Techno-Industrial Platform Development for Production of Probiotics: From Cell Bank to Bulk Powder
Prof. Dr. rer. Nat. Hesham A. El Enshasy DIRECTOR
INSTITUTE PEMBANGUNAN BIOPRODUK UNIVERSITI TEKNOLOGI MALAYSIA
E-mail: [email protected]
The 2nd. Science and Methematics International Conference (SMIC) 2020 8-9 August, Jakarta, Indonesia
Probiotics (Science and Business)
Probiotic yeast
Bioprocess manufacturing Platform Design Market and Future trends
‘Lecture content
Humans: 10% Human cells and 90% Bacteria
• The human body contains about 100 trillion cells, but only maybe one in 10 of those cells is actually
— human. The rest are from
bacteria, viruses and other
microorganisms.
Gut in Microflora
"The definition of a human microbiome is all the microbial microbes that live in and on our bodies but also all the genes
— all the metabolic capabilities they bring to supporting human health”
They belong in and on our bodies; they help support our
health; they help digest our food and provide many kinds of protective mechanisms for human health,"
Microflora In Human Body
Gut Microbes
- Extract vitamins and other nutrients we need to survive, - Teach our immune systems how to recognize dangerous
invaders
- Produce helpful anti-inflammatory compounds and
- Produce chemicals that fight off other bugs that could make us sick.
Microflora In Human Body
Microflora In Human Body
• Production of various bioactive compounds
PROBIOTIC
• Probiotic ( Greek Language) “ for life” .
• It was first used by Lilly and Stillwell in 1965 to describe
“substances secreted by one microorganism which stimulates the growth of another”.
• Parker was the first to use the term probiotic in the sense that it is used today “organisms and substances which
contribute to intestinal microbial balance”.
• In 1989, Fuller attempted to improve Parker’s definition of probiotic with the following distinction: “A live
microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial
balance.”
Administration of probiotic
• “The specific microorganisms shall be viable, active & abundant at the level of at least 10
7cfu /g in the product to the date of minimum durability”
• Minimum Consumption: 100g of a probiotic food with 10
7cfu/ g.
Administration of probiotic
New Formulations
Probiotic Skin Care Toothpaste Vaginal
Wash
Probiotic Foods
The total market of Probiotics reached 20 US$ Billions by 2020
Most of Probioics business focused on Pharma, Food, Feed Industries
Global Market for Probiotics
Global Probiotics Market is
primarily driven by the
following factors:
▪ Prevents the growth of harmful bacteria in the digestive tract.
▪ Rising demand in food and beverages for functional products.
▪ Increased awareness about the animal health and negative impact of extensive use of antibiotics.
▪ Rising disposable income in emerging countries.
Global Market for Probiotics
Lactobacillus strains Bifidobacteriums sp. Others L. casei
L. rhamnosus L.acidophilus L. bulgaricus L. fermentum L. lactis
L. johnsonii L. reuteri
B. bifidum B. breve B. infantis B. lactis B. longum
S. boulardii S. thermophilus E. faecium
K. lactis
Probiotic Business Parterns
Ablility to survive in the extreme conditions of digestive tract Adherence to epithelial cells and intestinal mucosa
Colonization potential in the human intestinal tract
Production of antimicrobial substances towards pathogens Safety in regard to human use
Stability during storage under normal conditions
Microorganis (Type, thermo-adabtation, Draughness-adaptation) Production facility (Up-stream) and control system
Product formulation (powder, micro-encapsulated, etc...) Cultivation mode / Scalability
Down stream requirements
Inhibit pathogen attachment
I nhibit the action of microbial toxins Stimulate immunoglobulin A
Trophic effects on intestinal mucosa
Saccharomyces cerevisiae
Help to feed 6,3 Billion people
S ave life for 150 million Diabetics
Produce drugs and vaccines for other hundred thousands
Global market size of 430,000 tons of dry solid yeast per year
High cell density of Saccharomyces boulardii ! Ethanol Inhibition Effect !
Ethanol Production during HCDC
Process Challenges
Adaptive strain to draughness and tolerate to moderate heat (to
change the downstream process from freeze dyring to spray drying)
Scaling up of the Process
Down Stream
(Separation/Isolation/Purification and increase of product Stability) Determination of process bottleneck(s)
Bioprocess Optimization in Lab. scale
(Development of cultivation strategy to reach the maximal productivity)
Glucose repression Oxygen limitation
Mixing Aeration
pH control
Foaming effect / Mixing
0.0 0.5 1.0 1.5 2.0 2.5
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
0 5 10 15 20
0.0 0.5 1.0 1.5 2.0 2.5
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
0 10 20 30 40 50 60 70 80 90 100
CDW [g L-1 ]
Shake Flask
(B)
pH
time [h]
CDW [g L-1 ]
Bioreactor
pH
(A)
DO [%]
Maximal Biomass 2.5 g L-1 !!!!
