Okafor N. Modern Industrial Microbiology and Biotechnology

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!! Modern Industrial Microbiology and Biotechnology

rhizobia being assessed. One seedling each is placed on nitrogen free slants of agar

containing appropriate mineral salts in a large test tube. (approx. 5 cm x 20 cm).

The surface of the agar is flooded with a heavy suspension of the rhizobium to be

tested. Control slants with nitrate and without rhizobia are prepared. The

nitrogen-fixing ability of the system is assessed by harvesting the seedlings and

determining their dry weight and nitrogen contents over the control after an

appropriate period of growth. Alternatively all the nodules may be excised from

the various test groups and assessed indirectly for nitrogen fixation by the

acetylene reduction method. This is a rapid and convenient method for assessing

nitrogen fixation. Nodules are exposed to acetylene and thereafter the gases are

checked in a gas chromatograph for ethylene production. The ability of nodules,

free-living bacteria or other biological systems to fix nitrogen is determined from

the extent of acetylene-ethylene conversion.

(ii) Soil cores: Undisturbed soil cores from the field may be planted with germinated

seedlings and inoculated with a culture of rhizobium in a glass house. Assessment

of nitrogen fixation may then be done as described above.

(iii) Field assessment: For field assessment the material in which the rhizobium is carried

should have been selected and the experiment laid out in such a way that; (a)

uninoculated plots are tested for the presence of naturally occurring rhizobia; (b)

plots are inoculated with the organism being tested; (c) plots are inoculated with

the test organism and simultaneously supplied with nitrogen fertilizers. The

plants are then harvested and checked for dry weight and nitrogen content.

While soil agar and soil tube tests are useful for rapid screening, there is no substitute

for field testing using the expected final carrier in soil as similar as possible to that in

which the rhizobium is destined to grow.

18.2 FERMENTATION FOR RHIZOBIA

(i) The inoculum: The inoculum is prepared from a stock culture preferably stored in

sterile soil or on agar overlain with oil. The organisms used in preparing the

inoculum are preferably those able to form nitrogen-fixing nodules with several

legumes (i.e., the so-called ‘broad-spectrum’ strains). Where these are not available

a mixture of several strains effective in a wide range of legumes are used. In this

way the need to prepare a large number of small amounts of the inoculant is

obviated. The inoculum added is preferably about 1.0% of the total volume and

should have a density of 10

-10

organisms per ml.

(ii) Medium: Rhizobia are not very demanding of nutritional requirements. Most

industrial media used consist of yeast extract or yeast hydrolysate, a carbohydrate

source and mineral salts. In some media yeast extracts supply all the nitrogen,

vitamins (especially biotin) and minerals required by the bacteria. Corn steep

liquour and hydrolyzed casein are sometimes used to supplement yeast extract. In

some media, one or more of potassium phosphate, magnesim sulfate, ferric

chloride, and sodium chloride may be added. For the fast growers the carbohydrate

source is usually sucrose; for the slow growers it may be mannitol, galactose or

arabinose. Fast growers (e.g. Rhizobium meliloti) have large gummy colonies which

form in three to five days. Slow growers (e.g. Rhizobium japonium) have small

The Manufacture of Rhizobium Inoculants !!

colonies taking 7 to 10 days to develop. The former are petrichuously flagellated

while the latter are sub-polarly flagellated.

(iii) Aeration: Rhizobia are aerobic organisms; nevertheless the type of intensive

agitation and aeration used for the production of yeasts or some antibiotics does

not seem necessary. Indeed rhizobia will grow quite well in an unaerated

fermentor if there is a broad enough surface area to permit oxygen diffusion. Very

low aeration, as low as 0.5 liters/hour, has been found satisfactory. In large

fermentors air sparging without agitation is usually satisfactory.

(iv) Time and temperature: The temperature used is about 20°C and while fast growers

attain high numbers (in excess of 140 x 10

/ml) in about two to three days, slow

growers in medium specially designed to facilitate their growth attain slightly less

than this number in three to five days.

(v) The fermentor: Fermentors used for rhizobium culture are small in comparison with

those used for antibiotics. The larger sizes range from 1,000 to 2,000 liters.

