718 International Journal of Food Science and Technology 2009, 44, 718–724

Original article
Stability of bifidobacteria in powdered formula

Fumiaki Abe,* Hirofumi Miyauchi, Ayako Uchijima, Tomoko Yaeshima & Keiji Iwatsuki
Food Science and Technology Institute, Morinaga Milk Industry Co., Ltd, Kanagawa Prefecture 228-8583, Japan
(Received 24 July 2008; Accepted in revised form 11 November 2008)

Summary Two stability tests of bifidobacteria in powdered formula were conducted. In a strain comparison test, three
kinds of bifidobacterial powders; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and
Bifidobacterium infantis M-63 powders, admixed in follow-up formula were used. In a commercial product
evaluation, powdered formulas for toddlers containing bifidobacteria sold in the Indonesian market were
analysed. When the inactivation rate constant of each sample, which was used as an index of the loss rate,
was determined from the stability tests, B. longum was the most stable strain. The mean inactivation rate
constant of commercial products was significantly lower than those obtained in strain comparison, although
the same strains (B. longum BB536 and B. breve M-16V) were used. A possible reason was the lower water
activity of commercial products compared to the follow-up formula. Also, higher storage temperature
yielded lower stability in all strains or samples, which obeys the Arrhenius theory.
Keywords Bifidobacterium, Bifidobacterium breve M-16V, Bifidobacterium infantis M-63, Bifidobacterium longum BB536, follow-up formula,
powdered formula, probiotic, stability.

Mah et al., 2007; Puccio et al., 2007). In particular,

Introduction
Nowadays probiotics are used in a wide variety of foods
such as yogurt, cheese, dietary supplement, milk, con-
fection and cereal (Champagne et al., 2005). As probi-
otics are known to have various beneficial effects on
human health, many manufacturers have developed
various health products containing probiotics for an
increasingly health conscious consumer market (Vinde-
rola et al., 2000; Charalampopoulos et al., 2002; Mat-
tila-Sandholm et al., 2002; Philips et al., 2006; Shah,
2007). Recently powdered formulas containing probiot-
ics for infants or young children have become popular
worldwide. In particular, bifidobacteria are often used in
these products. A possible reason for this trend is that
bifidobacteria are the dominant bacteria in infant
intestine (Mitsuoka, 1982; Magne et al., 2006). Many
investigations have examined the relationship between
intestinal microbiota and infant health or between
administration of probiotics and intestinal health, and
studies suggest that bifidobacteria are the most impor-
tant intestinal bacteria and that supplementation of
probiotics supports the intestinal health of infants
(Mevissen-Verhage et al., 1987; Penders et al., 2006;
*Correspondent: Fax: +81 46 252 3055;
e-mail: f_abe@morinagamilk.co.jp
probiotics have various beneficial effects for preterm
infants, such as improving the intestinal microbiota,
protecting against necrotising colitis, maintaining cyto-
kine balance and balancing intestinal fatty acids (Li
et al., 2004; Fujii et al., 2006; Barclay et al., 2007;
Deshpande et al., 2007; Wang et al., 2007). Also,
Hattori et al. (2003) reported that administration of
Bifidobacterium breve was effective in reducing the
allergy scores including atopic symptom in infants with
milk allergy. The accumulated evidence demonstrating
the useful actions of probiotics for infants has acceler-
ated the development and marketing of an increasing
number of powdered formulas containing probiotics.
However, a prerequisite for probiotic products is that
a sufficiently large number of viable probiotic bacteria
survive in the final product at the time of consumption,
as probiotics are expected to modify the intestinal
microbiota (Saxelin et al., 1999). Since the drying
process may cause significant damage to the membranes,
proteins and nucleic acids of bacteria, it is not easy to
maintain living cells in foods for a long time under
general conditions such as ambient temperature (Potts,
2001; Bill & Potts, 2002). Therefore there are some
issues of stability when using probiotics in powder
products such as powdered formula. Scientists have
tried to develop technologies to produce stable bacterial

doi:10.1111/j.1365-2621.2008.01881.x
 2009 Institute of Food Science and Technology

