Applied Microbiology and Biotechnology (2018) 102:2635–2644

https://doi.org/10.1007/s00253-018-8789-8

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Effect of ultrasound on lactic acid production by Lactobacillus strains

in date (Phoenix dactylifera var. Kabkab) syrup
Seyed Mohammad Bagher Hashemi 1 & Amin Mousavi Khaneghah 2 & Jorge A. Saraiva 3 & Anet Režek Jambrak 4 &
Francisco J. Barba 5 & Maria J. Mota 3

Received: 28 September 2017 / Revised: 12 December 2017 / Accepted: 16 January 2018 / Published online: 9 February 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Date syrup is rich in fermentable sugars and may be used as a substrate for different microbial fermentations, including lactic acid
fermentation processes. The beneficial effects of ultrasounds (US) on bioprocesses have been reported for several microorgan-
isms, due to the enhancement of cell growth, as well as improvements in yields and productivities. Therefore, US treatments
(30 kHz, 100 W, 10–30 min) were applied to two lactobacilli (Lactobacillus helveticus PTCC 1332 and Lactobacillus
acidophilus PTCC 1643), during fermentation using date syrup as substrate. The effects on lactic acid fermentation were
evaluated by analyzing cell growth (dry cell weight and viable cell count), substrate consumption (quantification of glucose
and fructose), and product formation (quantification of lactic acid) over time. The effects of US were also evaluated on cell
membrane permeability. Both lactobacilli were able to grow well on date syrup without the need for addition of further ingre-
dients. The US effects were highly dependent on treatment duration: treatments of 10- and 20-min stimulated lactobacilli growth,
while the treatment extension to 30 min negatively affected cell growth. Similarly, the 10- and 20-min treatments increased sugar
consumption and lactic acid production, contrarily to the 30-min treatment. All US treatments increased cell membrane perme-
ability, with a more pronounced effect at more extended treatments. The results of this work showed that application of
appropriate US treatments could be a useful tool for stimulation of lactic acid production from date syrup, as well as for other
fermentative processes that use date syrup as substrate.

Keywords Date syrup . Lactic acid . Fermentation . Lactobacillus acidophilus . Lactobacillus helveticus . Ultrasound

Introduction potassium, and calcium) (Biglari et al. 2008; Biglari et al.

Date palm (Phoenix dactylifera L.) is one of the oldest
trees in the Arabian Peninsula and mainly grows in dry
and semi-dry regions, including the Canary Islands, north-
ern Africa, Middle East (Iran), Pakistan, India, and
California (Homayouni et al. 2015; Jridi et al. 2015).
Date fruits provide a wide range of essential nutrients, in-
cluding carbohydrates, fat, and protein and dietary fiber, as
well as vitamins and minerals (e.g., iron, phosphorus,
2009; Jridi et al. 2015; Jalali et al. 2014). The production
of this fruit is accompanied by a substantial loss of dates
(up to 30%), due to degradation of important organoleptic
properties, as well as contamination with fungus or infes-
tation by insects (Masmoudi et al. 2008; Besbes et al.
2009; Abbès et al. 2015). These dates have low quality
for direct consumption, but may be used to prepare high
value-added products, such as jam, syrup, jelly, butter, liq-
uid sugar, and others (Abbès et al. 2015).

* Maria J. Mota 3
QOPNA, Chemistry Department, University of Aveiro, Campus
mjmota@ua.pt Universitário de Santiago, 3810-193 Aveiro, Portugal
4
1
Faculty of Food Technology and Biotechnology, University of
Department of Food Science and Technology, College of Agriculture, Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia
Fasa University, Fasa, Iran 5
2
Nutrition and Food Science Area, Preventive Medicine and Public
Department of Food Science, Faculty of Food Engineering, Health, Food Science, Toxicology and Forensic Medicine
University of Campinas (UNICAMP), Campinas, São Paulo, Department, Faculty of Pharmacy, Universitat de València, Avda.
Brazil Vicent Andrés Estellés, s/n 46100 Burjassot, Valencia, Spain
2636
Date syrup is one of the most attractive date by-products
in date processing (Ahdno and Jafarizadeh-Malmiri 2017),
with many food applications, including the use as flavoring
and sweetening agent in the formulation of sweets, snacks,
jams, butters, and dairy products, as well as confectionery
and bakery products (Elleuch et al. 2008; Jridi et al. 2015).
