. Without it the fermentation and leavening
of thedough will be impossible. The yeasts areimported into
Nigeria from foreign countries and are most times costly.
Since researcher had found out that our indigenous
beverages and ripe fruits are mostly fermentedbyyeastsand
other organisms, it is very needful that those yeasts to be
isolated and tested for use in different food industries.
Two local beverages and a fruit sample were examined for
the presence of indigenous Saccharomyces cerevisiae. Since
the media used to isolate the yeast strains were selective for
isolation of these specie only 30 colonies of tentative yeasts
was isolated. The isolation of S. cerevisiae in the study was
carried out using sterilized grape must (GM) broth and
followed by sub culturing in SDA. Biochemical and
physiological qualities are the important attributes of yeast
strains to be used as baker’s yeasts. During the courseofthis
study, the isolated yeasts were characterized and enriched
for their suitabilityasbaker’syeasts.The30coloniesofyeasts
isolated possess the morphological features of S. cerevisiae
species but 12 showed the attributes of baker’s yeast as
shown on the tables of results above. Of the30yeastcolonies,
18 isolates showed opaque, smooth,fluffy,regularcolonyand
creamy in colour while yeast strain isolated from BY2 and
BY3 showed similar characteristics except they were rough
compared to the others and the commercial S. cerevisiae.
Similar finding was observed by Kevin (2005) who reported
that typical S. cerevisiae colonies can be creamy or rough and
regular in shape. Colony formed by cells of wild S. cerevisiae
showed afluffy morphology as reported by Kockova (1990),
Cavalieri et al. (2001) and Kuthan et al. (2003). The
morphologicaldifferencesofyeasts colonies were verymuch
depending on the absence of an extracellular matrix, the
compactness ofcolonyandthedifferencesofcellshapewithin
the colony (Kuthan et al., 2003).
The cells morphology of thirteen yeast colonies observed
under a light microscope with100Xmagnificationshowedan
ellipsoid or ovoid shape which conformed to the
characteristic of S. cerevisiae isolated by Greame and Nia,
(2005). Ten of the yeast colonies have similar capability of
budding (budding ascus formed), however, yeast strains of
SY1 and SY10 (isolated from shaddock) showed extra
capability of budding which indicated a higher growth rate
when compared to other strains including the commercial
yeast but lack other characteristics of bakers yeasts. With
these characteristics shown, these strains should be able to
perform a vigorous fermentation, according to Hough et al.
(1971).
Asuitableconcentrationofalcoholisneededinbread making
in order to achieve the preferred flavor. As shown in Table1,
all yeast strains were able to grow in a medium containing
10%(v/v)ofethanol and13%(v/v)ofethanolconcentration,
while all the yeast strain including the commercial yeast
strain showed nogrowthat 15% ethanol concentration. High
concentration of alcohol is reportedtobetoxictotheyeastby
inhibiting the cells growth due to the destruction of the cell
membrane(IngramandButtke,1984).Inthisexperiment,the
highest concentration of ethanol that the commercial yeast
strain was able to survive was at 13% (v/v). Those strains
which were capable to grow in similar concentration were
expected tohaveability to producesimilarqualityofbreadas
commercial strain.
The selected yeast strains were alsotestedfortheirgrowthat
high temperature (Table 1). Most of the yeast strains could
tolerate the temperature up to 37°C. Yeaststrains of SY1 and
SY10 (isolated fromshaddock), FY9(isolatedfromfura),BY2
and BY3(isolated from burukutu) and CY (commercial yeast
strain) were able to grow at higher temperature of 45°C.
Thereforitcanbeconcludedthatthe yeasts isolated from the
beverages and fruit were able to grow at high ethanol
concentration (13%) and high temperatureof37°Cand45°C.
The flocculation abilities were also tested on the yeasts
strains. AccordingtoAmri et al. (1982) andMikietal.(1982),
yeast cells which have ability to flocculate caused by cell
adhesion process is an interesting characteristic in bread
making and brewing industry. Results showed all the chosen
strains isolated from “burukutu”and“fura”showedintensive
flocculation, those from shaddock and the commercial yeast
strain (CY)exhibitedmoderateflocculation as shown in Table1.
