Okafor N. Modern Industrial Microbiology and Biotechnology

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!$ Modern Industrial Microbiology and Biotechnology

19.8.3 Fermented Foods from Protein-rich Oil-seeds

Stew condiments made from oil-rich seeds are eaten in parts of West Africa, while

condiments from fish and fish products appear to be common in parts of Asia.

Stew condiments eaten in parts of Nigeria include dawadawa, know as iru in the

Southwest geopolitical zone of Nigeria which is produced from the seeds of Parkia

biglobosa. Cadbury Plc Nigeria now makes and markets dawadawa as Dadawa. A

condiment very similar to dawawa is okpeyi and comes from the Nsukka area of the

Southeast and is made from the seeds of Prosopsis africana. Another major soup

condiment popular in the Southeast zone is ogili which may be made from the seeds of

castor-oil seeds (Ricinus communis) or egusi (Citrullus lanatus sub-species colocynthoides).

Egusi, pumpkins and squashes are members of the family Cucurbitaceae or cucurbits and

their seeds contain about 50% oil and 35% protein after dehulling. Besides egusi, another

well-known cucurbit in Nigeria is ugu or Telfaria sp.; its seeds and those of soybeans are

sometimes used for making ogili.

A fermented delicacy and meat substitute, ugba or ukpaka is made from the seeds of the

African locust bean, Pentaclethra macrophylla Benth). This is generally eaten with

stockfish and it is very popular in the South-east zone of Nigeria. Bacillus spp. have been

implicated in the fermentation of the various oilseeds discussed above.

19.8.4 Food Condiments Made from Fish

Fish sauce is eaten in many parts of Asia including Japan, Thailand, Vietnam, the

Philippines, Indonesia and Malaysia, and some parts of Northern Europe, including

France.

Table 19.5 Methods of fish sauce preparation and countries of use

S/No Country Fish used Method

1 France Gobius spp. 2-8 weeks in 4:1 salt solution

2 Indonesia Clupea spp. 6 months in 6 :1 salt solution

3 Japan Clupeapilchardus 5-6 months in 5 :1 salt to rice

4 Malaysia Stelophorus spp. 4–12 months; 5 :1 salt to sugar

5 The Philippines Stelophorus spp. 3–12 months; 4: 1 salt solution

6 Thailand Stelophorus spp. 6-12 months; 4:1 salt solution

The fish is fermented in a solution of salt. Sometimes, the fish is fermented whole, or

sometimes only the viscera are fermented. Sometimes carbohydrate sources such as

malted rice may be added, as in Japan.

SUGGESTED READINGS

Amund, O.O., Ogunsina, O.A. 1987. Extracellular amylase production by cassava-fermenting

bacteria. Journal of Industrial Microbiology. 2, 123-127.

Bamforth, C.W. 2005. Food, Fermentation and Microorganisms. Blackwell Oxford, UK.

Banigo, E.O.I., de Man, J.M., Duitschaever, C.L. 1974. Utilization of high-lysine corn for the

manufacture of ogi, using a new improved proceessing system. Cereal Chemistry 51: 559–

573.

Production of Fermented Foods !$

Bankole, M.O., Okagbue, R.N. 1992. Properties of “nono”, a Nigerian fermented milk food.

Ecology of Food and Nuitrition. 27, 145-149.

Batock, M., Azam-Ali, S. 1998. Fermented Frutis and Vegetables. A Global Perspective. FAO.

Agricultural Services Bulletin No. 134. Food and Agriculture Organization of the United

Nations, Rome, Italy.

Enujiugha, V.N., Akanbi, C.T. 2005. Compositional Changes in African Oil Bean (Pentaclethra

macrophylla Benth) Pakistan Journal of Nutrition, 4. 27-31.

Ikediobi, C.O., Onyike, E. 1982. The use of linamarase in gari production. Process Biochemistry

17, 2-5.

Kobawila, S.C. Louembe, D., Keleke, S., Hounhouigan, J., Gamba, C. 2005. Reduction of the

cyanide content during fermentation of cassava roots and leaves to produce bikedi and ntoba

mbodi, two food products from Congo. African Journal of Biotechnology. 4 689-696.

Lei, V., Amoa-Awua, W.K.A., Brimer, L. 1999. Degradation of cyanogenic glycosides by

Lactobacillus plantarum strains from spontaneous cassava fermentation and other

microorganisms International Journal of Food Microbiology 53, 169-184.

