Encapsulation of Lactobacillus paracasei using Spray Gun technology

Maribel Jimnez a, Esmeralda Jimnez a, Ebner Azuaraa, Guadalupe Lunab Cesar I. Beristaina
a

Instituto de Ciencias Bsicas, Universidad Veracruzana, Xalapa ,Veracruz, Mxico

(maribjimenez@uv.mx).
b
DEPI, Instituto Tecnolgico de Orizaba, Orizaba, Veracruz, Mxico

ABSTRACT
There are many techniques in probiotic encapsulation, however most of them use high temperature and the
size of microcapsules obtained is big, and the novel technique Spray Gun can be used as a part of
encapsulation process which not include temperatures over 40 C. Encapsulation of Lactobacillus casei using
the spray gun technique, avoid high temperature used improved its viability and protected against the
undesirable conditions during metabolism and storage. Activated Lactobacillus casei with culture medium
MRS incubated for 12 hours at 37 C. Subsequently, were prepared the wall material using alginate, starch
2%, and canola oil 10%, homogenized for 15 minutes at 400 rpm, this matrix is added to the spray gun.
Spraying was performed at 1.5 kg f, at a distance of 15 cm, using a nozzle of 8 mm, wall material is
crosslinked on calcium chloride at 3%, thus obtaining capsules. To determine the viability capsules were
dissolved in phosphate buffer pH 7, 1M, in a grid of magnetic stirring at 100 rpm, incubate by MRS agar
plate, the same activation conditions, strain after 48 hours of being reported readings are performed colony
forming units (cfu). Capsules were obtained 0.5 mm in diameter on average and they had a mean viability of
10-5 cfu/g after the drying by fluidized bed drying, using this emerging technology of spray gun, got a better
drying capsules, this was achieved without using high temperatures and can thus improve their viability.
Keywords: Lactobacillus casei; fluidized bed drying; capsules; spray gun; drying; Viability
INTRODUCTION

Nowadays people prefer prevent rather than to use curative approaches toward diseases. Diet is a good
strategy for maintaining optimum health throughout life and preventing early onset of chronic diseases such
as gastrointestinal (GI) disorders, cardiovascular disease, cancer, osteoporosis, as well as promoting healthier
ageing [1]. In this sense, probiotic bacteria are defined as live microorganisms which, when administered in
adequate amounts, confer a health on the host. Probiotics may exert their beneficial effects in various
settings by different mechanisms. These include the production of antimicrobial factors such as bacteriocinas,
competitive exclusion of pathogen binding to the mucosal epithelium, competition for nutrients, conditioning
of the mucosal epithelium and sub epithelial structures, and modulation of the immune system. The growth
activity of lactic acid bacteria (LAB) is affected by fermentation conditions such as pH, temperature, medium
composition and the other factors [2]. On the other hand during processing and storage of foods, probiotic
microorganisms may be exposed to high temperatures, low pH, high osmotic pressure and high levels of
oxygen [3], the ability of microorganisms to survive and multiply in the host strongly influences their
probiotic benefits. The bacteria should be metabolically stable and active in the product, survive passage
through the upper digestive tract in large numbers, and have beneficial effects when have shown low viability
of probiotics in yoghurt and fermented milk [4], a good viability and activity of probiotics are considered
prerequisites for optimal functional. However, several studies have shown that non-viable probiotics can
have beneficial effects such as immune modulation and carcinogen binding in the host, a promising solution
to this problem is encapsulation [5]. The encapsulation is a process in which the cells are retained within an
encapsulating matrix or membrane. The physical protection of probiotics by encapsulation is a new approach
to improve the probiotic survival. Encapsulation helps to isolate the bacterial cells from the effects of the
hostile environment and gastrointestinal tract, thus potentially preventing cell loss [6]. A microcapsule
consists of a semi permeable or non-permeable, spherical, thin and strong membrane surrounding a
solid/liquid core, with a diameter varying from a few microns to 1 mm. Food grade polymers such as
alginate, chitosan, carboxymethyl cellulose, carrageenan, gelatin and pectin are mainly applied, using various
encapsulation technologies [7]. To keep the viability and extend their storage shelf life is convenient to
convert the capsules into dry capsules employing techniques such as spray drying, fluidized bed drying, etc.
The spray drying is an economic and effective technology, however, it causes high mortality as result of
simultaneous dehydration, thermal and oxygen stresses imposed to bacteria during the drying process [8].

