UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY

MEDICINE CLUJ-NAPOCA

FACULTY OF ANIMAL SCIENCE AND BIOTECHNOLOGY

DOCTORAL SCHOOL: PLANT AND ANIMAL RESOURCES

DOMAIN: BIOTECHNOLOGY

POP OANA LELIA (MUREAN)

DEVELOPMENT OF INNOVATIVE SYSTEMS FOR PROBIOTICS

ENCAPSULATION, WITH APPLICATIONS IN BIOMEDICINE

PhD THESIS ABSTRACT

PhD SUPERVISOR

Prof. Dr. Carmen Socaciu

CLUJ-NAPOCA

2014

II

CONTENTS

INTRODUCTION: AIMS AND OBJECTIVES .................................................................... VI

CHAPTER 1 ........................................................................................................................... IX

MICROENCAPSULATION OF LACTOBACILLUS PLANTARUM, WITH LUCERNE

GREEN JUICE, AS PREBIOTIC, IN ALGINATE-CHITOSAN MICROSPHERES, AND

THEIR BEHAVIOR IN SIMULATED GASTROINTESTINAL CONDITIONS ................ IX

INTRODUCTION ........................................................................................................... IX

1.1. MATERIALS AND METHODS .............................................................................. X

1.1.1. Materials ....................................................................................................... X

1.1.2. Lucerne green juice- obtaining process and characterization ...................... X

1.1.3. Probiotic strain: Lactobacillus plantarum .................................................... X

1.1.4. Preparation of alginate microspheres .......................................................... XI

1.1.5. Microsphere morphologic characterization ................................................. XI

1.1.6. Resistance to gastrointestinal media. Preparation of simulated gastric and

intestinal juices ........................................................................................................................ XI

1.1.7. Cell viability .............................................................................................. XII

1.1.8. Statistical analyses ..................................................................................... XII

1.2. RESULTS AND DISCUSSIONS .......................................................................... XII

1.2.1. Lucerne green juice obtaining process and characterization ..................... XII

1.2.2. Morphologic structure of microspheres containing L. plantatum ............. XII

1.2.3. Survivability of free and microencapsulated probiotic cells in Simulated

Gastric Juice (SGJ) at pH=1,5 ............................................................................................. XIII

1.2.4. Survivability of free and microencapsulated probiotic cells in Simulated

Intestinal Juice (SIJ) pH = 7,4 .............................................................................................. XIV

1.3. CONCLUSION ...................................................................................................... XV

CHAPTER 2 ........................................................................................................................ XVI

III

INFLUENCE OF ENCAPSULATION MATRIX AND TECHICAL PARAMETERS ON

PHYSICAL PROPERTIES OF MICROSPHERES AND SURVIVABILITY OF

PROBIOTIC CELLS ........................................................................................................... XVI

INTRODUCTION ........................................................................................................ XVI

2.1. MATERIALS AND METHODS .......................................................................... XVI

2.1.1. Polymers used in experiments as filler materials ..................................... XVI

2.1.2. Probiotic strain ........................................................................................ XVII

2.1.3. Encapsulation of B. lactis 300B in alginate based microspheres ........... XVII

2.1.4. Phisical properties of alginate filler mixtures ....................................... XVII

2.1.5. Entrapment efficiency of B. lactis 300B encapsulation ........................ XVIII

2.1.6. Determination of microspheres size and shape ..................................... XVIII

2.1.7. Determination of microspheres shape ...................................................... XIX

2.1.8. Microspheres liophilization ...................................................................... XIX

2.1.9. Determination of bulk density, tapped density and flowability ............... XIX

2.1.10. B. lactis 300B viability before and after encapsulation and liophilization

.............................................................................................................................................. XIX

2.1.11. Stability tests of the liophilized microsphers .......................................... XX

2.1.12. Statistical analyses ................................................................................... XX

2.2. RESULTS AND DISCUSSION ............................................................................ XX

2.2.1. Microencapsulation yield and entraptment efficiency .............................. XX

2.2.2. Alginate- filler mixture properties and microspheres size and shape .... XXII

2.2.3. Bulk, tapped density and flowability of the liophilized microspheres .. XXIV

2.2.4. Survival of Bifidobacterium lactis 300B in fresh microspheres ........... XXVI

2.2.5. Viability after liophilization ................................................................. XXVII

2.2.6. Survivability of entrapped probioticcells in freeze dried microspheres XXIX

2.3. CONCLUSIONS ................................................................................................. XXX

CHAPTER 3 ..................................................................................................................... XXXI

BEHAVIOR OF BIFIDOBACTERIUM LACTIS 300B AFTER ENCAPSULATION IN

DIFFERENT COATED ALGINATE/PULLULAN MICROSPHERES ......................... XXXI

IV

INTRODUCTION ..................................................................................................... XXXI

3.1. MATERIALS AND METHODS ...................................................................... XXXII

3.1.1. Materials ............................................................................................... XXXII

3.1.2. Probiotioc strain .................................................................................... XXXII

3.1.3. Preparation of alginate/pullulan microspheres ..................................... XXXII

3.1.4 Microspheres size .................................................................................. XXXII

3.1.5 Coating of AP microspheres ................................................................ XXXIII

3.1.6. Freeze drying process ......................................................................... XXXIII

3.1.7. Cell viability ....................................................................................... XXXIII

3.5.8. Evaluation of B. lactis 300B release in simulated intestinal media ... XXXIV

3.1.9. Statistical analyses .............................................................................. XXXIV

3.2. RESULTS AND DISCUSSIONS ................................................................... XXXIV

3.2.1. Characterization of microencapsulation process ................................ XXXIV

3.2.2. Microspheres shape and size. Entrapment efficiency ........................ XXXIV

3.2.3. Comparative survival of B. lactis 300B cells ...................................... XXXV

3.2.4. B. lactis 300 survivability during storage in freeze dried microspheres

........................................................................................................................................ XXXVI

3.2.5. The release of encapsulated B. lactis 300B in simulated intestinal media

....................................................................................................................................... XXXVII

3.3. CONCLUSIONS ............................................................................................ XXXVII

CHAPTER 4 .................................................................................................................. XXXIX

STABILITY COMPARATON OF FREE AND ENCAPSULATED LACTOBACILUS

CASEI IN YOGHURT FOR LONG TIME STORAGE ................................................ XXXIX

INTRODUCTION .................................................................................................. XXXIX

4.1. MATERIALS AND METHODS .................................................................... XXXIX

4.1.1. Microbial cultures, media and growth conditions .............................. XXXIX

4.1.2. Microencapsulation of L. casei................................................................... XL

4.1.3. Examination of alginate and alginate-pectin microspheres ........................ XL

4.1.4. Freeze drying of L. casei microspheres ...................................................... XL

V

4.1.5. Preparation of yoghurt including microspheres ......................................... XL

4.1.6. Dynamics of yoghurt acidification with L. casei, free and encapsulated . XLI

4.1.7. Enumeration of probiotic cells ................................................................. XLI

4.1.8. Statistical analyses ................................................................................... XLII

4.2. RESULTS AND DISCUSSIONS ........................................................................ XLII

4.2.1. Physical examination of alginate and alginate/pectin microspheres ....... XLII

4.2.2. Yoghourt with microspheres ................................................................... XLII

4.2.3. Dynamics of yoghourt acidification ........................................................ XLII

4.2.4. Viability of the entrapped L. casei in the yoghourt over 35 days .......... XLIII

4.3. CONCLUSIONS ................................................................................................ XLIV

GENERAL CONCLUSIONS ........................................................................................... XLVI

REFERENCES ................................................................................................................. XLVII

VI

INTRODUCTION: AIMS AND OBJECTIVES

Bioencapsulation is an emerging technology applied for the protection and controlled

release of valuable molecules or cells.

The pioneering work in the field of microencapsulation was carried out long time ago

by Chang (Chang, 1971) who used encapsulation in order to stabilize enzymes. Since then,

cell encapsulation has gained significant interest in a broad range of applications such as

pharmacy, medicine, food production, agriculture and environment protection. The

utilization of immobilized microbial cells in various biotechnological processes was found to

be advantageous over the use of free cells.

The aim of this PhD thesis was to develop new encapsulation recipes and techniques

adapted to probiotic microorganisms and investigate the behavior of probiotics before,

during and after encapsulation. Different types of encapsulation matrices were used in order

to develop an optimal formula for a high entrapment efficiency and high cell viability.

The behavior of the entrapped probiotic cells was studied in simulated gastrointestinal

conditions and, in terms of viability, in food products, as yogurt, for long time storage.

The main objectives of the experimental studies were:

Microencapsulation of Lactobacilus plantarum with lucene green juice, as prebiotic,

in alginate-chitosan microspheres, and their behavior in simulated gastrointestinal

conditions.

Influence of encapsulation matrix and technical parameters on physical properties of

microspheres and survivability of probiotic cells.

Behavior of Bifidobacterium lactis 300B after encapsulation in different coated

microspheres of alginate-pullulan.

Comparative stability of free and encapsulated Lactobacillus casei in yoghurt for long

time storage.

Thesis structure: The thesis is structured into two parts, a first one represented by

literature study and a second one, focused on original contributions.

The First part includes 2 chapters (1-2):

PhD Thesis Abstract

VII

Chapter 1 summarizes the basic informations about bioencapsulation; matrices used

as fillers and different types of encapsulation techniques.

