ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 25 (2008) 13–21
www.elsevier.com/locate/fm

Incorporation of citrus fibers in fermented milk

containing probiotic bacteria
Esther Sendra, Patricia Fayos, Yolanda Lario, Juana Fernández-López,
Estrella Sayas-Barberá, José Angel Pérez-Alvarez
Dpto. Tecnologı´a Agroalimentaria, Escuela Politécnica Superior de Orihuela, Universidad Miguel Hernández, Ctra. Beniel km 3.2,
03312 Orihuela (Alicante), Spain
Received 6 June 2007; received in revised form 4 September 2007; accepted 5 September 2007
Available online 11 September 2007

Abstract

The incorporation of Lactobacillus acidophilus CECT 903, Lactobacillus casei CECT 475 and Bifidobacterium bifidum CECT 870
together with lemon (LF) and orange (OF) fibers obtained from juice by-products were tested in (i) a model system: fiber enriched with de
Man Rogosa Sharp (MRS) broth cultured with each probiotic bacteria and (ii) evaluation of populations of probiotic bacteria in
fermented milks formulated with citrus fibers. Citrus fibers enhanced L. acidophilus CECT 903, and L. casei CECT 475 survival in MRS
during refrigerated storage, whereas erratic results were obtained for B. bifidum CECT 870, OF enhanced its growth and LF had
inhibitory effect. Populations of probiotic bacteria decreased with storage time in MRS broth. The presence of yogurt starter bacteria in
probiotic fermented milks favored the growth and survival of L. acidophilus and B. bifidum. Citrus fiber presence in fermented milks also
enhanced bacterial growth and survival of the tested probiotic bacteria. This study indicates that citrus fiber enriched fermented milk
have good acceptability and are good vehicles for a variety of commercial probiotics but survival of B. bifidum will need to be improved.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Probiotic; Citrus fiber; Fermented milk

1. Introduction effects for human health. The recommended daily intake of

fiber is about 38 g for men and 25 g for women (Trumbo et al.,
Production of citrus products in 2004 in FAO countries was 2002). Fibers may be used to fortify foods. Fermented milk
63.95 and 12.53 million tonnes of oranges, and lemons and products already have a positive healthy image due to the
limes, respectively (FAO, 2006). One-third of the citrus fruits beneficial action of its viable bacteria and they are good
is processed to obtain several products, mainly juice candidates to be enriched with citrus fibers. Fiber may interact
(Izquierdo and Sendra, 2003). The amount of residue obtained with other food components during processing. These
from citrus fruits accounts for 50% of the original amount of interactions may lead to changes in bioavailability of
whole fruit (Cohn and Cohn, 1997). The residue remaining nutrients, texture or flavor of the product. Fibers may also
after juice and essential oil extraction is mainly used to obtain interact with the microbial populations of yogurt, either
pectins and also as animal feed with little economic value. The residual essential oils may inhibit their growth or fiber
accumulation of these residues is an environmental problem. components as oligosaccharides may enhance their growth.
These by-products are rich in fiber. The possibility of To exert beneficial effects in the host, it is essential that
successfully including these by-products in the human food lactic acid bacteria and bifidobacteria be alive and abundant
industry would help in enhancing the economic development in the product at the time of consumption. No general
of citrus producers and processors. Fibers also have beneficial agreement has been reached on the concentration of
probiotics to achieve probiotic effects; usually counts from
Corresponding author. Tel.: +34 966749735; fax: +34 966749677. 106 to 108 CFU/g are recommended (Lourens-Hatting and
E-mail address: [email protected] (E. Sendra). Viljeon, 2001). In Japan, the Fermented Milks and Lactic

0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2007.09.003
 ARTICLE IN PRESS
14 E. Sendra et al. / Food Microbiology 25 (2008) 13–21

