Chapter II

REVIEW OF RELATED LITERATURE

I. INTRODUCTION

Monosodium glutamate is the sodium salt of glutamic acid. Glutamate is a naturally

occurring amino acid that is found in nearly all foods, especially high protein foods such as dairy

products, meat and fish and in many vegetables. Foods often used for their flavoring properties, such

as mushrooms and tomatoes, have high levels of naturally occurring glutamate. It is a flavor

enhancer commonly added to Chinese food, canned vegetables, soups and processed meats.

Monosodium glutamate added to foods produces a flavoring function similar to the glutamate that

occurs naturally in foods. It acts as a flavor enhancer and adds a fifth taste, called “umami”, which

is best described as a savory, broth-like or meaty taste. The Food and Drug Administration (FDA)

has classified MSG as a food ingredient that's "generally recognized as safe," but its use remains

controversial. For this reason, when MSG is added to food, the FDA requires that it be listed on the

label. In the European Union, monosodium glutamate is classified as a food additive (E621)and

regulations are in place to determine how and when it can be added to foods. Typically, monosodium

glutamate is added to savory prepared and processed foods such as frozen foods, spice mixes, canned

and dry soups, salad dressings and meat or fish-based products. In some countries, it is used as a

table-top seasoning. MSG has been used as a food additive for decades.

In the manufacture of monosodium glutamate, the transformation of the raw material

(cassava root crop) into the desired chemical product can be achieved by a multiple step procedure.

1|Page
This procedure is broken down into a number of steps that provide intermediate transformations,

and are carried out through chemical reaction, separation, crystallization, and drying.

This chapter attempts to predict how the process of the manufacture of monosodium

glutamate would behave if a manufacturing plant was constructed. The chemical process was

designed to utilize the raw materials as efficiently as possible. The process must be economically

wise and practical to prevent the production of waste that might be environmentally harmful, also,

to preserve the reserves of raw materials as much as possible.

The raw material must undergo thorough peeling, cleaning, crushing, separating, filtration,

and drying to excrete the starch. Fermentation is essential in producing the product itself. Enzymes

such as alpha amylase are utilized to catalyze the formation of the product. Moreover, liquefaction

is a key and essential handling of starch and if this procedure does not go well, inconveniences such

as poor filtration, turbidity of the procedure may occur. Additionally, when extracting from regular

sources, the standard method is hydrolysis with aqueous acid, followed by catch of the amino acids

by entry of the hydrolysate over an emphatically acidic ion exchange resin. The process of forming

the crystals is a kind of chemical precipitation reaction. It is based on the salt or ionic nature of the

products. Adding the sodium to the glutamate will further stabilize the product to undergo the

various harsh downstream reactions. It is of most importance that the solution of MSG from the

fermentation broth must be purified to produce clear and beautiful crystals. Ultimately the final steps

include centrifugation, decantation and filtration to obtain the crude crystals the crystallization

process are usually carried out using the crystallizer. Finally, the separated L-glutamic acid, α-form

2|Page
crystals were re-dissolved in water and placed into an enamel-jacketed ironware vessel. This

monosodium glutamate solution was then decolorized by adding activated carbon and filtering. The

filtered, clear solution was then concentrated by heating and cooled in the enameled vessel, causing

monosodium L-glutamate (MSG) crystals to form and precipitate. When separated from the

solution, the lump of MSG crystals was cracked by hammer into powder and separated from any

adhered mother liquor by centrifugation. The final MSG powder was dried, sieved, and packed as

the final product.

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II. LIST OF RELATED LITERATURE

A. BOOKS

Ahmed, J. &Rahman,S. (2012). Handbook of food process design. John Wiley & Sons,

2012.

Arnon, I. (2012). Modern methods of plant analysis / Modernemethoden der

pflanzenanalyse: Volume 7 of modern methods of plant analysis modern methoden

der pflanzenanalyse. Springer Science & Business Media.

Bassam, N. EL. (2013). Energy plant species: Their use and impact on environment and

development. New York, NY. Earthscan.

Bickerstaff, G.F., Ed. Immobilization of Enzymes and Cells, Totowa, N.J.: Humana Press,
1997.

Board, N. Modern Technology of Industrial Chemicals. Asia Pacific Business Press Inc.

Cassava production, processing and marketing in vietnam. (1992). CIAT

Dey, G., Singh, B., Banerjee, R. (2003) Immobilization of ·-Amylase Produced by Bacillus
circulans GRS 313, Brazilian Archives of Biology and Technology, 46(2): 167-176.

4|Page
Faurie, R &Thommel, J. (2003). Microbial production of l-amino-acids. New York.

Springer-Verlag Berlin Heidelberg.

Fellows, P. J. (2009). Food processing technology: Principles and practice. 3rd edition.

Elsevier.

Fleck, M. & Aram M. P. (n.d.) Salts of amino acids: Crystallization, structure and

properties.N.p.: Springer, n.d. Google Books. Web. 29 Jan. 2016.

Flickinger, M. C., & Drew, S. W. (1999). The encyclopedia of bioprocess technology:

Fermentation, biocatalysis, and bioseparation. New York: Wiley.

Gerhartz, Wolfgang. (1990). Enzymes in Industry. Weinheim, FRD.

Ikeda, M. (2003). Microbial production of l-amino acids.

Inui, M. & Yukawa, H. (Eds.) (2013). Corynebacterium glutamicum: Biology and

Biotechnology. London, Springer-Verlag Berlin Heidelberg.

Lea, D. (Ed). (2003). Agricultural and mineral commodities year book. First ed. United

Kingdom, Europa Publications.

5|Page
Lebot, V. (2008). Tropical root and tuber crops: Cassava, sweet potato, yams and aroids.

Encyclopedia of life and support systems.

Lee. B. (2014). Fundamentals of food biotechnology.

Luckner, M., et al. (1984). Secondary metabolism in microorganisms, plants and animals.

Springer Berlin Heidelberg.

Maga, J. A. &Tu, A. T. (1994). Food additive toxicology. New York, N.Y. Marcel Dekker.

Palaniswami, M.S. & Peter, K.V. (2008). Tuber and roots crops. New Publishing Agency.

Prescott, S.C. and Dunn, C.G. (1959), In: Industrial microbiology. C.G. Dunn (Ed),

McGraw Hill Book Company, Inc. New York, London, 710-722.

Reineccius, Gary (2005) Flavor Chemistry and Technology. New York. Taylor and Francis
Group.

Sadhukhan, R., Roy, S.K., and Chakrabarty, S.L. (1993) Immobilization of ·-amylase
fromMyceliophthorathermohila D-14 (ATCC 48104), Enzyme and Microbial
Technology 15(9): 801-804

6|Page
Speight, J. G. (2002). Chemical and process design handbook. New York. McGraw-Hill.

Spies JR. Colorimetric procedures for amino acids. In: Colowick SP, editor. Kaplan N.O.

methods in enzymology. III. New York: Academic Press; 1957. pp. 468–471.

Tatsumi, N. & Inui, M. (2013). Coryebacteriumglutamicum: Biology and biotechnology.

New York, NY. Springer-Verlag Berlin Heidelberg.

Vaughan, J. &Geissler, C. (2009). The new oxford book of food plants. New York. Oxford

University Press.

Wiseman, A., Ed. Handbook of Enzyme Biotechnology, 3rd Ed., Hertfordshire, Great
Britian: Ellis Horwood Limited, 1995.

Yoshioka T, Ishii T, Kawahara Y, Koyama Y, & Shimizu E. Method for producing L

glutamic acid by continuous fermentation. United States patent US 5; 1999. p. 300

Zieglar, H. &Zieglar, E. (Eds.). (2008). Flavourings: Production, composition,

applications, regulations. N.Y. John Wiley & Sons, 2008.

7|Page
 Hillocks, R.J.,Thresh, J.M., Bellotti, A. Cassava: Biology, Production and Utilization

Brennan, J., Grandison, A. Food Processing Book

B. PATENTS

Bulloff, J. J. & Novak, L. J. (1959). Synthesis of glutamic acid and salts thereof.

Cami, P., Nesle, CH. Et.al. (1999). Process for the preparation of monosodium glutamate.
United States.

G. Duan, Y. Q. (2010). Patent No. WO2010129648 A2.

J.M. Cervantes, A. S.-P. (2005). Patent No. US20050077029 A1.

K. Wolfgang, W. K. (1975). Patent No. US3860395.

Miescher, G. M. (1966). Process for producing monosodium glutamate. October 25,1966.

8|Page
Northbrook, Purvis, J. L. &Vassel, B. (1958). Process for producing monosodium

glutamate crystals. Deerfield, Ill. Assignors to International Minerals & Chemical

Corporation, a corporation of New York. May 13, 1958.

Novak, L. J. &Bulloff, J. J. (1959) Synthesis of glutamic acid and salts. United States

OHIO COMMW ENG CO 2905711.

Shildneck, P. R. (1942). Preparation of monosodium glutamate. United States Patent

2306646.

Tetsuwo, O. (1952). Manufacturing method of solid l-monosodium glutamate

monohydrate. United States. Ajinomoto KK 2584731.

Yoshiyuki, T. (1994). Pullulanase. Methods of producing pullulanase and methods of

saccharification of starch using pullulanase . Research Development Corporation

of Japan, assignee. Patent US5316924 A. March 31, 1994.

Hambrock, K. (1951). Production of Lysine and Monohydrochloride and Derivatives.

Niagara Falls, N. Y., as signor to E. I. du Pont de Nemours and Com pany,

Wilmington, Del., a corporation of Dela Ware. United States Patent Serial No. 148,
737

9|Page
C. WEBSITE

“AmbicaCrushtech PVT.LDT.” (n.d.) Retrieved from http://ambicacrushtech.com.

“Application of Microbial α-amylase in industry – a review” (2010). Retrieved from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769773/

Bagby, C. (n.d.). Highland woodworking. Retrieved from

http://www.highlandwoodworking.com.

Bangke, N. (2008). Introduction to cassava starch production. Retrieved from

http://www.who.int/medical_devices/innovation/hospt_equip_32.pdf.

Basic Fermentation Chemistry. (n.d.). Retrieved August 04, 2016, from

http://arbl.cvmbs.colostate.edu/hbooks/pathphys/digestion/herbivores/fer
ment.html

“Cassava” (1995). Retrieved from https://www.hort.purdue.edu.

“Cassava” (2003). Retrieved from

http://www.encyclopedia.com/topic/cassava.aspx.

“Cassava” (2014). Retrieved from http://www.food-info.net/uk/products/rt/cassava.htm.

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“Cassava flour and starch”. Retrieved from

http://www.fao.org/docrep/x5032e/x5032e02.htm.

“Corynebacterium glutamicum” (2007). Retrieved from

http://web.mst.edu/~microbio/BIO221_2007/C_glutamicum.htm.

“Double effect/Multi effect Falling Film Evaporator.” Retrieved from http://tomato-

machinery.com/8-2-falling-film-evaporator.html/118771

“Encyclopedia of chemical engineering equipment” (n.d.). Retrievedfrom

http://encyclopedia.che.engin.umich.edu.

“From tapioca starch to nua powder (MSG)” (2011). Retrieved from

http://www.thaitapiocastarch.org/article23.asp.

Hahn, S. K. (n.d.). An overview of traditional processing and utilization of cassava in

Africa. Retrieve from http://www.fao.org/wairdocs/ilri/x5458e/x5458e05.htm.

“komline-Sanderson.” (1946). Retrieved from

http://www.komline.com/docs/rotary_drum_vacuum_filter.html.

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LLC, B. P. (2000, March 23). Pusher Centrifuge: Operation, Applications and Advantages.
Retrieved from http://www.pharmaceuticalonline.com

“Microbial production of 7 types of amino acids.” (2015). Retrieved July 05, 2016, from

http://www.biologydiscussion.com.

“Monosodium glutamate (MSG) – The third spice” (2014). Retrieved from

http://www.starch.dk/isi/bio/msg.asp.

“Preparation and manufacturing of monosodium glutamate (MSG)”. (n.d.). Retrieved

January 26, 2016, from

http://formulation.vinensia.com/2011/03/preparation-and-manufacturing-of.html.

“Production process.” (n.d.). Retrieved January 26, 2016, from

https://www.ajinomoto.com/features/amino/lets/product/.

Protection, A. a. (n.d.). FAO corporate document repository. Retrieved from

http://www.fao.org/docrep/x5415e/x5415e01.htm#chapter.

