How do Streptococcus thermophilus and Lactobacillus bulgaricus precipitate the curd of swiss cheese?

How do Streptococcus thermophilus and Lactobacillus bulgaricus precipitate the curd of swiss cheese?

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I learned in my food microbiology class that Streptococcus thermophilus and Lactobacillus bulgaricus precipitate the curd of swiss cheese. However, I was wondering what type of mechanisms do these bacteria use in order to accomplish this process.

lactic acid bacteria ferment the disaccharide lactose present in high amounts in milk to the three-carbon compound lactic acid, which acidifies the milk. this causes the proteins also present in milk to denaturate and coagulate, forming curdles. other causes/methods of curdling can be application of heat, salts or by adding the enzyme chymosin (rennin).

Fermentation Process of Cheese | Microbiology

Cheese can be defined as a consolidated curd of milk solids in which milk fat is entrapped by coagulated casein. Unlike fermented milks, the physical characteristics of cheese are far removed from those of milk.

This is because protein coagulation proceeds to a greater extent as a result of the use of proteolytic enzymes and much of the water content of the milk separates and is removed in the form of whey. Typically the yield of cheese from milk is of the order of 10%.

Cheese making can be broken down into a number of relatively simple unit operations. Slight variations of these and the use of different milks combine to generate the huge range of cheeses available today said to include 78 different types of blue cheese and 36 Camembert’s alone.

Classification of cheeses is made difficult by this diversity and the sometimes rather subtle distinctions between different types. Probably the most successful approach is one based on moisture content, with further subdivision depending on the milk type and the role of micro-organisms in cheese ripening (Table 9.7).

Cheese is a valuable means of conserving many of the nutrients in milk. In many people, it evokes a similar response to wine, playing an indispensible part in the gastronome’s diet and prompting Brillat-Savarin (1755-1826) to coin the rather discomforting aphorism that ‘Dessert without cheese is like a pretty woman with only one eye’.

Despite this, the attraction of a well-ripened cheese eludes many people and it is sometimes hard to understand how something that can smell distinctly pedal can yield such wonderful flavours. This paradox was encapsulated by a poet, Leon-Paul Fargue, who described Camembert as ‘the feet of God’.

Today cheese making is a major industry worldwide, producing something approaching 10 million tonnes per annum. Much is still practiced on a relatively small scale and accounts for the rich diversity of cheeses still available.

Large-scale industrialized production is increasingly important, however, and is dominated by one variety, Cheddar, which is now produced throughout the world, far removed from the small town in Somerset where it originated.

Cheddar cheese is particularly valued for its smooth texture and good keeping qualities, although products sharing the name can vary dramatically in flavour. In what follows we will describe the basic steps in cheese making with particular reference to the manufacture of Cheddar cheese.

Cow’s milk for cheese production must be free from antibiotics and sanitizing agents that might interfere with the fermentation. Although it is not compulsory, a heat treatment equivalent to pasteurization is usually applied at the start of processing. This helps to ensure a safe product and a reliable fermentation, although cheeses made from raw (unpasteurized) milk have been claimed to possess a better flavour.

The milk is then cooled to the fermentation temperature which, in the case of Cheddar and other English cheeses such as Stilton, Leicester and Wensleydale, is 29-31 °C. The starter organisms used in most cheese making are described as mesophilic starters, strains of Lactococcus lactis and its subspecies.

Thermophilic starters such as Lactobacillus helveticus, Lb. casei, Lb. lactis, Lb. delbrueckii subsp. bulgaricus and Strep, salivarius subsp. thermophilic are used in the production of cheeses like Emmental and Parmesan where a higher incubation temperature is employed.

The role of starter organisms in cheese making is both crucial and complex. Their central function is the fermentation of the milk sugar lactose to lactic acid. This and the resulting decrease in pH contribute to the shelf-life and safety of the cheese and gives a sharp, fresh flavour to the curd.

The stability of the colloidal suspension of casein is also weakened and calcium is released from the casein micelles improving the action of chymosin. After the protein has been coagulated, the acid aids in moisture expulsion and curd shrinkage, processes which govern the final cheese texture.

There are two different systems for uptake and metabolism of lactose in LAB. In most lactobacilli and Strep, salivarius subsp. thermophilus, lactose is taken up by a specific permease and is then hydrolysed intracellularly by β-galactosidase.

The glucose produced is fermented by the EMP pathway which the galactose also enters after conversion to glucose-6-phosphate by the Leloir pathway (Figure 9.7). Most lactococci and some lactobacilli such as Lb. casei take up lactose by a phosphoenol- pyruvate (PEP)-dependent phosphotransferase system (PTS) which phosphorylates lactose as it is transported into the cell.

The lactose phosphate is then hydrolysed by phospho-β-galactosidase to glucose, which enters the EMP pathway, and galactose- 6-phosphate which is eventually converted to pyruvate via the tagatose-6-phosphate pathway.

These pathways are of practical import in cheese making in the lactococci, lactose utilization is an unstable, plasmid encoded characteristic and loss of these genes can clearly have serious consequences for milk fermentation.

Using transduction techniques, molecular biologists have produced strains of Lactococcus lactis in which this property has been stabilized by integration of the lactose utilization genes in the chromosome.

The thermophilic lactobacilli, which employ a lactose permease and β- galactosidase, metabolize the glucose produced preferentially, turning to galactose only when lactose becomes limiting. This can be a problem in some products. The accumulation of galactose can give rise to a brown discolouration during the heat processing of Mozzarella cheese.

In Swiss cheeses such as Emmental, residual galactose can affect product flavour since propionic acid bacteria ferment it in preference to lactate. In doing so they produce a preponderance of acetic (ethanoic) acid which does not confer the usual nutty flavour associated with the equimolar concentrations of acetate and propionate produced by the Propionibacterium from lactate.

Lactic acid bacteria are nutritionally fastidious and require preformed nucleo­tides, vitamins, amino acids and peptides to support their growth. To grow to high cell densities and produce acid rapidly in milk, dairy starters must have proteolytic activity to overcome the limitation imposed by the low non-protein nitrogen pool in native milk.

These systems are comprised of proteinases, associated with the surface of the bacterial cell wall, which can hydrolyse casein proteins. Peptidases in the cell wall degrade the oligopeptides produced down to a size that can be transported into the cell (4-5 amino acid residues) where they are further degraded and utilized.

While this ability is essential to starter function, it also plays an important role in the development of cheese flavour during ripening or maturation. Citrate fermentation to diacetyl is required in some cheese varieties and starter cultures for these include species such as Lactococcus lactis subsp. diacetylactis or Leuconostoc cremoris.

Carbon dioxide is another product of this pathway and is important in producing the small eyes in Dutch cheese like Gouda or giving an open texture that will facilitate mould growth in blue-veined cheeses. In other cheese, such as Cheddar, this would be regarded as a textural defect.

To produce Cheddar cheese, starter culture is added at a level to give 10 6 -10 7 cfu ml -1 . In the past these cultures were grown-up in the dairy from stock cultures or from freeze-dried preparations bought in from commercial suppliers. Nowadays frozen, concentrated cultures that are added directly to the cheese vat are increasingly used because of their ease of handling and the greater security they offer the cheese maker.

This applies particularly to the risk of bacteriophage inhibition of the fermentation which has been a major preoccupation of the cheese maker since it was first identified in New Zealand in the 1930s. Problems of phage infection are not confined to cheese making but have also been encountered in the production of yoghurt and fermented meats.

A bacteriophage is a bacterial virus which in its virulent state infects the bacterial cell, multiplies within it, eventually causing the cell to burst (lysis). When this occurs during a cheese fermentation, acidification slows or even stops causing financial losses to the producer as well as an increased risk that pathogens might grow.

An important source of phage in cheese making is thought to be the starter culture organisms themselves which carry within them lysogenic phages that can be induced into a virulent state. Problems occur particularly when starters contain a single strain or only a few strains and the same culture is re-used over an extended period.

During this time, phages specific to that organism build up in the plant and can be isolated from the whey and from environmental sources such as drains and the atmosphere, increasing the chance of fermentation failure.

In the past, control of this problem has been based on the observation of rigorous hygiene in the dairy, the rotation of starter cultures with differing phage susceptibilities and propagation of starters in phage-inhibitory media which contain phosphate salts to chelate Ca 2+ and Mg 2+ required for successful phage adsorption to the bacterial cell.

LAB possess their own resistance mechanisms to phage infection which include restriction/ modification of non-host DNA, inhibition of phage adsorption by alteration or masking of specific receptors on the cell surface, and reduction of burst size (the number of phages released per infected cell).

Most of these mechanisms appear to be plasmid encoded and this has opened the way for new strategies for phage control so that trans-conjugants with enhanced phage resistance are now available.

A time course for the production of Cheddar cheese showing pH changes and the timing of different process stages is shown in Figure 9.8. A good starter should produce around 0.2% acidity within an hours incubation. It will multiply up to around 10 8 -10 9 cfu g -1 in the curd producing an acidity of 0.6-0.7% before its growth is stopped by salting.

After about 45 min rennet is added. The time of renneting and the amount added are other important variables in cheese making which differ with cheese type. Rennet is a preparation from the fourth stomach or abomasum of suckling calves, lambs or goats.

Its most important component is the proteolytic enzyme rennin or chymosin which cleaves k-casein, the protein responsible for the stability of the casein micelle, between phenylalanine 105 and methionine 106.

This releases a 64 amino acid macro-peptide into the whey leaving the hydrophobic para-k-casein attached to the micelle. Loss of the macro-peptide leads to the formation of cross-links between the micelles to form a network entrapping moisture and fat globules.

Authentic chymosin is produced as a slaughterhouse by-product but microbial rennets are available, produced from fungi such as Mucor miehei, Mucor pusillus and Endothia parasitica. These lack the specificity of animal rennet and have been associated with the production of bitter peptides in the cheese.

Now however the genes for chymosin have been cloned into a number of organisms and nature-identical chymosin is available commercially, produced using the bacterium E. coli and yeasts.

After 30-45 min, coagulation of the milk is complete and the process of whey expulsion is started by cutting the curds into approximately 1 cm cubes. Whey expulsion is further assisted by the process known as scalding when the curds, heated to 38-42 °C, shrink and become firmer.

The starter organisms are not inhibited by such temperatures and continue to produce acid which aids curd shrinkage. Cheeses produced using thermophilic starters can be scalded at higher temperatures without arresting acid development. When the acidity has reached the desired level (generally of the order of 0.25%), the whey is run off from the cheese vat.

It is at this stage that the process known as cheddaring occurs. The curd is formed into blocks which are piled up to compress and fuse the curds, expelling more whey. Nowadays the traditional manual process is mechanized in a cheddaring tower.

At the end of cheddaring, the curd has a characteristic fibrous appearance resembling cooked chicken breast. The blocks of curd are then milled into small chips. This facilitates the even distribution of salt which, in Cheddar, is added at a level of between 1.5 and 2% w/w. The salted curd is formed into blocks which are then pressed to expel trapped air and whey.

Finally the cheese is ripened or matured at 10°C to allow flavour development. During this stage, which can last up to five months to produce a mild Cheddar, the microflora is dominated by non-starter lactobacilli and a complex combination of bacterial and enzymic reactions give the cheese its characteristic flavour.

In particular, proteases and peptidases from the starter culture .continue to act, even though the organism can no longer grow. With other proteases from the rennet, they release free amino acids (principally glutamic acid and leucine in Cheddar) and peptides which contribute to the cheese flavour.

