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Table of content
CHAPTER 1. OVERVIEW OF PROBIOTICS 1
I.History and definition of probiotics .1
1. History of probiotics.1
2. Definition of probiotic .1
 
II.Microbes used as probioticS .3
III. Charateristics of probiotics 4
1. Properties of trains of probiotics .4
Lactobacillus.4
Bifidobacterium .5
Streptococcus .6
Enterococcus .7
Lactococcus lactis .7
2. Technologiacal properties .8
 
IV. Effects probiotics on human health .9
 
1. Disorders associated with the gastrointestinal tract .9
1.1. Prevention of diarrhea caused by certain pathogenic bacteria and viruses.10
1.2. Helicobacter pylori infection and complications .11
1.3. Inflammatory diseases and bowel syndromes .11
1.4. Cancer .12
1.5. Constipation .12
2. Mucosal immunity .12
3. Allergy .13
4. Cardiovascular disease .14
5. Hypocholesterolemic effect .14
6. Urogenital tract disorder.15
6.1. Bacterial vaginosis . 15
6.2. Yeast vaginitis .16
6.3. Urinary tract infections .16
7. Use of probiotics in otherwise healthy people . 16
8. Lactose intolerance .17
9. Reduction of the risk associated with mutagenicity and carcinogenicity .18
 
CHAPTER 2: DRYING PROBIOTICS .20
 
I. GENERAL MECHANISM .20
1. Introduction .20
2.1. Freeze drying .21
2.2. Spray Drying .21
2.3. Fluidized Bed Drying 23
2.4. Vacuum Drying 24
2.5. Foam formation .24
2.6. Mixed Drying Systems .25
 
II. CURRENT DRYING METHODS .27
1. Freeze drying 27
1.1. Mechanism and procedure: 27
Purpose 27
Mechanism .28
Procedure .28
1.2. Equipments and technological parameters .29
1.2.1. Equipments .29
1.2.2.Processing parameters .32
1.3. Factors influence freeze drying process .33
1.3.1. Depth of product in container .33
1.3.2. Vapor pressure diferential .33
1.3.3. Amount of solid in the product, their particle size and their thermal conductance .34
 
1.4. The influence of processing factors on quality of product .34
2.Spray drying .42
2.1. Mechanism and material changes .42
2.2.Equipment and technology parameters .45
2.3. Factors influencing the spray drying .54
2.3.1. Kind of equipment and papameters .54
2.3.2. Dry matter content in the feed .57
2.3.3. Air temperature .58
2.3.4.Other factors .59
2.4. Products .60
 
III. COMPARISION .60
 
IV. ACHIEVEMENTS IMPROVE SPRAY DRYING AND QUALITY OF PRODUCT
1. Cell physiology .61
1.1. Application of mild stress prior to dehydration .61
1.2. Growth phase .63
1.3. Growth media .63
1.3.1. Carbohydrates .63
1.3.2.Compatible solutes: polyols, NaCl, amino acids and
amino derivatives .64
1.3.3. pH .64
1.4. Genetic-modification of probiotic strains .65
2. Protective agents .66
2.1. Amorphous glass forming .66
2.2. Eutectic crystallizing salts .66
3. Rehydration .67
4. Storage and packaging .68
5. Microencapsulation .70
 
V. PRODUCTS CONTAINED PROBIOTICS .72
1. Forms of probiotic powder .72
 
VI. Storage the products contained probiotics .73
 
 
 
 
 
 