Ordinarily they range from 5 liters through 40 liters to about 200 liters and are also

usually quite unsophisticated compared with those used for producing

antibiotics.

18.3 INOCULANT PACKAGING FOR USE

After the fermentation of the organism it is packaged for delivery in either of two forms: (a)

as a coating on the seeds, (or seed inoculants) from which the rhizobia develop at

planting and invade the roots; (b) direct application into soil with the seeds introduced

shortly before or after soil inoculants.

18.3.1 Seed Inoculants

Seed inoculants are more commonly used than soil inoculants. In seed inoculation the

rhizobia may be offered as a liquid or broth, frozen concentrates, freeze-dried or oil-dried

preparation. No specific carrier is used for these preparations. Although gum Arabic,

milk, and sucrose are sometimes added to these essentially liquid preparations, they still

do not offer sufficient protection to the bacterial from the environment. The bacteria

therefore die out quickly. By far the commonest preparations are offered with carriers.

A carrier is the material which binds the rhizobium to the seed. Carriers should have

a high water-holding capacity provided a nutritive medium for the growth of rhizobia

protect the bacteria from harmful environmental effect e.g. sunlight and favor their

survival on the seeds and in the soil, and in particular they should not be toxic to the

bacteria.

Agar may sometimes be used as a carrier but by far the most widely used carrier is peat.

Other locally available materials may be used.

(i) The use of peat as a carrier

Peat is the first stage in the formation of coal, which when freshly obtained is moist. It

must be dried and milled or shredded. Peats vary in their properties and each must be

studied and undesirable properties rectified before final use. For example peats mined in

some parts of the world have a high content of sodium chloride, which must be removed

!! Modern Industrial Microbiology and Biotechnology

by leaching with water before the peat is used. Peats are also usually acid, and finely

ground CaCO

up to about 5% is used to raise the pH to about 6.8.

The survival rate of rhizobia in unsterilized carriers is low because of competition and

antagonism from resident organisms. The peat is therefore usually sterilized by hot air,

steam (including autoclaving), gamma irradiation and chemical sterilants. Hot air and

steam seem to be favored, as gumma-irradiation facilities are not always easily accessible

and the post-treatment removal of chemical sterilants may sometimes be difficult. Care

must be taken to ensure that materials toxic to the rhizobia are not released by the use of

too high a temperature.

In order to introduce the organism into a carrier two methods are used. In the United

States the broth containing a heavy growth of rhizobium in excess of 10

/ml, is mixed

with CaCO

and sprayed onto sterilized ground peat. It is then incubated in thin layers at

26-28°C for two to three days to allow the heat generated during the wetting of the peat to

dissipate. Thereafter it is ground and bagged: adequate numbers are reached in the peat

in three to five weeks for fast growers and in about twice as long for slow growers. At the

end of this period the preparation is refrigerated till used. In other parts of the world, the

broth is inoculated into bottles or polythene bags containing sterile peat or a peat/soil

mixture. They are shaken after 24 hours and allowed to grow for one to two weeks at

26-30°C and thereafter stored at 2-4°C. For seed inoculation the rhizobium in broth or

with a carrier is brought in contact with the seeds, which then become coated with the

organisms.

(ii) Use of other carriers

Peat is not available in some parts of the world. Any carrier which meets the requirements

indicated above would do. A wide range of materials have in fact been tried with success

including lignite, coal, charcoal, bagasse, coir dust, composted straw plus charcoal,

ground wheat straw, rice husk, and ground talc.

The essential thing is that the carrier, be it peat or any other substance(s) must be

shown to be suitable both in laboratory experimentation, and also in the field.

18.3.2 Soil Inoculants

When seed inoculation is not practicable, soil inoculation is resorted to. The following

are conditions when seed inoculation is not efficient:

(a) In some epigeal legumes for example soybean, the seed coat of the seedling is lifted

out of the ground during the emergence of the cotyledons (seed leaves). Under this

condition, the rhizobia clinging to the seed coat are not deposited in the soil.

(b) Seeds coated with fungicides or insecticides cannot be successfully inoculated

with rhizobia.

are introduced, heavy soil inoculation is practiced, especially if very aggressive but

ineffective rhizobia exist in the soil.