Acid Beverages Association has already established a Fernández-López et al. (2004). To obtain powder with a
standard that requires more than 107 CFU/mL to be present particle size of 0.701–0.991 mm, a grinder and appropriate
in dairy products to claim to contain probiotic microorgan- sieves were used. The orange fiber product used has: 80.1%
isms, whereas the Swiss Food Regulation as well as the crude fiber (determined by Weende method, 962-09 AOAC,
Standard FIL/IDF requires that such products contain more 1995), 7.34% moisture, 11.3% water holding capacity
than 106 CFU/mL. (WHC), pH 3.92, 30 CFU/g aerobic mesophilic bacteria
Donkor et al. (2006) reviewed the factors that may affect and 25 CFU/g of molds and yeasts. The lemon fiber has:
the survival of Lactobacillus and Bifidobacterium spp. in 81.3% crude fiber, 3.71% moisture, 12.6% WHC, pH 3.98,
yogurt: strain of probiotic bacteria, inoculation level, 165 CFU/g aerobic mesophilic bacteria and 90 CFU/g of
incubation temperature, pH, growth promoters and inhibi- molds and yeasts.
tors, presence of hydrogen peroxide and oxygen, concentra- Pure strains of B. bifidum CECT 870, L. casei CECT 475
tion of metabolites, lactic and acetic acid, buffering capacity and L. acidophilus CECT 903 were obtained from the
of the media, storage temperature, and availability of Spanish Culture Collection (Burjassot, Valencia). For
nutrients. The main factors are ultimate pH and accumula- propagation sterile 10 mL aliquots of the Man Rogosa
tion of organic acids (Dave and Shah, 1997; Shah, 2000). Sharpe (MRS) broth were inoculated and incubated at
Ravula and Shah (1998) observed that when probiotic 30 1C for L. casei and at 37 1C for L. acidophilus. For
bacteria grow together with yogurt cultures the metabolic propagation of bifidobacteria, sterile MRS broth was
activity of Streptococcus thermophilus and Lactobacillus supplemented with 0.05% L-cysteine to provide anaerobic
delbrueckii ssp. bulgaricus during storage (organic acids and condition and stimulate their growth (Ravula and Shah,
amino acids release) may affect probiotics viability. 1998); inoculated vials were also introduced inside anaero-
Several authors have studied the interactions among bic jars for incubation at 37 1C. Incubation time was about
different fibers and probiotic bacteria. Capela et al. (2006) 72 h. The activated microorganisms after three successive
reported that the prebiotic ‘RaftilosesP95’ when added at transfers were used for the trials. Starter commercial
1.5% (w/v) to yoghurt improved the viability of the yogurt cultures contained S. thermophilus and L. delbrueck-
combined selected probiotic organisms (Lactobacillus acid- ii subsp. bulgaricus (Ezals MY900; Rhodia Food, Sasse-
ophilus, Lactobacillus casei, Lactobacillus rhamnosus and nage, France).
Bifidobacterium spp.) by 1.42 log during 4 weeks of storage at Skim milk powder used had 34% protein, 52% lactose,
4 1C. L. casei cells were immobilized on fruit (apple and pear) 1% fat, 6.8% ash and 5.2% moisture (Central Lechera
pieces and the immobilized biocatalysts were used separately Asturiana, CAPSA, Granda-Siero, Spain). Deionised
as adjuncts in probiotic Feta cheese making by Kourkoutas water was used to reconstitute the milk powder. For all
et al. (2006) with satisfactory results. Perez-Conesa et al. the tests the same batch of skim-milk powder was used.
(2005) proved the benefits of combining commercial Culture media and supplements used were: bacteriologi-
nondigestible oligosaccharides (NDO) (short-chain fructoo- cal peptone, MRS broth and MRS agar (Cultimed,
ligosaccharides, inulin, oligofructose, galactosyllactose) with Panreac, Castellar del Vallés, Barcelona, Spain), M-17
several species of Bifidobacterium in powder infant formula. and MRS fermentation broth (Scharlau, Barcelona,
As the pH of the reconstituted formula was always close to Spain); D-sorbitol (Acros Organics, Geel, Belgium);
neutrality (from 6.74 to 7.06), the number of bacteria was L-cysteine, NNLP (nalidixic acid, neomycine sulfate,
always over the recommended level. Martinez-Villaluenga lithium chloride, paromycine sulfate) and vancomycine
et al. (2005) reported that the raffinose family of oligosac- (Sigma Chemical Co., St Louis, MO).
charides (RFOs) isolated from lupin seeds applied to the
manufacture of probiotic fermented milk increased Bifido- 2.2. Survival of probiotic bacteria in a model system
bacterium lactis Bb-12 and L. acidophilus populations at the
final fermentation time compared with controls, and that Sterile 50 mL aliquots of MRS broth (control), MRS
time of fermentation was reduced from 12 to 10 h. broth with 1% OF and MRS broth with 1% LF were
The objective of the present study was to investigate the inoculated with each individual probiotic bacteria. Each
effect of lemon and orange fiber (obtained from citrus by- microorganism was always incubated at their recom-
products) on growth and survival of the probiotic bacteria mended conditions, after 72 h of incubation flasks were
Bifidobacterium bifidum CECT 870, L. casei CECT 475 and cooled and stored at 4 1C. Counts on MRS agar and pH
L. acidophilus CECT 903 in a model system and in were determined at days 1, 5, 10, 20 and 40 of cold storage.
fermented milk during cold storage. The whole experiment was run in triplicate, samples were
analyzed in duplicate.
2. Methods
2.3. Comparison of counts of probiotic bacteria: MRS agar
2.1. Materials vs selective enumeration media

Fiber was obtained from orange (OF) and lemon Up to this point only work with pure cultures had been
(LF) juice by-products by a procedure described by carried out, and so MRS agar had been used for bacterial
 ARTICLE IN PRESS
E. Sendra et al. / Food Microbiology 25 (2008) 13–21 15