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eptically to grow media
Growing
Centrifuging
Centrifuging
Washing
media
Prepare
29
1.2. Equipments and technological parameters:
1.2.1. Equipments:
Figure 3.1: Structure of lyophilization equipment
Freeze drying
Suspending
Adding cryoprotectant
Storing
Filling in glass vials or ampoules Loading onto trays
Loading onto freeze dry chamber
Ampoules,
glass vials
Sealing
30
1- Freeze dry chamber 2- Valve 3- Syphon 4- Hot water tank 5- Condenser
6- Liquid seperate tank 7- Amonium condenser 9- Compresser 10- Vacuum pump
11,12,13- Electric motors 14- Water pump 15- Filter 16- Heat exchanger
17- Vacuummeter 18- Control valve 19- Tray 20- Thermal net 21- Thermal
controler
Structure of freeze dryer:
Drying chamber:
The drying chamber of freeze dryer consist of a vacuum tight unit containing
loading door and one or more inspector wimdow. Each chamber contains aset of
shelves that has hydrolic stoppering mechanism and gas bleed system. Heat is
applied directly through electrical resistantce coilsor by circulating hot water,
silicon or glycol.
Condenser:
The condenser of freeze dryer is a vacuum tight unit separate from chamber by a
vacuum
Valve. They contain a set of cooling plate refrigerated by refrigeration
system.Condenser is supplied with hot waterdefrosting system. It was a cold trap
used to collect the moisture.
Vacuum pump :
Each freeze dryer is equipped with a two stage rotary gas ballast vacuum pump.
Vacuum pump is an essential component of the freeze dryer and is required for
evacuated emvironment around the product. Vacuum pump keeps the chamber
and condenser sufficiently free from the residual gases, water vapor stream to be
unable to flow from the drying meterial.
Refridgeration system :
The freeze dryer is provided with twin independent direct expansion, single or
multi-component refrigeration plant. The commonly used refrigerant is Freon R-
502 gas.
Control facilities :
31
The freeze dryer is instrumented to control and operate th various plant
components like shelf staking mechanism. Hydraulic stoppering device and
chamber condenser valve.
Instrumentation is also available to mesure and record the various process
variables like chamber pressure, shelf temperature, product temperature , and
condenser temperature
Figure 3.2: Freeze drier
1.2.2.Processing parameters:
Growing:
Temperature : 37C
Time : until the late log phase or the early stationary phase of each strain (17
hours for L. paracasei ssp. paracasei NFBC 338 and L. rhamnosus under
anaerobic conditions)
Centrifuging :
Purpose : to havest micro-organism
32
Temperature : 4C
Time : 15 minutes
Rotary velocity :9000 x g
Washing :
Purpose : to wash out fementation broth residues
Wash 3 times with distilled water
Suspend :
Purpose : to prepare feed solution for freeze drying (the suspension of micro-
organism with initial concentration from 108cfu/ml to 1011cfu/ml)
1% inoculum in MRS broth or 15% RSM (reconstituted skimmed milk; Golden
Vale, Cork,Ireland)
Often distilled water to make up to reach 20%(w/w) solid
Votex to get a homogenous suspension
Adding cryoprotectant :
Purpose : to protect cells from damage during freezing step
Figure 3.3: Filling suspension in glass vials
33
Freeze drying :
Freezing step : at –80 C for 21 hours
Freeze dried for 48 h ,P =0.08Pa ,(40 h of primary drying at -30ºC and 8 h of
secondary drying at -10ºC).
Moisture left:less than 1%
Storage:
Temperature :-18 C under vacuum, in dark place.
1.3. Factors influence freeze drying process
1.3.1. Depth of product in container:
The greater the depth of product in the container, the longer the drying process
will be
1.3.2. Vapor pressure diferential:
The actual driving force for the process is the vapor pressure diferential between
the vapor at the surface where drying of product is occuring and that at the
surface of the ice on the condenser
1.3.3. Amount of solid in the product, their particle size and their thermal
conductance :
Amount of solid in the product, their particle size and their thermal conductance
will affect the rate of drying. The more solid present, the more impedment will
be provided to the escape of the water vapor. The smaller of the ice crystal size,
the faster the drying rate is. The poor thermal conducting properties of the solid
in the product, the slower of heat transfer rate through the frozen material to the
drying boundary. Solid content must be appoximately between 5-25%, so
mannitol or gelatin are used as bulk agent
1.4. The influence of processing factors on quality of product:
The freeze drying medium, physiological state of the cells (Broadbent and Lin,
1999), freezing rate (Peter and Reichart, 2001), freeze drying parameters,
34
rehydration conditions, and initial cell concentration (Costa et al., 2000) are the
different parameters that account for freeze drying tolerance. The percentage
survival has been reported to increase with increasing initial cell concentration
up to 1011 cfu/ml (Costa et al., 2000); a minimal concentration 107cfu/ml is
generally recommended (Pocard et al., 1994). Bacterial cells in the stationary
phase are more resistant, indicating that the age of the culture has a
positiveeffect (Brashears and Gilliland, 1995). It has also been reported that the
addition of Tween 80 increases the proportion of unsaturated fatty acids in the
membrane (Beal et al., 2001), which in turn leads to modification of the
membrane permeability favoring better survival.
Intrinsic Tolerance of Cultures.
Although variation in viability after drying among different lactic acid starter
cultures is explicit, no clear explanation is available. Some studies found that
viability is correlated to cell morphology, i.e., cell size and shape. Bozog lu et al.
reported that small spherical streptococci cells are more resistant to freeze
drying than long rod lactobacilli cells because of the lower surface area to
volume ratio. It has been reported that the higher the surface area of the cell, the
higher the membrane damage owing to extracellular ice crystal formation during
freezing (Fonseca, Beal, & Corrieu, 2000). Consequently, cell size has a strong
influence on survival of probiotics during freeze-drying, with small spherical
cells such as enterococci being more resistant to freezing and freeze-drying than
larger rod shaped lactobacilli (Fonseca et al., 2000). While a number of stress
proteins induced by heat or oxidative stresses are known to vary among species
or strains, the viability of different species or subspecies of Bifidobacterium sp.
also varies with their heat or oxygen tolerance. The majority of species with high
heat and moderate oxygen tolerance have relatively high viabilities (68- 102%)
compared to species with a low tolerance to heat and a variety of tolerances to
oxygen (19.4 -38%) . In a more specific instance, different strains of L. lactis
can differ in their capability to accumulate glycine betaine as a compatible
solute, which is due to some genotypical differences . This accumulation in
lactic acid bacteria affects the ability to survive drying. Since the differences in
sensitivity to dehydration seem to be an individual trait, it poses a difficulty in
adopting drying conditions from one given strain to other strains.
Table 3.1: Viabilities of different Strains of Lactic Acid Bacteria of the same
species
35
Growth Media and Growth Conditions.
Growth media
Two recent studies have shown that the growth media can have a significant
effect on the freeze dried survival of Enterococcus faecalis, Enterococcus
durans and Lactobacillus bulgaricus (Carvalho et al., 2003, 2004). These studies
also highlighted that the effects can be strain and protectant dependent.
Lactobacillus bulgaricus showed the lowest decrease in viability after freeze-
drying when grown in the presence of mannose, compared to fructose, lactose or
glucose. Survival during room temperature storage was also found to be
dependent on the drying protectant medium used. Carvalho and co-workers
(2004) showed supplementation of the drying medium with any of the four
carbohydrates tested enhanced the desiccation protection during storage. Other
36
sugar types, such as fructose and sorbitol also provided better protection than the
standard growth media carbohydrate glucose (Carvalho et al., 2004). This is
consistent with a study on the survival of Bradyrhizobium japonicum by Streeter
and co-workers (2003) who showed presence of trehalose during growth was
much more effective in protection against desiccation compared to adding
trehalose to the culture a...

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