Introduction to Lipases Industrial Production of Lipases by Fermentation Industrial Significance of Lipases

1. ‘LIPASES’
PREPARED BY:
NAHAL JEHANGIR (18006231-011)
BS. BIOTECHNOLOGY
COURSE TITLE: Food Biotechnology

2. CONTENTS
 Introduction to Lipases
 Sources
 Classification
 Production by Fermentation Technology
 Applications
 Industrial & Medical Aspects
 Significance in Food Biotechnology

3. Introduction
• A lipase is any enzyme (extracellular) that catalyzes the hydrolysis of fats.
• Splits fats into fatty acids and glycerol. (Glycerol Ester Hydrolases)
• A subclass of Esterases.
• Lipases perform essential roles in digestion, transport and processing of dietary
lipids in most organisms.
• Genes encoding lipases are even present in certain viruses.

4. What are Lipases?

5. Enzymatic Activity
The mode of action of lipase is:

6. Sources
• Many microbial lipases have been commercially available in free or immobilized
form.
• Produced in pancreas, seeds, Euphorbiaceae (Ricinus communis).
• Numerous species of;
 Bacteria (Bacillus, Pseudomonas, and Burkholderia)
 Yeasts (Candida rugosa, Yarrowia lipolytica and Candida antarctica)
 Molds (Aspergillus, Trichoderma viride)
• Microbes are potent lipase producer.

7. Pancreatic Lipase
• Also known as pancreatic triacylglycerol lipase, is secreted from
the pancreas
• The primary lipase that hydrolyzes dietary fat molecules in the
human digestive system.
• Requires a high pH for its activation. That is why fats are the
hardest of all foods to digest.
• Mechanism: Bile salts secreted from the liver and stored in
gallbladder are released into the duodenum where they coat and
emulsify large fat droplets into smaller droplets, thus increasing
the overall surface area of the fat, which allows the lipase to break
apart the fat more effectively.

8. Classification

9. Industrial Production
The industrial production of lipases involves the following steps;
• Sterilization
• Isolation of pure culture
• Characterization & Screening
• Optimization
• Substrate addition
• Inoculation
• Automation of bioreactors
• Harvesting / recovery

10. Production Steps:
1. Isolation of Lipase Producing Bacteria:
• Sludge and oil contaminated samples are collected from effluents.
• STRAINS:
 Candida lipolytica
 Asperigillus niger (Fungus used mostly)
 Pseudomonas, Bacillus and Lactobacillus sp.
• Samples are subjected to serial dilution.
• Incubation at 37°C for 24-48 hours
• Nutrient medium Agar
• A clear zone around the colonies indicates the production of lipase.

11. 2. Screening
• Screening is done on Tributyrin agar rich media. (Primary Screening)
• P-NPP Assay for estimating lipolytic activity (Para-nitro phenyl phosphate method).
3. Characterization
• Effect of pH and Temperature
• Effect of organic solvent and detergents
• Analytical characterization methods are necessary to ensure the identity, purity, structural
and conformational integrity, and activity of an enzyme product.

12. 4. Substrate Addition
• Solid State Fermentation (SSF) is used for the commercial production of lipases due to;
 Higher yields and productivities
 Extended stability of products
 Lower production cost
• Different types of agricultural wastes and oily substrates.
• Rice bran, Wheat bran, Gingelly oil cake
Almond meal, Mustard oil cake,
Neem oil cake, Groundnut oil cake,
Gingerly seed and groundnut kernel & Olive oil.

13. 5. Fermentation Process
• Optimal medium composition: glucose 0.1, olive oil 3.0, NH4Cl 0.5, yeast extract 0.36,
K₂HPO₄0.1, MgCl2 0.01, and CaCl20.4 mM.
• Carbon source: Wheat bran, rice husk, lentil husk, banana waste, watermelon waste.
•Olive oil as inducer and yeast extract as nitrogen source.
• To maintain sufficient oxygen concentration for optimum production, fermentation has
been carried out at 600 rpm and at different aeration rates. Gas flow rate of 50.34 cm3/s
yielded optimum production of lipase.
6. Optimization
• Lipase production was optimized at different pH (7 - 10), temperature (30 - 60°C)
incubation period (1-5 days) with constant shaking at 120 rpm. Bacteria were cultured in
nutrient broth with 1% olive oil.
• During the cultivation, lipase activity will be measured every 12 h to determine the
maximum lipase producing period.

