Advances in Microbiology, 2012, 2, 364-367 Published Online September 2012 (
Influence of Micro-Encapsulated Probiotic Lactobacillus
acidophilus R0052 on the Characteristics of Plain Yogurt
Edwin Noland1, Kayanush J. Aryana1,2*
1School of Animal Science, Louisiana State University Agricultural Center, Baton Rouge, USA
2Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, USA
Email: *
Received July 1, 2012; revised July 30, 2012; accepted August 10, 2012
Micro-encapsulation is a method of providing probiotic living cells with a physical barrier against adverse environ-
mental conditions. Lactobacillus acidophilus is one of the most effective forms of probiotic bacteria and is commer-
cially available as pure culture and encapsulated form. It is not clear whether the use of micro-encapsulated L. aci-
dophilus will result in yogurt of a better quality co mpared to non micro-en capsulated L. acidophilus. The objective was
to determine the influence of micro-encapsulated L. acidophilus on the characteristics of fat free plain yogurt. Yogurt
mixes were pasteurized and at 37˚C were inoculated with Streptococcus thermophilus, Lactobacillus delbrueckii subsp.
bulgaricus and micro-encapsulated L. acidophilus R0052 or non micro-encapsulated L. acidophilus R0052. Yogurt
manufacture was replicated three times. Yogurts with micro-encapsulated L. acidophilus R0052 had significantly (P <
0.05) high er flavor scor es, compared to yogurts with n on micro-en capsulated L. acidophilus R0052. The L. acidophilus
counts, apparent viscosity, pH and syneresis, of the yogurts with micro-encapsulated L. acidophilus R0052 were not
significantly (P < 0.05) different from those of the yogurts with non micro-encapsulated L. acidophilus R0052. Use of
micro-encapsulated L. acidophilus R0052 resulted in better tasting yogurts probably because of the taste imparted by
the trace amounts of the micro-encapsulating material.
Keywords: Probiotic; Micro-Encapsulation; Fermented; Cultured; Shelf Life
1. Introduction
Probiotics are defined as “live microorganisms which
when administered in adequate amounts confer health
benefits to host” [1]. There has been an increasing inter-
est in the role of probiotic bacteria in human health.
Health advantages associated with the probiotic intake
include alleviation of symptoms of lactose malabsorption,
increase in natural resistance to infectious diseases of the
intestinal tract, suppression of cancer, redu ction in serum
cholesterol concentrations, improved digestion, and sti-
mulation of gastrointestinal immunity [2]. It is generally
accepted that successful delivery and colonization of
viable probiotic cells in the intestine are essential for
probiotics to be efficacious [3]. As a guide, the Intl.
Dairy Federation has recommended that the bacteria be
viable and abundant in the product and be present at a
population of at least 107 colony-forming units (CFU)/g
until the date of co nsumption [4]. However, studies indi-
cate that the bacteria may not survive in sufficient num-
bers when incorporated into dairy products and during
their passage through the gastrointestinal tract [5]. Sev-
eral factors influence the survival and colonization of
these bacteria, including resistance to low pH, bile acids,
and digestive enzymes [1].
Micro-encapsulation is a method of providing probi-
otic living cells with a physical barrier against adverse
environmental conditions [6]. Micro-encapsulation helps
to protect the beneficial bacteria from destruction by
stomach acid for example and thereby enhances its vi-
ability. Several methods of micro-encapsulation of pro-
biotic bacteria include spray drying, extrusion, emulsion
and phase separation [7]. The most commonly reported
micro-encapsulation procedure is based on the calcium-
alginate gel capsule formation, and materials for micro-
encapsulation include alginates, kappa-carrageenan, gel-
lan gum, gelatin and starch [7].
The two culture bacteria Streptococcus thermophilus
and Lactobacillus bulgaricus are required in yogurt
manufacture according to the legal description of yogurt
[8]. Lactobacillus acidophilus is one of the most effec-
tive forms of probiotic bacteria. Health benefits of Lac-
tobacillus acidophilus include reduction in occurrence of
diarrhea in humans, enhancement of the immune system,
reduction in cholesterol and improved symptoms of lac-
*Corresponding a uthor.
opyright © 2012 SciRes. AiM
tose intolerance [9] and antitumor effects [10]. Use of L.