(d) Finally fragile seeds such as peanuts (groundnuts) may break following prior

wetting during seed inoculation.

The Manufacture of Rhizobium Inoculants !!!

Besides the above difficulties soil inoculation has the additional advantage of relative

ease of application; furthermore, it may be used to inoculate the growing plant in

situations where earlier inoculation failed.

Rhizobia may be inoculated into soil using one of two ways:

(a) Frozen concentrate obtained from centrifugation is thawed and diluted with water

and applied to soil;

(b) Free flowing granules made from peat-rhizobium preparations may be applied to

soil.

18.4 QUALITY CONTROL

It is important that the number and nitrogen-fixing qualities of the inoculant meet the

expectation of the user at the time of application. The number of rhizobia required for

effective nodulation depends on a large number of factors including the size of the seed,

the presence or absence of competing rhizobia in the soil weather conditions; the

temperature moisture, and type of soil. Nevertheless the accepted standard is a minimum

of 1,000 cells of Rhizobium per seed, except in the case of soybean where the minimum is

10.

In some countries, such as Australia a government or university body supervises all

stages of the production of inoculants including the testing and selection of the strains,

maintenance and issuing of stock cultures to manufacturers, assessing the quality of the

broth and the final preparation of the peat-carried inoculum. In other countries notably

the United States the control is left to the producing companies. In either case the

performance of the inoculants in the hands of the farmer is the ultimate test. Wherever

rhizobia are inoculated some means of ensuring the quality of the product must exist.

Once the material has been prepared and the maximum number possible in the

preparation attained quality is most easily maintained by storing it under refrigeration

until use, provided that adequate content and some aeration is maintained in the

package. A system of expiry date based on experience is often indicated on the package.

SUGGESTED READINGS

Brockwell, J. 1977. In: A Treatise on Dinitrogen Fixation: Section IV: Agronomy and Ecology.

R.W.F. Hardy, A.H. Gibson, (eds). Wiley, New York, USA. pp. 277-309.

Flickinger, M.C., Drew, S.W. (eds), 1999. Encyclopedia of Bioprocess Technology - Fermentation,

Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.

Temprano, F.J., Albareda, M., Camacho, M., Daza, A., Santamaria, C., Rodrýguez-Navarro. 2002.

Survival of several Rhizobium/Bradyrhizobium strains on different inoculant formulations and

inoculated seeds. International Microbioliology. 5, 81–86.

!!" Modern Industrial Microbiology and Biotechnology

19.1 INTRODUCTION

Fermented foods may be defined as foods which are processed through the activities of

microorganisms but in which the weight of the microorganisms in the food is usually

small. The influence of microbial activity on the nature of the food, especially in terms of

flavor and other organoleptic properties, is profound. In terms of this definition,

mushrooms cannot properly be described as fermented foods as they form the bulk of the

food and do not act on a substrate which is consumed along with the organism. In

contrast, yeasts form a small proportion by weight on bread, but are responsible for the

flavor of bread; hence bread is a fermented food.

Fermented foods have been known from the earliest period of human existence, and

exist in all societies. Fermented foods have several advantages:

(a) Fermentation serves as a means of preserving foods in a low cost manner; thus

cheese keeps longer than the milk from which it is produced;

(b) The organoleptic properties of fermented foods are improved in comparison with

the raw materials from which they are prepared; cheese for example, tastes very

different from milk from which it is produced;

material; thus fermentation removes flatulence factors in soybeans, and reduces

the poisonous cyanide content of cassava during garri preparation (see below);

(d) The nutritive content of the food is improved in many items by the presence of the

microorganisms; thus the lactic acid bacteria and yeasts in garri and the yeasts in

bread add to the nutritive quality of these foods;

(e) Fermentation often reduces the cooking time of the food as in the case of fermented

soy bean products, or ogi the weaning West African food produced from fermented

maize.

Fermented foods are influenced mainly by the nature of the substrate and the

organisms involved in the fermentation, the length of the fermentation and the treatment

of the food during the processing.