enumeration. From now on mixed cultures (yogurt starters 2.5. pH

with individual probiotics) were going to be tested, so
we decided to compare counts of pure cultures of pH was determined by means of a pH meter GPL 21
probiotic bacteria on MRS agar compared to those Crison (Alella, Spain).
obtained on selective media. Media to be used and
incubation conditions were: MRS-vancomycine at 37 1C 2.6. Sensory evaluation
for 72 h and anaerobiosis for counts of L. casei CECT 475
(Tharmaraj and Shah, 2003), MRS-D-sorbitol at 45 1C for Sensory analysis was run on 2 and 30 days stored
72 h and anaerobiosis for L. acidophilus CECT 903 fermented milks, except for those containing B. bifidum,
(Tharmaraj and Shah, 2003), MRS-NNLP at 37 1C and which were not evaluated as the L-cysteine added to
72 h and anaerobiosis for B. bifidum CECT 870 (Dave and enhance its growth provokes a strong sulfur-like odor.
Shah, 1996). Panelists (30) were members of the staff and students of the
Miguel Hernández University, Alicante, Spain. All sensory
work was carried out in the sensory laboratory at the
2.4. Survival of starter cultures and probiotic bacteria in University, which fulfills requirements according to the
fermented milks containing citrus fibers International Standards (ISO, 1988). During evaluation,
the panelists were situated in private booths under
Procedure to prepare fermented milk: skim milk powder incandescent light, with an intensity of approximately
was reconstituted with water at 12% total solids and 10% 350 lx. Tap water was provided between samples to cleanse
sugar was added. Fiber powders were weighed and the palate. White plastic cups with 40 mL of fermented
distributed into 500 mL Pyrexs flasks (3 g to each flask) milks at 10 1C were provided. Six samples were evaluated in
except for control yogurts. 200 mL of milk were poured each session. The sample presentation order was rando-
into the flasks and were pasteurized at 80 1C for 30 min. mized for each panelist. A total of five sessions were run.
Pasteurization was ended by immersion of the flasks in ice- A seven-point scale (from imperceptible to extremely high)
water baths. When 43 1C was reached, the yogurt starter was used to rate the following parameters: citrus aroma,
culture was added and the flasks were gently shaken and citrus flavor, off-flavors, acidity, creaminess and fiber
brought to incubation at 43 1C till pH 4.7 was obtained, at content. A seven-point hedonic scale was used to evaluate
that moment gels were cooled down in a cabinet with air product acceptability.
convection at 4 1C.
The probiotic cultures were prepared separately, and 2.7. Data processing
then added into the yogurt, in order to obtain high counts
of probiotic bacteria and avoid microbial competition with SPSS 14.0 (Chicago, IL, USA) for Windows was used.
the starter cultures. 100 mL aliquots of skimmed milk (12% General Lineal Model procedures and Tukey test for
total solids) were pasteurized at 80 1C for 30 min and means comparison were used for the statistical analysis
cooled down to 40 1C. Individual probiotic bacteria were of data.
inoculated and incubated at their corresponding optimum
growing temperature: 30 1C for L. casei, 37 1C for 3. Results and discussion
L. acidophilus and for Bifidobacteria enrichment milk was
added 0.05% L-cysteine and afterwards incubated under 3.1. Survival of probiotic bacteria in a model system
anaerobic conditions. The cultured milks were incubated containing citrus fibers
until the onset of gelation (approximately 38 h).
Finally, pure cultures of probiotic bacteria (100 mL All the containers used in the present experiments were
aliquots) were added to the fermented milk flasks (already Pyrexs flasks. Glass containers are always preferred over
containing 200 mL of fermented milk) and gently shaken. plastic cups for the storage of fermented milks, as probiotic
Flasks containing 300 mL of probiotic fermented milk viability is about 30–70% higher when stored in glass
(final fiber dose 1%) were obtained and stored at 4 1C. So, bottles than in plastic cups (Shah, 2000). The main reason
three types of fermented milk (control, OF and LF) for that is the lower oxygen permeability of glass compared
inoculated with three individual probiotic cultures (L. casei to plastic cups that helps in maintaining microaerophilic
CECT 475, L. acidophilus CECT 903, B. bifidum CECT or anaerobic conditions.
870) were sampled at days 1 and 30 of cold storage. The In general, the viability of all probiotics in MRS broth
whole experiment was run in triplicate and samples were decreased during refrigerated storage (Figs. 1–3).
analyzed in duplicate. B. bifidum CECT 870: pH of citrus fiber enriched MRS
Counting of S. thermophilus was done in M17 agar and broth was lower than that of control (Table 1). Storage
aerobic incubation at 37 1C for 48 h (Dave and Shah, 1996) time did not significantly (P40.05) affect pH. Fiber
and counting of L. delbrueckii ssp. Bulgaricus, in MRS pH addition by itself may contribute to some decrease but an
5.2 and anaerobic incubation at 45 1C for 72 h (Tharmaraj increased metabolic activity of the Bifidobacteria would
and Shah, 2003). also have contributed to the dramatic pH decrease.
 ARTICLE IN PRESS
16 E. Sendra et al. / Food Microbiology 25 (2008) 13–21

1.00E+09 Table 1
Orange fiber Lemon fiber Control
1.00E+08 pH evolution of a culture of Bifidobacterium bifidum CECT 870 in MRS
1.00E+07 broth (0.05% cysteine) enriched with 1% citrus fibers and stored at 4 1C
1.00E+06 for 40 days (n ¼ 9)
CFU/g

1.00E+05
1.00E+04 Storage time Orange fiber Lemon fiber Control
1.00E+03 (days)
Mean Standard Mean Standard Mean Standard
1.00E+02
error error error
1.00E+01
1.00E+00 1 4.89 70.27 4.63 70.18 5.22 70.03
0 5 10 15 20 25 30 35 40 45
5 5.01 70.26 5.00 70.22 5.58 70.06
Storage time (days) 10 4.75 70.18 4.77 70.14 5.40 70.01
20 4.80 70.16 4.89 70.15 5.62 70.05
Fig. 1. Evolution of counts of Bifidobacterium bifidum CECT 870 kept in
40 4.77 70.08 4.90 70.11 5.65 70.05
MRS (0.05% cysteine) broth enriched with 1% citrus fibers and stored at
4 1C for 40 days.

1.00E+09 Table 2
Orange fiber Lemon fiber Control
1.00E+08 pH evolution of a culture of Lactobacillus acidophilus CECT 903 in MRS
1.00E+07 broth enriched with 1% citrus fibers and stored at 4 1C for 40 days (n ¼ 9)
1.00E+06
CFU/g

Storage time Orange fiber Lemon fiber Control

1.00E+05 (days)
1.00E+04 Mean Standard Mean Standard Mean Standard
1.00E+03 error error error
1.00E+02
1 3.95 70.03 3.95 70.03 4.95 70.01
1.00E+01 5 4.06 70.07 4.05 70.01 5.16 70.05
0 5 10 15 20 25 30 35 40 45
10 3.99 70.02 4.05 70.01 5.13 70.01
Storage time (days) 20 4.01 70.04 4.08 70.01 5.11 70.05
40 3.96 70.09 3.99 70.07 5.15 70.12
Fig. 2. Evolution of counts of Lactobacillus acidophilus CECT 903 kept in
MRS broth enriched with 1% citrus fibers and stored at 4 1C for 40 days.