Chapter 2 presents data about morphology features of probiotics, their classification,

metabolism, health effects and applications.

The Second part includes 4 chapters (3-6):

Chapter 3 includes experimental data about the encapsulation of Lactobacillus

plantarum, in lucerne green juice, incorporated in alginate-chitosan microspheres,

and their behavior in simulated gastrointestinal conditions.

Chapter 4 contains experimental data about the influence of encapsulation matrix

and other parameters on Bifidobacterium lactis 300B survivability.

Chapter 5 includes data about the incorporation and survival of Bifidobacterium

lactis 300B, before and after encapsulation, coating and freeze drying.

Chapter 6 describes the stability of free and encapsulated Lactobacilus casei in

yogurt during long time storage.

Part of this PhD thesis was achieved at Brace GmbH -the microsphere company, in

Karlstein Germany, in collaboration with our University of Agricultural Sciences and

Veterinary Medicine, Cluj-Napoca, Romania, with a financial support provided for six

months (2012) by Deutsche Bundesstiftung Umwelt grant.

PhD Thesis Abstract

VIII

EXPERIMENTAL RESULTS

Bioencapsulation is an emerging technology, used since 3 decades, continuously

developing and more accepted in the pharmaceutical, chemical, cosmetic and foods

industries, but not only (Augustin and Sanguansri, 2003; Augustin et al., 2001; Nedovic et

al., 2011; Poncelet et al., 2006; Vandamme et al., 2007). The encapsulation of probiotics has

become very attractive technology being adequate for protection of probiotics in the acidic

media of the stomach (Brachkova et al., 2010; Shahidi and Han, 1993), and ensure the target

delivery in the colon.

To improve the viability and resistance of probiotics in time and at different

temperatures, but furthermore, to ensure the minimum dose necessary to reach the

therapeutic level, after the passage throws upper and lower gastrointestinal tract, the

encapsulation of probiotics is needed (Burgain et al., 2011). The immobilization of cells is

made, more frequently, in alginate capsules.

The great potential of probiotic cells is recognized and appreciated in an extremely

wide range of areas related more or less with human health. Nevertheless, for optimum

action of these probiotic cells, it is essential that they are provided with appropriate

conditions for growth and metabolism, and also protect from rough environmental conditions

that they are susceptible to. Furthermore, in case of probiotic cells, achievement or

sustenance of high cell density, secure of cells activity for longer period of time and easy

mending of the cells from the products is oftentimes expected. For these achievements,

bioencapsulation is proposed.

PhD Thesis Abstract

IX

CHAPTER 1

MICROENCAPSULATION OF LACTOBACILLUS PLANTARUM, WITH

LUCERNE GREEN JUICE, AS PREBIOTIC, IN ALGINATE-CHITOSAN

MICROSPHERES, AND THEIR BEHAVIOR IN SIMULATED

GASTROINTESTINAL CONDITIONS

INTRODUCTION

The aim of this study was to demonstrate the lifespan increase of Lactobacillus

plantarum, lucerne green juice (LGJ) (as prebiotic additive) after the encapsulation in

different alginate-chitosan matrices.

Objectives

Entrapment of Lactobacillus plantarum suspension, containing LGJ, in an alginate

matrix, obtaining microspheres and tests on their survivability in acidic media of

the simulated gastric juice.

Preparation of chitosan coated capsules and evaluation of their controlled release

in the simulated intestinal juice.

Different methods were applied to demonstrate the efficacy of encapsulation:

The scanning electron microscopy (SEM) for the visualization of the internal

structure of the alginate microspheres and the entrapped L. plantarum.

The viability of the entrapped probiotic cells before and after exposure to the

simulated gastric juice (30, 60, 90 and 120 minutes).

The viability of the entrapped probiotic cells in chitosan coated capsules, before

and after exposure to the simulated intestinal juice after 60, 90 and 120 minutes.

PhD Thesis Abstract

X

1.1. MATERIALS AND METHODS

1.1.1. Materials

Alginate was supplied by FMC, Norway, chitosan, calcium chloride, nutrient agar and

MRS broth from Merck (Germany). All materials and solutions were sterilized at 121 C for

15 min. For the simulated gastrointestinal juices were used pepsin, pancreatin and bile salts

from Bioaqua, Romania.

1.1.2. Lucerne green juice- obtaining process and characterization

The lucerne green juice (LGJ) was obtained directly from fresh lucerne. The chemical

composition of the lucerne green juice is in Table 1. The juice was used immediately after

obtained in the proposed experiments. From 1 kg fresh lucerne we obtained approximately

300 ml LGJ The percentage of ash in the LGJ was 5,3% (w/w). Salt components (PO43-

,

Mg2+

, K+, Na

+) are needed by microorganisms for growth.

Table 1

Chemical composition of lucerne green juice (LGJ)

LGJ LGJ

Sugar [gl-1

] 8.280.1 NO2-

[mgl-1

]

PhD Thesis Abstract

XI

cells were harvested by centrifugation at 3000 g for 5 min at 4C washed twice with sterile 9

g/L sodium chloride solution and resuspended in 2.5 mL of sodium chloride solution 5 g/L.

1.1.4. Preparation of alginate microspheres

A Multinozzle Biotech Encapsulator from EncapBioSistems Inc. was used, with 350

m nozzle size, and crosslinked in calcium chloride (20 g/L). The condition were: 15 g/L

alginate, 75 g/L probiotic cells (equivalent of 1010

CFU/g) with or without LGJ.

Microspheres were hardened 30 min in CaCl2, and then rinsed with sterile NaCl (8.5 g/L).

The fresh rinsed alginate microspheres were immersed under continuous stirring in

1g/L water chitosan solution for 30 minutes, and then washed with sterile NaCl 8.5 g/L.

1.1.5. Microsphere morphologic characterization

The morphologic optical characterization of the obtained alginate microspheres was

done using Scanning electron microscopy. The dehydrated were covered in gold and the

measurement was performed at 100 and 2000 magnitude, using an E 302C SEM microscope.

1.1.6. Resistance to gastrointestinal media. Preparation of simulated gastric and

intestinal juices

The simulated juices was prepared according to Brinques ans Ayub (Brinques and

Ayub, 2011). The formulation of simulated gastric juice (SGJ) was prepared as follow:

pepsin was suspended in sterile sodium chloride solution (0.5%, w/v) to a final concentration

of 3 g /L and adjusting the pH to 1.5 with concentrated HCl or sterile 0.1 mol/L NaOH.

Simulated intestinal juice (SIJ) was prepared by suspending pancreatin in sterile

sodium chloride solution (0.5%, w/v) obtaining a desired final concentration of 1 g/L, with

4.5% bile salts and adjusting the pH to 7.4 with sterile 0.1 mol /L NaOH. Both solutions

were sterilized by filtration through a 0.22 m membrane.

The tolerance of free and entrapted probiotic cells of L. plantarum on simulated

gastric and intestinal juices was determined using the adapted method described in the

literature (Brinques and Ayub, 2011). Aliquots of 1 mL were removed at 0, 30, 60, and 120

min (for all trials) for the determination of total viable counts using the palte count method.

PhD Thesis Abstract

XII

1.1.7. Cell viability

The enumeration of viable probiotic cells was conducted in triplicates before/after

encapsulation and coating. The entrapped cells were released from the alginate microspheres

after their suspension in phosphate buffer with 7.40.2 pH, after stirring. The released cells

were analised using simple serial dillutions in sterile NaCl solution (8.5 g/L). Aliquots of 1

ml from the last three dilutions were used to measure the cell density using the plate

counting method on nutrient agar and expressed in colony forming units (CFU) per ml. After

72h of incubation at 37C the number of CFU/ml was counted and converted to log CFU.

1.1.8. Statistical analyses

. The statistical evaluation was carried out using Graph Prism Version5.0 (Graph Pad

Software Inc., San Diego, CA, USA).

1.2. RESULTS AND DISCUSSIONS

1.2.1. Lucerne green juice obtaining process and characterization

From 1 kg fresh lucerne we obtained approximately 300 ml LGJ The percentage of

ash in the LGJ was 5,3% (w/w).

1.2.2. Morphologic structure of microspheres containing L. plantatum

By scanning electron microscopy the internal structure of the alginate microspheres

containing the entrapped L. plantarum can be observed (Fig. 1) at different magnifications.

a) b)

Fig. 1 SEM micrograph of alginate microspheres containing L. plantarum at a) low ( x 100)

and b) high ( x 2000) magnifications.

PhD Thesis Abstract

XIII

The arrows (Fig. 1b) show the probiotic cells insertion in the alginate matrix. The size

of the obtained microspheres was determined, 1110.512.7 m. The mean diameter of the

coated microspheres were significantly (p

PhD Thesis Abstract

XIV

The decrease rate of microencapsulated cells in comparison with free cells was with

102 CFU/ml after 30 minutes in SGJ and respectively 10

5 CFU/ml after 120 minutes in SGJ.

The rate of decreased survivability was significantly lower for free bacteria FC (p

PhD Thesis Abstract

XV

the entrapped probiotic cells (Chavarri et al., 2010), because an ion exchange reaction takes

place when the microspheres are immersed in bile salt (Murata et al., 1999).

We also observed a similar effect in our study. However, there is no consistence in

the reported encapsulation procedure.

