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CHAPTER 3 :Investigation of the efficiency of encapsulation of probiotics in an interpolymer complex in supercritical carbon dioxide using scanning electron microscopy
ABSTRACT
Probiotics, beneficial microorganisms, must be available in certain numbers for them to produce their beneficial effects. The problem of low viability and stability of probiotics is well known worldwide. Microencapsulation, a technique for coating or protecting sensitive actives from detrimental environmental factors, has been used by various researchers in an attempt to solve this problem. However the methods used for microencapsulation still generally involve exposure of probiotics to water or other solvents, heat, oxygen, etc. during the encapsulation process, which compromises the stability of probiotic cultures. A novel method of encapsulation using formation of an interpolymer complex in supercritical carbon dioxide was developed. This study reports on the use of scanning electron microscopy (SEM) to investigate the efficiency of the newly developed encapsulation method in terms of the effect of the encapsulation process on the cell’s morphology. The effect of the encapsulation process on stability of the bacterial cells was also investigated. SEM images indicated liquefaction of both polymers (poly (vinyl pyrrolidone) (PVP) and poly (vinyl acetate-co-crotonic acid)) (VA-CA) in scCO2. Complete encapsulation of Bifidobacterium lactis cells was achieved, indicated by absence of bacterial surfaces on the encapsulated particles. Encapsulation of B. lactis within the interpolymer complex produced smooth textured particles that were less porous when compared to non-encapsulated freeze-dried bacteria powder. Pores may allow contact between cells and unfavourable environmental factors. No visual morphological changes to B. lactis cells were observed due to the encapsulation process. Survival of non-encapsulated cells and cells that were exposed to the encapsulation process was similar. Thus, the encapsulation process did not negatively affect stability and viability of bacterial cells. The successful encapsulation of the bacterial cells within the interpolymer complex, the absence of changes to cell morphology and the use of FDA-approved polymers give the technology potential for application in the food and pharmaceutical industries.
Keywords: interpolymer complex; supercritical carbon dioxide; encapsulation; probiotics, Bifidobacterium lactis, poly (vinylpyrrolidone), poly (vinyl acetate co-crotonic acid)
INTRODUCTION
The World Health Organization and Food and Agriculture Organization of the United Nations (FAO/WHO, 2001) define probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Leahy et al., 2005). High numbers of viable probiotic cultures are necessary for production of beneficial health effects (Salminen et al., 1996; Holzapfel et al., 1998; MacFarlane and Cummings, 1999; Miguel, 2001; Teitelbaum and Walker, 2002; Talwalkar et al., 2004). Maintanance of viability of the cultures during processing and storage presents a serious technological and marketing challenge for incorporation of these cultures in functional foods by industries.
It is difficult and sometimes even impossible for manufacturers to back up claims on their product labels due to unstable shelf lives of probiotic cultures (Siuta-Cruse and Goulet, 2001). Several market surveys reported a decline in the counts of Lactobacillus acidophilus and Bifidobacterium spp. during the shelf life of commercial products containing probiotics, with cell numbers significantly lower than the recommended levels at the end of shelf life (Micanel et al., 1997; Vinderola et al., 2000; Elliot and Teversham, 2004; Huff, 2004). The survey done in this project on fermented probiotic products available on South African retail store shelves indicated that there was a problem of survival of probiotics, especially bifidobacteria, in products.
Over the years, microencapsulation, the process whereby the core material is captured in a shell or coating for controlled release, has been used. Through microencapsulation, cells can survive processes such as freezing and freeze-drying better, as well as be protected from attack by bacteriophages (Krasaekoopt et al., 2003). Protection of probiotics by encapsulation in hydrocolloid beads has been investigated for improving their ability in food products and the intestinal tract. Researchers favour the use of extrusion and emulsion for encapsulating microbial cells (Krasaekoopt et al., 2003). The disadvantage of using emulsions is that production of large quantities of beads and washing them free of oil is difficult (Stormo and Crawford, 1992). It is also difficult to produce gel beads at a large scale due to a number of reasons as discussed in Section 1.7.2.2 (Krasaekoopt et al., 2003; Picot and Lacroix, 2004).
Several techniques such as spray drying and fluidized bed drying are used for encapsulating the cultures and converting them into a concentrated form. One of the disadvantages of these techniques is that the bacteria are completely released in the product. Thus the cells are not protected from the product environment and during passage through the stomach or intestinal tract (Krasaekoopt et al., 2003). Toxic organic solvents accumulate in microbial cells and kill them through destruction of the functional properties needed for their survival (Sardessai and Bhosle, 2002; Matsumoto et al., 2004).
