International Journal of Food Properties

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A comparative study on different methods for the

evaluation of baker’s yeast bioactivity

Aziz Homayouni Rad & Zahra Kasaie

To cite this article: Aziz Homayouni Rad & Zahra Kasaie (2017) A comparative study on different
methods for the evaluation of baker’s yeast bioactivity, International Journal of Food Properties,
20:1, 100-106, DOI: 10.1080/10942912.2016.1141297

To link to this article: https://doi.org/10.1080/10942912.2016.1141297

© 2017 Taylor & Francis Group, LLC

Accepted author version posted online: 17

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INTERNATIONAL JOURNAL OF FOOD PROPERTIES
2017, VOL. 20, NO. 1, 100–106
http://dx.doi.org/10.1080/10942912.2016.1141297

A comparative study on different methods for the evaluation of

baker’s yeast bioactivity
Aziz Homayouni Rada and Zahra Kasaieb
a
Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran; bEast Azerbaijan Research and Education
Center for Agriculture and Natural Resources, Tabriz, Iran

ABSTRACT ARTICLE HISTORY

Four samples of instant yeast powder were evaluated by epifluorescence light Received 15 September 2015
microscopy, microbial tests, gasography, the Henry-Simon pressure meter, and Accepted 9 January 2016.
baking. All of the results showed that yeast (A) showed the highest number of
KEYWORDS
green cells (178 ± 7), the highest number of viable yeast (15 × 1010 cfu/mg), the
Baker’s yeast;
highest amount of CO2 production (163 ± 2 mL/3 h and 875 ± 3 mbar/3 h), and Epifluorescence light
the highest volume (132 ± 1 cm3) and height of the bread (5.0 ± 0.3 cm) due to microscopy; Bioactivity;
its higher bioactivity when compared to types B, C, and D (p < 0.05). The Gasography; Henry-Simon
correlation study within the methods demonstrated that the Henry-Simon pressure meter
pressure meter was a faster, easier, inexpensive, and more accurate method
than others. Therefore, it could be considered as an appropriate method to
evaluate the fermentation activity of baker’s yeast.

Introduction
Saccharomyces cerevisiae has been a key ingredient in bread making. It is responsible for the
leavening of the dough, as well as for the formation of desired sensorial characteristics.[1]
Leavening activity by carbon dioxide (CO2) is an important quality index in the baker’s yeast
industry.[2] The most widely recognized role of yeasts is ascribable to their fermenting ability in
bread. Since CO2 is produced during fermentation by yeasts, measuring the volume of gas produced
can be a useful measurement for the evaluation of active yeast.[3] Baker’s yeast is required to have
high-leavening capability to ensure high-quality baking products.[4,5] The success of the technologi-
cal process in bread making is conditioned by the formation of gas in the final hours of the
technological process.[6] In several methods frequently used to evaluate the fermentation activity
of baker’s yeast, one of them measures the time required for dough to attain a given volume and the
other measures the volume of the dough for a given proof time.[4,5] The latter attribute is most
conveniently determined by measuring the volume increase of fermenting dough, whereas gas
production can be estimated by any of several available procedures, such as the oven rise recorder
method, alveography method, and pressure meter methods. The viability of yeast is important in
industrial microbiology. Traditional methods of assessing viability based on counting of colony
forming units (CFUs) typically take between 24 and 48 h, and are too slow for practical uses in
bakeries.[7] The sensitivity of this method tends to be low; in addition, the time necessary for
culturing the microorganisms makes it impossible to use the results for process control.
Alternatively, direct observation of stained cells by microscopy can be used to rapidly assess yeast
viability. The most widely used stain to measure yeast viability is the methylene blue. Viability
measurement using flow cytometry and fluorescent vital staining provides a rapid and objective
approach to directly measuring yeast cell viability.[8]

CONTACT Zahra Kasaie Tabrizu1986@yahoo.com East Azerbaijan Research and Education Center for Agriculture and Natural
Resources, Abbasi Avenue, Tabriz, Ghol-Ghasht Ave., P.O. Box 14711, Tabriz 5166813843, I. R. Iran.
Color versions of one or more figures in the article can be found online at www.tandfonline.com/ljfp.
© 2017 Taylor & Francis Group, LLC
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 101