Theflocculation characteristicwas determined byyeastcells
sticking together and provides easy separation from the
broth medium. This phenomenon has an economic effect on
the production of yeast biomass because it can reduce the
energy cost during biomass centrifugation (Iraj et al., 2002).
In addition, flocculation properties of S. cerevisiae ensure a
high cell densityand large volume of harvested cells and also
able to rise the ethanol productivity during the fermentation
process (Kevin, 2005).
The production of hydrogen sulfide by twelve isolated S.
cerevisiae strains was determined during fermentation of
lead acetatemedium(LA).HenschkeandLee(1994)reported
that hydrogen sulfide productionpropertywasnotusefulfor
wine yeasts. Yeasts with elevated production of hydrogen
sulfide are undesirable for bread making because it confers
flavor and taste that compromise the quality of the bread.
However, in this study all yeast strains produced hydrogen
except commercial yeast (CY), BY2, BY3, FY9, SY1 and SY10
(Table 2). Since their hydrogen sulphide production were of
low response and other attributes in bread making are
acceptable, they can be used in the industry without their
effect being noticed.
In stress exclusion test, thirteen yeast colonies including
commercial baker’s yeast were examined in order to select
the strains which were able to adapt in bread making
conditions. Using strains which were noteffectively adapted
to stress condition could be a mistake due to impossible
settlementduringfermentationprocess(Thaisetal.,2006).In
allstress analyses,theyeastcellswereinitiallygrownonYPG
medium to ensure yeast were in the similar state condition,
followed by growing onto medium YPG incubated at 37oC,
8% (v/v) of ethanol, 20 % (w/v) of glucose and 20%(w/v)of
sucrose with 8%(v/v) of ethanol. The yeastcellsweregrown
continuously for 15 days to observe the cell viability due to
each stress condition. Strain survival of baker’s yeast under
various stressconditionscouldprovideusefulinformationon
its ability to grow and carry out fermentation as impaired
yeast. The impaired yeast growth (Ivorraetal.,1999)during
the fermentation usually does not grow in optimal
conditions and continuously exposed to several stress
especially to osmotic and ethanol stress (Queroletal.,2003).
All the thirteen yeast strains including commercial yeast
showed the necessary characteristics suitable for bread
making as indicated in Table 2. Our findings were in
agreement with that of Pataro et al. (2000) who reported
that most of S. cerevisiae strains isolated from conventional
International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470
@ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 831
fermentation processes were physiologically adapted to
extreme conditions. In this case the strains wereabletogrow
on medium(YP) containing 20% (w/v)glucoseand8%(v/v)
ethanol and incubated at 37°C. Since the yeast cells in
bread making produce ethanol as secondarymetabolitesthe
ethanol stress were conducted to observe its tolerance to
ethanol.
The primary role of baker’s yeast in dough development,
fermentative capacity using carbon source simultaneously
with the productionofcarbondioxideisimportantparameter
for bread making (Benitez et al., 1996). All yeast strains
tested were identified as S. cerevisiae due to its ability to
ferment sucrose, maltose, fructose, glucose, galactose and
raffinosebutnotonlactose(Thaisetal.,2006).Table3 shows
the fermentative ability of thirteen S. cerevisiae strains
including commercial strain using7differentcarbonsources.
S. cerevisiae strains of BY2, BY3, FY9, SY1 and SY10 showed
their ability to change the broth medium color from green to
yellow (forsucrose,maltose,fructose,glucoseandgalactose)
within 2-5 hour fermentation. But other strains took more
than 24 hours incubation timetoproduce thesimilar results.
This indicated thattheywentthroughacidicfermentationand
was able to initiate the fermentation process faster than the
commercial strain. The commercial S. cerevisiae strain was
acidicfermenting yeast andthecolorchangestook18hoursof
fermentation. In contrast, for carbon source of raffinose S.
cerevisiae strains BY6, FY4, and SY6 showed the changes of
color from green to blue, which indicated an alkaline
fermentation. All yeast strains including commercial strain
did not ferment lactose. Tarek (2001) reported the S.
cerevisiae cells which were unable to ferment lactose were
actually due to lack of lactase or β-galactosidase system. It
was also showed that all yeast strains which utilized
respective sugars also produced carbon dioxide (Table 3).