Ramesh K. Shah, D. P. (2003). Fundamentals of heat exchanger design. John Wiley &
Sons, Inc.

Recycling Device Of Liquidized Waste Steam During Production Of Monosodium

Glutamate Manufacturing Method. Retrieved from http://www.technology-
x.net/A23L/201420219991.html

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“SilesFor.” (1970). Retrieved from http://www.silesfor.com/en/rotating-extractor/.

“Starch hydrolysis by amylase” (N.D.). Retrieved from

http://www.eng.umd.edu/~nsw/ench485/lab5.htm.

“Stedman.” (1834). Retrieved from

http://www.stedman-machine.com/hammer-mill-crushers.html.

“Sterilizing units, steam, bulk.” (n.d.) Retrieved from

http://www.who.int/medical_devices/innovation/hospt_equip_32.pdf.

“Technical memorandum on cassava starch”. (n.d.) Retrieved from

http://www.starch.dk/isi/starch/cassavastarch.asp

“The facts on monosodium glutamate” (2002). Retrieved from

http://www.eufic.org/article/en/artid/monosodium-glutamate/

“What is a vibrating screen?” (n.d.) Retrieved from

http://www.wisegeek.com/what-is-a-vibrating-screen.htm.

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D. JOURNAL

Agarwal, P. et. Al. (2013). Application of Heat Exchangers in Bioprocess Industry: A

Review. International Journal of Pharmacy and Pharmaceutical Sciences, vol. 5
issue 1, 2014.

Ajao, K.R. et. Al. (2009). Performance evaluation of a locally fabricated mini cassava
flashdryer.Ajao, K.R. and Adegun, I.K. (2009). Performance evaluation of a locally
fabricated mini cassava flash dryer. Journal of Agricultural Technology 5(2): 281-
289.

Ariff, A. B., Hii, L.S., Tan, J. S. & Ling, T. C. (2012). “Pullulanase: Role in Starch
Hydrolysis and Potential Industrial Applications,” Enzyme Research, vol. 2012,
Article ID 921362, 14 pages, 2012. doi:10.1155/2012/921362.

Baskar, R. et. Al. (2014). Production of L-glutamic acid with Corynebacterium glutamicum
and pseudomonas reptilivora: a study on immobilization and reusability. Avicenna
Journalof Medical Biotechnology, 6(3), 163-168.

Cordeiro, C. A., Martins, M., Leal, M. M. & Luciano, A. B. (2002). Production and

properties of alpha-amylase from thermophilic Bacillus sp. Brazilian Journal of

Microbiology, 33(1),57-61. https://dx.doi.org.

De Souza, P. M., & De Oliveira, M, P. (2010). Application of microbial α-amylase in

industry – A review. Brazilian Journal of Microbiology, 41(4), 850–861.

http://doi.org/10.1590/S1517-83822010000400004.

Gayte-Sorbier, C. B. (1985). Journal of Food Science. Stability of GlutamicAcid and

Monosodium Glutamate Under Model System Conditions: Influence of Physical
and Technological Factors, Vol.50.

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G.C.Grimwood, M. P. (554-558). Observations on Centrifugal Operation Part 2.

Gomes, E.; Guez, M.A.U. Martin, N. & Silva, R. (2007). Thermostable enzymes: sources,

production and industrial application. Quim. Nova, 30, 136-145.

"History of glutamate production1,2,3." (2009). The American Journal F Clinical Nutrition

728S-732S 90.3.

Ibrahim, N. A., M. El-Hossamy, M. S. &Morsy, B. M. Eid. (2004). Optimization and

Modification of Enzymatic Desizing of Starch-Size. Polymer-Plastics Technology
& Engineering. 43:519-539.

I.M. Demiate, V. K. (2011). Cassava starch in the Brazilian food industry, 388-397.

K., & A. (2015). Production, Characteristics and Industrial Application of Fungal

Glucoamylase. Brazilian Journal of Microbiology. Retrieved July 20, 2016, from

https://www.researchgate.net.

Kauffman, George. (2004). "The monosodium glutamate story: The commercial

production of MSG and other amino acids."

Liu, G. et. Al. (2014). Pullulanase. Hydrolysis Behaviors and Hydrogel Properties of

Debranched Starches from Different Sources.

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Marquet, M. et al. (1986). Glutamate excretion by Corynebacterium glutamicum: A study

of glutamate accumulation during a fermentation course. Volume 25, Issue 3, pp.

220-223. Springer-Verlag.

Norouzian, D. et. Al. (2006). Fungal glucoamylases. Biotechnology Advances 24 (2006),

80– 85.

Pratt, S. (n.d.). Thermo Fisher Scientific. Understanding Temperature Control in

Bioreactor System.

Shyamkumar, R., Moorthy, I. M. G., Ponmurugan, K., &Baskar, R. (2014). Production of

L-glutamic Acid with Corynebacterium glutamicum (NCIM 2168)
and Pseudomonas reptilivora (NCIM 2598): A Study on Immobilization and
Reusability. Avicenna Journal of Medical Biotechnology, 6(3), 163–168.

Sundarram, A. et. Al. (2014). α – Amylase Production and Applications: A Review.

Science and Education Publishing.

Torchia, Mark G. (1999). Enzymes can be used to hydrolyze starch at low temperatures.

Chemtech. 29:3-9

Ukwuru, M.U. &Egbonu, S.E. (2013). Recent development in cassava-based products

research. Academia of Journal of Food Research.

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Xu, X. et. Al. (2008). Expression of a fungal glucoamylase in transgenic rice seeds. Protein

Expression and Purification, 61 (2008), 113–116.

Zhang, H. et. Al. (2010). Preparation of resistant starch by hydrolysis of maize starch with

pullulanase. H. Zhang, Z. Jin / Carbohydrate Polymers, 83 (2011), 865–867.

Gruenberg, A. et. Al. (2013). Beyond Growth Rate 0.6: Corynebacterium Glutamicum

Cultivated in Highly Diluted Environments

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III. REVIEW OF RELATED LITERATURE

A. PRODUCT LITERATURE

Monosodium Glutamate

In an internet source entitled “The Facts on Monosodium Glutamate” retrieved from

http://www.eufic.org/article/en/artid/monosodium-glutamate/ (2002), Glutamate which is naturally

occurring in amino acid that can be found in almost all foods is the sodium salt of glutamic acid.

Also, the glutamate can also be produced by the body that is an essential role in normal body

function. The monosodium glutamate enhances the flavor of the food and adds the “umami” .

usually, monosodium glutamate is

Monosodium glutamate enhances the flavor and adds the “umami” taste in food. Typically,

it is added to savory prepared and processed foods. It contains about one-third of the sodium of table

salt and when used it can reduce the total sodium in recipe by 20% to 40%. (eufic.org)

According to the book entitled “Food Additive Toxicology” by Maga (1994).

Derived from glutamic acid is Monosodium Glutamate or MSG. MSG is naturally occurring

in any protein-containing food because usually, one of the major amino acids that is present in most

types of protein is glutamic acid. In food processing, protein can be hydrolyzed via numerous

common means. Free glutamic acid is then formed, which can readily react with sodium ions

resulting to the production of Monosodium Glutamate. Generally, MSG is also found in protein-rich

food.

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From an internet source entitled “Monosodium Glutamate (MSG)– Third Spice” retrieved from

http://www.starch.dk/isi/bio/msg.asp,

In the manufacturing process, a fermentation step is introduced in a chemical synthesis,

similar with manufacturing L-ascorbic acid. However, instead of a racemic mixture of left and right-

handed enantiomers as would be the result of a pure chemical reaction, microorganisms are utilized

to produce occurring L-form naturally. Bacterial species such as Corynebacterium glutamicum can

be used in the fermentation process. Manufactured MSG contains over 99% of naturally

predominant L-glutamate form.

The flavor-intensifying property of Monosodium glutamate (C5H3NO4Na H2O)3 was

discovered by Dr. Ikeda in 1908. He described that it is crystalline white, transparent and very

soluble in water (73% w/v). Now it is used commercially as a flavor enhancer, usually in

combination with nucleotides inosinate, to provide an expansion and extension of taste in processed

food such as soups, biscuits, noodles, Chinese food, meat and vegetable processing etc. There are

three types of monosodium 24 glutamates, the L, D, and LD type, but only the L-type has the flavor

intensifying property. The concentration of monosodium glutamate used in salted food is generally

between 0.2-0.5%. Glutamic acid mother liquor in MSG production is also used in the manufacture

of sauce and as soil conditioner, fertilizer etc. In the minds of many, the safety/toxicity of

monosodium glutamate (MSG) is considered a controversial subject. MSG is a natural component

in many food such as mushrooms, tomatoes and peas. The free glutamate consumed daily as MSG

typically equals about 1/1000 of the total glutamate present in the body. Generally, the main use of

MSG is as a food ingredient, and so its safety when used in the diet is the most important aspect

when consuming MSG.

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 Monosodium Glutamate (MSG) is an important flavoring agent, but has no flavor of its own.

It is used to improve the flavors of the food. There are three forms of glutamic acid, but monosodium

salt of L-glutamic acid has a flavor-accentuating capacity. The constituent of all common proteins

is the glutamic acid and it is produced by a process in which the principal steps are concentration

and collection of the filtrate, hydrolysis usually with caustic soda, neutralization and acidification

of the hydrolysate, partial removal of the inorganic salts, crystallization, separation, and purification

of the glutamic acid. L-glutamic acid can be acquired using fermentation of carbohydrates with

Micrococcus glutamics or Brevihacteriumdivaricatum. (Chemical and Process Design Handbook).

Vietnamese companies produced MSG using cassava starch (75%) and byproducts from the

sugar industry (25%) as raw material. They used old technologies that leads to low conversion rates

of cassava starch. To produce MSG, cassava starch was supplied from processing centers through

wholesalers. The required quality of starch is 90-92%. At that time, starch processing centers had

many problems, such as fluctuating starch quality due to different technologies and root availability.

MSG companies had to store starch for production, but absence of good storage facilities, variable

consistency and low quality of starch caused quality losses. (Cassava Production, Processing and

Marketing in Vietnam, CIAT).

MSG is known flavor enhancing agent. The largest producer and consumer of MSG in the

world is China. It is done by a microbial fermentation of starch or sugar (molasses) in the presence

of ammonium salts. The most commonly used bacteria is Brevibacterium glutamicum which

converts sugar to glutamic acid. (M.S. Palaniswami and K.V. Peter, 2008).

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 B. RAW MATERIALS LITERATURE

1. Cassava Root Crop Definition

Based on an internet source entitled “Cassava” retrieved from

https://www.hort.purdue.edu/newscrop/CropFactSheets/cassava.html,

Cassava is grown for its enlarged starch-filled roots that contains nearly the maximum

concentration of starch on a dry weight basis. Also, from Encyclopedia of Food and Culture (2003),

The freshly peeled cassava root crop contains about 30 to 35% of starch (carbohydrate) and very

little protein (1 to 2 percent) and fat (less than 1 percent).

It has naturally significant amounts of calcium (50 mg / 100 g), phosphorus (40 mg / 100 g)

and vitamin C (25 mg / 100 g). The quality of protein is relatively good, and the starch is highly

digestible.

From the journal entitled “Recent development in cassava-based products research”,

The bulky roots of the cassava contain 60 to 65% moisture. To lessen the moisture content,

it is processed to dry form, and in turn it will be converted into a more durable and stable product.

Similar to other root and tuber crops, cassava root crop contains up to 65% water which is the most

probable major limitation to improve its potential uses. However, the chemical composition of

cassava root crop shows that it is a major source of carbohydrates. Therefore, it is very crucial to

convert cassava into exploitable products.

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According to the book “Energy Plant Species: Their Use and Impact on Environment and

Development” by Bassam, N. EL.,

Cassava’s young tubers are known to contain much less starch than the older tubers. But as

the tuber becomes older, it tends to become more lignified and fibrous and the starch content tends

to decrease or remain constant as a percentage of the dry weight of the tuber.

Found from the book entitled “Cassava flour and Starch: Progress in Research and Development”

by Dufour, et.al.,

With the help of modern technology and by choosing the most suitable bacteria, the

conversion rate of cassava starch to MSG can be increased.