In some cases this can give rise to a flavour defect: casein proteins contain a high proportion of hydrophobic amino acid residues such as leucine, proline and phenylalanine and if they are degraded to produce peptides rich in hydrophobic residues, the cheese will have a bitter taste.

The lipolytic and proteolytic activities of moulds play an important role in the maturation of some cheeses. In blue cheeses such as Stilton, Penicillium roquefortii and P. glaucum grow throughout the cheese.

Both can grow at reduced oxygen tensions, but aeration is improved by not pressing the curds and by piercing the blocks of curd with needles. P. camembertii and P. caseicolum are associated with surface-ripened soft cheeses such as Camembert and Brie.

The keeping qualities of cheese vary with the type but are always much superior to those of milk. This is principally the result of the reduced pH (around 5.0 in Cheddar), the low water activity produced by whey removal and the dissolution of salt in the remaining moisture.

Under these conditions yeasts and moulds are the main organisms of concern. The latter are effectively controlled by traditional procedures to exclude air such as waxing or by modern refinements such as vacuum packing.

How do Streptococcus thermophilus and Lactobacillus bulgaricus precipitate the curd of swiss cheese? - Biology

Microorganisms found in cheese are responsible for much of the uniqueness and character that we all know and love

What are Microbes?

“Microbe” is short for microorganism -- any microscopic living organism*. There are many things found in this branch of the evolutionary tree. In cheese we usually find bacteria, yeasts, and molds. Even looking at just these categories leaves thousands upon thousands of microbes that could potentially be found cheese. For brevity's sake, we're going to cover just the basics (thus the “101”). There is a lot we won't cover here. If you're looking for a more in-depth review of microbes and cheese, check our

*To avoid excessive repetition we'll be using “microbe”, “organism”, and “bug” interchangeably. I'll try my best to get the spelling right and not say how important orgasms are to cheese making.

Microbes in Cheese

The bacteria, molds, yeasts, etc. that find their way into cheese can be added intentionally by the cheesemaker or affineur. And by intentionally, I mean a person made a judgement call and chose which organism to add to the cheese. Of more interest and import are the multitude of microbes that are introduced into the cheese without any direct decision making from the cheesemaker/affineur. This is where a cheese takes on its so-called “terroir”. Microbes native to the milk will be carried over to the cheese and as cheese is being made and as it is being aged there are many ambient organisms that weasel themselves in.

Microbes are introduced into cheese at every step in the cheese making process

Lactic Acid Bacteria

These are the microbes (bacteria) that are added to the milk very early in the cheese making process that induce the fermentation process. The main reaction taking place here is the conversion of lactose to lactic acid, acidifying the milk, which explains how they get their name. You may have also heard of these guys referred to as “starter cultures”. Examples from this category include:

    - Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris are common lactic acid bacteria that are used to make cheeses like cheddar - Streptococcus salivarius ssp. thermophilus is an example of a culture used in cheese like mozzarella - Lactobacillus helveticus is an example of a culture commonly used in Swiss and alpine cheeses. L. helveticus is also commonly used as an adjunct. (below)

Lactic acid bacteria are a ubiquitous starter culture

Adjunct Cultures

Adjuncts are microbes that are added for reasons other than just producing lactic acid. In many cases, adjuncts are added to encourage flavor development in the cheese. Lactobacillus helveticus (see above) is a common example, often giving cheeses a pleasant sweet flavor and promoting the growth of tyrosine crystals.

Lactobacillus helveticus often gives a sweet flavor to cheeses like aged Gouda


Related to adjuncts, Non-Starter Lactic Acid Bacteria are lactic acid bacteria that grow as cheese is ripened that weren’t added for the express purpose of acidifying the milk. Usually these microbes are present naturally in the milk or get picked up along the way during cheesemaking. As cheese ages, the numbers of NSLAB increase while starter cultures die off. Each of their exact roles in cheese flavor development is still trying to be understood completely. Examples include:

Eye Formers

Swiss cheese (and Gouda to a lesser degree) have pronounced eye (hole) formation due to the action of certain bacteria. Propionibacterium freudenreichii ssp. shermanii is a specific bacterium that converts lactic acid into carbon dioxide, propionic acid, and acetic acid. The carbon dioxide seeps into the cheese body and produces the eyes we all know and love. The other products of Propionibacterium metabolism also give the characteristic flavors commonly associated with Swiss cheeses.

Propionibacterium produce CO2 and form the eyes in Swiss

Gouda cheese often have eyes as well, although usually to a smaller degree than Swiss. In this case, it’s not propionibacteria that is responsible, but usually bacteria such as Leuconostoc mesenteroides and Lactococcus lactis ssp lactis biovar. diacetylactis. In this case it is citric acid that is converted to carbon dioxide and diacetyl (buttery flavor).


The two main molds that are found in/on cheese are blue and white, respectively. While there are specific molds cheesemakers add to get the cheese they want, there are many molds that grow naturally on the surfaces of cheese during affinage. We’ll be covering the former.

White Mold

Penicillium camemberti (fka Penicillium candidum) is the most popular mold species that is responsible for the nice white lawn on the surfaces of cheeses like camembert and brie. The metabolism of this mold is responsible for some of the characteristic aromas associated with white mold cheeses (mushroom, ammonia, etc.) as well as the texture (soupy goodness). You can often see this by looking at a cross section of a piece of young brie. You’ll observe a translucent soupy boarder surrounding a chalky center.

Penicillium camemberti covers Brie and Camembert

Blue Mold

Penicillium roqueforti and Penicillium glaucum are the big players in the blue mold worlds. These are what give bleus the blues. The pigments are created by the molds as well as the unique flavor and distinctive texture. As mentioned in the opening paragraph of the post, these molds are living breathing organisms. Starving them of oxygen will change their metabolism and they will change color as well as produce off-flavors. For this reason it’s best to never vacuum package blue cheeses (or any mold-contain cheese for that matter). A common misconception is that when blue cheeses are pierced during the aging process that mold is being injected. Actually, those needles are there to create air channels. The mold is usually added to the milk during the preliminary cheese making steps or to the curds before they're hooped. The piercing only serves to encourage mold growth by introducing oxygen.

Penicillium roqueforti is a common blue mold added to cheese
Bayley Hazen Blue - Jasper Hill

Mold-like Yeast

I’d also like to mention Geotrichum candidum which is a yeast that exhibits mold-like tendencies. This microbes is responsible for the “brainy” appearance some cheeses have.

Geotrichum candidum is responsible for the brainy appearance of some cheeses

Surface Ripened Bacteria

Brevibacterium linens is one of the most common bacteria that make up “smear” bacteria. It is also responsible for foot odor, which explains the smell of many surface ripened cheeses. This bacterium produces a multitude of compounds including ones that give rise to the distinctive aroma. Corynebacteria are another class of bacteria commonly found on these cheeses. It's important to remember that the combination of microorganisms is what makes the magic happen for many cheeses.

Brevibacterium linens and yeast often produces orange colored pigments


Often forgotten, but yeast are commonly used in the molded and surface ripened cheeses. They are also naturally present in many natural rind cheeses. These are important parts of the aging process of many cheeses. In many cases, there is a careful balance of yeast, mold, and bacteria that give rise to natural rinds. This will have its own post one day.

Here we talked about microbes seperately for the most part. It's important to remember cheese is often teaming with a whole slew of microbes interacting with each other and the environment. It's the combination of bacteria, yeast, molds, etc. that make the magic happen.

2] Coagulation of milk

In manufacture process of cheese the milk proteins are coagulated to form a solid curd. In milk the protein casein is around 82 % and 18 % is whey protein. In the curd formed after coagulation milk contains fat globulins, water soluble material and water. Coagulation of milk can be done by two ways:-

  • Coagulation of milk by using Lactic starter culture – The milk is acidified by fermentation of lactose to lactic acid by using some bacterial cultures. The bacterial cultures used in coagulation of milk are Streptococcus cremoris, Streptococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus helveticus. The bacterial culture is mixed properly with milk till acidity reaches to 0.17 to 0.2 % and then the coagulation enzyme are added in milk.
  • Coagulation of milk by using Enzyme – A number of different enzyme preparation are used commonly to clot milk. Enzyme like rennet, porcine, pepsin, and protease from selected micro-organism are most commonly used.In manufacture of cheddar cheese the most commonly used enzyme is rennet enzyme. Rennet enzyme coagulates casein in 20 to 40 minutes. Casein protein gets coagulated at its isoelectric point at pH OF 4.7.

Microbes in Human Welfare Important Extra Questions Very Short Answer Type

Question 1.
Which bacterium Is responsible for the formation of curd from milk?
Lactobacillus but Agaricus (Lactic acid bacteria).

Question 2.
What is brewing?
Brewing is a complex fermentation process, which involves the production of malt beverages such as beer, ale, porter, and stout with the help of strains of Saccharomyces cerevisiae.

Question 3.
Name the type of association that genus Glomus exhibits with the higher plant. (CBSE2014)
Mycorrhiza- Symbiotic association.

Question 4.
Which one of the following is the baker’s yeast used in fermentation-Saccharum Barberi, Saccharomyces cerevisiae or Sonalika? (CBSE2009)
Saccharomyces cerevisiae.

Question 5.
Milk starts to coagulate when Lactic Acid Bacteria (LAB) is added to milk as a starter. Mention two benefits that LAB provides. (CBSE 2009)

  1. LAB checks the growth of disease-causing microbes.
  2. LAB converts milk into curd and also increases nutritional quality by increasing vitamin B12.

Question 6.
Give the scientific name of the source organism from which the first antibiotic was produced. (CBSE Sample paper 2018-19)
Penicillium Notatum

Question 7.
Name the different vitamins which are produced by micro-organisms.

  1. Riboflavin or Vitamin B2 is produced by yeast and bacteria.
  2. Vitamin B12 or cobalamine is produced by bacteria and actinomycetes.

Question 8.
Name the original wild strain of the mold by which vitamin B2 is produced.
Ashbya Gossypii.

Question 9.
What is a single-cell protein (SCP)?
Single-cell protein (SCP) refers to any microbial biomass produced by uni and multi-cellular organisms and can be used as food or feed additives.

Question 10.
Name a microbe used for statin production. How do statins lower blood cholesterol levels?
Monascus Purpureus Mechanism: Statins are competitive inhibitors of enzymes required for cholesterol synthesis. Therefore, play role in decreasing cholesterol level in the body.

Question 11.
‘Swiss cheese’ is characterized by the presence of large holes. Name the bacterium responsible for it. (CBSE Delhi Outside 2019)
Propionibacterium sharmanii

Question 12.
What for Nudeopolyhedra viruses (NVP) are being used nowadays? (CBSE, Delhi 2014, 2019C)
Nudeopolyhedro viruses are being used to kill insects and other arthropods pests of crops. The viruses have no effect on plants and non-target animals. Thus used in biological control of pests.

Question 13.
How has the discovery of antibiotics helped mankind in the field of medicine?
Antibiotics have helped mankind in treating most of the deadly bacterial and fungal diseases of humans.

Question 14.
Why is distillation required for producing certain alcoholic drinks?
For increasing the alcohol strength or concentration of the drinks.

Question 15.
What is the primary sludge?
All the solids that settle from the sewage on primary treatment constitute primary sludge.

Question 16.
What is the relationship between BOD and organic matter in sewage?
The greater the BOD of wastewater more is the amount of organic matter in sewage.

Question 17.
Name two gases produced during secondary treatment by sewage.

Question 18.
What are bioreactors?
In the pilot plant, the glass vessels are replaced by stainless steel vessels. They are called bioreactors.

Question 19.
Name the bacteria which can be used for yogurt formation.