The carbon dioxide released during dough fermentation
process is prominent as a leavening agent of dough (Thais et
al., 2006).
CONCLUSIONS
In conclusion, local beverages (burukutu and fura) and fruit
(shaddock) could be sources of novel Saccharomyces
cerevisiaewhichcanbepotentiallyusedasleaveningagentfor
bread and other confectionary makings in our food
industries.
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[25] Pataro, C., Guerra, J. B., Petrillo-Peixoto, M. L.,
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[27] Rose, A. H. and Vijayalakshmi, G. (1993) In: Rose, A. H.
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[28] Tarek, M. E-N. (2001) Immobilization of recombinant
strainsofSaccharomycescerevisiaeforthehydrolysisof
lactose in salted Domiati cheese whey. European Food
Research and Technology 212:225-227
[29] Thais, M., Danilo G. and Tania, M.B. (2006) Isolation
and characterization of Saccharomyces cerevisiae
strains of winery interest. Brazilian Journal of
Pharmaceutical Sciences.42:119-126

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Novel Sources of Saccharomyces Species as Leavening Agent in Bread Making

  • 1. International Journal of Trend in Scientific Research and Development (IJTSRD) Volume: 3 | Issue: 2 | Jan-Feb 2019 Available Online: www.ijtsrd.com e-ISSN: 2456 - 6470 @ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 827 Novel Sources of Saccharomyces Species as Leavening Agent in Bread Making Umeh, S. O1., Okpalla, J.2, Okafor, J. N. C.1 1Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Nigeria 2Department of Microbiology, Chukwuemeka Odimegwu Ojukwu University Uli, Anambra, Nigeria ABSTRACT Mega biodiversity of Nigerian’s flora and fauna which include microorganism could be conserved and served as alternative source forbaker’s yeast, the leavening agent in bread making. This study was conducted in attempt to exploit the potential of Saccharomyces cerevisiae strains isolated from two local beverages (“burukutu” and “fura”) andatropicalfruit(shaddock)as a leavening agent in bread making. The enrichment was carried out by fermenting the samples (burukutu, fura and Shaddock juice) in medium containing sterilized grape must at 30°C for 3 days followed by isolation of tentative yeastsat30°C for3 days. The samples tested showed the presence of yeasts and thirty (30) isolates from the three samples was selected for identification of S. cerevisiae strains through biochemical andphysiologicaltests. Ofthethirty yeaststrainsidentified,12 strains were chosen as baker’s yeast due to their potential to tolerate ethanol concentration of up to 13 % (v/v), grow at temperature of 30 - 45°C and low or no hydrogen sulphide production. They are able to flocculate intensively, grow at high stress levels and even showed better fermentative performance compared to commercial baker’s yeast(CY).These attributesthusindicatethat the local beverages and fruits could be potential sources of indigenous S. cerevisiae strains for leaveningagentin breadmaking and for other confectionary industries. Keywords: Grape fruits, Burukutu, Fura, Shaddock, Bread making, Saccharomyces cerevisiae, leavening agent INTRODUCTION As a predominant species it is well known that Saccharomyces cerevisiae plays an important role as a leavening agent in bread making. The leavening step is essential in the fermentationofdough.Itdoesnotonlyinduce and increase the volume of dough through gas incorporation buthelps creating the desiredflavorandtexture(Fleuryetal., 2002). PreviousresearcherhavearguedthatS.cerevisiaedoes notlive in other environmentexcept nature,butcanbefound only in vinery environmentas a wild or domesticatedspecies (Martini et al., 1996). In many instances, the strains are clearly specializedfor use in the laboratory (Mortimer and Johnston, 1986) and in the production of wine (Kunkee and Bisson, 1993), beer (Hammond, 1993) and bread (Rose and Vijayalakshmi, 1993). This has supported the common view that S. cerevisiae is a domesticated species that has continuously evolved in association with the production of alcoholicbeverages(Mortimer,2000;Martini,1993;Naumov, 