Nwachukwu, S.U., Edwards, A.W.A. 1987. Microorganisms associated with cassava

fermentation for lafun production. Journal of Food and Agriculture. 1, 39-42.

Okafor, N., Okeke, B., Umeh, C., Ibenegbu, C. 1999. Secretion of Lysine by Lactic Bacteria and

Yeasts Associated with Garri Production Using a Synthetic Gene. Letters in Applied

Microbiology, 28, 419-422.

Okafor, N., Uzuegbu., J.O. 1993. Studies on the Contributions of Factors other than micro-

organisms to the Flavour of Garri. Journal of Agricultural Technology, 1, 36-38.

Okafor, N., Umeh, C., Ibenegbu, C. 1998. Carriers for starter cultures for the production of garri,

a fermented food derived from cassava. World Journal of Microbiology and Biotechnology.

15, 231-234.

Okafor, N., Umeh, C., Ibenegbu, C. 1998 Amelioration of garri, a fermented food derived from

cassava, Manihot esculenta Crantz, by the inoculation into cassava mash, of micro-organisms

simultaneously producing amylase, linamarase, and lysine. World Journal of Microbiology

and Biotechnology, 14, 835-838.

Okafor, N., Umeh, C., Ibenegbu, C, Obizoba., I.C., Nnam, P. 1998. Improvement in garri quality

by the inoculation of micro-organisms into cassava mash. International Journal of Food

Microbiology. 40, 43-49.

Okafor, N., Ejiofor, M.A.N. 1985. Microbial breakdown of linamarin in fermenting cassava pulp.

MIRCEN Journal of Applied Microbiology and Biotechnology. 2, 269-276.

Okafor, N., Ejiofor, M.A.N. 1985. The linamarase of Leuconostoc mesenteroides: Production,

isolation and some properties. J. Sci. Food Agric. 36, 668-678.

Okafor, N., Ejiofor, A.O. 1990. Rapid detoxification of cassava mash fermenting for garri

production following inoculation with a yeast simultaneously producing linamarase and

amylase Process Biochemistry International. 25, 82-86.

Okafor, N., Uzuegbu, J. 1987. Studies on the contributions of micro organisms on the organo-

leptic properties of garri, a fermented food derived from cassava (Manihot esculenta Crantz).

J. Food Agric. 2, 99-105.

Okafor, N. 1977. Microorganisms associated cassava fermentation for garri production. J. Appl.

Bact. 42, 279-284.

Okafor, N., Oyolu, C., Ijioma, B.C. 1984. Microbiology and biochemistry of foo-foo production.

J. Appl. Microbiol., 55, 1-13.

Omofuvbe, B.O. Abiose, S.H., Shonukan, O.O. 2002. Fermentation of soybean (Glycine max) for

soy-daddawa production by starter culturesof Bacillus. Food Microbiology, 19, 187-190.

!$ Modern Industrial Microbiology and Biotechnology

Oteng-Gyang, K., Anuonye, C.C. 1987. Biochemical studies on the fermentation of cassava

(Manihot utilissima Pohl). Acta Biotechnologica. 7, 289-292.

Oyewole, O.B. 2001. Characteristics and significance of yeasts’ involvement in cassava fermentation

for ‘fufu’ production. International Journal of Food Microbiology. 65, 213-218.

Oyewole, O.B., Odunfas 1990. Characterization and distribution of lactic acid bacteria in cassava

fermentation during fufu production. Journal of Applied Bacteriology, 68, 145-152.

Popoola, T.O.S., Akueshi, C.O. 1986. Nutritional value of dawadawa, a local spice made from

soybean (Glycine max) MIRCEN Journal. 2, 405-409.

Sanni, A.I., Onilude, A.A., Fadahunsi. S.T., Ogunbanwo, S.T., Afolabi, R.O. 2002. Selection of

starter cultures for the production of ugba, a fermented soup condiment. European Food

Research and Technology, 215, 176-180.

Tamime, A.Y., Robinson, R.K. 1999. Yoghurt Science and Technology. CRC Press.

Wood, B.J.R. (ed) 1985. Microbiology of Fermented Foods, Vol 1 Elsevier Applied Science

Publishers. London and New York.

Wood, B.J.R. (ed) 1985. Microbiology of Fermented Foods, Vol 2 Elsevier Applied Science

Publishers. London and New York.

Production of Metabolites as

Bulk Chemicals or as Inputs in

Other Processes

Section.