Organic solvents are not only toxic to cells, they are expensive as well. Negative effects of environmental factors such as moisture, temperature and oxygen on probiotic cultures must be minimized. Medical and food industries require ultra pure products. All these reasons indicate that new peocessing techniques that can fulfil all these requirements must be developed (Vasishtha, 2003). An encapsulation technology that would overcome the problems posed by current technologies, enabling protection and preservation of sensitive substances, improvement of their viability, effectiveness and shelf lives should therefore be developed.
The challenges in developing commercially viable encapsulated products depend on:
- Selection of appropriate shell formulation from FDA-approved GRAS (generally recognized as safe) materials
- Selection of the most appropriate process to provide the desired morphology
- Stability and release mechanism
- Economic feasibility of large scale production, including capital, operating and other miscellaneous expenses, such as the transportation cost, regulatory cost and downtime losses.
Supercritical fluids (SCFs) are fluids heated to temperatures and pressures above their critical temperature and pressure. They are able to solubilize compounds and can penetrate low porosity materials (Demirbas, 2001). SCFs have gas-like diffusivity and liquid-like densities (Reverchon and Porta, 2001). Though they were originally used for extraction, experience accumulated in recent years on their use and processes indicated the possibility to explore and envision their use beyond the common practice of extraction (Reverchon and Porta, 2001; Sarrade et al., 2003). Supercritical fluid technologies can also be applied in making new innovative products (Sihvonen et al., 1999; Reverchon and Porta, 2001). Encapsulation of drugs for release at specific sites in the human body is one of the new areas for application of supercritical fluid technology (Sihvonen et al., 1999).
Supercritical carbon dioxide (scCO2), has received increasing attention due to its cost effectiveness and environmental friendliness (Bae et al., 2004; Novik et al., 2006). The relatively low critical parameters (Tc = 31.1 ëC and Pc = 73.8 bar1) of scCO2 lends it towards processing of pharmaceuticals and other materials that are sensitive to temperature, solvents, oxygen, water, etc. such as proteins, labile drugs and bacteria (Reverchon and Porta, 2001; Bae et al., 2004). An additional advantage of using SCFs as solvents, particularly in pharmaceutical applications is that there is no residual solvent in the final product (Corrigan and Crean, 2002). Typical applications of scCO2 in biotechnology include micronization of drugs and encapsulation of sensitive actives for controlled release of the immobilized material (Jung and Perrut, 2000; Fages et al., 2004; Ginty et al., 2005; Yeo and Kiran, 2005).
Most polymers are not sufficiently soluble in or compatible with scCO2 and can thus not be processed using it as a solvent or plasticiser. Different approaches can be used to overcome the problem of insolubility and incompatibility between polymers and scCO2, though they are typically not allowed in food and pharmaceuticals. The different approaches used and reasons for their limitation in these industries are given in Table 3.1.
Polymers with complementary sites/ molecular groups can interact with each other in solution to form physical networks by interpolymer complexation (Tsuchida, 1994; Henke et al., 2005). Interpolymer complex assemblies form through any of four fundamental attractive interactions, namely electrostatic attraction, hydrogen bonding, hydrophobic interaction and Van der Waals forces (Henke et al., 2005). It has been shown that hydrogen bonding (Tilly et al., 1994) and dipole-dipole interactions (Ekart et al., 1993) can occur in scCO2. scCO2 has largely been used in the food industry for extraction of labile food components and in pharmaceutical industries for extraction and purification of vitamins. Recently Novik et al. (2006) reported the use of scCO2 in the probiotics field for extraction of glycolipids from Bifidobacterium adolescentis 94 BIM. However, the formation of interpolymer complexes in scCO2 and its application in encapsulation of probiotic bacterial cells was to our knowledge, reported for the first time by Moolman et al. (2005).
The main objectives of this study were therefore to investigate the efficacy of this novel encapsulation technique based on interpolymer complexation in scCO2 using SEM, the effect of encapsulation on cell morphology and to determine the effect of the encapsulation process on stability of bacteria using conventional plating techniques.
MATERIALS AND METHODS
Bacterial cultures
Bifidobacterium lactis Bb-12 and Bifidobacterium longum Bb-46 were obtained as DVS sachets from CHR- Hansen.
