UNIVERSITY OF AGRICULTURAL SCIENCES AND … · UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY...
Transcript of UNIVERSITY OF AGRICULTURAL SCIENCES AND … · UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY...
-
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.