JFS S: Sensory and Nutritive Qualities of Foods

Sensory Acceptability and Stability of Probiotic

Microorganisms and Vitamin C in Fermented
Acerola (Malpighia emarginata DC.) Ice Cream
CARMEN SLVIA FAVARO-TRINDADE, SABRINA BERNARDI, RENATA BARBOSA BODINI, J
ULIO C
ESAR DE CARVALHO BALIEIRO, AND
EDUARDO DE ALMEIDA

ABSTRACT: Six fermented acerola ice creams were produced, containing different starter cultures (Bifidobacterium
longum, Bi.lactis, and traditional yogurt starter cultureStreptococcus thermophilus and Lactobacillus delbrueckii
spp. bulgaricus) and final pH (5 and 4.5). The ice creams were evaluated for probiotic culture viability, vitamin C
stability, and sensory acceptance. Mix fermentations were stopped when pH 5.0 and 5.5 were attained. However,
after the addition of acerola pulp the determined pH were 4.5 and 5, respectively. Mixes were frozen and stored for
15 wk at 18 C. The viable counts for probiotic cultures remained above the recommended minimum limit of 106
cfu/g during 15 wk storage even in products with pH 4.5. Vitamin C concentration remained around 140 mg/100 g of
product. The attributes of aroma, taste, texture, and overall acceptance obtained scores in the range of 5.15 to 7.22.
The fermented acerola ice cream was a suitable food for the delivery of vitamin C and Bifidobacterium strains with
excellent viability and acceptable sensory characteristics.
Keywords: Bifidobacterium lactis, Bifidobacterium longum, dairy dessert, frozen yogurt

Introduction

S: Sensory & Nutritive Qualities of Foods

odern consumers are increasingly interested in their personal

health, and expect the food they eat to be healthy or even
capable of preventing illness (Mattila-Sandholm and others 2002).
Probiotics are live microbial food supplements, which benefit the
health of consumers by maintaining or improving their intestinal
microbial balance (Fuller 1989).
Lactobacillus and Bifidobacterium are the most common species
of bacteria used as probiotics for the production of fermented milks
and other dairy products (Fuller 1989).
Though, nonviable microorganisms could have probiotic properties, in most of cases viable cultures have better effects (Ouwehand
and Salminen 1998). Therefore, in order to maintain consumer confidence in probiotic products, it is important to ensure a high survival rate of the bacteria during the product shelf life (Saxelin and
others 1999). The ingestion of 106 to 109 viable cells per day is necessary for humans in order to develop beneficial effects (Lee and
Salminen 1995). In addition, a pleasant taste and an attractive texture are essential for all dairy products, regardless of the health
message connected to them (Saxelin and others 1999).
Ice cream seems, therefore, suitable for delivering probiotics in
human diet. Several studies (Hekmat and Mcmahon 1992; Davidson
and others 2000; Akin 2005) have demonstrated that it is possible
to produce ice cream type frozen yogurt using different ratios of
fermented mixes. In order to ensure that the product provides an
adequate content of microorganisms, cells must survive freezing
and refrigerated storage.

Freezing and thawing cause various degrees of damage to cells,

including microorganism death (lethal effect), inhibition of its development, and reduction or interruption of metabolic activity (sublethal effect) (Davies and Obafemi 1985). Some studies have proved
that lower temperatures can ensure a higher survival rate, and that
mortality increases with storage time (Smittle and others 1974;
Thunell and others 1984; Foschino and others 1992). However, the
direct enumeration of active cells by plating after thawing has not
been thoroughly investigated.
Acerola (Malpighia emarginata DC.) is a plant originated in Central America that has been propagated in South America including
Brazil due to its adaptation to soil and climate. It is nutritionally important, mainly due to its very high level of vitamin C (2000 to 3000
mg/100 g of juice) (Marino Neto 1986). According to Yamashita and
others (2003), acerola products that have undergone pasteurization
and freezing processes presented greater vitamin C retention at the
end of the storage period.
The aim of this work was to evaluate the resistance of Bifidobacterium longum, Bi. lactis, and vitamin C to the process employed in
the manufacturing of acerola ice cream, as well as to determine the
stability of probiotic cultures and vitamin C during a 15 wk storage
period at 18 C, and to evaluate the sensorial acceptance of probiotic products in comparison with a product elaborated with the
traditional starter culture of yogurt.