acidophilus in rats reduced the number of colon cancer
cells in a dose dependent manner [11]. Lactobacillus
acidophilus is widely used as an adjunct culture in yogurt
manufacture in the United States [12] and these L. aci-
dophilus cells are in the non micro-encapsulated form. It
is not clear if the use of micro-encapsulated L. acidophi-
lus cells would result in a yogu rt of a better quality com-
pared to non micro-encapsulated L. acidophilus. The ob-
jective was to study the influence of micro-encapsulated
L. acidophilus on the characteristics of fat free plain yo-
2. Materials and Methods
2.1. Yogurt Manufacture
Yogurts were manufactured using standard procedure [13,
14] with slight alteration. Yogurt mixes were homoge-
nized, pasteurized and temperature lowered to 40˚C and
inoculated with yogurt culture bacteria Streptococcus
thermophilus and Lactobacillus delbrueckii ssp. bulga-
ricus (Chr. Hansen Milwaukee, WI) at a constant rate of
20 g per gallon (3.785 L). Encapsulated L. acidophilus
R0052 or non encapsulated L. acidophilus R0052 (Insti-
tut Rosell-Lallemand Inc. Montreal, Quebec, Canada)
were individually incorporated in the yogurt mixes at the
same rate of 20 g per 3.785 L. Inoculated yogurt mixes
were poured into 355 mL containers (Reynolds RD C212—
Del-Pak Combo-Pak, Alcoa, Inc., Pittsburgh, PA) and in-
cubated at 40˚C to pH 4.5 before cooling to 4˚C. Samples
were stored at 4˚C until analyzed. Product manufacture
was replicated three times.
2.2. Lactobacillus acidophilus Enumerations
Counts of L. acidophilus were enumerated as reported
earlier [15] but with modifications. The appropriate
amount of distilled water was added to a 500 mL or 1 L
graduated cylinder. MRS base medium without dextrose
was prepared by weighing the appropriate proportion of
10.0 g of proteose peptone #3 (United States Biological,
Swampscott, MA), 10.0 g of beef extract (Becton, Dick-
inson and Co., Sparks, MD), 5.0 g of yeast extract (Bec-
ton, Dickinson and Co., Sparks, MD), 1.0 g of polysor-
bate 80 (Tween 80) (Sigma-Aldrich Inc., St. Louis, MO),
2.0 g of ammonium citrate (Fisher Scientific, Fair Lawn,
NJ), 5.0 g of sodium acetate, anhydrous (EMD Chemi-
cals Inc., Gibbstown, NJ), 0.1 g of magnesium sulfate,
anhydrous (EMD Chemicals Inc., Gibbstown, NJ), 0.05 g
of manganese sulfate, monohydrate (Sigma-Aldrich Inc.,
St. Louis, MO), 2.0 g of dipotassium phosphate (Fisher
Scientific, Fair Lawn, NJ), and 15.0 g of agar (EMD
Chemicals Inc., Gibbstown, NJ) and diluting these ingre-
dients to the appropriate proportion of 1 L with distilled
water. This mixture was heated to boiling with agitation
before autoclaving at 121˚C for 15 min. A 10% (w/v)
sorbitol (EMD Chemicals Inc., Gibbstown, NJ) solution
was prepared and filtered sterilized with Nalgene Mem-
brane Filter Units (Nalge Co., Rochester, NY), and the
appropriate amount of this so lution was aseptically add ed
to the MRS base medium to form a 10% sorbitol solution
(final concentration of 1 % sorbitol i.e. 1 g sorbitol in 100
mL of final medium) and 90% MRS base medium mix-
ture immediately before pouring the plates. The appro-
priate dilutions of yogurt were made with 99 mL of ster-
ilized peptone (or sterilized Butterfield buffer in pre-
filled dilution bottles (Weber Scientific, Hamilton, NJ)).
The pour plate method with this MRS-sorbitol agar was
performed. Petri dishes were placed in BBL GasPaks
(BBL, Becton, Dickinson and Co., Cockeysville, MD)
and incubated anaerobically at 37˚C for 72 h. A Quebec
Darkfield Colony Counter (Leica Inc., Buffalo, NY) was
used to assist in enumerating the colonies.
2.3. pH
The pH of the yogurts at 4˚C was determined using an
UltraBasic B e nchtop pH Meter (Denver Inst rument Com p-
any, Arvada, CO, USA) calibrated using commercial pH
4.00 and 7.00 bu ffer solution s .
2.4. Apparent Viscosity
The apparent viscosities were determined at 4˚C using a
Brookfield DV II+ viscometer (Brookfield Engineering
Lab Inc., Stoughton, MA, USA) with a helipath stand. A
T-C spindle was used at 10 rpm. The data were acquired
using the Wingather® software (Brookfield Engineering
Lab Inc., Stoughton, MA, USA). One hundred data poin ts
were averaged per sample.
2.5. Syneresis
The release of whey from the yogurt samples was meas-
ured by inverting a 300 g sample at 4˚C on a fin e cheese
cloth placed on top of a funnel. The quantity of whey
collected in a graduated cylinder after 2 h of drainage
was used as an index of syneresis.
2.6. Sensory Evaluation
Sensory evaluations were conducted using a seven mem-
ber experienced panel. The panelists had over 4 months
of training in judg ing yogurts. Samples were provided to
panelists in three digit random number co ded plastic cups.
