Survival of encapsulated probiotics through spray drying and non-refrigerated storage for animal feeds application

doi:10.4236/as.2013.45B015

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Vol.4, No.5B, 78-83 (2013) Agricultural Sciences

doi:10.4236/as.2013.45B015

Survival of encapsulated probiotics through spray

drying and non-refrigerated storage for animal feeds

application

Norfahana Abd-Talib1,2, Siti Hamidah Mohd-Setapar2*, Aidee Kamal Khamis1, Lee Nian-Yian2,

Ramlan Aziz1

1Institute Bio Product Development, Universiti Teknologi Malaysia, UTM Johor Bahru, Johor, Malaysia

2Centre of Lipid Engineering and Applied Research (CLEAR), Universiti Teknologi Malaysia, UTM Johor Bahru, Johor, Malaysia;

*Corresponding Author: [email protected]

Received 2013

ABSTRACT

The objective of this study is to verify potential of

various types of microorganisms during spray

drying and non-refrigerated storage that can be

enhanced substantially by selecting suitable pro-

tective colloids. Four selected probiotics tested

are Lactbacillus plantarum B13 and B18, which

are the bacteria probiotics and Kluyveromyces

lactis and Saccharomyces blouradii, non-bac-

teria probiotics. Two levels of experiment occur

starting with formulation study of encapsulation

agent followed by the viability study of different

probiotics after spray dry and two weeks nonre-

frigerated storage. The formulation of 30% of

gum Arabic, 15% of gelatin and 45% of coconut

oil can homogenize well at least for two hours

and can produce acceptable dried product (be-

low 4% of moisture content) at low outlet tem-

perature (70℃ - 75℃). K. lactis, S. blouradii

gives 2.57% and 2.4% of viability percentage

after spray drying process and 25.84% and

2.04% after two weeks nonrefrigerated storage

respectively. The colonies of non-probiotics

bacteria after both conditions are between 1010

and 106 cfu/mL which is among the accepted

level for industrial application. However, the

survival of probiotics in a spray-dried form dur-

ing non-refrigerated storage is higher at low of

moisture content compared to others.

Keywords: Probiotics; Survival Rate; Spray Drying;

Nonrefrigerated Storage

1. INTRODUCTION

Probiotics have been recently defined as “live microbes

which transit to the gastro-intestinal tract and having posi-

tive effect s to th e health for the consu mers [1]. I t have b een

applied in aquaculture for controlling diseases,

enhancing the immune response, supplementing or even

in some cases replacing the use of anti microbial. The

use of probiotics as farm animal feed supplements dates

back to the 1970' and originally incorporated into feed to

increase the animal's growth and to improve its health by

increasing its resistance to disease. It was assumed that

the effectiveness of probiotics were related to the

gastroin-testinal tract and effects on incidence of diarrhea

and other gut infection s. According to literatu res show in

Table 1, there are some probiotic strains which can give

positive effect to various typ es of animals.

Some of other probiotics which used in animal feed

are microscopic fungi such as strain of yeasts. There are

billion strains of yeasts that are supposed to be explored

in animal feed study. The strains like Saccharomyces

boulardii and Kluveromyces lactis are having high po-

tential as a probiotic that can improve animal health.

Yeast seemed to facilitate increased mobilization of body

reserves and to increase milk fatty acid production of

ruminants. Therefore, it was possible to apply yeast’s

strain as probiotics strain in powder form of animal feed

in line with the effectiveness in animal health. For exam-

ple camel calves can improve weight gain, average daily

gain and feed utilizations after apply yeast as supple-

mentation diet in different forms [9].

It is known that probiotics were very useful for animal

but the preparation of animal feed that containing probi-

otic especially in powder form are not easy. To ensure

the amount of probiotics cu lture required can achiev e the

target; some of the factors are to be tested first in order to

increase stability or viability of the probiotic products.