Materials and Methods

Materials

MS 20050707 Submitted 12/1/2005, Accepted 5/12/2006. The Authors are with

Univ. de Sao Paulo, Faculdade de Zootecnia e Engenharia de alimentos,
Dept. de Engenharia de alimentos. Avenida Duque de Caxias Norte, 225,
Caixa Postal 23, CEP 13 635 900, Pirassununga SP, Brazil. Direct inquiries
to author Favaro-Trindade (E-mail: carmenft@usp.br).

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JOURNAL OF FOOD SCIENCEVol. 71, Nr. 6, 2006

Commercial freeze-dried probiotic cultures of B. longum (BL-04)

and B. lactis (BL-01) were obtained from Rhodia (Brazil) and Danisco
(Brazil), respectively. Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus were obtained from Chr. Hansen
(Brazil).


C 2006 Institute of Food Technologists
doi: 10.1111/j.1750-3841.2006.00100.x
Further reproduction without permission is prohibited

Sensory acceptability and stability . . .

samples. Samples were stored at 18 C in a freezer to maintain inSix fermented acerola ice cream samples were prepared contain- tegrity during sensory analyses. The ice creams were sensory evaling either (1) S. thermophilus and L. delbrueckii spp. bulgaricus and uated after 2 wk of storage.
final pH 5, (2) S. thermophilus and L. delbrueckii spp. bulgaricus and
final pH 4.5, (3) B. longum and final pH 5, (4) B. longum and final Statistical analysis
The obtained data were statistically analyzed by SAS (2001), 8.02
pH 4.5, (5) B. lactis and final pH 5, or (6) B. lactis and final pH 4.5.
Ice cream mix was manufactured with 15.7 kg pasteurized milk version, through PROC ANOVA procedure. Tukeys Honestly Signif(3% fat), 3.94 kg sucrose, 1.08 kg pasteurized cream milk (35% fat), icant Difference (HSD) was adopted as multiple comparisons pro0.048 kg emulsifier lecithin, 0.038 kg stabilizer guar gum, and 0.038 kg cedures.
For sensorial evaluation, the statistical model of cross classifistabilizer carboxymethylcellulose.
Ice cream mix was homogenized, pasteurized at 85 C for 10 min cation showed the main effects of culture (B. longum, B. lactis
in a hot water bath (RCamargo, Brazil), aged at 4 C for 16 h in traditional culture) and pH samples (4.5 and 5.0), besides double
a vat (RCamargo, Brazil) and divided into 6 parts. Each part was interaction (Culture pH).
preheated at 40 C in a bath hot water, inoculated with appropriate
culture (0.01% of mix weight) and then fermented at 40 C until pH
Results and Discussion
reached 5.5 or 5.0. After fermentation, 0.53 kg acerola pasteurized
pulp was incorporated into each mix. The mixes were frozen using pH
the ice cream freezer (RCamargo, Brazil), with the product being
The mix fermentations were interrupted when pH 5.5 and 5.0
drawn and mixed with air. The outlet temperature of the frozen mix were achieved. However, with the addition of acerola pulp before
was 6 C. The frozen mixes were extruded and packaged (1 L plastic freezing, pH values were reduced to 5.0 and 4.5, respectively.
containers) and stored in a freezer (18 C).
Ice creams pH did not vary during storage period.
Processes were carried out in triplicate for each of the 6 evaluated
samples. After each process all the analyses were repeated except Probiotics viability
for the sensory analysis.
Table 1 show the results of counts of the B. longum and B. lactis

Procedure for ice cream manufacturing

pH
The pH of mixes and ice creams were measured using a digital
pH meter (Marte, Brazil).