0 5 10 15 20 0
1 2 3 4 5 6
0 2 4 6 8 10
0 20 40 60 80 100
0.0 0.1 0.2 0.3 0.4 0.5 0.6
time [h]
CDW [g/L] Glucose [g/L]
DO [%] Y X/S [g/g]
Maximal Biomass 5.0 g L-1 !!!!
0 5 10 15 20 25 30 0
10 20 30
0 5 10 15 20 25 30 35 40 45 50
0 20 40 60 80 100
0 4 8 12 16 20
CDW [g L-1 ]
Time [h]
Glucose [g L-1 ]
Fed-Batch Batch
DO [%] FR glucose [g L-1 h-1 ]
Maximal Biomass 35 g L-1
0 5 10 15 20 25 30 35 40 0
10 20 30 40 50 60 70 80 90
0 5 10 15 20 25 30 35
0 20 40 60 80 100
0 4 8 12 16 20
CDW [g L-1 ]
Time [h]
Glucose [g L-1 ]
Fed-Batch Batch
DO [%] FR glucose [g L-1 h-1 ]
Maximal Biomass 85 g L-1
(*) data obtained from the previous batch culture.
) /
.
().
( .
. 1 .
) (
/
tF t
e
setX t
V Y m
t
m
set E L FS X s
+
=
Where:
ms Mass flow of substrate [g h-1]
t Cultivation time [h]
tF: Start time of feeding phase [h]
µset Adjusted specific growth rate [h-1]
mE Maintenance coefficient [g g-1 h-1]
YX/S The biomass/substrate yield coefficient [g g-1]
XF The biomass concentration at the start time of feeding phase [g]
VL The culture volume [L]
Exponential feeding strategy is the most suitable for microbial cells for High cell density cultivaiton (Korz et al., 1992; El-Enshasy et al., 1997)
0 5 10 15 20 25 30 35 40 45 0
30 60 90 120
0 2 4 6 8 10 12 0 14
20 40 60 80 100
Fed-Batch Phase Batch Phase
time [h]
CDW [g/L] Glucose [g/L]
DO [%]
Maximal Biomass 120 g L-1
[1] [2] [3]
[4] [5]
[7] [6]
[8]
[9]
Production of Probiotics in Large Scale
1- Efficient Design for maximal oxygen transfer
2- Efficient cooling design to overcome high heat production rate in HCD culture 3- Efficient control system for aeration and agitation
4- Oxygen supply and high quality gas mixer 5- High quality out-gas analysis system
5- Efficient control system for DO cascading with different parameters
6- Highly efficient control system for aeration, agitation and substrate supply\
7- On-line turbidity meter for on-line cell growth determination
Se-enriched S. boulardii
production
Different Se Concentrations
0 1 2 3 4 5 6 7 0
5 10 15 20 25 30
-20 0 20 40 60 80 100 120 140 160
0 1 2 3 4 5 6
CDW [g L-1 ]
Selenium [mg mL-1]
pH
Selenium [g g-1 ]
90 mg mL ¯ ¹ was selected as the optimal Se concentration
Resulting 21.93 µg g ¯ ¹ Se accumulation in cell.
4.3 g L¯ ¹ biomass production
Study of Addition Time and Treatment Period for Selenium Enrichment
0 10 20 30 40 0
1 2 3 4 5 6 7
0 10 20 30 40
0 5 10 15 20
0 1 2 3 4 5 6 7
CDW [g L-1 ]
Selenium Addition time [h]
Data after 12 h cultivation
Selenium [g g-1 ]
Data after 24 h cultivation
CDW [g L-1 ] Selenium [g g-1 ] The best addition time was 16 h.
Followed by cultivation for 24 h.