1996). Nowadays there are controversial arguments regarding the nature of S. cerevisiae. Kurtzman and Fell (1998) reported thatfruits, vegetables, drink andagriculture products are among important microhabitats for yeast species in thewild. Adequate work has been reported to look at the potential of indigenous S. cerevisiae as leavening agent during bread making from local sources. The leavening agents (yeasts) currently used by bakery industries in Nigeria are mostly imported fromEurope and America. Thus, in this work the presence of yeasts from Nigerian local beverages and fruits are exploited, thus S. cerevisiae strains isolatedfrom them was characterizedtobe used as baker’s yeast as leavening agent in bread making. “Burukutu” and “Fura” are two cheap beverage drinks that are produced by rural women using their local technology. These beverages are produced and consumed all year round and are always available. They are produced from local sorghum and millet (Sorghum beer and Millet meal) respectively. They cannot be preserved for a long time even in the refrigerator due to their perishable nature. On the other hand, shaddock fruits are wild fruits from wild grape that has no recognizable use attributed to it. It ripes and wastes in the forests. Due to the report of Kurtzman and Fell (1998) thatfruits, vegetables, drink and agricultureproducts areamong important microhabitats for yeast species in the wild, the shaddock fruits were harvested, peeled and their juice extracted and fermented to check if they contain wild yeasts specifically Saccharomyces cerevisiae. Having isolated the yeasts from these local sources, they were enriched and tested for their ability to serve as baker’s yeast. The isolated yeasts were compared with the attributes of commercial baker’s yeast and found to be very close and even batterthan the imported yeasts. They are fresh and directly isolated but the imported yeasts had stayed for so long before reaching the bakers. This work has recommended the isolation and enrichment of yeasts from indigenous products to beused in our baking industries as this will encourage our local production of bread and other confectionaries. MATERIALS AND METHODS SAMPLE COLLECTION Millet meal (Fura), Sorghum beer (Burukutu), Commercial bakers’ yeast (Saccharomyces cerevisiae), flour and other baking ingredients were purchased from Eke-Awka market, Anambra State using sterile plastic containers and were transported to the laboratory. Shaddock fruits were harvested from a farm at Umuzocha Village Awka and identified by a botanist in theDepartmentof Botany,Nnamdi Azikiwe University Awka, Anambra State, Nigeria. Other chemicals and reagents used were obtained from the Department of Applied Microbiology and Brewing of the same institution and are of analytical grade.
  • 2. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 828 PREPERATION OF SAMPLES The shaddock fruits were washed with water containing sanitizers and peeled. The juice was aseptically extracted using a hand juice extractor, filtered with muslin cloth and the filtrate collected in a sterile plastic container. ISOLATION OF THE YEASTS The enrichment procedure to detect and isolate fermenting yeast species from the collected samples was carried out by adding 1 ml of each sample into high-sugar medium (grape must, pH 3.2, with sugar added to a final concentration of 27%, w/v). All the micro-fermentations were carried out at 27°C in 100ml Erlenmeyer flasks containing 50 ml sterilized grape must (Thais et al., 2006) and incubated for 3 days. After incubation 1ml of each of the enriched samples was serially diluted in triplicates rangingfrom101 to104 and 1ml of each dilution was inoculated onto Sabouraud dextrose agar (SDA) containing chloramphenicol (to avoid bacterial growth) by a pour plate method and subsequentlyincubated for 3 days. The colonies were then counted and selected according totheirmorphologicalcharacteristicsasdescribed by Martini et al. (1996). Tentative yeast isolates from the SDA plates of 102and 104 dilutions were then sub- cultured onto Yeast Peptone Dextrose (YPD) medium (10 g /L Yeast