Based on the journal entitled “Recent development in cassava-based products research”,

The processing of cassava tubers in large scale production of starch yields waste in solid and

liquid form. Fibrous slurry contains about 15 to 20% of the cassava tubers processed. On a dry

weight basis, though, this contains 15 to 70% starch. The starch is hydrolyzed into glucose by boiling

with hydrochloric or sulphuric acid solution in closed converters under pressure. The glucose is

filtered and converted into glutamic acid by bacterial fermentation. The resulting glutamic acid is

refined, filtered and treated with caustic soda to produce monosodium glutamate, which is then

centrifuged and dried in drum driers. The finished product (MSG) is usually at least 99% pure.

From an internet source, it says that Cassava (ManihotesculentaCrantz), popularly known as

tapioca, contains about 80% starch of which 60% can be recovered on a dry weight basis. Starch

22 | P a g e
hydrolyzates of different dextrose equivalents (D.E.) were peppered by the enzymatic hydrolysis of

dried cassava starch. The rate of cell growth was directly related to the D.E. values of the

hydrolysate. The higher the D.E. value, the lesser the time to achieve optimum growth. Maximum

glutamic acid production at 8.8 mg/ml was obtained when a suitably diluted hydrolysate having the

dextrose equivalent (D.E.) 85-90, was supplemented with NaNO3 (0.7%), KHZPO4 (0.18 w/v%)

and a mineral solution containing Mg, Mn, Fe, Zn, NaCl and 0.1% v/v corn steep liquor, and was

fermented for 60 hours under agitation at 30°C. Highest conversion rate at 33.9% was obtained with

the hydrolysate having 45-50 D.E. value. In line with this, supplementation of the hydrolysate with

inorganic nitrogen alone was not effective.

According to the book entitled “Quality Management Manual for Production of high quality Cassava

flour” by Dziedzoave, et. al (2010).,

Cassava root crops are cheap and excellent source of dietary carbohydrate due to its high

starch content of about 60%. Although the roots are rich in niacin, riboflavin, thiamine, and calcium,

they nevertheless contain low levels of protein. It can only be improved by incorporating high

protein-containing root crops such as soybeans and cowpeas in cassava diets as a means of protein

fortification.

Lastly, according to an internet source entitled “Cassava Nutrient Requirements,”

The growth of the root crop is favourable at a pH range of 5.5 to 6.5.

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 2. Corynebacterium Glutamicum

According to an internet source entitled “Corynebacterium Glutamicum” retrieved from

http://web.mst.edu/~microbio/BIO221_2007/C_glutamicum.htm,

The Corynebacterium Glutamicum is an important fermentative bacterium most widely used

for the production of Monosodium Glutamate or MSG. It is a natural producer of glutamic acid. C.

glutamicum is a Gram positive, facultatively anaerobic, heterotrophic bacterium with an irregular

rod shape in a V-formation. It is found in soil, animal feces, fruits and vegetables, and also non-

pathogenic. Though it was originally isolated for its ability to produce massive amounts of glutamic

acid, C.glutamicum and closely related organisms have been developed for the production of most

of the biogene amino acids, nucleotides, and vitamins.

From the book entitled “Microbial Production of L-Amino Acids” by Faurie, et.al.,

The glutamate producing bacteria Corynebacterium glutamicum has a unique ability to

produce significant amounts of L-glutamate directly from cheap sugar and ammonia. A fermentation

process emerged and is used, and this allow drastic reduction of the production cost of MSG which

has been produced industrially by extraction from protein-hydrolysates or chemical synthesis until

then.

Moreover, for many years now, MSG has been widely used in the food industry as a food

enhancer. MSG is produced by growing C. glutamicum in a medium containing molasses, sugars or

starch as a fermentation substrate. The excreted glutamic acid from the fermentation process is

filtered out of the medium and neutralized, producing Monosodium Glutamate. Further purification,

crystallization and drying yields a white powder of MSG which is then ready to use as a flavor

24 | P a g e
enhancer. MSG is one of the most common products made using C. glutamicum. However, C.

glutamicum is also used for the production of many other amino acids and vitamins.

From an article entitled “Production of L-glutamic acid with Corynebacterium glutamicum and

Pseudomonas reptilivora: A Study on Immobilization and Reusability” by Shyamkumar, et. al

(2014),

In a biotechnological process, the species of Corynebacterium are used for production of L-

glutamic acid by the aid of submerged fermentation, and a number of fermentation techniques have

been used for the production of L-glutamic acid.

Gathered from the book entitled “Corynebacterium glutamicum: biology and biotechnology” by

Tatsumi, et. al (2003).,

Currently, the utilization of C. glutamicum to produce glutamate is a well-established

technology. The carbon sources usually used to produce bulk quantities of L-glutamate or L-lysine

are beet molasses or cane and starch hydrolysates, as well as raw sugars. This is necessary to meet

the market demand for L-glutamate or L-lysine.

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Also, stated in an article entitled “Microbial Production of 7 Types of Amino Acids from

http://www.biologydiscussion.com/amino-acids/microbial-production-of-7-types-of-amino

acids/10356,”

L-Glutamic acid was the first amino acid to be produced by microorganisms. The

original bacterium, Corynebacterium glutamicum, that was first used for large scale manufacture of

glutamic acid, continues to be successfully used even today. The other important organisms,

although used to a lesser extent due to low yield, employed for glutamic acid production belong to

genera Micro bacterium, Brevibacterium and Arthrobacter.

All these organisms have certain morphological and physiological characters comparable to

C. glutamicum. Biochemically, glutamic acid-producing bacteria have a high activity of glutamate

dehydrogenase and a low activity of α-ketoglutarate dehydrogenase. They also require the vitamin

biotin.

From the book “Encyclopedia of Food Microbiology,”

Industrial amino acid fermentation using C. glutanicum is performed using batch, fed batch,

repeated fed- batch or continuous fermentation. In all cases, the concentration of the carbon source

is maintained at low levels to limit oxygen uptake rate and to avoid excessive formation of by-

products. The major advantage of the fed-batch process is the high product concentration that can

be achieved. when the maximum filling degree is reached, the vessel is not emptied completely, but

an appropriate volume (10-20%) retains in the reactor as inoculum for the next cycle.

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 Corynebacterium glutamicum, a bacterium discovered in 1956 for its natural capacity to

accumulate L-glutamate extracellularly, is currently used for the industrial production of L-

glutamate (about 3 million tons in 2014) and, thus, is considered an excellent host for the production

of glutamate-derived amino acids such as L-ornithine, L-citrulline, L-argi-nine, L-proline and

GABA.

From the book entitled “Corynebacterium glutamicum: Biology and Biotechnology,”

C. glutamicum has several industrially important characteristics such as its high growth yield

even under condtions of high sugar concentration. It has one drawback; its optimal growth

temperature is around 30 C which is lower than that of E.coli. For this reason, the use of C.

glutamicum may be economically disadvantageous, especially in tropical regions, because of the

substantial cost of utilities necessary to maintain the optimum fermentation temperature.

From the journal entitled “Production of L-Glutamic acid by Corynebacterium glutamicum DSM

20300T and Arthrobacter globiformis MTCC 4299 using fruits of Muntingia calabura Linn.,”

After optimization of physicochemical parameters, the highest production of glutamic acid

was obtained at 15.1 mg/ml with Corynebacterium glutamicum DSM 20300T at pH 7.0, temperature

300C was maintained for 48h incubation time with urea, biotin and penicillin; optimum

concentration were 2.0g/l, 1µg/l and 1U/ml respectively.

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From a journal entitled “Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly

diluted environments,”

“Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct

amplifier for the productivity of any growth coupled or decoupled production process. Recently, it

has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a

50% higher growth rate compared to a 1 L batch cultivation [Grünberger et al. (2012) Lab Chip].

We here compare growth of C. glutamicum with glucose as substrate at different scales covering

batch cultivations in the liter range down to single cell cultivations in the picoliter range. The

maximum growth rate of standard batch cultures as estimated from different biomass quantification

methods is mu = 0.42 ± 0.03 h(-1) even for microtiter scale cultivations. In contrast, growth in a

microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth

rate of mu = 0.62 ± 0.02 h(-1). When in the same perfusion system cell-free supernatant from

exponentially grown shake flask cultures is used the growth rate of single cells is reduced to mu =

0.47 ± 0.02 h(-1). Likewise, when fresh medium is additionally supplied with 5 mM acetate, a

growth rate of mu = 0.51 ± 0.01 h(-1) is determined. These results prove that higher growth rates of

C. glutamicum than known from typical batch cultivations are possible, and that growth is definitely

impaired by very low concentrations of byproducts such as acetate.”

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 3. Alpha-amylase

Based on a study by Sundarram et al. entitled “α – Amylase Production and Applications: A

Review”,

α-Amylase (E.C.3.2.1.1) is a hydrolase enzyme that catalyzes the hydrolysis of internal α-1,

4-glycosidic linkages in starch to yield products like glucose and maltose. It is a calcium

metalloenzyme; it depends on the presence of a metal co-factor for its activity. There are 2 types of

hydrolases: endohydrolase and exohydrolase. Endohydrolases act on the interior of the substrate

molecule, meanwhile exohydrolases act on the terminal non-reducing ends.

Based on an internet source entitled “Starch Hydrolysis by Amylase” gathered from

http://www.eng.umd.edu/~nsw/ench485/lab5.htm,

“The fungal alpha-amylase belongs to the saccharifying category and attacks the second

linkage from the non- reducing terminals (i.e. C4 end) of the straight segment, resulting in the

splitting off of two glucose units at a time. Of course, the product is a disaccharide called maltose.

The bond breakage is thus more extensive in saccharifying enzymes than in liquefying enzymes.

The starch chains are literally chopped into small bits and pieces. Finally, the amyloglucosidase

(also called glucoamylase) component of an amylase preparation selectively attacks the last bond

on the non-reducing terminals. The type to be used in this experiment can act on both the alpha-1,4

and the alpha-1,6 glucosidic linkages at a relative rate of 1:20, resulting in the splitting off of simple

glucose units into the solution. Fungal amylase and amyloglucosidase may be used together to

convert starch to simple sugars. The practical applications of this type of enzyme mixture include

the production of corn syrup and the conversion of cereal mashes to sugars in brewing.”

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From an internet source entitled “Application of Microbial α-amylase in industry – a review”,

gathered from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769773/,

“α-Amylases have potential application in a wide number of industrial processes such as

food, fermentation, textile, paper, detergent, and pharmaceutical industries. Fungal and bacterial

amylases could be potentially useful in the pharmaceutical and fine-chemical industries. However,

with the advances in biotechnology, the amylase application has expanded in many fields such as

clinical, medicinal and analytical chemistry, as well as their widespread application in starch

saccharification and in the textile, food, brewing and distilling industries. The most widespread

applications of α-amylases are in the starch industry, wich are used for starch hydrolysis in the starch

liquefaction process that converts starch into fructose and glucose syrups The enzymatic conversion

of all starch includes: gelatinization, which involves the dissolution of starch granules, thereby

forming a viscous suspension; liquefaction, which involves partial hydrolysis and loss in viscosity;

and saccharification, involving the production of glucose and maltose via further hydrolysis.”

According to the Journal entitled Production and properties of alpha-amylase from thermophilic

Bacillus sp.,

It states that the a-amylases produced by different Bacillus species vary not only in their

types (saccharifying or liquefying) but also in the range of pH and temperature for their optimal

activity. The bacterial source of the enzyme is usually from either Bacillus amyloliquefaciens or

Bacillus licheniformis, the latter now being of greater industrial importance.

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 Torchia (1999) found that starch could be hydrolyzed at low temperatures with the help of

α-amylase. His study showed that with the help of α-amylase, starch could be hydrolyzed at

temperatures where the reaction would not normally occur.

Ibrahim et al. (2004) varied the pH of the solution in order to find the optimal condition for

starch hydrolysis. They found a pH of 7 was optimal for their solution mixture and application. The

optimal ranges of pH and temperature could change depending on substrate concentration, the

amount of enzyme present, and the other molecules present in the solution.

From an internet source retrieved from https://www.megaessays.com/viewpaper/65926.html,

It states that the enzyme alpha-amylase is a catalyst to break up starch to from glucose.

Without being consumed in the reaction, enzymes are catalysts that speed up the rate of the reaction.

They achieve this by combining with a substrate and then separating from it form products.

Environment factors that affect enzyme reactions include temperature and pH. The optimal

temperature and pH at which the reaction will occur coincides with the point where the rate of

reaction is at maximum value. The purpose of this experiment was to determine the ideal

temperature, pH, and fastest rate for alpha-amylase. This was performed using I KI as an indicator

of the complex and a spectrophotometer to read the measurements at a temperature range from 15-

70°C and a pH range of 4.0-6.5. Readings of starch concentration were then taken over a twenty-

minute interval. The experimental results indicate that the optimal temperature is 50° and the optimal

pH is 5.0.

