Microencapsulation of Probiotic Bacteria with Alginate and Prebiotic and Evaluation

of Survival in Ice Cream

Cynthia Jurkiewicza, Milena P. M. Boscariolia, Rubia G. Ferreiraa, Eliana P. Ribeiroa, Wesley H. Prietoa,
Leo Kunigka
a

Maua Institute of Technology, So Caetano do Sul, Brazil (cynthia@maua.br)

ABSTRACT
The aim of this study was to evaluate the influence of prebiotics, resistant starch and acacia gum,
incorporated into alginate beads, on the survival of microencapsulated bacteria in ice cream over a period of
180 days of storage at -18 C. For microencapsulation of Lactobacillus acidophilus NCFM and
Bifidobacterium lactis BI-04, 1.0% sodium alginate containing 0.1% of each freeze-dried culture and 2.0% of
prebiotic (acacia gum or resistant starch) was atomized in a 0.1 M calcium chloride. Dairy ice cream was
produced with the following composition (w/w): 59% of pasteurized milk, 17% cream, 8.0% skim milk
powder, 12% sucrose, 3.0% glucose, 0.5% guar gum and carrageenan, 0.2% emulsifier, and 0.3% vanilla
flavor. The mixture was ripened for 24 h at 5 C and microencapsulated probiotics were added before
freezing in order to achieve approximately 10 8 cfu.g-1. As controls, ice cream with free probiotics and
microencapsulated probiotics without prebiotics were produced. Microencapsulation and the addition of
acacia gum and resistant starch did not significantly (p > 0.05) influenced the survival of probiotic cultures.
At the end of 180 days of storage, L. acidophilus and B. lactis population remained above 107 and 108 cfu.g-1,
respectively, even in ice cream with free probiotics. The microencapsulation had no effect on sensorial
acceptability (p > 0.05) of probiotic ice cream. The results indicate that dairy ice cream can be considered a
suitable vehicle for incorporating probiotic microorganisms.
Keywords: microencapsulation; resistant starch; acacia gum; probiotic; ice-cream

INTRODUCTION

Functional foods are those that promote health benefits beyond basic nutritional functions, when consumed
in usual diet. Prebiotics and probiotics are current examples of functional food ingredients (1).
Probiotics are defined as live microorganisms, which administered in adequate amounts, confer a health
benefit to the host (2, 3). Species of Lactobacillus and Bifidobacterium, normal components of the intestinal
microbiota, are usually employed in many probiotic foods.
Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the
growth and/or activity of populations of bacteria in the colon (4, 5, 6). Prebiotics might also enhance the
growth and survival of probiotics in foods (7).
Due to de wide range of therapeutic benefits associated with the consumption of probiotic microorganisms,
there has been an increase in the variety of probiotics foods, including fermented milk, many tips of cheese,
juice, chocolate and also ice cream. Although dairy ice cream seems to be a good vehicle for probiotic
cultures due to its composition and pH near to 6.0, the viability of these microorganisms can be affected by
freezing process and oxygen toxicity. In order to overcome these problems, microencapsulation methods can
be applied to increase the survival of probiotic cultures in frozen dairy products (8, 9, 10).
The aim of this study was to evaluate the influence of prebiotics, resistant starch and acacia gum,
incorporated into alginate beads, on the survival of microencapsulated probiotic bacteria in ice cream over a
period of 180 days of storage at -18 C.

MATERIALS & METHODS

Probiotic strains and encapsulation materials
Freeze-dried Lactobacillus acidophilus NCFM and Bifidobacterium lactis BI-04, were provided by Danisco,
Brazil. The cultures were kept at -18C until required for microencapsulation and addition to ice-cream. The

