Lactic acid, cheese, glutamic production

1. Lactic acid Production:
• Cheese production
• Glutamic acid production

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3. Introduction:
 Lactic acid present in two isomeric forms i.e: L (+), D(-) isomers.
 Used in Lactic acid is used as a food preservative, and flavouring
agent.
 Used in intestinal treatment and textile industry.

4. Microorganisms for production of lactic acid:
Lactic acid producing bacteria are broadly categorized in two types.
Homo-fermentative bacteria: Only production of lactic acid and
therefore suitable for industrial purpose.
Hetero-fermentative bacteria: Produce other by products, not
useful for industrial production of lactic acid.

5. Microorganism selection:
 In 1856, Louis Pasteur discovered Lactobacillus and its role in the
making of lactic acid.
 Lactobacillus sp are used for lactic acid production:
 L. lactis: Maltose
 L. acidophilus: Starch
 L. bulgaricus : Lactose
 L. delbrueckii: Glucose

6.  L(+) lactic acid is produced commercially in fermentation
processes by lactic acid bacteria or fungi such as Rhizopus
oryzae in submerged fermentation.
 Rhizopus sp. can manufacture L (+) lactic acid the yield is
very low as compared to lactic acid bacteria.
Fungi as Inoculum:

7. Medium and Manufacturing Process of Lactic Acid:
 Molasses, starch, maltose, lactose, sucrose and agricultural
wastages used as raw materials for the production of
Lactic acid.

8. Biosynthesis of lactic acid:
Pyruvate on
reduction gives
lactic acid molecules

9. Biosynthesis of lactic acid:
 Synthesis of lactic acid occurs through glucose oxidation by
glycolysis to produce pyruvate.
 Pyruvate on reduction gives lactic acid molecules.
 Most microorganisms normally produce only one isomer of
lactic acid L or D isomer form.

10.  Batch and fed-batch fermentation generally used for lactic
acid production.
 The size of inoculum is usually 5–10% of the liquid volume
in this fermenter.
 The fermentation is usually kept at 35–45 °C and at pH 6–6.5.
 The fermentation is carried out for 5-10 days

11. Recovery of Lactic Acid:
 To the fermentation medium, CaCO3 is added; pH adjusted to 10,
broth is heated and filtered.
 Lactic acid is converted to calcium lactate.
 Calcium lactate. decomposes remain sugar and kills bacteria.
 The H2SO4 is added to remove Ca as CaSO4.
 Finally lactate is purified by ion exchange chromatography.

12. Cheese production:

13. Introduction:
 Cheese is a food derived from milk that is produced by coagulation
of the milk protein casein.
 Cheese consists of proteins and fat from milk.
 During cheese production, the milk is usually acidified by bacteria or
fungi, and adding the enzyme rennet causes coagulation.

14.  It contains a good amount of proteins, Vitamin A, riboflavin,
calcium, phosphorous, zinc, and Vitamin B12.
 It is used to make pizzas.
 It is a very rich source of calcium and Vitamin B (Bone Strength).
 Cheese is considered excellent for your skin health, since it
contains Vitamin B.
 It has loads of natural fats that can lead to weight gain.
Applications of Cheese:

15. The five main steps involved in the production of cheese.
1. Pasteurization of Milk
2. Microorganism selection
3. Coagulum Formation
4. Separation of Curd from Whey
5. Ripening of Cheese.

16. Step 1: Pasteurization of Milk:
 Pasteurization is a process that kills microbes (mainly
bacteria) in milk or in juice.
 In these process the milk is heated to 72 °C for 15 seconds
(HTST, also known as "flash).

17. Step 3: Microorganism selection
Streptococcus lactis
 S. cremoris
S. thermophilus
Lactobacillus lactis.
L. bulguricus

18. Step 4: Coagulum Formation:
 Milk coagulation occurs due to two distinct activities:
Inoculation with bacterial cultures:
 Milk inoculated with bacterial cultures Streptococcus lactis or S.
cremoris for incubated at 31°C.
 S. thermophilic with Lactobacillus lactis. (for incubation at 50°C),
results in lactose degradation to produce lactic acid, which lowers
the pH to about 4.6.

19. Incubation with rennet:
 (ii) Incubation with rennet cleaves
Casein protein into para-K-casein
and caseino macropeptide.
 This cleavage occurs at a specific
peptide bond between
phenylalanine and methionine (-
phe 105-met 106-), these leads to
coagulation.

20. Rennet enzyme:
 Rennet is extracted from the inner mucosa of the stomach of
young calf.
 But the rennet obtained from Mucor miehei is relatively more
thermostable and hence remains active during ripening.