Question 20.
What is Bacitracin?
It is an antibiotic obtained from Bacillus Licheniformis.

Question 21.
Name the group of organisms and the substrate they act on to produce biogas. (CBSE 2009)
Methanogens such as Methanol bacterium act on activated sludge to produce biogas.

Question 22.
WrIte the scientific name of the microbe used for fermenting malted cereals and fruit juices. (CBSE 2011)
Saccharomyces cerevisiae

Question 23.
Write an alternate source of protein for animal and human nutrition. (CBSE 2014)
Single-cell proteins.

Question 24.
How are the members of the genus Glomus useful to organic farmers? (CBSE Delhi Outside 2019)
Many members of the genus Glomus form mycorrhizae- symbiotic associations with roots of higher plants. The fungal component of these associations helps in the absorption of phosphorus from soil. It also makes the plant drought-resistant.

Microbes in Human Welfare Important Extra Questions Short Answer Type

Question 1.
Expand the ‘LAB’. How are LABs beneficial to humans? (Write any two benefits) (CBSE 2019 C)
LAB-Lactic Acid Bacteria Benefits:

  • Found in curd. They improve the nutritional quality of food.
  • Yogurt is prepared from milk by Lactobacillus Bulgaricus.

Question 2.
What is cyclosporin A? What is its importance?
Cyclosporin A. It is an eleven-membered cyclic oligopeptide obtained through the fermentative activity of fungus Trichoderma Polysporum.

Importance. It has antifungal, anti-inflammatory, and immunosuppressive properties. It inhibits the activation of T-cells and therefore, prevents rejection reactions in organ transplantation.

Question 3.
How do antibiotics act?
Antibiotics do not have identical effects on all harmful microbes. All of them inhibit growth or destroy bacteria, viruses, and fungi. Actually, antibiotic molecules should disrupt a vital link in the microbe’s metabolism and this link is their target or point of impact.

Question 4.
Write the various steps of fermentation.
The major steps of fermentation are:

  1. Sterilization of the fermenter and medium in steam. It is carried out under pressure and high temperature.
  2. Inoculation of a selected strain of the yeast.
  3. Recovery of the product.

Question 5.
What are the two ways by which micro-organisms can be grown in bioreactors?
Micro-organisms can be grown in the bioreactors in two ways:

  1. As a layer or film on the surface of the nutrient medium. It is known as a support growth system.
  2. By suspending cells or mycelia in a liquid medium contained in the growth vessel. It is known as a suspended growth system.

Question 6.
What is sewage? In which way can this be harmful?
Sewage is used and wastewater consisting of human excreta, wash waters, industrial and agricultural wastes that enter the sewage system. In general, sewage contains 95.5% water and 0.1 to 0.5% organic and inorganic matter. They are very harmful to us due to the presence of a variety of micro¬organisms in them, most of which are highly pathogenic. Sewage has a high BOD value, which develops anaerobic conditions in water resulting in the death of water animals and emitting foul smell due to incomplete oxidation of organic materials in the sewage.

Question 7.
What is the key difference between primary and secondary sewage treatment?
Primary treatment of wastes is the screening and removal of insoluble particulate materials, by addition of alum and other coagulants. It is the physical removal of 20-30% of organic materials present in sewage in particulate form. Secondary treatment of waste is the biological removal of dissolved organic matter through trickling filters, activated sludge, lagoons, extended aeration systems, and anaerobic digestors.

Question 8.
Draw a simple diagram to show an anaerobic sludge digester.

Anaerobic sludge digester.

Question 9.
GIve the full form of Bt. Name the insects killed by It.
The full form of Bt is Bacillus Ttiuringiensis. It kills a wide range of Insects Like moths, beetles, mosquitoes, aphids, and termites.

Question 10.
Why are biofertilizers or biopesticides preferred to chemical fertilizers or pesticides? (CBSE Delhi 2011)
Biofertilizers or biopesticides are preferred to chemical fertilizers or pesticides because

  • They are safe to use and are biological in origin.
  • They do not spoil the quality of the soil and are target-specific.
  • They do not pollute the atmosphere and are non-poisonous.
  • They are less expensive and are biodegradable.

Question 11.
Name the blank spaces a, b, c, and d from the table given below: (CBSE 2008)

Type of microbe Scientific name Product Medical application
(i) Fungus a Cyclosporin B
(ii) c Mascus Purpureus Statin d

(a) Trichoderma polypore
(b) Organ transplantation (Immunosuppressant)
(c) Yeast
(d) Blood cholesterol-lowering agent

Question 12.
How does the addition of a small amount of curd to fresh milk help the formation of curd? Mention a nutritional quality that gets added to the curd. (CBSE Delhi 2010 and Outside Delhi 2019)

  1. Curd is prepared from milk.
  2. Microorganisms such as Lactobacillus and others commonly called lactic acid bacteria (LAB) grow in milk and convert it to curd.
  3. During growth, the LAB produces acids that coagulate and partially digest the milk proteins.
  4. A small amount of curd added into the fresh milk as inoculum or starter contains millions of LAB which at suitable temperatures multiply, thus converting milk to curd, which also improves its nutritional quality by increasing vitamin B12.
  5. In our stomach too, the LAB plays a very beneficial role in checking disease-causing microbes.

Question 13.
Name a free-living and symbiotic bacterium that serves as a biofertilizer. Why are they called so? (CBSE Outside Delhi 2016)
Free-living nitrogen-fixing bacteria Azotobacter and Bacillus Polymyxa Symbiotic nitrogen-fixing bacteria. Rhizobium.

These micro-organisms enrich the soil by fixing nitrogen. They enhance the availability of nutrients to crops, thus called biofertilizers.

Question 14.
(i) Why are fruit juices bought from the market clearer as compared to those made at home?
Bottled juices are clarified by the use of pectinases and proteases.

(ii) Name the bioactive molecules produced by Trichoderma Polysporum and Monascus Purpureus. (CBSE Delhi 2013)
(a) Bioactive molecules produced by Trichoderma polypore are cyclosporin A. It is used as an immunosuppressive agent in organ- transplant patients.
(b) Bioactive molecules produced by Monascus Purpureus are statins. It is a blood cholesterol-lowering agent.

Question 15.
Your advice is sought to improve the nitrogen content of the soil to be used for the cultivation of a non-leguminous terrestrial crop.
(i) Recommend two microbes that can enrich the soil with nitrogen.
Azospirillum, Azotobacter, Anabaena, Oscillatoria (Any two)

(ii) Why do leguminous crops not require such enrichment of the soil? (CBSE 2018)
Leguminous crops do not require such enrichment of the soil because they have a symbiotic association with Rhizobium bacteria which traps nitrogen directly from the atmosphere and provides it to the plant and in turn gets food and shelter.

Question 16.
What are ‘floes’, formed during secondary treatment of sewage? (CBSE Delhi 2019)
Floes are masses of bacteria, associated with fungal filaments to form mesh-like structures.

Question 17.
Write any two places where methanogens can be found. (CBSE Delhi 2019)
Methanogens can be found in the following places:

  1. In anaerobic sludge (digester) of a sewage treatment plant
  2. In rumen (gut/stomach) of cattle or ruminants
  3. Marshy areas
  4. Flooded paddy fields
  5. Biogas plant Methane, H2S, and C02 are produced during microbial digestion of organic compounds in case of secondary treatment of sewage.
  6. The dung of the cattle produces methane gas in the biogas plants.

Microbes in Human Welfare Important Extra Questions Long Answer Type

Question 1.
Give examples to prove that microbes release gases during metabolism.

  1. Large holes in ‘Swiss Cheese’ are due to the production of a large amount of C02 by a bacterium named Propionibacterium shamanic.
  2. The puffed-up appearance of dough is due to the production of C02 gas by yeast, Saccharomyces cerevisiae.
  3. Methane, H2S, and CO2 are produced during microbial digestion of organic compounds in the case of secondary treatment of sewage.
  4. The dung of the cattle produces methane gas in the biogas pLants.

Question 2.
Make a table showing industrial products obtained from activities of bacteria.
industrial products obtained from use activities of Bacteria:

Question 3.
What are Baculo viruses? Write their significance.
Baculoviruses are those viruses, which attack insects and other arthropods, e.g. Nuclepolyhedrovirus.

  • Baculoviruses are species-specific and narrow-spectrum insecticides.
  • They have no negative impacts on plants, birds, mammals, or even other non-target insects.
  • The desirable aspect In conservation of beneficial insects in overall integrated pest management (IPM) program as in an ecologically sensitive area.

Question 4.
Which nitrogen fixers are available on a commercial basis In the market? Also, name the beneficial crop.

Beneficial crop

Question 5.
Distinguish between the roles of floes and anaerobic sludge digester in sewage treatment. (CBSE Delhi 2016)
Floes are masses of bacteria associated with fungal filaments to form mesh-like structures. These microbes digest a lot of organic matter, converting it into microbial biomass and releasing a lot of minerals. Anaerobic sludge digester is a large tank in which anaerobic microbes digest the anaerobic mass as well as aerobic microbes of sludge. Biogas is produced by methanogens. It is inflammable and a source of energy.

Question 6.
Tabulate the list of common antibiotics, organisms producing them, and organisms sensitive to these antibiotics.

Name of Antibiotic

Sensitive Organisms

Question 7.
Give a flow chart of sewage treatment.
Flow chart of sewage treatment:

Flow chart of sewage treatment

Question 8.
List the events that lead to the production of biogas from wastewater whose BOD has been reduced significantly. (CBSE Dethi 2016)

  1. During secondary treatment of wastewater, sewage fungus forms focus.
  2. BOD decreases. As it decreases to 10-15% of originaL sewage, the wastewater Is taken to a Large settling tank where the focus of sewage fungus settles down.
  3. The supernatant can be passed into water bodies or treated further.
  4. The organic sediment is passed into an anaerobic sludge digester where anaerobic microbes methanogens decompose organic matter.
  5. It is accompanied by the production of blogs and the formation of manure or compost.

Question 9.
Explain the basis of biological control of weeds.
Basis of biological control of weeds:

  1. Biological control of weeds involves breeding of insects that would feed selectively a weed or use of certain micro-organisms which will produce diseases in the weeds and eliminate them.
  2. Certain crop plants do not allow the growth of weeds nearby. They are called smoother plants such as Barley, Rye, Sorghum, Millet, etc. They eliminate weeds through chemicals.
  3. In some cases, specially tailored plants called transgenic plants have been introduced which have tolerance against weeds.
  4. In India and Australia, the overgrowth of cacti was checked by the introduction of the cochineal insect (Cactoblastis cactorum).
  5. The latest technique is to use fungal spores to control weeds. These are suitable because they can be kept for a long time and also resist adverse conditions.

Question 10.
What are biofertilizers? What are the main sources of biological nitrogen fixation? Name two organisms that fix nitrogen symbiotically and two organisms that fix symbiotically.
Biofertilizers are organisms that can bring about soil nutrient enrichment by their biological activity.

  • Sources of biofertilizers: Bacteria, cyanobacteria, and fungi.
  • Biological nitrogen fixation: The conversion of atmospheric nitrogen into nitrogenous compounds through the agency of living organisms is called biological nitrogen fixation.

Symbiotically nitrogen-fixing organisms:

  • Rhizobium leguminosarum, Frankia Bacillus radicicola.
  • Free-living/Asymbiotic nitrogen-fixing organisms-Cyanobacteria, Azotobacter.

Question 11.
(a) What is biogas? What are its components? What is the calorific value of biogas? (CBSE Outside Delhi 2013)
Biogas is a methane-rich fuel gas produced by anaerobic breakdown or digestion of biomass with the help of methanogenic bacteria.