Encapsulation agent is the formulation of single or com-

bination of wall material that can resist the condition

along the journey. Encapsulation is a process in which

tiny droplets or particles are wrapped with a protective

N. Abd-Ta lib et al. / Agricultural Sciences 4 (2013) 78-83 79

coating yielding capsules for countless application. Sev-

eral methods of encapsulation of probiotic have been

reported by [10]. The objective of the research is to dem-

onstrate the various types of probiotics microorganisms

during spray drying and storage at room temperature by

selecting suitable protective colloids as an encapsulation

agent.

2. MATERIALS AND METHODOLOGY

2.1. Stability of the Feed Composition

The stage one of this experiments was conducted by

the conventional try and error method. The several feed

formulations were choose which with high advantages in

spray dryer process and animal feed industry. At the first

stage, the homogenize mixture without probiotic cell was

hold for 2 hours to test stability of the mixture by using

homogenizer (Heidolth, DIAX 900, Germany). This is

holding time of the feed formulation in the feed bottle

during spray dry (Pilot Spray Drying Plant PSD-00,

Hemray Enterprise, Bombay). The homogenized mix-

tures were proceed for drying process in the spray dryer

at inlet temperature 110℃ and outlet temperature of air

70℃- 75℃ and should give the moisture content below

4%. Details of the various wall materials used are shown

in Table 2.

Table 1. Some probitiocs strain with the benefits to animals.

Animal Probiotics Functions References

Fresh

water

fish

Lactobacilli spp.

Lactobacillus sakei) Enhance the host protection

against pathogens. [2]

Broiler

chicken

Lactobacillus

reuteri (33%)

Lactobacillus

crispatus (18.7%)

Lactobacillus

salivarius (12.3%)

To maintain balanced

microbiota and to reduce

potential pathogens [3]

Pig Enterococcus

aecium

Great production of specific

antibodies against

salmonella enteric [4]

Broiler

chicken

Lactobacillus

acidphilus and

Streptococcus

aecium

Reducing population of

Clostridium perfringers

which can lead to necrotic

enteritis prevention

[5]

Fish

Lactobacillus

lan

arum

To promote growth a n d

enhance immunity and

resistance against

Streptococcus sp. and an

iridovirus

[6]

Piglets Lactobacillus

lan

arum

Improve gro wt h

performance without

affecting the

gastrointestinal ecology.

[7]

Young

dairy

calves

Lactobacillus

acidophilus and

Lactobacillus

lan

arum

Body weight increased [8]

Table 2. Formulation of emulsion.

FormulationsIngredient

A 30% gum Arabic, 15% gelatin, 45% coconut oil

B 30% gum Arabic, 15% gelatin, 45% lecithin

C 30% gum Arabic, 15% lecithin, 45% coconut oil

D 15% gelatin, 30% lecithin, 45% coconut oil

E 15% gelatin, 30% maltodextrin, 45% coconut oil

F 30% maltodextrin, 15% le ci th in , 45% coconut oil

G 15% gelatin, 30% maltodextrin, 45% lecithin

H 15% gum Arabic, 45% gelatin, 7.5% lecithin,

67.5% coconut oil

I 45% gum Arabic, 15% gelatin, 7.5% lecithin,

67.5% coconut oil

2.2. Survival of Different Types of Probiotics

During Spray Dry

The second stage of stud y was focused on the viability

of different type of probiotics on the spray dry condition

and storage at 25℃ in two weeks. Four different types

of probiotics microorganisms were chosen to investigate

the viability, stability with wall material used, condition

of spray dry, and two weeks non-refrigerated condition.

The chosen probiotics were Lactobacillus plantarum B13

and B18, Kluyveromyces lactis and Saccharomyces

blouradii. The mixtures of wall material s were mi xed wi t h

deionised water and autoclave a 121℃ for 15 minutes

(water phase). The coconut oil was sterilized filter and

then the probiotic culture was dispersed into coconut oil

using ho- mogenizer at the lowest speed (3000 rpm) for

10 - 15 minutes (Zen tr ifugen , Hettich,D-78532,Tuttlinge

n, Ger- many). Then, the coconut oil were mixed up with

water phase mixture by using homogenizer at 3000 rpm

for 10 minutes in sterilized condition.