Table 3
1.00E+11 pH evolution of a culture of Lactobacillus casei CECT 475 in MRS broth
Orange fiber Lemon fiber Control enriched with citrus fibers and stored at 4 1C for 40 days (n ¼ 9)
1.00E+10
Storage time Orange fiber Lemon fiber Control
1.00E+09
(days)
CFU/g

1.00E+08 Mean S.E. Mean S.E. Mean S.E.

1.00E+07 1 3.65 70.04 3.68 70.05 3.72 70.05

5 3.84 70.01 3.85 70.02 3.88 70.01
1.00E+06 10 3.71 70.02 3.71 70.04 3.78 70.03
1.00E+05 20 3.86 70.02 3.88 70.03 3.96 70.04
0 5 10 15 20 25 30 35 40 45 40 3.87 70.01 3.93 70.01 4.02 70.03
Storage time (days)

Fig. 3. Evolution of counts of Lactobacillus casei CECT 475 kept in MRS

broth enriched with 1% citrus fibers and stored at 4 1C for 40 days. Bifidobacteria (Table 2). LF and OF addition significantly
enhanced L. acidophilus viability (Fig. 2), the increased
survival rate ranged from 1 to 2 log CFU/mL. Fiber-
However, pH values were always over 4.5 which is the enriched MRS broth maintained L. acidophiulus counts
critical value for Bifidobacteria survival (Vinderola et al., over 6 log CFU/mL for at least 10 days, whereas at 5 days
2002). Dissolved oxygen and acidity are reported to be the of cold storage control media counts were below this
key factors for Bifidobacteria survival (Dave and Shah, target.
1997). L. casei CECT 475: pH slightly decreased with fiber
Commercial shelf life of fermented milks is about 28 addition (Table 3). Metabolic activity of L. casei was
days, at this time B. bifidum CECT 870 viability was quite probably similar in all MRS broths. Since there was no
low (Fig. 1): in LF media their populations were reduced significant difference (P40.05) in pH or decline in pH
1 log CFU/mL whereas in OF media populations increased values among batches during storage, it was unlikely that
3 log CFU/mL when compared to control values. these changes would have affected the viability of L. casei
L. acidophilus CECT 903: pH of MRS broth containing probiotics due to termination pH. Citrus fiber addition
L. acidophilus presented the same behavior as that of significantly (Po0.05) increased L. casei survival, which
 ARTICLE IN PRESS
E. Sendra et al. / Food Microbiology 25 (2008) 13–21 17

Table 4
Counts of probiotic bacteria in milk obtained on MRS agar and selective media (n ¼ 6)

Probiotic microorganism Culture media and conditions Mean S.E.

Lactobacillus casei MRS: at 30 1C, 72 h 2.73E+09 73.90E+08

MRS-vancomycine: at 37 1C, 72 h anaerobiosis 2.97E+09 71.10E+08
Lactobacillus acidophilus MRS: at 37 1C, 72 h anaerobiosis 1.85E+08 73.50E+07
MRS-D-sorbitol: at 45 1C, 72 h anaerobiosis 3.89E+07 75.00E+05