1.3. CONCLUSION

The study summarizes the co-encapsulation of Lactobacillus plantarum with a

lucerne green juice, in different concentrations, in comparison to the free and entrapped

probiotic cells, on the gastrointestinal passage survivability.

The chapter conclusions are:

Lucerne green juice was succesfuly obtained in the laboratory (30% yeld) with a

percentafe of 5,3% ash.

The size of the microspgeres containing Lactobacillus plantarum range drom 1110

m (for the microspheres without coating) to 1269 m (for the chitosan coated

ones).

The SEM micrograph image revealed the morphologic structure of alginate

microspheres containing L. plantarum. The probiotic cells could be observed in

the alginate matrix.

After exposure to SGJ the highest survivability level was observed for the sample

where L. plantarum was entrapped with LGJ 20%.

The most effective formulation for protection of entrapped probiotic cells in SIJ

was the one with 20% LGJ after 60 min incubation.

PhD Thesis Abstract

XVI

CHAPTER 2

INFLUENCE OF ENCAPSULATION MATRIX AND TECHICAL

PARAMETERS ON PHYSICAL PROPERTIES OF MICROSPHERES AND

SURVIVABILITY OF PROBIOTIC CELLS

INTRODUCTION

The aim of this study was to find the optimal formula for the entrapment of

Bifidobacterium lactis 300B in alginate based microspheres, in order to obtain adequate

physical and biochemical properties that sustain the viability of the cells. Seven different

encapsulation filler materials were used: three types of celluloses, two types of starch,

dextrin and pullulan.

Objectives

We aimed to compare seven different solid fillers, used for the entrapment of

Bifidobacterium lactis 300B in alginate based microspheres, in terms of production,

entrapment efficiency, micrometric properties and after the encapsulation procedure.

As procedures we applied:

A freeze drying method in order to obtain long time and temperature stability for

the entrapped probiotic cells.

The cell viability determination after freeze drying.

Survivability determination of the entrapped microorganisms in the freeze dried

microspheres after 3, 6, 9 and 15 days at room temperature and at 4 C.

2.1. MATERIALS AND METHODS

2.1.1. Polymers used in experiments as filler materials

A commercially available Manguel GMB sodium-alginate was supplied by FMC,

Norway, Starch BR-07, Starch BR-08 from BRACE, Dextrin Crystal from Sigma, Na-CMC

from Dow Chemicals, HPMC from Harke Pharma, Microcrystalline Cellulose Viva-pur 105

type (MCC) from Rettenmayer and Pullulan from Hayashibara were used and compared as a

filler in alginate based microspheres. Calcium chloride was purchased from Brenntag,

PhD Thesis Abstract

XVII

sodium phosphate from Merck (Germany), bifidus selective medium agar and peptone from

Sigma-Aldrich (Germany). ll materials and solutions, including the CaCl2 solution were

sterilized at 121 C for 15 min.

2.1.2. Probiotic strain

Bifidobacterium lactis 300B was used as probiotic strain. The strain was purchased as

lyophilized probiotics powder from Howaru, Germany. The probiotic was used as received

from the supplier. A viability test was performed before each trial. All materials and

solutions were sterilized by autoclaving at 121C for 15 min. prior utilization.

2.1.3. Encapsulation of B. lactis 300B in alginate based microspheres

Lyophilized B. lactis 300B, 75 g/L, were encapsulated using cross linking gelation.

The encapsulation formulation consists in 15 g/L alginate (FMC, Norway) and 15 g/L from

eatch filler materia. The Spherisator M, type 2002SP-AE5-D0 was used in the microspheres

formulation process, at Brace GmbH Germany. The microspheres were hardened for 30 min

in CaCl 40 g/L (Brenntag , Australia) , and then rinsed with sterile sodium chloride 8.5 g/L

(Sigma-Aldrich, Germany). The filler used for encapsulation of the probiotic powder, and

each sample codification are shown in Table 2

Table 2

Bifidobacterium lactis 300B encapsulation formulation

Filler material Code of sample

Microspheress prepared

with sodium alginate 1.5%

(w/v) and different filler

materials 1.5% (w/v)

HPMC AHPMC

Na CMC ACMC

Microcrystalline cellulose AMCC

Starch BR-07 AS07

Starch BR-08 AS08

Dextrin AD

Pullulan AP

2.1.4. Phisical properties of alginate filler mixtures

The density of the alginate-filler mixture was calculated using the mass (m) and

volume (V) ratio: (1) = m/V g/cm3

PhD Thesis Abstract

XVIII

The viscosity was measured with an HAAKE viscometer VT-02 (ThermoFisher,

Germany)at 23 1C. The value for the surface tension of the alginate based solutions at 15

g/L was obtained from Chan et al. (Chan et al., 2011a) and considered constant for all seven

samples.

2.1.5. Entrapment efficiency of B. lactis 300B encapsulation

The entrapment efficiency of B. lactis in the fresh microspheres was determined

according to (Sandoval-Castilla et al., 2010) with slight change as follows: Entrapment

efficiency = (aF/b) 100 (CFU/g)

Where a is CFU/g in the microspheres, and b is CFU/g in the biopolymer slurry before

production, and F is the sphere packing factor (Aste and Weaire, 2008). We considered the

cubical densest package for all calculations F=0.70 (Aste and Weaire, 2008; Holleman et al.,

1985).

2.1.6. Determination of microspheres size and shape

The size of the microspheres was determined based on different formulas described

by Chan et al. (Chan et al., 2011b) with slight change as follows.

Theoretical diameter of detached liquid drop, Dl(T) (mm): (2) DI(T)= (6dT/g)1/3

, were

dT is the capillary tip diameter (mm); is surface tension (g/s2); is density (g/mm

3); and g

is gravitational acceleration (mm/s2).

Corrected diameter of detached liquid drop, Dl(C) (mm): (3) Dl(C) = kLF * Dl(T), where

kLF is the liquid lost factor, kLF = 0.980.04dT.

Corrected diameter of Caalginate microspheres after gelation, Db(C) (mm): (4) Db(C) =

kSF(gelation) * Dl(C), where kSF is the shrinkage factor attributed to the gelation process, which

was found to be for Ca- alginate microspheres kSF(gelation) = 0.88 (Chan et al., 2011b).

The reduction in microspheres size after lyophilization was calculated and expressed

by a shrinkage factor, as shown below: (5) kSF(lyophilization) =(Db Db(lyophilized))/Db, where

kSF(lyophilization) is the shrinkage factor attributed to the lyophilization process; Db is the

diameter of the microspheres obtained as described above before lyophilization (mm); and

Db(lyophilized) is the diameter of the microspheres obtained after lyophilization (mm).

PhD Thesis Abstract

XIX

2.1.7. Determination of microspheres shape

The microspheres shape was quantified using the sphericity factor (SF), which is

given by the following equation: Sphericity factor (6) (SF) =(dmax dmin)/(dmax + dmin),

where dmax is the largest diameter and dmin is the smallest diameter perpendicular to dmax.

Tweenty microspheres were used for each determination.

2.1.8. Microspheres liophilization

The fresh obtained microspheres were liophilized using a VaCo 5 freeze dryer from

Zirbus (Germany) at once and freeze dried at -50C , 0.05 mbar for 24h. Samples were

analyzed immediately and after 3, 6, 9 and 15 days at 23C1C and 4C1C.

2.1.9. Determination of bulk density, tapped density and flowability

The bulk density (BD) of the lyophilized microspheres was determined by pouring a

known mass of microspheres (mp) into a calibrated cylinder, and it was calculated by

dividing the mass (mp) by the bulk volume (vB), as shown in following equation:

(7) BD =mP/vB

The tapped density (TD) was determined by tapping a calibrated cylinder containing

microspheres until the equilibrium tap volume (vT) was obtained. The tapping was performed

until no volume change was observed. Hausners ratio of microcapsules was computed

according to the following equation: (8) Hausner ratio = TD/BD

The Hausner ratio is a parameter that inversely influence the microsphere flowability.

2.1.10. B. lactis 300B viability before and after encapsulation and liophilization

Non-encapsulated and encapsulated Bifidobacterium lactis 300B were enumerated

immediately after the encapsulation, and freeze drying process respectively, using the plate

counting method, on BSM agar (Sigma-Aldrich, Germany). The microspheres were

dissolved completely in sodium citrate (20 g/L) with an adjusted pH=7.3, before

enumeration of viable cells. Dilutions steps 1:10 were performed in saline solution (8.5 g/L).

From the last three dilutions, one ml of the dilution was introduced in the Petri dish where

the nutrient agar medium was added. The operation was repeated three times for each

dilution. After 72 h incubation at 37C in the anaerobic jar (Sigma-Aldrich, Germany) the

number of colony-forming units (CFU) was counted and converted to log10 CFU/g.

PhD Thesis Abstract

XX

The survival of B. lactis in each of the freeze-dried samples was determined using the

formula: (9) survival = (n/n0), where n0 is the number of bacteria per gram of wet

microspheres before drying, and n is the number of the freeze-dried microspheres right

away after drying (Simpson et al., 2005).

2.1.11. Stability tests of the liophilized microsphers

The stability of the probiotic cells in the microspheres, as a function of storage time (3, 6, 9

and 15 days) at room temperature and at 4C was obtained by calculating the ratio of CFU/g

of microspheres storage/CFU/g of microspheres immediately after freeze-drying.