Encapsulation of bacteria
Bifidobacterium cells were encapsulated using a Particles from Gas-Saturated Solution (PGSS) reactor (Fig. 3.1). All equipment was wiped with 70 % ethanol using a paper towel, and allowed to dry before contact with the materials. 2 g of PVP (Kollidon 12PF, mass-average molar mass 2 000 – 3 000 g/mol, BASF) was dried for 5 h at 80 ëC and 60 mbar (absolute) in a vacuum oven (Model VO65, Vismara) and immediately placed in a dessicator to prevent moisture absorption. A sealed packet of either B. longum Bb-46 (Chr. Hansen) or B. lactis Bb-12 (Chr. Hansen) was removed from storage at -12 ëC and allowed to warm to room temperature while sealed. 2 g of the bacteria was then ground to a powder passing through a 150 µm sieve using a coffee grinder (Model CG100, Kenwood). 6 g of VA-CA (Vinnapas C305, mass-average molar mass 45 000 g/mol, Wacker) was then added to the bacteria (together with any additives (e.g. glyceryl monostearate – Croda Chemicals, in reactions were additives were included) and the dried PVP. The blend was then ground and mixed for 1 min. The powder blend was then immediately transferred to the pre-heated 1 reaction chamber. The chamber was then sealed and flushed and pressurized with sterile filtered CO2 (99.995% purity, Air Products) up to a pressure of 300 bar, with the temperature controlled at 40 ëC. The material was left to equilibrate for 2 h with intermittent stirring, after which the liquefied product was sprayed through a 500 µm capillary with length 50 mm, into a 10 expansion chamber that was pressure-controlled at 15 bar (gauge). Fig. 3.2 is a simplified flow diagram showing steps occurring in the encapsulation process. A clear description of the encapsulation technology is outlined in Moolman et al. (2006).
Scanning electron microscopy (SEM)
SEM was used to verify encapsulation of B. lactis cells into the polymer and release of the cells from the polymer into solution during subsequent suspension of the encapsulated material. The freeze-dried and encapsulated bifidobacteria were suspended in ¼ strength Ringer’s solution. The suspended cells were filtered out using a 0.2 m Millipore filter membrane. The cells were fixed to the 0.2 m membrane using 2.5 % gluteraldehyde for 30 min. The fixed cells were then washed 3 x 15 min in 0.15 M-phosphate buffer. Then dehydration of the sample was done in an increasing series of ethanol as follows: 50 % (1 x 15 min), 70 % (1 x 15 min), 90 % (1 x 15 min) and 100 % (3 x 15 min). The filter membrane was then dried in a critical point dryer for 24 h, mounted on stainless steel studs and then coated with gold plasma. The freeze-dried and encapsulated powder were put on a sticky tape on the studs and directly coated with gold plasma without undergoing any treatments for SEM. The samples coated with gold were then examined using JEOL 840 scanning electron microscope.
Bacterial counts
1 g of Bifidobacteria was suspended in 9 m of sterile ¼ strength Ringers solution (pH 7). A series of dilutions up to 10-10 was prepared from this suspension. 0.1 m of appropriate dilutions was pour plated onto De Man, Rogosa and Sharpe (MRS) agar (Merck, Pty.(Ltd)), supplemented with 0.05 % cysteine hydrochloride. Each dilution was plated out in triplicate. The plates were incubated anaerobically in anaerobic jars with Anaerocult A gaspaks (Merck Pty (Ltd.), at 37 oC for 72 h. Anaerobisis inside the jars was indicated by inclusion of Anaerocult C test strips (Merck, Pty (Ltd)). The numbers of colonies grown were counted and from these the numbers of viable cells were calculated (cfu/g). Reported values are averages of the three replicates.
RESULTS AND DISCUSSION
Liquefaction of polymers in scCO2
SEM was used to examine whether the developed SCF encapsulation method was efficient and whether the polymers used were liquefied during exposure to scCO2. SEM images of PVP and VA-CA before and after exposure to scCO2 are shown (Fig. 3.3). These images indicated that liquefaction of both polymers occurred during exposure to scCO2 (Fig. 3.3). Images before exposure to scCO2 showed polymers as individual granules (separate loose particles with individual particles/ granule’s three dimensional structure visible showing that the particles were separate) (Fig. 3.3 A, C) while those after exposure appeared as a monolithic foam (Fig. 3.3 B, D). No individual particles similar to those observed before exposure to scCO2 were present. The continuous appearance (compact layer) was the result of liquefaction of the polymers by the dissolution of scCO2 in the polymers. Dissolution of scCO2 in polymers is known to lower glass transition temperature (Tg) and facilitates formation of a smooth morphology (Yue et al., 2004).
Acknowedgements
Conference Contributions
List of abbreviations
List of tables
List of figures
Summary
INTRODUCTION
REFERENCES
Chapter 1: LITERATURE REVIEW
1.1 Normal intestinal microflora
1. 2 Probiotics
1. 2.1 Bifidobacteria
1. 2.1.1 Bifidobacterium bifidum
1. 2.1.2 Bifidobacterium longum
1. 2.1.3 Bifidobacterium adolescentis
1. 2.1.4 Bifidobacteriun infantis
1. 2.1.5 Bifidobacterium breve
1. 3 Prebiotics
1. 3.1 Non digestible oligosaccharides (NDO’s)
1. 3.2 Fructooligosaccharides(FOS)
1. 3.3 Galactooligosaccharides (GOS)
1. 3.4 Soy oligosaccharide.
