Influence of Micro-Encapsulated Probiotic <i>Lactobacillus acidophilus</i> R0052 on the Characteristics of Plain Yogurt

doi:10.4236/aim.2012.23045

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Advances in Microbiology, 2012, 2, 364-367

http://dx.doi.org/10.4236/aim.2012.23045 Published Online September 2012 (http://www.SciRP.org/journal/aim)

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: *[email protected]

Received July 1, 2012; revised July 30, 2012; accepted August 10, 2012

ABSTRACT

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.

E. NOLAND, K. J. ARYANA 365

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-

gurt.

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

E. NOLAND, K. J. ARYANA

366

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

syneresis.

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).

E. NOLAND, K. J. ARYANA 367

characteristic hence the use of micro-encapsulated bacte-

ria should be considered in yogurt manufacture.

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