2.1.12. Statistical analyses

The mean values and the standard error were calculated from the triplicate data using

Graph Prism Version4.0 (Graph Pad Software Inc., San Diego, CA, USA). For the size

determination, all the calculations were performed using Microsoft Excel 2010.

2.2. RESULTS AND DISCUSSION

2.2.1. Microencapsulation yield and entraptment efficiency

Different processing conditions were investigated in order to evaluate the possibility

of industrial scale production of probiotic entrapment in one of the formulations described

above. It was found that the production of microspheres, in terms of quantity and quality,

was influenced by the nature of the seven types of filler materials. The production of 600

g/h, the value obtained in our study, is an easily achievable outcome. Future attempts

resulting in the production of higher outputs ranging from 3.6 kg/h to 10 kg/h may be

reasonably expected. As can be observed in Fig. 4, in terms of production per minute, the

highest productiont is observed for the samples AS08, AP and AS07 in this order.

PhD Thesis Abstract

XXI

Fig. 4 Comparative alginate-based microspheres

production with different filler materials. (() sample

AHPMC, () sample ACMC, () sample AMCC,

() sample AS07, () sample AS08, () sample AD,

() sample AP) (for abbreviations see Table 2).

Fig. 5 Entrapment efficiency

(CFU/g %) of B. lactis 300B in

alginate based microspheres

correlated to the filler type.

The production for the samples AHPMC was 580 g/h, ACMC-537,4 g/h and AD-

552,4 g/h are similar while the sample AMCC- 441 g/h, shows the lowest production rate.

A high entrapment capacity was observed for all formulations, as is represented in

Fig. 5. In our study, the entrapment efficiency of Bifidobacterium lactis 300B into the

microspheres varied between 57.20 and 69.96%. In the literature (Jyothi et al., 2010; Reid et

al., 2005) the entrapment efficiency data is linked to the viability losses in the microspheres.

The type of polymer influences the encapsulation efficiency, mainly through smaller

shrinkage and by extended entanglement of the polymer chains. The highest entrapment

efficiencies were obtained for the samples AHPMC, AP and AS07 in a decreasing order.

Due to their excellent physicochemical and mechanical properties, HPMC, pullulan and

starch enhanced the alginate action. The microspheres filled with microcrystalline cellulose

(sample AMCC) and dextrin (sample AD) showed the lowest entrapment efficiency, but not

statistically significantly lower than the other samples. The rest of the alginate based

microspheres, respectively the ones using the two types of cellulose and starch BR-08 as

sam

ple

AHPM

C

sam

ple

ACM

C

sam

ple

AM

CC

sam

ple

AS07

sam

ple

AS08

sam

ple

AD

sam

ple

AP

0.0

0.2

0.4

0.6

0.8

En

tra

pm

ent

effi

cien

cy (

CF

U/g

%)

sam

ple

AHPM

C

sam

ple

ACM

C

sam

ple

AM

CC

sam

ple

AS07

sam

ple

AS08

sam

ple

AD

sam

ple

AP

0.0

0.2

0.4

0.6

0.8

En

tra

pm

ent

effi

cien

cy (

CF

U/g

%)

PhD Thesis Abstract

XXII

fillers showed similar trends. Our results concerning the entrapment efficiencies when using

the different cellulose types and starch as fillers are in agreement with those previously

found by Nochos et al. (Nochos et al., 2008) and Sultana et al. (Sultana et al., 2000).

2.2.2. Alginate- filler mixture properties and microspheres size and shape

The various microsphere formulations show an average particle size in the range of

1,054 1,066 mm. As can be seen in Table 3, similar trends can be observed for all

formulations in the dripping and gelation processes regarding the size of the microspheres.

Furthermore, in Table 3 it can observed that the drop size decreased consistently along the

hardening process.

Table 3

Density and viscosity of the mixture, nozzle size and the calculated diameters for each

sample

Sample Density

(g /cm3)

Viscosity

(mPas)

Tip diameter

(mm)

Theoretical

diameter of

detached liquid

drop (mm)

Corrected

diameter of

detached liquid

drop (mm)

Corrected

diameter of

microsphere after

gelation (mm)

Experimental

diameter of

microsphere after

gelation (mm)

AHPMC

1,0370.01 19011.8 0.3

1,2248 1,1907

1,099

1,0668

ACMC

1,0400.01 49020.6 0.3

1,2061 1,1765

1,084

1,0539

AMCC

1,0390.015 26023.2 0.3

1,2166 1,1827

1,092

1,0596

AS07

1,0380.018 25517.61 0.3

1,2169 1,183

1,090

1,0599

AS08

1,0270.019 25018.9 0.3

1,2208 1,1868

1,095

1,0633

AD

1,0190.01 24816.8 0.3

1,224 1,1899

1,097

1,0661

AP

1,0170.014 31012.4 0.3

1,2158 1,1819

1,091

1,0589

Values are mean (n =3) standard deviation

The factors affecting the size of the microspheres involve the viscosity of the polymer

solution, the diameter of the nozzle and the distance between the outlet and the coagulation

solution (Anal et al., 2003; Anal and Singh, 2007; Anal and Stevens, 2005) and the

manufacturing methods used (Grabnar and Kristl, 2011). In our study for all the samples the

same size diameter of the nozzle was used. A correlation between the size of the obtained

microspheres and the viscosity can be observed. The sample AHPMC, the less viscous from

the samples, with 190 (mPas) viscosity hade the biggest diameter, 1.0668 mm. This kind of

PhD Thesis Abstract

XXIII

correlation between the viscosity and the microspheres size underlined also by other studies

(Chan et al., 2011b; Chandramouli et al., 2004). Similar trends were observed for the

samples AD, AS07, AS08, ACMC and AP with no significant differences neither when the

viscosity is discussed nor the diameter of the obtained microsphere.

In the vibration dip casting, the drop was formed by a vibration system. When the

droplet was extruded by the flow rate, it broke up with the vibration under resonance, the

liquid drop detached from the nozzle and immerse into the hardening bath where bound ions

and create linkages lead to the gel formation. Microspheres were smaller than the drop

detached from the nozzle, a phenomenon attributed to the syneresis effect happened in the

formed gel. The calculated diameters of the microspheres after gelation were found to give a

nigh approximation to the obtained experimentally as can be observed in Fig. 6

Previous reports (Donati et al., 2005) have shown that the shrinkage factor, can be

used to correct the diameter of the microspheres after gelation. Chan et al. (Chan et al.,

2011a) have shown that low viscosity of the filler leads to high shrinkage factor. In

accordance to what was previously found, this trend is also observed in the present work as

can be seen in Fig. 6. In our study, the highest amount of shrinkage of the lyophilized

microspheres is attributed to AHPMC and AD, the samples that proved the lowest viscosity

of the mixture. The least amount of shrinkage can be attributed to the samples ACMC and

AP due to the same motive regarding the viscosity (Nienaltowska et al., 2010).

Fig. 6 Effect of viscosity of the samples on the shrinkage factor of lyophilized Ca-alginate

based microspheres after liophilization.

100 200 300 400 5000.75

0.80

0.85

0.90

0.95

AHPMC

ACMC

AMCC

AS07

AS08

AD

AP

Viscosity (mPa s)

Sh

rin

ka

ge f

acto

r, k

sf (

lio

ph

iliz

ati

on

)

PhD Thesis Abstract

XXIV

The shape of the alginate based microspheres was delineating using the sphericity

factor due to its effectiveness in determining shape changes. A perfect sphere is defined by a

sphericity factor equal to 0; meanwhile, the elongated objects have values of the sphericity

factor approaching to unity. According to Goh et al. (Goh et al., 2012) a high concentration

of polymer leads to an increased sphericity. In the present study, all the obtained

microspheres have hade spherical shape in spite of the type of the filler. The lyophilization

process induced a deformation in the structure of the microspheres caused by the sublimation

of the water from the hydrogel matrix. This fact resulted in microspheres with an

unpredictable and irregular shape, occurrence observed in former studies (Chan et al., 2011b;

Rassis et al., 2002; Zohar-Perez et al., 2004). Nevertheless, in our study, the deformation of

the microspheres was attenuated by the different fillers used as can be observed in Table 4

were the sphericity factor of the lyophilized microspheres is presented.

Table 4

Sphericity factor (SF) of the lyophilized microspheres

Sample AHPMC ACMC AMCC AS07 AS08 AD AP

(SF) 0.2030.003 0.1490.008 0.0920.009 0.1570.005 0.1540.005 0.1830.003 0.1030.004

Values are mean (n =3) standard deviation

2.2.3. Bulk, tapped density and flowability of the liophilized microspheres

Generally, entrapment efficiency is used as a quality parameter for the dried

microspheres. Nevertheless, other assessed quality control parameters as bulk density,

tapped density and the Hausner Ratio provided the powder flowability (Kennedy and

Panesar, 2006). The obtained results are presented in Table 5.

The bulk density can be defined as the mass of microspheres divided by the total

volume occupied, which includes the microspheres volume, the inter-particle void volume

and the internal pore volume. In the present study, the results indicate that the bulk density of

the samples ranges from 0.18 to 0.28 g/cm3. As it is well-known, a dry product with a high

bulk density can be stored in a smaller container than a product with a relatively lower bulk

density.