2.3. Viability Analysis

1 mL of each formulation was transfer into a universal

bottle and 9 mL sterilized water is added. The solution is

serially diluted until dilution factor of 1010. 0.01 mL is

taken from all the dilution factors and transferred into the

petri dish contain medium agar for each type of probiotic

respectively. After that, petri dish is incubate for 24 hours

at 37℃ for Lactobacillus plantarum and Kluyveromyces

lactis and 30℃for 24 hours for Saccharomyces bloura-

dii. After 24 hours, the number of colony forming is cal-

culated with naked eyes. The moisture content of spray

dried powders was determined by oven drying at 102℃

until constant weight was obtained.

N. Abd-Ta lib et al. / Agricultural Sciences 4 (2013) 78-83

3. RESULTS AND DISCUSSION

3.1. Formulations study of Encapsulation

Agent

Try and error method carried out to get the suitable

emulsion formulation in order to get the good product.

Based on previous studies, four basic materials which

can contribute on production of good powder product

were selected. After doing some try and error formula-

tion and stability checked, the observ ation were shown in

Table 3.

After doing the observation, the stability test resulted

that Formulation A and Formulation D show a highest

potential in maintaining the stable characteristics after

keeping for two hours. The maximum duration of spray

drying is about two hours. So, at least the formulation

can homogenized well at the duration time is more than

enough. Lecithin was an economic an effective wall ma-

terial but unfortunately it cannot homogenize well with

other wall material selected. The ability of solid per-

centage used in the solution has been checking up after

selection of the suitable emulsion formulation successful.

Formulation A and Formulation D were the only two

formulations that have been selected to the next part of

study.

Table 3. Stability of different of encapsulation agent formula-

tions.

Formulations Ingredients Stability Observations

A 30% gum Arabic,

15% gelatin,

45% coconut oil

After homogenize, milky whit e

and stable solution produced

B 30% gum Arabic,

15% gelatin,

45% lecithin

After homogenize, 70 percent

from solution precipitated.

C 30% gum Arabic,

15% lecithin,

45% coconut oil

After homogenize, 50 percent

from solution precipitated.

D 15% gelatin,

30% lecithin,

45% coconut oil

After homogenize, m ilk y y ell ow

and stable solution produced

E 15% gelatin,

30% maltodextrin,

45% coconut oil

After homogenize, milky whit e

and unstable solution produced

F 30% maltodextrin,

15% lecithin,

45% coconut oil

After homogenize, 70 percent

from solution precipitated

G 15% gelatin,

30% maltodextrin,

45% lecithin

After homogenize, 50 percent

from solution precipitated.

15% gum Arabic,

45% gelatin,

7.5% lecithin,

67.5% coconut oil

After homogenize, 30 percent

from solution precipitated.

45% gum Arabic,

15% gelatin,

7.5% lecithin,

67.5% coconut oil

2 layer of milky a n d p l a i n

solution produced

3.2. The Effect of Feed Formulation on

Moisture Content

Formulations A and D were the only two emulsions

that can be homogenize well in the long time given. After

spray drying process, Formulation A give more positive

result than Formulation D but still cannot achieve the

target. According to Desmond et al.,[11], the level of

moisture content required for prolonged powder storage

life and stability are at least four percent. Different of

solid percentage selected and being tested to impro ve the

dried dairy product and the parameters used also can

contribute to the improvement of dried product quality.