Extract, 20 g/L Peptone, 20 g/L dextrose and 20g/L agar) and incubated at 30°C for 3 days. Representative colonies were picked from the plates and their pure cultures preserved in slants and stored in the refrigerator at 40C.The yeast isolates were coded as follows; SY (shaddock yeast) BY (burukutu yeast) FY (fura yeast) and the commercial baker’s yeasts were designated as CY. MICROSCOPIC OBSERVATION OF THE YEASTS MORPHOLOGY A loop full of each colony of the isolates was supplemented in a drop of sterile distilled water placed on glass slide and smeared until the smear dry off. The smear was then stained with dilute methylene blue dye, air dried and observed under light microscope at 100 X magnification according to Thais et al., 2006. ETHANOL TOLERANCE OF THE ISOLATES The ability of the isolates to grow in higher ethanol concentrations was testedbygrowingthemin Yeast peptone glucose(YPG) broth containing threedifferentconcentration of ethanol (Thais et al., 2006). A loop full of each isolate was inoculated into a freshly prepared YPG broth containing different concentrationsofethanol10%,13% and15%(v/v) respectively and observed after 72 hours. TEMPERATURE TOLERANCE OF THE ISOLATED YEASTS The ability of the yeast isolates to grow at higher temperatures was verified by plating the yeast isolates onto YPG medium and incubated at three different temperatures (Thais et al., 2006). A loop full of each isolate was streaked on a freshly prepared and dried YPG medium and incubated at three different temperatures of 27oC, 37oC and 45ºC and observed for 72 hours. HYDROGEN SULFIDE PRODUCTION TEST The ability of the yeast to produce hydrogen sulphide (H2S) was examined by growing the yeast isolates on lead acetate medium (40 g/L glucose, 5 g/L yeast extract, 3 g/L peptone, 0.2 g/L ammonium sulfate, 1 g/L lead acetate and 20 g/L agar) and incubated 30ºC for 10 days (Ono et al., 1991). FLOCCULATION ABILITY OF THE ISOLATES In this test, isolates were inoculated in 10 ml of freshly prepared YPG broth and incubated at 30ºC for 3 days (Thais et al., 2006). A loop full of each isolates was inoculated intoa freshly prepared YPG broth and observed for 72 hours after which the tubes were agitated to observe flocculation formation. STRESS EXCLUSION TEST Stress exclusion test was conducted as described by Thais et al., (2006). The ability of the isolates to grow at different stress conditions was done for 15 days’ incubation onto different media. This was done by first growing the yeast isolates onto Yeast Peptone Glucose (YPG) (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 20 g/L agar) medium at 30ºC for 3 days, then on YPG medium containing 8% (v/v) ethanol, YPG with 20% (w/v) glucose and then in YPG medium supplemented with 2% (w/v) sucrose and 8% (v/v) ethanol and incubated under the same conditions. FERMENTATIVE CAPACITY OF THE ISOLATED YEAST STRAINS In this test, the fermentative capacity media was prepared and the test was conducted as described by Atlas and Parks (1996). Prior to yeast cells growth into a freshly prepared Yeast fermentation broth (YFB) containing Peptone 7.5 g/L, yeast extract 4.5g/L; 1ml of 1.6%(w/v)bromothymoblue as an indicator, 6% (w/v) each of glucose, sucrose, fructose, galactose, raffinose, lactose and maltose were autoclaved separately. The yeast cells were grown at 30oC for 3 days. The YFB was added with respective sugar, and then yeast cells were examined on the fermentative ability of the different carbon sources. The Durham tubes were also placed into the media to trap the carbon (iv) oxide released and the indicator to determine acid production by a colour change (green to yellow) if the yeast cells have the ability to ferment the respective sugar. RESULTS The following results were obtained from the study. Table 1 showed the effect of ethanol concentrations, different temperatures, flocculation ability and Hydrogen sulphide production of the isolated yeasts revealing their potential as leavening agent. Table 2 presents theabilityof theisolates to grow on different media and Table 3 showed the fermentative capacity of the yeast on different carbon sources.