Production of

Fermented Foods

+0)26-4

Production of Fermented Foods !!#

The fermented foods discussed in this chapter are arranged according to the

substrates used:

Wheat

Bread

Milk

Cheese

Yoghurt

Maize

Ogi, Akamu, Kokonte

Cassava

Garri

Foo-foo, Akpu, Lafun

Vegetables

Sauerkraut

Pickled cucumbers

Stimulant beverages

Coffee, Tea and Cocoa

Legumes and oil seeds

Soy sauce, Miso, Sufu

Oncom. idli

Ogili, Dawa dawa, Ugba

Fish

Fish sauce

19.2 FERMENTED FOOD FROM WHEAT: BREAD

Bread has been known to man for many centuries and excavations have revealed that

bakers’ ovens were in use by the Babylonians, about 4,000 B.C. Today, bread supplies

over half of the caloric intake of the world’s population including a high proportion of the

intake of Vitamins B and E. Bread is therefore a major food of the world.

19.2.1 Ingredients for Modern Bread-making

The basic ingredients in bread-making are flour, water, salt, and yeasts. In modern bread-

making however a large number of other components and additives are used as

knowledge of the baking process has grown. These components depend on the type of

bread and on the practice and regulations operating in a country. They include ‘yeast

food’, sugar, milk, eggs, shortening (fat) emulsifiers, anti-fungal agents, anti-oxidants,

enzymes, flavoring, and enriching ingredients. The ingredients are mixed together to

form dough which is then baked.

!!$ Modern Industrial Microbiology and Biotechnology

19.2.1.1 Flour

Flour is the chief ingredient of bread and is produced by milling the grains of wheat,

various species and varieties of which are known. For flour production most countries

use Triticum vulgare. A few countries use T. durum, but this yellow colored variety is more

familiarly used for semolina and macaroni in many countries. The chief constituents of

flour are starch (70%), protein (7-15%), sugar (1%), and lipids (1%).

In bread-making from T. vulgare the quality of the flour depends on the quality and

quantity of its proteins. Flour proteins are of two types. The first type forming about 15%

of the total is soluble in water and dilute salt solutions and is non-dough forming. It

consists of albumins, globulins, peptides, amino acids, and enzymes. The remaining 85%

are insoluble in aqueous media and are responsible for dough formation. They are

collectively known as gluten. It also contains lipids.

Gluten has the unique property of forming an elastic structure when moistened with

water. It forms the skeleton which holds the starch, yeasts, gases and other components of

dough. Gluten can be easily extracted, by adding enough water to flour and kneading it

into dough. After allowing the dough to stand for an hour the starch can be washed off

under a running tap water leaving a tough, elastic, sticky and viscous material which is

the gluten. Gluten is separable into an alcohol soluble fraction which forms one third of

the total and known as gladilins and a fraction (two thirds) that is not alcohol-soluble

and known as the glutenins. Gladilins are of lower molecules weight than glutenins; they

are more extensible, but less, elastic than glutenins. Glutelins are soluble in acids and

bases whereas glutenins are not. The latter will also complex with lipids, whereas

glutelins do not. ‘Hard’ wheat with a high content of protein (over 12%) are best for

making bread because the high content of glutenins enables a firm skeleton for holding

the gases released curing fermentation. ‘Soft’ wheat with low protein contents (9-11%)

are best for making cakes.

19.2.1.2 Yeast

The yeasts used for baking are strains of Saccharomyces cerevisiae. The ideal properties of

yeasts used in modern bakeries are as follows:

(a) Ability to grow rapidly at room temperature of about 20-25°C;

(b) Easy dispersability in water;

rather than alcohol in flour dough;

(d) Good keeping quality i.e., ability to resist autolysis when stored at 20°C;

(e) Ability to adapt rapidly to changing substrates such as are available to the yeasts

during dough making.

(f) High invertase and other enzyme activity to hydrolyze sucrose to higher

glucofructans rapidly;

(g) Ability to grow and synthesize enzymes and coenzymes under the anaerobic

conditions of the dough;

(h) Ability to resist the osmotic effect of salts and sugars in the dough;

(i) High competitiveness i.e., high yielding in terms of dry weight per unit of substrate

used.