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 4. Fungal Glucoamylase

According to the study of Norouzian, et al., entitled “Fungal Glucoamylase”,

“Glucoamylase (GA), also known as amyloglucosidase or g-amylase (EC 3.2.1.3), is a

biocatalyst capable of hydrolyzing a-1,4 glycosidic linkages in raw (sparsely soluble) or soluble

starches and related oligosaccharides with the inversion of the anomeric configuration to produce h-

glucose. In addition to acting on a-1,4 linkages, the enzyme slowly hydrolyzes a-1,6 glycosidic

linkages of starch (Weil et al., 1954; Pazur and Ando, 1960; Koshland, 1953; Fierobe et al., 1998).”

“Glucoamylases of fungal origin usually occur in multiple forms (Manjunath et al., 1983;

Miah and Ueda, 1977; Pazure et al., 1971) and these multiplicities may be related to either the

activity of protease produced along with GA concomitantly or, as pointed out by Svensson et al.

(1986), the forms may be derived by different secondary processing. Fungal glucoamylases have

two domains, namely a catalytic domain and a starch binding domain. The two domains are

connected by an O-glycosylated polypeptide linker located at the N-terminus. The starch binding

domain of GA plays an active role in hydrolyzing raw starch and supports the enzyme adsorption to

the cell wall where local increase of enzyme concentration may result in enhanced glucose flow to

the cell (Kaneko et al., 1996; Neustroev et al., 1993). Partial or total proteolytic excision of the starch

binding domains leads to the formation of GA capable of degrading soluble starch only (Cutinho

and Reilly, 1997). Therefore, an important objective to enhance GA activity toward raw starch is to

reduce protease production during GA fermentation. To accomplish this, the operational approaches

that have been successful include: a) cell immobilization (Liu et al., 1998); b) growth morphology

(Gregg et al., 2001); c) pH control (Bertolin et al., 2003); d) nutrient control (Pedersen et al., 2000);

e) bioreactor configuration (Pandy and Radhakrishna, 1992); and f) the use of protease inhibitors

(Xu et al., 2000).”

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From the study of Xu, et al. entitled “Expression of a Fungal Glucoamylase in Transgenic Rice

Seed,”

“Glucoamylase, which catalyses the hydrolysis of the a-1,4 glycosidic bonds of starch, is an

important industrial enzyme used in starch enzymatic saccharification. In this study, a gluco-amylase

gene from Aspergillus awamori, under the control of the promoter of seed storage protein Gt1, was

introduced into rice by Agrobacterium-mediated transformation. Significant glucoam-ylase activity

was detected specifically in the seeds but no other tissues of the transgenic rice lines. The highest

enzymatic activity was found in the transgenic line Bg17-2, which was estimated to have about

500units per gram of seeds (one unit is defined as the amount of enzyme that produces 1l mol of

reducing sugar in 1 min at 60°C using soluble starch as substrate).

For industrial applications, starch is usually required to be converted to glucose, the

fermentable sugar. This conversion process of starch is normally achieved by two enzymatic

hydrolysis steps using two key enzymes, a-amylase and glucoamylase. In the first step, starch slurry

is liquefied into maltodextrins at high temperature by a-amylase, and in the second step, the

maltodextrins are further converted to glucose by glucoamylase.

We found that the enzyme has a broad range of optimum pH, from 3.6 to 5.4. The activity

reduced rapidly when pH got higher than 5.4. However, there was still significant residue activity

around pH 7.0. The enzymatic activity increased with the increase of temperature up to 60 °C.

The enzyme produced by its native host fungi was reported with optimum activity at pH 5

and optimum temperature at 60 °C”

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According to a journal entitled “Production, Characteristics and Industrial Application of Fungal

Glucoamylase,”

It states that the optimum production of glucoamylase is observed at a range of pH from 4.0

to 5.0 and temperature 30 to 40°C with the incubation period of 4 to 5 days. Optimum activity is

also found at wide range of pH 4 to 8 and temperature 40 to 70°C. Most of the fungi produce several

isoenzymes that have different molecular weights ranges from 40 to 90 kDa. The majority of fungal

glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-

binding domain by an O-glycosylated linker region. The catalytic domain folds as a twisted (α/α)6-

barrel containing a hexa-helical hairpin toroidal structure while starch binding domain folds as an

antiparallel β-barrel having two independent substrate binding sites. The present review focuses on

recent findings on glucoamylase production, characteristics, structure, mechanism of action and

industrial applications.”

Another journal entitled “Production and characterization of glucoamylase from fungus Aspergillus

awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources,”

Also says that glucoamylase is widely used in the food industry to produce high glucose

syrup, and also in fermentation processes for production beer and ethanol. In this work the

productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces

cerevisiae, produced in submerged fermentation using different starches, was evaluated and

characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mg protein

or 2.9 U/mg biomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase

presented optimum activity at temperature of 55°C, and, in the substratum absence, the

34 | P a g e
thermostability was for 1h at 50°C. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability

between 5.0 and 7.0. The half-life at 65°C was at 30.2 min, and the thermal energy of denaturation

was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme’s preference for the

substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most

susceptible to the enzymatic action.”

According to Gomes et al. (2007), in the industrial processes to hydrolyze the starch, at the

saccharification step, the system must have the pH between 4.2-5.0. Additionally, the enzymes

needthe material to be cooled to temperatures below 60 ºC.

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 5. Pullulanase

Based on a study by Liu, et al. entitled “Pullulanase Hydrolysis Behaviors and Hydrogel Properties

of Debranched Starches from Different Sources,”

“Pullulanase treatment resulted in an obvious reduction in viscosity for all tested starches

except debranched pea starch (DBPeS). The average molecular weight of small fragments of

debranched potato starch (DBPoS) was higher than that of the other samples, and longer lateral

chains tended to be cleaved by pullulanase for DBPoS. The results of the solubility and water-

holding capacity analyses indicated the DBS starches were capable of capturing water to form

hydrogels. DBPoS was more easily digested by gastrointestinal amylase, whereas its pea-source

counterpart showed the opposite trend. The results confirmed that generation of short linear glucan

chains by pullulanase hydrolysis effectively improved solubility and water-holding capacity,

which contributed to their hydrogel-forming properties.”

According to the study by Zhang et al. entitled “Preparation of resistant starch by hydrolysis of

maize starch with pullulanase,”

“When the amount of pullulanase was lower than 12 ASPU/g, The RS content showed a

steady increase as the level of pullulanase in the reaction mixture increased. The maximum RS

content was observed at the level of 12 ASPU/g of pullulanase (44.7 g/100 g). Results of these

experiments indicated that at this level of pullulanase, all maize starch was saturated by

pullulanase. It is therefore that the optimum amount of pullulanase under this condition was judged

to be 12 ASPU/g. In a previous report, optimum amount of pullulanase was 10 ASPU/g (Wu et

al., 2009), probably due to the difference in substrate.”

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From a journal entitled “Pullulanase: Role in Starch Hydrolysis and Potential Industrial

Applications,”

It states that Pullulanase with EC 3.2.1.41 or also known as α-dextrin 6-glucanohydrolase,

pullulan 6-glucanohydrolase, limit dextrinase, and amylopectin 6-glucanohydrolase is derived

from various microorganisms such as Bacillus acidopullulyticus, Klebsiellaplanticola, Bacillus

deramificans, thermophilic Bacillus sp. AN-7, Bacillus cereus FDTA-13,

and Geobacillusstearothermophilus. Microbial pullulanase attracts more interest because of its

specific action on α-1,6 linkages in pullulan, a linear α-glucan consisting essentially of

maltotriosyl units connected by 1,6-α-bonds. The structure of pullulan produced by the

fungus, Aureobasidium pullulans. Enzymes hydrolysing pullulan are classified into groups based

on the substrate specificities and reaction products. Pullulanases type I, which are able to hydrolyse

efficiently the α-(1,6) glucosidic bonds in pullulan and branched polysaccharides, have been

extensively studied. Pullulanases type II, also called amylopullulanases, are prominent in starch

processing industry due to the specific debranching capacity of hydrolysing either α-(1,6) or α-

(1,4) glucosidic linkages. This enzyme debranch pullulan and gives maltotriose as final product

and it also attacks α-(1,4) bonds in starch, amylose, and amylopectin. Both pullulanases type I and

type II attach α-1,6 glucosidic linkages in pullulan, producing maltotriose while these enzymes are

unable to degrade cyclodextrin.”

According to a patent entitled “Pullulanase, methods of producing pullulanase and methods of

saccharification of starch using pullulanase,”

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 It says that the pullulanase of the invention is enzymatically active in a wide pH range and

heat stable. The pullulanase of the invention can be used along with glucoamylase produced by

Aspergillus niger in the same condition, a combination which promotes saccharification of

liquefied starch and gives an increased yield of glucose. In addition, the pullulanase of the

invention can be used along with β-amylase or various oligosaccharide-producing amylase to

efficiently produce various oligosaccharides. The method of producing pullulanase should

comprise of the following (a) culturing in a culture medium the bacterium, accession number Ferm

BP-3761, or mutants thereof Microbacteriumimperiale capable of producing said pullulanase

wherein the pullulanase has the following characteristics:i) a working pH of 4-9; ii) an optimum

temperature of about 60° C.; iii) a pH stability of pH 4.5-10; and iv) being inactivated by about

10% at 50° C., by about 30% at 55° C or by about 60% at 60° C. when a solution containing

pullulanase is heated for 10 minutes; andv) not being inactivated when a pullulanase solution

containing 1×10-2M CaCl2 is heated at 50° or 55° C. for 10 minutes; b) allowing the pullulanase

to accumulate in the culture medium; an c) isolating the pullulanase from the culture medium.

Pullulanase is produced by various microorganisms such as B. acidopullulyticus,

Klebsiellaplanticola, B. deramificans, B. cereus FDTA-13, and Geobacillusstearothermophilus

(Hii et al., 2012).

According to a research paper by Arbakariya, B. A., et al., entitled “Pullulanase: Role in Starch

Hydrolysis and Potential Industrial Applications”,

“Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyze the

α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which

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enables a complete and efficient conversion of the branched polysaccharides into small

fermentable sugars during saccharification process. The industrial manufacturing of glucose

involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action

of α-amylase; saccharification, which results in further transformation of maltodextrins into

glucose. During saccharification process, pullulanase has been used to increase the final glucose

concentration with reduced amount of glucoamylase.”

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 C. PROCESS LITERATURE

RAW MATERIAL PREPARATION LITERATURES

1. Cassava Roots Preparation

Cassava starch production is a physical procedure in which starch in cassava root is

separated from fiber, protein and inorganic and organic impurities. In this procedure, cassava

starch is separated from its suspension in water. (Bangke N., 2008).

Peeling and washing

Food and Agriculture Organization of the United Nations has developed a process in

washing and peeling the roots of Cassava Root Crops. In order to remove the outer skin of the

roots and also the adhering dirt, washing is done provided that the roots are sufficiently ripe. Only

the outer skin is removed even without the use of brushes. The mechanical washer that is immersed

in water is a perforated cylindrical tank. A spiral brush propels the roots while they are subjected

to vigorous scrubbing in order to remove all dirt. A centrifugal pump is fitted to one end of the

machine and connected to a series of jets arranged along the carrying side of the brush. These jets

produce a countercurrent to the flow of the roots, ensuring that they receive an efficient washing.

(Bencini,1991) stated that peeling must be done to remove the inedible outer parts of the

roots consisting of the corky and the cortex that are known to contain most of the toxic cyanogenic

glucosides, the ratio of glucocides compared to the starchy flesh varying between 5-10:1. Hence,

for a root composed of 15% peel with a total cyanide content of 950 mg/kg (fresh weight basis)

and 35 mg/kg in the flesh, 83% of the total cyanide is removed by peeling.
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 Dziedzoave, et. al (2010) emphasized in his book entitled “Quality Management Manual

for Production of high quality Cassava flour” . that no matter what method is used, there must be

no fragments of peel after the process of peeling. After peeling , it is transferred into washing tank

, drums or pans and immerse peeled roots under water for immediate washing. Prolonged exposure

to air of the peeled roots may cause discoloration. Rewashing of the peeled roots is also

recommended until complete absence of dirt, sand, sticky mud, fecal matter, or offensive odor.