Modern Industrial

Microbiology and Biotechnology

20.1 ORGANIC ACIDS

A large number of organic acids with actual or potential uses are produced by micro-

organisms. Citric, itaconic, lactic, malic, tartaric, gluconic, mevalonic, salicyclic,

gibberelic, diamino-pimelic, and propionic acids are some of the acids whose microbial

production have been patented. In this chapter the production of only citric and lactic

acids will be discussed.

20.1.1 Production of Citric Acid

Citric acid is a tribasic acid with the structure shown in Fig. 20.1.

C - COOH

OH - C - COOH

C - COOH

Fig. 20.1 Structure of Citric Acid

It crystallizes with the large rhombic crystals containing one molecule of water of

crystallization, which is lost when it is heated to 130°C. At temperatures as high as 175°C

it is converted to itaconic acid, aconitic acid, and other compounds.

20.1.2 Uses of Citric Acid

Citric acid is used in the food industry, in medicine, pharmacy and in various other

industries.

Uses in the food industry

(i) Citric acid is the major food acidulant used in the manufacture of jellies, jams,

sweets, and soft drinks.

Production of Organic Acids

and Industrial Alcohol

+0)26-4

!$$ Modern Industrial Microbiology and Biotechnology

(ii) It is used for artificial flavoring in various foods including soft drinks.

(iii) Sodium citrate is employed in processed cheese manufacture.

Uses in medicine and pharmacy

(iv) Sodium citrate is used in blood transfusion and bacteriology for the prevention of

blood clotting.

(v) The acid is used in efferverscent powers which depend for their efferverscence on

the CO

produced from the reaction between citric acid and sodium bicarbonate.

(vi) Since it is almost universally present in living things, it is rapidly and completely

metabolized in the human body and can therefore serve as a source of energy.

Uses in the cosmetic industry

(vii) It is used in astringent lotions such as aftershave lotions because of its low pH.

(viii) Citric acid is used in hair rinses and hair and wig setting fluids.

Miscellaneous uses in industry

(ix) In neutral or low pH conditions the acid has a strong tendency to form complexes

hence it is widely used in electroplating, leather tanning, and in the removal of iron

clogging the pores of the sand face in old oil wells.

(x) Citric acid has recently formed the basis of manufacture of detergents in place of

phosphates, because the presence of the latter in effluents gives rise to eutrophication

(an increase in nutrients which encourages aquatic flora development).

20.1.3 Biochemical Basis of the Production of Citric Acid

Citric acid is an intermediate in the citric acid cycle (TCA) (Fig. 20.2). The acid can

therefore be caused to accumulate by one of the following methods:

(a) By mutation – giving rise to mutant organisms which may only use part of a

metabolic pathway, or regulatory mutants; that is using a mutant lacking an

enzyme of the cycle.

(b) By inhibiting the free-flow of the cycle through altering the environmental

conditions, e.g. temperature, pH, medium composition (especially the elimination

of ions and cofactors considered essential for particular enzymes). The following

are some of such environmental conditions which are applied to increase citric

acid production:

(i) The concentrations of iron, manganese, magnesium, zinc, and phosphate must

be limited. To ensure their removal the medium is treated with ferro-cyanide or

by ion exchange fresins. These metal ions are required as prosthetic groups in

the following enzymes of the TCA: Mn

or Mg

by oxalosuccinic

decarboxylase, Fe

+++

is required for succinic dehydrogenase, while phosphate

is required for the conversion of GDP to GTP (Fig. 20.2).

(i) The dehydrogenases, especially isocitrate dehydrogenase, are inhibited by

anaerobiosis, hence limited aeration is done on the fermentation so as to

increase the yield of citric acid.

(ii) Low pH and especially the presence of citric acid itself inhibits the TCA and

hence encourages the production of more citric acid; the pH of the fermentation

Production of Organic Acids and Industrial Alcohol !$%

must therefore be kept low throughout the fermentation by preventing the

precipitation of the citric acid formed.

(iii) Many of the enzymes of the TCA can be directly inhibited by various

compounds and this phenomenon is exploited to increase citric acid

production. Thus, isocitric dehydrogenase is inhibited by ferrocyanide as well

as citric acid; aconitase is inhibited by fluorocitrate and succinic

dehydrogenase by malonate. These at enzyme antagonists may be added to the

fermentation.

Citric acid can be caused to accumulate by using a mutant lacking an enzyme of the cycle or

by inhibiting the flow of the cycle

Fig. 20.2 The Tricarboxylic Acid Cycle

!$& Modern Industrial Microbiology and Biotechnology

20.1.4 Fermentation for Citric Acid Production

For a long time the production of citric acid has been based on the use of molasses and

various strains of Aspergillus niger and occasionally Asp. wenti. Although several reports

of citric acid production by Penicillium are available, in practice, organisms in this group

are not used because of their low productivity. In recent times yeasts, especially Candida

spp. (including Candida quillermondi) have been used to produce the acid from sugar.

