Suwimol Chockchaisawasdee - Costas E. Stathopoulos: Brueckii
Suwimol Chockchaisawasdee - Costas E. Stathopoulos: Brueckii
Suwimol Chockchaisawasdee - Costas E. Stathopoulos: Brueckii
125–132
SUMMARY
Commercial yoghurts and yoghurt-type products do not always fulfill the minimum requirement of viable culture cell
counts at the time of consumption, which is the most important factor in order for such products to exhibit therapeutic
effects. This work aimed to investigate the survivability of Streptococcus thermophilus, combined with Lactobacillus del-
brueckii ssp. bulgaricus, and Lactobacillus acidophilus, combined with Lactobacillus casei, in two fermented milk samples
supplemented with isomalto-oligosaccharides (IMO) prepared from banana flour. The preferential fermentation of
oligosaccharides by the combined starter cultures was also investigated. After 28 days of storage at 4 °C, the viable cell
numbers of all bacterial strains in both samples were not changed (p > 0.05). Lactose, isomaltotriose, isomaltotetraose,
and maltooligoheptaose and larger oligomers, were depleted by approximately 50%, 40%, 20% and 20%, respectively.
The decrease of lactose and IMO in both fermented milk samples did not differ (p > 0.05). In this study, IMO could
maintain the viable cell numbers of all bacteria used in the experiments. The order of oligosaccharide fermentation
preference of the cultures was lactose > isomaltotriose > maltooligoheptaose and larger oligomers > isomaltotetraose.
Keywords
fermented milk; probiotic yoghurt; isomalto-oligosaccharides; lactic acid bacteria; prebiotics
Cultured or fermented milks are probably Portugal and USA, this concept has been adopted
the oldest dairy products known to humans [1]. as product identification. However, in the United
According to the Codex Standard [2], fermented Kingdom, the whole range of yoghurt-type fer-
milks are products derived by fermentation of milk mented milk products are labelled as ‘yoghurt’ [6].
by suitable organism(s), in which the milk’s struc- Since the 1980s, therapeutic yoghurts and other
ture is altered by the reduction of pH. Among the fermented milk products containing probiotic bac-
fermented milk products range, yoghurt is one teria, in particular lactobacilli and bifidobacteria,
of the most well-known and developed product have been increasingly studied and marketed [7].
world-wide [3, 4]. Several factors, such as acidity, The manufacturing process of therapeutic or pro-
production of aroma compounds, textural charac- biotic yoghurts is similar to that of the convention-
teristics, sensory attributes, nutritional value and al type; the main difference is the culture starters
therapeutic properties contribute to the creation used in the process. There have been many studies
of desirable products [5]. Strictly speaking, ‘yo- reporting that some Lactobacillus spp., and in par-
ghurt’ must contain two specific microorganisms ticular Bifidobacterium spp., contained in yoghurt
belonging to the lactic acid bacteria (LAB) group, can colonize the large intestine, reduce its pH
namely, Streptococcus thermophilus and Lacto- and control the growth of undesirable microor-
bacillus delbrueckii ssp. bulgaricus, as dominant ganisms [3, 4, 8]. Therefore, bacterial cultures in-
organisms [2]. In many countries, such as France, cluding L. rhamnosus, L. reuterii, L. acidophilus,
Suwimol Chockchaisawasdee, Faculty of Science and Technology, Loei Rajabhat University, Loei, 42000, Thailand.
Costas E. Stathopoulos, School of Environmental and Life Sciences, University of Newcastle, Ourimbah 2258, New South
Wales, Australia.
Correspondence author:
Suwimol Chockchaisawasdee, e-mail: csuwimol@gmail.com
L. plantarum, L. casei, B. bifidum and B. longum kok, Thailand). The enzyme trans-glucosidase L
are widely used as inocula in therapeutic ferment- (E.C. 3.2.1.20) was a gift from Amano Enzyme
ed milks in various combinations [7, 9]. However, (Nagoya, Japan). Commercial isomalto-oligosacc-
to exhibit the desirable therapeutic effects, starter ahride (IMO) syrup was kindly provided by Corn
culture(s) must be present in the product in high Products International (Seoul, Korea). Skimmed-
viable counts at the time of consumption, which milk and whole-milk powder were kindly supplied
is at least 107 CFU × ml-1 of combined organisms by S&P Syndicate (Bangkok, Thailand) and Pepsi-
and at least 106 CFU × ml-1 of other supplemen- Cola (Thai) Trading (Bangkok, Thailand), respec-
tary microorganisms [2]. Many researchers recom- tively.