powders, and many studies have investigated the effects
of drying on microbial cell survival using various kinds
of microorganisms including lactic acid bacteria and
starter cultures (Kets et al., 1996; Castro et al., 1997; To
& Etzel, 1997; Carvalho et al., 2004; Zhao & Zhang,
2005). Furthermore, the effects of storage conditions on
survival of some bacterial species have been discussed
recently (Hernandez et al., 2007; Higl et al., 2007;
Portner et al., 2007). However, there is no report on
the stability of probiotics in powdered formulas for
infants or small children, especially in commercial
formulas sold in the retail market, although the stability
of bifidobacteria in yogurt and cheese has been inves-
tigated in detail (Daigle et al., 1999; Adhikari et al.,
2000; Vinderola et al., 2000; Champagne et al., 2005;
Philips et al., 2006). It is very important for both
manufacturers and consumers to know the stability of
probiotics in powdered formulas. In particular, the
viable counts of probiotics in powdered formulas at the
end of shelf life and the effect of storage temperature on
stability of probiotics in final product are interesting
topics of investigation.
This paper is the first report on the stability of
bifidobacteria in powdered formula evaluated by two
long-term stability tests. One is a strain comparison test
that compares the stability of three bifidobacterial
strains admixed in follow-up formula. Another test is
commercial product evaluation that analyses the stabil-
ity of bifidobacteria in commercial powdered formulas
for toddlers, which are labelled to contain bifidobacteria
and are being sold in the stores. Furthermore, based on
kinetic analyses of the data from both stability tests, the
characteristics of stability of bifidobacteria in powdered
formula are discussed.
Materials and methods
Bifidobacteria powder
Three kinds of bifidobacteria powder: Bifidobacterium
longum subsp. longum BB536 powder (B. longum
powder), Bifidobacterium breve M-16V powder (B. breve
powder), and B. longum subsp. infantis M-63 powder
(Bifidobacterium infantis powder) were used in the strain
comparison test. All three bifidobacterial powders were
manufactured by mixture with starch following lyophil-
isation at Morinaga Milk Industry Co., Ltd (Tokyo,
Japan) and sold as ingredients for probiotics products.
All strains were isolated from infant and deposited into
culture collections; B. longum biotype longum BB536 as
American Type Culture Collection strain BAA-999,
B. breve M-16V as Belgian Co-Ordinated Collections of
Micro-organisms (BCCM) strain LMG23729 and
B. longum biotype infantis M-63 as BCCM strain
LMG23728. Standard viable counts of the bifidobacte-
rial powders are more than 8.0 · 1010 colony forming
 2009 Institute of Food Science and Technology
Stability of bifidobacteria in formula powder F. Abe et al. 719
unit (CFU) g)1 for B. longum, more than
1.0 · 1011 CFU g)1 for B. breve, and more than
8.0 · 1010 CFU g)1 for B. infantis.
Strain comparison tests
For the strain comparison test, a follow-up formula
‘Chil-mil’ (Morinaga Milk Industry Co., Ltd), which
contains no probiotic bacteria and is a general com-
mercial product widely sold in the Japanese powdered
formula market, was used. The standard composition
was 18.0% fat, 14.5% protein, 60.5% carbohydrate,
4.0% ash, 3% moisture and 460 kcal 100 g)1 energy.
The water activity, which is a very important indicator
for stability of probiotics, was 0.25 ± 0.025 (aver-
age ± SD) as measured by the Rotronic Hygroskop
DT (Rotronic Instrument Corp., Huntington, NY,
USA). Each of the three bifidobacterial powders; B.
longum, B. breve and B. infantis powder, was mixed
thoroughly with the follow-up formula Chil-Mil to
obtain powdered formula containing 0.05–1.0% (w ⁄ w)
bifidobacteria. After mixing, 6–9 g of bifidobacteria-
containing powdered formula which contained at least
more than 4.8 · 107 CFU g)1 was put into an alumin-
ium bag (PET ⁄ AL ⁄ PE, 70 lm thick) and hermetic
sealed for stability testing. The aluminium bag was used
to maintain the same moisture level of the sample during
storage period, because moisture cannot pass through
the material. Aluminium bag samples containing each
powdered formula were stored at temperatures of 5, 25,
37 and 45 C. Viable counts of each sample were
monitored continuously during a storage period for a
maximum of 24 months to observe bifidobacterial
survival under various temperature conditions. Eighteen
tests using eighteen lots of B. longum BB536 powder, five
tests using three lots of B. breve M-16V powder, and two
tests using one lot of B. infantis M-63 powder were
performed to observe the stability of the three strains in
powdered formula. To compare the stabilities, the
common logarithms of cell counts during storage were
plotted with time, and the slopes of the regression lines
were compared.
Commercial product evaluation
For the commercial product evaluation, Chil-Kids
Platinum with vanilla flavour, Chil-Kids Platinum with
honey flavour, Chil-Kids School Platinum with vanilla
flavour, and Chil-Kids School Platinum with chocolate
flavour were used. All the products were manufactured
by PT Sanghiang Perkasa (Jakarta, Indonesia) and were
sold in the Indonesian market as powdered formula.
Chil-Kids Platinum with two flavours were for toddlers
aged 1–2 years, and the standard composition was
13.5% fat, 18% protein, 61% carbohydrate, 4.5% ash,
3% moisture, and 438 kcal 100 g)1 energy. Chil-Kids
International Journal of Food Science and Technology 2009, 44, 718–724
