• Starch, a glucose polymer is one of the most widely available plant polysaccharides which is hydrolysed by an enzyme called amylase.
• One of the main uses of amylases is in the production of sweetener for the food industry.
• The hydrolysis of starch with amylase result first in the production of short chain polymers called dextrins then disaccharide maltose and finally glucose.
• The most important enzyme in the starch saccharification process are
• α-amylases, -amylases, glucoamylases, glucose isomerases, pullulanases, and isoamylases.
• α-amylase: (1-4 α-Dglucan-glucanohydrolases)
  • α-amylases are extracellular enzymes which hydrolyse α-1,4-glycosidic bond.
  • These enzymes are endoenzyme splitting the substrate in the interior of the molecule.
  • Bacteria which produces α-amylases are Bacillus subtilis, B. cereus, B. amyloliquefaciens, B. coagulens, B. polymyxa, B. acidocaldarins, B. lichiniformis, Lactobacillus micrococcus, Pseudomonas, Arthrobacter, Escherichia, Proteus, Thermomonospora, Serretia
  • Fungi- Aspergillus, Penicillium, Cephalosporium, Neurospora and Rhizopus.
  • The most important -amylase produced are Bacillus amyloliquefaciens, B. lichiniformis, and Aspergillus oryzae.
• β-amylase: (α-1,4-D glucan-maltohydrolases)
  • β-amylases are usually of plant origin but some micro-organisms also produce.
  • This enzyme catalyzes the hydrolysis of the second α-1,4-glycosidic bond from non-reducing end.
  • E.g. of β-amylase production micro-organisms are B. polymyxa, B. cereus, B. megaterium, Streptomyces spp., Pseudomonas spp., Rhizopus spp. etc.
• Gluco-amylase: (α-1,4-D glucan-glucohydrolase)
  • This enzyme acts on starch by splitting glucose unit from the non-reducing end.
  • Micro-organism involved are Aspergillus niger, A. oryzae, Rhizopus spp.

Fungal α-amylase production:

• Inoculum- spores of Aspergillus oryzae obtained from DDA or SDA plate at 28^oC for 5-6 days.
• Medium

Application of α-amylase:

• Amylases have potential application in a number of industrial processes such as in food, textiles, paper industries, bread making, glucose and fructose syrup, detergents, fuel, ethanol, from starches, fruit juices, alcoholic beverages, sweatness digestive aid, spot remover in dry cleaning.
• Bacterial α-amylase are now also used in areas of clinical, medicinal and analytical chemistry.
• In most wildly used thermostable enzymes are the amylases in starch industry.

Industry (field)	Applications
Starch industry	Liquefaction of starch for production of glucose, fructose, maltose
Milling	Modification of -amylase deficient flour
Alcohol	Liquefaction of starch before the addition of malt for saccharification
Bakery	Increase in the production of fermentable carbohydrates
Brewing	Preparation of barley, liquefaction of additives, enhanced fermentability of grains, modification of beer characteristics
Paper industry	For the liquefaction of starch without sugar production for sizing of paper
Textile industry	Continuous de-sizing at high temperature
Feed industry	Improvement of utilization of enzymatically barley in poultry and calf raising
Sugar industry	Improvement of filter-ability of can sugar juice through breakdown of starch in juice.
Laundry and detergent	Increase in cleaning power additive in dish washer detergent

Microbial α and β amylase- Production process and industrial applications

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Objective:

• to test whether the organism produces the starch hydrolyzing enzyme amylase or not

Introduction:

The main aim of this test is to detect the ability of organism to produce enzyme amylase. Starch is a polysaccharide composed of monomers unit α-D-glucose. These monomers units are arranged in repeated fashion giving two types of polymers, ie. Amylose and amylopectin.

Amylose is the linear polymer of α-D-glucose linked by α (1,4) glycosidic bond. Amylopectin is linear as well as branched polymer of α-D-glucose linked by α (1,4) glycosidic bond at linear point and α(1,6) glycosidic bond at branch point.

The enzyme amylase is an extra cellular enzyme produced by microorganism which hydrolyses the starch breaking α (1,4) glycosidic bond to produce dextrin, maltose and glucose molecules.