mended ranges of minimum daily dose higher than Lyophilized bacterial cultures Lactobacil-
107–108 CFU × ml-1 [10, 11], or even as high as lus acidophilus TISTR450, Lactobacillus casei
108–109 CFU × ml-1 [12]. Despite the importance TISTR1463, Lactobacillus delbrueckii ssp. bul-
of viability of probiotics in the products, many garicus TISTR451 and Streptococcus thermophilus
surveys have reported poor viability of probiotics TISTR458 were purchased from Thailand In-
in some preparations of commercially available stitute of Science and Technological Research
products, especially when the products were stored (TISTR; Bangkok, Thailand).
for a long period of time (28–35 days) or kept at
a higher temperature (10–12 °C) [3, 11, 13, 14]. Preparation of isomalto-oligosaccharides
Some studies took interest in combining pro- Isomalto-oligosaccharides (IMO) were pre-
biotics and prebiotics in order to solve the bacte- pared from banana flour. Banana flour slurry
rial survivability problem, and reported various (250 g·kg-1) was hydrolysed by applying Term-
combinations of pro-prebiotics that could improve amyl SC (0.15 ml, 95 °C, pH 5.5–6.0, 2 h) and Fun-
the viability of the cultures [15–18]. Prebiotic has gamyl 800L (0.3 ml, 50 °C, pH 5.5–6.0, 24 h), and
been widely known since 1995 after GIBSON and subsequently used for IMO synthesis by applying
ROBERFROID [19] gave its definition as ‘a non- trans-glucosidase l (0.3 ml, 50 °C, pH 5.5–6.0, 12 h).
digestible food ingredient that beneficially affects The synthesized mixture was subjected to baker’s
the host by selectively stimulating the growth yeast fermentation (10 g·l-1, 24 h). Ethanol pro-
and/or activity of one, or limited number of, bacte- duced during fermentation was removed by rotary
ria in the colon that can improve the host health’. evaporation [22]. Of total oligosaccharides with
Since the 1990s, oligosaccharides with various various degrees of polymerization (DP), the final
structures and production technologies have been IMO mixture was composed of 53% isomalto-
available on the market, especially in Japan [20, triose/panose (IMO DP3), 21% isomaltotetraose
21]. (IMO DP4) and 26% maltooligoheptaose and
This study aimed to investigate the application larger oligomers (MO DP 5). The syrup was di-
of isomalto-oligosaccharides, which were prepared luted to get a solution of 30 g·l-1 of total oligosac-
from banana flour, on survivability of probiotic charides and filtered through 0.2 mm membrane
cultures and their preferences of oligosaccharide filters before applying into milk bases.
fermentation in fermented milk.
Starter culture preparation
Lyophilized bacterial cells S. thermophilus (ST),
MATERIALS AND METHODS Lb. acidophilus (LA), Lb. delbrueckii ssp. bulgari-
cus (LB), and Lb. casei (LC) were revitalized as
Materials follows. ST was rehydrated by M17 broth and all
Chemicals used were of analytical grade and lactobacilli were rehydrated by MRS broth. The
were obtained from BDH (Poole, United King- bacterial suspensions were then transferred onto
dom). All chemicals used in high performance their respective agar media. The plates were incu-
liquid chromatography (HPLC) were of HPLC bated anaerobically at 37 °C for 72 h in anaerobic
grade obtained from Merck (Darmstadt, Ger- jars with gas-generating kits (Anaerobic system
many). Diluents and culture media were obtained BR 38; Oxoid). Cultures were transferred three
from Difco (Detroit, Michigan, USA). Selective times successively for activation. A single colony of
media de Man Rogosa and Sharpe (MRS) and each strain was selected and inoculated into 10 ml
M17 broth were obtained from Oxoid (Cambridge, aliquot of 100 g·l-1 sterile reconstituted skim milk
United Kingdom). Two -amylase enzymes, (RSM) supplemented with 20 g·l-1 glucose and
Termamyl SC (E.C. 3.2.1.1) and Fungamyl 800L 10 g·l-1 yeast extract [23]. The cultures were anaer-
(E.C. 3.2.1.1; Novozymes, Bagsvaerd, Denmark), obically incubated at 37 °C for 24 h. The cultures
were kindly provided by East Asiatic (Bang- were subsequently transferred into fresh RSM me-
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Viability of probiotics in fermented milk supplemented with isomalto-oligosaccharides
dium at an inoculation level of 50 ml·l-1 and were California, USA) was used. Oligosaccharide pro-
anaerobically incubated at 37 °C for 24 h before file was determined using Sugar-Pak 1 resin-based
being used as starters. column (6.5 300 mm; Waters, Milford, Massa-
chusetts, USA); injection volume was 10 μl. The
Fermented milk production column was heated to 70 °C and degassed HPLC
Two fermented milks were made using dif- water was used as a mobile phase at a flow rate
ferent combinations of starter cultures. The first of 0.5 ml·min-1. Glucose, maltose, a commer-
combination was conventional yoghurt starters cial IMO, and a commercial MO were used as
(ST and LB), and the second combination was standards. Their ratios of peak areas to concentra-
two probiotic strains (LA and LC). Milk base tions were used to convert peak areas of oligosac-
(150 ml) was prepared by dissolving whole milk charides found in the samples into concentrations.