720 Stability of bifidobacteria in formula powder F. Abe et al.
School Platinum with two flavours were for children
older than 3 years, and the standard composition was
12.0% fat, 15% protein, 66% carbohydrate, 4.0% ash,
3% moisture and 432 kcal 100 g)1 energy. All powdered
formulas had labels stating that the product contained
more than 420 million of B. longum BB536 and B. breve
M-16V in 100 g. The mean water activity (aver-
age ± SD) of six samples was 0.20 ± 0.02. All pow-
dered formula products were tested before the end of
shelf life. To observe bifidobacterial survival in com-
mercial powdered formulas, a total of six lots of
powdered formula products comprising three lots of
Chil-Kid Platinum with vanilla flavour, one lot of Chil-
Kid Platinum with honey flavour, one lot of Chil-Kid
School Platinum with vanilla flavour, and one lot of
Chil-Kid School Platinum with chocolate flavour were
used. Eleven to fifteen g of each powdered formula was
put into aluminium bags (PET ⁄ AL ⁄ PE, 70 lm thick)
and hermetic sealed as for the strain comparison test.
Aluminium bags containing powdered formulas were
stored at 5, 25, 30 and 37 C, and the survival of
bifidobacteria in each sample was measured by the
following enumeration method.
Enumeration of viable counts of bifidobacteria
Bifidobacterial counts were enumerated using pour plate
technique. In the strain comparison test, 5 g of storage
sample was weighed and suspended in 95 mL of suspen-
sion buffer (4.5 g potassium dihydrogenphosphate, 6.0 g
disodium hydrogen phosphate, 0.5 g l-cysteine and 0.5 g
Tween-80 in 1000 mL distilled water). All ingredients of
the buffer were purchased from Kanto Chemical Co., Inc.
(Tokyo, Japan). After mixing thoroughly in the buffer,
standard pour plate technique was performed using BL
agar medium (Nissui, Tokyo, Japan) as plating media.
The agar plates were incubated at 37 C for 72 h under
anaerobic condition (N2: 80%, H2: 10%, CO2: 10%) in an
anaerobic chamber (Anaerobox; Hirasawa Works Co.,
Ltd, Tokyo, Japan). Viable count was expressed in CFU.
In commercial product evaluation, the enumeration
procedures were the same as for the strain comparison
test except that 10 g of storage sample was weighed and
dissolved in 90 mL of suspension buffer.
Inactivation rate constant of bifidobacteria
To compare bifidobacteria stability in each sample, the
logarithm of reduction in cell count versus storage period
was plotted and the regression line was obtained. The log
reduction in cell count at 1 year on the regression line
(equivalent to slope of regression line) was set as the
inactivation rate constant (log10 CFU g)1 year)1) which
was used an index of reduction in cell number during
storage. Also, for each sample, the natural logarithm of
inactivation rate constant was plotted against the inverse
International Journal of Food Science and Technology 2009, 44, 718–724
of absolute storage temperature to test for agreement with
the Arrhenius theory.
Statistical treatment of the results
To evaluate the difference in slope of the regression line
defined as inactivation rate constant, analyses of covari-
ance were used to compare the inactivation rate
constants between strains, storage temperatures and
commercial products. Unpaired Student’s t-test was
performed to evaluate the difference in mean water
activity between the powdered formulas used in strain
comparison test and commercial product evaluation.
Also, regression analyses were used to examine the
correlation between natural logarithm of inactivation
rate constant versus inverse of absolute temperature for
storage, and the regression coefficient (R2) were com-
pared. Furthermore, analyses of covariance for natural
logarithm of inactivation rate constant and inverse of
storage temperature were performed to observe the
differences in the Arrhenius plots.
Results
Strain comparison tests
To observe the stability of B. longum BB536, B. breve
M-16V and B. infantis M-63 admixed in powdered
formula, the log10 reductions in cell count were plotted
against storage periods for the three bifidobacterial
strains, and the regression lines were obtained (Fig. 1a–
c). For all three strains, the reduction in cell count
increased with time of storage. Also, there were signif-
icant (P < 0.05) differences in the slope of the regres-
sion line between all temperature conditions in all three
strains. The slopes became significantly steeper as
temperature increased. Since the slope of the regression
line reflects the loss rate of the strain under that
condition, the slope obtained from Fig. 1 was defined
as the inactivation rate constant (log10 CFU g)1 year)1)
for each condition. The inactivation rate constants
obtained from the respective regression lines at 5, 25,
37 and 45 C were used for comparisons between strains
(Fig. 2a–d). When the inactivation rate constants were
compared between strains by covariance analyses, the
constants were significant different among species at all
storage temperatures except 5 C. The inactivation rate
constant of B. longum was significantly lower than that
of B. breve at 25 and 37 C and than that of B. infantis at
37 and 45 C, indicating that B. longum was the most
stable strains among the three strains tested.
Commercial products evaluation
Six commercial powdered formula products for tod-
dlers, which contained bifidobacteria as probiotics, were
 2009 Institute of Food Science and Technology
 Stability of bifidobacteria in formula powder F. Abe et al. 721