PhD Thesis Abstract

XXV

Tapped density refers to the bulk density of the microspheres after a specified

compaction process. The variation of tapped density in our study was from 1.20 to 0.32

g/cm3. In this study, it was found that the tapped density was the highest in samples AMCC,

AP, ACMC and AS07, and the tapped density was found to be higher than the bulk density.

A correlation between the viscosity of the sample and the densities was found. The sample

AMCC and AP show the highest values of viscosity, and also the highest values for the

densities. The same tendency was observed in all the samples. This behavior was also

observed in previous work (Chan et al., 2011b) where the difference of the samples viscosity

was due to various concentrations of the filler.

Table 5

Bulk density, Tapped density and flowability of the lyophilized microspheres obtained from

mixtures of alginate filler materials Table 2)

Values are mean (n =3) standard deviation

The Hausner ratio of a granular material is defined as a measure of the interparticle

friction or cohesiveness of the material (Kennedy and Panesar, 2006). The description of

microspheres degree of compaction is defined by this ratio, and it can be defined as the ratio

of the tapped density to the bulk density. The presence of high inter-particle friction is

indicated by a larger value (Abdullah and Geldart, 1999). The friction is affected by the class

of material used; the microspheres size and shape, the surface, the size distribution, the

atmospheric conditions (humidity and temperature) and the inter-particle forces (e. g.

cohesion and electrostatics).

Sample Bulk density (g/cm3) Tapped density (g/cm

3) Flow ability (kg/s)

AHPMC 0.18 0.015 0.20 0.013 1.06 0.003

ACMC 0.26 0.014 0.28 0.019 1.07 0.005

AMCC 0.27 0.016 0.32 0.021 1.16 0.008

AS07 0.25 0.019 0.26 0.017 1.09 0.002

AS08 0.20 0.014 0.21 0.016 1.13 0.001

AD 0.19 0.013 0.21 0.015 1.09 0.004

AP 0.28 0.011 0.30 0.019 1.06 0.006

PhD Thesis Abstract

XXVI

A higher Hausner ratio means that the material is more cohesive and less able to flow

freely. A Hausner ratio of less than 1.5 has been used to indicate good flowability (Thalberg

et al., 2004) since particles at this ratio show little potential for further consolidation. In this

study, all lyophilized microspheres were free flowing as indicated by Hausner ratios ranging

from 1.07 to 1.16. In spite of this quality, the highest Hausner ratio was observed for the

sample AMCC, which shows an increased inter-particle friction. The sample AHPMC

showed the lowest Hausner ratio value folowed by the sample AP. However, the Haunsner

ratio of the microspheres were similar despite the type of the filler. Chan et al. (Chan et al.,

2011b) correlate the values of the Haunsner ratio with the concentration of the polymer

rather than with the type of the filler. In our study, the similarity can be derived from the fact

that was not a significant difference in regarding the size of the obtained microspheres.

2.2.4. Survival of Bifidobacterium lactis 300B in fresh microspheres

The entrapment of cells is influenced by the encapsulation process. The density ofB.

lactis encapsulated cells in microspheres were calculated based on the ratio of viable cells

after encapsulation over the initial number of viable cells in the slurry.

According to the data presented in Fig. 7 the probiotic cells survival after the

encapsulation process decreased in all the cases. However, the microspheres matrix

composition provided a different degree of protection to the entrapped cells resulting in

different survivability values (expresed as log CFU/g). The encapsulation procedure was the

same for all seven samples. It can be observed a greater number of surviving cells, by

providing Bifidobacterium lactis 300B with a proper covering matrix. It is reported in the

literature (Rodriguez-Huezo et al., 2007) that the oxygen protection immediately after

gelation has a beneficial effect by reducing the decaying rate of cells consistently. The

specific property of pullulan to form strong, oxygen-impermeable films is consistent

reported (Leathers, 2003; Singh et al., 2008).

This statement supports our results, shown in Fig. 7 where the survival rate of the

probiotic cells under the same conditions is observed, indicating that the formulation of

sample AP, were the pullulan filler was used, offer the best protection, providing a value of

1013

CFU/g immediately after encapsulation. The microspheres where the HPMC filler was

PhD Thesis Abstract

XXVII

used, reduce, to some extent, the protective effect, but the survivability is still high compared

to the rest of the samples: 1012

CFU/g compared to 109

CFU/g the mean of the rest samples.

It is interesting to observe that this microspheres variation was also positive for the freeze

drying process and storage conditions.

Fig. 7 Viability of B. lactis 300B in the slurry after encapsulation in the seven types of

alginate based microspheres, expresed as log CFU/g.

The number of live cells found in the microspheres after the encapsulation process

influences the number of living cells that will be found in any final product. In the literature

was found results that (Capela et al., 2007; Chan et al., 2011b) correlate the survival of

entrapped cells after the encapsulation process to the physical properties of the microspheres.

Our results fell in between because the samples with HPMC, pullulan and starch showed the

best physical properties (in this order), while the best survival rate was observed in the

alginate/pullulan formulation. In this specific case, the oxygen protection of pullulan

balanced the expense of the physical properties.

2.2.5. Viability after liophilization

The application of shock freeze to the fresh microspheres caused considerably less

shrinkage (data not shown). For all the samples, the mass reduction was higher than 92%.

This value is important on the scaling up process.

slur

ry A

HPM

C

AHPM

C

slur

ry A

CM

C

ACM

C

slur

ry A

MCC

AM

CC

slur

ry A

S07

AS07

slur

ry A

S08

AS08

slur

ry A

DAD

slur

ry A

PAP

0

5

10

15

Su

rviv

al

(lo

g C

FU

/g)

PhD Thesis Abstract

XXVIII

The freezing rate controls the nucleation and growth of ice crystals that are

to initiate the freezing process (Maa and Prestrelski, 2000). Slow freezing creates

that allow the ice nuclei to grow into larger crystals. Rapid freezing affects mainly the

number of the nuclei and not their size. However, fast freezing creates smaller ice

than slow freezing (Maa et al., 1999; Maa and Prestrelski, 2000). These findings are

associated with changes of protein state, as well as of the cells phospholipid

during the freeze drying process. The deteriorative reactions are: damages created by

crystals to the cell membrane, and freezing induced unfolding of proteins. This

affects the survivability of the entrapped cells as is evident in

Fig. 8

Fig. 8 Survival of B. lactis 300B before and after freeze drying in samples AHPMC

(alginate/HPMC cellulose) and AP (alginate/pullulan).

Since the AP and AHPMC samples proved to be the best formulae for

B. lactis 300B we decided to analyse the survival rate in these freeze dried

order to prevent the rupture of the probiotic membrane by the large ice big crystals

a slow freezing process, the samples were shock frozen at -18C for 30 minutes. The

of encapsulated cells in the freeze dried microspheres were calculated based on the

viable cells after freeze drying over the initial number of viable cells in the fresh made

microspheres is shown in

0 5 10 15

Alginate/Pullulan

Alginate/Pullulan FD

Alginate/HPMC cellulose

Alginate/HPMC cellulose FD

Survival (log CFU/g)

PhD Thesis Abstract

XXIX

Fig. 8. A higher survival rate of Bifidobacterium is observed in both samples. Such

behavior can be attributed to the cell wall and membrane composition of Bifidobacterium

(Carvalho et al., 2004). A 14.16% and 17.98% loss of cell viability was registered in the

AHPMC respectively AP microspheres in the freeze drying process. For the freeze drying of

the nonencapsulated Bifidobacterium, the literature (Capela et al., 2006) reports a mean of

77.78% survival.

2.2.6. Survivability of entrapped probioticcells in freeze dried microspheres

Survivability of Bifidobacterium lactis 300B loaded in AP and AHPMC microspheres

has the tendency to decline during storage. The survival was maintained at 1010

CFU/g after

15 days of storage at room temperature and 4C for alginate/pullulan based microspheres

and for the alginate/HPMC cellulose based microspheres at 107 CFU/g after 15 days at room

temperature and 109 CFU/g after 15 days at 4C. The influence of the temperature on freeze

dried alginate/pullulan and alginate/HPMC cellulose based microspheres is shown in Fig. 9.

Fig. 9 Survivability of probiotic cells in freeze dried microspheres after 3, 6, 9 and 15 days

kept at room temperature and at 4C. Symbols: () 4C alginate/pullulan microspheres, ()

room temperature alginate/pullulan microspheres, () 4C alginate/HPMC microspheres,

() room temperature alginate/HPMC microspheres

A temperature close above 0C generally leads to higher survival compared to more

elevated storage temperatures (Heidebach et al., 2009; Picot and Lacroix, 2004; Weinbreck

et al., 2010), because lower temperatures result in reduced rates of detrimental chemical

reactions, such as fatty acid oxidation (Tanghe et al., 2003). In addition, the freeze dried

0 2 4 6 8 10 12 14 16

8

10

12

14

Time (days)

Su

rviv

al

(lo

g C

FU

/g)

PhD Thesis Abstract

XXX

microspheres kept at refrigeration temperature demonstrated better protection for the

entrapped anaerobic bacteria compared to the microspheres stored at room temperature.

Holayoni et al. (Homayouni et al., 2008) affirms that the survival of bacteria against

unfriendly conditions is species dependent also. Our studys results show that B. lactis 300B

after 15 days at room temperature and at refrigeration, is maintained to the level of the

therapeutic minimum (>107 CFU/g) or higher. The HPMC cellulose filling was found to

increase the survivability of B. lactis 300B during storage at room temperature (Klayraung et

al., 2009).