Some studies also report the use of this syrup as a fermen-
tation substrate (Karbasi et al. 2015). In fact, date by-
products are rich in fermentable sugars (glucose and fruc-
tose) and many essential elements suitable for microbial
growth. As a result, date syrup can be used as a culture
medium, in order to produce value-added compounds
through fermentation processes (Nancib et al. 2005;
Nancib et al. 2009; Tang et al. 2014; Karbasi et al. 2015).
The utilization of date-based products as fermentation sub-
strates was already studied for a wide variety of microor-
ganisms, as reviewed by Tang et al. (2014). Some of the
processes studied in this context include the production of
citric acid (Mehyar et al. 2005; Mostafa and Alamri 2012),
cellulose (Moosavi-Nasab and Yousefi 2011), xanthan gum
(Moosavi-Nasab et al. 2010; Salah et al. 2011a, 2011b),
and others. Some studies also report the use of date prod-
ucts as substrates for lactic acid fermentations—either for
lactic acid production or for development of fermented
dairy foods—using different lactobacilli (Nancib et al.
2001, 2005; Chauhan et al. 2007; Elsanhoty et al. 2012;
Karbasi et al. 2015). Chauhan et al. (2007) reached a lactic
acid concentration of 2.6 g L−1 with Lactobacillus sp.
KCP01 using date juice as a carbon source, and a concen-
tration of 15.1 g L −1 when salts and organic nitrogen
sources were supplemented into the medium. Nancib
et al. (2005) investigated the effect of different nitrogen
sources for the production of lactic acid by Lactobacillus
rhamnosus and reported the highest production with yeast
extract, reaching a final concentration of 24.8 g L−1. In
another study with L. rhamnosus, Nancib et al. (2001)
reached a lactic acid concentration of 46.8 g L −1 by
supplementing the medium with a combination of ammoni-
um sulfate and yeast extract in a proportion of 3:2, respec-
tively. Date syrup was also used as a substrate to prepare a
non-dairy probiotic beverage by Lactobacillus acidophilus
and L. rhamnosus. Higher sugar consumption and higher acid
production were reached in fermentation by L. acidophilus,
compared to L. rhamnosus. Both strains were also able to
produce gluconic acid (4.19 and 4.29 g L−1 for L. acidophilus
and L. rhamnosus, respectively) and lactic acid (3.71 and
3.80 g L−1 for L. acidophilus and L. rhamnosus, respectively)
(Karbasi et al. 2015).
The improvement of fermentation yields and productiv-
ities is an important issue for the fermentation industries.
There are innumerous strategies for stimulation of micro-
bial growth and manipulation of their metabolic behavior.
One of these approaches involves the application of
Appl Microbiol Biotechnol (2018) 102:2635–2644
different (sub-lethal) stress conditions prior or during fer-
mentation, in order to affect cell growth and metabolism,
but without compromising cell viability. These stress con-
ditions can be generated by high pressure, electric fields,
or ultrasounds (Mota et al. 2017). The beneficial effects of
ultrasounds (US) on bioprocesses have been reported for
several microorganisms, but the mechanisms behind these
improvements have not been clearly identified. Some au-
thors attributed these promoting effects to electroporation,
which increases cell membrane permeability and acceler-
ates the exchange of substances (Chuanyun et al. 2003).
However, some other factors may also play a relevant
role, such as the improvement of mass transfers (Herrán
et al. 2008), or the pressure fluctuations generated during
US treatments that induce stress on cells, possibly pro-
moting growth and metabolic changes (Herrán et al.
2008; Hua et al. 2011).
There are some studies analyzing the effects of US on
lactic acid fermentation, particularly for the production of
fermented dairy products. In most cases, application of US
was shown to improve microbial growth and/or fermenta-
tive activity, thereby accelerating the fermentation process-
es (Nguyen et al. 2009). US treatments (20 kHz, 90–450 W
for 1–10 min) applied on milk samples inoculated with L.
bulgaricus promoted faster acid production compared to
the samples without sonication and resulted in a reduction
of fermentation time by 0.5 h. The acceleration of fermen-
tation may result from the release of intracellular enzymes
(such as β-galactosidase) from the cells, which may lead to
increased lactic acid production (Wang et al. 1996). The
effects of US (20 kHz, 100 W for 7–30 min) on lactic acid
fermentation were also investigated using different
Bifidobacterium strains, and sonication was found to re-
duce the fermentation time for almost all bifidobacteria
strains (Nguyen et al. 2009). In addition, US caused chang-
es in the organic acid profiles of these strains, since fer-
mentation with B. lactis showed a decrease in the ratio of
acetate to lactate, while fermentation with B. longum had a
decrease in the ratio of acetate and propionate to lactate
(Nguyen et al. 2009).