  • 3. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 829 Table 1: Effect of ethanol concentrations, temperatures, flocculation and hydrogen sulfide production of Saccharomyces cerevisiae strains Yeast Strains Ethanol Concentration (V/V) Temperature °C Flocculation Hydrogen Sulfide Production Potential To Be Employed As Leavening Agent10% 13% 15% 25 30 37 45 BY2 +++ ++ - +++ +++ +++ +++ +++ - Yes BY3 +++ ++ - +++ +++ +++ +++ +++ - Yes BY6 +++ ++ - +++ +++ +++ + +++ + Yes BY8 +++ ++ - +++ +++ +++ + +++ + Yes FY4 + + - +++ +++ ++ - +++ + Yes FY7 ++ + - +++ +++ ++ - +++ + Yes FY8 ++ + - +++ +++ ++ - +++ + Yes FY9 +++ ++ - +++ +++ ++ +++ +++ - Yes SY1 +++ ++ - +++ +++ ++ ++ +++ - Yes SY3 +++ +++ - +++ +++ +++ - ++ + Yes SY6 +++ +++ - +++ +++ +++ - ++ + Yes SY10 +++ +++ - +++ +++ +++ +++ ++ - Yes CY +++ ++ - +++ +++ +++ + ++ - Yes Key: Intensive response (+++);moderate response (++);low response (+);no response (-);CY- Saccharomyces cerevisiae commercial as control YPG – yeast peptone glucose medium YPS – yeast peptone sucrose medium Yes - potentially use as leavening agent No – could not being use as leavening agent Table 2: Stress exclusion tests on isolated yeasts for temperature and cell osmotic pressure in high concentration of ethanol and sugar Yeast strains Source Growth on different Media Yeast strains YPG Temperature 37°C Ethanol (8%, v/v) YPG (20%, v/v) YPS (20%, w/v) + ETOH (8%,v/v) Potentially use as leavening agent BY2 Burukutu +++ +++ +++ +++ ++ Yes BY3 Burukutu +++ +++ +++ +++ +++ Yes BY6 Burukuru +++ +++ +++ +++ +++ Yes BY8 Burukutu +++ +++ +++ +++ ++ Yes FY4 Fura +++ +++ +++ +++ +++ Yes FY7 Fura +++ +++ +++ +++ ++ Yes FY8 Fura +++ +++ +++ +++ ++ Yes FY9 Fura +++ +++ +++ +++ +++ yes SY1 Shaddock +++ +++ +++ +++ ++ Yes SY3 Shaddock +++ +++ +++ +++ +++ Yes SY6 Shaddock +++ +++ +++ +++ ++ Yes SY10 Shaddock +++ +++ +++ +++ +++ Yes CY Commercial yeast +++ +++ +++ +++ +++ yes KEY: Intensive response (+++);moderate response (++);low response (+);no response (-);CY- Saccharomyces cerevisiae commercial as control YPG – yeast peptone glucose medium YPS – yeast peptone sucrose medium Yes - potentially use as leavening agent No – could not being use as leavening agent Table 3 Fermentative capacity of the isolated yeasts Yeasts strains Car Glucose bon sources Maltose and carbon dioxide relea Fructose Sucrose se in each sugar fermentationa Lactose Galactose Raffinose Yeast strains identified as Saccharomyces cerevisiae BY2 + + + + - + + Yes BY3 + + + + - + + Yes BY6 + + + + - + *+ Yes BY8 + + + + - + + Yes FY4 + + + + - + *+ Yes FY7 + + + + - + + Yes FY8 + + + + - + + Yes FY9 + + + + - + + Yes SY1 + + + + - + + Yes SY3 + + + + - + + Yes SY6 + + + + - + *+ Yes SY10 + + + + - + + Yes C.Y cerevisiae + + + + - + + Yes Key: aAssimilate carbon and simultaneously release of carbon dioxide indicated (+); did not assimilate carbon source andnot release carbon dioxide indicated as (-) Acidic fermentation (Broth turn into yellow), *Alkaline fermentation (Broth turn into blue), Yes - potentially use as leavening agent , No – could not being use as leavening agent
  • 4. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 830 DISCUSSIONS Baker’s yeast is one of the most important ingredients in baking industries. Without it the fermentation and leavening of thedough will be impossible. The yeasts areimported into Nigeria from foreign countries and are most times costly. Since researcher had found out that our indigenous beverages and ripe fruits are mostly fermentedbyyeastsand other organisms, it is very needful that those yeasts to be isolated and tested for use in different food industries. Two local beverages and a fruit sample were examined for the presence of indigenous Saccharomyces cerevisiae. Since the media used to isolate the yeast strains were selective for isolation of these specie only 30 colonies of tentative yeasts was isolated. The isolation of S. cerevisiae in the study was carried out using sterilized grape must (GM) broth and followed by sub culturing in SDA. Biochemical and physiological qualities are the important attributes of yeast strains to be used as baker’s yeasts. During the courseofthis study, the isolated yeasts were characterized and enriched for