Moreover, new cassava root is put into the drying cleaner of soil-removal and peeling, in

which a screw guide plate settled on an internal mass of the chamber enclosure, it pushes forward

the cassava root in the barrel confine pivots. Cassava roots collide and rub themselves further

against the divider to remove soil and peel off skin. After soil removal and peeling of cassava

roots, these go into the tumult water cleaner. The U-tank water cleaner with fastened propeller is

utilized in the initial step of water cleaning. Water goes into the U-tank in which a pole, with screw

propeller, is equipped. Cassava roots are fed, pivoted to the pole and pushed forward to clean soil

and peel once more. From that point, cassava roots proceed to the second step water cleaning.

Cylinder cleaner is utilized as a part of the second water cleaning. The cylinder cleaner comprises

of three clearing areas: rough washing, bathe washing and intensive washing, where water is

sustained and cassava roots keep running alongside the barrel and proceed to take out soil and peel

in the status of spaying, flushing, bathing, rubbing, and cleaning. Water which serves as a cleaning

media is utilized at the rate of 1:4. Subsequent to cleaning the soil completely, 95% of peel is

removed and the clean cassava roots move to squashing or crushing area (Bangke N., 2008).

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Crushing

Based on an article by the Food and Agriculture Organization of the United Nations , they

emphasized the importance of crushing the cassava root crops . It is necessary break all the cell

walls in order to release the starch granules. There are ways this can be done . it can be by

biochemical or mechanical action . in biochemical method which does not give complete yield and

its quality is not good, an old method, allows the roots to ferment at a certain stage . then they are

pounded to a pulp and the starch is washed with water. While mechanical action is done by slicing

the roots and then rasping , grating or crushing them, which breaks the flesh into a fine pulp.The

cell walls are torn up and the whole of the root is turned into a mass in which the greater part, but

not all, of the starch granules is released By pressing the roots against a swiftly moving surface

provided with sharp protrusions. The percentage of starch set free is called the rasping effect. Its

value after one rasping may vary between 70 and 90 percent: the efficiency of the rasping operation

therefore determines to a large extent the overall yield of starch in the processing. It is difficult to

remove all the starch, even with efficient rasping devices, in a single operation. Therefore, the pulp

is sometimes subjected to a second rasping process after screening

Bangke (2008) uses a hammer crusher to crush the tissue of cassava makes the little

granular of starch deteriorate and leave from the roots. The hammer knives, hammer jaw, teeth

,grill disc and rubbing plate strike, rasp cut and squeeze the continuous fed cassava roots which

release starch particles that turn into raw starch milk when mixing with the water as a media in the

proportion of 1:1. The second step of crushing process is applied for full breaking down cassava

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root tissue which becomes finer granular of starch that separates thoroughly to increase the

extraction rate. The second step of crushing process is applied for full breaking down cassava root

tissue which becomes finer granular of starch that separates thoroughly to increase the extraction

rate. The requirement for the first crushing ensures the starch milk to go throughΦ8.0 to 16.0mm

pore of basket sieve of centrifuge while the second crushing enables starch milk to flow through

Φ1.2~1.4mm pore of basket sieve of centrifuge.

Separation

A shaking screen will be utilized to separate the starch from the pulp. It consists of a slightly

inclined horizontal frame, 4 meters in length and covered with gauze, which is put into a lengthwise

shaking motion in short strokes by means of an eccentric rod. The fresh pulp, after being mixed

with water in distribution tanks, is transferred by pipes to the higher end of the screen. In the

process of screening, the pulp on top of the screen is slowly pushed downward by the shaking

motion.

It is preferable to let the suspensions pass a series of shaking screens of increasing fineness

(80-, 150-, and 260-mesh). The first screen retains the coarse pulp, and the succeeding screens the

finer particles. The coarse pulp is sent back to the second hammer mill for further grinding and

then returned to the shaking screen. Effective rising of the pulp on the screens is done by inserting

one or more shallow transverse channels in the surface of the screen, where the strong vibrating

movements caused by the shaking of the screen effectively loosen the starch granules from the

pulp (http://www.fao.org/docrep/x5032e/x5032E02.htm).

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 “Screening is accomplished by passing the material over a surface provided with openings

of the desired size. The equipment may take the form stationary or moving bars, punched metal

plate, or woven wire mesh. Screening consists in separating a mixture of various sizes of particle

into two or more portions, each of which is more uniform in size of particle than is the original

mixture” (Brown, et al., 1951).

“Vibrating screens are screens that are rapidly vibrated with small amplitude and are less

likely to blind than are gyrating screens. Vibrations can be mechanically or electrically.

Mechanical vibrations are usually transmitted from high-speed eccentrics to the casing of the unit

and from there to steeply inclined screens. Electrical vibrations from heavy duty solenoids are

transmitted to the casing or directly to the screens. Ordinarily no more than three decks are used

in vibrating screens, between 1800 and 3600 vibrations per minute as usual. A 48- by 120- in draws

about 4 hp” (McCabe, Smith, & Harriot, 1993).

Mixing

According to Elizabeth Christian “Cold Water may be used to physically separate starch

granules. When mixed insoluble starch, water puts starch granules in a suspension known as a

“slurry.” The cold water suspension is then slowly mixed into the hot liquid for thickening. Hot

water is not a successful separating agent as hot water partially gelatinizes the starch. Adding

starch to cold water and stirring it up will allow the granules to be dispersed throughout the liquid.

Now when you heat the solution all the granules are hydrated and will thicken beautifully.”

According to Chiu & Solarek (2009), “the general approach to overcome some of the

shortcomings of traditional pre-gelatinized starch is to maintain starch granule integrity while

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providing cold water thickening. Two major classes of technology have been developed. One

controls the swelling of starch granules in a mixture of water and an organic solvent. The other

involves spray drying aqueous starch slurry under carefully controlled conditions.”

Filtration

Bangke(2008) uses a Rotational filter in his design in which impurities is further cleaned

out to ensure that no block happens. Impurities are held by a separating drum and conveyed to the

bottom of filter by hair brush, then discharged when starch milk is input into the filtering drum.

After filtration, starch milk streams out from the pipe, which associate with the second phase of

separation section.

45 | P a g e
2. Starch Processing

PROCESS REACTION LITERATURE

Hydrolysis

Liquefaction is a key and essential handling of starch of this procedure does not go well,

inconveniences, for example, poor filtration, turbidity of the procedure arrangement, and so on the

active arrangement having the accompanying creation of the starch: 2% maltose, 0.30% glucose

and 97.7% oligosaccharide (Nampoothikiri et al, 1999).

According to the patent J.L. Purvis et al (1958),

“Glutamic acid in the amount of about 261 parts obtained by hydrolyzing concentrated

Steffen’s filtrate and separating glutamic acid at its isoelectric point was slurried in about 400 parts

H2O and sufficient sodium hydroxide added to dissolve the glutamic acid and raise the pH of the

solution to about 7. The temperature of the solution was raised to about 50° C. and one part L-

alanine was dissolved therein. This solution was agitated moderately while maintaining the

temperature constant at 50°C. The vessel containing the solution was not covered. After about 6

hours at 50°C water, in amount of 50 grams had evaporated and about one part of fine monosodium

glutamate crystals, was added as seed crystals. The seeded solution was agitated moderately for

about 12 more hours at 50° C, during which time the total weight of the crystallizer mixture

diminished to about 500 parts. The monosodium glutamate crystals which separated were removed

by filtration and were found to be short, stout crystals in which the longest axis was about equal in

length to the shortest axis.”

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 However, Sano (2009) stated that the problems with the initial hydrolysis process were

largely environmental, deriving from the corrosion of the materials in the facility by the evolution

of vapors containing hydrogen chloride gas. To solve this problem, a sulfuric acid hydrolysis

process was conducted. However, this approach failed, due to amino acid racemization produced

by the heat generated in the neutralization process. Consequently, the method returned to the use

of hydrochloric acid. To scale up hydrolysis, the Domyojigame vessel was replaced by a granite

stone chamber with enameled steam pipes. Finally, in the 1930s, the corrosion problem was

completely solved through the development of a rubber-lined iron vessel.

Starch is a large complex carbohydrate used as a way for plants to store excess glucose.

The hydrolysis of 1-4 glycosidic bonds in starch molecules leads to a conversion of starch into

simple sugars (Wiseman, 1995). The α-amylase is one enzyme that plays a key role in the starch

liquefaction process.

According to a journal entitled “Effect of Immobilization Method on Activity of Alpha-Amylase,”

“Immobilization or enzyme entrapment allows for easy separation of the enzyme from the

starch hydrolysis products which in turn can save the enzyme, labor, and overhead costs (Gerhartz,

1990). The enzyme catalyzed degradation of starch into smaller sugars is important in the

production of syrups in food industry This process, which has used α-amylase as an industrial

biocatalyst since the 1940’s in order to thin starch to less viscous liquid and lead to syrup

production, has limitations because of not being able to recover the catalyst after the process.”

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Figure 2.1. Flow chart of starch liquefaction process

A number of methods have been developed to immobilize the enzyme α-amylase for easy

recovering, they are: entrapment within cross linked polyacrylamide gel, covalent binding to the

surface of sepharose gel beads activated by cyanogen bromide (CNBr), and entrapment within

calcium alginate beads (Sadhukhan et al., 1993).

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From a journal entitled “Effect of Immobilization Method on Activity of Alpha-Amylase,”

“Among the methods, physical entrapment in calcium alginate beads has shown to be

relatively easy, rapid, and safe in comparison with other immobilization methods (Bickerstaff,

1997). A comparison of 12 stabilization of alpha-amylase using immobilization by entrapment

within polyacrylamide gel, calcium alginate beads, and covalent binding to sepharose beads

demonstrated that enzyme activity of α-amylase was best maintained when the enzyme was

entrapped in calcium alginate beads (Sadhukhan et al., 1993).

The preparation of alginate beads involves mixing of sodium alginate and alpha-amylase

solutions in buffer, followed by dripping of the solution into calcium chloride solution to form a

gel (Sadhukhan et al., 1993). Calcium alginate physical entrapment at pH 5 and at 60 °C reported

to be optimum conditions for α-amylase activity immobilized by physical entrapment (Dey et al.,

2003).”

With regards to adjusting the pH, based from the journal entitled “Surface Chemical Compositions

and Dispersity of Starch Nanocrystals Formed by Sulfuric and Hydrochloric Acid Hydrolysis” by

Wei et al. (2014),

The pH of starch solution can be adjusted using HCl or NaOH to either make the solution

acidic or basic.

Additionally, to support the previous statement, according to a study entitled “Starch Hydrolysis

by Amylase” by Wang (2011),

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 After the addition of amylase in their starch solution in the hydrolyzer, 1N HCl was added

to adjust the pH of the solution to 4.7.

Saccharification

Nedwin et.al explained stated that glucoamylases catalyze the hydrolysis of alpha-1,4-

glucosidic linkages of maltodextrins formedafter liquefaction from non-reducing ends, releasing

D-glucose. Saccharification produces high glucose syrup debranching enzymes, such as

pullulanases which aids saccharification.

According to the study of Nedwin et.al entitled Alpha-amylase blend for starch processing

and method of use thereof, “Alpha-amylases are isolated from a wide variety of bacterial, fungal,

plant, and animal sources. Many industrially important alpha-amylases are isolated from Bacillus

sp., in part because of the generally high capacity of Bacillus to secrete amylases into the growth

medium. In addition, Bacillus alpha-amylase variants with altered while more desirable properties

are obtained through genetic engineering. Furthermore, there is a need for blends of alpha-

amylases, or variants thereof, which can capitalize on the best properties of at least two alpha-

amylases of different origins.” Glenn E. Nedwin, Alpha-amylase blend for starch processing and

method of use thereof, 2014.

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Figure 2.2. The use of enzymes in processing starch. Typical conditions are given.

Inoculum Preparation

From the book of “Encyclopedia Of Bioprocess Technology: Fermentation, Biocatalysis, And

Bioseparation,”

It states that microorganism used for L-glutamate fermentation are usually preserved under

lyophilization below -80°C or for a short period, by keeping the stock culture below 10-15°C. To

refresh the microorganisms, stocked in either form, they are inoculated on an agar medium

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composed of 1% yeast extract and polypeptone, 0.5% sodium chloride, and 2% agar, at an

optimum temperature of microorganisms. The refreshed microorganisms are those that are

cultivated in liquid medium, shaken vigorously and transferred to a small fermenter to allow them

to propagate to about several kiloliters for seed culture.

