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Manufacturing of Ethyl Alcohol from Fruit Waste

INTRODUCTION

In general, alcohol means colorless volatile inflammable liquid especially

intoxicant in wire, beer, spirits etc. and as a solvent, fuel etc. Depending on the

interest of the person involved, for alcohols are many things to many people. To

most non technical people, "alcohol" in the broad sense is used to describe any

intoxicating beverage; thus, a heavy or habitual drinker is called "alcoholic" the case

is some what different in the industrial manufacturing fields which produce finished

goods, intermediates, or raw materials, alcohols play a key role as important

organic solvents and rank second only to water in terms of their almost universal

application. Hence to these people alcohols mean solvents.

Take a spoonful of medicine; feel the smoothness of the lacquer on the pine

pralines in the play room ; look at the tyres of your car, smell the window cleaner

spray, make use of a hair spray, deodorant stick, or are antiperspiral; carry your

water proof cloth covered books to school etc. Alcohols play their solvent role in all

these personal events. Alcohols can be regarded as hydroxyl derivatives of

hydrocarbons, one the basis of several types of classification, di-hydric. There are

various types of alcohol belonging to the homologues series of alcohols, of like

methanol, ethanol, propanol etc. teach member in the series have its own specificproperty and most active member of the series is "Ethanol".

Here the fig. Below shows the chemicals from ethanol.

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Acetaldehyde

Ethylene

AlkylatedAeromatics

Diethyl Ether

Mixed ether

Organic Esters

ButadieneEthoxides

Halides

Esters

Ethanol


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distillation came into the mind of the people workers over the put of alcohol for

India.

In India, the concept of fermentation was commercialized in 1900 to 1910

after the discovery of Louis Pastern and the process came in to use before 1930 to

about 1948 saw the development of indirect Hydration process to manufacture

Ethanol synthetically.

OBJECTIVE :

Growth in potable alcohol industries in liked with the demand for the

consumption of the people and the chemical products in industries in which alcohol

plays an impatant role.

Fruits ripe within 4-5 days, the unripe fruits can be stored at 50% 52 F forfive weeks while ripe fruits can be stored at 320 to 350F.

Yield up to 10 years 750 fruits/ha.

Field up to 5 years 1000 1500 fruits/ha.

After 15 years, yield was 2000 2500 fruits or 18 20 tons /ha.

Especially, in Maharashtra, Sapota was grown in area of 3900ha and the

production was obtained 10,920.

HANDLING AND STORAGE :

Unripe Sapota is kept into the large capacity containers and the containers

are packed with the straw. So, that the surface of the two fruits could not touch with

each other. It should be stored away from all ignition sources and also from high

and low temp. The temp is kept moderate ( depending upon the atmosphere

conditions of the place ).

Under these conditions, the unripe Sapota gets ripen and ready to eat.

All the sources for ethanol production

The three types of sources of ethanol production

1) SACCHARINE ( Sugar containing ) materials in which the carbohydrate ( the

actual substance from which the alcohol is made ) is present in the form of simple,

directly fermentable six and twelve carbon sugar cane, sugar beets, fruit ( Fresh or

dried ), citrus molasses, cane sorghum why and skim milk.

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2) STARCHY MATERIALS : That contain more complex carbohydrates such as

starch and insulin that can be broken down into the simplex sex and twelve carbon

sugars by hydrolysis with acid or by the action of enzymes in a process called

malting. Such materials includes corn, grain sorghum, barley, wheat, potatoes,

sweet potatoes, Jerusalem artichokes, cacti manioc, arrowroot and so in.

3) CELLULOSE MATERIALS :- Such as wood, wood waste, paper, straw, corn

stalks, corn cobs, cotton, etc. which contain material that can be hydrolyzed with

acid, enzymes or otherwise converted into fermentable sugars called glucose.

Different type of fruits likely to produce ethanol.

Fruit composition in Municipal solid waste :-

The fruit pertaining to solid.The Municipal solid waste pertaining to fruit waste in Akola City is 25%.

Now, the composition of waste, in Hampshire waste the energy content

cones from :

Articles % total of energy content

Paper/Card 46%

Plastics 22%

Putrescibles 11%

Fires 5%

Textiles 3%

Misc. 13%

Table for carbohydrate content in other fruits.

Fruit production and Acreage in Maharashtra :

The fruit production and acreage in Maharashtra State is given below in table form

as.

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FRUIT CHOSEN FOR EXPERIMENTATION

We have chosen the Chiku for experimentation. Botanical Name of Chiku is

Acharas sapota and the Family name is Mallinkara acaracs.

Chiku is called sapota. It is cheafly grown in moist coastal tracts of

paninsular India, but in recent years it has spread in zones of the Deccan Plateau

and also in sub mountain tracts of North India. In India the main centers of its

cultivation are coastal tract and Maharashtra, coastal areas of Andhra Pradesh,

Tamil Nadu, Karnataka, Saurashtra and sub-mountain areas of U.P. and Bengal. In

Maharashtra it is grown over the area of 100 hectares mainly concentrated in

Thana, Poona, Ahmednagar and Aurangabad districts. More than 70% of the area

is is in Thana district. In India, it is mainly grown for fruits, but in other countries themilky latex from the bark of the tree yields an important commercial product, which

forms the base for manufacture of Chewing-gum. It has very attractive shape and

used as decorative tree.

Origin:- It is a native of Maxico in Tropica, America, like that of guava. It is so

established in India, that one can hardly believe that it is foreign to India.

Tree characters:- It is slow growing evergreen tree, forms a good grown does not

require any training and pruning, height of the tree is 8-12 m bears terminaly.

Climate:- It is a tropical fruit crop which likes strictly tropical climate warm and moist

weather with high annual rainfall of 250 cms (80"-100"). It thrives in places where

maximum and minimum temperatures do not go beyond 340C and 110C. It does not

very hot and dry summers and temperature below 50C. If temperature go above

430C. dropping of blossom and scortching of fruits takes place in dry zones. Rain

cloudy weather is not any way harmful to the plant. It can be grown from sea level

to an elevation of 1000 m, but does not do well above 500 m. The best chiku

plantations are found within a short distance from sea shore ( in heavy rainfall area).

It can also be grown in drier tracts, chiku flowers all the year round in June-July,

Oct-Nov, and Jan.Feb. While under Poona conditions it flowers in June-July and

crops harvested in Dec-January.

Soil :- Chiku can be grown in soils having a minimum depth of 1 m. having water

table below 3-4 m. Chiku requires wall drained soils, alluvial loams on the river

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banks, sandy loans sea, red lateritic soils of high rainfall area and well drained

medium black soils are suitable for its cultivation. Chiku will not thrife well on ill

drained soils, soils having hard pan below, loamy soils very deep and stickly soils,

soils containing high per cent of lime are not suitable. The pH range should be 5.5

to 7.5.

Propagation:- Chiku is commercially propagated by vegetative methods like are

layering or inarch grafting, Inarch grafts are prepared on the root stock seedlings of

Rayans(Khirni) or chiku itself. In India, Khirni is very commonly used as root stock

for chiku. The gooti is shallow rooted the majority of roots concentrating in upper

30-40 cms. Of soils while roots of graft go as deep as 90 cms so gooti can thrive

well in both light and deep soils while grafts are suitable for planting only in soilshaving 1 m depth, gooti is reported to hear sweeter and mellower pulp, Grafts on

Khirni bear havily than on layers.

Planting:- In drier part planting is done on the onset of monsoon where as in heavy

rainfall areas it is planted after the heavy showers are over. Before planting the

land should be ploughed harrowed and brought to a fine tilth. The pits of size 1 x 1

x 1 m. are opened at a distance of 10 x 10 m. or 12 x 12 m. The pits should be

filled in with 2 kg phosphate, FYM and good soil. After planting the plant should be

supported with bamboo.

Varieties:- There are three main varieties grown in Maharashtra.

1. Kalipatti:- Leaves are dark green, leading variety of the state level shaped fruits,

or excedant spreading branches. The quality is best, pulp is very sweet, mellowing,

yields heavily. It contains one-two seeds, does well in climate of Konkan.

2. Pillipatti:- Next best variety of the State. It is also called as 'Chatri' due to

peculiar habit of growth in whorls, leaves lighter green, fruits are oval and round

small fruits. It is less sweet as compared to Kalipatti. Does best in humid climate.

3.Cricket ball :- Fruits large sized, round variety having granular flesh of market

sweetness grown away from the see coast in drier areas. Bears heavily as

compared to above two varieties.