Paraffins became used as substrate from about 1970. In the processes described mainly

by Japanese workers bacteria and yeasts have been used. Among the bacteria were

Arthrobacter paraffineus and corynebacteria; the yeasts include Candida lipolytica and

Candida oleiphila.

Fermentation with molasses and other sugar sources can be either surface or

submerged. Fermentation with paraffins however is submerged.

(a) Surface fermentation: Surface fermentation using Aspergillus niger may be done on

rice bran as is the case in Japan, or in liquid solution in flat aluminium or stainless

steel pans. Special strains of Asp. niger which can produce citric acid despite the

high content of trace metals in rice bran are used. The citric acid is extracted from

the bran by leaching and is then precipitated from the resulting solution as calcium

citrate.

(b) Submerged fermentation: As in all other processes where citric acid is made the

fermentation the fermentor is made of acid-resistant materials such as stainless

steel. The carbohydrate sources are molasses decationized by ion exchange,

sucrose or glucose. MgSO

, 7H

O and KH

at about 1% and 0.05-2%

respectively are added (in submerged fermentation phosphate restriction is not

necessary). The pH is never allowed higher than 3.5. Copper is used at up to 500

ppm as an antagonist of the enzyme aconitase which requires iron. 1-5% of

methanol, isopranol or ethanol when added to fermentations containing

unpurified materials increase the yield; the yields are reduced in media with

purified materials.

As high aeration is deleterious to citric acid production, mechanical agitation is not

necessary and air may be bubbled through. Anti-form is added. The fungus occurs as a

uniform dispersal of pellets in the medium. The fermentation lasts for five to fourteen

days.

20.1.5 Extraction

The broth is filtered until clear. Calcium citrate is precipitated by the addition of

magnesium-free (Ca(OH)

. Since magnesium is more soluble than calcium, some acid

may be lost in the solution as magnesium citrate if magnesium is added. Calcium citrate

is filtered and the filter cake is treated with sulfuric acid to precipitate the calcium. The

dilute solution containing citric acid is purified by treatment with activated carbon and

passing through iron exchange beds. The purified dilute acid is evaporated to yield

crystals of citric acid. Further purification may be required to meet pharmaceutical

stipulations.

Production of Organic Acids and Industrial Alcohol !$'

20.1.6 Lactic Acid

Lactic acid is produced by many organisms: animals including man produce the acid in

muscle during work.

20.1.6.1 Properties and chemical reactions of lactic acid

(i) Lactic acid is a three carbon organic acid: one terminal carbon atom is part of an

acid or carboxyl group; the other terminal carbon atom is part of a methyl or

hydrocarbon group; and a central carbon atom having an alcohol carbon group.

Lactic acid exists in two optically active isomeric forms (Fig. 20.3).

L (+) Lactic acid D (-) Lactic acid

Fig. 20.3 Optical Isomers of Lactic Acid

(ii) Lactic acid is soluble in water and water miscible organic solvents but insoluble in

other organic solvents.

(iii) It exhibits low volatility. Other properties of lactic acid are summarized in Table

20.1.

(iv) The various reactions characteristic of an alcohol which lactic acid (or it esters or

amides) may undergo are xanthation with carbon bisulphide, esterification with

organic acids and dehdrogenation or oxygenation to form pyruvic acid or its

derivatives.

(v) The acid reactions of lactic acid are those that form salts and undergo esterification

with various alcohols.

(v) Liquid chromatography and its various techniques can be used for quantitative

analysis and separation of its optical isomers

Technical grade lactic acid is used as an acidulant in vegetable and leather tanning

industries. Various textile finishing operations and acid dyeing of food require low cost

technical grade lactic acid to compete with cheaper inorganic acid. Lactic acid is being

used in many small scale applications like pH adjustment, hardening baths for

cellophanes used in food packaging, terminating agent for phenol formaldehyde resins,

alkyl resin modifier, solder flux, lithographic and textile printing developers, adhesive

formulations, electroplating and electropolishing baths, detergent builders.

Lactic acid has many pharmaceutical and cosmetic applications and formulations in

topical ointments, lotions, anti acne solutions, humectants, parenteral solutions and

dialysis applications, and anti carries agents. Calcium lactate can be used for calcium