Fluidized bed drying is a method use to dry different kind of food, is economic. The spray gun is a technique
novel in the pharmaceutical industry with good results of encapsulation, this technique is cheap, with the
advantage that uses room temperature is beneficial to maintain the viability. The main objective of this study
is to evaluate the application a novel technique spray gun to encapsulating as well as the application of
fluidized bed drying to produce dry capsules of long shelf life, highly viable probiotics from LAB strain,
Lactobacillus paracasei.
MATERIALS & METHODS
Materials
The bacterial strain used in this study was pure lyophilized culture of L. paracasei LBC81 LY0 provided by
DANISCO, Mxico. Encapsulation aids were alginato (Sigma Aldrich) Starch (Food Zave). All other
reagents were of analytical grade.
Bacteria, culture conditions and preparation of cell concentrate
A lyophilized culture of L. Paracasei was cultured for 12 h at 37 C in sterile MRS broth in anaerobic
environment. Cells were harvest at the late log phase by centrifugation at 1500 rpm for 20 min, washed once
with sterile saline solution 0.1 % [9].
Encapsulation procedure
All glassware and solutions used in the protocol were sterilized at 121 C for 15 min. Alginate beads were a
produced using a modified encapsulation method originally by Sheu and Marshall [10] and Sultana et al.
[11]. A 2 % alginate mixture in distilled water was prepared containing 2 % maize starch and 0.1 % culture.
The mixture was added into 10 % canola oil. The mixture was stirred vigorously until was fully emulsified.
The operating conditions of spray gun were as follows; spray pressure, 30 PSI, nozzle diameter, 0.7 mm.
Then the solution was sprayed on the 3% CaCl2 solution.
Fluidized bed drying
The capsules was subjected to a drying process by the operating conditions of fluidized bed drying were as
follows; fluidization velocity, 99 m3/s, inlet temperature 30 C
Entrapped L. paracasei enumeration
Viable cells were counted according to the standard plate method. Samples of 1ml of the broken emulsions
were suspended in 20 ml of 0.2 M phosphate buffer and applied on MRS agar plates, and colonies were
counted after 48 h of incubation at 37 C.
Physicochemical properties of the capsules
The moisture measurement is by the method of drying in a vacuum oven. The particle density was measured
by a pycnometric method using toluene. The particle density is the total mass of the particle divided by its
total volume. Bulk density was determined in two grams of powder, which were loosely weighed into 10 mL
graduate cylinder. The final volume was recorded and bulk density was calculated by dividing the sample
weight by the volume [12]. The compact density was determined by the method of "Tappin 'in which 2-5 g of
capsules were placed in a 10 mL test tube. The probe is hit on the flat surface to volume constant. The
compact density calculated by dividing the weight of the sample by the constant volume occupied by the
sample.
RESULTS & DISCUSSION
The initial physical properties of capsules prepared and obtained by spray gun are shown in Table 1. Initial
bulk density was 0.458 g/cm3, compact density was 0.67 g/cm3, and particle density was 0.76 g/cm3. This
important because, physical properties are directly related to the quality and utilization of food products,
causing changes in their structure and porosity. The physical characteristics of the individual particles are
mainly determined by the encapsulating material and physical properties and indirectly, these properties can
provide predictions about storage stability. The initial moisture was 80 % and after 30 minutes of drying the
moisture was 16 %. The physical characteristics on the individual particles are mainly determined by the

material from which are encapsulated. Powder property measurement is important because these properties
intrinsically affect powder behavior during storage, handling and processing such as transportation, mixing
and packaging.

Time (Min)
0
5
10
15
20
25
30

Table 1. Physical properties during fluidized bed drying process

Particle density (g/cm3)
Moisture
Bulk density (g/cm3)
Compact density
3
(g/cm )
(g/cm3)
80 0.707
0.76 0.001
0.458 0.006
0.67 0.013
64.5 0.910
0.67 0.013
0.438 0.005
0.63 0.011
54.3 0.777
0.64 0.002
0.428 0.005
0.54 0.077
40.6 2.333
0.62 0.001
0.418 0.003
0.52 0.000
36.2 0.707
0.57 0.000
0.416 0.000
0.49 0.007
23.1 0.848
0.55 0.000
0.414 0.000
0.47 0.000
16.3 0.070
0.52 0.000
0.410 0.000
0.43 0.000

The viability of the solution before encapsulation was 6.80 X 109cfu, and after encapsulation by Spray Gun
was 2.40 X 108 CFU, which represent 3.38 % of the viability with respect to the initial solution. These results
were similar to report by Semyonov et al. [13], when they used coacervation. The vialibility of
microorganism decreases with increased drying time, reaching values of 5.40 x105 corresponding to 9 %
from initial viability (Figure
1).