Based on these results, application of US seems to be a
promising approach to modify and possibly improve lactic
acid fermentation processes. Therefore, the present work
intends to investigate the effects of US throughout lactic
a c i d f e r m e n t a t i o n b y t w o d i ff e r e n t l a c t o b a c i l l i
(Lactobacillus helveticus PTCC 1332 and Lactobacillus
acidophilus PTCC 1643), using date syrup as culture me-
dium. The effects of US on biomass concentration, viable
cells count, and membrane permeability were evaluated.
Product formation was also analyzed by pH measurement
and quantification of lactic acid, while substrate consump-
tion was analyzed by quantification of the main ferment-
able sugars in date syrup, i.e., glucose and fructose.
Appl Microbiol Biotechnol (2018) 102:2635–2644
Material and methods
Date syrup and chemicals
Twenty kilograms of date (Phoenix dactylifera var. Kabkab)
syrup was purchased (January 2017) from a local market in
Shiraz city (Fars, Iran). Date syrup was supplemented with 1%
(w/v) yeast extract, and the pH of the supernatant was adjusted
to pH 7 (Model 520A, Orion Research Inc., Boston, MA,
USA) with 1 N NaOH. After that, date syrup was heated at
105 °C for 15 min. All chemicals used were supplied by
Merck Co. (Darmstadt, Germany) and Sigma Chemical Co.
(St. Louis. MO, USA).
Evaluation of date syrup chemical composition
The analysis of date syrup’s content (ash, water, and protein)
was done according to the Institute of Standards and Industrial
Research of Iran (Iranian National Standards Organization
2013). The sample was ashed by heating for 5 h in an electric
oven at 550 °C. Total protein content was calculated by mul-
tiplying Kjeldahl nitrogen by 6.25. Moisture content was de-
termined using a vacuum oven at 70 °C. Glucose and fructose
were determined by HPLC (Knauer, Azura, Germany)
equipped with a Eurokat H column (250 × 30 mm, 5 μm,
Knauer) and K-2310 refractive index (RI) detector. Sulfuric
acid 0.02 M in ultrapure water at 0.6 mL min−1 was used as
eluent.
Bacterial strains and cultures
L. helveticus PTCC 1332 and L. acidophilus PTCC 1643 were
purchased from Iranian Research Organization for Science
and Technology, Tehran, Iran. The strains were reactivated
overnight in MRS broth (Oxoid, Altrincham, UK) under an-
aerobic conditions at 37 °C.
Fermentation process
Inoculums were prepared by transferring the stock culture of
L. helveticus or L. acidophilus to an Erlenmeyer flask contain-
ing 100 mL of MRS broth. Cells were grown statically at
37 °C until the cell population reached 7.00 log (CFU
mL−1), which was next used to initiate the lactic acid fermen-
tation (Hashemi et al. 2016, 2017a). Date syrup (200 mL) was
inoculated with ≈4.20 log (CFU mL−1) of L. helveticus or L.
acidophilus. For US treatment, a Hielscher ultrasonic device
(UP100H model, Germany; 100 W, 30 kHz) with a titanium
horn (M6 × 0.75) was used. The device was adjusted at 25%
amplitude of waves and duration of the sonication was 10, 20,
and 30 min. US probe was placed at the approximately 2 cm
from the top into the sample in Erlenmeyer flasks. Application
of US promoted a temperature increase of 2, 4, and 5 °C, for
2637
10-, 20-, and 30-min treatment, respectively. Based on the
difference between the two temperatures (ΔT), ultrasonic
power (P) was calculated by Eq. 1, where t is the treatment
time, C is the specific heat of water, and M is its mass. As a
result, the ultrasonic power of 3.97 W was estimated for 10-
and 20-min treatments, and 3.25 W for the 30-min treatment.
P ¼ ΔT =t  C  M ð1Þ
Fermentations were carried out in Erlenmeyer flasks for
14 h at 35 °C, with temperature controlled by a water bath.
Fermentation time of the sonicated samples was calculated at
the beginning of ultrasound treatment. Samples without any
US treatment (but fermenting at the same conditions) were
used as controls. All fermentations, as well as analyses, were
conducted in triplicate.
Viable cells count
For determination of viable cells, serial dilutions (using pep-
tone water) of date syrup containing L. helveticus or L.
acidophilus were prepared, and aliquots of 0.1 mL of proper
dilutions were plated in MRS agar plates followed by incuba-
tion at 37 °C for 72 h.
Biomass concentration and pH measurement
Biomass concentration was measured by optical density mea-
surement at 570 nm (UV/visible, Philips, Cambridge, UK)
and calibrated to the cell dry weight (Nancib et al. 2001).