Bifidobacterium bifidum MRS-0.05% L-cysteine: at 37 1C, 72 h 1.09E+07 71.00E+05

MRS-NNLP: at 37 1C, 72 h anaerobiosis 9.25E+06 75.50E+05

were always over 8 log CFU/mL during 40 days of cold fiber-enriched fermented milks although pH was below 4.5
storage, 1 unit higher than control counts (Fig. 3). and in control yogurt pH was over 4.7. Vinderola et al.
(2002) reported that pH 4.5 or lower negatively affects the
3.2. Counts of probiotic bacteria: MRS agar vs selective cell viability of bifidobacteria in yogurt at 5 1C. Counts in
enumeration media fermented milks were much higher than those in MRS
broth after 30 days; it seems that yogurt starter cultures
Selective media was going to be used for the next favored the growth of B. bifidum CECT 870. Dave and
experiment. So we decided to evaluate the differences in the Shah (1997) stated that S. thermophilus has antagonistic
counts of probiotic bacteria in MRS and selective media effect(s) on the growth of Bifidobacteria. Shankar and
(Table 4). MRS-vancomycine allowed the recovery of Davies (1976) reported that B. bifidum grows better in the
even more colonies of L. casei than MRS: 108.79%. presence of L. bulgaricus due to their symbiotic relation.
MRS-NNLP also showed a good performance, 84.86% of The free amino acids resulting from the proteolytic activity
the counts of Bifidobacteria detected in MRS. MRS- of L. bulgaricus may promote the growth of Bifidobacteria
sorbitol only allowed the recovery of 21.02% of the (Singh et al., 1980).
L. acidophilus counted in MRS. It is well known that there Several authors have studied the interactions among
is no general accepted or fully adequate selective media for Bifidobacteria and commercial fibers in probiotic fermen-
most of the probiotic bacteria (Dave and Shah, 1996; Shah, ted milks: Varga et al. (2006) reported that the presence
2000; Masco et al., 2005). of inulin at 1–5% (w/v) did not influence significantly
(P40.05) the survival rates of either S. thermophilus or
3.3. Survival of starter cultures and probiotic bacteria in L. acidophilus. However, the addition of inulin at 5% (w/v)
fermented milks containing citrus fibers had a significant beneficial effect (Po0.05) on the viability
of Bifidobacteria after 28 days of refrigerated storage. The
Based on the available published data, we decided to largest increase in Bifidobacteria was seen on xylo-oligo
develop a liquid probiotic fermented milk as Lin et al. saccharides and lactulose, the largest in Lactobacilli was on
(2006) reported that the viable cell densities of probiotic fructooligosaccharides (Rastall and Maitin, 2002). Martı́-
bacteria in liquid products were higher than those in the nez-Villaluenga et al. (2006) observed that RFOs extracted
solid products. Probiotics were grown as sole starters as from lupin seeds had beneficial effects on the survival of
Shah (2000) recommended a two-step fermentation proce- B. lactis Bb-12 and L. acidophilus La-5 in fermented milk
dure in order to obtain high counts of probiotics and avoid during 21 days of storage in refrigerated probiotic
the inhibitory effect of hydrogen peroxide and acids on the fermented milk. However, the refrigerated storage de-
probiotic bacteria. When probiotic strains were used on creased significantly the viable cell counts of probiotic
their own to obtain fermented milk with high counts of bacteria.
probiotic bacteria, the acidification was slow (about 38 h) The inhibitory effect of LF on B. bifidum that was
as has been previously reported by Saxelin et al. (1999). observed in the experiment in MRS broth was not detected
B. bifidum CECT 870: Milk was supplemented with in fermented milk. Counts of Bifidobacteria were always
0.05% L-cysteine to favor the growth of B. bifidum as below 6 log CFU/mL. Carr and Ibrahim (2005) evaluated
Güler-Akin and Akin (2007) recommended cysteine addi- the viability of Bifidobacteria among commercial bio-
tion and incubation lower than 43 1C for the manufacture yogurts and reported counts ranging from 0.0 to 6.0 log
of bio-yogurt. Dave and Shah (1998) reported that the CFU/mL, 76% of the samples were found to contain viable
addition of cysteine adversely affected the viability of cultures. Masco et al. (2005) reported that by conventional
S. thermophilus but in our study such inhibition was not cultivation, 70.7% of the commercial bio-yogurts analyzed
evident (Table 5). Surprisingly, all microbial populations were found to contain culturable Bifidobacteria whereas
increased after 30 days of storage compared to the first by culture-independent qualitative analysis members of
day. Counts were always higher (but not significantly) in the genus Bifidobacterium could be detected in 96.5%
 ARTICLE IN PRESS
18 E. Sendra et al. / Food Microbiology 25 (2008) 13–21

Table 5
pH and average microbial counts of fermented milk containing Bifidobacterium bifidum CECT 870

Storage Type of S. thermophilus L. delbrueckii subsp. B. bifidum CECT 870 pH

time (days) yogurt bulgaricus

Mean Standard Mean Standard Mean Standard Mean Standard

error error error error

1 Orange 5.38E+07 76.00E+05 8.15E+05 73.50E+04 4.25E+03 76.50E+02 4.37 70.001

fiber
Lemon fiber 4.65E+06 71.65E+06 4.05E+05 71.50E+04 2.20E+03 73.00E+02 4.34 70.003
Control 3.10E+06 72.83E+05 6.68E+05 74.40E+04 1.65E+03 71.50E+02 4.72 70.001
30 Orange 4.40E+08 71.50E+08 9.40E+05 72.00E+04 2.20E+05 75.00E+03 4.26 70.003
fiber
Lemon fiber 9.45E+07 72.50E+06 8.80E+05 72.00E+04 2.08E+05 72.00E+03 4.26 70.003
Control 5.22E+07 71.00E+06 7.05E+05 71.15E+05 1.73E+05 77.50E+03 4.71 70.002

Table 6
pH and average microbial counts of fermented milk containing Lactobacillus acidophilus CECT 903

Storage Type of S. thermophilus L. delbrueckii subsp. L. acidophilus CECT 903 pH

time (days) yogurt bulgaricus

Mean Standard Mean Standard Mean Standard Mean Standard

error error error error

1 Orange 6.80E+08 71.40E+08 4.57E+07 76.70E+06 2.55E+07 75.90E+06 4.41 70.001

fiber
Lemon fiber 4.70E+08 76.00E+07 5.42E+07 74.39E+07 3.13E+07 73.30E+06 4.33 70.009
Control 8.90E+07 72.00E+06 2.13E+07 75.75E+06 2.07E+07 74.15E+06 4.60 70.003

30 Orange 2.90E+08 72.00E+07 1.26E+07 76.00E+05 1.20E+07 73.00E+05 4.27 70.003

fiber
Lemon fiber 3.40E+08 78.00E+07 1.86E+07 76.00E+05 9.20E+06 75.00E+05 4.31 70.002
Control 1.40E+07 71.90E+06 9.65E+06 75.00E+04 8.85E+06 74.50E+05 4.54 70.003