(a) 3.0 in Fig. 3. Although a great decrease was observed at the

highest temperature condition (37 C), bifidobacteria
Reduction cell counts

2.5
were stable in the powdered formulas tested at temper-
(Log10 CFU g–1)

2.0 atures lower than 30 C, as evidenced by the mainte-

nance of bifidobacteria cell counts of more than
1.5
1 · 107 CFU g)1 even after 24 months of storage. When
1.0 the inactivation rate constants of the commercial prod-
0.5
ucts at various storage temperatures were determined in
the same manner as in the strain comparison test, there
0.0 were significant differences between the inactivation
0 6 12 18 24
constants obtained from the commercial product eval-
Storage period (month)
uation and those obtained from the strain comparison
tests (Fig. 2). Commercial powdered formulas had
(b) 3.0 significantly (P < 0.01 or P < 0.05) lower inactivation
rate constants compared to both B. longum and B. breve
Reduction cell counts

2.5
obtained from the strain comparison test at both 25 and
(Log10 CFU g–1)

2.0 37 C, although there were no significant differences at

1.5
low storage temperature (5 C).
1.0
The Arrhenius plot
0.5
The relationship between natural logarithm of the
0.0
0 6 12 18 24
inactivation rate constant for each sample and inverse
Storage period (month) of absolute storage temperature is illustrated in Fig. 4.
High correlation coefficients (R2 > 0.91) were observed
in all strains or commercial products. Also, the slope of
(c) 3.0 B. infantis ()22,102 ± 2443) was significantly different
(P < 0.05) from B. breve ()13,832 ± 2443) and com-
Reduction cell counts

2.5
(Log10 CFU g–1)