14. 7. Purification / Harvesting (DOWNSTREAMING)
 Calcium Chloride precipitation
 Ammonium Sulphate precipitation
 Colum Chromatography
• Supernatant containing extracellular lipase obtained from
fermented broth is treated with 0.4M CaCl2 to precipitate fatty acids
followed by centrifugation at 4°C and 12000rpm for 30 min.
• The supernatant was collected in a glass beaker and to it chilled
acetone was added slowly to allow protein precipitation. The
precipitates were then harvested by centrifugation at 4°C and
12000rpm for 30 min.
• Supernatant then subjected to ultrafiltration and dialyzed
overnight.

15. Experimental Design

16. APPLICATIONS
• Processing of fats and oils
• Detergents industry
• Food Processing (Improvement of flavor)
• Synthesis of fine chemicals and pharmaceuticals
• Paper manufacture (Control of pitch)
• Production of cosmetics & beverage
• Waste water treatment (By removing oil)
• Biocatalysts in industrial applications
• Diagnostic and medical use

17. Industrial Aspects:
• Lipases serve important roles in human practices as ancient as yogurt and
cheese fermentation.
• A biotechnology company has brought recombinant lipase enzymes to market
for use in applications such as baking, laundry detergents.
• As biocatalysts in alternative energy strategies to convert vegetable oil into
fuel. (Biodiesel)
• Can be more environmentally friendly and safe.
• Detergent formulations (Minimize toxic phosphate usage)
• Food, pharmaceutical, leather, textile, cosmetic, and paper industries.

18. Medical & Diagnostic Aspects
• Blood tests for lipase may be used to help investigate and diagnose acute
pancreatitis and other disorders of the pancreas.
• Cystic fibrosis, Alzheimer's disease, atherosclerosis
• Measured serum lipase values may vary depending on the method of analysis.
• Lipase can also assist in the breakdown of fats into lipids in those
undergoing pancreatic enzyme replacement therapy (PERT).
• Lipase is used for indigestion, heartburn, allergy to gluten in wheat
products (celiac disease), Crohn's disease, and cystic fibrosis.

19. Lipases in Food Biotechnology:
• Blood tests for lipase may be used to help investigate and diagnose acute
pancreatitis and other disorders of the pancreas.
• Cystic fibrosis, Alzheimer's disease, atherosclerosis
• Measured serum lipase values may vary depending on the method of analysis.
• Lipase can also assist in the breakdown of fats into lipids in those
undergoing pancreatic enzyme replacement therapy (PERT).
• Lipase is used for indigestion, heartburn, allergy to gluten in wheat
products (celiac disease), Crohn's disease, and cystic fibrosis.

20. REFRENCES
• https://labpedia.net/lipase-serum/
• https://www.mdpi.com/2077-0472/11/6/540/htm
• https://www.researchgate.net/figure/Application-of-lipases-in-different-industries_fig2_340974234
• https://www.informit.com/articles/article.aspx?p=2783636&seqNum=4
• https://www.slideshare.net/LIKHITHK1/lipase-production-and-urification-likhith-k?qid=e9f4792d-
cab5-41be- 994b-0788ddf6a047&v=&b=&from_search=8
• https://www.slideshare.net/ponkalaivanis/production-of-lipases-and-cellulase
• https://www.slideshare.net/MDCrules/enzymes-their-production
• https://microbenotes.com/lipase-test-objectives-principle-procedures-and-results/
• https://www.researchgate.net/profile/Ali-Nawaz-
3/publication/288933945/figure/fig4/AS:669283327696898@1536580988204/Effect-of-lipid-
substrate-for-lipase-production-by-Aspergillus-niger-in-solid-state.ppm

21. Thankyou
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