Components of biogas: Methane, Carbon dioxide, Hydrogen sulfide, hydrogen, and nitrogen.
Calorific value 23-28 MJ/m 3 .

(b) Why is a slurry of cattle dung (gobar) added to bio-wastes in the tank of a gobar gas plant for the generation of biogas? (CBSE Delhi 2019 C)
Slurry consisting of excreta dung of cattle commonly called gobar is rich in methanogen bacteria. It is used for the generation of biogas. These bacteria called methane bacterium grow anaerobically and break down the cellulose of dung to liberate gases such as methane, C02, and H2.

Question 12.
(?) Name the toxin produced by B. Thuringiensis.
∝-exotoxin, β-exotoxin, γ-exotoxin, and louse factor

(ii) Nitrogen fixers are available on a commercial basis in the market? Also, name the beneficial crop and microbes used in the following table.

Beneficial crop

A. Rhizobium B. Legume C. Legume

(iii) Expand BOD and COD
BOD- Biological Oxygen Demand COD- Chemical Oxygen Demand

Question 13.
By a flow chart showing the stages in anaerobic digestion during the production of biogas.

Stages in Anaerobic Digestion during biogas formation

Question 14.
Given below is a list of six microorganisms. State their usefulness to humans.
(i) Nucleopolyhedrovirus
(ii) Saccharomyces cerevisiae
(iii) Monascus Purpureus
(iv) Trichoderma polypore
(v) Penicillium Notatum
(vi) Propionibacterium shamanic. (CBSE Delhi 2016)

Name of Micro-organisms

Question 15.
Explain the different steps involved in the secondary treatment of sewage. (CBSE Sample paper 2018—19)
Secondary treatment of sewage is also called biological treatment. Justify this statement and explain the process. (CBSE 2018)

  1. Secondary treatment of sewage is a biological process that employs the heterotrophic bacteria naturally present in the sewage.
  2. The effluent from the primary treatment is passed into large aeration tanks, where it is constantly agitated and the air is pumped into it.
  3. This allows the rapid growth of aerobic microbes into ‘floes’ which consume the organic matter of the sewage and reduce the biological oxygen demand (BOD). The greater is the BOD of wastewater, the more is its polluting potential.
  4. When the BOD of sewage is reduced significantly, the effluent is passed into a settling tank, where the ‘floes’ are allowed to sediment forming the activated sludge.
  5. A small part of the activated sludge is pumped back into the aeration tanks.
  6. The remaining major part of the sludge is pumped into anaerobic sludge digesters, where the anaerobic bacteria digest the bacteria and fungi in the sludge-producing methane, hydrogen sulfide, and carbon dioxide,
    i. e. biogas. This is why secondary treatment of sewage is also called biological treatment.
  7. The effluent after secondary treatment is released into water-bodies like streams or rivers.

Question 16.
Microbes can be used to decrease the use of chemical fertilizers. Explain how this can be accomplished. (CBSE Delhi 2019)

  1. Rhizobium bacteria present in the root nodules of leguminous plants (pea family) forms a symbiotic association and fixes atmospheric nitrogen into organic forms as nitrates/nitrites which are used by the plant as nutrient.
  2. Free-living bacteria in the soil Azospirillum and Azotobacter can fix atmospheric nitrogen thus enriching the nitrogen content of the soil.
  3. Many members of the genus Glomus (Fungi) form mycorrhizal symbiotic associations with higher plants. In these, the fungal symbiont absorbs phosphorus from soil and passes it to the plant.

Question 17.
(?) Organic farmers prefer biological control of diseases and pests to the use of chemicals for the same purpose. Justify.
Chemical methods often kill both useful and harmful living beings indiscriminately. The organic farmer holds the view that the eradication of the creatures that are often described as pests is not only possible but also undesirable, for without them the beneficial predatory and parasitic insects which depend upon them as food or hosts would not be able to survive. Thus, the use of biocontrol measures will greatly reduce our dependence on toxic chemicals and pesticides.

(ii) Give an example of a bacterium, a fungus, and an insect that are used as biocontrol agents. (CBSE 2018)
Insects = Ladybird and Dragonflies. Bacteria = Bacillus thuringiensis. Fungus = Trichoderma

Question 18.
The three microbes are listed below. Name the product produced by each one of them and mention their use.
(i) Aspergillus niger
(ii) Trichoderma polypore
(iii) Monascus Purpureus (CBSE Delhi 2018C)
(i) A patient had suffered myocardial infarction and clots were found in his blood vessels. Name a ‘clot buster’ that can be used to dissolve clots and the microorganism from which it is obtained.
(ii) A woman had just undergone a kidney transplant. A bioactive molecular drug is administered to oppose kidney rejection by the body. What is the bioactive molecule? Name the microbe from which this is extracted.
(iii) What do doctors prescribe to lower the blood cholesterol level in patients with high blood cholesterol? Name the source organism from which this drug can be obtained. (CBSE Outside Delhi 2019)
(i) Aspergillus niger produces citric acid. Citric acid is used as a flavoring agent and as a food preservative.
(ii) Trichoderma Polysporum produces a bioactive molecule cyclosporin A. It is used as an immunosuppressive agent in organ transplant patients.
(iii) Monascus Purpureus produces statins. Statins are capable of competitive inhibition of enzymes required for cholesterol synthesis. Hence, it is used as blood cholesterol-lowering agents.
(i) Streptokinase-‘Clot buster’ can be used to dissolve clots. It is obtained from the bacteria Streptococcus.
(ii) The bioactive molecule is Cyclosporin A which is used as an immunosuppressive agent in organ transplantation. It is produced by the fungus Trichoderma Polysporum.
(iii) Doctors prescribe Statins to lower blood cholesterol. It is obtained from the fungus Monascus Purpureus.

Question 19.
Baculoviruses are good examples of biocontrol agents. Justify giving reasons. (CBSE Delhi 2018C)
Baculoviruses kill insects and other arthropods, hence they are used as biocontrol agents especially Nucleopolyhedrovirus.

  • These viruses are species-specific and have narrow spectrum insecticidal applications.
  • They do not harm non-target organisms like other harmless insects, birds, animals, etc.
  • It is very useful in integrated pest management programs or treatment of ecologically sensitive areas.

Question 20.
Describe the primary and secondary treatment of domestic sewage before it is released for reuse. (CBSE, 2014)
Treatment of domestic sewage. The municipal wastewaters are treated in Effluent Treatment Plant (ETP) prior to disposal in water bodies.

It consists of 3 steps: primary, secondary, and tertiary.
1. Primary treatment. It includes physical processes, such as sedimentation, floatation, shredding (fragmenting and filtering). These processes remove most of the large debris.

2. Secondary treatment. It is a biological method. Activated sludge method. Sewage, after primary treatment, is pumped into aeration tanks or oxidation ponds. Here, it is mixed with air and sludge containing algae and bacteria. Bacteria consume organic matter. The process results in the release of C02 and the formation of sludge or biosolid. Algae produce oxygen for the bacteria. The water, which is now almost clear of organic matter, is chlorinated to kill microorganisms.

3. Tertiary treatment. It involves. removal of nitrates and phosphates. The water, after the above treatment, is then released. It can be reused.

Question 21.
Explain biological control of pests and plant pathogens with examples.
The very familiar beetle with red and black markings the Ladybird, and Dragonflies are useful to get rid of aphids and mosquitoes, respectively.

Role of Bacillus Thuringinesis:
Bt Coming to microbial biocontrol agents that can be introduced in order to control butterfly caterpillars is the bacteria Bacillus thuringiensis (often written as Bt). These are available in sachets of dried spores which are mixed with water and sprayed onto vulnerable plants such as Brassica and fruit trees, where these are eaten by the insect larvae. In the gut of the larvae, the toxin is released and the larvae get killed.

The bacterial disease will kill the caterpillars, but leave other insects unharmed. Because of the development of the methods of genetic engineering in the last decade or so, scientists have introduced B. thuringiensis toxin genes into plants. Such plants are resistant to attack by insect pests. Bt-cotton is one such example which is being cultivated in some states of our country.

Biological control of plant pathogens: A biological control developed for use in the treatment of plant disease is the fungus Trichoderma. Trichoderma sp. are free-living fungi that are very common in soil and root ecosystems. They are effective biocontrol agents of several plant pathogens.

Baculoviruses are pathogens that attack insects and other arthropods. The majority of baculoviruses used as biological control agents are in the genus Nucleopolyhedrovirus. These viruses are excellent candidates for species-specific, narrow spectrum insecticidal applications.

They have been shown to have no negative impacts on plants, mammals, birds, fish, or even on non-target insects. This is especially desirable when beneficial insects are being conserved to aid in an overall IPM (integrated pest management) program, or when an ecologically sensitive area is being treated.

Question 22.
How do biofertilizers enrich the soil?
Biofertilizers play a vital role to solve the problems of soil fertility and soil productivity.

  1. Anabaena azollae, a cyanobacterium, lives in symbiotic association with the free-floating water fern, Azolla. The symbiotic system Azolla-Anabaena complex is known to contribute 40-60 mg N ha-1 per rice crop. In addition to this, cyanobacteria add organic matter, secretes growth-promoting substances like auxins and vitamins, mobilizes insoluble phosphate, and thus improves the physical and chemical nature of the soil.
  2. Rhizobium Leguminoserum and Azospirillum fix atmospheric nitrogen as nitrates and nitrites.
  3. Mycorrhizae formed by an association of bacteria and roots of higher plants increase soil fertility.

Question 23.
Discuss the role of Microbes as Biofertilizers. (CBSE Delhi 2011, 2015, 2019)
Role of microbes as biofertilizers:
Bacteria, cyanobacteria, and fungi (mycorrhiza) are the three groups of organisms used as biofertilizers.
1. Bacteria:
(a) Symbiotic bacteria Rhizobium.
(b) Free-living bacteria Azospirillum and Azotobacter.
(c) They fix the atmospheric nitrogen and enrich soil nutrients.

2. Cyanobacteria, e.g. Anabaena, Nostoc, Aulosira, Oscillatoria, etc.
(a) They function as biofertilizers by fixing atmospheric nitrogen and
(b) Increasing the organic matter of the soil through their photosynthetic activity.

3. Fungi/mycorrhizae:
(a) Fungi form a symbiotic association with roots of higher plants (mycorrhizae), e.g. Glomus.
(b) The fungus absorbs phosphorus and passes it on to the plant.
(c) Other benefits of mycorrhizae are :

  • resistance to root-borne pathogens.
  • tolerance to salinity.
  • tolerance to drought.
  • the overall increase in the plant growth and development

Question 24.
You have been deputed by your school principal to train local villagers in the use of biogas plants. With the help of a labeled sketch explain the various parts of the biogas plant. (CBSE Outside Delhi 2013)
Biogas plant:


Several bioactive compounds are released as a result of microbial growth and metabolism during cheese production and digestion, which include bioactive peptides, EPS, SCFAs, and CLA. These compounds define the functional profile of cheese, and by designing the fermentation and ripening parameters, the availability of these compounds can be increased. Besides, extensive studies are required to demonstrate the health beneficial effects of such designer functional cheese products. Numerous studies have suggested possible solutions for the enhancement of bioactive compounds in cheese, as described in the subsequent sections.