By increasing the solid percentage used in the solu tion,

it shows the positive result for formulation A. The mois-

ture content decreased to a very stable value. Because of

the suitable outlet temperature have been proven by

Meng et al.,[12] which the best value to dry the probiotic

culture optimally is around 70℃ - 75℃. Inlet tempera-

ture have been controlled based on the outlet temperature

selected. The inlet temperature was maintained at 110℃

in order to obtain an outlet air temperature in the range of

70℃ - 75℃. Therefore, the only parameters that can be

manipulated here is feed flowrate. Based on previous

part of study, wet product produced if using 120 rpm of

feed rate. It preferred to decrease the feed rate to produce

the better form of powder. By using 100 rpm of feed rate,

the product form gives a very low moisture content per-

centage that was below four percent.

For formulation D, when the amount of the solid par-

ticle in the formulation increases, the moisture content

was increased up to 15%. The high stickiness of the

powder on the walls of drying chamber was observed.

The results were unreliable and the formulation D dis-

qualified to the n ext part of study automatically.

3.3. Spray Drying of Different Types of

Probiotic Microorganisms

Different types of microorganisms selected based on

the priority in animal health enhancement. Other re-

searcher has been proved that Lactobacillus strain and

yeast strain can improve animal health. Encapsulations

of the entire microorganisms by the best-selected wall

material and the best-feed flowrate from the previous

part of study have been done. The objective of this part

of the study is to check the viability of different probiot-

ics such as L. plantarum strain 13 and 18, K.lactis and

S.blouradii.

Figure 1 shows the viability of the different types of

microorganisms through spray dryer condition and two

weeks of non-refrigerated storage at 25℃. It directly

shows the yeast strain of Kluyveromyces lactis give

highest survival rate on both situation. The recom-

mended minimum numbers of viable microorganisms in

N. Abd-Ta lib et al. / Agricultural Sciences 4 (2013) 78-83

probiotic food for efficacy are 106 cfu/g [13]. The reten-

tion of high viability during drying and storage presents

particular challenged and can be classified as a major

problem in commercial probiotic prod uction [12]. In this

research, the inlet, outlet temperature and feed rate was

constant at 110℃, 75℃ and 100 rpm respectively. Gen-

erally, it was found that percentage of survival of Lacto-

bacillus plantarum B18 strain was higher than others

during spray dry with the similar carrier. However, it

gives lowest viab ility dur ing storag e. It was believed that

better survival of Lactobacillus plantarum B18 may be

attributed to less sensitivity of this organism when ex-

posed to heat. Only Kluyveromyces lactis can gives a sta-

ble survival characteristic than others.

Openly accessible at

Figure 1 proved the low potential of Lactobacillus

plantarum B13 strain through spray dry and two weeks

storage. The number of cells reduced from 1.28 × 108

cfu/ ml to 2.1 × 106 cfu/ml and final ly to 3.0 × 105 cfu/ml.

This type of bacteria not effective and not suitable for

comercial used with the wall material of gum Arabic, gela-

tin an d coconut oil. The resu lt will be better with the other

mi xture as an e ncapsulation agent. The resistance was very

low even only in the spray dry condition. It was not appli-

cable to select this type of bacteria to improve the animal

feed if the carrier cannot match well with the bacteria.

Lactobacillus plantarum B18 resulted the best viabil-

ity after spray drying and storage at room temperature

with combination of gum Arabic, gelatin and co conut oil

as a wall material. Therefore, it was only reliable in spray

dry condition not during storage. The figure also shows

how the cells drastically decrease just after spray dry and

after two weeks of storage compared to before. The

number of cells reduced from 3.25 × 107 cfu/ml to 2.15 ×

107 cfu/ml and after two weeks storage, the total de-

creased to 4 ×102 cfu/mL. The viability of the bacteria

along the storag e depends on the sensibility of the b acte-

ria to oxygen and the ability of carrier material as a pro-

tector. This is because of the low moisture content of the

powder about to affect the viability of the bacteria of

Lactobacillus Plantarum B18 to be decreased drastically.