Probiotics viability
Prior to and immediately after freezing the product, samples were
taken to assess the viability of probiotic microorganisms by plate
count. During the next 15 wk of storage, the product was regularly
examined for probiotic cell viability. Samples of ice cream (25 g)
were serially diluted (w/w) with 2% citrate solution and the spread
inoculated in duplicate onto plates of MRS agar (Acumedia, Lansing, Mich., U.S.A.). Inoculated plates were incubated at 37 C for 72
h, anerobically (Anaerobac, Probac, Brazil). The resulting colonies
were counted and total viable populations calculated per gram of
product (Favaro-Trindade and Grosso 2002).
Tests were carried out in triplicate.

Determination of vitamin C

strains, respectively, in mix, newly produced and acerola ice cream

stored for up to 15 wk.
There was no significant difference (P < 0.05) in cell populations
before and after product manufacture. These results indicated that
these cultures were resistant to freezing, churning, and air incorporation and were very similar to that reported by Christiansen and
others (1996), Hagen and Narvhus (1999), and Alamprese and others
(2002) for different probiotic microorganisms and ice cream production conditions. Heenan and others (2004) reported that there was
no significant difference in cell populations of B. lactis BDBB2 and
Bb-12 before and after freezing and churning of a frozen soy dessert.
After 15 wk, the populations of B. longum and B. lactis showed
little decrease (0.1 to 0.6 log unit), but these variations were not statistically significant for both culture samples, except for B. longum
in acerola ice cream at pH 5.0. The numbers of viable bifidobacteria
in all the samples remained above 106 cfu/g during 15 wk storage at
18 C. These results indicated that these cultures were resistant to
frozen storage.
Small variations existed in measuring viable numbers of microorganisms at regular intervals, for example, at the 9th week for B.
longum at pH 5.0, in which the variation could be attributed to

Prior to, immediately after freezing, and after 15 wk of storage

the product samples were taken to assess the stability of vitamin C
(ascorbic acid) by the method of iodometric titration (Andrade and
Table 1 --- Viable counts (cfu/g) of B. longum and B. lactis
others 2002).
in fermented acerola mix and ice cream samples with pH
Tests were carried out in triplicate.
4.5 and 5.0 after 1, 3, 6, 9, 12, and 15 wk of storage at
18 C
The sensory evaluation was carried out in laboratory scale, in individual booths under fluorescent white light and with 66 untrained
panelists.
The panelists, students, and staffs from USPFood Engineering
Dept. (Pirassununga, Brazil) were selected based on willingness to
participate of this project.
The consumer acceptance tests were conducted according to
Meilgaard and others (1999) for evaluation of taste, aroma, texture,
and overall acceptance. A 9-point structured hedonic scale was used,
with 1 as dislike extremely and 9 as like extremely. The samples
were presented nomadically in plastic glasses coded with 3 digit random numbers. Panelists were instructed to rinse the palate among

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Storage
weeks
Mix
0
1
3
6
9
12
15

B. longum
pH 4.5
5.4 108aA
2.6 108aA
3.6 108aA
3.5 108aA
3.4 108aA
3.8 108aA
2.7 108aA
3.2 108aA

pH 5.0
2.3 108aB
1.7 108abA
1.2 108bcB
1.1 108bcB
1.2 108bcB
9.6 107cA
9.3 107cB
1.3 108bcB

B. lactis
pH 4.5
6.5 108aA
1.1 108aA
4.3 108aA
6.4 108aA
5.7 108aA
5.0 108aA
5.1 108aA
4.5 108aA

pH 5.0
2.1 108bB
1.0 108bB
5.1 107aB
1.3 108bA
1.6 108bA
1.4 108bB
1.3 108bA
1.4 108bB

a,b,c

Means within the same column with different letters are significantly
different (P < 0.05).
Means within the same row with different letters are significantly different
(P < 0.05).