their suitabilityasbaker’syeasts.The30coloniesofyeasts isolated possess the morphological features of S. cerevisiae species but 12 showed the attributes of baker’s yeast as shown on the tables of results above. Of the30yeastcolonies, 18 isolates showed opaque, smooth,fluffy,regularcolonyand creamy in colour while yeast strain isolated from BY2 and BY3 showed similar characteristics except they were rough compared to the others and the commercial S. cerevisiae. Similar finding was observed by Kevin (2005) who reported that typical S. cerevisiae colonies can be creamy or rough and regular in shape. Colony formed by cells of wild S. cerevisiae showed afluffy morphology as reported by Kockova (1990), Cavalieri et al. (2001) and Kuthan et al. (2003). The morphologicaldifferencesofyeasts colonies were verymuch depending on the absence of an extracellular matrix, the compactness ofcolonyandthedifferencesofcellshapewithin the colony (Kuthan et al., 2003). The cells morphology of thirteen yeast colonies observed under a light microscope with100Xmagnificationshowedan ellipsoid or ovoid shape which conformed to the characteristic of S. cerevisiae isolated by Greame and Nia, (2005). Ten of the yeast colonies have similar capability of budding (budding ascus formed), however, yeast strains of SY1 and SY10 (isolated from shaddock) showed extra capability of budding which indicated a higher growth rate when compared to other strains including the commercial yeast but lack other characteristics of bakers yeasts. With these characteristics shown, these strains should be able to perform a vigorous fermentation, according to Hough et al. (1971). Asuitableconcentrationofalcoholisneededinbread making in order to achieve the preferred flavor. As shown in Table1, all yeast strains were able to grow in a medium containing 10%(v/v)ofethanol and13%(v/v)ofethanolconcentration, while all the yeast strain including the commercial yeast strain showed nogrowthat 15% ethanol concentration. High concentration of alcohol is reportedtobetoxictotheyeastby inhibiting the cells growth due to the destruction of the cell membrane(IngramandButtke,1984).Inthisexperiment,the highest concentration of ethanol that the commercial yeast strain was able to survive was at 13% (v/v). Those strains which were capable to grow in similar concentration were expected tohaveability to producesimilarqualityofbreadas commercial strain. The selected yeast strains were alsotestedfortheirgrowthat high temperature (Table 1). Most of the yeast strains could tolerate the temperature up to 37°C. Yeaststrains of SY1 and SY10 (isolated fromshaddock), FY9(isolatedfromfura),BY2 and BY3(isolated from burukutu) and CY (commercial yeast strain) were able to grow at higher temperature of 45°C. Thereforitcanbeconcludedthatthe yeasts isolated from the beverages and fruit were able to grow at high ethanol concentration (13%) and high temperatureof37°Cand45°C. The flocculation abilities were also tested on the yeasts strains. AccordingtoAmri et al. (1982) andMikietal.(1982), yeast cells which have ability to flocculate caused by cell adhesion process is an interesting characteristic in bread making and brewing industry. Results showed all the chosen strains isolated from “burukutu”and“fura”showedintensive flocculation, those from shaddock and the commercial yeast strain (CY)exhibitedmoderateflocculation as shown in Table1. Theflocculation characteristicwas determined byyeastcells sticking together and provides easy separation from the broth medium. This phenomenon has an economic effect on the production of yeast biomass because it can reduce the energy cost during biomass centrifugation (Iraj et al., 2002). In addition, flocculation properties of S. cerevisiae ensure a high cell densityand large volume of harvested cells and also able to rise the ethanol productivity during the fermentation process (Kevin, 2005). The production of hydrogen sulfide by twelve isolated S. cerevisiae strains was determined during fermentation of lead acetatemedium(LA).HenschkeandLee(1994)reported that hydrogen sulfide productionpropertywasnotusefulfor wine yeasts. Yeasts with elevated production of hydrogen sulfide are undesirable for bread making because it confers