powder (110 g·l-1) in the 30 g·l-1 IMO solution in
250 ml bottles. The milk base was then pasteurized Statistical analysis
by immersion in a water bath at 90 °C for 30 min Differences of oligosaccharide depletions in
[23]. After cooling down to approximately 35 °C, two fermented milk samples during storage were
40 ml·l-1 of starter cultures, which were composed treated by t-test. Analysis of variance (ANOVA)
of ST and LB, or LA and LC, in a ratio of 1:1, with Duncan’s new multiple range test ( = 0.05)
were inoculated into the milk bases. The mixes was used to compare the bacteria counts during
were gently stirred for 30 s before incubation at storage time using SPSS for Windows version
42–44 °C for ST-LB fermented milk, and 37–40 °C 11.01 software (IBM, Somers, New York, USA).
for LA-LC fermented milk. When the pH of the
milk reached 4.5, the fermentation was stopped by
refrigeration at (4 ± 1) °C. The fermented milks RESULTS AND DISCUSSION
were maintained at such temperature thereafter.
Samples were taken at days 1, 7, 14, 21, and 28 to Fermentation of IMO by probiotic strains
determine the viability of the cultures. The experi- Two IMO-added set fermented milk samples
ments were carried out in triplicate. were made using different combination of lac-
tic acid producers (ST and LB; LA and LC). The
Enumeration of bacteria ST-LB fermented milk was incubated at 42–44 °C,
Fermented milk samples (1 g) were taken at which was the optimum temperature of traditional
days 1, 7, 14, 21, and 28. Each sample was 10-fold yoghurt making [26]. The LA-LC fermented milk
serially diluted in 1.5 g·l-1 sterile peptone water. was incubated at 37–40 °C since Lb. casei has the
Cultures were enumerated by the pour plate tech- optimum growth temperature of 37–40 °C and the
nique using a presumptive medium and incubation upper limit of growth at 43–48 °C [24, 27]. Fig. 1
conditions for each culture. ST colonies were enu- shows viable cell counts of all strains monitored
merated on M17 agar and anaerobic incubation for 28 days. It was found that in ST-LB fermented
at 37 °C for 48 h [23]. LB and LC were enumer- milk, the numbers of ST were comparable to those
ated on MRS agar and MRS-NaCl agar, respec- of LB (8.13 and 8.17 log10 CFU·g-1, respectively).
tively, with anaerobic incubation at 37 °C for 72 h. The viability of ST was maintained for 14 days
LA cells were grown on pH-modified agar with before slightly decreasing afterwards (0.11 log cy-
anaerobic incubation at 43 °C for 72 h [24]. Plates cle). The numbers of LB, on the other hand, were
containing 25–250 colonies were counted and bac- slightly increased during the first 21 days (0.09 log
terial numbers calculated as colony forming units cycle) and then slightly decreased at the end of the
(CFU) per gram of product. storage time. In LA-LC fermented milk, the loga-
rithms of viable cells counts of both strains were in
Oligosaccharide analysis by high performance the range of 7.9–8.0 throughout the storage time.
liquid chromatography The population changes of all cultures were not
Proteins were removed from the samples different over the observation period (p > 0.05).
by means of a membrane filter (10 k molecular The results showed that the synthesized IMO
weight cut-off), and the saccharide composition of mixture could not increase the viable cell numbers
the samples was determined by high performance of all starter cultures, but rather maintained them.
liquid chromatography (HPLC) as described else- Other studies on probiotic viability of other strains
where [25] with modifications as follows. of lactobacilli (such as Lb. reuteri, Lb. rhamnosus,
An HPLC unit equipped with an isocratic Lb. acidophilus) and bifidobacteria (B. infantis,
pump, a refractive index (RI) detector and an B. longum, B. pseudolongum, B. animalis, B. bifi-
autosampler (1100 Series; Agilent, Santa Clara, dum) in fermented milk products supplemented
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Chockchaisawasdee, S. – Stathopoulos, C. E. et al. J. Food Nutr. Res., 50, 2011, pp. 125–132
Fig. 1. The viability of starter cultures in fermented milk samples containing 30 g·l-1 IMO mixture during storage
at 4 °C.
A – Streptococcus thermophilus (ST) and Lactobacillus bulgaricus (LB) fermented milk; B – Lactobacillus acido-
philus (LA) and Lactobacillus casei (LC) fermented milk.