prebiotics, acacia gum, Fibregum B, was provided by Colloides Naturels, Brazil, and resistant starch, Hi
Maize 260, was provided by National Starch, Brazil. Sodium alginate, I-3G-150 from Kimica Chile Ltda,
was obtained by Vogler Ingredients, and calcium chloride was obtained by Vetec Qumica Fina Ltda, Brazil.
Encapsulation procedure
Alginate beads were produced using a modified extrusion technique originally reported by Liserre et al. (11).
Solutions of sodium alginate (1% w/v) containing approximately 109 cfu/mL of Bifidocacterium lactis and
Lactobacillus acidophilus, with or without 2% of prebiotic (acacia gum or resistant starch), were atomized in
0.1 M calcium chloride. The atomization was achieved by forcing the sodium alginate solution to the
stainless steel tube with the aid of a peristaltic pump at a flow rate of 2.5 mL/min and compressed air at a
flow rate of 2.5 m3/min. The solution of calcium chloride remained under constant magnetic stirring until the
end of the encapsulation. Alginate beads remained at rest for 30 minutes, after that were separated from the
calcium chloride solution with stainless steel sieves (mesh of 250, 355, 500, 710 and 1000 mm), washed with
distilled water and stored at 5C overnight.
Ice cream Production
Dairy ice cream was formulated with the following composition (w/w): 59% of pasteurized milk, 17% cream,
8.0% skim milk powder, 12% sucrose, 3.0% glucose, 0.5% guar gum and carrageenan, 0.2% emulsifier, and
0.3% vanilla flavor. The mixture was ripened for 24 h at 5 C and microencapsulated probiotics were added
before freezing in order to achieve approximately 10 8 cfu.g-1. As controls, ice cream with free probiotics and
microencapsulated probiotics without prebiotics were produced. Each type of ice-cream was produced in
triplicate.
Enumeration of probiotic bacteria
The enumerations of probiotic microorganisms were performed before freezing and at 1, 30, 60, 90,120 and
180 days of storage at -18C. Samples of ice cream (25 g) were diluted in 225 mL phosphate buffer (pH 7.5)
and homogenized for 5 min in a lab-blender 400 stomacher (Seward). Ten milliliter of this dilution was used
to obtain serial dilutions in physiological solution. Lactobacillus acidophilus was counted by spread-plating
0.1 mL of each dilution in DeMan-Rogosa-Sharpe agar (MRS, oxoid) and incubation at 43C for 72 h under
anaerobic conditions. Bifidobacterium was counted by pour-plating 1 mL of each dilution in DeMan-RogosaSharpe agar (MRS, Oxoid) supplemented with 0.5% of L-cysteine solution (10%), 0.5% of dicloxacillin
solution (10 mg/100 ml) and 1.0% of lithium chloride solution (10%) and incubation at 37C for 72 h under
anaerobic conditions.
Sensory analyses
Sensory analysis to compare ice cream with free microorganisms (SA) and ice cream with microorganisms
encapsulated in calcium alginate (SB) was performed with 74 untrained panelists using a hedonic scale (12).
The panelist received samples of approximately 25g of each ice cream and were asked to evaluate the overall
liking using a structured 9 points hedonic scale (1 = dislike extremely and 9 = like extremely). The sensory
data were analyzed by ANOVA.
Physical and chemical analysis
The pH of the ice cream was determined with a pH meter (Tec-2, Tecnalise). The mean composition of ice
creams (dry matter, ash, fat and protein content) was determined according with standard methods (13). All
measurements were replicated four times. The overrun of the final product was determined using equation 1.

Overrun

mix weight weight of equal volume of ice cream

100
weight of equal volume of ice cream

(1)

Statistical analysis
Analysis of variance (ANOVA) and Duncan test were applied for determination of significant difference
(p < 0.05) between means of cell counts.

RESULTS & DISCUSSION

Table 1 show B. lactis counts in ice creams during storage. No significant reduction (p > 0.05) in B. lactis
counts was observed after freezing and during storage at -18C of all types of ice creams. Encapsulation in
calcium alginate with and without prebiotics (acacia gum and resistant starch) had no effect on survival of
B. lactis in ice cream. At the end of 180 days of storage, ice cream with free microorganism (SA) revealed
mean counts of B. lactis of 8.19 0.06 log cfu/g, and no significant difference (p > 0.05) were detected
between population of B. lactis in ice cream SA, SB, SC and SD.
Table 1. Counts of Bifidobacterium lactis in ice cream during 180 days of storage at -18C.
Storage
Bifidobacterium lactis (Log UFC/g)
days
SA
SB
SC
SD
0*
8,20 0,03Aa
8,2 0,2 Aa
8,0 0,1 Aa
8,0 0,2 Aa
Aa
Aa
Aa
1
8,16 0,06
8,15 0,09
8,10 0,07
7,8 0,1Ab
Aa
Aa
Aa
30
8,3 0,2
8,20 0,06
8,20 0,04
8,0 0,2 Aa
Aa
Aa
Aa
60
8,3 0,2
8,2 0,2
8,22 0,07
8,0 0,1 Aa
Aa
Aa
Aa
90
8,3 0,2
8,2 0,2
8,21 0,05
8,0 0,3 Aa
Aa
Aa
Aa
120
8,2 0,1
8,1 0,2
8,3 0,1
8,1 0,2 Aa
Aa
Aa
Aa
150
8,10 0,05
8,1 0,2
8,20 0,07
7,9 0,1 Aa
Aa
Aa
Aa
180
8,19 0,06
8,1 0,2
8,09 0,05
7,96 0,09 Aa
* Number of B. lactis in ice cream mix before freezing.
A: Means in the same column with different capital letters were significantly different (p < 0.05).
a: Means in the same line with different lower case were significantly different (p < 0.05).
SA: ice cream with free cells, SB: ice cream with microorganisms encapsulated with calcium alginate, SC: ice cream with
microorganisms encapsulated with calcium alginate and acacia gum, SD: ice cream with microorganisms encapsulated
with calcium alginate and resistant starch.