2.3. CONCLUSIONS

Seven different types of natural polymers namely hydroxypropyl methylcellulose

(HPMC), sodium-carboxymethyl cellulose (Na-CMC), microcrystalline cellulose (MCC),

starch BR-07, starch BR-08, dextrin and pullulan were used in order to develop the optimal

formula for the entrapment of Bifidobacterium lactis 300B in Ca-alginate based

microspheres. Laminar flow drip casting with Brace-Encapsulator was used in order to

prepare the microspheres. The results showed that alginate/pullulan and alginate/HPMC

formulation provide high protection for the bacterial strain used for encapsulation. These two

formulations were further used to obtain freeze dried microspheres, for which the viability in

time and at different temperatures was tested. The final results showed a higher viability than

the level of the therapeutic minimum (>107 CFU/g) after 15 days of storage. Other

parameters like entrapment efficiency, production rate, sphericity, flowability were also

discussed.

PhD Thesis Abstract

XXXI

CHAPTER 3

BEHAVIOR OF BIFIDOBACTERIUM LACTIS 300B AFTER

ENCAPSULATION IN DIFFERENT COATED ALGINATE/PULLULAN

MICROSPHERES

INTRODUCTION

The aim of this study was to develop a novel protection system for

Bifidobacterium lactic 300B based on encapsulation in alginate/pullulan matrix by cross-

linking gelation and coating with three different biopolymers. Furthermore, the physico-

chemical properties of the resulting fresh and freeze-dry microspheres, as well as their ability

to protect probiotic cells during exposure to different temperatures and periods of time, were

evaluated.

Objectives

Preparation of an encapsulated formula for Bifidobacterium lactis 300B using

alginate/pullulan (AP) mixture as matrix.

Application of three types of biopolymers coating on the AP microspheres in order to

increase the protection for the entrapped B. lactis 300B.

The use of freeze drying method to obtain minimum therapeutic level, time and

temperature stability of the uncoated and coated microspheres.

The methods used to monitor the results are:

Optical visualization of the efficiency of coating application.

Comparative survivability of the probiotic cells after encapsulation and coating.

Comparative probiotic cell release and viability in simulated intestinal media from the

coated vs. uncoated microspheres.

PhD Thesis Abstract

XXXII

3.1. MATERIALS AND METHODS

3.1.1. Materials

A commercially available Manguel GMB sodium-alginate was supplied by FMC,

Norway. Chitosan and gelatin, calcium chloride, pullulan, E-poly-L-lysine, glutaraldehyde

and sodium phosphate were purchased from Merck (Germany). Bifidobacterium selective

medium (BSM) agar and peptone from Sigma-Aldrich Chemie GmbH (Germany) were also

used. All materials and solutions were sterilized at 121 C for 15 min.

3.1.2. Probiotioc strain

The strain used for the trial was Bifidobacterium lactis 300B, lyophilized probiotics

powder purchased from Howaru. The probiotic was used as lyophilized powder, as received

from the supplier. A viability test of the powder was performed before each trial.

3.1.3. Preparation of alginate/pullulan microspheres

The microspheres were prepared aseptically using a Spherisator M, type 2002SP-

AE5-D0 at Brace GmbH Germany with a nozzle size of 300 m, and crosslinked in calcium

chloride (40 g/L). Before encapsulation the viscozity of the mixture was measured using a

Haake Viscotester VT-2. The standard condition used for encapsulation were: 15 g/L

alginate, 75 g/L B. lactis 300B powder, and 15 g/L pullulan. Microspheres were hardened for

30 min in calcium chloride solution.

The entrapment efficiency was determined according to (Sandoval-Castilla et al.,

2010) with small changes as follows:

Entrapment efficiency = (axF/b)100

Where a is CFU/g in the microspheres, b is CFU/g in the mixture before

encapsulation and F is the sphere packing factor (Aste and Weaire, 2008), which was

considered the dense packing for all calculations 0.70.

3.1.4 Microspheres size

The theoretical and the corrected diameter of the AP B. lactis 300B microspheres was

determinated using (Chan et al., 2011b) method. The bead shape was quantified using the

sphericity factor (SF), which is given by the following equation:

PhD Thesis Abstract

XXXIII

Sphericity factor (SF) =dmax dmin/dmax + dmin, where dmax is the largest diameter and

dmin is the smallest diameter perpendicular to dmax.

3.1.5 Coating of AP microspheres

Three types of dip coatings were applied to the wet AP based microspheres. After the

microspheres preparation, three types of coationg were applied (Error! Reference source

not found.29).

1) The first coating with 0.8 g/L E-poly-L-lysine mixed with 1 g/L alginate solutions

were used (coating 1). Fresh rinsed microspheres were immersed in E-poly-L-lysine solution

under continuous stirring for 30 minutes, after that the microspheres were separated from the

solution and wash with sterile NaCl. In the next step, alginate coating was applied by stirring

the microspheres for another 30 minute in the 1 g/L alginate solution. Finally, the coated

microspheres, were washed as described above.

2) Chitosan coating (coating 2): fresh rinsed AP microspheres were immersed under

continuous stirring in 1g/L chitosan solution for 30 minutes, and washed with sterile NaCl.

3) Gelatin chemical cross linked (coating 3): the fresh alginate/pullulan microspheres

were stirred for 60 minutes at 32C in 100 g/L gelatin solution in a 1:1.5 ratio (w/w). Then,

microspheres were aseptically separated from the gelatin solution and mixed 2 minutes in

glutaraldehyde solution (5 g/L), ending with a sterile NaCl (0,85 g/L) washing solution.

3.1.6. Freeze drying process

The fresh coated microspheres were shock frozen at -18C in isopropanol before

liophilization. The microspheres were freeze dried at -50C and 5x10-2

mbar for 24h using a

VaCo 5 freeze dryer from Zirbus (Germany). The freeze dried material was collected in

sterile recipients and analyzed immediately the process was complete.

3.1.7. Cell viability

The enumeration of viable B. lactis 300B cells was conducted before/after AP

encapsulation, AP microspheres coating and freeze drying. The entrapped cells were

released from the microspheres using phosphate buffer with 7.40.2 pH. After that 10-fold

dilutions were made in peptone water (casein peptone 1g/L, sodium chloride 5g/L and

Tween 80 ml/L). Aliquots 1 ml from the last three dilutions was used in the plate counting

PhD Thesis Abstract

XXXIV

method on BSM agar for the colony forming units (CFU) determination. After 72h of

incubation in anaerobic jars at 37C the number of CFU was counted and converted to log10

CFU. All determinations were done in triplicate.

3.5.8. Evaluation of B. lactis 300B release in simulated intestinal media

The intestinal media was mimed by the phosphate buffer used for the release of the

entrapped cells in order to determinate the cell viability inside the microspheres. The first

trial was with a 40.2 pH, but the release did not happen. To mime the lower gastrointestinal

tract the pH of the buffer was modified using steril sodium hydroxide to 7.40.2..

3.1.9. Statistical analyses

The statistical evaluation was carried out using Graph Prism Version5.0 (Graph Pad

Software Inc., San Diego, CA, USA). All the calculations were performed using Microsoft

Excel.

3.2. RESULTS AND DISCUSSIONS

3.2.1. Characterization of microencapsulation process

The bioencapsulation technique used to obtain alginate/pullulan microspheres were

described before (Brandau, 2002).

The production was made at laboratory scale recording an average production of

11.56 g/min AP microspheres, the characteristics of the obtained microspheres were

infuenced by the viscosity of the mixture and technical parameters used in the process, like

pressure and frequency (Chan et al., 2009).

3.2.2. Microspheres shape and size. Entrapment efficiency

The obtained microspheres were characterized in terms of size and shape using an

optical microscope.

Table 6 shows size results for the four types of microspheres and encapsulation yields

of the alginate/pullulan microspheres. The mean diameters of the coated microspheres were

significantly (p

PhD Thesis Abstract

XXXV

Table 6

Size and encapsulation yield for the obtained uncoated and coated microspheres

Capsule type Capsule size (m)

(n=10)

Encapsulation yield (%) (n=10)

Alginate/Pullulan 1110.512.7a 66.870.28

After Coating 1 (polylisine/alginet) 1269.514.4bd

nd

After Coating 2 (chitosan) 12459.2c nd

After Coating 3 (gelatin) 12755.9d nd

Note: Means with different letters in a column are significantly different (p

PhD Thesis Abstract

XXXVI

in the coated microspheres is shown in Fig. 11. Freeze-dried Bifidobacterium-loaded will

contain both intact, viable and damaged cells. Under suitable conditions the injured cells

may repair and become viable, i.e. capable of colony formation on suitable media (Cui et al.,

2000). The release from the gelatin-coated alginate/pullulan microspheres was not possible.

One can observe that encapsulation and respectively coating process did not significantly

influence the cell viability. The alginate coating proved the highest viability of 115

CFU/g,

similar to non-coated AP microspheres.

powde

r

slur

ry

AP

mic

rosp

here

s

coat

ing

1

coat

ing

2

coat

ing

3

0

5

10

15

20

Via

ble

cells (

log

CF

U/g

)

Fig. 11 Comparison of the viability of probiotic cells after encapsulation and coating process

(microspheres alginate/pullulan; coating 1 alginate-coating alginate/pullulan

microspheres; coating 2- chitosan-coating alginate/pullulan microspheres; coating 3- gelatin-

coating alginate/pullulan microspheres).