Water was provided to panelists to rinse their palate be-
tween samples. Panelists were instructed not to talk dur-
ing the sensory evaluation. The official American Dairy
Science Association intercollegiate dairy products evalua-
tion contest score card was used to evaluate flavor on a 1
Copyright © 2012 SciRes. AiM
Copyright © 2012 SciRes. AiM
to 10 point scale (10 = no criticism).
2.7. Statistical Analysis
Data were analyzed by Analysis of Variance using Proc
Mixed of the Statistical Analysis Systems. Significant dif-
ferences between means were determined using Fisher’s
protected Least Significant Difference test. Significant
differences we re determined at α = 0.05.
3. Results and Discussion
The pH values are reported in Table 1. At week 5 the pH
was an average of 4.4 pH units. There were no differ-
ences in pH between the yogurts made using the mi-
cro-encapsulated and non micro-encapsulated bacteria.
Micro-encapsulation being just a physical coating on the
microorganism [7] and did not play a role in influencing
product pH.
Lactobacillus acidophilus counts were converted to
log10 scale before the data were analyzed by SAS. The L.
acidophilus counts are reported in Table 1. There were
no differences in counts of the micro-encapsulated and
non micro-encapsulated bacteria. The reason for the mi-
cro-encapsulation of L. acidophilus was to increase bac-
terial viability by protection against the acidic environ-
ment of the stomach having pH’s between 1.50 - 2.00.
Viability of bacteria in yogurt declines when the yogurt
pH drops below 4.3 [16] hence since the pH of the yo-
gurts at 5 weeks was 4.4 there were no drop in counts of
non micro-encapsulated L. acidophilus compared to the
micro-encapsulated L. acidophilus.
Syneresis is the serum released from the product. The
syneresis values are presented in Tab le 2. There were no
differences between the two different types of yogurts.
The microencapsulating material is a fatty acid and does
not play a role in binding water hence did not influence
Apparent viscosity values are reported in Table 2.
There were no differences in apparent viscosity. The L.
acidophilus was used in trace amounts of 0.1% v/v of
yogurt mix, hence the microencapsulating material was
also present in trace amounts. Micro-encapsulating mate-
rial can be a starch [7] which would have a partial thick-
ening effect on the yogurt. The micro-encapsulating ma-
terial in the present study was a fatty acid hence there
was no change in viscosity of the yogurts.
Flavor scores are reported in Table 3. Yogurts with
micro-encapsulated bacteria had significantly higher fla-
vor scores compared to yogurts with non encapsulated
bacteria. Microencapsulating material was a fatty acid
which probably was the reason for making the fat free
plain yogurts taste diff erent.
4. Conclusion
Use of micro-encapsulated L. acidophilus improved pro-
duct flavor but did not have any effect on the remaining
characteristics studied. Product flavor is an important
Table 1. Mean ± SE of pH values and L. acidophilus counts of the various yogurts over a storage period of 5 weeks.
Treatments pH at weeks L. acidophilus counts ( log cfu/mL) at weeks
1 3 5 1 3 5
Micro-Encaps ulated 4.55 ± 0.09A 4.48 ± 0.03A 4.41 ± 0.11A 7.87 ± 0.18A 7 .56 ± 0.19A 7.07 ± 0.10A
Non Micro-Enc apsulated 4.52 ± 0.03A 4.43 ± 0.09A 4.40 ± 0.07 A 7.86 ± 0.29A 7.52 ± 0.28A 7.05 ± 0.16A
AMeans in each column with the same letter did not differ significantly (P < 0.05).
Table 2. Mean ± SE of syneresis and apparent viscosity of the various yogurts over a storage period of 5 weeks.
Treatments Syneresis (mL) at weeks Apparent viscosity (×104 cP) at weeks
1 3 5 1 3 5
Micro-encapsulated 139.3 ± 8.1A 123.7 ± 9.8A 119.3 ± 6.4A 3.58 ± 0.08A 3.68 ± 0.20A 2.22 ± 0.05A
Non Micro-Enc apsulated 140.7 ± 11.9A 125. 7 ± 5.1A 121.7 ± 5.8A 3.64 ± 0.16A 3.73 ± 0.30A 2.25 ± 0.19A
AMeans in each column with the same letter did not differ significantly (P < 0.05).
Table 3. Mean ± SE of flavor scores of the various yogurts over a storage period of 5 weeks.
Treatments Flavor scores at weeks
1 3 5
Micro-encapsulated 7.53 ± 0.50A 7.69 ± 0.53A 7. 67 ± 0 .51A
Non Micro-Enc apsulated 7.00 ± 0.10B 7.10 ± 0.15B 7.13 ± 0.15B
A,BMeans in each column with the same letter did not differ significantly (P < 0.05).
characteristic hence the use of micro-encapsulated bacte-
ria should be considered in yogurt manufacture.
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