Manuring:- When the plants are one year old it should receive about 5 kg of FYM

and about 150 gm of nitrogen. The doses should be increased with the

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advancement in age. At the age of 7th years it starts bearing fruits. There are two

main seasons Oct-Nov. and Jan-Feb, so far each flowering the tree should receive

50 kg FYM + 1 kg N, 0.5 kg P2O5 and 0.5 kg of K2O. In drier areas the flowering will

be mostly at beginning of monsoon, so manuring should be done in the month of

May. In case of Chiku there is not bahar treatment (i.e. with holding of water

practices are followed ). However, immediately after harvest orchard hygienic

practices are followed.

Training and Pruning:- Naturally chiku trees assumes a very attractive shape. It is

an ever green tree and requires hardly and pruning, training is done by allowing the

plant to grow upto and height of 1 m above which 3-4 well spaced branches are

allowed to arise.Harvesting and Yield:- Fruits are ready for harvest in about 4 months i.e. Oct-Nov.

flowering fruits matures by Jan-Feb. or flower matures fruits by April-May.

Harvesting is done when fruits is fully developed matured fruit are not allowed to

ripe on the fruit. At maturity it develop deep chocolate color. If you take a streak on

the fruit immature fruit shown a green below while matured fruit shows yellow colour

below the skin.

Fruits ripe within 4-5 days. The unripe fruits can be stored at 500-520F for

five weeks while ripe fruits can be stored at 320-350F.

Yield up to 10 years- 750 fruits/ha.

Yield 15 years - 1000 1500 fruits/ha.

After 15 years yield - 2000-2500 fruits or 18-20 tons/ha.

Highly susceptible to water logging and very densative to stagnation. It

however tolerates considerable drought. The plant can tolerate extremes but the

yield goes down as shadding of flowers occurs above 39 0C. Fair distribution or

rains with mild summer helps in increasing the set. Ramphal cannot withstand

severe summers or cold as that of sustard apple.

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Fruit carbohydrates table:

Fruit Carbohydrates Calories per piece

Apple 10.5 grams 44

Apple cooking 9 grams 35

Apricot 6.7 grams 30

Avocado 2 grams 150

Banana 26 grams 107

Blackberries each 0.2 grams 1

Blackcurrant each 0.25 grams 1.1

Cherry each 0.6 grams 2.4

Clementine 7.5 grams 30

Currants 1.4 grams 5

Damson 7.2 grams 28

Dates 3.3 grams 12.5

Gooseberries 0.65 grams 2.6

Grapes each 0.6 grams 2.4

Grapefruit whole 23 grams 100

Guava 4.4 grams 24

Kiwi 8 grams 34

Lemon 3.4 grams 20

Lychees 0.7 grams 3

Mango 9.5 grams 40

Melon 26 grams 110

Nectarines 9 grams 42

Olives trace 6.8

Orange 8.5 grams 35

Passion Fruit 3 grams 30

Paw Paw 6 grams 28

Peach 7 grams 35

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Pear 12 grams 45

Pineapple 12 grams 50

Plum 6 grams 25

Prunes 2.2 grams 9

Raisins 1.4 grams 5

Raspberries each 0.2 grams 1.1

Rhubarb 0.8 grams 8

Satsumas 8.5 grams 35

Strawberries (1 average) 0.6 grams 2.7

Sultanas 1.4 grams 5

Tangerine 6 grams 26

Values for carbohydrates in fruit may vary between different sized pieces!

Fruit Production and Acreage in Maharashtra

Sr.

No.

Fruit Area Production

1 Grapes 10,000 79,000

2 Banana 53,800 14,18,7003 Mango 35,400 1,45,1404 Sweet orange 5,700 20,5205 Mandarin orange 33,600 1,51,2006 Kagzi lime 13,200 29,0407 Other citrus fruits 800 1,6008 Pomegranate 7,700 40,0409 Guava 8,500 39,95010 Custard apple 2,800 6,72011 Fig 200 16012 Jackfruit 300 326

13 Papaya 1,500 11,25014 Sapota 3,900 10,92015 Cashew nut 19,000 5,89016 Others 23,700 18,480

Total 2,20,100 19,78,936

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Physical Properties of Ethanol

Constants Absolute 95% (by

vol)

Boiling point,0

C 78.3 78.0Electrical conductivity at 250C, Ohm-1/cm 1.35 x 10-9 -Explosive limits in air, vol % 4.3-19.0 -Flash point ( ASTM Tag Open Cup ), 0C 21 22Freezing point, 0C 114.1 -Heat of combustion of liquid, Kcal/mole 328 -Heat of fusion, cal/g 25.0 -Heat of vaporization at bp and 1 atm, cal/g 204.3 -Ignition temp.(apparent) in air, 0C 371-427 -Refractive index, n2D0 1.3614 1.3651Specific gravity at 20/20 0C 0.7905 0.8038

Specific tension at 200C, dynes/cm 0.579 0.618Surface tension at 200C, dynes/cm 22.3 22.8Vapour pressure at 200C, mm Hg 44 43Viscosity at 20 0C, cps 1.22 1.41

Chemical Properties : The chemical properties of ethanol are typical of n-

saturated monohydric alcohols, especially in reactions which are concerned with the

hydroxyl group. The ethyl group, however, does undergo several unique reactions,

(as was previously noted for the methyl group of methanol).

Alkylation. Ethanol is an important alkylating agent, especially with ammonia

and the amines. This particular unit process is known ammonolysis and aminolysis,

and the reaction with ammonia is shown below :

CH3 CH2OH + NH3 CH3 CH2NH2 + H2O ethyl amine

Sulfuric acid is known to dehydrate ethanol to ether in the manufacturing

process which is based on hydration. Hydrochloric acid and alumina get are also

commonly used as dehydratic catalysts.

The aromatic nucleus may be alkylated with ethanol in the presence of a

Friedel-Crafts catalyst :

CH2 CH3+ H2O

+ CH3 CH2OH

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ethyl benzene

Concentrated sulfuric acid may from ethyl hydrogen sulfate if added slowly to

ethanol :

CH3 CH2OH + H2SO4 CH3 CH2 H2SO4 + H2O

Slow distillation at reduced pressure will then form diethyl sulfate, an

ethylating agent :

2CH3 CH2 H2SO4 (CH3 CH2 )2SO4 + H2SO4

Sulfur trioxide adds to ethanol and forms carbyl sulfate :

CH3 CH2OH + 2SO3 CH2 CH2 O| | |

SO2 O SO2 Carbyl sulfate

Complex Formation. Ethanol behaves like water of crystallization and forms

complexes with various inorganic compounds. The alcohol combines with calcium,

magnesium, and platinum chlorides to form crystalline products.

Dehydration. This reaction may proceed in two directions with ethanol. The

intramolecular route to ethylene is favored when high temperatures and large

catalyst ratios are employee :

CH3 CH2OH CH2 = CH2 + H2O

Intermolecular dehydration forms diethyl ether :

2CH3 CH2OH CH3 CH2 O CH2 CH3 + H2O

Ester Formation. Both inorganic and organic acids will form esters with

ethanol :

CH3 CH2OH + HNO3 CH3 CH2 NO3 + H2O ethyl nitrate

Organic acids form organic esters and water :

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CH3 CH2OH + RCOOH RCOOCH2 CH3 + H2O ester

Since esterification is an equilibrium reaction, completion is favored by alarge excess of alcohol, by a large excess of acid, or by removal of water, Ethanol is

quite low in cost; therefore, it is ordinarily the compound used in excess. Acid

derivatives (such as acid chlorides, acid anhydrides, and simple alkyl esters of

acids) will also form esters.

Ethanol condenses with the carbonyl group of aldehydes and ketones at

about 1000C to form acetals :

2CH3 CH2OH + RCHO RCH(OCH2 CH3)2+ H2O

The process of ester interchange is used to prepare ethyl esters from natural

fats and oils :

CH2OOCR CH2OH| |

2CH3 CH2OH + CH2OOCR CH2OH + 3 RCOOCH2CH3| | ethyl esters

CH2OOCR CH2OHglycerol

Ethers. Ethyl ether formation is the result of an intermolecular catalytic

dehydration of ethanol at low temperature :

2CH3 CH2OH CH3 CH2OCH2 CH3 + H2O

Vinyl ethyl ether or diethyl acetal are formed by the addition of ethanol to

acetylene, the final product being dependent on whether an alkaline or acid catalyst

is used :

NaOC2H5

CH3 CH2OH + CH CH CH3 CH2OCH2= CH2 Vinyl ethyl ether

acid

CH3 CH2OH + CH3 CH2OCH CH2 CH3 CH(OCH2CH3)2 catalyst diethyl acetal

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Haloform Reaction, Ethanol may be oxidized by hypohalites to acetaldehyde

and will then undergo the "haloform" reaction :

CH3 CH2OH + NaOCl CH3 CHO + NaCl + H2O

CH3 CHO + 3NaOCl CCl3 CHO + 3NaOH

CCl3 CHO + NaOH CHCl3 + HCOONa

Halogenation. Ethanol may undergo a typical reaction in which hydrogen

halides, phosphorous halides, and similar reagents replace the hydroxyl group by

halogen :

3CH3 CH2OH + PCl 3CH3 CH2Cl + P(OH)3

Oxidation. Conversion of ethanol to acetaldehyde can be accomplished byoxidation or dehydration. Air oxidation is carried out in the presence of copper and

silver wire catalysts:

Cu, Ag

CH3 CH2OH + (O) CH3 CHO + H2O

Dehydrogenation of ethanol is catalyzed by chromium-activated copper :

Cu

CH3 CH2OH CH3 CHO + H2

Further oxidation of acetaldehyde leads to acetic acid :

Cu

CH3 CHO+ (O) CH3 COOH

Direct chemical oxidation of ethanol to acetic acid in one step is not carried

out commercially because of appreciable decomposition to carbon dioxide, carbon

monoxide, methane and other low molecular weight compounds. However, one

step oxidation to acetic acid is achieved industrially by fermentation.