21. Step # 4. Separation of Curd (coagulum ):
 The coagulum is heated to 38°C and cooled.
 It eliminates the remaining rennet activity and separates the
watery fluid called whey.
 The curd is separated from whey, salted, and mixed with
proteases and/or lipases; or inoculated with specific fungi,
e.g., Penicillium, and Aspergillus etc.

22. Step 5: Ripening of cheese:
 These cheeses can be unripened or ripened.
 Unripened cheeses are made by coagulating milk proteins (casein) with
acids and enzymes.
 Examples include soft cheeses, cottage cheese.
 Ripened cheeses are made by coagulating milk proteins with enzymes
(rennet) and acids.
 These cheeses are then ripened (aged) by bacteria or mold.
 Cheddar, Swiss, Roquefort, Camembert examples of mold-ripened
cheeses.

23. Step 5: Ripening of cheese:
 The cheese bricks are inoculated with specific strains of fungi for
the development of appropriate flavours through protease and
lipase activities.
 Proteases hydrolyse proteins to produce peptides of variable
sizes.

24.  Peptides having terminal acidic amino acid residues produce
meaty flavours.
 But hydrophobic amino acid residues produce bitter flavours.
 The stronger flavours of Italian cheeses are produced
by a lipid hydrolysis.

26.  Camembert Cheese: Penicillium camembertii used as inoculum
during ripening process.
 Roquefort Cheese: The cut surface of this cheese shows a
characteristic greenish blue colour due to the presence of a specific
mould, Penicillium roquefortii.
 Swiss Cheese: Streptococcus thermophilus some thermophilic
lactobacilli used as inoculum.
What are the different types of cheeses?

27. Glutamic acid (Amino acid) production:

28. Introduction:
 L-glutamic acid is one of the major amino acids that is present in
a wide variety of foods.
 It is mainly used as a food additive and flavor enhancer and used
as food preservative.
 Corynebacterium glutamicum (also known as Brevibacterium
flavum) is the most widely used for production.

29. Food Production:
As flavor enhancer
As nutritional supplement.
Beverage
As flavor enhancer: in soft drink and wine.
Cosmetics
As Hair restorer: in treatment of Hair Loss.
As Wrinkle: in preventing aging.
Other Industries
As intermediate: in manufacturing of various organic chemicals.

30.  The C. glutamicum have high activity of glutamate
dehydrogenase and low activity of alpha ketoglutarate
dehydrogenase.
 They also require the vitamin biotin.
Micro organism for the product of Glutamic acid:

31. Properties of C. glutamicum.
 Produces spores or can be easily inoculated.
 Grows rapidly on a large scale in inexpensive medium.
 Produces desired product quickly.
 Should not be pathogenic

32. Raw materials for the production of Glutamic acid:
Carbon sources:
 Glucose, sucrose, fructose, maltose and molasses used as carbon
sources.
Nitrogen sources:
 The concentration of ammonia is very crucial for converting carbon
source to glutamic acid.
 Sometimes, urea is also used as a nitrogen source.

33. Biosynthesis of L-glutamic Acid:

35. Biosynthesis of L-glutamic Acid:
 Glucose is broken down to pyruvate.
 Pyruvate is converted to acetyl CoA.
 Phosphoenol pyruvate and pyruvate (by the enzyme
phosphoenol pyruvate carboxylase or pyruvate carboxylase) can
be independently converted to oxaloacetate.
 Both these carboxylation reactions are quite critical, and require
biotin as the cofactor.

36.  The next series of reactions that follow are the familiar citric acid
(Krebs) cycle reactions wherein the key metabolite namely α-
ketoglutarate is produced.
 In the routine citric acid cycle, α – ketoglutarate is acted upon by
the enzyme α- ketoglutarate dehydrogenase to form succinyl CoA.
 For the production of glutamic acid, α-ketoglutarate is converted to
L-glutamic acid by the enzyme glutamate dehydrogenase (GDH).

37. Release of Glutamic Acid:
 Glutamic acid is synthesized intra-cellularly, and therefore its
release or export is equally important.
 There are several ways of increasing the membrane
permeability for exporting glutamic acid:
 Addition of penicillin
 Biotin limitation

38. Recovery:
 As the fermentation is complete, the cells are separated.
 The culture broth is passed through anion exchanger (+ binds to -
Negative ions).
 The glutamic acid bound to the resins is eluted by using NaOH.
 With NaOH, glutamic acid forms monosodium glutamate (MSG).
 Which can be purified by passing through anion exchanger.
 Finally MSG can be subjected to evaporation and crystallization.

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