The survival of Kluyveromyces lactis comparing with

the other bacteria, it gives the most reliable where the

rate of survival of the cell during spray dry and even

storage were higher. Number of cells reduced from 2.99

× 109 cfu/ml to 7.4 × 107 cfu/ml and 1.9 × 107 cfu/mL

after two weeks storage at 25℃. The ability of this cell

with the carrier which consists of gum Arabic, gelatin

and coconut oil can be commercialized. Different from

human, a probiotic dose between the ranges of 106 to 107

cfu/g in the animal feeds is more than enough [13]. Be-

cause of that, the value resulted for Kluyveromyces lactis

shows the ability to improve the probiotic in the animal

feeds. The other strain of yeast name Saccharomyces

blouradii also gives positive result for this study. The

Figure 1 proved the amount of cell culture that can

maintained in heat and storage. The value of cell culture

can give good impact on animal feed industry which

were reduced from 2.1 × 1010 cfu/ml to 3.9 × 108 cfu/ml

and after two weeks of storage at 25℃, it becomes 7.82

× 106 cfu/ml.

LP B13LP B18K.LS.B

0.0

5.0x10

1.0x10

1.5x10

2.0x10

2.5x10

3.0x10

3.5x10

4.0x10

4.5x10

5.0x10

5.5x10

6.0x10

6.5x10

7.0x10

7.5x10

2.0x10

4.0x10

6.0x10

8.0x10

1.0x10

1.2x10

1.4x10

1.6x10

1.8x10

no. of bacteria (cfu/mL )

type of bacteria

before spray

after spray

after 2 weeks storage

Figure 1. The colony of different probiotics after spray dry and two weeks storage.

N. Abd-Ta lib et al. / Agricultural Sciences 4 (2013) 78-83

LP B13LP B18K.LS.B

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

mo istu r e c o n te n t ( %)

type of bacteria

Figure 2. Moisture percentage of powder form of different probiotics after drying using spray dry.

3.4. Moisture Content of Different

Spray-Dried Probiotics

The carrier used during spray drying of probiotics is

known to have an influence on storage ability. Because

of this study deal with the different types of probiotics,

the ability of the encapsu lation agent to protect probiotic

microorganisms during drying is important characteristic.

Figure 2 had shown the graph of moisture content per-

centage in the different type of dried form probiotics.

Moisture content values give the big impact to viability

of probiotic microorganisms during storage. Spraydried

cultures retain viability for longer at low temperature;

however, refrigeration is expensive to supplier and re-

tailer of product. There is a need to produce the probiotic

bacteria cultures that are stable in ambient temperature

[11].

4. CONCLUSIONS

This study demonstrated the possibility of producing

dry probiotic that suitable as an animal feed using spray

drying. Using combination of gum Arabic, gelatin and

coconut oil with inlet and outlet temperature, feed rate

and solid percentage in the solution is 110℃, 75℃, 100

rpm and 13.5% respectively give the best powder form

of product which is below 4%. A bacterial survival rate

during both spray dry and two weeks non-refrigerated

storage condition have a wide range of percentages.

Kluyveromyces lactis and Saccharomyces blouradii gives

the big potential result for this study, wh ich is suitable to

use in animal feed industry. Survival of probiotic in a

spray-dried form during storage is higher at lower mois-

ture content.

5. ACKNOWLEDGEMENTS

The Universiti Teknologi Malaysia is thankful for the grant given to

Dr. S.H. Mohd-Setapar’s project (Q.JI3000.7125.00H02) under Re-

search University Grant Scheme, part of which enabled this review

article to be prepared. We are grateful for the contribution of all people

involved in this paper pr ep aration.

REFERENCES

[1] Tannock, G.W., Munro, K., Harmsen, H.J.M., Welling,

G.W., Smart, J. and Gopal, P.K. (2000) Analysis of the

fecal microflora of human subject consuming a probiotic

product containing lactobacillus rhamnosus DR 20.