3.1 Production of bioactive peptides

Bioactive peptides are specific protein fragments that demonstrate a positive impact on health conditions (Santiago-López et al., 2018 Wu et al., 2020 ). These peptides affect primary body function and physiological systems, including gastrointestinal, nervous, cardiovascular, and immune systems (Daliri, Oh, & Lee, 2017 ). The functionality of bioactive peptides differs depending on the specific hydrolysis of protein and amino acid sequences of the peptides, which lead to the expression of varying bioactive properties (Table 1). Milk-derived bioactive peptides are generated during milk fermentation and further processing stages, such as ripening and storage conditions (Figure 2). The major cheese bioactive peptides include the two tripeptides, Isoleucine-Proline-Proline (IPP) and Valine-Proline-Proline (VPP), which demonstrate Angiotensin I converting enzyme inhibitory (ACEI) activity (Fan et al., 2019 ). Quantification of IPP and VPP in 44 traditional soft, semihard, and hard cheeses by HPLC followed by triple mass spectrometry revealed the maximum concentration of IPP and VPP as 95 and 224 mg/kg, respectively. ACEI activity of the tripeptides was determined, which ranged between IC50 value of 2.0 to 29.5 mg/ml cheese (Ueli Bütikofer, Meyer, Sieber, & Wechsler, 2007 ). Similarly, the IPP and VPP concentration in 11 varieties of Swiss cheese ranged from 1.6 to 424.5 mg/kg cheese (Bütikofer et al., 2008 ).

Peptide Bioactivity Organism Source Reference
β-CN f(194−209) ACE-inhibition Lactobacillus helveticus LH-B02 Prato cheese Baptista et al., 2018
β-CN f(43−52) ACE-inhibition Lactococcus lactis ssp. lactis Saare Cheese Taivosalo et al., 2018
αs1-CN f(1−6) ACE-inhibition Lactobacillus acidophilus Scamorza cheese Albenzio et al., 2015
β-CN f(70−77) Antidiabetic Lactococcus lactis ssp. lactis ATCC19435 Gouda cheese Uenishi, Kabuki, Seto, Serizawa, and Nakajima, 2012
αs1-CN f(24-32) ACE-inhibition Lactobacillus casei 279 Cheddar cheese Ong, Henriksson, and Shah, 2007
β-CN f(99-101) ACE-inhibition Lactobacillus helveticus Grana Padano cheese Stuknyte et al., 2015
κ-CN f(33-38) ACE-inhibition Lactobacillus casei Cheddar cheese Lu et al., 2016
αs1-CN f(157-164) ACE-inhibition Penicillium roqueforti PR-R Danish cheese Mane, Ciocia, Beck, Lillevang, and McSweeney, 2019
β-CN f(159-169) Antimicrobial Lactobacillus delbrueckii ssp. bulgaricus Minas Cheese Fialho et al., 2018
β-CN f (193-209) ACE-inhibition Lactobacillus helveticus LHB02 Prato cheese Baptista, Negrão, Eberlin, and Gigante, 2020
αs1-CN f(30-37) Antimicrobial Lactobacillus acidophilus Parmigiano Reggiano cheese Martini et al., 2020
β-CN f(193−207) Immunomodulation Lactobacillus rhamnosus GG Camembert-type cheese Galli et al., 2019

The proteolytic activity of microorganisms associated with the ripening process affects ACEI properties of cheese. Aged Cheddar cheese has been identified as a significant source of ACEI inhibitory peptides that include EKDERF, VRYL, YPFPGPIPN, FFVAP, apart from VPP and IPP. ACEI peptide types and concentration in Wisconsin Cheddar cheese were found to increase with extension in ripening time, reaching maximum values of 2.8, 7.4, and 5.3 mg/100 g cheese for IPP, VPP, and EKDERF, respectively. However, ACEI activity of water-soluble extract (WSE) fractions of Cheddar cheese decreased with an increase in ripening time up to 2 months as compared to young (3 to 6 days) cheese, which was followed by stable or increased ACEI activity (Lu et al., 2016 ). These results highlight the importance of ageing in the antihypertensive potential of cheese peptides.

The capability of proteolytic activity and biosynthesis of bioactive peptides is studied by applying individual LAB for cheese production. High ACEI activity and antioxidant peptide production were reported in Pecorino Siciliano cheese fermented by Lb. rhamnosus PRA331 and Lactobacillus casei PRA205 (Solieri, Rutella, & Tagliazucchi, 2015 ). Lb. casei PRA205 fermented milk has been examined to contain VPP and IPP, with concentrations of tripeptides reaching up to 32.88 mg/L, which confer significant ACE-inhibitory property (IC50 value: 54.57 µg/mL Solieri et al., 2015 ). Similarly, Lb. helveticus LH-B02 has been reported to enhance the release of the ACEI peptide β-CN f(194−209) during Prato cheese ripening (Baptista et al., 2018 ). MALDI ToF/MS-based peptide profiling of Camembert-type cheese upon addition of Lb. rhamnosus GG as adjunct culture, revealed the presence of 15 peptides with bioactive potential. The peptide β-CN f(193−209) has been found at maximum intensity, which is known for ACEI and antimicrobial properties (Galli et al., 2019 ).

The peptide profile of cheese changes upon gastrointestinal digestion, which may lead to alteration in the extent of bioactivity of the peptides. The ACEI activity of Gamalost and Norvegia cheese had been shown to be increased during gastrointestinal digestion, resulting in the production of bioactive peptides, aromatic amino acids such as Trp, Tyr, and Phe, and positively charged amino acids, Lys and Arg (Qureshi, Vegarud, Abrahamsen, & Skeie, 2013 ). The digestion also led to the degradation of certain bioactive peptides, but this does not affect the overall activity due to the formation of novel peptides, maintaining the balance of ACEI activity. Antihypertensive peptides, for example, AYFYPEL, VKEAMAPK, EMPFPK, LHLPLP, and YQEPVL, had been identified in Valdeòn cheese after simulated gastrointestinal digestion (Sánchez-Rivera et al., 2014 ). In vitro gastrointestinal digestion of Grana Padano cheese could increase in bioactive casein phosphopeptides by about two folds (Cattaneo, Stuknytė, Ferraretto, & De Noni, 2017 ). In another study, the fate of eight ACE-inhibitor peptides was followed after the in vitro static gastrointestinal digestion by employing UPLC/HR-MS (Stuknyte, Cattaneo, Masotti, & De Noni, 2015 ). During gastrointestinal digestion, the tripeptides, IPP, VPP, along with HLPLP and LHLPLP prevailed the proteolysis, with unchanged ACEI activity. These reports imply the role of gastrointestinal digestion in bioactive peptide profiles of cheese and their functionality upon consumption. The challenge in bioactive peptides rich cheese lies in the stability of these peptides during ripening and gastrointestinal digestion. Therefore, starter culture can be selected for the production of bioactive peptides, which are resistant to digestive enzymes or their successive digestion can result in the production of peptides, with higher activity.

The release of immunomodulatory peptides, αs1-CN f(194-199), β-CN f(193-207), and αs1-CN f(1-23), has been observed during the ripening of Camembert cheese using Lb. rhamnosus GG (Galli et al., 2019 ). Peptide profiling of Coalho cheese by LC-MS/MS revealed the presence of immunomodulatory peptides, β-CN f(193-209) and αs1-CN f(1-23) (Fontenele, Bastos, dos Santos, Bemquerer, & do Egito, 2017 ). The hard cow milk cheese produced using rennet of different origins was found to be enriched with antioxidant peptides, EIVPN, DKIHPF, and VAPFPQ, with high metal chelating activity (Timón, Andrés, Otte, & Petrón, 2019 ). Ripening and in vitro gastrointestinal digestion resulted in the release of several bioactive peptides from Parmigiano-Reggiano cheese, for example, immunomodulators (QEPVL and YPFPGPI), dipeptidyl peptidase IV-inhibitory peptides (INNQFLPYPY, IPIQY, YPFPGPIPN), antioxidant peptides (YFYPEL, TQTPVVVPPFLQPE, VYPFPGPIPN, KVLPVPQK, FYPEL, AVPYPQR), and hypocholesterolemic peptides (GLDIQK and IIAEK) (Martini, Conte, & Tagliazucchi, 2020 ). The β-casein-derived hexapeptides (EAMAPK and AVPYPQ) were found abundantly present in gastrointestinal digests of Stracchino soft cheese (Pepe et al., 2016 ). The peptides showed antioxidant activity along with the upregulation of superoxide dismutase, Nrf2 antioxidant response, and reduced reactive oxygen species level. Studies on many unexplored cheese products can lead to the identification of novel peptides with specific, as well as multifunctional health benefits.

3.2 Microbial EPS

EPS are bioactive carbohydrate macromolecules produced by the starter and adjunct microbial strains during food fermentation. EPS polymers are present as long chains consisting of branched, substituted sugar derivatives, repeated sugar units, or substituted sugars, including phosphate or acetyl substituting groups (Sanlibaba & Çakmak, 2016 ). EPS are produced in fermented dairy products, including yogurt, cheese, buttermilk, and kefir by the action of LAB (Pessôa et al., 2019 ). In addition to being prebiotic, EPS could also impart various health beneficial effects upon consumption (Jiang et al., 2018 Nampoothiri et al., 2016 ). Various studies have reported the application of EPS releasing LAB strains in the production of cheese with bioactive properties. Prato cheese could be prepared by using EPS producing strains, for example, L. lactis ssp. cremoris, L. lactis ssp. lactis, and S. thermophilus, without changing the sensory characteristics and physiological properties of the cheese (Nepomuceno, Costa Junior, & Costa, 2016 ). Cheddar cheese had been manufactured using EPS producing strains, L. lactis, Lb. plantarum SKT109, and Lb. plantarum JLK0142 with improved yield and antioxidant potential (Costa et al., 2010 Wang, Wu, Fang, & Yang, 2019 ). Cheddar cheese prepared using Lb. plantarum JLK0142 in combination with non-EPS-producing cheese starter showed significantly high 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activities in addition to inhibitory effects on α-amylase, ACE, and HT-29 tumor cell growth (Wang et al., 2019 ).

In a comparative study, EPS producing probiotic, Lb. plantarum, was isolated from camel milk, which, when used for the production of Akawi cheese, resulted in a product having high radical scavenging properties in comparison to cheese produced using non-EPS producing strains (Al-Dhaheri et al., 2017 ). Besides, the storage of cheese with EPS-producing strains resulted in a product having high ACEI and antiproliferative activities (Al-Dhaheri et al., 2017 ). The presence of EPS in cheese is associated with increased water and fat retention, resulting in increased cheese yield, in addition to improved health benefits (Xu et al., 2019 ). Capsular EPS producing S. thermophilus MTC360 along with starter LAB, Lb. helveticus LH100 had been used to produce Mozzarella cheese with increased moisture levels, and subsequently increasing cheese yield (Mohamed, 2015 ). The low-fat Caciotta type cheese manufactured by using S. thermophilus ST446 along with Lb. rhamnosus LRA and Lb. plantarum LP as adjunct cultures demonstrated high moisture retention, higher yields, and significantly elevated levels of free amino acids, as compared to the full-fat variant (Di Cagno et al., 2014 ). EPS enriched cheese products need further investigation and validation for specific health benefits by animal studies and clinical trials.