A,B

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S: Sensory & Nutritive Qualities of Foods

Sensory evaluation

Sensory acceptability and stability . . .

the inherent errors within microbiological methods, or the possible clumping of microorganisms in inadequately mixed samples
causing variations within the different tubs tested on each occasion
(Haynes and Playne 2002).
The total viable populations of B. longum and B. lactis in products with pH 4.5 were significantly higher than in products with
pH 5.0 in almost all period analyzed (Table 1), probably due to the
mixes being fermented for a longer time (approximately 1 h more) to
reach a lower pH; consequently, the microorganisms had more time
for multiplication. On the other hand, this lower pH did not have a
harmful effect, as expected. Laroia and Martin (1990) showed that
bifidobacteria did not survive well in fermented products at pH below 4.6.

Determination of vitamin C
Vitamin C was monitored in the mix and ice creams to verify
the effect of freezing and frozen storage on this nutrient. The results for vitamin C concentration in mix fermented by B. longum,
B. lactis, or traditional yogurt starter culture and acerola ice creams
newly produced and stored for up to 15 wk, varied between 143.4
and 137.0 mg/100 g, with average values of 141.7 mg/100 g and
standard deviation of 2.13. The concentration of vitamin C was not
affected by the process employed in the production, pH, frozen storage, and by the microorganism used as starter culture, once the
concentration did not change significantly (P < 0.05) for any of the
treatments tested. According to Yamashita and others (2003), acerola
products going through a process of pasteurization and freezing
showed greater vitamin C retention at the end of the storage period.

Sensory analysis
A good sensory quality is required by all food products and very
limited information has been reported on the effects of probiotic
cultures in ice cream or frozen yogurts and sensory quality of these
products.
Acerola fermented ice creams were evaluated by an untrained
panel composed of 40 women and 26 men, aged 18 to 50.
The results presented in Table 2 show the means attributed by
the panelists for the acceptance test. In general, the samples had
good acceptance, range of 6.53 to 5.61, or liked slightly to liked
regularly.
There was significant difference (P < 0.05) among means attributed to taste and global acceptance only for sample 4. It probably
means that the panelists considered the taste as the most important
attribute, so the means were similar.
Texture obtained superior significant means (P < 0.05) if compared to the other attributes for all treatments. This result agrees
with the responses given by the panelists. Related to the ones liked
the most, 76% of them quoted texture as the most appreciated attribute. Reduction in pH caused by fermentation process can result
Table 2 --- Means values obtained with sensorial analysis:
comparison among attributes
Ice cream
types

S: Sensory & Nutritive Qualities of Foods

1 (Traditional cultures/pH 5.0)

2 (Traditional cultures/pH 4.5)
3 (B. longum/pH 5.0)
4 (B. longum/pH 4.5)
5 (B. lactis/pH 5.0)
6 (B. lactis/pH 4.5)

Overall
Aroma Taste Texture acceptance
5.88c
6.00c
5.73b
5.15c
5.44c
5.47c

6.48b
6.58b
5.72b
5.21c
5.74b
6.00b

7.14a
7.22a
5.83a
6.92a
6.30a
7.17a

a,b,c

6.53b
6.42b
5.71b
5.61b
5.68b
5.92b

Means within the same row with different letters are significantly different (P
< 0.05).

Scores are based on 9-point hedonic scale with 1 as dislike extremely and 9
as like extremely.