mercial product ()12,242 ± 3,006), although there was

2.0
no difference between the other two combinations
1.5 (P > 0.05).
1.0

0.5
0.0
0 6 12 18
Storage period (month)
Figure 1 Changes of log reduction of cell count during storage for
B. longum (a), B. breve (b), and B. infantis (c) admixed in powdered
formula. Temperature conditions were 5 C ()), 25 C (s), 37 C (4)
and 45 C (h).
used to test stability of bifidobacteria in commercial
products. The average water activity of the commercial
products was 0.20 ± 0.020 (SD), which was signifi-
cantly (P < 0.05) lower than that of the powdered
formula (0.25 ± 0.025) used in the strain comparison
test. When the stability of bifidobacteria in each
commercial product was analyzed statistically, there
was no significant difference (P > 0.05) in the inactiva-
tion rate constant between products at all storage
temperatures (5, 25, 30 and 37 C) (data not shown).
The changes in viable cell counts of six commercial
powdered formulas during 24-month storage at various
storage temperatures, and the regression lines are shown
Discussion
Recent studies on probiotics and the roles of intestinal
24 microbiota in infants have indicated that probiotics,
especially bifidobacteria, are very important for infant
health (Mah et al., 2007). Powdered formulas for infant
or young children containing probiotics have been
developed and many clinical trials of these products
have confirmed not only the effects of probiotics on
infant health, but also their safety (Puccio et al., 2007;
Chouraqui et al., 2008). On the other hand, before
manufacturing or launching a commercial product
containing probiotics, long-term stability test using the
actual product is necessary to confirm the stability of
probiotics and to determine the shelf life, because the
manufacturer has to guarantee a sufficiently high viable
cell count in the product until the end of shelf life.
Especially, powdered formulas are stored at ambient
temperature and it is possible that the products are
stored at higher temperature conditions during trans-
port or interim storage, particularly in summer season
or in hot countries like Indonesia. Generally, it is known
that the viability of live bacteria such as probiotics
decreases during storage and is sensitive to higher

 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009, 44, 718–724
722 Stability of bifidobacteria in formula powder F. Abe et al.

P < 0.01

(a) (b) P < 0.05

0.25 0.25

Inactivation rate constant

(Log10 CFU g –1 year –1)
Inactivation rate constant
(Log10 CFU g –1 year –1)

0.20 0.20

0.15 0.15

0.10 0.10

0.05 0.05

0.00 0.00
B. longum B. breve B. infantis Commercial B. longum B. breve B. infantis Commercial
Products Products

P < 0.05

P < 0.01
(c) (d)
40 P < 0.01
2.50 P < 0.01
Inactivation rate constant
Inactivation rate constant

(Log10 CFU g –1 year –1)

(Log10 CFU g –1 year –1)

2.00 30
Figure 2 Inactivation rate constants of B. lo-
1.50 ngum (grey), B. breve (white) and B. infantis
20
(diagonal stripe) obtained from strain com-
1.00
parison test and of bifidobacteria in com-
10
0.50 mercial powdered formulas (black) obtained
N.T. from commercial product test at four tem-
0.00 0
B. longum B. breve B. infantis Commercial B. longum B. breve B. infantis Commercial perature conditions: (a) 5 C, (b) 25 C, (c)
Products Products 37 C and (d) 45 C.
Logarithm of inactivation rate constant

8.0 4
o10 CFU g –1)

[Ln(Log10 CFU g–1 year –1)]

7.0 0
Cell counts (Log

–2
6.0 –4

–6
5.0
–8
0 6 12 18 24 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036
Storage period (month) –1
Inverse temperature (°K )
Figure 3 Changes of viable bifidobacteria counts in commercial
Figure 4 Arrhenius plots for the inactivation rate constants of B. longum
powdered formulas stored at 5 C ()), 25 C (h), 30 C ( ) and 37 C
(¤), B. breve ( ), B. infantis (d) and commercial product ( ).
(s).

temperature conditions. If a large reduction in cell count

occurs in probiotic products such as powdered formulas products. However, to the best of our knowledge, there
during storage, the commercial values would be lost and are only a few reports on the stability of probiotics in
children would not be able to benefit from the expected powdered formulas for infants or toddlers (Perez-
probiotic effects due to insufficient viable cells in the Conesa et al., 2006).
products. Therefore the change in stability at various In this study, we conducted a strain comparison test to
storage temperatures should be considered in probiotic compare the stability of three Bifidobacterium strains
products. Also, it is extremely important for manufac- admixed in powdered formula, and a commercial product
turers and consumers that the tests should include not test to evaluate the stability of bifidobacteria in several
only samples made in the laboratory but also commer- commercial bifidobacteria-containing powdered for-
cial products manufactured at the factory to be sold in mula, using long-term stability tests. Although all three
the stores, because sometimes there are large differences strains showed reduction of cell counts during storage as
of the quality between laboratory samples and factory shown Fig. 1, there were significant differences in the