Production of Fermented Foods !!%

The amount of yeasts used during baking depends on the flour type, the ingredients

used in the baking, and the system of baking used. Very ‘strong’ flours (i.e., with high

protein levels) require more yeast than softer ones. High amount of components

inhibitory to yeasts e.g., sugar (over 2%), antifungal agents and fat) usually require high

yeast additions. Baking systems which involve short periods for dough formation, need

more yeast than others. In general however yeast amounts vary from 2-2.75% (and

exceptionally to 3.0%) of flour weight. The roles of yeasts in bread-making are leavening,

flavor development and increased nutritiveness. These roles and the factors affecting

them are discussed more fully below.

Yeast ‘food’ The name yeast ‘food’ is something of a misnomer, because these

ingredients serve purposes outside merely nourishing the yeasts. In general the ‘foods’

contain a calcium salt, an ammonium salt and an oxidizing agent. The bivalent calcium

ion has a beneficial strengthening effect on the colloidal structure of the wheat gluten.

The ammonium is a nitrogen source for the yeast. The oxidizing agent strengthens gluten

by its reaction with the proteins’ sulfydryl groups to provide cross-links between protein

molecules and thus enhances its ability to hold gas releases during dough formation.

Oxidizing agents which have been used include iodates, bromates and peroxide. A well-

used yeast food has the following composition: calcium sulfate, 30%, ammonium

chloride, 9.4%, sodium chloride, 35%, potassium bromate, 0.3%; starch (25.3%) is used as

a filler.

19.2.1.3 Sugar

Sugar is added (a) to provide carbon nourishment for the yeasts additional to the amount

available in flour sugar (b) to sweeten the bread; (c) to afford more rapid browning

(through sugar caramelization) of the crust and hence greater moisture retention within

the bread. Sugar is supplied by the use of sucrose, fructose corn syrups (regular and high

fructose), depending on availability.

19.2.1.4 Shortening (Fat)

Animal and vegetable fats are added as shortenings in bread-making at about 3% (w/w)

of flour in order to yield (a) increased loaf size; (b) a more tender crumb; and c) enhanced

slicing properties. While the desirable effects of fats have been clearly demonstrated their

mode of action is as yet a matter of controversy among bakery scientists and cereal

chemists. Butter is used only in the most expensive breads; lard (fat from pork) may be

used, but vegetable fats especially soy bean oil, because of its most assured supply is now

common.

19.2.1.5 Emulsifiers (Surfactants)

Emulsifiers are used in conjunction with shortening and ensure a better distribution of

the latter in the dough. Emulsifiers contain a fatty acid, palmitic, or stearic acid, which is

bound to one or more poly functional molecules with carboxylic, hydroxyl, and/or

amino groups e.g., glycerol, lactic acid, sorbic acid, or tartaric acid. Sometimes the

carboxylic group is converted to its sodium or calcium salt. Emulsifiers are added as 0.5%

flour weight. Commonly used surfactants include: calcium stearyl- 2-lactylate, lactylic

stearate, sodium stearyl fumarate.

!!& Modern Industrial Microbiology and Biotechnology

19.2.1.6 Milk

Milk to be used in bread-making must be heated to high temperatures before being dried;

otherwise for reasons not yet known the dough becomes sticky. Milk is added to make the

bread more nutritious, to help improve the crust color, presumably by sugar

cearamelization and because of its buffering value. Due to the rising cost of milk, skim

milk and blends made from various components including whey, buttermilk solids,

sodium or potassium caseinate, soy flour and/or corn flour are used. The milk

substitutes are added in the ratio of 1-2 parts per 100 parts of flour.

19.2.1.7 Salt

About 2% sodium chloride is usually added to bread. It serves the following purposes:

(a) It improves taste;

(b) It stabilizes yeast fermentation;

(d) Helps retard proteolytic activity, which may be related to its effect on gluten;

(e) It participates in the lipid binding of dough.

Due to the retarding effect on fermentation, salt is preferably added towards the end of

the mixing. For this reason flake-salt which has enhanced solubility is used and is added

towards the end of the mixing. Fat-coated salt may also be used; the salt becomes

available only at the later stages of dough or at the early stages of baking.