Reaction with Metals. Sodium, potassium and calcium may replace the

hydroxyl hydrogen in ethanol to form a metal alkoxide (alcoholate):

Cu, Ag

2CH3 CH2OH + 2N 2CH3 CH2 ONa + H2 Sodium ethoxide

Sodium and aluminum ethoxide are valuable agents in the field of organic

synthesis.

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Thermal Decomposition. Hearing ethanol above 800 C forms ethylene,

acetaldehyde, water, and hydrogen. The use of a finely divided catalyst (such as

alumina or metals) will cause decomposition at lower temperatures.

FERMENTATION

Fermenting fruits and vegetables can bring many benefits to people in

developing countries. Fermented foods play an important role in providing food

security, enhancing livelihoods and improving the nutrition and social well being of

millions of people around the world, particularly the marginalised and vulnerable.

Improving food security

Eight hundred million people do not have enough food to eat. If we include those

not free from hunger the figure rises to 1.2 billion people. This is one fifth of the

World's population. A further two billion people are deficient in one or more micro-

nutrients (Anon, 1996). In the seventies, food security was viewed mainly in terms

of food supply at the global and national levels. Since then there has been a major

shift in understanding of food security with more emphasis on access to food rather

than purely on production. The Food and Agriculture Organisation of the United

Nations (FAO), amongst other influential organisations, has recognised that the

problem of food security cannot be tackled in isolation. Moreover that it is an

integral component of other development issues. FAO highlights the fact that the

world food insecurity problem is a result of undemocratic and inequitable distribution

of and access to resources rather than a problem of global food production (Anon,

1995), (Anon, 1996).

Fermentation technologies play an important role in ensuring the food security of

millions of people around the world, particularly marginalised and vulnerable

groups. This is achieved through improved food preservation, increasing the range

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of raw materials that can be used to produce edible food products and removing

anti- nutritional factors to make food safe to eat.

Food preservation

Fermentation is a cheap and energy efficient means of preserving perishable raw

materials. When harvested, fruit and vegetables, undergo rapid deterioration,

especially in the humid tropics where the prevailing environmental conditions

accelerate the process of decomposition. There are several options for preserving

fresh fruit and vegetables including drying, freezing, canning and pickling. However

many of these are inappropriate for use on the small-scale in developing countries.

For instance the canning of vegetables at the small-scale has serious food safetyimplications and contamination with botulism is a possibility. Freezing of fruits and

vegetables is not economically viable at the small-scale. Fermentation requires very

little sophisticated equipment, either to carry out the fermentation or for subsequent

storage of the fermented product. It is a technique that has been employed for

generations to preserve food for consumption at a later date and to improve food

security. There are examples from around the world of the role fermented foods

have played in preserving food to enhance food security.

Increasing income and employment

The production of fermented fruit and vegetable products provides income and

employment to millions of people around the world.

Food processing is probably the most important source of income and employment

in Africa, Asia and Latin America. The Food and Agriculture Organisation of the

United Nations has stated that value added through marketing and processing rawproducts can be much greater than the value of primary production (Anon, 1995).

For instance in sub-Saharan Africa more than 60% of the workforce is employed in

the small scale food processing sector, and between one third and two thirds of

value added manufacturing is based on agricultural raw materials (World Bank,

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1989), (Conroy et al, 1995). This is particularly important as agriculture and the

formal sector are unable to absorb the growing labour force in many countries.

Fermented foods are popular throughout the world and the production of fermentedfood products is important in many countries in providing income and employment.

In Africa, fermented cassava products (like Gariand Fufu) are a major component

of the diet of more than 800 million people and in some parts of Africa it constitutes

over 50% of the diet (Oyewole, 1992). In Asia the preparation of fermented foods is

a widespread tradition. Kimchi (a fermented cabbage product) is the major food

product of Korea. Soy sauce (a fermented legume product) is economically

important from Indonesia to Japan. Over a billion litres are produced each year in

Japan alone. Over 2000 million litres are produced each year in Korea and over 150

million litres in Taiwan. Miso (a fermented legume product) is also very important in

Asia with over 560,000 tons produced a year in Japan alone (Anon, 1982). In Latin

America, fermented cereal products, alcoholic drinks and fermented milk products

are three of the most important sectors of the economy.

Improving nutrition

The optimum health and nutrition of individuals is dependent upon a regular supply

of food and a balanced diet. When diets are sub-optimal, the individual's capacity

for work and achievements are greatly reduced. The most vulnerable groups are

women, children and weaning infants. Availability of food, dietary restrictions and

taboos, misconceptions, limited time available for feeding or eating compound to

create a group of individuals who are nutritionally disadvantaged. Approximately

30% of women consume less than their daily requirements of energy and at least

40% of women world-wide suffer from iron-deficiency anaemia. Fermentation can

enhance the nutritional value of a food product though increased vitamin levels and

improved digestibility.

Vitamins

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Fermentation processes can result in increased levels of vitamins in the final

product. Saccharomyces cerevisiae is able to concentrate large quantities of

thiamin, nicotinic acid and biotin and thus form enriched products.

Sorghum beer in Southern Africa contains relatively high levels of riboflavin

and nicotinic acid, which are important for people consuming a high maize

diet. Pellagra (a vitamin deficiency disease associated with high maize diets)

is unusual in communities in which sorghum beer is consumed. Even

children benefit from consuming the dregs which contain relatively little

alcohol but are rich in vitamins.

Palm wine in West Africa is high in vitamin B12, which is very important for

people with low meat intake, and who subsist primarily on a vegetarian diet.

Pulque (a fermented plant sap) is an important source of vitamins for the

economically deprived in Mexico. The fermentation process involved in

Pulque production increases its vitamin content. For instance the vitamin

content (milligrams of vitamins per 100g of product) of pulque increases from

5 to 29 for thiamine, 54 to 515 for niacin and 18 to 33 for riboflavin

(Steinkraus, 1992) during fermentation.

Idli (a lactic acid bacteria fermented product consumed in India) is high in

thiamine and riboflavin.

Digestibility

Micro-organisms contain certain enzymes, such as cellulases, which are incapable

of being synthesised by humans. Microbial cellulases hydrolyse cellulose into

sugars which are then readily digestible by humans. Similarly pectinases soften the

texture of foods and liberates sugars for digestion. Fermented foods are often moreeasily digestible than unfermented foods (Kovac, 1997), (Parades-Lopez, 1992).

Lactic acid fermented weaning foods are traditionally produced in developing

countries, both to improve the safety of the food and to improve its digestibility.

Starchy porridges are commonly fed to weaning infants in developing countries. The

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consistency of these gruels, combined with the small capacity of the infants

stomach, means that it is physically impossible for the child to consume adequate

energy to meet its high demands. By acidifying the porridge through lactic acid

fermentation, starch is hydrolysed into shorter chains of glucose and dextrose,

which reduce the viscosity of the porridge and increase its energy density. Thus the

child is more able to meet its energy requirements.

Medicinal benefits

There are many traditional beliefs about the medicinal properties of fermented food

products. The Fur ethnic group in Sudan strongly believe that the consumption of

fermented foods protects them from disease (Dirar, 1992). Koumiss (a fermented

milk product in Russia) has been used to treat tuberculosis. Pulque (a fermented

fruit sap) is felt to have medicinal properties in Mexico.

There is a sound scientific basis to these assertions:

The lowering of the pH inhibits the growth of food spoiling or poisoning

bacteria and destroys certain pathogens (Hammes, and Tichaczek, 1994).

Certain lactic acid bacteria (e.g. Lactobacillusacidophilus) and moulds have

been found to produce antibiotics and bacteriocins (Wood and Hodge, 1985)

(Matususaki et al, 1997) (Adams and Nicolaides, 1997), (Gourama and

Bullerman, 1995), (Nout, 1995)..