of the analyzed products. So, there is a possibility that real essential growth factors in the form of peptides and amino
counts may be higher. Further studies may be directed to acids in the yogurts may have promoted the growth of
increase Bifidobacteria viability, may be through increasing L. acidophilus and sustained the growth of B. lactis and
inoculum’s dose or microencapsulation. Kailasapathy Lactobacillus paracasei (Donkor et al., 2006).
(2006) observed that microencapsulation increased survival The interaction of L. acidophilus with several fibers and
of L. acidophilus and B. lactis by 2 and 1 log cell numbers, fiber extracts has also been studied by several authors:
respectively. Kailasapathy and Masondole (2005) reported Desai et al. (2004) tested several prebiotic substances
that microencapsulation did not offer protection to the and observed best viability of Lactobacilli with inulin
probiotic bacteria L. acidophilus DD 910 and B. lactis DD and raftilose whereas lactulose was the least effective
920 in feta cheese. in maintaining viability. Hi-maize was also tested. The
L. acidophilus CECT 903: Counts of L. acidophilus were control samples containing no prebiotics had average
over 6 log CFU/mL in all evaluated samples (Table 6) and survival rate of 33% at 28 days of cold storage. Addition
pH values were higher for control fermented milks. of prebiotics improved the growth rates and decreased
Donkor et al. (2006) reported that Lactobacilli strains fermentation time, there was also slight improvement in
showed a good cellular stability maintaining constant survival rate with prebiotics. Survival of Lactobacilli was
concentration throughout storage period regardless of strain specific. Özer et al. (2005) successfully used lactulose
termination pH. Counts of all three evaluated populations and inulin as growth promoters for B. bifidum and
were slightly lower in control than in fiber-enriched L. acidophilus to keep probiotic counts over 107 during cold
fermented milks (although not significantly). The combina- storage. Lactulose did not promote the viability of Lactoba-
tion of L. acidophilus with yogurt starter cultures clearly cillus spp. Gokavi et al. (2005) in an oat-based symbiotic
enhanced its growth as counts in fermented milk were beverage containing 0.9% dietary fiber observed that
much higher than in MRS broth at 30 days. When L. plantarum and L. casei survived for 10 weeks whereas
probiotics are mixed with yogurt cultures, higher levels of L. acidophilus survived for about 4 weeks over 107 CFU/g.
 ARTICLE IN PRESS
E. Sendra et al. / Food Microbiology 25 (2008) 13–21 19

L. casei CECT 475: Counts of all microbial populations perception after 30 days of storage, probably due to the
were over 6 log CFU/mL in L. casei containing probiotic diffusion of compounds from the fiber. The perception of
fermented milks except for L. delbrueckii subsp. bulgaricus citrus flavor tended to be higher than that of citrus aroma.
in LF and control samples after 1 day of storage (Table 7) Acidity and off flavors did not show significant differences
and all populations increased after 30 days of cold storage, due to any of the studied variables and presented average
probably due to synergisms among microbial populations. values of 3.53 (S.D. ¼ 1.21) and 1.58 (S.D. ¼ 0.72),
No significant differences were detected among pH due to respectively, the results are not presented in the table.
storage time or fiber presence. L. casei CECT 475 was the Creaminess perception was highest in control probiotic
best-performing probiotic bacteria judging from its highest milks (Po0.05), storage time also enhanced the perception
counts in all the experiments. of creaminess whereas no effect could be attributed to
probiotic bacteria. Fiber particles may be responsible for
3.4. Sensory analysis the reduced creaminess of fiber-fermented milks. The
fermentation process which includes pH decrease and
Sensory evaluation results of probiotic milks containing formation of exopolysaccharides may be responsible for
L. casei and L. acidophilus are presented in Table 8. There the increased creaminess of fermented milks stored for 30
were significant differences (Po0.05) in citrus aroma and days. Garcı́a-Pérez et al. (2006) reported by instrumental
flavor as well as fiber content perception between control and sensory analysis, increased viscosity with storage time
and fiber added yogurts, whereas no differences were due in orange fiber-enriched yogurts. Fiber content and overall
to the probiotic bacteria used. There was a tendency (not acceptability are quite balanced as fiber and sugar contents
significant P40.05) to increase citrus aroma and flavor as well as yogurt cultures had been previously established

Table 7
pH and average microbial counts of fermented milk containing Lactobacillus casei CECT 475

Storage Type of S. thermophilus L. delbrueckii subsp. L. casei CECT 475 pH

time (days) yogurt bulgaricus

Mean Standard Mean Standard Mean Standard Mean Standard

error error error error

1 Orange 6.00E+07 71.00E+06 1.30E+06 71.00E+05 1.11E+08 71.00E+06 3.99 70.001

fiber
Lemon fiber 4.65E+07 71.65E+06 9.65E+05 71.50E+04 9.90E+07 72.50E+05 4.01 70.003
Control 3.82E+07 73.80E+06 5.00E+05 71.00E+05 9.92E+07 71.50E+05 4.16 70.001
30 Orange 1.10E+09 71.50E+08 1.08E+08 72.00E+06 3.04E+08 76.80E+07 3.92 70.003
fiber
Lemon fiber 9.80E+08 74.00E+07 9.65E+07 71.50E+06 1.93E+08 75.50E+06 3.97 70.003
Control 1.17E+08 74.50E+06 4.85E+06 73.50E+05 1.42E+08 73.50E+06 4.00 70.003

Table 8
Scores obtained by sensory evaluation of probiotic-fermented milks with lemon and orange fibers at days 2 and 30 after manufacture (1–7 points)