Values are means of triplicate analyses with standard deviation.
with various prebiotics (inulin, fructooligosaccha- may have resulted in better protection of probiotic
rides, galactooligosaccharides, oligosaccharides of cells [18], or probably because the bacteria were
raffinose family) have been reported with a con- supplied with plenty of carbon sources that they
sensus to exhibit a higher retention of bacterial could metabolize over the storage time.
numbers in the respective medium compared to The changes of the concentrations of oligosac-
control [16–19]. The mechanism of maintaining charides available in the fermented milk samples
viability of probiotics in dairy products in the pres- are illustrated in Tab. 1 and Fig. 2. It was found
ence of prebiotics is not thoroughly elucidated. It that, in both samples, all oligosaccharide classes
is probably due to higher viscosity of the medium decreased over storage time, particularly lactose
after addition of prebiotics, and such conditions and isomaltotriose/panose, which decreased sig-
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Viability of probiotics in fermented milk supplemented with isomalto-oligosaccharides
Fig. 2. Comparison of percentage changes of (A) lactose and (B) IMO in ST-LB and LA-LC fermented milk samples
containing 30 g·l-1 IMO mixture during storage at 4 °C.
Values are means of triplicate analyses with standard deviation. Bars with the different superscript letters are
significantly different (p < 0.05).
nificantly during 14–21 days. Isomaltotetraose and and a three-stage continuous gut culture model
high DP maltooligosaccharides were also changed using IMO as a sole carbon source to maintain lac-
significantly but not to such high extent as those tic acid flora, and also to facilitate lactate, acetate,
of lactose and isomaltrose/panose. The result pre- propionate and butyrate generation [35, 36]. As
sented in Fig. 2B shows that IMO concentration for carbon utilization preference in both samples
in LA-LC fermented milk was lower than that in (Fig. 3), lactose was the most preferred carbon
ST-LB fermented milk. However, the differences source. Comparing IMO fermentation preference,
were not statistically significant (p > 0.05). Lac- it seems that isomaltotriose/panose was preferred
tose is a fermentable disaccharide for streptococ- to isomaltotetraose. This is probably due to the
ci and lactobacilli. These bacteria initiate lactose less complicated structure, so it was easier me-
fermentation by either hydrolysis catalysed by - tabolized by the bacteria. The percentage changes
D-galactosidase, or hydrolysis of phosphorylated of isomaltotetraose and maltooligoheptapse and
lactose by -D-phosphogalactosidase [28]. Several larger oligomers were comparable in LA-LC fer-
species of lactobacilli including Lb. bulgaricus pos- mented milk (Fig. 3B), but this was not the case in
sess both enzymes [29]. ST-LB fermented milk (Fig. 3A).
Homofermentative conversion of lactose to With regards to ST-LB fermented milk,
lactic acid is the most important fermentative SANDERS and co-workers [37] reported that all
reaction in dairy processing including yoghurt S. thermophillus and most Lb. bulgaricus strains
production. The fate of lactose in fermentation have a high -D-galactosidase activity, which could
of conventional yoghurt starters has been well explain the higher depletion levels of lactose com-
documented [28]. Lactic acid formation result- pared to those in LA-LC fermented milk (Fig. 2A).
ing from the fermentation plays a very important The interaction of Lb. bulgaricus and S. ther-
role in yoghurt characteristics, it reduces the pH mophilus has been described as mutual interac-
of the yoghurt mix, preserves the flavour and con- tion [38], since in a mixed culture S. thermophilus
tributes to it, and modifies the texture of yoghurt produces formic acid that stimulates the growth of
by causing coagulation of casein micelles [1, 30]. Lb. bulgaricus, while Lb. bulgaricus produces cer-
It has been reported that lactic acid bacteria, es- tain amino acids (glycine, histidine, valine, leucine,
pecially lactobacilli and streptococci, express the isoleucine) that stimulate the growth of S. ther-
ability to metabolize various carbon sources avail- mophilus. The decrease of viable cell counts of
able in their environments [31–34]. The reduction S. thermophilus after 14 days, although not signifi-
of IMO in the fermented milk samples suggested cantly different from those of Lb. bulgaricus, was
that the cultures used in the experiment could me- in agreement with an earlier study which reported
tabolize them. This is in agreement with previous that the growth rate of Lb. bulgaricus was higher
reports using a static batch culture fermentation than that of S. thermophilus when the concentra-
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Viability of probiotics in fermented milk supplemented with isomalto-oligosaccharides
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Chockchaisawasdee, S. – Stathopoulos, C. E. et al. J. Food Nutr. Res., 50, 2011, pp. 125–132
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