International Journal of Food Science and Technology 2009, 44, 718–724  2009 Institute of Food Science and Technology

inactivation rate constant (an index of loss rate) between
storage temperatures and between strains. The results of
all stability tests indicate that higher storage temperature
yields higher inactivation rate constant for bifidobacteria,
which is consistent with previous investigations of
bifidobacteria stability (Simpson et al., 2005). The inac-
tivation rate constant of B. longum was lower than that of
B. breve or that of B. infantis in many conditions. These
results suggest that among the three species tested,
B. longum is the most suitable bifidobacterial powder
for use in powdered formula. This storage test will be
helpful for manufacturers to consider the extra amount of
bifidobacteria to be added in their products at the time of
manufacturing because expected survival rates at the end
of shelf life of the products can be estimated using the
inactivation rate constants indicated in Fig. 2. Many
studies have demonstrated various beneficial effects of
B. breve M-16V on infant health (Hattori et al., 2003; Li
et al., 2004). The survival data shown in this study will
help manufacturers decide the number of bifidobacteria
to add during manufacturing when using this strain in
powdered formulas.
For commercial probiotic products, it is very impor-
tant to maintain a high enough number of viable
probiotics at the end of shelf life in order to achieve the
probiotic effect obtained clinical investigations. Mah
et al. used powdered infant formula supplemented with
1 · 107 CFU g)1 of B. longum BB536 in their clinical
test and observed probiotic effects, although the other
lactobacillus was also administered. We confirmed that
the stability of bifidobacteria in Indonesian powdered
formulas were high enough because more than
1 · 107 CFU g)1 of bifidobacteria survived after
24 months storage at 30 C which is presumed to be
the ambient temperature in Indonesia (Fig. 3). These
results will provide confidence to Indonesian consumers.
Also, although all powdered formulas contained the
same strains (B. longum BB536 and B. breve M-16V)
used in the strain comparison test of this study, which
were confirmed by randomly amplified polymorphic
DNA (RAPD) techniques (data not shown), the inac-
tivation rate constants were significantly different
between the commercial product test and the strain
comparison test (Fig. 2). The stability of bifidobacteria
in commercial products was significantly higher than
that of B. longum and B. breve in the strain comparison
test at both 25 and 37 C. One possible reason is the
difference in water activity between the powdered
formulas used in the two tests. The follow-up formula
Chil-Mil used in the strain comparison test had water
activity of 0.25 ± 0.025, and was significantly
(P < 0.05) higher than that (0.20 ± 0.02) of the com-
mercial products. Some investigators have reported that
water activity affects the stability of dried microorgan-
isms and that higher water activity results in lower
survival rate (Schoug et al., 2006; Higl et al., 2007). Also
 2009 Institute of Food Science and Technology
Stability of bifidobacteria in formula powder F. Abe et al. 723
Nagawa et al. (1987) reported that water content was
influenced with bifidobacteria stability in powder form.
These reports suggested that there was a relationship
between moisture level and the stability. In this study, if
the tested powders had the same water activity, the
difference in stability might not exist. Also, the differ-
ences in composition of powdered formula may be
another reason, although the powdered formula tested
were similar products and there was no great difference
in composition between products.
On the other hands, logarithm of the inactivation rate
constants showed a good relationship with the inverse of
absolute temperature in all products; i.e. obeying the
Arrhenius rate law. This result was supported by
previous report (Damajanovic & Rdulovic, 1968), sug-
gesting that the residual bifidobacteria counts upon
storage at room temperature in not only probiotic
powder but also commercial powdered formula can be
predicted by an accelerated stability test conducted at
higher temperatures. However there were significant
differences in the slope of the Arrhenius plot between
strains. Simpson et al. (2005) reported that there were