Type Citrus aroma Citrus flavour Creaminess Fiber content Overall acceptability

2 days 30 days 2 days 30 days 2 days 30 days 2 days 30 days 2 days 30 days

L. casei control 0.23a 0.36a 1.17a 1.21a 2.82b 3.32b 0.80a 0.69a 5.22a,b 5.48b
(0.16) (0.28) (0.97) (0.76) (0.44) (0.39) (0.62) (0.34) (0.19) (0.18)
L. casei lemon 2.76b 3.43b 3.56b 3.83b 1.98a 2.47b 3.91b 3.78b 5.03a 5.36b
(0.65) (0.63) (0.84) (0.71) (0.52) (0.51) (0.73) (0.93) (0.21) (0.27)
L. casei orange 3.55b 3.96b 3.99b 4.12b 2.10a 2.67b 3.55b 3.42b 5.43b 6.01c
(0.95) (0.87) (0.85) (0.93) (0.54) (0.49) (0.82) (0.87) (0.24) (0.18)
L. acidophilus control 0.27a 0.32a 0.97a 1.03a 2.72b 3.19b 0.76a 0.71a 5.13a,b 5.59b
(0.16) (0.23) (0.57) (0.79) (0.44) (0.53) (0.39) (0.31) (0.18) (0.28)
L. acidophilus lemon 2.76b 3.63b 3.47b 3.75b 1.78a 2.39a,b 3.71b 3.56b 4.78a 5.23a,b
(0.90) (0.78) (0.63) (0.98) (0.55) (0.61) (0.89) (0.91) (0.23) (0.24)
L. acidophilus orange 3.65b 4.12b 3.67b 3.96b 2.03a 2.94b 3.63b 3.47b 4.96a 5.79b,c
(0.98) (0.89) (0.87) (0.93) (0.64) (0.37) (0.96) (1.02) (0.28) (0.23)

Mean values for each parameter (combining 2 and 30 days) with different superscript letters (a, b, c) are significantly different (Po0.05).
 Values in parentheses denote standard deviation.
 ARTICLE IN PRESS
20 E. Sendra et al. / Food Microbiology 25 (2008) 13–21

based on sensory studies (Garcı́a-Pérez et al., 2005, 2006). Desai, A.R., Powell, I.B., Shah, N.P., 2004. Survival and activity of
All types of fermented milks received average scores of at probiotic Lactobacilli in skim milk containing prebiotics. J. Food Sci.
69 (3), 57–60.
least 4.78 on a seven-point scale. Orange enriched
Donkor, O.N., Henriksson, A., Vasiljevic, T., Shah, N.P., 2006. Effect of
fermented milks got the best results for overall accept- acidification on the activity of probiotics in yoghurt during cold
ability followed by control and lemon milks. It has to be storage. Int. Dairy J. 16 (10), 1181–1189.
pointed out that acceptability increased with storage time FAO, 2006 /www.fao.orgS.
and that is probably due to the increased perception of Fernández-López, J., Fernández-Ginés, J.M., Alesón-Carbonell, L.,
creaminess. Sendra, E., Sayas-Barberá, E., Pérez-Alvarez, J.A., 2004. Application
of functional citrus by-products to meat products. Trends Food Sci.
Technol. 15, 176–185.
4. Conclusions Garcı́a-Pérez, F.J., Lario, Y., Fernández-López, J., Sayas, E., Pérez-
Alvarez, J.A., Sendra, E., 2005. Effect of orange fiber addition on
yogurt color during fermentation and cold storage. Color Res. Appl.
Citrus fibers enhanced L. acidophilus CECT 903 and 30 (6), 457–463.
L. casei CECT 475 survival in MRS broth during Garcı́a-Pérez, F.J., Sendra, E., Lario, Y., Fernández-López, J., Sayas, E.,
refrigerated storage, but uneven results were obtained for Pérez-Alvarez, J.A., 2006. Rheology of orange fiber enriched yogurt.
B. bifidum CECT 870. OF enhanced its growth and LF had Milchwissenschaft 61 (1), 55–59.
Gokavi, S., Zhang, L., Huang, M.-K., Zhao, X., Guo, M., 2005. Oat-
inhibitory effect. Populations of probiotic bacteria de- based symbiotic beverage fermented by Lactobacillus plantarum,
creased with storage time in MRS broth. The combination Lactobacillus paracasei ssp. casei, and Lactobacillus acidophilus.
of yogurt starter cultures and the tested probiotic bacteria J. Food Sci. 70 (4), 216–223.
resulted in a symbiotic interaction that favored the growth Güler-Akin, M.B., Akin, M.S., 2007. Effects of cysteine and different
and survival of all probiotic bacteria in fermented milks. incubation temperatures on the microflora, chemical composition and
sensory characteristics of bio-yogurt made from goat’s milk. Food
Citrus fiber presence in fermented milks also enhanced Chem. 100 (2), 788–793.
bacterial growth and survival of the tested probiotic ISO, 1988. International Standard 8589. Sensory analysis general guidance
bacteria. This study indicates that citrus fiber-enriched for the design of test rooms. Ref. No. ISO 8589:1988 (E). International
fermented milks are good vehicles for a variety of Organization for Standardization, Geneva.
commercial probiotics, as proved by bacterial counts and Izquierdo, L., Sendra, J.M., 2003. Citrus fruits: composition and
characterization. In: Caballero, B., Trugo, L., Finglas, P. (Eds.),
sensory scores, but survival of B. bifidum will need to be Encyclopedia of Food Sciences and Nutrition. Academic Press,
improved and the use of L-cysteine suppressed or reduced Elsevier Science Ltd., Oxford.
to avoid off-flavors. Kailasapathy, K., 2006. Survival of free and encapsulated probiotic
bacteria and their effect on the sensory properties of yoghurt. Food
Sci. Technol. 39 (10), 1221–1227.
Acknowledgment Kailasapathy, K., Masondole, L., 2005. Survival of free and micro-
encapsulated Lactobacillus acidophilus and Bifidobacterium lactis and
The financial support by Bancaja-UMH through the their effect on texture of feta cheese. Aust. J. Dairy Technol. 60 (3),
252–258.
Project ‘‘seguridad alimentaria y propiedades funcionales
Kourkoutas, Y., Bosnea, L., Taboukos, S., Baras, C., Lambrou, D.,
de leches fermentadas formuladas con subproductos Kanellaki, M., 2006. Probiotic cheese production using Lactobacillus
cı́tricos y bacterias probióticas: interacciones fibra de casei cells immobilized on fruit pieces. J. Dairy Sci. 89 (5), 1439–1451.
cı́tricos-bacterias probióticas’’ is gratefully acknowledged. Lin, W.-H., Hwang, C.-F., Chen, L.-W., Tsen, H.-Y., 2006. Viable counts,
characteristic evaluation for commercial lactic acid bacteria products.
Food Microbiol. 23 (1), 74–81.
References Lourens-Hatting, A., Viljeon, B.C., 2001. Yogurt as probiotic carrier
food. Int. Dairy J. 11, 1–17.
AOAC, 1995. Official Methods of Analysis, 15th ed. Association of Martinez-Villaluenga, C., Frias, J., Vidal-Valverde, C., Gomez, R.,
Official Analytical Chemist, Washington, DC. 2005. Raffinose family of oligosaccharides from lupin seeds as
Capela, P., Hay, T.K.C., Shah, N.P., 2006. Effect of cryoprotectants, prebiotics: application in dairy products. J. Food Prot. 68 (6),
prebiotics and microencapsulation on survival of probiotic organisms 1246–1252.
in yoghurt and freeze-dried yoghurt. Food Res. Int. 39 (2), 203–211. Martı́nez-Villaluenga, C., Frias, J., Gomez, R., Vidal-Valverde, C., 2006.
Carr, J.P., Ibrahim, S.A., 2005. Viability of bifidobacteria in commercial Influence of addition of raffinose family oligosaccharides on probiotic
yogurt products in North Carolina. (2005). Milchwissenschaft 60 (4), survival in fermented milk during refrigerated storage. Int. Dairy J. 16
414–416. (7), 768–774.
Cohn, R., Cohn, A.L., 1997. Subproductos del procesado de las frutas. In: Masco, L., Huys, G., De Brandt, E., Temmerman, R., Swings, J., 2005.
Arthey, D., Ashurst, P.R. (Eds.), Procesado de frutas. Acribia, Culture-dependent and culture-independent qualitative analysis of
Zaragoza, pp. 213–228. probiotic products claimed to contain bifidobacteria. Int. J. Food
Dave, R.I., Shah, N.P., 1996. Evaluation of media for selective Microbiol. 102 (2), 221–230.
enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii Özer, D., Akin, S., Özer, B., 2005. Effect of inulin and lactulose on
spp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium spp. survival of Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum
J. Dairy Sci. 79, 1529–1537. BB-02 in acidophilus–bifidus yogurt. Food Sci. Technol. Int. 11 (1),
Dave, R.I., Shah, N.P., 1997. Viability of yogurt and probiotic bacteria 19–24.
in yoghurts made from commercial starter culture. Int. Dairy J. 7, Perez-Conesa, D., Lopez, G., Ros, G., 2005. Fermentation capabilities of
31–41. bifidobacteria using nondigestible oligosaccharides, and their viability
Dave, R.I., Shah, N.P., 1998. Ingredient supplementation effects as probiotics in commercial powder infant formula. J. Food Sci. 70 (6),
on viability of probiotic bacteria in yogurt. J. Dairy Sci. 81, 2804–2816. 279–285.
 ARTICLE IN PRESS
E. Sendra et al. / Food Microbiology 25 (2008) 13–21 21