Because of the extreme importance of yeast bioactivity, it has attracted the attention of scientists
and their efforts come in two main parts: The first part reviews the amount of CO2 produced by the
yeast and the other is to examine the viability of them. The measurement of gas production amount
was done by assessing the volume and pressure of gas production.[9] Also, microscopic techniques
and microbiologically tests have been used for counting and observation of yeasts viability using
various methods and special media cultures. Presently, there are few articles that have been
published on yeast viability. It was shown as a positive correlate between dough volume increasing
and capabilities of yeast viability, which can be demonstrated by methylene blue staining and
electrophoresis.[10] Several staining methods, in both fluorescent and non-fluorescent, are available
for determining the viability of yeast cells, but there is no absolute method. A staining protocol is
reported for the assessment of viability in yeast by using the fluorescent method.[11] Flow cytometric
methods for assessing cell viability conducted frequently by using one or more fluorescent probes for
parameters such as membrane integrity, enzymatic activity, nucleic acid content, and membrane
potential.[12]
This study focused on comparison of different methods for the evaluation bioactivity of
Saccharomyces cerevisiae and assesses the correlation between different methods. Another purpose
of this study is to present an appropriate technique to measuring baker’s yeast fermentation activity.
The aim of this investigation is to help industrial bakers and allow for researchers to select the best
way to evaluate the fermentation activity of baker’s yeast.

Material and Methods

Four samples of instant dry yeast were obtained from the various companies of Iran including Fariman
(Mashhad, Iran), Razavy (Mashhad, Iran), Golmaye (Tabriz, Iran), and Dezmaye (Ahvaz, Iran) that were
named A, B, C, and D. Flour was obtained from the Athar Company (Maragheh, Iran).

Evaluation of Chemical Properties of Flour

American Association of Cereal Chemists (AACC) approved measuring methods were used to assess
the amounts of moisture content, ash, wet gluten, and Zeleny sedimentation, respectively, following:
44–16, 09–07, 38–12, 56–11.

Gasography Test
A direct measurement of the dough volume as a result of the CO2 production during proofing
was carried out using a simple measuring device. Immediately after processing, a small piece of
dough (5 g) was gently rounded and placed inside a flask, which was submerged in a water
bath at a constant temperature. The flask is connected via tubing to an inverted graded
cylinder to collect the produced CO2. To avoid any CO2 dissolution in water, the water in
the beaker was acidified to a pH of 2.5. The temperature of water bath was kept constant at 40°
C to mimic the dough proofing process. The amount of gas produced was reported as cubic
centimeters for 5 g of dough. The gas volume was recorded for the four samples of yeasts every
minute during total testing time (180 min).

Henry-Simon Pressure Meter

The amount of CO2 pressure that yeasts produced during 180 min was measured. Yeast (0.38 g), 10 g
flour, and 2 mL of distilled water were added and mixed together. The water temperature was 30°C.
The mixture of flour, water, and yeast as a dough was rounded into a small ball and placed into a
pressure meter. The door of the pressure meter closed quickly. The Henry-Simon pressure meter was
102 A. H. RAD AND Z. KASAIE

placed in a water bath at 30°C. Then, each hour, the gauge of pressure meter was read and reported
as mbar for a total of 180 min.

Epifluorescence Light Microscopy Test

In this method, yeast suspensions were prepared and standardized (1 g yeast powder dissolved in 100
cc distillated water then diluted to 10−6), then the slide samples were stained with fluorescein di-
acetate (FDA) and were observed using an epifluorescence microscope with a 100-W Hg vapor arc
lamp as a light source. The active yeast cells in the suspension appeared brilliant green. The yeast–
water suspension was stained with FDA in a tube and was gently smeared out onto an object glass.
The suspension of yeast cells was filtered through a membrane, and the yeast cells collected on the
filter were stained and the number of active yeast cells was counted. Cells were counted within a
10 mm2 area. Real color images were acquired using a two-stage Peltier cooled charge-coupled
device (CCD) camera (Nikon, Eclipse, Evolution MP) with a spectral range of 290–1000 nm. The
dynamic range of red, green, and blue was 14 bit. Staining was performed by addition of 0.02% FDA
per 1 mL of sample and held for 10 min at room temperature in the dark.