3.3 Enhancement of CLA

Among the conjugated fatty acids in the natural environment, CLA isomers are regarded as functional lipids molecules. CLA consists of a group of geometric and positional isomers of linoleic acid with a conjugated double bond. Major CLA isomers include the biologically active cis-9, trans-11 CLA, trans-10, and cis-12 CLA (de Almeida et al., 2015 ). Cheese-derived CLA exhibit antioxidative, antihypertensive, anticarcinogenic, antiadipogenic, antiinflammatory, antidiabetic, and antiobesity properties (Table 2 Bassaganya-Riera et al., 2012 Florence et al., 2009 Gutiérrez, 2016 Koba & Yanagita, 2014 Murru et al., 2018 Renes et al., 2019 ). CLA isomers are naturally found in milk and are formed upon incomplete dietary fatty acids biohydrogenation in the rumen of cows. The CLA content in cheese ranges from 0.05% to 2.86% of total fatty acid, which depends on environmental, geographical, and physiological parameters, apart from the ability of the bacterial strains present during fermentation (Abd El-Salam & El-Shibiny, 2014 ). The formation of CLA depends on fermentation time, the composition of CLA in raw milk, and linoleate isomerase activity of LAB (Salsinha, Pimentel, Fontes, Gomes, & Rodríguez-Alcalá, 2018 ). LAB use hydratase, dehydrogenase, isomerase, and reductase enzymes for the biohydrogenation of fatty acids, similar to Propionibacterium and rumen bacteria (Salsinha et al., 2018 ).

CLA isomers Bioactivity Source Lactic acid bacteria Enzyme Reference
c9, t11-CLA Cardioprotective Cheddar cheese Lactobacillus plantarum ZS2058 Linoleate isomerase Yang et al., 2014
c9, t11-CLA Antioxidant Pico cheese Lb. plantarum L3C1E8 Linoleate isomerase Ribeiro et al., 2018
c9, t11-CLA Antioxidant Miniature cheese Lb. plantarum L200 Linoleate isomerase Ares-Yebra et al., 2019
t10, c12-CLA Antidiabetic White pickle cheese Lb. paracasei E10 Linoleate isomerase Gursoy et al., 2012
t10, c12-CLA Antidiabetic White pickle cheese Lb. acidophilus O16 Linoleate isomerase Gursoy et al., 2012
c9, t11-CLA Antioxidant Arzua-Ulloa cheese Lb. paracasei L45 Linoleate isomerase Ares-Yebra et al., 2019
c9, t11-CLA Antioxidant Scamorza cheese Bifidobacterium longum and Bifidobacterium lactis Linoleate isomerase Albenzio et al., 2013
c9, t11-CLA Antioxidant Scamorza cheese Lb. acidophilus Lipase, Linoleate isomerase Albenzio et al., 2013
c9, t11-CLA Antioxidant Coalho cheese Lb. acidophilus LA5 Linoleate isomerase Barbosa et al., 2016
c9, t11-CLA Antioxidant Sheep milk cheese Lb. plantarum TAUL 1588 Linoleate isomerase Renes et al., 2019



Amino acids, α-keto acids, NADH, pyridoxal 5-phosphate, KG, erythromycin (ERY), lysozyme, diethyl ether, N-undecalactone, and tridecane were purchased from Sigma Chemical Co. (St. Louis, Mo.).

Bacterial strains and plasmids.

Lactococcus lactis D11 (8) and L. casei LC202 were obtained from Rhodia, Inc. (Madison, Wis.), L. casei ATCC 334 was obtained from the American Type Culture Collection (Manassas, Va.), and Escherichia coli SURE was obtained from Promega Corp. (Madison, Wis.). Stocks of each culture were maintained at �ଌ, and working samples were prepared from frozen stocks by two transfers in appropriate broth medium. Lactococcus lactis D11 was propagated at 30ଌ in sterile reconstituted skim milk, while lactobacilli were grown at 37ଌ in MRS broth (Difco, Detriot, Mich.). E. coli was grown at 37ଌ in Luria-Bertani broth (30) with shaking. Plasmid pTRKH2 (29) was obtained from T. R. Klaenhammer of North Carolina State University, Raleigh.

Isogenic strain construction.

The gene encoding L. casei LC202 d -Hic (dhic) was isolated by PCR and cloned into the high-copy-number vector pTRKH2. Amplification was performed with Expand High Fidelity DNA polymerase (Roche Diagnostics, Indianapolis, Ind.), using 31-mer forward (5′-AAGCACTCGAGATACCGGTGACTTACCATGG-3′) and reverse (5′-CGTTATCTGCAGATTGCCGTCTCCTTGTTCG-3′) oligonucleotide primers designed from the L. casei dhic sequence (24) and concatenated with XhoI and PstI linkers, respectively. Template DNA for PCR was isolated as previously described (25), and then amplification of a 1.5-kbp DNA fragment encoding dhic was performed in a Hybaid Thermal Reactor (National Labnet Co., Woodbridge, N.J.) programmed for 35 cycles of 92ଌ for 30 s, 55ଌ for 30 s, and 68ଌ for 180 s. The amplicon was cut with XhoI and PstI, ligated into XhoI and PstI double-digested pTRKH2, and then transformed into E. coli SURE by electroporation using standard laboratory methods (30). Transformants were selected on Luria-Bertani agar that contained 500 μg of ERY per ml, plasmid DNA was isolated from Ery r CFU by the alkaline lysis method (30), and the presence of dhic insert DNA was confirmed by agarose gel electrophoresis and DNA sequence analysis. The pTRKH2:dhic plasmid construct from a representative clone was selected for further work and designated pHADH.

Transformation of L. casei ATCC 334 was performed essentially as described by Ahrne et al. (2). Briefly, stationary-phase cells were inoculated at 2% into 500 ml of MRS (Difco) broth and incubated at 37ଌ until the suspension reached an A600 of 0.8. The cells were harvested by centrifugation at 5,000 × g, washed twice with sterile, distilled water, and suspended in 2.5 ml of ice-cold, sterile 30% polyethylene glycol 1450 (Sigma Chemical Co.). Three microliters of pHADH or pTRKH2 was mixed with 100 μl of cell suspension in a 0.2-cm electroporation cuvette and placed on ice for 3 min. An electric pulse was delivered in a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set to the following parameters: 2.5 kV, 25 㯏, and 200 Ω. After electroporation, 0.9 ml of warmed (37ଌ) MRS broth was added, and the cells were incubated at 37ଌ for 2 h. Transformants were collected on MRS agar that contained 5 μg of ERY per ml, and then cell lysates were prepared by the method of Anderson and McKay (3) and uptake of pTRKH2 or pHADH was confirmed by agarose gel electrophoresis. Representative isolates of L. casei ATCC 334 transformed with pTRKH2 or pHADH were selected for further work and designated L. casei 334e and the L. casei HADH strain, respectively.

D -Hic activity.

The d -Hic activity in cell lysates from L. casei 334e and the L. casei HADH strain was measured spectrophotometrically as previously described (20) with phenylpyruvic acid (PPA), indole pyruvic acid, p-hydroxyphenylpyruvic acid, and 2-ketoisocaproate as substrates. Specific activity was expressed as micromoles of NADH consumed per milligram of protein per minute, and the values reported represent the means from duplicate experiments replicated on two separate days.

Cheese manufacture.

Frozen cell preparations of L. casei 334e and the L. casei HADH strain (approximately 10 8 CFU per ml after thawing) were prepared by Rhodia, Inc., and then duplicate vats of 50% reduced-fat Cheddar cheese were manufactured on the same day and from the same milk supply at the University of Wisconsin—Madison from 250-kg lots of pasteurized milk (1.3% fat) as described previously (8). Cheese was made with three different starter culture blends: 1.5% (wt/wt) Lactococcus lactis D11 bulk starter grown overnight in skim milk without pH control (pH 𢏄.6), 1.5% D11 starter plus 25 ml of L. casei 334e cell preparation, and 1.5% D11 starter plus 25 ml of L. casei strain HADH cell preparation. After milling, one half of the curd from each vat was dry salted with 2.8% sodium chloride (wt/wt), while the other half was salted with 2.8% NaCl plus 2% (wt/wt) KG. The cheeses were hooped into 9-kg blocks, pressed overnight, and then vacuum packaged and ripened at 7ଌ.

Samples of each cheese (approximately 20 g) were collected at monthly intervals for enumeration of starter and nonstarter bacteria as described previously (8). Enumeration of the L. casei 334e and HADH strains in cheese was performed by incubation at 37ଌ on MRS agar that contained 5 μg of ERY per ml.

Cheese volatile analysis.

Investigation of cheese volatile compounds was performed using gas chromatography and mass spectrometry (GC-MS) by the method of Colchin et al. (10). Approximately 100 g of each sample was collected after 3 months of ripening and stored in glass jars at �ଌ until needed. Samples for GC-MS were prepared from 10 g of shredded cheese mixed with 40 ml of distilled water. N-Undecalactone and tridecane were added at 1 μg per g of cheese as internal standards, and cheese extracts were purged with nitrogen gas at a rate of 800 ml per min for 40 min in a circulating water bath (35 ± 1ଌ). Adsorbent traps (ORBO-100 Supelco, Bellefonte, Pa.) used during the sample purge were subsequently eluted with distilled diethyl ether. The first 2 ml of solvent eluate was collected and concentrated under nitrogen to approximately 100 μl for sample injection. Separation of volatile compounds collected from cheese samples was achieved using a Hewlett-Packard (Avondale, Pa.) model 6890 gas chromatograph equipped with a 60-m by 0.25-mm (inside diameter) capillary StabilWax DA column (Restek, Bellefonte, Pa.) with a 0.5-μm film thickness. The chromatography parameters included an initial temperature of 40ଌ for the first 4 min, which was increased at a rate of 7ଌ per min to a final temperature of 220ଌ. A column flow rate of 1.5 ml per min was maintained following injection of a 2-μl sample.

MS (Hewlett-Packard 5973 series) of cheese volatiles was performed in electron impact ionization mode with an ion source temperature of 230ଌ, ionization voltage of 70 eV, and mass-scan range between 29 and 400 m/z. Identities and quantities of volatile compounds were determined by internal-standard corrected integration responses of known standards and by comparison of mass spectra against those of a standard database (database mass spectral library, version 1.6d National Institute of Standards and Technology, Gaithersburg, Md).

Sensory evaluation.

Sensory attributes of 3-month-old cheeses were evaluated in duplicate (2 samples from each treatment × 2 evaluations per sample) by judges with more than 150 h of individual training in descriptive sensory analysis of cheese. Cheeses were evaluated for 16 flavor attributes defined by a descriptive sensory language for Cheddar cheese flavor (13, 14).


The effects of culture treatment or addition of KG on cheese volatiles and sensory character were evaluated by statistical analysis of variance (ANOVA) with SAS software (SAS Institute, Inc., Cary, N.C.) using standardized peak areas from GC-MS data. When treatment effects were significant, least-significant-difference pairwise comparison tests were performed to identify the treatment that produced the effect. Some data were subject to nonlinear log transformations to normalize data and meet assumptions of homogenous variance.

Chapter 9. Fermentation as a Method for Food Preservation

this chapter, particular emphasis is placed on how these changes are also beneficial in terms of extending the shelf life of the product.

Humans are unable to survive without food and drink therefore, the supply of these essentials has had a major

impact on the development of the human species and continues to do so even today. The rapidly increasing

world population necessitates that the amount of food wasted due to spoilage is kept to a minimum. Food

production is only one part of the process to ensure continuous, diverse, safe, food supplies to meet the consumer demands. Food must also be stored and preserved to achieve this objective. The requirement to store

and preserve foods has long been recognized, from the time well before there was any knowledge of microbiology. Fermentation, along with salting, cooking, smoking, and sun drying, is one of the earliest ancient traditions developed by cultures all around the world to extend the possible storage time of foods. Before the

initiation of preservation technology, humans frequently had to choose between starvation and eating spoilt

foods and then suffer the possible consequences of this. For thousands of years, raw animal and plant ingredients have been fermented. Fermented fruits were probably among the first fermented foods eaten [1,2]. The

methods for fermentations were developed by trial and error and from the experiences of many generations.