The beneficial health effects of lactic acid bacteria on the intestinal flora are

well documented (Ottogalli and Galli, 1997), (Motarjemi et al, 1996).

Substances in fermented foods have been found to have a protective effect

against the development of cancer (Frohlich et al, 1997).

Fermentation is a traditional method of reducing the microbial contamination of

porridges in Kenya (Watson, Ngesa, Onyang, Alnwick and Tomkins, 1996) A study

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in Tanzania has shown that children fed with fermented gruels had a 33% lower

incidence of diarrhoea than those fed unfermented gruels, owing to the inhibition of

pathogenic bacteria by lactic acid forming bacteria (Svanberg, 1992).

Improving cultural and social well being

Fermentation can improve the flavour and appearance of food. One important area

is the creation of meat-like flavour. Over the years, Sudanese women have

developed products to replace meat in their diets. These include "kawal", fermented

wild legume leaves, "sigda" (fermented sesame press-cake) and "furundu"

(fermented red sorrel seeds). The strong flavours of fermented food products can

enhance a dull diet. Fermented vegetables such as pickles, gundrukand sauerkraut

are used as condiments to enhance the overall flavour of the meal. A small amount

of pickle can make a bland starchy diet (like dahl and rice in Asia) much more

appealing (Battcock, 1992).

The diversity of fermented foods

Numerous fermented foods are consumed around the world. Each nation has its

own types of fermented food, representing the staple diet and the raw ingredientsavailable in that particular place. Although the products are well know to the

individual, they may not be associated with fermentation. Indeed, it is likely that the

methods of producing many of the worlds fermented foods are unknown and came

about by chance. Some of the more obvious fermented fruit and vegetable products

are the alcoholic beverages - beers and wines. However, several more fermented

fruit and vegetable products arise from lactic acid fermentation and are extremely

important in meeting the nutritional requirements of a large proportion of the worlds

population. Table 2.1 contains examples of fermented fruit and vegetable products

from around the world.

Organisms responsible for food fermentations

The most common groups of micro-organisms involved in food fermentations are:

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Bacteria

Yeasts

Moulds

1 Bacteria

Several bacterial families are present in foods, the majority of which are concerned

with food spoilage. As a result, the important role of bacteria in the fermentation of

foods is often overlooked. The most important bacteria in desirable food

fermentations are the lactobacillaceae which have the ability to produce lactic acid

from carbohydrates. Other important bacteria, especially in the fermentation of fruits

and vegetables, are the acetic acid producing acetobacterspecies.

2 Yeasts

Yeasts and yeast-like fungi are widely distributed in nature. They are present in

orchards and vineyards, in the air, the soil and in the intestinal tract of animals. Like

bacteria and moulds, yeasts can have beneficial and non-beneficial effects in foods.

The most beneficial yeasts in terms of desirable food fermentation are from the

Saccharomyces family, especially S. cerevisiae. Yeasts are unicellular organisms

that reproduce asexually by budding. In general, yeasts are larger than most

bacteria. Yeasts play an important role in the food industry as they produce

enzymes that favour desirable chemical reactions such as the leavening of bread

and the production of alcohol and invert sugar.

Table 1 : Fermented foods from around the world.

Name and region Type of product

Indian sub-continent

Acar, Achar, Tandal achar, Garam nimboo achar Pickled fruit and vegetables

Gundruk Fermented dried vegetable

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Lemon pickle, Lime pickle, Mango pickle

South East Asia

Asinan, Burong mangga, Dalok, Jeruk, Kiam-chai,

Kiam-cheyi, Kong-chai, Naw-mai-dong, Pak-siam-

dong, Paw-tsay, Phak-dong, Phonlami-dong,

Sajur asin, Sambal tempo-jak, Santol, Si-sek-chai,

Sunki, Tang-chai, Tempoyak, Vanilla,

Pickled fruit and vegetables

Bai-ming, Leppet-so, Miang Fermented tea leaves

Nata de coco, Nata de pina Fermented fruit juice

East Asia

Bossam-kimchi, Chonggak-kimchi, Dan moogi,

Dongchimi, Kachdoo kigactuki, Kakduggi, Kimchi,

Mootsanji, Muchung-kimchi, Oigee, Oiji, Oiso

baegi, Tongbaechu-kimchi, Tongkimchi, Totkal

kimchi,

Fermented in brine

Cha-tsai, Hiroshimana, Jangagee, Nara senkei,Narazuke, Nozawana, Nukamiso-zuke, Omizuke,

Pow tsai, Red in snow, Seokbakji, Shiozuke,

Szechwan cabbage, Tai-tan tsoi, Takana, Takuan,

Tsa Tzai, Tsu, Umeboshi, Wasabi-zuke, Yen tsai


Hot pepper sauce

Africa

Fruit vinegar Vinegar

Hot pepper sauce

Lamoun makbouss, Mauoloh, Msir, Mslalla, Olive Pickled fruit and vegetables

Oilseeds, Ogili, Ogiri, Hibiscus seed Fermented fruit and vegetable

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seeds

Wines Fermented fruits

Americas

Cucumber pickles, Dill pickles, Olives, Sauerkraut, Pickled fruit and vegetables

Lupin seed, Oilseeds, Pickled oilseed

Vanilla, Wines Fermented fruit and vegetable

Middle East

Kushuk Fermented fruit and

vegetables

Lamoun makbouss, Mekhalel, Olives, Torshi,

Tursu


Wines Fermented fruits

Europe and World

Mushrooms, Yeast Moulds

Olives, Sauerkohl, Sauerruben Pickled fruit and vegetables

Grape vinegar, Wine vinegar Vinegar

Wines, Citron Fermented fruits

(Taken from G Campbell-Platt (1987))

Moulds

Moulds are also important organisms in the food industry, both as spoilers and

preservers of foods. Certain moulds produce undesirable toxins and contribute to

the spoilage of foods. TheAspergillus species are often responsible for undesirable

changes in foods. These moulds are frequently found in foods and can tolerate high

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concentrations of salt and sugar. However, others impart characteristic flavours to

foods and others produce enzymes, such as amylase for bread making. Moulds

from the genus Penicillium are associated with the ripening and flavour of cheeses.

Moulds are aerobic and therefore require oxygen for growth. They also have the

greatest array of enzymes, and can colonise and grow on most types of food.

Moulds do not play a significant role in the desirable fermentation of fruit and

vegetable products.

When micro-organisms metabolise and grow they release by-products. In food

fermentations the by-products play a beneficial role in preserving and changing the

texture and flavour of the food substrate. For example, acetic acid is the by-product

of the fermentations of some fruits. This acid not only affects the flavour of the final

product, but more importantly has a preservative effect on the food. For food

fermentations, micro-organisms are often classified according to these by-products.

The fermentation of milk to yoghurt involves a specific group of bacteria called the

lactic acid bacteria (Lactobacillus species). This is a general name attributed to

those bacteria which produce lactic acid as they grow. Acidic foods are less

susceptible to spoilage than neutral or alkaline foods and hence the acid helps to

preserve the product. Fermentations also result in a change in texture. In the case

of milk, the acid causes the precipitation of milk protein to a solid curd.

Enzymes

The changes that occur during fermentation of foods are the result of enzymic

activity. Enzymes are complex proteins produced by living cells to carry out specific

biochemical reactions. They are known as catalysts since their role is to initiate and

control reactions, rather than being used in a reaction. Because they areproteinaceous in nature, they are sensitive to fluctuations in temperature, pH,

moisture content, ionic strength and concentrations of substrate and inhibitors. Each

enzyme has requirements at which it will operate most efficiently. Extremes of

temperature and pH will denature the protein and destroy enzyme activity. Because

they are so sensitive, enzymic reactions can easily be controlled by slight

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adjustments to temperature, pH or other reaction conditions. In the food industry,

enzymes have several roles - the liquefaction and saccharification of starch, the

conversion of sugars and the modification of proteins. Microbial enzymes play a role

in the fermentation of fruits and vegetables.

Nearly all food fermentations are the result of more than one micro-organism, either

working together or in a sequence. For example, vinegar production is a joint effort

between yeast and acetic acid forming bacteria. The yeast convert sugars to

alcohol, which is the substrate required by the acetobacter to produce acetic acid.

Bacteria from different species and the various micro-organisms - yeast and moulds

-all have their own preferences for growing conditions, which are set within narrow

limits. There are very few pure culture fermentations. An organism that initiates

fermentation will grow there until its by-products inhibit further growth and activity.

During this initial growth period, other organisms develop which are ready to take

over when the conditions become intolerable for the former ones.

In general, growth will be initiated by bacteria, followed by yeasts and then moulds.