The pH of samples was determined using a bench-top pH
meter, which was before calibrated with pH 4.0 and 7.0
buffers.
Quantitative determinations of glucose, fructose,
and lactic acid
Fermented samples were centrifuged at 5000g for 20 min, and
the supernatants containing glucose, fructose, and lactic acid
were analyzed by an HPLC (Knauer, Azura, Germany)
equipped with a K-2600 UV-visible and K-2310 RI detector.
The separation columns for sugars and lactic acid were a
Eurokat H (250 × 30 mm, 5 μm, Knauer column) and an
Ultrasep ES-FS special (250 × 30 mm, 5 μm, Knauer col-
umn), respectively. Sulfuric acid 0.02 M in ultrapure water
at 0.6 mL min−1 was used as eluent, and the detector was a
RI detector and temperature was set at 37 °C. Lactic acid was
identified at 210 nm. Each of the examined compounds was
quantified by comparing its measured peak area against the
standard curves which were obtained specifically for the ref-
erence solutions containing that compound. The standard
curves were obtained at five different concentrations (1–
10 μg/100 μL) of each compound. The detection limit was
2638
defined as the concentration corresponding to a peak height
three times the baseline noise level. The ChromGate Software
A1493 was used as software to determine targeted compounds
(Mousavi et al. 2013; Hashemi et al. 2017b).
Membrane permeability of nucleic acids and proteins
Effect of ultrasound treatment on membrane permeability of L.
helveticus and L. acidophilus was measured by determining the
contents of nucleic acid and extracellular protein. After the
fermentation process, changes of nucleic acid and extracellular
protein contents were determined on the basis of their maxi-
mum absorption wavelength at 260 and 280 nm, respectively.
The absorbance (A) of the fermented samples (treated with US
or not) was measured at 260 and 280 nm after centrifugation at
4000g for 20 min. The nucleic acid or extracellular protein
contents were calculated by the following equation:
The nucleic acid or extracellular protein ð%Þ
¼ ðUltrasound −Awithout ultrasound =Awithout ultrasound Þ  100
(Dai et al. 2017).
Statistical analyses
One-way ANOVA and Duncan’s multiple range test using
SPSS software (SPSS 20.0 for Windows; SPSS Inc.,
Chicago, IL, USA) were used for measurement of differences
between groups for all times at p < 0.05.
Results
Date syrup composition
Date syrup composition is highly determinant for the fermen-
tative process since it affects not only the bacterial ability to
grow in that medium but also the metabolic products formed
during fermentation. The composition of the date syrup used
in this work is presented in Table 1. Besides water, sugars
were the most predominant components of the syrup, with
concentrations of approximately 67.41 and 55.12 g L−1 for
glucose and fructose, respectively. This richness in
Table 1 Chemical
composition of date Component Concentration (g L−1)
syrup. Reported values
represent the mean ± Water 851.1 ± 4.48
standard deviation of Glucose 67.41 ± 0.13
three independent Fructose 55.12 ± 0.08
replicates (n = 3)
Ash 14.23 ± 0.18
Protein 9.15 ± 0.14
Appl Microbiol Biotechnol (2018) 102:2635–2644
fermentable sugars makes date syrup a suitable medium for
microbial growth and fermentation. The estimated protein
content was approximately 9.15 g L−1.
Effect of US on lactic acid fermentation
Biomass concentration and viable cell counts
In order to understand the US effects on cell growth, dry cell
weight and viable cell counts were determined. Although both
parameters allow cell quantification, cell dry weight detects
viable and non-viable cells, while viable cell counts only de-
tect the viable ones. Figure 1 shows the variation of these two
parameters over time, in samples subjected to different US
treatments (with duration of 10, 20, or 30 min) at the begin-
ning of fermentation, performed by L. acidophilus (Fig. 1a, b)
or L. helveticus (Fig. 1c, d).