differences in bifidobacterial stability between species
and between strains. In view of the inter-strain difference
in the slope of the Arrhenius plot, when trying to predict
bifidobacterial survival in an acceleration test at higher
temperature conditions, the slope of the Arrhenius plot
should be considered for each strain individually.
This investigation indicated that there were some
differences in stability between Bifidobacterium strains,
that the bifidobacteria in the Indonesian commercial
products were survived well at ambient temperature
condition, and that the stability in powdered formula
was in accordance with the Arrhenius theory. These
results will be useful for manufacturers and consumers
to understand probiotic products. We hope that this
investigation will support further development of pro-
biotic products with good stability in the world.
Acknowledgments
We thank our technical staff in Food Science & Technical
Institute of Morinaga Milk Industry Co., Ltd for deter-
mining viable counts of bifidobacteria in this study. We
are also indebted to Mr Hamano of Nutritional Science
Laboratory in Morinaga Milk Industry Co., Ltd for his
kind assistance in statistical analysis.
References
Adhikari, K., Mustapha, A., Grun, I.U. & Fernando, L. (2000).
Viability of microencapsulated bifidobacteria in set yogurt during
refrigerated storage. Journal of Dairy Science, 83, 1946–1951.
Barclay, A.R., Stenson, B., Simpson, J.H., Weaver, L.T. & Wilson,
D.C. (2007). Probiotics for necrotizing enterocolitis: a systematic
review. Journal of Pediatric Gastroenterology and Nutrition, 45, 569–
576.
International Journal of Food Science and Technology 2009, 44, 718–724
724 Stability of bifidobacteria in formula powder F. Abe et al.
Bill, D. & Potts, M. (2002). Life and death of dried prokaryotes.
Research in Microbiology, 153, 7–12.
Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X. & Gibbs,
P. (2004). Relevant factors for the preparation of freeze-dried lactic
acid bacteria. International Dairy Journal, 14, 835–847.
Castro, H.P., Teixeira, P.M. & Kirby, R. (1997). Evidence of
membrane damage in Lactobacillus bulgaricus following freeze
drying. Journal of Applied Microbiology, 82, 87–94.
Champagne, C.P., Gardner, N.J. & Roy, D. (2005). Challenges in the
addition of probiotic cultures to foods. Critical Review Food Science
and Nutrition, 45, 61–84.
Charalampopoulos, D., Wang, R., Pandiella, S.S. & Webb, C. (2002).
Application of cereals and cereal components in functional foods: a
review. International Journal of Food Microbiology, 15, 131–141.
Chouraqui, J.P., Grathwohl, D., Labaune, J.M. et al. (2008). Assess-
ment of the safety, tolerance, and protective effect against diarrhea
of infant formulas containing mixture of probiotics or probiotics
and probiotics in a randomized controlled trial. The American
Journal of Clinical Nutrition, 87, 1365–1373.
Daigle, A., Roy, D., Bélanger, G. & Vuillemard, J.C. (1999).
Production of probiotic cheese (cheddar-like cheese) using enriched
cream fermented by Bifidobacterium infantis. Journal of Dairy
Science, 82, 1081–1091.
Damajanovic, V. & Rdulovic, D. (1968). Predicting the stability of
freeze-dried Lactobacillus bifidus by the accelerated storage test.
Cryobiology, 5, 101–104.
Deshpande, G., Rao, S. & Patole, S. (2007). Probiotics for prevention
of necrotizing enterocolitis in preterm neonates with very low birth
weight: a systematic review of randomized controlled trials. Lancet,
369, 1614–1620.
Fujii, T., Ohtsuka, Y., Lee, T. et al. (2006). Bifidobacterium breve
enhances transforming growth factor beta1 signaling by regulating
Smad7 expression in preterm infants. Journal of Pediatric Gastro-
enterology and Nutrition, 43, 83–88.
Hattori, K., Yamamoto, A., Sasai, M. et al. (2003). Effects of
administration of bifidobacteria on fecal microflora and clinical
symptoms in infants with atopic dermatitis. Arerugi, 52, 20–30.
Hernandez, A., Weekers, F., Mena, J. et al. (2007). Culture and spray-
drying of Tsukamurella paurometabola, C-924: stability of formu-
lated powders. Biotechnology. Letters, 29, 1723–1723.
Higl, B., Kurtmann, L., Carisen, C.U. et al. (2007). Impact of water
activity, temperature, and physical state on the storage stability of
Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose
matrix. Biotechnology Progress, 23, 784–800.