There are definite reasons for this type of sequence. The smaller micro-organisms

are the ones that multiply and take up nutrients from the surrounding area mostrapidly. Bacteria are the smallest of micro-organisms, followed by yeasts and

moulds. The smaller bacteria, such as Leuconostocand Streptococcusgrow and

ferment more rapidly than their close relations and are therefore often the first

species to colonise a substrate (Mountney and Gould, 1988).

Table 2 : Micro-organisms commonly found in fermenting fruit and vegetables

Organism Type Optimum

conditions

Reactions

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Acetobacter genus

A. aceti

A. pasteurianus

A. peroxydans

Aerobic

rods

aw > =0.9 Oxidise organic compounds

(alcohol) to organic acids

(acetic acid). Important in

vinegar production.

Streptococcaceae

Family

Gram

positive

cocci

Acid

tolerant

aw > =0.9

Streptococcus genus

S. faecalis

S. bovis

S. thermophilus

Homofermentative. Most

common in dairy

fermentations, but S. Faecalis

is common in vegetable

products. Tolerate salt and

can grow in high pH media.

Leuconostoc genus

L. mesenteroides

L. dextranicum

L.

paramesenteroides

L. oenos

Gram

positive

cocci

Heterofermentative. Produce

lactic acid, plus acetic acid,

ethanol and carbon dioxide

from glucose. Small bacteria,

therefore have an important

role in initiating fermentations.

L. oenos is often present in

wine. It can utilise malic acid

and other organic acids.

Pediococcus genus

P. cerevisiae

P. acidilactici

P. pentosaceus

Saprophytic organisms found

in fermenting vegetables,

mashes, beer and wort.

Produce inactive lactic acid.

Lactobacillaceae Gram Acid Metabolise sugars to lactic

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Family positive

rods. Non-

motile

tolerant

aw > =0.9

acid, acetic acid, ethyl alcohol

and carbon dioxide.

Lactobacillus genus The genus is split into two

types homo- and hetero-

fermenters. Saprophytic

organisms. Produce greater

amounts of acid than the cocci

Homofermentative

Lactobacillus spp.L. delbrueckii

L. leichmannii

L. plantarum

L. lactis

L. acidophilus

Produce only lactic acid. L.

plantarum important in fruitand vegetable fermentation.

Tolerates high salt

concentration.

Heterofermentative

Spp.L. brevis

L. fermentum

L. buchneri

Produce lactic acid (50%) plus

acetic acid (25%), ethylalcohol and carbon dioxide

(25%). L. brevis is the most

common. Widely distributed in

plants and animals. Partially

reduces fructose to mannitol.

Yeasts Tolerate

acid, 40%sugar

aw > =0.85

Saccharomyces

Cerevisiae

Many

aerobic,

pH 4-4.5

20-30 C

S. cerevisiae can shift its

metabolism from a

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S. pombe some

anaerobes

fermentative to an oxidative

pathway, depending on

oxygen availability. Most

yeasts produce alcohol and

carbon dioxide from sugars.

Debaromyces

Zygosaccharomyces

rouxii

Candida species

Geotrichum

candidum

Tolerant of high salt

concentrations

Tolerates high salt

concentration and low aw

Desirable fermentation

It is essential with any fermentation to ensure that only the desired bacteria, yeasts

or moulds start to multiply and grow on the substrate. This has the effect of

suppressing other micro-organisms which may be either pathogenic and cause food

poisoning or will generally spoil the fermentation process, resulting in an end-

product which is neither expected or desired. An everyday example used to

illustrate this point is the differences in spoilage between pasteurised and

unpasteurised milk. Unpasteurised milk will spoil naturally to produce a sour tasting

product which can be used in baking to improve the texture of certain breads.

Pasteurised milk, however, spoils (non-desirable fermentation) to produce an

unpleasant product which has to be disposed of. The reason for this difference is

that pasteurisation (despite being a very important process to destroy pathogenic

micro-organisms) changes the micro-organism environment and if pasteurised milk

is kept unrefrigerated for some time, undesirable micro-organisms start to grow and

multiply before the desirable ones. In the case of unpasteurised milk, the non-

pathogenic lactic acid bacteria start to grow and multiply at a greater rate that any

pathogenic bacteria. Not only do the larger numbers of lactic acid bacteria compete

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more successfully for the available nutrients, but as they grow they produce lactic

acid which increases the acidity of the substrate and further suppresses the bacteria

which cannot tolerate an acid environment.

Most food spoilage organisms cannot survive in either alcoholic or acidic

environments. Therefore, the production of both these end products can prevent a

food from spoilage and extend the shelf life. Alcoholic and acidic fermentations

generally offer cost effective methods of preserving food for people in developing

countries, where more sophisticated means of preservation are unaffordable and

therefore not an option.

The principles of microbial action are identical both in the use of micro-organisms in

food preservation, such as through desirable fermentations, and also as agents of

destruction via food spoilage. The type of organisms present and the environmental

conditions will determine the nature of the reaction and the ultimate products. By

manipulating the external reaction conditions, microbial reactions can be controlled

to produce desirable results. There are several means of altering the reaction

environment to encourage the growth of desirable organisms. These are discussed

below.

Manipulation of microbial growth and activity

There are six major factors that influence the growth and activity of micro-organisms

in foods. These are moisture, oxygen concentration, temperature, nutrients, pH and

inhibitors (Mountney and Gould, 1988). The food supply available to the micro-

organisms depends on the composition of the food on which they grow. All micro-

organisms differ in their ability to metabolise proteins, carbohydrates and fats.

Obviously, by manipulating any of these six factors, the activity of micro-organisms

within foods can be controlled.

Moisture

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Water is essential for the growth and metabolism of all cells. If it is reduced or

removed, cellular activity is decreased. For example, the removal of water from cells

by drying or the change in state of water (from liquid to solid) affected by freezing,

reduces the availability of water to cells (including microbial cells) for metabolic

activity. The form in which water exists within the food is important as far as

microbial activity is concerned. There are two types of water - free and bound.

Bound water is present within the tissue and is vital to all the physiological

processes within the cell. Free water exists in and around the tissues and can be

removed from cells without seriously interfering with the vital processes. Free water

is essential for the survival and activity of micro-organisms. Therefore, by removing

free water, the level of microbial activity can be controlled. The amount of wateravailable for micro-organisms is referred to as the water activity (aw). Pure water

has a water activity of 1.0. Bacteria require more water than yeasts, which require

more water than moulds to carry out their metabolic activities. Almost all microbial

activity is inhibited below aw of 0.6. Most fungi are inhibited below aw of 0.7, most

yeasts are inhibited below aw of 0.8 and most bacteria below aw 0.9. Naturally, there

are exceptions to these guidelines and several species of micro-organism can exist

outside the stated range. See table for further information on water activity and

microbial action. The water activity of foods can be changed by altering the amount

of free water available. There are several ways to achieve this drying to remove

water; freezing to change the state of water from liquid to solid; increasing or

decreasing the concentration of solutes by adding salt or sugar or other hydrophylic

compounds (salt and sugar are the two common additives used for food

preservation). Addition of salt or sugar to a food will bind free water and so

decrease the aw. Alternatively, decreasing the concentration will increase the

amount of free water and in turn the aw. Manipulation of the aw in this manner can be

used to encourage the growth of favourable micro-organisms and discourage the

growth of spoilage ones.

Table 3: Water activity for microbial reactions

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Aw Phenomenon Examples

1.00 Highly perishable foods

0.95 Pseudomonas, Bacillus,

Clostridium perfringens and some

yeasts inhibited

Foods with 40% sucrose or 7%

salt

0.90 Lower limit for bacterial growth.

Salmonella, Vibrio

parahaemolyticus, Clostridium

botulinum, Lactobacillus and some

yeasts and fungi inhibited

Foods with 55% sucrose, 12% salt.

Intermediate-moisture foods (aw =

0.90-0.55)

0.85 Many yeasts inhibited Foods with 65% sucrose, 15% salt

0.80 Lower limit for most enzyme

activity and growth of most fungi.

Staphylococcus aureus inhibited

Fruit syrups

0.75 Lower limit for halophilic bacteria Fruit jams

0.70 Lower limit for growth of mostxerophilic fungi

0.65 Maximum velocity of Maillard

reactions

0.60 Lower limt for growth of osmophilic

or xerophilic yeasts and fungi

Dried fruits (15-20% water)

0.55 Deoxyribose nucleic acid (DNA)

becomes disordered (lower limit

for life to continue)

0.50 Dried foods (aw=0-0.55)

0.40 Maximum oxidation velocity

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0.25 Maximum heat resistance of

bacterial spores

Taken from Fellows (1988).

Oxidation-Reduction potential

Oxygen is essential to carry out metabolic activities that support all forms of life.