Meanwhile, Aguinaldo et al. (2008) stated that microorganism inoculum preparation

requires careful proliferation of relatively few cells to a dense suspension of bacteria (especially

strains of Brevibacterium). These are grown aerobically in a liquid nutrient medium containing

carbon source and a nitrogen source such as ammonium ion and growth factors. The bacteria

selected for this process have the ability to excrete glutamic acid; they synthesize outside of their

cell membrane into the medium and accumulate there.

Additionally, in the laboratory, Corynebacterium is grown in agar slant for 24 hours and

then transferred in 10mL of the sterile liquid medium for 48 hours under continuous shaking at

room temperature. the starter medium is prepared by diluting an aliquot of hydrolyzate with

distilled H2O to make a 3% sugar solution. Nutrients such as ammonium sulfate, sodium

phosphate, magnesium sulfate are added. After adjusting the pH to 4.8 – 5.0 the starter medium is

sterilized at 15 psi steam pressure for 15 minutes. The seed culture is transferred into the

fermentation medium and allowed to propagate for 15-30 minutes before being sent to the

fermenter.

According to Harry Wood et. Al (1974), “As a practical matter, however, it is necessary

first to thin the starch before subjecting it to the action of glucoamylase. This thinning step may
52 | P a g e
be accomplished either by means of acid or enzyme. The starch is thinned to a D.E. of about -20,

then treated with glucoamylase. This two-stage process is referred to as an acid-enzyme process

or an enzyme-enzyme process, depending upon the nature of the thinning step employed.”

In the Liquefaction of starch there is a formation of reversion products known as

oligosaccharides linked by α-1,6 linkages, and thus to obtain glucose from the gelatinized starch,

addition of glucoamylase together with pullulanase which de-branches these α-1,6 linkages are

required. These two enzymes have the same range for pH optima and blending them in the

liquefaction process proves to be practical since experimental results showed that combining the

two in the hydrolysis of starch gave higher amounts of product than that when the two enzymes

were used separately. The reason why blending the two enzymes gave a more efficient hydrolysis

of starch is that there is an immediate contact of pullulanase to the α-1,6 linkages when the

glucoamylase opened up the starch structure and exposed the α-1,6 linkages. (Roy, 2003)

From the journal entitled “Production of L-glutamic Acid with Corynebacterium

glutamicum (NCIM 2168) and Pseudomonasreptilivora (NCIM 2598): A Study on

Immobilization and Reusability,”

They were able to study how inoculum was prepared. It was stated that inoculum was

prepared by transferring cells from agar slant into 250 ml flask containing 100 ml of the culture

medium. Half (0.5) ml of each culture was taken and inoculated in the production medium and

also used for immobilization studies. The constitution of the medium for preparing agar slant was

kept at pH = 7.0 and incubated at 30°C for at least three days. The slants were preserved at 4°C and

subculture twice in a month. The medium composition for the production of glutamic acid was as

the following: (g/l) Glucose-50.0, Urea-8.0, Biotin-0.002, K2HPO4-1.0, MgSO4.7H2O-2.5,

53 | P a g e
MnSO4. 7H2O-0.1, CaCO3-1.6. The medium pH was adjusted to 7.0 with 1N sodium hydroxide or

1N hydrochloric acid. The fermentation was carried out in 250ml Erlenmeyer flask. The

fermentation medium was inoculated with 1% (v/v) of the overnight culture (C. glutamicum and

equal volume of C. glutamicum and P. reptilivora mixed culture). The production medium was

kept in an orbital incubator shaker at 30°C at 120 rpm for 48 hr. Then the cells and debris were

removed by centrifugation at 10000 g at 4o C for 10 min. Supernatants were used as the crude

glutamic acid source for estimation.

Fermentation

Miescher et al (1966) stated that monosodium glutamate can be produced through

hydrolysis of vegetable protein, synthesis via chemical means, and by fermentation. The

mentioned processes all produce glutamic acid, which is an essential ingredient to produce

monosodium glutamate.

According to a book source entitled "Secondary Metabolism in Microorganisms, Plants and

Animals,"

L-glutamic acid, L-proline and L-ornithine are amino acids with five C-atoms. The

secondary products derived from these compounds either possess the aliphatic carbon chain of the

amino acids L-glutamic acid and L-ornithine, e.g., γ-methylglutamic acid, ornithuric acid and

phenylacetyl-L-glutamine, are pyrrolidine derivatives like L-proline, cf. the formulas of N-methyl-

Δ1-pyrrolinium cation and cuscohygrine orpossess the more complicated ring systems of

54 | P a g e
quinolizidine and tropane. L-glutamic acid is formed from α-ketoglutaric acid, an intermediate of

the tricarbonic acid cycle by glutamate dehydrogenase. It is transformed reversibly to L-proline

and L-ornithine.

Fig. 2.3. Biosynthesis and transformation of the amino acids of the glutamic acid-proline-ornithine
family; 1Glutamyl kinase, glutamate semialdehyde dehydrogenase; 2 spontaneous cyclization; 3
pyrroline-5-carboxylate reductase; 4 proline oxidase; 5 acetylglutamate synthase;
6acetylglutamate kinase, acetyl-γ-glutamylphosphate reductase; 7acetylornithine
aminotransferase; 8 acetylornithine deacetylase; 9 glutamine synthetase; 10glutaminase; 11
ornithine decarboxylase

55 | P a g e
Fig. 2.4. Formation of substituted L-glutamine derivatives; 1 Glutamine synthetase, which has a
relatively low substrate specificity

According to internet source entitled “An overview of traditional processing and utilization of

cassava in Africa” gathered from http://www.fao.org/wairdocs/ilri/x5458e/x5458e05.htm,

“Fermentation consists of two distinct methods: aerobic and anaerobic fermentation. For

aerobic fermentation, the peeled and sliced cassava roots are first surface-dried for 1-2 hours and

then heaped together, covered with straw or leaves and left to ferment in air for 3-4 days until the

pieces become moldy. The fermented moldy pieces are sun-dried after the mold has been scraped

off. The processed and dried pieces (called "Mokopa" in Uganda) are then milled into flour, which

is prepared into a "fufu" called "kowan" in Uganda. The growth of mold on the root pieces,

increases the protein content of the final products three to eight times (Amey 1987, Sauti et al.

1987). This fermentation method is also very popular in other parts of East Africa such as

Tanzania, Rwanda, and Zaire. In anaerobic fermentation, grated cassava for processing into "gari"

is placed in sacks and pressed with stones or a jack between wooden platforms. Whole roots or

pieces of peeled roots for processing into "fufu" are placed in water for 3-5 days. During the first

stage of gari production, the bacterium Corynebacteriamanihot attacks the starch of the roots,

leading to the production of various organic acids (such as lactic and formic acids) and the lowering

56 | P a g e
of substrate pH. In the second stage, the acidic condition stimulates the growth of a mold,

Geotrichum candida, which proliferates rapidly, causing further acidification and production of a

series of aldehydes and esters that are responsible for the taste and aroma of gari (Odunfa 1985).

The optimum temperature for the fermentation for gari processing is 35°C, increasing up to 45°C.”

Meanwhile, according to a book entitled "Fundamentals of Food Biotechnology,"

The amino acid L-glutamate and its salt, MSG, are well-known flavor enhancers, and the

industry produces more than 500,000 tons/year. L-glutamate production has been found to occur

in a wide variety of bacteria, streptomyces, yeasts, and fungi. Some Brevibacteriumflavum and C.

Glutamicum species are major glutamate producers, but most commonly glutamate is produced by

the fermentation of glucose and sucrose as carbon and energy sources by C. Glutamicum. This

organism accumulates α-ketoglutaric acid because it is incapable of performing a complete

tricarboxylic acid (TCA) cycle. As a consequence, the excess α-ketoglutaric acid is channeled

toward the synthesis of glutamic acid. In addition, growth under biotin-limiting conditions results

in an increased ability of the organism to excrete glutamic acid into the media. This makes the

purification process simpler. If biotin is limiting, phospholipid synthesis is limited, and thus the

cell membranes become leaky. Thus, all glutamate producers require biotin, lack L-glutarate

dehydrogenase, and show increased activity of glutamate dehydrogenase. Under optimal culture

conditions, glutamate-producing bacteria convert about 50-60% of the added carbon source to the

L-glutamate. In a typical fermentation from glucose with Brevibacteriumdivaricatum, 0.65 mL/L

of oleic acid is added. After beginning the growth of culture at pH 7.8 and 38oC, a fed batch is

57 | P a g e
operated and the fermentation process is stopped after 30-35 hours with a glutamate yield of 100

g/L.

According to the internet source entitled “Amino acids are made from natural material” retrieved

from https://www.ajinomoto.com/features/amino/lets/product/,

“The amino acid fermentation method is a method for the production of amino acids

utilizing the phenomenon that microorganisms convert nutrients to various necessary vital

components. With the fermentation method, raw materials such as syrups are added to

microorganism culture media, and the proliferating microorganisms are allowed to produce amino

acids. These are enzymes that play an important role in the process. Enzymes, which are proteins

to catalyze chemical reactions in the living body, are indispensable to degrade and synthesize

substances. Consecutive reactions by 10 to 30 kinds of enzymes are involved in the process of

fermentation, and various amino acids are produced as results of these reactions. Generally, amino

acids cannot be manufactured in quantities without deactivating the regulatory mechanism that

microorganisms possess. However, the glutamate-producing microorganism has such a rare

characteristic that glutamate can be produced solely by setting special fermentation conditions

without improving the strain. A fermentation tank is fed with raw materials such as syrup derived

from sugar cane, and glutamate-producing microorganisms are fermented under the appropriate

conditions. During this fermentation process, glutamate is excreted from the microorganisms into

the fermented broth. This is how glutamate is obtained in large amounts.”

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Based from the journal by Toshikiri et al (n.d.),

Amino acid fermentation is conducted by fermenting bacterial cells in a culture medium in

a fermenter and separating fermentation solution withdrawn from the fermenter into a solution

containing said bacterial cells and a solution not containing bacterial cells by a cell separator. The

solution containing said bacterial cells being circulated from the said cell separator to the said

fermenter by circulating means to perform amino acid fermentation continuously, and bubbles

being removed from the said fermentation solution by a bubble separator before said fermentation

solution is fed to said circulating means and said cell separator.

Sano (2009) stated that the fermentation method is a production process in which a specific

amino acid is synthesized in large amounts by a specially selected microorganism in culture. The

selected microorganism is cultured with carbohydrates and ammonia and releases the L-form of

the amino acid into the culture medium. The cell produces glutamate from 2-oxo-glutarate (2-oxo-

pentanedioic acid) by reductive ammonia fixation that uses the enzyme glutamate dehydrogenase,

a normal cellular constituent. In 1956 Kyowa Hakko Kogyo Co Ltd succeeded in developing the

first industrial fermentation technology for L-glutamate. The L-glutamate-producing bacterium

was reported in 1957 by Kinoshita et al (15). Since that report, many bacteria useful in glutamate

production have been isolated, including Corynebacterium glutamicum,

brevibacteriumlactofermentum, and brevibacteriumflavum. These glutamate-producing bacteria

are all coryneform bacteria, which are gram positive, non-spore-forming, and non-motile and

require biotin for growth. Glutamate accumulation in the medium occurs only under biotin-limiting

conditions. The requirement for biotin limitation prevented the use of standard raw materials such

59 | P a g e
as sugar molasses because they contained biotin. Significant efforts were thus expended to

overcome this difficulty. Ultimately, methods were discovered, such as the addition of a surfactant

or of penicillin or the use of microorganisms auxotrophic for glycerol or oleate that allowed the

bacteria to produce large amounts of glutamate without biotin limitation.