100

80

60

40

20

0
0

10

15
20
Time (min)

25

30

35

Figure 1.Viability of Lactobacillus paracasei encapsulating by spray gun and by fluidized bed drying.

One principal criterion for the stability of capsules is the moisture content. The higher viability was when the
capsules had moisture high 70% this corresponding to 0.96-0.94 of water activity. Figure 2 shows the
decreased to the moisture as they are drying the capsules, obtaining a major viability when the moisture
content of capsules was high, the viability after 30 minutes of drying was 5.40x105, this viability was similar
to reported by Semyonov et al [13] when they used spray freeze to drying method and coacervation method
of encapsulation.

120

100

80

60

40

20

0
10

20

30

40

50

60

70

80

90

Moisture content (%)

Figure 2. Relation between moisture content (%) and viabilidad of Lactobacillus paracasei.

CONCLUSION
The present study demonstrate that fluidized bed drying is an appropriate process to generate dried capsules
of defined dimensions containing probiotic bacteria L. Paracasei, that retain viability during the
encapsulation by spray gun. We conclude that encapsulating by spray gun and drying by fluidized bed drying
are a good option to maintain viable L. paracasei to incorporate new food systems.
REFERENCES
[1]

[2]

[3]
[4]
[5]
[6]
[7]

[8]
[9]

[10]

Sandoval-Castilla O., Lobato-Calleros C., Garcia-Galindo H.S., Alvarez-Ramirez J. & Vernon-Carter E.J. 2009.
Textural properties of alginate-pectin beas and survivability of entrapped L.b casei in simulated gastrointestinal
conditions and in yogurt. Food Research International, 43, 111-117.
Annan N.T., Borza N.T. &Trueistrup Hansen L. 2007. Encapsulation in alginate-coated gelatine microspheres
improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastrointestinal
conditions. Food Research International, 41, 184-193
Capela P., Hay T. & Shah N.P. 2007. Effect of homogenization on bead size and survival of encapsulated probiotic
bacteria. Food Research International, 40, 1261-1269.
Krasaekoop W., Bhandari B. & Deeth H. Evaluation of encapsulation techniques of probiotics for yogurt.
International Dairy Journal, 13, 3-13.
Homayouni A., Azizi A., Ehsani M.R., Yarmand M.S. & Razavi S.H. 2008. Effect of microencapsulation and
resistant starch on the probiotic survival and sensory properties of symbiotic ice cream. Food Chemistry, 111, 50-55
Mattilan-Sandholm T., Myllarinen P., Crittenden R., Mogensen G., Fondn R. & Saarela, M. 2002.Technological
challenges for future probiotic foods. International Dairy Journal, 12, 173-182.
Pimentel-Gonzalez D.G., Campos-Montiel R.G., Lobato-Calleros C., Pedroza-Islas R. & Vernon-Carter E.J. 2009.
Encapsulation of Lactobacillus rhamnosus in doubl emulsions formulated with sweet whey as emulsifier and
survival in simulated gastrointestinal conditions. Food Research International, 42, 292-297.
Anal A. & Singh KY. 2007. Recent advances in microencapsulation of probiotics for industrial applications and
targeted delivery. Trends in Food Science and Technology, 5, 240-251.
Leslies, S.B, Israeli, E. Lighthart, B., Crowe, J.H & Crowe, L.M. 1995. Threhalose and sucrose protect both
membranes and proteins in intact bacteria during drying. Applied and Environmental Microbiology, 61(10), 35923597.
Sheu T. Y. & Marshall, R.T., 1993. Improving survival of culture bacteria in frozen desserts by microentrapment.
Journal of Dairy Science, 76(7), 1902-1907

[11]

[12]
[13]

Sultana K., Goodward, G., Reynolds N., Arumugaswamy R., Peiris P. & Kailasapathy K, (2000). Encapsulation of
probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in
yoghurt. International Journal of Food Microbiology, 62, 47-55.
Bhadari B,R., Dumouling H.M.J., Richard H.M.J. Noleau & Lebert.1992. Flavor encapsulation by spray drying
application to citral and linaly acetate. Journal of Food Science, 57, 217-221.
Semyonov D., Ramon O., Kaplun Z., Levin-Brener L., Gurevich N. & Shimoni E. 2010. Microencapsulation of
Lactobacillus paracasei by spray freeze drying. Food Research International, 43, 193-202.