Both lactobacilli were able to grow well on date syrup
without the need of any further ingredients. The control sam-
ples (without US treatment) reached cell counts of 9.6 and 9.2
ð2Þ
log (CFU mL−1), and biomass concentrations of 5.0 and
4.7 g L−1, after 14 h of fermentation. Regarding the effects
of US on the process, the results in Fig. 1 showed similar
behavior for the two lactobacilli. In both cases, the US treat-
ment of 20 min resulted in the highest cell growth during
fermentation, with these samples reaching biomass concentra-
tions of 5.2 and 5.0 g L−1, and cell counts of 10.0 and 9.8 log
(CFU mL−1) after 14 h, for L. acidophilus and L. helveticus,
respectively. These values were significantly higher (p < 0.05)
than the ones observed for the control samples. In general, the
US treatment of 10 min also increased biomass concentra-
tions, and cell counts relative to the controls, but at a lower
extent compared to the 20-min treatment. In the case of L.
acidophilus, similar (p > 0.05) biomass concentration and cell
count values were observed in the control and the 10-min-
treated samples, after 14 h. In contrast, the 30-min treatment
negatively affected cell growth, compared to the controls. In
fact, a slight reduction of viable cells was detected at the be-
ginning of the process, between the 0 and 2 h, which corre-
sponds to the period when US treatments were applied (during
the first 30 min). After that period, cells were able to recover
the ability to grow, but after 14 h, biomass concentrations and
viable cell counts were still significantly lower (p < 0.05) than
the controls.
Fermentative activity
Measurement of pH over time is a rapid and simple method for
monitoring lactic acid fermentation since lactic acid is usually
the primary product of this process. Figure 2 shows the pH
variation over time for US-treated samples fermented by L.
acidophilus (Fig. 2a) or by L. helveticus (Fig. 2b). For both
microorganisms, US treatments of 10 and 20 min promoted
Appl Microbiol Biotechnol (2018) 102:2635–2644 2639

Fig. 1 Biomass concentration

and viable cell counts for the
samples of L. acidophilus (a, b)
and L. helveticus (c, d) over
fermentation time, according to
the US treatment applied: 10 min
(diamonds ), 20 min
(circles ), or 30 min
(triangles ). Control samples
(no US treatment) are represented
as stars ( ). All fermentation
experiments were performed with
three independent replicates (n =
3)

more accentuated pH variation and consequent lower final pH
values (in the range of 2.4–2.6), compared to the control samples
(in the range of 3.0–3.4). In L. acidophilus samples, the pH after
14 h was similar (p > 0.05) for the 10- and 20-min treatments,
but the same was not observed for L. helveticus. For the 30-min
US treatment, pH showed lower variation over time compared to
controls, showing pH values of 4.6 and 5.0 after 14 h, for L.
acidophilus and L. helveticus, respectively.
In order to understand the effects of US on fermentation, a
more detailed analysis was needed, not only for the production
of lactic acid but also to the consumption of glucose and fruc-
tose. These results are presented in Fig. 3, with glucose con-
sumption in Fig. 3a and Fig. 3d, and fructose consumption in
Fig. 3b and Fig. 3e, for L. acidophilus and L. helveticus, respec-
tively. For L. acidophilus, US treatments of 10 and 20 min in-
creased glucose and fructose consumption during fermentation,
relative to the controls. After 14 h, residual glucose concentra-
tions of 22.8 and 17.8 g L−1, and residual fructose concentrations
of 18.6 and 14.8 g L−1 were detected for samples sonicated for
10 and 20 min, respectively. Control sample showed a residual
glucose concentration of 26.8 g L−1 and a residual fructose con-
centration of 22.3 g L−1. For L. helveticus, the stimulating effect
of the 10-min treatment was less pronounced, with the residual
concentration of glucose and fructose (27.1 and 22.3 g L−1, re-
spectively) close to the control (28.5 and 23.1 g L−1, respective-
ly). In contrast, US treatment of 20 min caused a substantial
increase of sugar consumption, reaching residual concentrations
of glucose and fructose of 17.8 and 14.8 g L−1, respectively.
Lactic acid concentrations over time are showed in Fig. 3c,
f. US treatments of 10 and 20 min significantly increased
(p < 0.05) lactic acid concentration for the two lactobacilli,
compared to the controls. For both samples, the highest lactic

Fig. 2 pH variation for the samples of L. acidophilus (a) and L. helveticus Control samples (no US treatment) are represented as stars ( ). All
(b) over fermentation time, according to the US treatment applied: 10 min fermentation experiments were performed with three independent
(diamonds ), 20 min (circles ), or 30 min (triangles ). replicates (n = 3)
2640
Fig. 3 Glucose, fructose, and lactic acid concentrations for the samples of
L. acidophilus (a, b, c) and L. helveticus (d, f, e) over fermentation time,
according to the US treatment applied: 10 min (diamonds ), 20 min
acid concentrations were reached when treated for 20 min
(45.37 and 41.66 g L−1 for L. acidophilus and L. helveticus,
respectively). However, the US treatment of 30 min signifi-
cantly decreased (p < 0.05) the production of lactic acid rela-
tive to controls: after 14 h, lactic acid concentrations were
24.51 and 20.47 g L−1 for L. acidophilus and L. helveticus,
respectively.