Free atmospheric oxygen is utilised by some groups of micro-organisms, while

others are able to metabolise the oxygen which is bound to other compounds such

as carbohydrates. This bound oxygen is in a reduced form.

Micro-organisms can be broadly classified into two groups - aerobic and anaerobic.

Aerobes grow in the presence of atmospheric oxygen while anaerobes grow in the

absence of atmospheric oxygen. In the middle of these two extremes are the

facultative anaerobes which can adapt to the prevailing conditions and grow in

either the absence or presence of atmospheric oxygen. Microaerophilic organisms

grow in the presence of reduced amounts of atmospheric oxygen. Thus, controlling

the availability of free oxygen is one means of controlling microbial activity within a

food. In aerobic fermentations, the amount of oxygen present is one of the limiting

factors. It determines the type and amount of biological product obtained, the

amount of substrate consumed and the energy released from the reaction.

Moulds do not grow well in anaerobic conditions, therefore they are not important in

terms of food spoilage or beneficial fermentation, in conditions of low oxygen

availability.

Temperature

Temperature affects the growth and activity of all living cells. At high temperatures,

organisms are destroyed, while at low temperatures, their rate of activity is

decreased or suspended. Micro-organisms can be classified into three distinct

categories according to their temperature preference (see table2.4).

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Table 4 Classification of bacteria according to temperature requirements.

Temperature required for growth 0C

Type of

bacteria

Minimum optimum maximum General sources of

bacteria

Psychrophilic 0 to 5 15 to 20 30 Water and frozen foods

Mesophilic 10 to 25 30 to 40 35 to 50 Pathogenic and non-

pathogenic bacteria

Thermophilic 25 to 45 50 to 55 70 to 90 Spore forming bacteria

from soil and water

(Taken from Mountney and Gould, (1988).

Nutritional requirements

The majority of organisms are dependent on nutrients for both energy and growth.

Organisms vary in their specificity towards different substrates and usually only

colonise foods which contain the substrates they require. Sources of energy varyfrom simple sugars to complex carbohydrates and proteins. The energy

requirements of micro-organisms are very high. Limiting the amount of substrate

available can check their growth.

Hydrogen ion concentration (pH)

The pH of a substrate is a measure of the hydrogen ion concentration. A food with a

pH of 4.6 or less is termed a high acid or acid food and will not permit the growth of

bacterial spores. Foods with a pH above 4.6. are termed low acid and will not inhibit

the growth of bacterial spores. By acidifying foods and achieving a final pH of less

than 4.6, most foods are resistant to bacterial spoilage.

The optimum pH for most micro-organisms is near the neutral point (pH 7.0).

Certain bacteria are acid tolerant and will survive at reduced pH levels. Notable

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acid-tolerant bacteria include the Lactobacillus and Streptococcus species, which

play a role in the fermentation of dairy and vegetable products. Moulds and yeasts

are usually acid tolerant and are therefore associated with spoilage of acidic foods.

Micro-organisms vary in their optimal pH requirements for growth. Most bacteria

favour conditions with a near neutral pH (7). Yeasts can grow in a pH range of 4 to

4.5 and moulds can grow from pH 2 to 8.5, but favour an acid pH. The varied pH

requirements of different groups of micro-organisms is used to good effect in

fermented foods where successions of micro-organisms take over from each other

as the pH of the environment changes. For instance, some groups of micro-

organisms ferment sugars so that the pH becomes too low for the survival of those

microbes. The acidophilic micro-organisms then take over and continue the

reaction. The affinity for different pH can also be used to good effect to occlude

spoilage organisms.

Inhibitors

Many chemical compounds can inhibit the growth and activity of micro-organisms.

They do so by preventing metabolism, denaturation of the protein or by causing

physical damage to the cell. The production of substrates as part of the metabolic

reaction also acts to inhibit microbial action.

Controlled fermentation

Controlled fermentations are used to produce a range of fermented foods, including

sauerkraut, pickles, olives, vinegar, dairy and other products. Controlled

fermentation is a form of food preservation since it generally results in a reduction of

acidity of the food, thus preventing the growth of spoilage micro-organisms. The twomost common acids produced are lactic acid and acetic acid, although small

amounts of other acids such as propionic, fumaric and malic acid are also formed

during fermentation.

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It is highly probable that the first controlled food fermentations came into existence

through trial and error and a need to preserve foods for consumption later in the

season. It is possible that the initial attempts at preservation involved the addition of

salt or seawater. During the removal of the salt prior to consumption, the foods

would pass through stages favourable to acid fermentation. Although the process

worked, it is likely that the causative agents were unknown. Today, there are

numerous examples of controlled fermentation for the preservation and processing

of foods. However, only a few of these have been studied in any detail - these

include sauerkraut, pickles, kimchi, beer, wine and vinegar production. Although the

general principles and processes for many of the fermented fruit and vegetable

products are the same -relying mainly on lactic acid and acetic acid formingbacteria, yeasts and moulds, the reactions have not been quantified for each

product. The reactions are usually very complex and involve a series of micro-

organisms, either working together or in succession to achieve the final product.

What are yeasts?

A yeast is a unicellular fungus which reproduces asexually by budding or division,

especially the genus Saccharomyces which is important in food fermentations

(Walker, 1988). Yeasts and yeast-like fungi are widely distributed in nature. They

are present in orchards and vineyards, in the air, the soil and the intestinal tract of

animals. Like bacteria and moulds, they can have beneficial and non-beneficial

effects in foods. Most yeasts are larger than most bacteria. The most well known

examples of yeast fermentation are in the production of alcoholic drinks and the

leavening of bread. For their participation in these two processes, yeasts are ofmajor importance in the food industry.

Some yeasts are chromogenic and produce a variety of pigments, including green,

yellow and black. Others are capable of synthesising essential B group vitamins.

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Although there is a large diversity of yeasts and yeast-like fungi, (about 500

species), only a few are commonly associated with the production of fermented

foods. They are all either ascomycetous yeasts or members of the genus Candida.

Varieties of the Saccharomyces cervisiae genus are the most common yeasts in

fermented foods and beverages based on fruit and vegetables. All strains of this

genus ferment glucose and many ferment other plant derived carbohydrates such

as sucrose, maltose and raffinose. In the tropics, Saccharomycespombe is the

dominant yeast in the production of traditional fermented beverages, especially

those derived from maize and millet (Adams and Moss, 1995).

Conditions necessary for fermentation

Most yeasts require an abundance of oxygen for growth, therefore by controlling the

supply of oxygen, their growth can be checked. In addition to oxygen, they require a

basic substrate such as sugar. Some yeasts can ferment sugars to alcohol and

carbon dioxide in the absence of air but require oxygen for growth. They produce

ethyl alcohol and carbon dioxide from simple sugars such as glucose and fructose.

C6H12O6 2C2H5OH + 2CO2

Glucose yeast ethyl alcohol carbon dioxide

In conditions of excess oxygen (and in the presence of acetobacter) the alcohol can

be oxidised to form acetic acid. This is undesirable if the end product is a fruit

alcohol, but is a technique employed for the production of fruit vinegars (see later

section on mixed fermentations).

Yeasts are active in a very broad temperature range - from 0 to 50 0 C, with an

optimum temperature range of 200 to 300 C.

The optimum pH for most micro-organisms is near the neutral point (pH 7.0).

Moulds and yeasts are usually acid tolerant and are therefore associated with the

spoilage of acidic foods. Yeasts can grow in a pH range of 4 to 4.5 and moulds can

grow from pH 2 to 8.5, but favour an acid pH (Mountney and Gould, 1988).

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In terms of water requirements, yeasts are intermediate between bacteria and

moulds. Bacteria have the highest demands for water, while moulds have the least

need. Normal yeasts require a minimum water activity of 0.85 or a relative humidity

of 88%.

Yeasts are fairly tolerant of high concentrations of sugar and grow well in solutions

containing 40% sugar. At concentrations higher than this, only a certain group of

yeasts the osmophilic type can survive. There are only a few yeasts that can

tolerate sugar concentrations of 65-70% and these grow very slowly in these

conditions (Board, 1983). Some yeasts for example the Debaromyces - can

tolerate high salt concentrations. Another group which can tolerate high salt

concentrations and low water activity is Zygosaccharomyces rouxii, which is

associated with fermentations in which salting is an integral part of the process.

Production of fruit alcohol

Alcohol and acids are two primary products of fermentation, both used to good

effect in the preservation of foods. Several alcohol-fermented foods are preceded

by an acid fermentation and in the presence of oxygen and acetobacter, alcohol can

be fermented to produce acetic acid. Most food spoilage organisms cannot survive

in either alcoholic or acidic environments. Therefore, the production of both these

end products can prevent a food from undergoing spoilage and extend its shelf life.

