XXI SIMPÓSIO NACIONAL DE BIOPROCESSOS

XII SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSA

3 a 6 de setembro de 2017
Aracaju, Sergipe, Brasil

SOYBEAN MOLASSES AS SUBSTRATE AND PREBIOTIC SOURCE FOR

GROWTH OF PROBIOTIC Saccharomyces boulardii

Maria Helena Queiroz1, Ismael Maciel de Mancilha2, Juan Daniel Rivaldi1,3*

1
Centro Universitário Teresa D’Ávila, Lorena-SP, Brasil
2
Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena-SP, Brasil
3
Facultad de Ciencias Químicas, Universidad Nacional de Asunción, San Lorenzo, Paraguay
E-mail: danielrivaldi@gmail.com

ABSTRACT

The purpose of this study was to evaluate the growth of therapeutic relevance yeast
Saccharomyces boulardii in a medium containing soybean molasses, a byproduct of protein
extraction, rich in sucrose and oligosaccharides (raffinose and stachyose). Commercial lyophilized
cell of the yeast strain was inoculated in medium containing 80 g/L soy molasses and cultivated in a
cylindrical glass reactor at 0.5 and 1.5 vvm of sterile air flux. The highest cell concentration (14.8
g/L) and the best biochemical parameters were obtained at the highest aeration rate. The results
show the potential of the soybean molasses as feedstock for yeast growth and future formulation of
a symbiotic product.

1. INTRODUCTION
Brazil, as the second largest soybean producer in the world, accounts 30% of the total
soybean production, which has been transformed mainly in oil, protein and derivatives industrial
products. Soy molasses, a byproduct of soy industries, is a brown viscous liquid containing
carbohydrates (dextrose, sucrose, fructose, stachyose and raffinose), proteins, flavonoids and
minerals. Currently, soy molasses results from protein extraction from defatted soybean meal using
an aqueous solution of ethanol (70%) as solvent, followed by evaporation and distillation. The
residues of distilled process are concentrated into a viscous liquid to yield soy molasses (Fig 1).
Biotechnological processes require inexpensive substrate and nutrient source to minimize
the cost of medium formulation and achieve economic viability. In this context, soy molasses
represents an alternative substrate for microbial growth and biomolecules production. Nowadays,
the most important studies on soybean molasses utilization have been focused on ethanol
production using Saccharomyces cerevisiae as a biological agent (Siqueira et al., 2008).
On the other hand, several microorganisms, including species of bacteria and yeast have
received much attention due to their probiotic characteristics, regarding its nutritional and
therapeutic benefits to the human and animal host (Rivaldi et al., 2013).
 XXI SIMPÓSIO NACIONAL DE BIOPROCESSOS
XII SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSA

3 a 6 de setembro de 2017
Aracaju, Sergipe, Brasil

Figure 1. Integrated process for protein extraction and soybean Figure 2. Schematic representation of the
molasses production cultivation system. 1. Air pump, 2. Filter,
3. Glass reactor, 4. Magnetic stirrer 5.
Recovery flask
The efficacy of the yeast Saccharomyces boulardii as a probiotic microorganism for the
prevention and therapy of gastrointestinal disorders has been demonstrated (Kelesidis,
Pothoulakis, 2012). Currently there are several commercial products formulated with this yeast
strain. Therefore, the objective of the present study was to evaluate soybean molasses as substrate
for formulation of a growth medium of the mentioned probiotic yeast at different aeration
conditions.

2. MATERIAL AND METHODS

Soybean molasses was kindly donated by the industry Selecta S.A located at Araguari, Minas
Gerais State, Brazil. The soybean molasses contained sucrose, 199 g/kg; fructose 26 g/kg; glucose 6
g/kg; raffinose 50 g/kg, stachyose 120 g/Kg, crude protein 50 g/kg and trace of minerals (calcium,
sodium, magnesium, potassium and sulfur). Lyophilized commercial yeast Saccharomyces boulardii
that presents probiotic properties was utilized in this study.
The reaction system (Fig.2) consisted of a cylindrical glass reactor (Ø = 5.0 cm, H= 20 cm)
with a working volume of 250-mL. The growth medium formulation contained soy molasses 80 g/L
at pH 5.5 and added of ceftriaxone sodium (0.1 g/L) as antibiotic and silicone oil as antifoam agent.
Air previously filtered (Millipore membrane, 0.22 μm), was injected in the reactor at a flux of 0.5 or
1.5 vvm. The yeast cells were inoculated at an initial concentration of 0.01 g/L and the cultivation
was undertaken at 25 °C for 48 hours. Aliquots (5 mL) were obtained at different period of time.
Afterwards, the cells were collected by centrifugation (2000 x g, 15 min), washed twice with sterile
distilled water, and re-suspended in sterile water to obtain a cell suspension. Cell concentration
was determined by optical density (OD) measurements at 600 nm and OD correlated with the cell
concentration (g/L) by means of a calibration curve previously established. Total sugar was
determined according to the methodology described by DuBois (1956). All experiments were
carried out, at least, in duplicate.
 XXI SIMPÓSIO NACIONAL DE BIOPROCESSOS
XII SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSA

3 a 6 de setembro de 2017
Aracaju, Sergipe, Brasil

3. RESULTS AND DISCUSION

Soybean molasses is a low cost byproduct usually used as animal feed due to its high
concentration of disaccharides, oligosaccharides and other nutrients. Therefore, this byproduct can
be useful in bioprocess to obtain food-grade products. In this context, the growth of Saccharomyces
boulardii was evaluated in medium containing exclusively soybean molasses as carbon source at
different aeration conditions. The profiles of cell growth and substrate consumption are shown in
Figure 3. It can be noted that at low aeration rate (0.5 vvm), the cell growth achieved low
concentration (2.8 g/L) at the first 24 hours of cultivation with a maximum biomass of 7.8 g/L after
48 hours. The biomass, and the final carbohydrate concentrations, biomass productivity and others
biochemical parameters are shown in Table 1.
16 90 16 90
a
14 80 14 80

70 70
12 12

Total sugars (g/L)

Biomass (g/L)

Biomass (g/L)
Total Sugar (g/L)

60 60
10 10
50 50
8 8
40 40
6 6
30 30
4 4
20 20
2 10 2 10

0 0 0 0
0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50

Time (h) Time (h)

Figure 3. Profile of biomass and sugar concentration in medium containing soybean molasses as substrate at
aeration rates of 0.5 vvm (a) and 1.5 vvm (b).

The experiments carried out at aeration rate of 1.5 vvm resulted in the highest biomass
production (14.8 g/L) that is almost four-fold higher than the cultivation undertaken at 0.5 vvm
(Table 1). Concerning the substrate consumption, the strain showed considerable carbohydrates
assimilation capacity during the 29 hours of cultivation (61.6 g/L), which corresponds to 77 % of the
initial carbon source.
The remained sugar (23%) corresponds to stachyose, a tetrasaccharide consisting of two α-
D-galactose residue, one α-D-glucose residue, and one β-D-fructose residue sequentially linked.
This oligosaccharide is unassimilable by yeast, but it can be consumed by bacteria and filamentous
fungi through α-galactosidase synthesis. On the other hand, the trisaccharide raffinose can be
utilized by yeast species of the genus Saccharomyces (Mitterdorfer et al., 2001).
The yield regarding the conversion of substrate in biomass (YX/S) and biomass productivity
(QX) values were 0.24 g/g and 0.51 g/L.h, respectively. These values are at least 3.8-fold higher than
those observed at 0.5 vvm (Table 1). Furthermore, when the cultivation was carried out at aeration
rate of 1.5 vvm, the maximum specific growth rate (μmax) was higher (0.18 h-1) than that observed
at low aeration. A positive effect was also noted concerning the cell doubling time (tg) at higher
oxygen flux.
 XXI SIMPÓSIO NACIONAL DE BIOPROCESSOS
XII SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSA

3 a 6 de setembro de 2017
Aracaju, Sergipe, Brasil

Table 1. Biochemical parameters of Saccharomyces boulardii growing in soybean molasses for 29 hours
Aeration
0.5 vvm 1.5 vvm
Biomass (g/L) 3.3 14.8
Substrate* (g/L), (%) 30.2(52) 18.4(77)
YX/S (g/g) 0.06 0.24
QX (g/L/h) 0.11 0.51
QS (g/L/h) 1.71 2.12
µX max (1/h) 0.09 0.17
tg (h) 8.2 4.2
*Final substrate concentration, (%): consumed substrate. Tg (h) generation time

Muller et al. (2007) described the growth of Saccharomyces boulardii in an airlift reactor
using glucose as substrate, and they reported a positive effect of the aeration rate (1.0 to 1.5 vmm)
on the specific growth rate and biomass conversion yield.
Furthermore, it has also been observed a production of ethanol in the medium,
independently of the aeration rate that adversely affected the biomass yields (data not shown).
This physiological behavior derives from the activation of a respiro-fermentative metabolism that is
a characteristic of Saccharomyces sp., growing in medium with high substrate concentration and
presence of oxygen (Siqueira et al, 2008).

4. CONCLUSION
These results demonstrated the potential use of soybean molasses as a substrate for
formulation of a growth medium for the probiotic yeast Saccharomyces boulardii. Future work will
be undertaken in order to optimize the conditions for biomass production and to formulate a
symbiotic product, as well.

5. REFERENCES
Siqueira, P.F., Karp, S.G.; Carvalho, J.C.; Sturm, W.; Rodríguez-León, J. A.; Jean-Luc Tholozan, Singhania, R.R.;
Pandey, A.; Soccol, C. R, 2008. Production of bio-ethanol from soybean molasses by Saccharomyces
cerevisiae. Bioresource Technol. 99, 8156-8163.
Kelesidis, T.; Pothoulakis, C. 2012. Efficacy and safety of the probiotic Saccharomyces boulardii for the
prevention and therapy of gastrointestinal disorders. Ther Adv Gastroenterol. 5 (2), 111–125
Mitterdorfer, G.; Kneifel, W.; Viernstein, H. (2001). Utilization of prebiotic carbohydrates by yeast of
therapeutic relevance. Letters App. Microbiol, 33, 251-255
Muller, J.L.; Protti, K.L.; Machado, M.S.; Lacerda, L.L.V.; Bresolin, T.M.B.; Podlech, P.S. 2007. Comparison of
Saccharomyces boulardii growth in an airlift fermentor and in a shaker. Ciênc. Tecnol. Aliment.
Campinas, 27, 6888-693.
Rivaldi, J. D; Sousa-Silva, M. Duarte, L.C.; Ferreira, A.; Cordeiro, C.; Felipe, M.G.A.; Freire, A.P.; Mancilha, I.M
2013. Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains. Appl Microbiol
Biotechnol, 97:1735–1743