Ap-plication Enviroment Microbial, 66, 2578-2588.

doi:10.1128/AEM.66.6.2578-2588.2000

[2] Bucio, A., R. Hartemink., J.W. Schrama., J. Verreth. and

Rombouts, F.M (2005). Presence of lactobacilli in the in-

testinal content of freshwater fish from a river and from a

farm with a recirculation system. Food Mcrobiology, 23,

476- 482. doi:10.1016/j.fm.2005.06.001

[3] Hilmi, H.T.A., Surakka, A., Apajalahti, J. and Saris, P.E.J.

(2007) Identification of the most abundant lactobacillus

species in the crop of 1- and 5- week- old broiler chickens.

American Society for Microbiology, 73, 7867-7873.

[4] Szabó, I., Wieler, L.H, Tedin, K., Tedin, L.S., Taras, D.,

Hensel, A., Appel, B. and Nöckler, K. (2009) Influence of

a probiotic strain of enterococcus faecium on salmonella

enteric serovar typhimurium DT104 infection in a porcine

animal infection model. American Society for Microbiol-

N. Abd-Ta lib et al. / Agricultural Sciences 4 (2013) 78-83 83

ogy, 75, 2621- 2628.

[5] Dahiya. J.P, Wilkie, D.C., Van-Kessel, A.G. and Drew,

M.D. (2005) Potential strategies for controlling necrotic

enteritis in broiler chicken in post-antibiotic era. Animal

Feed Science and Technology, 129, 60-88.

doi:10.1016/j.anifeedsci.2005.12.003

[6] Son, V.M., Chang, C., Wu, M., Guu, Y., Chiu, C. and

Cheng, W. (2009) Dietary administration of the probiotic,

lactobacillus plantarum, enhanced the growth, innate

immune responses, and disease resistance of the grouper

epinephelus coioides. Fish & Shellfish Immunology, 26,

691- 698. doi:10.1016/j.fsi.2009.02.018

[7] Canibe, N., Miettinen, H. and Jensen, B.B. (2008) Effect

of adding lactobacillus plantarum or a formic acid con-

taining-product to fermented liquid feed on gas-

troin-testinal ecology and growth performance of piglet.

Live-stock Science, 114, 251- 262.

doi:10.1016/j.livsci.2007.05.002

[8] Abu-Tarboush, H.M., Al-Saiady, M.Y. and El-Din, A.H.K.

(1996) Evaluation of diet containing lactobacilli on per-

formance, fecal coliform, and lactobacilli of young dairy

calves. Animal Feed Science and Technology, 57, 39- 49.

doi:10.1016/0377-8401(95)00850-0

[9] Mohamed, M.I., Maareck, Y.A., Abdel-Majid, S. and

Awadalla, I.M. (2009) Feed intake digestibility, rumen

fermentation and groeh performance of camels fed diets

supplemented with a yeast culture or zinc bacitracin.

Animal Feed Science and Technology, 149,341-345.

doi:10.1016/j.anifeedsci.2008.07.002

[10] Kailasapathy, K. (2002) Microencapsulation of Probiotic

Bacteria: Tecnology and Potential Applications. Current

Issues Intestinal, Microbial, 3, 39-48

[11] Desmond, C., Ross, R.P., O'Callaghan, E., Fitzgerald, G.

and Stanton, C. (2002) Improve survival of lactobacillus

parasei NFBC 338 in spray-dried powders containing

gum Acacia. Journal of Applied Microbiology, 93, 1003-

1011. doi:10.1046/j.1365-2672.2002.01782.x

[12] Meng, X.C., Stanton, C., Fitzgerald, G.F., Daly, C. and

Ross, R.P. (2008). Anhydrobiotics: The challenges of

dring probiotics cultures. Food Chemistry, 106, 1406-

1416. doi:10.1016/j.foodchem.2007.04.076

[13] Guillot, J.F. (1998) Les probiotiques er dimenation ani-

male. Cahiers Agricultur es, 7, 49-54.