Starch when treated with iodine solution, it gives blue color complex. The iodine intercalates in the helical portion of starch molecule and produces blue color complex. When starch is hydrolyzed by amylase produce by microorganism, adding iodine does not change color of the media as there is no starch available for reaction. This indicates positive hydrolysis test. Usually soil bacteria gives positive hydrolysis test whereas members of Enterobacteriaceae gives negative starch hydrolysis test.

Requirements:

1. Media: 1% starch agar media
2. Gram’s iodine solution
3. Culture: 24 hours culture of Bacillus spp and E. coli

Procedures:

1. prepared agar media with 1% starch.
2. Inoculate the starch agar plate with the given organism ( E. coli and Bacillus in separate plate) using inoculating loop making small central line or streak in the plate.
3. Incubate the plates at 37 °C for 24 hours.
4. Flood the plates with gram’s iodine solution
5. Observe the plates after few minutes. A clear zone around the inoculated area indicates starch hydrolysis; positive test

Starch hydrolysis test: objectives, introduction, requirements and procedure

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• The human salivary gland is an exocrine gland.
• The salivary gland includes- the paired parotid, submandibular, and sublingual glands.
• The major function of the salivary glands is to secrete saliva, which plays a significant role in lubrication, digestion, immunity, and the overall maintenance of homeostasis within the human body.

1. Parotid gland

• Parotid gland is the largest salivary glands
• Ocured in pair
• weight:15–30 gm
• Location: It lies on each side of face in front of the ears, covering the masseter muscle at posterior.
• The boundaries of each parotid gland-
  • Superior border –Zygoma
  • Inferior border –Styloid Process, Styloid Process musculature, Internal Carotid Artery, Jugular Veins
  • Posterior border –External Auditory Canal
  • Anterior border –a diagonal line drawn from the Zygomatic root to the External auditory canal
• The long parotid ducts ( Sternsen’s duct) pass forward over masseter and open in the vestibule alongside the second upper molar tooth.
• constitute of about 20% of total salivary secretion
• The parotid gland secrete water, salts and salivary amylase, but unlike other salivary glands, they do not secret mucus, so the saliva produced by parotid gland is clear and watery.

2. Sub mandibular gland

• Size: half the size of parotid gland
• Location: located on the medial side of the mandible.
• Their ducts called submandibular ducts (warton’s duct), open on the floor of the mouth beside the lingular frenulum behind the lower incisors.
• Constitute of about 70% of total salivary secretion
• Secretes water, salts, salivary amylase and mucus

3. Sub lingual gland

• Size: smallest salivary gland
• Location: located in the floor of mouth beneath the tongue.
• They have numerous small ducts opening at the floor of the mouth.
• The sublingual duct are collectively known as duct of Rinivus.
• constitute of about 5% of total salivary secretion.
• Their secretion mostly contain water, salts and mucus.

Histology of salivary gland

• All the salivary glands are surrounded by a fibrous capsule.
• The gland are highly branched and consists of a number of lobules made up of small alveoli
• each alveoli is lined with secretory cells.
• Salivary glands are classified as exocrine glands that secrete saliva through ducts.
• The secretory structure is known as Salivary acinus.

Saliva

• Saliva is viscous, colourless, cloudy fluid which is the combined fluid of salivary gland and small mucus secreting glands of oral cavity.
• pH: 7.4-7.6 (slightly Alkaline)
• Amount: about 750-1500 ml of saliva is secreted in a day
• Composition: 98-99% water and 1-2 % (mineral salts, amylase, mucus, lysozyme, immunoglobulins, blood clotting factors etc)

Function of Saliva

1. Moistens oral mucosa: Saliva moisturize the oral cavity and it is an important non immune defense mechanism in oral cavity.
2. Swallowing: Saliva moistens dry food and cools hot food and make easy for swallowing and digestion
3. Stimulation of taste bud: Saliva provides a medium for dissolved foods to stimulate the taste buds.
4. Buffer: Saliva has a high concentration of bicarbonate ions and acts as physiological buffer
5. Digestion: Saliva contains Ptylin (salivary amylase) that digest starch
6. Antimicrobial activity: Saliva controls bacterial flora of the oral cavity. Lysozyme, Secretory IgA, and Salivary Peroxidase present in saliva have antimicrobial property.
7. Mineralization: Saliva contains high concentration of calcium and phosphate, which helps in mineralization of teeth.
8. Oral hygiene: saliva helps mouth and teeth clean
9. Protection: Saliva protects the teeth from dental carries.

Salivary gland

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