A selection of the most common fermented foods that have wide geographical distributions are shown in

Table 9.1. The key type of microorganisms associated with these foods are also included.

Examples of the More Common Fermented Foods

Note: LAB, lactic acid bacteria.

Fermentation as a Method for Food Preservation

Fermentation as a Preservation Method

As new preservation techniques have been developed, the importance of fermentation processes for food

preservation has declined. Yet fermentation can be effective at extending the shelf life of foods and can often

be carried out with relatively inexpensive, basic equipment. Therefore, it remains a very appropriate method

for use in developing countries and rural communities with limited facilities. In addition, the nondependence of fermentation on the use of chemical additives to the food appeals to the “more aware” consumer

market. The chemical composition of most foods is relatively stable therefore, generally preservation is

based on eliminating microorganisms or controlling their growth and the overall composition of the

microflora. To reduce or prevent microbial spoilage of food, four basic principles can be applied:

1. Minimize the level of microbial contamination onto the food, particularly from “high-risk”

2. Inhibit the growth of the contaminating microflora

3. Kill the contaminating microorganisms

4. Remove the contaminating microorganisms

Fermentations use a combination of the first three principles. Fermentations should not be expected to sterilize substandard raw products, but rather should use high-quality substrates. Microorganisms can improve

their own competitiveness by changing the environment so that it becomes inhibitory or lethal to other organisms while stimulating their own growth, and this selection is the basis for preservation by fermentation. A

number of different bacteriocidal and bacteriostatic factors that can be produced by lactic acid bacteria (LAB)

are shown in Table 9.2. Fermentation improves the safety of foods by decreasing the risks of pathogens and

toxins achieving the infective or toxigenic level, and extends the shelf life by inhibiting the growth of spoilage

agents, which cause the sensory changes that make the food unacceptable to the consumer.

Microbial Contamination of Foods

Foods are derived from other living organisms and during their development and preparation they are

continuously exposed to microbial contamination. The resultant contaminating microflora can have different effects on the food. These include negative effects such as spoilage, where the food becomes unfit

for human consumption or health risks when infectious or toxigenic microorganisms are present.

Negligible effects on the food occur when the microflora does not cause disease or any detectable

changes in the food. However, benefits can also be reaped from the action of the microorganisms when

their activity brings about improvements in the appeal of the food. In developed countries, the improved

appeal is the major reason for microbial

fermentations of foods continuing today.

The nutrient content and intrinsic

properties of many raw foods make them

Factors Produced by the Metabolic Activity of

ideal environments for microbial replicaMicroorganisms That Can Contribute to the Increased

tion. The rate at which the microorganisms

Stability and Safety of Fermented Foods

grow depends not only on the intrinsic

properties of the food (pH, redox potential,

Organic acids, e.g., lactic acid, acetic acid, and formic acid

water activity, etc.) but also on the condiLow redox potential

tions under which it is being stored, the

Accumulation of inhibitors, e.g., toxins, bacteriocins [117],

extrinsic factors, for example, temperature.

antibiotics, lactococcins, nisin, natamycin, hydrogen peroxide

Therefore, many raw food types need to be

consumed soon after production to be of

high nutritional value. Without preservaCarbon dioxide

tion measures, delays lead to the nutrients

Source: Adams, M.R. and Moss, M.O., Food Microbiology, The

being degraded and utilized by the contaRoyal Society of Chemistry, Cambridge, UK, 2000.

Handbook of Food Preservation, Second Edition

Examples of Microbial Metabolic End Products

Potential Benefits of Fermented Foods

Levels of antinutritional

A major consideration needs to be that under ideal conditions microorganisms can grow very rapidly,

being able to double in number in a short period of time. It must also be noted that there is a variation in

the optimum environmental conditions for different types and species of microorganisms, for example,

microorganisms can be categorized into broad groups such as aerobes and anaerobes depending on their

tolerance and use of oxygen and psychrophiles, mesophiles, and thermophiles based on the temperature

range optimum for their growth. In addition, the biochemical activity of different microorganisms varies

and may change in response to fluctuations in environmental factors, leading to a range of metabolic end

products (Table 9.3). By manipulating the environmental conditions, it is possible to select for specific

kinds of microorganisms that impart a particular taste, odor, texture, or appearance to the food. This is the

Benefits of Fermented Foods

Microorganisms per se can be used as food sources, but in many instances it is their effects on other

food sources that are of major interest. The acceptability of a food to the consumer is based mainly on

its sensory properties. The sought-after sensory properties of fermented foods are brought about by the

biochemical activity of microorganisms. Fermented foods were developed simultaneously by many cultures for two main reasons: (i) to preserve harvested or slaughtered products, which were abundant at

certain times and scarce at others and (ii) to improve the sensory properties of an abundant or unappealing produce [1,3].

However, a range of benefits can be obtained from food fermentations, some of which are shown in

Table 9.4. Consequently, fermented foods and drinks still retain an important role in the human diet.

Fermentation has low energy demands and can often be carried out without sophisticated technology and

designated plants. The simple techniques mean that the procedures can often be carried out in the home

[4]. Also, a number of studies have shown that consumers regard fermented food products as healthy and

natural, increasing consumer demands and their profitability [5].

Microorganisms Used in Food Fermentations

A variety of groups of microorganisms are frequently used in fermented foods. The principal groups are

LAB perform an essential role in the preservation and production of wholesome foods. Examples of lactic acid fermentations include (a) fermented vegetables such as sauerkraut, pickled cucumbers, radishes,

carrots, and olives (b) fermented milks such as yogurt, kefir, and cheeses (c) fermented/leavened breads

such as sourdough breads and (d) fermented sausages (Table 9.1). LAB have been grouped together as

Fermentation as a Method for Food Preservation

Principal Groups of Microorganisms Used for

Characteristics Common to Lactic Acid Bacteria

Fermentative anaerobes that are aerotolerant

Produce most of their cellular energy from the fermentation

Produce lactic acid from hexoses

Genera of Lactic Acid Bacteria Commonly Used in Food Fermentations

Oval cocci—pairs or chains

Oval cocci—pairs or chains

Cocci—single, pairs, or short chains

Source: Modified from Axelsson, L. in Lactic Acid Bacteria: Microbiology and Functional Aspects, Marcel Dekker, New

York, 1998, 1–72 Adams, M.R. and Moss, M.O., Food Microbiology, The Royal Society of Chemistry, Cambridge, UK, 2000.

they possess a range of common properties (Table 9.6), and all produce lactic acid that can kill or inhibit

many other microorganisms [6]. The primary use of lactic acid in the food industry is as a preservative,

an acidulant, or a dough conditioner. The principal genera of LAB are shown in Table 9.7. In general,

excluding some streptococci, they are harmless to humans. This makes LAB ideal agents for food preservation. LAB are subdivided based on their products from glucose fermentation. Homofermenters produce lactic acid as the major or sole product from glucose, while heterofermenters produce equimolar

amounts of lactate, carbon dioxide, and ethanol. Heterofermenters have an important role in producing

aroma components such as acetaldehydes and diacetyl. LAB have a range of methods for outcompeting

other microorganisms (Table 9.2). Their most effective mechanism is to grow readily in most foods, producing acid, which lowers the pH rapidly to a point where other competing organisms can no longer grow

[3]. Lactobacilli also have the ability to produce hydrogen peroxide [7], which is inhibitory to spoilage

organisms [3], while lactobacilli are relatively resistant to hydrogen peroxide [8]. The role of hydrogen

peroxide as a preservative is likely to be minor, especially when compared with acid production. Carbon

dioxide produced by heterofermenters also has a preservative effect, resulting partially from its contribution to anaerobiosis [3].

Consumers are taking a greater interest in the quality of foods and are creating a demand for chemicalfree, “natural health” foods. This has stimulated extensive research into the applications of LAB for both

the control of pathogenic and spoilage microorganisms and also for health promotion. A range of potential

health benefits has been associated with the consumption of LAB. Some benefits are as a consequence of

their growth and activity during food fermentations, and some from the resultant colonization of the gastrointestinal tract (Table 9.8). Many of these health claims are still controversial [9] and are the subject of

research to identify and substantiate specific roles [9–11].

A second group of bacteria with importance in food fermentations are the acetic acid producers. Acetic

acid is one of the oldest chemicals known it is named after the Latin word for vinegar “acetum.”

Handbook of Food Preservation, Second Edition

The acetic acid bacteria are acid tolerant, grow well at pH levels

below pH 5.0, are Gram-negative, motile rods, and are obligate

aerobes. They derive energy from the oxidation of ethanol to

acetic acid following the reaction shown below.

They are found in nature where ethanol is produced from the

fermentation of carbohydrates by yeasts, such as in plant nectars

and damaged fruits. Other good sources are alcoholic beverages

like fresh cider and unpasteurized beer. In liquids, they grow as a

surface film because of their demand for oxygen.

The acetic acid bacteria consist of two genera, Acetobacter

and Gluconobacter. Acetobacter can eventually oxidize acetic

acid to carbon dioxide and water using Krebs cycle enzymes

referred to as overoxidation. This is not the case with

Gluconobacter. The most desirable action of acetic acid bacteria

is in the production of vinegar. The same reaction can also occur

in wines, when oxygen is available, and here the oxidation of

alcohol to acetic acid is an undesirable change, giving the wine

Potential Health Benefits from Lactic

Improved nutritional value, e.g., production

of vitamins or essential amino acids

Reduced toxicity, e.g., by degradation of

Increased digestibility and assimilability

Control of intestinal infections

Improved digestion of lactose

Inhibition of tumor growth

Lowering of serum cholesterol levels

Source: Drouault, S. and Corthier, G., Vet. Res.,

Yeasts are widely distributed in natural habitats that are nutritionally rich and high in carbohydrates, such

as fruits and plant nectars [12]. Yeasts are rarely toxic or pathogenic and are generally acceptable to consumers [13]. After extensive study, yeasts have been classified into about 500 species [14]. However, only

a small number are regularly used to make alcoholic beverages [12]. Saccharomyces cerevisiae is the most

frequently used and many variants are available. Saccharomyces cerevisiae ferments glucose but does not

ferment lactose or starch directly. Yeasts are used to produce ethanol, CO2, flavor, and aroma. The reaction

can be represented by the following equation:

ethyl alcohol and carbon dioxide

Other metabolic products include minor amounts of ethyl acetate, fusel alcohols (pentanol, isopentanol, and

isobutanol), sulfur compounds, and leakage of amino acids and nucleotides that can all contribute to the sensory changes induced by yeasts [13].

The majority of fungal species have filamentous hyphae and are referred to as molds. They are grouped

into four main classes based on the physiology and production methods of their spores. Molds

are aerobic and have the greatest array of enzymes. Some molds are used in the food industry to produce

specific enzymes such as amylases for use in bread making. They are relatively tolerant to extreme

environments and are able to colonize and grow on most foods. Molds are important to the food industry, both as spoilers and preservers of foods and in particular in fermentations for flavor development.

Certain molds produce antibiotics [15,16], while mycotoxin production by others is an emerging cause

of concern in the food industry.

The Aspergillus species are often responsible for undesirable changes in foods, although some species

such as A. oryzae are used in fermentations of soybeans to make miso and soy sauce. Mucor and

Rhizopus are also used in some traditional food fermentations. Rhizopus oligosporus is considered essential in the production of tempeh from soybeans. Molds from the genus Penicillium are associated with

the ripening and distinctive flavor of cheeses. For example, during ripening of Roquefort and blue

cheeses, P. roqueforti is grown in air veins throughout the curd, and the distinctive flavors develop as the

milk lipids are broken down into methyl ethyl ketone and proteins are structurally altered.