With regard to L. acidophilus population (Table 2), no significant reduction (p> 0.05) during freezing of ice
cream was observed for all types of ice creams. However, L. acidophilus counts was significantly reduced (p
< 0.05), by approximately 0.6 Log cfu/g, during 180 days of storage at 18C in all treatments. Although
significant differences (p < 0.05) were detected between L. acidophilus counts in ice cream SA, SB, SC and
SD on days 120, 150 and 180, the difference were less than 0.5 Log cfu/g.
Table 2. Counts of Lactobacillus acidophilus in ice cream during 180 days of storage at -18C.
Storage
Lactobacillus acidophilus (Log UFC/g)
days
SA
SB
SC
SD
0*
8,33 0,08Aa
8,2 0,2Aa
8,0 0,2Aa
8,0 0,1Aa
Aa
Aa
Aa
1
8,26 0,08
8,19 0,02
8,05 0,06
8,0 0,3Aa
Ba
Ba
Ba
30
7,95 0,08
7,8 0,1
7,78 0,09
7,7 0,2ABa
Ba
BCa
BCa
60
7,8 0,1
7,7 0,2
7,7 0,1
7,60 0,07BCa
Ba
Ba
Ba
90
7,8 0,1
7,8 0,2
7,82 0,08
7,4 0,3BCb
Ba
BCb
Ba
120
7,8 0,1
7,47 0,06
7,8 0,1
7,5 0,1BCb
Ca
BCa
Da
150
7,5 0,2
7,5 0,2
7,51 0,02
7,2 0,2Cb
Ba
BCab
Dab
180
7,77 0,05
7,6 0,2
7,53 0,01
7,3 0,3BCb
* Number of L. acidophilus in ice cream mix before freezing.
A, B, C, D: Means in the same column with different capital letters were significantly different (p < 0.05).
A, b: Means in the same line with different lower case were significantly different (p < 0.05).
SA: ice cream with free cells, SB: ice cream with microorganisms encapsulated with calcium alginate, SC: ice cream with
microorganisms encapsulated with calcium alginate and acacia gum, SD: ice cream with microorganisms encapsulated
with calcium alginate and resistant starch.

To provide health benefits, a minimum of 106 -107 cfu g-1 of probiotic bacteria must be presented in food in
the moment of consumption (FAO). After 180 days of storage, the number of B. lactis and L. acidophilus in
all types of ice creams were above 108 and 107 cfu/g, respectively. This results shows that the dairy ice cream
is an adequate vehicle for probiotic incorporation even without microencapsulation of microorganisms.

Criscio et al. (14) also observed high population of probiotic bacteria in ice cream stored for 16 weeks at
-20C
Contrary to what was observed in the present study, Homayouni et al. (15) reported that encapsulation in
calcium alginate increased approximately 30% the survival of L. acidophilus and B. lactis in ice cream stored
for 180 days at -20C. However in the present study the overrun value was 41%, while in Hamayouni et al.
(15), the overrun was 95%. The greater incorporation of air could be responsible for the decrease of viable
counts of free microorganisms in ice cream during storage.
The high rate of survival of probiotic microorganisms in this study, especially free cells, can be justified by
the high total solids (33.6%), protein (3.4%) and fat (6.7%), that could protect or even encapsulate the
probiotics. Furthermore, the ice cream pH (6.28), is considered favorable for the survival of probiotic
microorganisms.
The sensory score (mean of 74 panelists) of ice cream with free probiotics (SA) and ice cream with
encapsulated probiotic (SB) was 7.61 and 7.75, respectively. Encapsulation had no significant (p > 0.05)
effect on sensorial acceptability of probiotic ice cream.
CONCLUSION
This study showed that encapsulation of Bifidobacterium lactis and Lactobacillus acidophilus in calcium
alginate and in calcium alginate with acacia gum or resistant starch did not interfere in the survival of
microorganisms in ice cream during 180 days of storage at -18C. Moreover encapsulation had no effect on
sensorial acceptability of probiotic ice cream. The numbers of probiotic bacteria in all types of ice cream
were above 107 cfu/g at the end of 180 days of storage. Dairy ice cream can be considered a suitable vehicle
for incorporating probiotic microorganisms.
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