Rastall, R., Maitin, V., 2002. Prebiotics and synbiotics: towards the next Tharmaraj, N., Shah, N.P., 2003. Selective enumeration of Lactobacillus
generation. Curr. Opin. Biotechnol. 13, 490–496. delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus
Ravula, R.R., Shah, N.P., 1998. Viability of probiotic bacteria in acidophilus, Bifidobacteria, Lactobacillus casei, Lactobacillus rhamno-
fermented frozen dairy desserts. Food Aust. 50, 136–139. sus, and Propionibacteria. J. Dairy Sci. 86, 2288–2296.
Saxelin, M., Grenov, B., Svensson, U., Fondén, R., Reniero, R., Mattila- Trumbo, P., Schlicker, S., Yates, A., Poos, M., 2002. Dietary
Sandholm, T., 1999. The technology of probiotics. Trends Food Sci. reference intakes for energy, carbohydrate, fiber, fat, fatty acids,
Technol. 10, 387–392. cholesterol, protein, and amino acids. J. Am. Diet. Assoc. 102 (11),
Shah, N.P., 2000. Probiotic bacteria: selective enumeration and survival in 1621–1630.
dairy foods. J. Dairy Sci. 83, 894–907. Varga, L., Szigeti, J., Gyenis, B., 2006. Influence of chicory inulin
Shankar, P., Davies, F., 1976. Associative bacterial growth in yogurt on the survival of microbiota of a probiotic fermented milk during
starters: initial observations on stimulatory factors. J. Soc. Dairy refrigerated storage. (2006). Ann. Microbiol. 56 (2), 139–141.
Technol. 30, 31–32. Vinderola, C.G., Mocchiutti, P., Reinheimer, J.A., 2002. Interactions
Singh, J., Khanna, A., Chandar, H., 1980. Effect of incubation among lactic acid starter and probiotic bacteria used for fermented
temperature and heat treatments of milk from milk of cow and dairy products. J. Dairy Sci. 85, 721–729.
buffalo on acid and flavour production by St. termophilus and L.
bulgaricus. J. Food Prot. 43, 399–400.