3.2.4. B. lactis 300 survivability during storage in freeze dried microspheres

Stability of B. lactis 300B loaded in alginate/pullulan based microspheres,

respectively in the coated microspheres at room temperature and 4C is shown in Fig. 12.

PhD Thesis Abstract

XXXVII

Fig. 12 Stability in freeze dried microspheres during 3, 6, 9 and 15 days at a) room

temperature and b) 4C () alginate/pullulan microspheres, () alginate-coated

alginate/pullulan microspheres, () chitosan-coated alginate/pullulan microspheres.

The survival was maintained at about 109 CFU/g after approximately four-week

storage even at the room temperature. The storage at refrigeration demonstrated higher

protection than the storage at room temperature, a result confirmed by literature (Cui et al.,

2000; Saarela et al., 2011).

3.2.5. The release of encapsulated B. lactis 300B in simulated intestinal media

The alginate/pullulan microspheres and the alginate-coating alginate microspheres

and chitosan-coating alginate microspheres have shown complete degradation after 20

minutes of intense shaking in the phosphate buffer (pH 7,4). This degradation is due to

phosphate ions which chelate calcium ions, the alginate and chitosan coating being dissolved

and releases the entrapped cells. The microspheres coated with gelatin coating 3, showed

no release either in the acid or basic pH, even after 24 h.

3.3. CONCLUSIONS

The encapsulation of B. lactis 300B can be done in alginate/pullulan matrix, with

excellent results, the oxygen protection being ensured by pullulan.

Room temperature 4 C

0 3 6 9 12 158

9

10

11

12

a)Time (days)

Su

rviv

al

(lo

g C

FU

/g)

0 3 6 9 12 158

9

10

11

12

b)

Time (days)

Su

rviv

al

(lo

g C

FU

/g)

PhD Thesis Abstract

XXXVIII

Alginate, chitosan and gelatin are suitable for coating application on

alginate/pullulan based microspheres.

Our study has indicated that the survival of alginate/pullulan immobilized cells is

higher than the therapeutically minimum (107 CFU/g) before and after freeze

drying.

The freeze drying method proved to be suitable in order to obtain minimum

therapeutic level, time and temperature stability in the uncoated and coated

microspheres.

The coated microspheres proved a higher protection for the entrapped probiotic

cells but not statistically significant higher compared with alginate/pullulan

uncoated microspheres, over 15 days at room temperature and/or at 4C.

PhD Thesis Abstract

XXXIX

CHAPTER 4

STABILITY COMPARATON OF FREE AND ENCAPSULATED

LACTOBACILUS CASEI IN YOGHURT FOR LONG TIME STORAGE

INTRODUCTION

The aim of this study was to investigate the effect of encapsulation on the survival

of L. casei in yoghurt during long time storage, free or encapsulated in alginate and alginate

pectin microspheres, and influence over yoghurt acidification.

Objectives

Encapsulation of L. casei in alginate matrix and lyophilization of microspheres.

Incorporaton the freeze dried microspheres in the yoghurt obtained in the

laboratory.

Testing the probiotic cells viability over a 35 days period.

Determination of dynamics of yoghurt acidification, related to L. casei viability.

To achieve the proposed objectives, the following methods were used:

Cross linking gelation for the entrapment of L. casei in alginate and alginate pectin

matrix

The viability of the entrapped probiotic cells in alginate and alginate pectin matrix

and stored in yoghourt at day 5, 10, 25, 20, 25, 30 and 35.

4.1. MATERIALS AND METHODS

4.1.1. Microbial cultures, media and growth conditions

In the trial was used the strain Lactobacillus casei, purchased from Bioaqua,

Romania. The lyophilized probiotic cells were planted in 5 ml MRS broth purchased from

Merck, Germany. The process was followed by 24 h incubation at 37C, and then cultivated

in the same conditions in 95 ml broth. The probiotic cells suspension was separated from the

broth by centrifugation at 3000 rpm for 5 minutes and 25C. The obtained pellet was rinsed

twice with sterile peptone water and suspended in samples of 30 ml broth, with 1010

CFU/ml

PhD Thesis Abstract

XL

density. All glassware, and solutions utilized in the protocols were sterilized at 121C for 15

min.

4.1.2. Microencapsulation of L. casei

For the encapsulation of L. casei two situations were addressed: encapsulation in

alginate matrix and in a mixture of alginate pectin. The conditions used in the experimental

work for the probiotic cells encapsulation were: a) 1.5% alginate; b) 1.5% alginate + 1,5%

pectin. In the obtained mixture was added with the probiotic cells having 1010

CFU/ml

density. After a proper mixing, Multinozzle Biotech Encapsulator (EncapBioSistems Inc.)

was used in order to obtain the microspheres. In the process a 300 m nozzle was used. The

microspheres were crosslinked in calcium chloride (Sigma Aldrich, Germany), (40 g/L), the

hardening bath, for 30 min, and then rinsed with sterile sodium chloride (8.5 g/L).

4.1.3. Examination of alginate and alginate-pectin microspheres

The dimensions, area, perimeter and diameter, of the obtained microspheres were

determined using an Axio Observer Zeiss microscope.

4.1.4. Freeze drying of L. casei microspheres

Based on the previous research, before incorporation of L. casei microspheres, in the

yoghurt, the microspheres were freeze dried. The conditions used for the microspheres freeze

drying were: -50C and 0.05 mbar for 24h. For the process was used a CHRIST freeze drier.

The freeze dried material was collected in sterile recipients. After the freeze drying, the

freeze dried microspheres were mixed with the prepared yoghourt.

4.1.5. Preparation of yoghurt including microspheres

A single trial of yoghurt was prepared in order to test the incorporation of

encapsulated probiotic cells. Milk whit 3,5% fat was inoculated with yoghurt starter culture

and well homogenized. The obtained mixture was incubated at 37C for 6 hours, followed

by the incorporation of the alginate and alginate/pectin microspheres. After homogenization,

the mixture was incubated at 37C for another 18 hours. Process flow diagram is presented

in Fig. 13.

PhD Thesis Abstract

XLI

Fig. 13 Flow diagram to prepare yoghurt with microencapsulated L. casei.

4.1.6. Dynamics of yoghurt acidification with L. casei, free and encapsulated

Dynamics of acidification to obtain yoghourt was comparatively studied with L. casei

as free or encapsulated. The rate of acidification was established by monitoring the pH

evolution in the three samples of milk to obtained yoghourt over a period of 45 h, incubated

at 37C.

4.1.7. Enumeration of probiotic cells

The microspheres were separated from the yoghourt by washing the yoghourt of the

microspheres with saline water (0,85%) on a sterile sieve. The entrapped probiotic cells were

released from the capsules using phosphate buffer. The enumeration of viable probiotic cells

was conducted on each sample in triplicate from day 0 to day 35 from 5 to 5 days.

milk whit 3,5% fat

+

yoghurt culture

1,5% alginate solution

or

1,5% A + 1,5 % pectine

Mixing for 3 min

Incubation at 37C for 6

hours

Incorporate microspheres

11,11%

Homogenization using

magnetic stirrer

Incubation at 37C for 18

hours

Addition L. casei cells

having 1010

CFU/ml

density

30 minutes in the

hardening bath

Rinsed with sterile

sodium chloride (8.5 g/L)

Freeze dried at -50C and

0.05 mbar for 24h

Storage at 4C for further

viability studies

PhD Thesis Abstract

XLII

4.1.8. Statistical analyses

The statistical evaluation was carried out using Graph Prism Version4.0 (Graph Pad

Software Inc., San Diego, CA, USA). For the size determination all the calculations were

performed using Microsoft Excel 2010.

4.2. RESULTS AND DISCUSSIONS

4.2.1. Physical examination of alginate and alginate/pectin microspheres

The size and the shape of the obtained microspheres were determined by optical

microscopy. The resulted product of the encapsulation process used in this study was

microspheres with a size range from 1.3 to 1.7 mm. The shape of the microspheres was

generally spherical. The area of the microspheres ranges from 4 to 4.3 mm2 and the

perimeter from 8 to 8.3 mm. Pectin grains were lay out in the microsphere mass,while the

probiotic cells were distributed randomly in the alginate matrix.

4.2.2. Yoghourt with microspheres

The approach adopted for the incorporation of the encapsulated probiotic cells in the

yoghourt in this study was: the microspheres were added after 6 hours of incubation at 37C

with yoghourt started cultures. In the literature (Sultana et al., 2000) is reported that free

probiotic cells like Lactobacilus strains have a low tolerance to the environmental factors

when are grown in mixture with other yoghourt starter cultures.

4.2.3. Dynamics of yoghourt acidification

Acidification dynamics was followed over a period of 48 h, after inoculation with

encapsulated and non-encapsulated L. casei in the milk used for yoghourt preparation. The

incipient inoculum for the cultures was around 1010

CFU/ml. The acidification ratio for the

encapsulated probiotic cells was slower than that notice for the free cells incubated under

comparable conditions (Fig. 14). The necessary time for the encapsulated probiotic cells to

reach at the same end point of acidity level is longer than that achieve by the free probiotic

cells. For example, the non-encapsulated probiotic cells reached to pH of 5.2 after 6 h

meanwhile in the encapsulated sample this pH was reached in more than 25 h. Resembling

model was also noticed by (Sultana et al., 2000). They used alginate starch matrix for the

PhD Thesis Abstract

XLIII

encapsulation and reached to the conclusion that the encapsulated cells took 20% longer

compared with free cells to reduce the pH of milk to 5. This fact leads us to the conclusion

that the assimilation and the release of metabolites across the encapsulated alginate pectin

matrix are slower. No statistical significant difference was notice between alginate and

alginate pectin encapsulation model.