From a journal source entitled "Glutamate excretion by Corynebacterium glutamicum: a study of

glutamate accumulation during a fermentation course,"

The excretion of the amino acid was induced by the addition of two surfactants at a very

precise step during the exponential growth. Some effects of the surfactants are reported and their

role on excretion triggering is discussed. They followed the glutamate repartition between

intracellular and extracellular media during a fermentation course in various conditions leading to

either high or low levels of excreted glutamate. The intracellular concentration of glutamate has

been determined taking into account variations of the cell volume reflecting modifications of

physiological state. To support this, an industrial strain of C. Glutamicum was used. The inoculum

medium contained (g/L): beet molasses 80, H3PO4 3, urea 8, MgSO4 0.5, desthiobiotine 10-4; pH

was adjusted to 5.75 before autoclaving (20 mn at 121oC). The inoculum culture was grown

overnight at 30oC on a rotary shaker. Fermentations were carried out in a 201 fermenter (Chemap)

with a working volume of 151. The medium had the following composition (g/L): beet molasses

150, H3PO4 1.65, (NH4)2SO4 1, MgSO4 0.5, MnSO4 0.005, desthiobiotine 10-4. Culture conditions

were pH 7.3, temperature +34.5oC, agitation 550 RPM, aeration 0.6 VVM. Surfactant S1 was a

polyethylene glycol acylated by stearic and palmitic acids. Surfactant S2 was laurylamine. The

mode of fermentation was a fed batch. The initial concentration of sucrose in the medium was

about 75 g/L; in the stage when the concentration fell to 30 g/L, a glucose solution (600 g/L) was

supplied to keep the sugar concentration constant in the medium. The results show that the acylated
60 | P a g e
surfactant S1 did not significantly modify the growth curve, while the amine surfactant S2 slowed

it down and induced the excretion of glutamate. It was a second type of fermentation according to

the Gaden classification since growth and production are uncoupled.

Moreover, glutamate fermentation is a typical aerobic type and the aerobic oxidation of

glucose involves both EMP and HMP pathways followed by TCA cycle. Two enzymes have been

shown to play a key role in the biosynthesis of L-glutamic acid. (i) Phosphoenolpyruvate

carboxylase and (ii) ot-Ketoglutarate dehydrogenase. The low activity and high instability of a-

ketoglutarate dehydrogenase favors the conversion of a-ketoglutarate by glutamate dehydrogenase

(GDH), especially in the presence of high NH4‘concentration. In microorganisms, 158 L-

glutamate can be formed either by the glutamate dehydrogenase or by the coupled reactions of

glutamine synthetase (GS) and glutamate synthase (GOGAT) (Yoshioka, 1999).

The raw cassava starch with the following composition: 83% starch, 15% H2O and 1.6%

inert is blended with water to create slurry containing 30% solid. The alpha amylase is likewise

included with the supplements it expected to get by in the bioreactor. The mixture undergoes

warming or cooking in a steam jacketed bioreactor with a mechanical shear or mixing. The starch

is gelatinized by the treatment in the reactor keeping up the blend pH around 6-6.5 (pH solidness).

The slurry is quickly warmed to 100 deg C and held at a temperature for ten minutes before it is

cooled to 90 deg C which is kept up for 1-3 hours to further hydrolyze the starch. This is then

diminished by constrained hydrolysis with an alpha amylase, 0.6 kg for every ton dry solid starch.

The alpha amylase are endo-enzymes that hydrolize alpha 1-4 glycosidic linkages in the starch to

frame less gooey arrangement of shorter oligosaccharides and last dextrose comparable up to 15%.

61 | P a g e
These compounds are restricted by the vicinity of the alpha 1-6 informal breakfast focuses

(glycosidic linkages), which the chemicals cannot hydrolyze. The alpha amylase comes in

structure Bacillus Licheniformis and is framed to be an extremely thermostable protein. This

catalyst requires 5ppm Ca- 2 that demonstrate to settle chemical against warmth denaturation and

really go about as a compound cofactor to upgrade its movement. The alpha amylase is ordinarily

utilized as solvent proteins and it is profoundly accessible in vast amounts (Nampoothikiri et al,

1999).

Moreover, according to the internet source entitled “Preparation and Manufacturing of

Monosodium Glutamate (MSG)” retrieved from

http://formulation.vinensia.com/2011/03/preparation-and-manufacturing-of.html#more,

Fermentation of molasses-sugar solely produces monosodium glutamate with the help of a

species of bacteria (Brevibacteriumlactofermentum). Glutamic acid is initially produced in this

manner. In the addition of soda (sodium carbonate), monosodium glutamate (MSG) is formed. It

is then purified and crystallized, resulting to a crystal-pure powder

For the process of fermentation itself, Aguinaldo et al (2008) studied that in the

fermentation tower, the solution that contains 66.76% H2O, 31.73% glucose, 0.62% maltose, 0.30

% oligosaccharides and 0.56% inert is fed in a fermenter. The working temperature is 26°C-30°C,

pH keeping up at 7-8. The droplets of the blend from the blending tank is always circulated air

through and disturbed to abbreviate the maturation time to 40-60 hours. The hatching of a huge

scale fermenter is completed by transferring microorganism from a seed age, which is 10-20% of
62 | P a g e
the volume of large scale fermenter. In this stage, Corynebacterium glutamicum consumes glucose

and produces glutamic corrosive with 70% yield, discharges carbon dioxide, gas and heat. The

unconverted glucose is 30%. Glutamic acid will stay in solution while the carbon dioxide gas will

rise through the fluid and fill the head space of the fermenter and gathered for the utilized as a part

of green houses or gathered in tank for the production of dry ice. To promote quick fermentation,

the Corynebacterium glutamicum ought to be added to a starter tank. This tank ought to contain a

little portion of fermentable fluid blended with H2O and conformed to the ideal fermenter

condition. The medium ought to be permitted to engender in the starter for 15-30 minutes before

being sent to the fermenter. The pH is kept at 7-8 while temperature is at 30°C. Aging will require

48-60 hours. Aging is finished when there is no further development of CO 2 gas. Fermenters are

outfitted with agitator and heat exchanger to uproot heat created amid fermentation. Agitation is

required for homogeneous substance to enhance mass exchange and for making high shear

conditions, which can separate air pockets. The fermenter will be required for consistent operation,

each to work at 60 hours and will have a cleaning time of 12 hours. In cleaning, the fermenter

ought to be discharged totally, washed with H2O and afterward steam cleaning of the vessel and

its parts is finished. Media segment froth an extraordinary arrangement and overabundance froth

can piece channels and prompt develop weight inside the vessel. The froth produced can be

controlled through expansion of hostile to froth operators.

Liquefaction process always involves a starch gelatinization process, which the aqueous

starch slurry is heated. Because of that, the granular starch in the slurry swells and bursts,

dispersing starch molecules into the solution. During the gelatinization process, there is an increase

in viscosity and during the remaining process steps, the starch must be thinned or “liquefied”. This
63 | P a g e
decrease in viscosity can be achieved by enzymatic degradation in a process referred to as

liquefaction. During liquefaction, the long-chained starch molecules are degraded into smaller

branched and linear chains of glucose units (dextrins) by an enzyme, such as alpha-amylase (US

Patent No. US7727726 B2, 2010).

In a patent by Miescher et al. (1966), they created a process which produced monosodium

glutamate directly in the fermentation medium and recover it as a concentrated solution of

monosodium glutamate which can be used in the same manner as crystalline monosodium

glutamate. In other variations of their process, the whole fermentation mixture containing the

monosodium glutamate can be dried, and produce a new composition which not only contains the

desirable qualities of monosodium glutamate but also contains vitamins, minerals and

proteinaceous material and has tasty qualities of its own. This can be used as a condiment for many

varieties of food, and also in feeds. Generally, the new process involves production of glutamic

acid by fermentation and conversion of the glutamic acid produced in the fermentation medium to

monosodium glutamate by adding sodium ions to the culture medium during the latter part of the

fermentation. The sodium ions can be added after a significant amount of the contemplated L-

glutamic acid production is achieved, usually after about 50 percent, and preferably after about 55

and up to about 70 percent or more of the contemplated glutamic acid production is achieved. The

monosodium glutamate is then recovered as a concentrate in solution or as a dry monosodium

glutamate containing powder.

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 With regards to the pH of the solution in the fermenter, the active arrangement of glutamic

acid is changed in accordance to pH 3 - 4 which is over the isoelectric point. The H+ is changed

over into an extremely solvent Na+ salts. The pH estimation of 3.2 is known as the isoelectric point,

the time when the acid is fit for precipitating crystals. On the off chance that the pH is beneath 3.2,

the acid dissolves in water. In the wake of changing the pH arrangement is sustained to pasteurizer

to deactivate the chemicals and microorganisms from the bioreactor (Chemical Engineering

Journal, 2004). Moreover, Dutta (2008) mentioned that the pH in a fermenter can be sustained by

utilizing either a buffer solution or a pH controller. Meanwhile, the temperature is controlled by

heating or cooling as the system requires.

From the internet source entitled “tapioca starch to Nua powder (MSG),”

For the nitrogen source, urea or ammonia was added. The cultivation is processed until

glutamic acid is excessively released in broth. Then, the glutamic acid broth is evaporated with

vacuum steaming for concentration.

From the journal “Fermentation and Recovery of L-Glutamic Acid from Cassava Starch

Hydrolysate by Ion-Exchange Resin Column,”

An experiment was conducted and the optimal parameters that was obtained were set to be

constant and maintained throughout the fermentation period. These parameters are such: a pH of

7.5, temp of 30o C, and agitation rate of 180 rpm.

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 SEPARATION PROCESS LITERATURE

Filtration

Based on “History of glutamate production,”

Filtration was used to separate the L-glutamic acid hydrochloride and it was dissolved

again in water. The solution was filtered to eliminate humus. After that, the pH was adjusted to the

isoelectric point of L-glutamic acid (pH 3.2) with sodium or potassium hydroxide. The solution

was stored for 1 week to allow the L-glutamic acid to crystallize.

NEUTRALIZATION PROCESS LITERATURE

Ikeda studied that the crystals of L-glutamic acid hydrochloride were separated from the

liquid by filtration and dissolved in water. This solution was again filtered to eliminate humus.

The pH was then adjusted to the isoelectric point of L-glutamic acid (pH 3.2) with sodium or

potassium hydroxide, and this solution was stored for 1 week to allow L-glutamic acid to

crystallize out. This step notably increased the purity of the crystals for the following reason. There

are 2 polymorphs in L-glutamic acid crystals: a metastable, granular α-form and a stable, thin,

plate-like β-form. The α-form grows better than the β-form in solutions containing other amino

acids. And, growing by its specific hydrogen bonding network, the dominant (001) face of the α-

form selectively incorporates L-glutamic acid molecules at both the L-α-amino acid and the γ-

carboxyl residues. Because the solution of the crude L-glutamic acid hydrochloride salt created in

the early production method (described above) still contained other amino acids, the α-form of

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glutamic acid was the dominant crystal formed at pH 3.2. Purity was improved because the grown

α-form crystals did not contain other amino acids.

According to a patent entitled “Preparation of Monosodium Glutamate” by Shildneck,

If the pH of the monosodium glutamate solution when diluted tenfold with water does not

lie within the range of pH 5.7 to 6.8, it is adjusted to a value within that range by the proper addition

of caustic soda or glutamic acid.

PURIFICATION PROCESS LITERATURE

From an article entitled “History of Glutamate Production” retrieved from

http://ajcn.nutrition.org/content/90/3/728S.full,

The separated L-glutamic acid, α-form crystals were re-dissolved in water and placed into

an enamel-jacketed ironware vessel. Sodium bicarbonate was added to adjust the solution to a

neutral pH (litmus paper was used). This monosodium glutamate solution was then decolorized by

adding activated carbon and filtering. The filtered, clear solution was then concentrated by heating

and cooled in the enameled vessel, causing monosodium L-glutamate (MSG) crystals to form and

precipitate. When separated from the solution, the lump of MSG crystals was cracked by hammer

into a powder and separated from any adhered mother liquor by centrifugation. The final MSG

powder was dried, sieved, and packed as the final product.

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 CRYSTALLIZATION PROCESS LITERATURE

According to the patent J.L. Purvis et al (1958),

“In a process for crystallizing monosodium glutamate from a supersaturated aqueous

solution of monosodium glutamate which normally deposits long, needle-like crystals thereof, the

improvement which comprises adding to said solution prior to said crystallization a small amount,

at least about 0.1% by weight, of alanine based on the weight of monosodium glutamate in said

solution, and maintaining a relatively low rate of crystal growth during said crystallization, said

rate of crystal growth being at least as low as that which occurs when a super-saturated solution

containing about 50% by weight of monosodium glutamate in water at room temperature is

allowed to undergo spontaneous crystallization at room temperature without evaporation, seeding

and agitation, whereby crystals of monosodium glutamate are obtained in which the longest axis

is less than about 5 times the length of the shortest axis”.