Microbial Test
Yeast survivability is determined as CFUs per gram of dry matter. A microbial test can be used to
measure the viability and survivability of yeast cells. Twenty-five grams of yeast was mixed with 175 mL
of water. The viability of the suspensions was checked by plate counting. Yeast cell suspensions were
counted on yeast extract-glucose-chloramphenicol (YGC) agar (YGC Agar, Merck) after 5 days of
incubation at 25°C. Logarithmic dilutions were carried out in saline, and the diluted suspensions were
cultured on YGC agar and incubated at 25°C for 5 days.

Baking Test
With the aim of correlation between yeast viability results with bread volume and height, all of the
dough samples were baked. The loaves were prepared using 530 g of flour, 280 mL of water, 1.5%
yeast solution, and 5 g of sodium chloride. Fermentation was carried out at 30°C for 85 min with a
relative humidity of 80%. Cooking time was 25 min at 200°C. Volume and height were recorded in
all loaves. We used the rapeseed displacement method for measuring the volume in the bread. For
measuring the height, after the cut, the bread height is calculated by a measuring instrument.

Results and Discussions

Wheat Flour Properties
The amounts of moisture content, ash, wet gluten, and Zeleny sedimentation is summarized in
Table 1. Flour humidity is acceptable and the amount of wet gluten showed that the flour protein
quality is mediocre. This flour, as a standard base, was used to formulate the dough from all of the
yeast samples.

Table 1. Mean values of wheat flour properties.

Moisture content Ash Wet gluten Zeleny sedimentation
12.7 ± 0.01 0.58 ± 0.01 22.12 ± 0.01 23.6 ± 0.02
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 103

Gas producion volume average (ml/3h)

163 162

154

139

Yeast A Yeast B Yeast C Yeast D

Yeast sample

Figure 1. The effect of yeasts type on the gas production volume (mL/3 h). Different letters indicate a significant difference (p <
0.05) between the treatments.

879
Gas production presure average (mbar/3h)

874

813

772

Yeast A Yeast B Yeast C Yeast D

Yeast sample

Figure 2. The effect of yeasts type on the gas production pressure (mbar/3 h). Different letters indicate a significant difference (p <
0.05) between the treatments.

Gasography and Henry-Simon Pressure Meter

The results with four samples of yeasts (A, B, C, and D) was obtained on a gasograph and a Henry-
Simon pressure meter and are presented in Figs. 1 and 2. As shown in these figures, for all yeasts, the
gas production rate increased over time, but it increase in yeast A more than other samples. The
yeast gas production power was: A > B > C > D. Yeast sample A has the best fermentative activity
due to its higher bioactivity. In both tests there was no significant difference between yeasts A and B.
But yeasts C and D have significant differences in the level of 5% with yeasts A and B. Therefore, a
higher bioactivity of yeast leads to higher gas production ability.

Correlation Between Gasography Methods and Henry-Simon Pressure Meter

The results of gasography and the Henry-Simon pressure meter with four treatments and three
replications during 180 min were obtained from Statistical Analysis System data analysis and shown
a high possetive correlation together (0.98***). The correlation between these two methods is
expressed by the following equation:
G ¼ 0:17373P þ 15:8235
where P stands for pressure meter and G stands for gasograph.
The Henry-Simon pressure meter method is easier and less expensive than the gasography
method. With the above equation (G = 0.17373P + 15.8235), and without needing to gasograph, it
can be achived by pressure meter data and changed milibar data to milliliter.
104 A. H. RAD AND Z. KASAIE

Figure 3. Live yeasts in yeast–water suspention shown in epifluorescence light microscopy after staining with FDA. Magnification: × 10.

Epifluorescence Light Microscopy Test

The images obtained from epifluorescence light microscopy (Fig. 3) confirmed that gasography and the
Henry-Simon pressure meter results revealed that yeast A had more living cells than the other yeasts,
and yeast D had lowest number of living cells. The yeast population that fluoresced green was shown to
be viable (>95%). Yeast vitality had a direct correlation with their gas production ability. Comparing
the vitality of yeasts showed that the yeast vitality was: A > B > C > D. Therefore, yeast sample A was
the best sample for the bread-dough making industry. The more vitality of the yeast (more cells that
fluoresced green in epifluorescence light microscopy test) leads to more bioactivity and gas powering.