Effect on membrane permeability of nucleic acids
and proteins
Membrane permeability was evaluated by measuring the
changes in the content of nucleic acids and proteins in
Appl Microbiol Biotechnol (2018) 102:2635–2644
(circles ), or 30 min (triangles ). Control samples (no US
treatment) are represented as stars ( ). All fermentation experiments
were performed with three independent replicates (n = 3)
the extracellular medium, relative to control samples. The
higher the increase of these compounds in the extracellular
medium, the higher the loss of cell constituents caused by
the US treatment, indicating higher permeability of the cell
membrane. Figure 4 shows the increment of nucleic acids
and protein contents in the extracellular medium after US
treatments of 10, 20, and 30 min, relative to the control
samples. For the two lactobacilli, the increase of US treat-
ment time led to increasing of cell permeability both to
nucleic acids and to proteins. As a result, the 30-min treat-
ment was the one with more effect on cell permeability,
with a 5.3–6.1% increase of extracellular nucleic acids,
and an 8.1–9.3% increase of extracellular proteins.
Appl Microbiol Biotechnol (2018) 102:2635–2644
Fig. 4 Increment ratio (%) of extracellular protein and nucleic acid content in the samples of L. acidophilus (a) and L. helveticus (b) relative to the
controls. All fermentation experiments were performed with three independent replicates (n = 3)
Discussion
Date syrup is reported in the literature as a rich culture medium
for microbial growth and fermentation, due to its composition
with a high concentration of fermentable sugars and many other
essential elements (Tang et al. 2014). Therefore, both lactobacilli
used in this study were able to grow well on date syrup without
need of any further ingredients, with the control samples (with-
out US treatment) reaching cell counts of 9.6 and 9.2 log (CFU
mL−1), and biomass concentrations of 5.0 and 4.7 g L−1, after
14 h of fermentation. Karbasi et al. (2015) reported similar con-
clusions for fermentation with L. rhamnosus and L. acidophilus
on date syrup, but in this case, cell counts in the range of 8.3–8.7
log (CFU mL−1) were reported for both microorganisms after
20 h of fermentation. A possible explanation for these lower cell
counts (compared to the present study) may be related to differ-
ences in the composition of the date syrup, which certainly af-
fects growth and fermentation.
Application of US during fermentation may exert different
effects on microbial cell growth, according to the intensity of the
treatment and the sensitivity of the microorganism to US. In this
case, application of US treatments of 10 and 20 min was found
to stimulate L. acidophilus and L. helveticus growth on date
syrup. For both lactobacilli, the US treatment of 20 min resulted
in the highest cell growth during fermentation, with these sam-
ples reaching biomass concentrations of 5.2 and 5.0 g L−1, and
cell counts of 10.0 and 9.8 logs (CFU mL−1), for L. acidophilus
and L. helveticus, respectively. The US treatment of 10 min also
stimulated cell growth compared to the controls, but at a lower
extent than the 20-min treatment. Growth stimulation by US is
reported for several microorganisms (Mota et al. 2017), includ-
ing some lactobacilli species (Wang and Sakakibara 1997). This
is generally attributed to electroporation, which corresponds to
the formation of pores in the cell membrane in response to US,
increasing cell permeability, and consequent acceleration of sub-
stance exchange (Chuanyun et al. 2003). Other mechanisms
may also be involved (Mota et al. 2017): (i) the improvement
2641
of gas-liquid mass transfer, which may be important in oxygen-
dependent fermentation processes, and solid-liquid mass trans-
fer; (ii) the generation of pressure fluctuation during US treat-
ments that can induce stress on cells, possibly promoting cell
growth and proliferation, together with changes in metabolic
processes.
In contrast, there are also several studies reporting the de-
crease of cell viability caused by US, possibly due to the
inhibition of cell propagation or even cell disruption
(Dakubu 1976; Mett et al. 1988; Sakakibara et al. 1994).