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JOURNAL OF FOOD SCIENCEVol. 71, Nr. 6, 2006

Table 3 --- Valuation means obtained with sensorial analysis: comparison of main effect culture
Cultures

Aroma

Taste

Texture

Overall
acceptance

Traditional
B. longum
B. lactis

5.94a
5.44b
5.45b

6.53a
5.47b
5.87b

7.14a
6.38b
6.73a,b

6.47a
5.66b
5.80b

a,b

Means within the same column with different letters are significantly different
(P < 0.05).

in structural alterations of proteins, which probably influenced positively the development of a pleasant texture on the ice cream.
Aroma was the least acceptable attribute in 4 of the treatments.
Means varied from 6 to 5.15. This attribute could probably be improved with the addition of acerola aroma; many panelists commented that they did not notice the aroma of the fruit, although the
samples had been prepared with about 13% of acerola pulp in their
composition.
ANOVA test could verify, through statistic treatment of data obtained with sensorial analysis, that double interaction (pH culture)
and main effects analyzed, it only detected significant results (P <
0.05) for the main effect of culture, for all variables (aroma, taste,
texture, and global acceptance) and variable texture for pH (Table 3
and 4).
Table 3 shows that samples elaborated with the traditional culture
of yogurt were better accepted (P < 0.05) in terms of aroma, taste,
and global acceptance than those produced with probiotic cultures.
There were no differences among probiotic cultures. This is a very
important result and proves that is not easy to elaborate fermented
foods with probiotic cultures with the same sensorial acceptance of
those obtained with traditional culture of yogurt.
S. thermophilus and L. delbrueckii ssp. bulgaricus produce as the
main metabolic only lactic acid while bifidobacterias are heterofermentatives, produce lactic and acetic acids besides formic and
succinic acids as secondary metabolic (Laroia and Martin 1990). Ice
creams elaborated with bifidobacteria probably had lower acceptance because of the presence of these metabolic. The presence of
acerola pulp in the ice creams was not able to mask the taste and
aroma given by produced metabolic. In addition, most consumers
are usually habituated to ingest dairy fermented products with sensorial characteristics of yogurt, it means, taste and aroma attributed
to metabolic produced by S. thermophilus and L. delbrueckii ssp.
bulgaricus. Otherwise, they tend to reject fermented products with
different characteristics.
Davidson and others (2000) reported that, in general, ice creams
fermented with probiotic cultures had presented less aroma and
taste of yogurt than those produced with traditional culture. The
characteristics of yogurt were favored by a lower pH. In this study,
the samples produced with traditional culture were not commented
by the panelists as the ones with characteristic aroma or taste of
yogurt although had been significantly better accepted.
The panelists did not report strange taste or aroma in the samples.
According to Hagen and Narvhus (1999), the results for sensorial
Table 4 --- Valuation means obtained with sensorial analysis: comparison of pH main effect
pH

Aroma

Taste

Texture

Overall
acceptance

4.5
5.0

5.54a
5.68a

5.93a
5.98a

7.11a
6.42b

5.98a
5.97a

a,b

Means within the same column with different letters are significantly different
(P < 0.05).
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Sensory acceptability and stability . . .

Conclusions

he results of our study indicate that it is feasible to use both

B. longum and B. lactis for mix fermentation aiming the production of ice cream with good consumer acceptance and probiotic properties, since, these bacteria survived at high levels under
unfavorable conditions such as freezing, churching, high oxygen
concentration, low pH, and low temperature storage. Besides, with
the use of acerola pulp, the ice cream also was a good source of
vitamin C.

Acknowledgments
The provision of probiotic cultures from Danisco and Rhodia, Brazil.
This work was financially supported by Fundaca o de Amparo a`
Pesquisa do Estado de Sao Paulo (FAPESP) (Project 03/06993-0).

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evaluation of ice creams produced with probiotic cultures were considered satisfactory because it was not noticed the presence of probiotic flavor in the samples.
Related to pH main effect, there was a significant effect only for
texture. Table 4 shows that pH 4.5 resulted in significantly superior
sensory acceptance (P < 0.05) for this attribute. The pH 4.5 is below
of the isoelectric point of casein, what probably provoked bigger alterations in the protein structures which resulted in a more pleasant
texture for the consumers.
The fermented acerola ice cream was very acceptable to consumers, with 64% of the panelists responding that they would be
interested in purchasing the product.