Kets, E.P.W., Teunissen, P.J.M. & Bont, J.A.M. (1996). Effect of
compatible solutes on survival of lactic acid bacteria subjected to
drying. Applied and Environmental Microbiology, 62, 259–261.
Li, Y., Shimizu, T., Hosaka, A., Kaneko, N., Ohtsuka, Y. &
Yamashiro, Y. (2004). Effects of Bifidobacterium breve supplemen-
tation on intestinal flora of low birth weight infants. Pediatrics
International, 46, 509–515.
Magne, F., Hachelaf, W., Suau, A. et al. (2006). A longitudinal study
of infant faecal microbiota during weaning. FEMS Microbiology
Ecology, 58, 563–571.
Mah, K.W., Chin, V.I., Wong, W.S. et al. (2007). Effect of a milk
formula containing probiotics on the fecal microbiota of Asian
infants at risk of atopic diseases. Pediatric Research, 62, 674–679.
Mattila-Sandholm, T., Myllärinen, P., Crittenden, R., Mogensen,
G., Fondén, R. & Saarela, M. (2002). Technological challenges
International Journal of Food Science and Technology 2009, 44, 718–724
for future probiotic foods. International Dairy Journal, 12, 173–
182.
Mevissen-Verhage, E.A., Marcelis, J.H., de Vos, M.N., Harmsen-van
Amerongen, W.C. & Verhoef, J. (1987). Bifidobacterium, Bactero-
ides, and Clostridium spp. in fecal samples from breast-fed and
bottle-fed infants with and without iron supplement. Journal of
Clinical Microbiology, 25, 285–289.
Mitsuoka, T. (1982). Recent trends in research on intestinal flora.
Bifidobacteria and Microflora, 1, 3–24.
Nagawa, M., Nakabayashi, A. & Fujino, S. (1987). reparation of the
bifidus milk powder. Journal of Dairy Science, 71, 1777–1782.
Penders, J., Thijs, C., Vink, C. et al. (2006). Factors influencing the
composition of the intestinal microbiota in early infancy. Pediatrics,
118, 511–521.
Perez-Conesa, D., Lopez, G. & Ros, G. (2006). Fermentation
capabilities of bifidobacteria using nondigestible oligosaccharides,
and their viablitiy as probiotics in commercial powder infant
formula. Journal of Food Science, 70, M279–M285.
Philips, M., Kaliasapathy, K. & Tran, L. (2006). Viability of
commercial probiotic cultures (L. acidophilus, Bifidobacterium sp.,
L. casei, L. paracasei and L. rhamnosus) in cheddar cheese.
International Journal of Food Microbiology, 108, 276–280.
Portner, D.C., Reuschner, G.K.L. & Murry, B.S. (2007). Optimising
the viability during storage of freeze-dried cell preparations of
Campylobacter jejuni. Cryobiology, 54, 265–270.
Potts, M. (2001). Desiccation tolerance: a simple process. Trends in
Microbiology, 9, 553–559.
Puccio, G., Cajozzo, C., Meli, F., Rochat, F., Grathwohl, D. &
Steenhout, P. (2007). Clinical evaluation of a new starter formula for
infants containing live Bifidobacterium longum BL999 and prebiot-
ics. Nutrition, 23, 1–8.
Saxelin, M., Grenov, B., Svensson, U., Fonden, R., Reniero, R. &
Mattila-sandholm, T. (1999). The technology of probiotics. Trends
in Food Science & Technology, 10, 387–392.
Schoug, Å., Olsson, J., Carlfors, J., Schnürer, J. & Håkansson, S.
(2006). Freeze-drying of Lactobacillus coryniformis Si3 – effect of
sucrose concentration, cell density, and freezing rate on cell survival
and thermophysical properties. Cryobiology, 53, 119–127.
Shah, N.P. (2007). Function cultures and health benefits. International
Diary Journal, 17, 1262–1277.
Simpson, P.J., Stanton, C., Fitzgerald, G.F. & Ross, R.P. (2005).
Intrinsic tolerance of Bifidobacterium species to heat and oxygen and
survival following spray drying and storage. Journal of Applied
Microbiology, 9, 493–501.
To, B.C.S. & Etzel, M.R. (1997). Survival of Breveibacterium linens
(ATCC9174) after spray drying, freeze drying, or freezing. Journal of
Food Science, 62, 167–189.
Vinderola, C.G., Prosello, W., Ghiberto, T.D. & Reinheimer, J.A.
(2000). Viability of probiotic (Bifidobacterium, Lactobacillus aci-
dophilus and Lactobacillus casei) and nonprobiotic microflora in
Argentinian Fresco cheese. Journal of Dairy Science, 83, 1905–1911.
Wang, C., Shoji, H., Sato, H. et al. (2007). Effects of oral adminis-
tration of Bifidobacterium breve on fecal lactic acid and short-chain
fatty acids in low birth weight infants. Journal of Pediatric
Gastroenterology and Nutrition, 44, 252–257.
Zhao, G. & Zhang, G. (2005). Effect of protective agents, freezing
temperature, rehydration media on viability of malolactic bacteria
subjected to freeze-drying. Journal of Applied Microbiology, 99, 333–
338.
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