flavor and taste that compromise the quality of the bread. However, in this study all yeast strains produced hydrogen except commercial yeast (CY), BY2, BY3, FY9, SY1 and SY10 (Table 2). Since their hydrogen sulphide production were of low response and other attributes in bread making are acceptable, they can be used in the industry without their effect being noticed. In stress exclusion test, thirteen yeast colonies including commercial baker’s yeast were examined in order to select the strains which were able to adapt in bread making conditions. Using strains which were noteffectively adapted to stress condition could be a mistake due to impossible settlementduringfermentationprocess(Thaisetal.,2006).In allstress analyses,theyeastcellswereinitiallygrownonYPG medium to ensure yeast were in the similar state condition, followed by growing onto medium YPG incubated at 37oC, 8% (v/v) of ethanol, 20 % (w/v) of glucose and 20%(w/v)of sucrose with 8%(v/v) of ethanol. The yeastcellsweregrown continuously for 15 days to observe the cell viability due to each stress condition. Strain survival of baker’s yeast under various stressconditionscouldprovideusefulinformationon its ability to grow and carry out fermentation as impaired yeast. The impaired yeast growth (Ivorraetal.,1999)during the fermentation usually does not grow in optimal conditions and continuously exposed to several stress especially to osmotic and ethanol stress (Queroletal.,2003). All the thirteen yeast strains including commercial yeast showed the necessary characteristics suitable for bread making as indicated in Table 2. Our findings were in agreement with that of Pataro et al. (2000) who reported that most of S. cerevisiae strains isolated from conventional
  • 5. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Reference Paper ID – IJTSRD21526 | Volume – 3 | Issue – 2 | Jan-Feb 2019 Page: 831 fermentation processes were physiologically adapted to extreme conditions. In this case the strains wereabletogrow on medium(YP) containing 20% (w/v)glucoseand8%(v/v) ethanol and incubated at 37°C. Since the yeast cells in bread making produce ethanol as secondarymetabolitesthe ethanol stress were conducted to observe its tolerance to ethanol. The primary role of baker’s yeast in dough development, fermentative capacity using carbon source simultaneously with the productionofcarbondioxideisimportantparameter for bread making (Benitez et al., 1996). All yeast strains tested were identified as S. cerevisiae due to its ability to ferment sucrose, maltose, fructose, glucose, galactose and raffinosebutnotonlactose(Thaisetal.,2006).Table3 shows the fermentative ability of thirteen S. cerevisiae strains including commercial strain using7differentcarbonsources. S. cerevisiae strains of BY2, BY3, FY9, SY1 and SY10 showed their ability to change the broth medium color from green to yellow (forsucrose,maltose,fructose,glucoseandgalactose) within 2-5 hour fermentation. But other strains took more than 24 hours incubation timetoproduce thesimilar results. This indicated thattheywentthroughacidicfermentationand was able to initiate the fermentation process faster than the commercial strain. The commercial S. cerevisiae strain was acidicfermenting yeast andthecolorchangestook18hoursof fermentation. In contrast, for carbon source of raffinose S. cerevisiae strains BY6, FY4, and SY6 showed the changes of color from green to blue, which indicated an alkaline fermentation. All yeast strains including commercial strain did not ferment lactose. Tarek (2001) reported the S. cerevisiae cells which were unable to ferment lactose were actually due to lack of lactase or β-galactosidase system. It was also showed that all yeast strains which utilized respective sugars also produced carbon dioxide (Table 3). The carbon dioxide released during dough fermentation process is prominent as a leavening agent of dough (Thais et al., 2006). CONCLUSIONS In conclusion, local beverages (burukutu and fura) and fruit (shaddock) could be sources of novel Saccharomyces cerevisiaewhichcanbepotentiallyusedasleaveningagentfor bread and other confectionary makings in our food industries. REFERENCES [1] Amri, M. 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