1. 3.5 Cereals.
1.4 Synbiotics
1. 5 Application of probiotics in gastrointestinal dysfunctions associated with gut microflora imbalance
1. 5.1 Lactose indigestion.
1. 5.2 Constipation
1. 5.3 Antibiotic associated and rotaviral diarrhoea.
1. 5.4 Crohn’s disease
1.5.5 Other application of probiotics
1.5.5.1 Food allergy
1.5.5.2 Atopic dermatitis
1.5.5.3 Cholesterol and heart disease
1.5.5.4 Cancer.
1.6 Shelf life stability of probiotics
1.7 Moving towards improving shelf life of probiotics
1.7.1 Cell immobilisation
1.7.1.1 Entrapment method
1.7.1.2 Covalent attachment
1.7.1.3 Ionic attachment
1.7.2 Microencapsulation
1.7.2.1 Extrusion
1.7.2.2Emulsion
1.7.2.3 Spray drying
1.7.3 Freeze drying of probiotics
1.8 Supercritical fluids
1.9 Methods for detection of probiotics cultures
1.10 REFERENCES
Chapter 2: VIABILITY OF PROBIOTIC CULTURES FROM YOGHURT SAMPLES RANDOMLY SELECTED FROM SOUTH AFRICAN RETAIL STORES
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1 Sample collection and storage
2.3.2 Bacterial enumeration
2.4 Results and Discussion
2.5 Conclusions
2.6 References
Chapter 3: INVESTIGATION OF THE EFFICIENCY OF THE NOVEL METHOD OF ENCAPSULATION OF PROBIOTICS IN AN INTERPOLYMER COMPLEX IN SUPERCRITICAL CARBON DIOXIDE USING SCANNING ELECTRON MICROSCOPY
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Bacterial cultures
3.3.2 Encapsulation of bacteria
3.3.3 Scanning electro microscopy
3.3.4 Bacterial counts
3.4 Results and Discussion
3.4.1 Liquefaction of polymers in scCO2
3.4.2 Interpolymer complexation and bacterial encapsulation
3.4.3 Appearance of non-encapsulated and encapsulated cells upon suspension
3.4.4 Viability of Bifidobacterium longum Bb-46 cells after scCO2 processing
3.5 Conclusions
3.6 References
Chapter 4: SIMULATED GASTRIC AND INTESTINAL FLUID SURVIVAL OF BIFIDOBACTERIUM LONGUM BB-46 ENCAPSULATED IN DIFFERENT INTERPOLYMER COMPLEXES
4.1 Abstract
4.2 Introduction
4.3 Materials and Methods
4.3.1 Bacterial cultures
4.3.2 Polymer formulations
4.3.3 Encapsulation of bacteria
4.3.4 Determination of total bacteria encapsulated
4.3.5 Preparation of simulated gastric and intestinal fluids
4.3.6 Survival of bacteria in simulated gastric fluid
4.3.7 Survival of bacteria in simulated intestinal fluid
4.3.8 Enumeration of Bifidobacteria
4.4. Results and Discussion
4.4.1 Survival in PVP:VA-CA (normal system) and PVP:PEOPPO-PEO
4.4.2 Survival of bacteria in polycaprolactone (PCL)
4.4.3 Effect of GMS incorporation on survival in simulated gastrointestinal fluids
4.4.4 Effect of higher GMS loading on protective properties of GMS:PVP:VA-CA interpolymer matrix
4.4.5 Effect of gelatine capsules on survival of GMS:PVP:VACA encapsulated bacteria in simulated gastrointestinal fluids
4.4.6 Effect of beeswax on survival in simulated gastrointestinal fluids
4.4.7 Comparing protection efficiencies of the different formulations
4.4.8 The effect of encapsulated B. longum Bb-46 on the microbial community of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) model
4.5 Conclusions
4.6 References
Chapter 5: SHELF LIFE STUDIES OF BIFIDOBACTERIUM LACTIS BB-12 ENCAPSULATED IN INTERPOLYMER COMPLEXES IN SUPERCRITICAL CO2 STORED UNDER DIFFERENT STORAGE CONDITIONS
5.1 Abstract
5.2 Introduction
5.3 Materials and Methods
5.4 Results and Discussion
5.5 Conclusions
5.6 References
Chapter 6: SHELF LIFE STUDIES OF BIFIDOBACTERIUM LONGUM BB-46 ENCAPSULATED IN INTERPOLYMER COMPLEXES IN SUPERCRITICAL CO2 STORED UNDER DIFFERENT STORAGE CONDITIONS
6.1 Abstract
6.2 Introduction
6.3 Materials and Methods
6.4 Results and Discussion
6.5 Conclusions
6.6 References
Chapter 7: GENERAL CONCLUSIONS
APPENDIX