Primitive wines and beers have been produced, with the aid of yeasts, for

thousands of years, although it was not until about four hundred years ago thatmicro-organisms associated with the fermentation were observed and identified. It

was not until the 1850s that Louis Pasteur demonstrated unequivocally the

involvement of yeasts in the production of wines and beers (Fleet, 1998). Since

then, the knowledge of yeasts and the conditions necessary for fermentation of wine

and beer has increased to the point where pure culture fermentations are now used

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to ensure consistent product quality. Originally, alcoholic fermentations would have

been spontaneous events that resulted from the activity of micro-organisms

naturally present. These non-scientific methods are still used today for the home

preparation of many of the worlds traditional beers and wines.

Alcoholic drinks fall into two broad categories: wines and beers. Wines are made

from the juice of fruits and beers from cereal grains. The principal carbohydrates in

fruit juices are soluble sugars; the principal carbohydrate in grains is starch, an

insoluble polysaccharide. The yeasts that bring about alcoholic fermentation can

attack soluble sugars but do not produce starch-splitting enzymes. Wines can

therefore be made by the direct fermentation of the raw material, while the

production of beer requires the hydrolysis of starch to yield sugars fermentable byyeast, as a preliminary step (Stanier, Dourdoff and Adelberg, 1972).

Raw fruit juice is usually a strongly acidic solution, containing from 10 to 25 percent

soluble sugars. Its acidity and high sugar concentration make it an unfavourable

medium for the growth of bacteria but highly suitable for yeasts and moulds. Raw

fruit juice naturally contains many yeasts, moulds, and bacteria, derived from the

surface of the fruit. Normally the yeast used in alcoholic fermentation is a strain of

the species Saccharomyces cerevisiae (Adams, 1985).

The fermentation may be allowed to proceed spontaneously, or can be "started" by

inoculation with a must that has been previously successfully fermented by S.

cerevisiae var. ellipsoideus. Many modern wineries eliminate the original microbial

population of the must by pasteurisation or by treatment with sulphur dioxide. The

must is then inoculated with a starter culture derived from a pure culture of a

suitable strain of wine yeast. This procedure eliminates many of the uncertainties

and difficulties of older methods. At the start of the fermentation, the must is aerated

slightly to build up a large and vigorous yeast population; once fermentation sets in,

the rapid production of carbon dioxide maintains anaerobic conditions, which

prevent the growth of undesirable aerobic organisms, such as bacteria and moulds.

The temperature of fermentation is usually from 25 to 30 oC, and the duration of the

fermentation process may extend from a few days to two weeks. As soon as the

desired degree of sugar disappearance and alcohol production has been attained,

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the microbiological phase of wine making is over. Thereafter, the quality and

stability of the wine depend very largely on preventing further microbial activity, both

during the "aging" in wooden casks and after bottling (Stanieret al, 1972).

At all stages during its manufacture, fruit juice alcohol is subject to spoilage by

undesirable microorganisms. Pasteur, whose descriptions of the organisms

responsible and recommendations for overcoming them are still valid today, first

scientifically explored the problem of the "diseases" of wines. The most serious

aerobic spoilage processes are brought about by film-forming yeasts and acetic

acid bacteria, both of which grow at the expense of the alcohol, converting it to

acetic acid or to carbon dioxide and water. The chief danger from these organisms

arises when access of air is not carefully regulated during aging. Much more seriousare the diseases caused by fermentative bacteria, particularly rod-shaped lactic acid

bacteria, which utilise any residual sugar and impart a mousy taste to the wine.

Such wines are known as turned wines. Since oxygen is unnecessary for the growth

of lactic acid bacteria, wine spoilage of this kind can occur even after bottling. These

risks of spoilage can be minimised by pasteurisation after bottling.

Fermentation pathways

The initial steps are identical to those of respiration. For example, forcarbohydrate fermentation, the pathway begins with glycolysis. In EM glycolytic

pathway, there are generated two pyruvate molecules, two reduced coenzyme

NADH molecules, and two ATP molecules for each molecule of glucose. The

remainder of the fermentation pathway is concerned with reoxidising the coenzyme.

In fermentation, reoxidation of NADH to NAD+ depends on the reduction of

pyruvate molecules formed during glycolysis. Different microorganisms have

developed different pathways for utilising the pyruvate for reoxidising the reduced

coenzyme with different terminal sequences of the various fermentation pathways

resulting in the formation of various end products (Fig). The different fermentation

pathways are named for the characteristic end products that are formed. The most

common ones are as follows :

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C.O.E.& T.,Akola 39


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Batch Growth of Micro-Organism

The batch growth of micro-organisms involves adding a small quantity of the

micro-organisms or their spores (the seed culture or inoculum) to a quantity of

nutrient material in a suitable vessel. In the case of an aerobic fermentation (i.e. a

growth process requiring the presence of molecular oxygen) the contents of the

vessel (or fermenter) are aerated and the growth of the micro-organism allowed to

proceed. For convenience, the case where the feed material is present in aqueous

solution is considered and, furthermore, it is assumed that in the feed there is

contained a carbon and energy source which is the limiting substrate for the growth

of the culture. Whilst for an aerobic culture aeration is of prime importance, the factthat air enters the vessel and leaves enriched in carbon dioxide will be ignored in

this discussion and the analysis focused on the changes occurring in the liquid

phase.

C.O.E.& T.,Akola 40

Fig. The ethanolicfermentation pathway

Fig. The homolactic acid fermentation

pathway End product is lactic acid (lactate)


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After inoculation, assuming no lag phase, the resultant growth can be

analysed by considering the unsteady-state material balances for the substrate and

biomass. The general form of this balance for a fermenter is :

+ = Accumulation

Since a batch process is being considered, the flow in and out of the fermenter are

both zero and the expression reduces to:

= Accumulation

So, for the case of the biomass:

VdT

dxVXVRx == (1)

where is the specific growth rate, V is the volume of the vessel and X is the

instantaneous concentration of the biomass. IfY is the overall yield coefficient for

the formation of biomass and the limiting substrate concentration is 5, then the

equivalent expression for substrate is:

VdTdSVRS = (2)

where Rs is the rate of conversion of substrate per unit volume of the reactor.

Equation 5.116 makes no assumptions regarding the uniformity of the yield

coefficient Yx/s, but if that can be taken to be constant then equation may be used to

relate equations (1) and (2). This condition is met when is large in comparison

with m so that, dispensing with the subscript, the differential form of equation can be

written:

dt

dX

dt

dSY =

(3)

which gives:

YRs = - X (4)

Equation 2 thus becomes:

C.O.E.& T.,Akola 41

Flow of

material inFormation by

biochemical

Flow of

material out

Formation by

biochemical


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dt

dSXY

= 1

(5)

The yield coefficient may also be expressed in its integral form as:

SS

XXY

=0

0

(6)

which can be re-arranged:

Y

XXSS 00

= (6)

If the growth follows the Monod kinetic model, then equation may be substituted into

equation 1 to give:

SK

SX

dt

dX

s

m

+=

(7)

The condition of the fermentation after any time / would then be given by:

=+ tX

X m

s dtX

dX

S

SK

00

(8)

However, S is a. function ofX and substitution using equation 6 must be made

before carrying out the integration. The result is:

++

+

+++

)XXYS(

YSln

)XYS(

YK

X

Xln

)XYS(

XYSYK

m

S

m

S

00

0

00000

00

(9)

A similar expression can be obtained for the substrate concentration:

tS

Sln

)YSX(

YK

X

)SS(Yln

)XYS(

XYSYK

m

S

m

S =

+

+

+++

0000

0

00

001

(10)

These rather unwieldy equations can be used to generate a graph showing the

changes in biomass and substrate concentrations during the course of a batch

fermentation (fig). Their main disadvantage is that they are not explicit in Xand Sso

that a trial and error technique has to be used to determine their values at a

particular value of t.

C.O.E.& T.,Akola 42


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Ethyl Alcohol by Fermentation

Chemical reactions

(a) Main reaction

InvertaseC12H22O11 + H2O 2C6H12O6

Zymase

C6H12O6 2C2H5OH + 2CO2

(b) Side reactions

2C6H12O6 + H2O ROH + R' CHOhigher mol. Wt. Alcohols

Quantitative requirements

(a) Basis : 1 ton of 100% alcohol (1.26 kiloliters) and 90%

yield from total sugar

Molasses (50-55% total sugar) 5.6 tons

Sulfuric acid (600 Be) 27 kg

Ammonium sulfate 2.5 kg

Coal 0.7-1.5 tons

Process water 12 tons

Cooling water 50 tons

Electricity 35 KWH

By-products : CO2 0.76 ton

Fusel oil (higher mol. wt. Alcohols )

Residual cattle feed or fertilizer 0.20 0.60 ton

(b) Plant capacities : 10-100 tons/day of ethyl alcohol

Process description

Molasses is diluted to a 10-15% sugar concentration and adjusted to a pH of

4-5 to support yeast growth which furnishes invertase and zymase catalyticenzymes. Nutrients such as ammonium and magnesium sulfate or phosphate is

added when lacking in the molasses. This diluted mixture called mash is run into

large wooden or steel fermentation tanks.