39.5 µg g ¯ ¹ of Se concentration was achieved with 5.58 g L¯ ¹ biomass production
Se Absorption Kinetic in the Optimized Medium
0 10 20 30 40
0 1 2 3 4 5 6 7 8 2 3 4 5 6 7 8
0 10 20 30 40
CDW [g L-1 ]
time [h]
Selenium addition time (16 h)
pH Selenium [g g-1]
Se accumulation reach maximum at 34.25 µg g¯ ¹ after 39 hours of cultivation (Selenium added 16 h post inoculation)
Rate of Se absorption was at 1.43 µg g¯ ¹ h¯ ¹
Post Harvest Se Enrichment
0 5 10 15 20 25 30 35
0 5 10 15 20 25 30
0 1 2 3 4 5 6 7
CDW [g L-1 ]
time [h]
Selenium [g g-1 ]
Maximum Se accumulation was achieved at 28.53 µg g¯ ¹ After 21 hour of treatment time
Rate of absorption at 1.01 µg g ¯ ¹ h ¯ ¹
Production of Se-Yeast in Batch and Fed-
Batch in semi-industrial scale
Production of Se yeast in 16-L batch bioreactor under uncontrolled pH
0 6 12 18 24 30 36 42 48
0 1 2 3 4 5 6 7 8 9
-5 0 5 10 15 20 25 30 35 40 20
40 60 80 100
0 5 10 15 20 25 30 35
0 6 12 18 24 30 36 42 48
3.0 3.5 4.0 4.5 5.0 5.5 6.0
CDW (g L-1 )
time [h]
Glucose (g L-1 ) Se addition (16h)
DO (%) Se (g g-1 ) pH
Maximal biomass achieved was 7.81 g L ¯ ¹ after 30 h of cultivation
Maximal Se accumulation reached 31.81 µg g ¯ ¹ Se absorption rate at 1.83 µg g ¯ ¹ h¯ ¹
Production of Se Yeast in 16-L Fed-batch Bioreactor with Complete Medium Feeding
0 10 20 30 40 50 60
0 5 10 15 20 25
0 5 10 15 20 25 30 35 40 0
20 40 60 80 100
3.0 3.5 4.0 4.5 5.0 5.5 6.0
0 5 10 15 20 25 30 35 40 45
time [h]
CDW (g L-1 )
Fed Batch Batch
Glucose (g L-1 )
DO (%)
Se addition at (16 h)
pH
Se (g g-1 )
Maximal cell mass 24.97 g L¯ ¹
(After 54 h cultivation)
Maximal Se content was 41.65 µg g¯ ¹
Se absorption rate was 2.35 µg g ¯ ¹ h¯ ¹
Parameters Batch Fed-Batch
Max Biomass [g L-1] 7.69 24.97
Max Se content [µg g-1] 31.81 41.65
Specific growth rate µ [h-1] 0.137 0.177 Se absorption rate [µg g -1 h-1] 1.83 2.35
Batch and Fed-Batch Se-yeast production
High Biomass Production of
Marine Probiotic Yeast
Isolation from Red Sea
(27.2578ºN, 33.8117º E)
Isolation of Marine Yeast
Fig. 1: Bootstrap consensus phylogenetic tree based on ITS1-5.8s-ITS2 sequence analyses by neighbor joining method, showing the relationship of Cryptococcus sp. strain YMHS with the most closely related yeasts. The scale bar represents 0.1 substitutions per nucleotide position.
Yeast Identification
Chlorphorm Ethyl acetate Xylol 0
10 20 30 40 50 60 70
Hydrophobicity [%]
Type of solvent
Hydrophobic properties of Cryptococcus sp. YMHS in different solvent systems. Data represent the means
standard deviation of triplicate assays.
Probiotic Yeast Functionality (Hydrophobicity)
Kinetics of cell growth and substrate consumption during submerged cultivation of Cryptococcus sp. YMHS in shake flask. Data represent the means standard deviation of triplicate assays.
Kinetics of the cell growth and substrate consumption during submerged cultivation of Cryptococcus sp. YMHS in 16-L bioreactor.
Bioprocess Optimization and Scaling up
0 5 10 15 20 25 30 35 40 45 0
5 10 15 20 25 30 35
0 2 4 6 8 10 12 14 16 18 0
1 2 3 4 5 6
0 10 20 30 40 50 60 70 80 90 100 110
Fed-Batch Batch
CDW [g/l]
time [h]
glucose [g/l]
Feeding rate [g/l/h]
DO [%]
Simple Fed-batch cultivation for HCDC
Biomass Production (30 g L-1)
- Controlled DO
- Increased mono-glucose feeding strategy
Five in situ 16 L STR
16 L STR
150 L STR
16 L STR
150 L STR
1500 L STR
R&D Industrial (I) Industrial (II)
Cell Bank (MCB, WCB)- WICC
Microbiological and Analytical Laboratories
Downstream
I - High Pressure Homogenizer - Self Clean SeparatorDownstream II - Spray Dryer
- Freeze Dryer
Microbial Bioprocess at IBD
New Book: Jan 2021
CRC Press
Probiotic Partneers
(Malaysia, Sweden, Egypt, Indonesia, Saudi Arabia)
Dr. Daniel Joe Dailin
Mr. Shanmuga Selvamani Ms. Roslinda Malik
Ms. Afif Najeha Kibly Ms. Jennifer Eyahmalay Mr. Solleh Ramli
Ms. Siti Zulaiha Hanapi - Many Others
(UTM-IBD, Malaysia) Dr. Ong Mei Leng
(Harita Go Green Sdn. Bhd., Malaysia) Mr. Vick Swaripagam
(SBG Feed Sdn. Bhd., Malaysia) Prof. Dr. Mukti Nurjayadi
Dr. Dalia Sukmawati
(Universitas Negeri Jakarta, Indonesia)
Prof. Maha El Demilawy Dr. Doaa Abdel Rashid Eng. Salah El Sayed (CRTA, Egypt)
Prof. Ashraf El Baz
(Sadat University, Egypt) Prof. Yosrya Shatia
(Ain Shams University, Egypt)
Prof. Rajni Hatti-Kaul
(Lund University, Sweden)
Dr. Elsayed Ahmed Elsayed
(King Saud University, Saudi Arabia)
INSTITUTE OF BIOPRODUCT DEVELOPMENT Universiti Teknologi Malaysia
81310 UTM Skudai
Johor Darul Takzim. Malaysia Tel : 07-5532499 Fax : 07-5569706