19.2.1.8 Water

Water is needed to form gluten, to permit swelling of the starch, and to provide a medium

for the various reactions that take place in dough formation. Water is not softened for

bread-making because, as has been seen, calcium is even added for reasons already

discussed. Water with high sulphide content is undesirable because gluten is softened by

the sulphydryl groups.

19.2.1.9 Enzymes

Sufficient amylolytic enzymes must be present during bread-making to breakdown the

starch in flour into fermentable sugars. Since most flours are deficient in alpha-amylase

flour is supplemented during the milling of the wheat with malted barley or wheat to

provide this enzyme. Fungal or bacterial amylase preparations may be added during

dough mixing. Bacterial amy1ase from Bacillus subtilis is particularly useful because it is

heat-stable and partly survives the baking process. Proteolytic enzymes from Aspergillus

oryzae are used in dough making, particularly in flours with excessively high protein

contents. Ordinarily however, proteases have the effect of reducing the mixing time of the

dough.

19.2.1.10 Mold-inhibitors (antimycotics) and enriching additives

The spoilage of bread is caused mainly by the fungi Rhizopus, Mucor, Aspergillus and

Penincillium. Spoilage by Bacillus mesenteroides (ropes) rarely occurs. The chief anti-

mycotic agent added to bread is calcium propionate. Others used to a much lesser extent

are sodium diacetate, vinegar, mono-calcium phosphate, and lactic acid.

Production of Fermented Foods !!'

Bread is also often enriched with various vitamins and minerals including thiamin,

riboflavin, niacin and iron.

19.2.2 Systems of Bread-making

Large-scale bread-making is mechanized. The processes of yeast-leavened bread-making

may be divided into:

(a) Pre-fermentation (or sponge mixing): At this stage a portion of the ingredients is

mixed with yeast and with or without flour to produce an inoculum. During this

the yeast becomes adapted to the growth conditions of the dough and rapidly

multiplies. Gluten development is not sought at this stage.

(b) Dough mixing: The balance of the ingredients is mixed together with the inoculum

to form the dough. This is the stage when maximum gluten development is sought.

rounded by machines.

(d) First (intermediate) proofing: The dough is allowed to rest for about 15 minutes

usually at the same temperature as it has been previous to this time i.e., at about

27°C. This is done in equipment known as an overhead proofer.

(e) Molding: The dough is flattened to a sheet and then moulded into a spherical body

and placed in a baking pan which will confer shape to the loaf.

(f) Second proofing: This consists of holding the dough for about 1 hour at 35-43°C and

in an atmosphere of high humidity (89-95°C)

(g) Baking: During baking the proofed dough is transferred, still in the final pan, to the

oven where it is subjected to an average temperature of 215-225°C for 17-23

minutes. Baking is the final of the various baking processes. It is the point at which

the success or otherwise of all the previous inputs is determined.

(h) Cooling, slicing, and wrapping: The bread is depanned, cooled to 4-5°C sliced

(optional in some countries) and wrapped in waxed paper, or plastic bags.

The Three Basic Systems of Bread-making

There are three basic systems of baking. All three are essentially similar and differ only in

the presence or absence of a pre-fermentation. Where pre-fermentation is present, the

formulation of the pre-ferment may consists of a broth or it may be a sponge (i.e., includes

flour). All three basic types may be sponge i.e includes flour. All three basic types may

also be batch or continuous.

(i) Sponge doughs: This system or modification of it is the most widely used worldwide.

It has consequently been the most widely described. In the sponge-dough system of

baking a portion (60-70%) of the flour is mixed with water, yeast and yeast food in a slurry

tank (or ‘ingridator’) during the pre-fermentation to yield a spongy material due to

bubbles caused by alcohol and CO

(hence the name). If enzymes are used they may be

added at this stage. The sponge is allowed to rest at about 27°C and a relative humidity of

75-80% for 3.5 to 5 hours. During this period the sponges rises five to six times because of

the volatile products released by this yeast and usually collapses spontaneously. During

the next (or dough) stage the sponge is mixed with the other ingredients. The result is a

dough which follows the rest of the scheme described above. The heat of the oven causes