This negative effect of US was also observed in the present
work, since the extension of the treatment time to 30 min
decreased cell growth during fermentation, compared to the
controls: after 14 h, biomass concentrations of 4.6 and
4.2 g L−1, and cell counts of 8.9 and 8.3 logs (CFU mL−1),
were estimated for L. acidophilus and L. helveticus, respec-
tively. These results suggest that this longer US treatment may
have caused extensive damage to lactobacilli cells, which
were not able to completely recover during the remaining time
of fermentation without sonication. The primary mechanism
responsible for microbial inactivation during sonication is re-
lated to the physical forces generated by cavitation. This phe-
nomenon promotes the uninterrupted formation of
microbubbles, which expand and implode violently after
reaching a critical size (Zinoviadou et al. 2015). The hydro-
phobic surfaces on microorganisms assist the collapse of the
cavitation bubbles, which lead to severe damage to the cell
wall. Moreover, erosion of cell walls and consequently micro-
bial inactivation may be caused by micro-streaming effects as
well as intracellular cavitation (Chandrapala et al. 2012;
Chandrapala and Leong 2015). Some chemical aspects may
also be related to microbial inactivation by US, such as the
formation of reactive species and free radicals that act on the
cell envelope by disrupting the cell walls, thus allowing the
cell lysis (Sango et al. 2014).
Regarding substrate consumption, both lactobacilli were
able to consume glucose and fructose from date syrup during
2642
fermentation, and apparently without preferential utilization
of any of the sugars. For the two bacterial strains, US treat-
ments of 10 and 20 min increased glucose and fructose con-
sumption during fermentation, relative to the controls.
However, in the case of L. helveticus, the stimulating effect
of the 10-min treatment was considerably less pronounced,
with the residual concentration of glucose and fructose (27.1
and 22.3 g L−1, respectively) close to the control (28.5 and
23.1 g L−1, respectively).
Comparing the two lactobacilli, lower pH values were
usually achieved by L. acidophilus (pH in the range of 2.4–
4.6) compared to L. helveticus (pH in the range of 2.5–5).
However, it is important to consider that pH provides in-
formation about the production of all organic acids present
in the samples, and not only lactic acid. In this particular
case, a similar effect was detected when considering the
concentration of lactic acid produced by both lactobacilli,
with the values ranging between 24.51 and 45.37 g L−1 for
L. acidophilus, and between 20.47 and 41.66 g L−1 for L.
helveticus. These results suggest that L. acidophilus is
more suitable for lactic acid production from fermentation
of date syrup.
It is also interesting to note that lactic acid concentration in
control samples (36.11 and 34.21 g L−1 for L. acidophilus and
L. helveticus, respectively) is considerably higher than that
described in the literature for other Lactobacillus strains grow-
ing in date juice. For example, Chauhan et al. (2007) reported
a lactic acid concentration of 2.6 g L−1 with Lactobacillus sp.
KCP01, and reached a concentration of 15.1 g L −1 by
supplementing the date juice with salts and organic nitrogen.
For L. rhamnosus, Nancib et al. (2005) reported a lactic acid
concentration of 24.8 g L−1, but also with supplementation of
date juice, in this case with yeast extract. Using a combination
of ammonium sulfate and yeast extract, Nancib et al. (2001)
reached a lactic acid concentration of 46.8 g L−1. These con-
centrations are closer to the values we observed for L.
acidophilus and L. helveticus, but in this case, date juice was
used as the substrate without further supplementation. This
higher lactic acid production may be explained by different
factors, such as the utilization of different bacterial strains,
differences in the composition of date juice, or different fer-
mentation conditions.
As expected, pH variation (and the production of lactic
acid) was also affected by sonication. For both microorgan-
isms, US treatments of 10 and 20 min promoted more accen-
tuated pH variation and consequent lower final pH values (in
the range of 2.4–2.6), compared to the control samples (in the
range of 3.0–3.4), which suggests that these US treatments
stimulated fermentation and production of lactic acid by these
Lactobacillus species in date syrup, which was confirmed by
the lactic acid concentration results. In fact, for both bacteria,
the highest lactic acid concentrations were reached after the
treatment of 20 min (45.37 and 41.66 g L−1 for L. acidophilus
Appl Microbiol Biotechnol (2018) 102:2635–2644
and L. helveticus, respectively). Stimulation of lactic acid fer-
mentation was also observed by Wang et al. (1996) by apply-
ing US treatments (20 kHz, 90–450 W for 1–10 min) on L.
bulgaricus in milk. A similar effect was reported by Nguyen
et al. (2009) for application of US (20 kHz, 100 W for 7–
30 min) on lactic acid fermentation by different
Bifidobacterium strains. In these cases, the authors explained
the increase in lactic acid production with the release of intra-
cellular enzymes (such as β-galactosidase) from the cells due
to sonication, which would promote lactose hydrolysis and
consequent acceleration of fermentation. Since the sugars
present in date syrup are glucose and fructose, the effect of
US on substrate hydrolysis is not a relevant factor for the
present work. Nevertheless, electroporation may have an es-
sential role in the stimulation of lactic acid production from
date syrup, as the increase of cell permeability accelerates the
exchange of substances (Chuanyun et al. 2003), including the
diffusion of nutrients across the cell membrane. This is one of
the mechanisms usually indicated in literature for enhance-
ment of different fermentative processes by sonication (Mota
et al. 2017). However, application of a 30-min US treatment
was found to decrease the pH variation (4.6 and 5.0 after 14 h,
for L. acidophilus and L. helveticus, respectively.) and the
production of lactic acid (24.51 and 20.47 g L−1 for L.