Fig. 14 Dynamics of yoghourt acidification with encapsulated and free probiotic cells of L.

casei..

4.2.4. Viability of the entrapped L. casei in the yoghourt over 35 days

Monitoring the viability of the probiotic cells was made over a period of 35 days, the

tests being performed at each 5 days, during storage at 4C. The study of survival of viable

probiotic cells in the alginate and alginate-pectin microspheres demonstrated a pattern. There

was a decrease of about 1 log as compared to the original number of probiotic cells present

in the firs day, over 35 days period, in both encapsulated forms.

In the Fig. 15 it is represented the survival rate (log CFU/g) of L. casei in the alginate

microspheres, stored in yoghourt over 35 days.

0 10 20 30 40

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

free cells

algiante encapsulated cells

alginate/pectin encap cells

Time (hours)

pH

PhD Thesis Abstract

XLIV

Fig. 15 Survival (log CFU/g) of encapsulated L.

casei in the alginate matrix, in yoghourt over 35

days

Fig. 16. Survival (log CFU/g) of

encapsulated L. casei in alginate/pectin

matrix, in yoghourt over 35 days.

The Fig. 16 shows the decrease of viability in the alginate-pectin microspheres. It can

be observed that the decrease of probiotic cells viability was not more than 2 log in the first

15 days in the alginate pectin microspheres. However, during the other 20 days, the decrease

is more significant, L. casei cells being found in a number of only 104 CFU/g in samples of

yoghourt.

4.3. CONCLUSIONS

The summarized chapter conclusions are:

The encapsulation of L.casei proved to give best results when alginate/pectin

mixture was used, instead of alginate.

The size of the obtained microspheres sizes range from 1.3 to 1.7 mm.

The acidity of yoghourt with added L. casei proved to be higher for the free cells,

meanwhile the pH of the yoghourt with encapsulated probiotic cells did not show

values under 5 even after 30 days of storage.

L. casei survivability was nearly under to 104 CFU/g in samples were alginate

matrix was used for the encapsulation, after 20 days of storage at 4C1C, men

PhD Thesis Abstract

XLV

while the samples where alginate/pectin was used, after the same period of time

the viability was almost 105 CFU/g.

Regarding the viability of entrapped L. casei in freeze dried microspheres, proved

to be higher than the minimum therapeutic level after 15 days at 4C in yoghourt.

The accessibility of encapsulated probiotic cells, for the consumer, can be

facilitated by adding the microspheres in usual dairy products as yoghourt.

PhD Thesis Abstract

XLVI

GENERAL CONCLUSIONS

Considering the main objectives of the research and the results obtained by different

vertical experimental investigations, there are pointed out the main 5 achievements:

The successful microencapsulation of probiotic Lactobacilus plantarum in

alginate-chitosan-coated microspheres, using lucerne green juice as prebiotic

The evaluation of the behavior of these microcapsules in simulated

gastrointestinal conditions ( gastric followed by intestinal simulated media)

Influence of five different encapsulation matrices on physical properties of

microspheres and survivability of probiotic cells encapsulated in these

microspeeres

Evaluation of stability dynamics of free and encapsulated Lactobacillus casei

inside yoghurt for long time storage ( 45 days)

Outlook:

As future plans, it may be useful to find new value-added, polymer combinations,

which are suitable for the bioencapsulation of specific probiotic cells with health promoting

properties and improved proprieties (i.e. encapsulation yield, entrapment efficiency, good

micrometric proprieties of the microspheres, targetted and controlled release).

The application of innvative encapsulation techniques to obtain microspheres with

entrapped bioactive agents, whose targeted and controlled release is aimed.

PhD Thesis Abstract

XLVII

REFERENCES

1. Abdullah, E.C., & Geldart, D. (1999). The use of bulk density measurements as

flowability indicators. Powder Technology, 102(2), 151-165.

2. Albertini, B., Vitali, B., Passerini, N., Cruciani, F., Di Sabatino, M., Rodriguez, L., &

Brigidi, P. (2010). Development of microparticulate systems for intestinal delivery of Lactobacillus

acidophilus and Bifidobacterium lactis. European Journal of Pharmaceutical Sciences, 40(4), 359-

366.

3. Anal, A.K., Bhopatkar, D., Tokura, S., Tamura, H., & Stevens, W.F. (2003).

Chitosan-alginate multilayer beads for gastric passage and controlled intestinal release of protein.

Drug Development and Industrial Pharmacy, 29(6), 713-724.

4. Anal, A.K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics

for industrial applications and targeted delivery. Trends in Food Science & Technology, 18(5), 240-

251.

5. Anal, A.K., & Stevens, W.F. (2005). Chitosan-alginate multilayer beads for

controlled release of ampicillin. International Journal of Pharmaceutics, 290(1-2), 45-54.

6. Annan, N.T., Borza, A.D., & Hansen, L.T. (2008). Encapsulation in alginate-coated

gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during

exposure to simulated gastro-intestinal conditions. Food Research International, 41(2), 184-193.

7. Aste, T., & Weaire, D., (2008). The Pursuit of Perfect Packing, Second Edition (2nd

ed). Taylor and Francis group, Northwestern.

8. Augustin, M.A., & Sanguansri, L., (2003). Encapsulation of food ingredients.

9. Augustin, M.A., Sanguansri, L., Margetts, C., & Young, B. (2001).

Microencapsulation of food ingredients In Proceedings of the Conference Name|, Conference

Location|.

10. Brachkova, M.I., Duarte, M.A., & Pinto, J.F. (2010). Preservation of viability and

antibacterial activity of Lactobacillus spp. in calcium alginate beads. European Journal of

Pharmaceutical Sciences, 41(5), 589-596.

11. Brandau, T. (2002). Preparation of monodisperse controlled release microcapsules.

International Journal of Pharmaceutics, 242, 179184.

PhD Thesis Abstract

XLVIII

12. Brinques, G.B., & Ayub, M.A.Z. (2011). Effect of microencapsulation on survival of

Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration, and yogurt. Journal

of Food Engineering, 103(2), 123-128.

13. Burgain, J., Gaiani, C., Linder, M., & Scher, J. (2011). Encapsulation of probiotic

living cells: From laboratory scale to industrial applications. Journal of Food Engineering, 104(4),

467-483.

14. Capela, P., Hay, T.K.C., & Shah, N.P. (2006). Effect of cryoprotectants, prebiotics

and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt.

Food Research International, 39, 203211.

15. Capela, P., Hay, T.K.C., & Shah, N.P. (2007). Effect of homogenisation on bead size

and survival of encapsulated probiotic bacteria. Food Research International, 40(10), 1261-1269.

16. Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., & Gibbs, P. (2004).

Relevant factors for the preparation of freezedried lactic acid bacteria. International Dairy Journal,

14, 835847.

17. Chan, E.-S., Lee, B.-B., Ravindra, P., & Poncelet, D. (2009). Prediction models for

shape and size of ca-alginate macrobeads produced through extrusion-dripping method. Journal of

Colloid and Interface Science, 338(1), 63-72.

18. Chan, E.-S., Lim, T.-K., Voo, W.-P., Pogaku, R., Tey, B.T., & Zhang, Z. (2011a).

Effect of formulation of alginate beads on their mechanical behavior and stiffness. Particuology,

9(3), 228-234.

19. Chan, E.-S., Wong, S.-L., Lee, P.-P., Lee, J.-S., Ti, T.B., Zhang, Z., Poncelet, D.,

Ravindra, P., Phan, S.-H., & Yim, Z.-H. (2011b). Effects of starch filler on the physical properties of

lyophilized calcium-alginate beads and the viability of encapsulated cells. Carbohydrate Polymers,

83(1), 225-232.

20. Chandramouli, V., Kailasapathy, K., Peiris, P., & Jones, M. (2004). An improved

method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric

conditions. Journal of Microbiological Methods, 56(1), 27-35.

21. Chang, T.M.S. (1971). Stablisation of enzymes by microencapsulation with a

concentrated protein solution or by microencapsulation followed by cross-linking with

glutaraldehyde. Biochemical and Biophysical Research Communications, 44(6), 1531-1536.

PhD Thesis Abstract

XLIX

22. Chavarri, M., Maranon, I., Ares, R., Ibanez, F.C., Marzo, F., & Villaran, M.d.C.

(2010). Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves

survival in simulated gastro-intestinal conditions. International Journal of Food Microbiology,

142(1-2), 185-189.

23. Cui, J.-H., Goh, J.-S., Kim, P.-H., Choi, S.-H., & Lee, B.-J. (2000). Survival and

stability of bifidobacteria loaded in alginate poly-l-lysine microparticles. International Journal of

Pharmaceutics, 210(1-2), 51-59.

24. Donati, I., Holtan, S., Morch, Y.A., Borgogna, M., Dentini, M., & Skjak-Braek, G.

(2005). New hypothesis on the role of alternating sequences in calcium-alginate gels. Biomolecules,

6(2), 1031-1040.