Yeast solution, grown by inoculating sterile mash, is added and fermentation

ensues with evolution of heat which is removed via cooling coils. The temperature

C.O.E.& T.,Akola 44


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is kept at 20-300C over a 30-70 hr period, rising near the end of 35 0C. Carbon

dioxide may be utilized as a by-product by water scrubbing and compressing;

otherwise it is vented after water scrubbing.

Separation of the 8-10% alcohol in the fermented liquor called beer is

accomplished by a series of distillations. In the beer still, alcohol (50-60% conc.)

and undesirable volatiles such as aldehydes are taken off the top and fed to the

aldehyde still. Alcohol is pulled off as a side-stream split to the rectifying column. In

this final column, the azeotropic alcohol-water mixture of 95% cannot is taken off as

a top side streams conde and run to storage where it is split into three parts :

(1) direct sale as potable, government controlled alcohol

(2) denatured by small additions of mildly toxic ingredients and sold for industrialuses

(3) made anhydrous by ternary azeotropic distillation using benzene or

extractive distillation using ethylene glycol

When fusel oil recovery is practiced, side-streams are drawn off near the bottom of

the aldehyde and rectifying columns and are separated by decantation. These

higher molecular weight alcohols are sold directly for solvents or are fractionated to

give predominantly amyl alcohol.

The bottoms from the beer still, known as slops, are either discharged as

waste or concentrated by evaporation to cattle feed depending on fuel and by-

product sales economics.

C.O.E.& T.,Akola 45


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Major engineering problems Collection and storage of molasses

Maintenance of sterile and specific yeast culture conditions.

Batch versus continuous operation; continuous molasses dilution in the head

end of the process and continuous distillation are incorporated to save

space, equipment and operating costs

Waste disposal problem : if uneconomic to concentrate for cattle feed, must

use trickling filters, activated sludge or anaerobic digestion to lower the

biological oxygen demand (BOD) before discharging to water run-off

Fuel economy in the series of distillations : use of preheat exchangers

Development of methods to produce anhydrous alcohol from the 95% alcohol

azeotrope.

C.O.E.& T.,Akola 46


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Alcohol from waste sulfite liquior.

Figure gives a flow sheet of the Bellingham, Washington, plant. Removal of

sulfur dioxide from the waste sulfite liquor is accomplished by stripping the liquor

with stream, by countercurrent flow in a column, approximately 8 ft. in diameter by

45 ft. tall, fabricated of stainless steel to the general pattern of a distillation column

having 20 plates. Liquor enters at the top and steam at the bottom. Sulfur dioxide

and steam are discharged out the top and are salvaged by injection into the digester

cooking acid. The sulfur recovery amounts to about 20 lb.of sulfur per ton of pulp

and offsets the cost of the steam required. The composition of the sulfite waste

liquor discharged at the bottom varies depending on the stream input. Stripping

results in complete removal of the free sulfur dioxide and part of the loosely

combined sulfur dioxide. The pH of the stripped liquor varies between 3.8 and 4.2

depending upon the grade of pulp being cooked. Although the column is equipped

with control instruments to deliver automatically a product of constant pH, it is

normally operated at a fixed steam liquor ratio of 1 lb of stream per 2 gal.of liquor

feed. Part of the heat input is recovered in the digester cooking acid. The sulfite

waste liquor from the base of the stripping column is pumped to the alcohol-plant

building for continuation of liquor preparation.

C.O.E.& T.,Akola 48


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The acidity of the sulfite waste liquor can be sufficiently reduced by stripping

alone to make further treatment unnecessary, but for best economy it usually

proves desirable to add lime. The lime is added as a 10% water slurry injected into

the sulfite waste liquor at a point just ahead of the coolers. The equipment consists

of an outside lime-storage bin, two agitated lime-slurry tanks, and the necessary

pump. A pH controller regulates the amount of lime addition. The sulfite waste

liquor is adjusted to a pH of 4.5 for fermentation. The quantity of lime average

about 3 lb.of lime per 1000 gal.of sulfite waste liquor treated.

After lime addition, the liquor is cooled to a temperature of 320C. Cooling is

accomplished by two-stage flash evaporation under high vacuum, as this method

has the advantage of concentrating the liquor at the same time that it is beingcooled and also of eliminating additional amounts of sulfur dioxide. Concentration is

in the order of 12% which results in proportional reduction of costs during the

subsequent steps of processing.

The essential feature of this process is that after fermentation, the fermented

work is run through a centrifugal separator to remove the yeast. This yeast is then

re-used in a following fermentation in the operating cycle. The basis of this process

is that when yeast is present in a suitable medium containing sugar, the course of

the resulting fermentation tends to divide into two stages. In the first stage, the

yeast cells multiply, using sugar for food, until they become crowded. During this

stage there is maximum growth of yeast and minimum production of alcohol. In the

second stage, the yeast reduces its rate of budding, or division, and continues to

feed on the remaining sugar present. During this stage there is minimum growth of

yeast and maximum production of alcohol. The purpose of re-using the yeast is to

establish at once in each new fermentation the same high concentration of yeast

cells that was present at the end of the previous fermentation. In this way the

fermentation is limited to the second stage and the alcohol yield, therefore, is

improved.

Under any given set of conditions there is a fairly narrow concentration range

of yeast cells per unit volume above which the rate of yeast growth diminishes every

rapidly. Operation throughout the entire fermenting cycle at yeast concentration in

C.O.E.& T.,Akola 49


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excess of such critical value offers important advantages in the case of waste sulfite

liquor. Since large volumes of fermenting liquor are involved, profile yeast growths

must ensue before the critical concentration is reached if the initial inoculum is

small. This yeast growth is obtained at the expense of sugar, which would

otherwise produce alcohol. At the same time, because waste sulfite liquor is, at

best, a dilute sugar solution, the sugar required for yeast growth represents a

greater proportion of the total than in the case of more concentrated sugar solutions

from molasses or grain. The advantages of the yeast re-use process in increasing

alcohol yields are, therefore, much greater in the case of waste sulfite liquor than in

application to the more conventional raw materials.

Fermentation is carried out in eight interconnected fomenter of 90,000 galcapacity each. To the liquor being pumped from storage is injected measured

proportionate amounts of urea and yeast. The liquor enters the first fomenter,

overflows into the second and so on through to the last fomenter whereupon

fermentation is complete. From 70 to 80% of the fermentable sugars are fermented

in the first two fomenters and about 95% of the fermentable sugars are converted to

alcohol in the complete cycle. Fermentation time has been varied between 12 and

20 hours. This short fermentation time, principally due to the elimination of the

yeast-multiplication stage, can also be attributed partly to the adequate mixing

provided in the fermenters, which keeps the yeast cells in suspension and partly to

the fact that the yeast is acclimatized through re-use.

Control of fermentation consists of regular measurement of the sugar

concentration of the liquor entering and leaving the fermented and the alcohol

content of the fermented liquor. The yeast is examined daily under the microscope

for viability and cell count.

From starch substrate

Technology for manufacturing alcohol from starch based raw materials such

as grains (sorghum grains) on laboratory scale which is scaled up to 15,000 LPD

pilot plant, the process involved in brief is as follows: The chemical equation which

takes place is as follows:

C.O.E.& T.,Akola 50


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(C6H10O5)n + H2O > n(C6H1206)Starch glucose water Free glucose

C6H12O6 > 2(C2H5OH) + 2CO2Glucose Ethanol Carbon dioxide

A) Milling and Gelatinization

The starch containing amylaceous material is first cleaned, purified, dried

and milled/ground in small particle and then charged into a steam chest where it is

cooled by steam at 140C and then enzymes are added. The steam injection

breaks down the starch and make it more water soluble. The starch swells to many

times the original size and become gelatinized. A liquefying enzyme also breaks the

starch into smaller molecular chain.B ) Hydrolysis and saccharification

The mash is then blown into a flash tank and cooled at around 90C. The

sudden expansion dissolves the starch out of it bond and disengages it so that it

can be decomposed more quickly and more completely. It is now ready for

fermentation in mash tabs. Here the addition of appropriate enzymes i.e. -

amylaze carries out composition of polysaccarides to the extent required under

controlled condition.

C) Fermentation

The fermented mash of saccharides is then charged to pre-fermenter where

yeast is added, (yeast is cultivated in a separate yeast culture vessel) The function

of pre-fermenter is to allow the yeast cells to multiply and reduce the chances of

bacterial contamination from t

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