According to the internet source entitled “Producing Crystals of Monosodium Glutamate”

retrieved from http://fermentationtechnology.blogspot.com/2011/10/producing-crystals-of-

monosodium.html,

“The process of forming the crystals is in simplification a kind of chemical precipitation

reactions. It is based on the salt or ionic nature of the products. Adding the sodium to the glutamate

will further stabilize the product to undergo the various harsh downstream reactions. While it is

true that the process of crystallization is based on the nucleation and crystal growth of the crystal

from a super saturated solution as it cools down, it is of most importance that the solution of MSG

from the fermentation broth must be purified so as to produce clear and beautiful crystals. Various
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downstream methods are used to obtain this clear broth such as cell separations which include

centrifugation and membrane separation. This is followed by various preparatory steps such as ion

exchange resin treatment, chromatography and crystallization. And in the crystallization stage

there is concentration, neutralization and cooling. Ultimately the final steps include centrifugation,

decantation, and filtration to obtain the crude crystals. The crystallization process is usually carried

out using the crystallizer”.

Meanwhile, according to a patent entitled “Preparation of Monosodium Glutamate” by Shildneck,

The crystallization of monosodium glutamate is satisfactory at a pH range of 5.7 to 6.8.

Crystallization at pH values above 1 is undesirable because it is accompanied by formation in the

product of a yellow color, an undesirable ammoniacal odor, and considerable hygroscopicity. The

degrees of these undesirable qualities increase rapidly with increase of pH above 7, and at pH 8

the product becomes useless as a condiment.

Additionally, Aguinaldo et al. (2008) also studied that the clear solution is transferred to

monosodium glutamate crystallizer. The solution containing 25.12% H2O, 73.92% monosodium

glutamate, and impurity is heated to super saturation or evaporation. Surfaced cooled crystallizer

is used in the manufacture of monosodium glutamate with 90% crystals. Super saturation can be

achieved by the evaporation and cooling of the heated stream exiting a heat exchanger within the

recirculation loop and then relieved by passing the supersaturated liquor through a fluidized bed

of crystals. The solubility of monosodium glutamate at 20°C is 38%. Feed inlet is to be surged

enough in the suspensions to prevent flashing at the feed point, as well as across the heat
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exchanger. Typically, it evaporates 7% of solvent and is designed to operate within the metastable

zone for the desired level of super saturation and with the mesh separator to maintain the desired

size of the crystals produced.

PRODUCT PREPARATION LITERATURE

Centrifugation

Centrifugation is one of the widely used process in industry mostly in food and

pharmaceutical industry. From the book entitled “Modern Technology of Industrial Chemicals” by

NIIR Board, states that the slurry form the crystallizer is dropped into a centrifuge, where the

mother liquor is evaporated and crystallized in separate units. The crystals from these units are

returned to the treating tank, and the final mother liquor is oxidized in a subsequent batch of

quinone.

According to a book entitled “Handbook of Sugar Refining: A Manual for the Design and

Operation of Sugar Refining Facilities” by Chung Chi Chou, lumps are formed, comprise of

smaller sugar crystals with a higher proportion of syrup than that found in the bulk of sugar. By

setting the industrial centrifuge to a speed of 700 to 1000 rpm for 15 to 25 seconds, >0.5mm of

particle size is obtained.

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From a book entitled “Handbook of Industrial Drying, Third Edition” by Arun S. Mujumdar,

“An example of such a product is crystalline lysine, a highly-heat sensitive material with

tendency to lump and with a large amount of bound water (up to 17% wb). The crystal size after

centrifugation is 0.2 to 2.5mm.”

Screening

Based on a patent entitled “Production of Lysine Monohydro Chloride and Derivatives” by

Hambrock, K.,

“Under identical crystallization conditions the optically active lysine monohydrochloride

dihydrate crystals produced are many times larger than those of the anhydrous dl-lysine

monohydrochloride and from a mixture the two can be separated to a large extent by mechanical

methods such as screening, that is by passing the material through a sieve or screen of such mesh

size that the larger crystals of the optically active dihydrate are retained while the smaller crystals

of the form pass through the sieve.”

D. EQUIPMENT LITERATURE

1. Preparation of Cassava Starch

Based from an internet source entitled “Wastewater Technology Fact Sheet Screening and Grit

Removal” retrieved fromhttp://water.epa.gov (1993),

Cassava must be peeled to evacuate the unpalatable external parts of the root comprising

of the corky periderm and the cortex. These are known to contain most of the toxic cyanogenic

glucosides, the ratio of glycosides contrasted with the boring substance changing between 5-10:1.

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Water Cleaner

According to N. Bangke (2008),

Water goes into the U-tank in which a shaft with screw propeller is equipped.

Cassava roots collide and rub against each other and also against the wall to remove soil and peel

off under the centrifugal force. At that point Cassava roots go into the second step water cleaning.

Hammer Mill

As stated by N. Bangke (2008),

Hammer mill crusher destroys the tissue of cassava and makes the very small granular of

starch decompose and depart from the roots. This crusher employs a rain of hammer blows to

shatter and disintegrate the material. Hammer mills produce a finish product size that is dependent

upon the openings in perforated screens or grate bars, Number, size and type of hammers, grinding

plate setting and motor speed.

Hammer mills are designed to be driven at either constant or variable rotor speeds. The

rotor is forged in one piece, the fixing holes for the beater arms are arranged in a special device.

The precisely balanced design of the opposing beater arms and beater heads ensures smooth

running.

There are 2 deign for hammer mill, the reversible and non-reversible hammer mill. The

reversible hammer mill (type 1212) allows bi-directional operation so that both sides of the beater

heads can be used while non-reversible hammer mill (type 1211) allows the beater head to be

reversed when one side is worn out.

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Shaking Screen

According to an internet source retrieved from http://www.wisegeek.com/what-is-a-vibrating-

screen.htm,

“A vibrating screen is a large mechanical tool used to separate solids, liquids and powders.

Industries as diverse as mining operations, chemical companies and construction firms utilize these

tools to help sort and clean items. Using gravity, motion and mesh screens, these tools perform the

work of several people in a fraction of the time.”

From the book entitled “Handbook of Food Process Design” by Rahman (2012),

“Screens or sieves are probably the most widely used mechanical separation device

(barrier). Screening processes may be wet or dry, depending on process requirements and

capabilities. Pressurized air or gas is often used as a combined separation technique and to dislodge

particles captured in screens.”

Screening is a method that may produce incomplete separation of contaminants or products

and also often used as a preliminary cleaning stage before other methods. The efficiency of screens

to separate materials can be improved by vibrating it, or by using abrasive discs or brushes to

remove the material from the aperture in the screen. (Fellows, 2009).

Mixing tank rrl!!!

Rotary Vacuum Filter

G. Duan (2010) stated that rotary vacuum filter will be used to reduce the water content to

about 40 to 45%. Rotary vacuum filter (RVF) is applied to process and waste slurries for filtering,

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clarifying, cake washing, extraction and dewatering. RVF is a continuous separation of solid and

liquid where both products are recovered. Clarification of liquid product can also be done by rotary

vacuum filter.

2. Cassava Starch to Monosodium Glutamate

Jacketed Bioreactor

According to S. Pratt (n.d.), bioreactors are typically made from glass or stainless steel,

which must be sterilized before use. Sterile culture medium is subsequently added to the bioreactor

and temperatures are stabilized before inoculation with cultured cells. Depending on the culture

requirements, there may be provisions to agitate the medium using a stirrer and/or by providing a

flow of gas (oxygen, nitrogen) and waste products, such as CO2 are removed. This stirring also

facilitates fluid mixing for an even distribution of temperature for increased uniformity.

Crystallizer

From an internet source entitled “Encyclopedia of Chemical Engineering Equipment” retrieved

from http://encyclopedia.che.engin.umich.edu/,

Crystallizers are used in industry to achieve liquid-solid separation. They are an important

piece of chemical processing equipment because they are capable of generating high purity

products with a relatively low energy input.

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 Forced-circulation crystallizers are evaporative crystallizers. It creates a super-saturated

solution by evaporating the solvent of a saturated solution. The solute of this supersaturated

solution then cools, forming crystals. These type of crystallizers are classified as mixed-

suspension, mixed-product-removal crystallizers.

Gathered from an internet source linked at http://www.swensontechnology.com/forced-

circulation-crystallizer/,

“Forced-circulation crystallizers can be operated on a batch basis, but the most frequent

use is in the continuous processing of such materials as sodium chloride, sodium sulfate, sodium

carbonate monohydrate, citric acid, monosodium glutamate, urea and other similar crystalline

materials.”

Moreover, according to an internet source at http://www.gea.com/en/products/forced-

crystallization-cryst.jsp,

The FC consists of four basic components: the crystallizer vessel, which provides most of

the volume dictated by the residence time requirements, the circulating pump, which provides the

mixing energy, the heat exchanger, which supplies energy to the crystallizer (in a typical

evaporative crystallization operation), and the vacuum equipment, which handles the vapors

generated in the crystallizer. Slurry from the crystallizer vessel is circulated, in plug flow fashion,

through the heat exchanger, and returned to the crystallizer vessel again, where its supersaturation

is relieved by deposition of material on the crystals present in the slurry. The supersaturation is

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controlled so as to avoid spontaneous nucleation, by sufficient circulation capacity. The evaporated

solvent is conducted to the vacuum system, where it is condensed and removed.

The average FC crystallizer evaporates solvent; thus, it increases the supersaturation in the

process liquor. This causes crystallization to occur. Moreover, most conventional FC units operate

under vacuum, or at slight super atmospheric pressure.

Batch vacuum crystallizer is a special case requiring very low operating temperatures

achieved only by very high vacuum. This application involves relatively small amounts of material

and when the material is being processed, it must be handled on less than a continuous basis.

From the patent entitled “Preparation of monosodium glutamate” by Shildneck,

The monosodium glutamate solution having the desired pH value is brought if necessary

to a pH of 5.7. It is the lowest pH value at a temperature between 20 C. and 50 C. In fact, it is

preferred to employ a temperature of between 30 C and 40 C. Increase of temperature above the

range specified may accelerate crystallization but results in the formation of an undesirable yellow

color in the solution and this color persists in the crystallized product. Below 20 C, the

crystallization is too slow for practical purposes.

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Pusher Centrifuge

Based from an internet source entitled “Pusher Centrifuge: Operation, Applications and

Advantages” retrieved from http://www.pharmaceuticalonline.com/,

The pusher centrifuge has been applied to a wide variety of industries. Pusher centrifuge is

a continuous filtering type centrifuge used for solid-liquid separation in the chemical and mineral

industries. Pushers have been used for more than 60 years for dewatering relatively large, free-

draining crystals. The pusher centrifuge has a unique design that minimizes moisture, impurity and

crystal breakage in discharged cake.

Crystal Crusher

Based from an internet source entitled “Solution for Crystal Growth” retrieved from

https://www.hamptonresearch.com,

The Crystal Crusher is designed to crush macro crystals into micro crystals for seeding and

other applications. The hemispherical end of the tool fits round; concave bottomed sitting drop

crystallization plates for efficient crystal crushing. The Crystal Crusher is molded from solid,

borosilicate glass. One end of the tool features a larger diameter, cylindrical handle, while the

opposite end features a smooth, hemispherical end for crushing crystals without damaging

crystallization plates.

Screener

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 According to an internet source the particle size of commercially available Monosodium

glutamate ranges from 60 to 80 mesh. Given the particle size, the particle diameter of 0.248-

0.175mm. Screener size of mesh 60 to 80 is to be used.

Tray Dryer

Fig. D.2.1. Tray Dryer

From the book entitled “Unit Operations in Food Processing”

“In tray dryers, the food is spread out, generally quite thinly, on trays in which the drying takes

place. Heating may be by an air current sweeping across the trays, by conduction from heated trays

or heated shelves on which the trays lie, or by radiation from heated surfaces. Most tray dryers are

heated by air, which also removes the moist vapors.

From the journal entitled “Design of Tray Dryers For Food Dehydration” by Kiranoudis et. al,

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 When the humidity level is high, the drying conditions become less intense resulting to longer

periods of treatment of the product in the dryer.

Storage

From an internet source entitled “Food Chem” retrieved from

http://www.foodchemadditives.com/products/monosodium-glutamate,

Monosodium Glutamate should be kept in dry, cool, and shaded place with original

packaging, avoid moisture, store at room temperature.

According to A. Gayte-Sorbier (1985), whatever the storage conditions, the applied

processing and the pH values, glutamic acid and monosodium glutamate were only converted to

pyroglutamic acid. It was shown that glutamic acid was converted to pyroglutamic acid as soon as

it was no longer under extreme pH conditions (pH 0 or pH 14).

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