Counting of Live Yeasts

Counting the numbers of live yeast was performed. The counting of live yeasts was obtained by
epifluorescence light microscopy done in five replicates at the microscopic field of 650 × 850
microns. Results showed that a high number of green cells after staining with FDA lead to high
gas powering ability of yeasts. The counting of live yeasts showed that live the yeast number was: A >
B > C > D (A: 178 ± 7, B: 163 ± 8, C: 78 ± 9, and D: 48 ± 8). The results represent the means of
duplicate measurements. Therefore, the yeast sample A was the best sample for the bread-dough
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 105

Table 2. Pearson’s simple correlation between all of the tests.

Test name Henry simon Gasography Microbial test Epifluorescence light microscopy Bread volume
Gasography 0.98***
Microbial test 0.96** 0.97**
Epifluorescence light microscopy 0.95** 0.95** 0.97**
Bread volume 0.94** 0.97** 0.97*** 0.96**
Bread height 0.95** 0.96** 0.94** 0.95** 0.98***
***Significant level of 0.001. **Significant level of 0.01. *Significant level of 0.01. Ns: non-significant.

making industry. The higher of a number of live yeast means more viability and bioactivity which
leads to more gaspowering.

Microbial Test
Colony counting of the samples showed that the number of cfu/mg yeasts is different between them.
Yeast A has a higher number of cfu/mg than others and yeast D has the lowest. The gasography and
epifluorescence light microscopy (EFLM) tests confirmed the results of the microbial test. In normal
conditions, if the cfu/mg in the dry matter of the yeast is higher than others, that yeast has a higher
amount of live cells, and has a higher gas production power than other yeasts. These results are
similar to Autio et al.[3] The microbial test showed that a high number of CFUs per mg dry matter of
yeasts leads to a higher performance of them. Colony counting of the samples was: A > B > C > D
(A: 15 × 1010, B: 14.6 × 1010, C: 12 × 1010, and D: 10 × 1010). The results represent the means of
duplicate measurements. Therefore, yeast sample A was the best sample for the bread-dough making
industry. So if the CFUs per milligram in the dry matter of yeast is higher than others, that yeast has
a higher amount of live cells, and had a higher gas production power than the other yeasts.

Bread Baking: Comparing Breads Height and Volumes

Baking test results also confirmed the previous tests results. Yeast A has the highest volume and
height of the breads, and yeast D has lowest volume and height of the breads. This result showed a
positive direct correlation between yeast survivability, the amount of cfu/mg, yeast gas production
power, and the height and volume of the breads. The heights of the breads were: A ˃ B ˃ C ˃ D (A:
4.8 ± 0.3 cm, B: 4.6 ± 0.3 cm, C: 3.8 ± 0.2 cm, and D: 3.1 ± 0.2 cm) and the volume of the breads
were: A ˃ B ˃ C ˃ D (A: 132 ± 10, B: 127 ± 6, C: 112 ± 5, and D: 102 ± 6). Therefore, yeast sample A
was the best sample for the bread-dough making industry.

Pearson’s Simple Correlation

As shown in Table 2 all of the tests have a positive correlation together, but the Henry-Simon
pressure meter is faster, easier, less expensive, and more accurate than the other methods. So
according to the statistical studies, the Henry-Simon’s pressure meter is presented as the best way
to evaluate the fermentation activity of baker’s yeast.

Conclusions
The total comparison of the gasography, the microbial tests (cfu/mg), the EFLM test (microscopy
staining), and the baking test showed the same results. In all of the tests, yeast A had the best
fermentation activity because of its higher bioactivity and survivability when compared with the
others. According to the very high positive correlation between different methods, it can be said that
there is an appropriate method for assessing fermentation activity of baker’s yeast without needing to
baking process. The Henry-Simon pressure meter is faster, easier, less expensive, and more accurate
106 A. H. RAD AND Z. KASAIE

than other methods. Therefore, according to the statistical studies, the Henry-Simon pressure meter
is presented as the most appropriate method to evaluate the fermentation activity of baker’s yeast.

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