acidophilus and L. helveticus, respectively) relative to con-
trols. The lower acid production may be a result of metabolic
inhibition (and consequent inhibition of fermentation) caused
by the US treatment of 30 min. This is in accordance with the
negative effects of this treatment on cell growth and viability.
As already discussed in this section, electroporation (and
the consequent increase of membrane permeability) may be
the main mechanisms behind the effects of sonication on these
fermentative processes. In order to confirm that possibility, the
permeability of lactobacilli cells was evaluated throughout
fermentation. For the two strains, US treatments of 10, 20,
and 30 min increased membrane permeability, promoting the
exchange of nutrients and metabolites through the cell mem-
brane. These results show that electroporation may have an
important role in the stimulation of cell growth and the in-
crease of fermentative activity observed for both lactobacilli
when subjected to US treatments of 10 and 20 min. In con-
trast, the extension of treatment time to 30 min led to an
increase in cell permeability, which seems to affect cell growth
and fermentation negatively. This adverse effect may be due to
the higher loss of cell constituents that compromised cell
structure and function or, in more extreme cases, due to cell
rupture. Therefore, the effects of US on the production of
lactic acid by L. acidophilus and L. helveticus were found to
be highly dependent on the duration of the treatments, since
short treatments (10 and 20 min) stimulated fermentation,
while longer treatments (30 min) had an inhibitory effect on
the process. Many authors reported variability in the sensitiv-
ity of different microorganisms to US, even between
Appl Microbiol Biotechnol (2018) 102:2635–2644
organisms from the same species (Sakakibara et al. 1994;
Nguyen et al. 2009). In the present work, the effect of US on
membrane permeability was more pronounced in L.
acidophilus than in L. helveticus, which suggests that the latter
is more resistant to sonication treatments. However, the effects
of US on cell growth, substrate consumption, and lactic acid
production were generally similar for both microorganisms.
In conclusion, L. acidophilus and L. helveticus were
both able to grow in date syrup and to use the fermentable
sugars present in this substrate to produce lactic acid.
Application of US treatments at the beginning of fermen-
tation was found to affect not only lactobacilli cell growth
but also the fermentative process, by changing the profiles
of substrate consumption and lactic acid production. The
effects of US on fermentation were highly dependent on
the treatment duration, with the shorter treatments (10 and
20 min) positively affecting the process, promoting higher
biomass and lactic acid concentrations after 14 h of fer-
mentation. The higher fermentative enhancement was ob-
served for the samples treated for 20-min treatments,
which achieved lactic acid concentrations of 45.37 and
41.66 g L−1 for L. acidophilus and L. helveticus, respec-
tively. Stimulation of growth and fermentation by US may
be promoted by several different mechanisms, but one of
the most commonly referred in literature is the increase of
membrane permeability, which promotes the exchange of
nutrients and metabolites through the cell membrane,
leading to higher cell growth and productivity improve-
ments. However, when the US treatment was extended to
30 min, cell growth and fermentation were negatively af-
fected relative to the control samples, possibly due to the
higher loss of cellular content (mediated by the increased
membrane permeability), or even cell rupture. Overall,
this work showed that the appropriate US could be a use-
ful tool for stimulation of lactic acid production from date
syrup when the intensity and the duration of the treat-
ments are adapted to that purpose. Also, this opens the
way for the application of this approach to other fermen-
tations that use date syrup as a substrate, for the produc-
tion of a wide variety of compounds.
Funding Amin Mousavi Khaneghah wishes to thank the support of
CNPq-TWAS Postgraduate Fellowship (grant no. 3240274290). Author
Maria J. Mota thanks Fundação para a Ciência e a Tecnologia for the
grant SFRH/BD/97061/2013 and Fundação para a Ciência e a
Tecnologia/Ministério da Educação e Ciência for the financial support
to the QOPNA research Unit (FCT UID/QUI/00062/2013), through na-
tional funds and where applicable co-financed by the Fundo Europeu de
Desenvolvimento Regional (FEDER), within the PT2020 Partnership
Agreement.
Compliance with ethical standards
The support from the laboratory of Food Science and Technology
Department, Fasa University, is also appreciated.
2643
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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