Fermentation as a Method for Food Preservation

Fermented foods may be produced by the action of fermentative microorganisms naturally found on

the raw materials or in the production environment. However, to improve reliability “starter cultures”

are frequently used. Starter cultures may be pure or mixed cultures. Using mixed starter cultures can

reduce the risks of bacteriophage infection [17] and improve the quality of the foods when the organisms are mutually beneficial. Food fermentations frequently involve a complex succession of microorganisms induced by dynamic environmental conditions. Fermentative microorganisms must be safe to

eat even in high numbers and must produce substantial amounts of the desired end product(s). For

practical reasons, the organisms should be easy to handle and should grow well, enabling them to outcompete undesirable microorganisms. The organism also needs to be genetically stable with consistent

performance both during and between food batches. In many traditional fermentations, the natural

microflora were used for the fermentation. Even so, some form of inoculation was frequently performed using simple techniques such as the use of one batch of food to inoculate the next batch, or the

repeated use of the same container [18]. Natural fermentations have a degree of unpredictability,

which may be unsatisfactory when a process is industrialized. Starter cultures are increasingly used to

improve not only the reliability, but also the reproducibility and the rate at which the fermentation is

initiated. Failed, poor-quality, or unsafe products lead to loss of customers and revenue, therefore their

incidence must be minimized.

The composition of starter cultures is based on knowledge of food-grade microbial genetics [19,20],

metabolism, and physiology as well as their interactions with foods [20]. Starter cultures are now developed mainly by design rather than by screening [21,22]. The overall objective is to exploit the properties

of the starter cultures to ensure reproducible standards of safety and quality [23].

Classification of Fermented Products

Fermented foods are classified in a number of different ways. They may be grouped based on the microorganisms, the biochemistry, or on the product type [24]. Campbell-Platt (1987) identified seven groups for

classification, namely, (1) beverages, (2) cereal products, (3) dairy products, (4) fish products, (5) fruit and

vegetable products, (6) legumes, and (7) meat products [25], whereas Steinkraus (1997) classified

fermentations according to the type of fermentation, for example, alcoholic wines and beers, and alkaline

Nigerian dawadawa [26]. In this chapter, the fermentations are grouped in terms of the biochemical products used to transform the food, for example, production of lactic acid, acetic acid, ethanol, and CO2.

Throughout history alcoholic beverages have had a place in most cultures. They require the alcoholic

fermentation of fruits or other high-sugar materials by yeasts. The alcohol content of the beverage acts

as a preservative and many of these products have long shelf lives. Over the years, brewing yeasts have

evolved by selection and mutations, and have been developed by genetic engineering. Major advances

have been made in improving the characteristics of the fermentation strains driven by the high revenue

associated with the alcoholic beverage industry.

Beer is produced by the fermentation of partially germinated cereal grains, referred to as malt, by yeasts.

Beers have a final ethanol content of about 3%–8% a huge variety of beers exists and they include ales,

lagers, and stouts. Both lagers and ales can be either light or dark in appearance. Ale is produced using

Saccharomyces cerevisiae, a top fermenter yeast, whereas lagers are produced using pure cultures of

Saccharomyces carlsbergensi, a bottom fermenter yeast. Ales are produced using warm fermentation

temperatures, 12°C–18°C and lager fermentation temperature is generally cold, 8°C–12°C [12]. Most

beer produced is of the lager variety.

Handbook of Food Preservation, Second Edition

Several steps are needed to make beer. First, the barley is soaked in water for 5–7 days to make malt

[27,28]. During this step, the grains partially germinate and produce enzymes, mainly amylases and proteases that are essential to the brewing process. Amylases degrade starch to glucose, a sugar needed for

the yeast fermentation, and proteases solubilize compounds in the grain and hops, which is important for

the quality of beer. Following germination, heat is applied to stop further sprouting and to dry the grain.

To develop color and aroma, the malt is roasted for 4–5 h at a temperature of 80°C–105°C. Maillard reactions are responsible for the color and aroma formation during kilning. The dried and crushed malt is suspended in water and mixed with boiled malt adjuncts, such as ground rice and corn. Amylase is generally

added at this stage to assure complete hydrolysis of starch. The mash is then incubated at 65°C–70°C for

a short time to allow the amylase to degrade the starch to glucose. The temperature is subsequently raised

to 75°C to inactivate the enzymes and the medium is allowed to settle. Insoluble matter sinks to the

bottom and serves as a filter as the liquid, called wort, is taken from the container. Hops or hop extracts

are then added to the wort. Hops are an indispensable ingredient as they act as a clarifier causing protein

to precipitate they give a specific aroma and bitter taste. Hops also possess antibiotic properties and

together with ethanol and carbon dioxide contribute to the stability of beer [29–31]. In addition, the

protein content of hops enhances the foam-building ability of beer. The mixture is boiled for 1.5–2.5 h

to obtain the correct delicate hop flavor [32]. The wort/hops mixture is then boiled to concentrate the

wort, kill many spoilage microorganisms, inactivate enzymes in the mash, and solubilize important compounds in the hops and mash. The wort is then separated, cooled, and fermented.

Fermentation is initiated by adding the appropriate yeast to the wort. Ale fermentation is completed

when the pH is lowered to around 3.8, generally in 7–12 days lager with pH values of 4.1–4.2 is

completed in 5–7 days [33]. During fermentation, the glucose in wort is converted into ethanol and CO2.

The fermented wort is then aged at 0°C for a period of weeks or months. During this period, the yeast

settle to the bottom of the vessel, bitter flavors are mellowed, and other compounds are formed that

enhance flavor. The beer is then filtered or centrifuged to remove yeast cells before packing and pasteurization. The beer is finished by addition of CO2 to a final content of 0.45%–0.52%. Finally, pasteurization of the beer at 60°C or higher may be carried out to destroy spoilage microorganisms [34].

There are a number of factors that protect beer from the growth of contaminating microorganisms.

These include low pH, redox potential, and levels of readily available carbon sources, the isohumulones

of hops that inhibit Gram-positive bacteria and the alcohol produced by the yeast [35]. The spoilage of

beer is caused mainly by acetic acid bacteria, LAB, and wild yeasts. The industrial spoilage of beers is

commonly referred to as beer infections [34].

Wine can be produced from any fruit juice with sufficient levels of fermentable sugars, in most cases

wine is a beverage obtained by full or partial alcoholic fermentation of fresh, crushed grapes or grape

juice (must), with an aging process. Wine-type grapes from cultivars of Vitis vinifera vines are most commonly used to produce wines [29]. Wine making involves a series of steps. First, grape clusters are

cleaned of rotten and dried berries and then separated from the stems. The grapes are subsequently

crushed and pressed to release juice, the must. The remaining grape skins and seeds, called pomace, are

then removed after a second press. In red wine making, the must is fermented together with the skin to

extract the red pigments from the skin, which are released only during fermentation. The extraction of

the red pigments is sometimes facilitated by raising the temperature to 50°C prior to fermentation of the

mash, or to 30°C after the main fermentation, followed by a short additional fermentation.

The fresh sweet must is treated with sulfur dioxide to suppress the growth of undesirable microorganisms, and prevent enzymatic browning and oxidation thus stabilizing wine color. The must is then

inoculated with Saccharomyces cerevisiae var. ellipsoides or pastorianus and allowed to ferment for 3–5

days at temperatures between 21°C and 32°C. During this period, ethanol level may reach 14%–18%.

Fermentation of red wine is longer than that of white wine, until the correct amount of color is extracted

from the skin. The wine is racked to get rid of the sediments. The wine is drawn-off or decanted into barrels, vats, or tanks for aging, the length of which could vary between 3 and 9 months. During this stage,

the wine clears and develops flavors. The wine is then removed from vats and poured into bottles in

which aging continues [36]. Following the alcoholic fermentation, a malolactic fermentation can be

Fermentation as a Method for Food Preservation

initiated to reduce the acidity and mellow the wine. During the malolactic fermentation, malic acid is

degraded to lactic acid by many LAB, mainly of the genera Lactobacillus, Leuconostoc (L. oenos), and

Pediococcus (P. cerevisiae) [35]. Lactic acid is not as acidic as malic acid hence, the acidity of the wine

is reduced. Wine can be subjected to some microbial and chemical defects. Microbial spoilage can

be caused by molds [37], LAB [38], and acetic acid bacteria [39,40]. Chemical defects lead mainly to

the browning of wine as a result of oxidative reactions of phenolic compounds, which in red wines, may

result in complete flocculation of the color pigments [41].

The fermentations discussed above can only produce a maximum alcohol content of about 17%.

Concentrations in excess of this inhibit the metabolism of the yeasts. To obtain higher alcohol concentrations, the fermented product must be subsequently distilled. Whiskey, gin, vodka, rum, and liqueurs

are examples of distilled spirits. Although the process for producing most products of these types is quite

similar to that for beers, the content of alcohol in the final products is considerably higher.

Yogurt is a coagulated milk product obtained by lactic acid fermentation through the action of

Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus. Yogurt is prepared using either whole

or skim milk, where the nonfat milk solids are increased to 12%–15% by concentrating the milk, or adding

powdered skim milk or condensed milk. The concentrated milk is pasteurized at 82°C–93°C for 30–60

min and cooled to the starter incubation temperature of 40°C–45°C. Yogurt starter is then added at a level

of around 2% by volume and incubated for 3–5 h, or until the titratable acidity of the final product reaches

0.85%–0.90% or a pH of 4.4–4.6 [34]. The yogurt is then cooled to 5°C to inhibit further acid production.

The symbiotic growth of the two organisms of the yogurt starter culture has been reviewed by many

authors [42–44]. The symbiotic growth of the two organisms is better observed when they exist in a 1:1

ratio and this results in lactic acid production and acetaldehyde at a rate greater than that produced by

either when growing alone [42]. Streptococci produce lactic acid, formic acid, and carbon dioxide. Formic

acid stimulates the growth of lactobacilli. The lactobacilli liberate some amino acids needed for the growth

of the streptococci, and produce acetaldehyde and more lactic acid to bring the pH to 4.4–4.6.

Acetaldehyde is the compound that contributes mostly to the typical flavor of yogurt, while acetoin,

diacetyl, and ethanol are produced in lower concentrations [45]. Yogurt flavor continuously changes during manufacture and storage. Flavor changes may vary depending on the cultures, mix formulation, and

incubation and storage conditions [46]. Lactobacillus acidophilus may be added with yogurt culture to

reduce excessive aldehyde and for health benefits. The type of yogurt starter used can change the physical characteristics of the final yogurt product. For example, ropy cultures used to enhance the viscosity of

“stirred” types of yogurt comprise Streptococcus salivarius ssp. thermophilus, and Lactobacillus strains

[47]. “Nonropy” starters are used for the manufacture of “set” types of yogurt. Other ways to increase the

viscosity of yogurt and subsequently decrease the syneresis of the whey include the addition of stabilizers, increasing nonfat milk solids, extending the time, and increasing the temperature of pasteurization.

Cheese is a concentrated milk product obtained after coagulation and whey separation of milk, cream or

partially skimmed milk, buttermilk, or a mixture of these products. Cheese may be consumed fresh or

after ripening. Cheese is commonly made from cow, ewe, goat, or buffalo milk. The majority of cheeses

are made from pasteurized milk. The use of subpasteurization heat treatment of milk or thermization is

also practiced to limit heat-induced changes in milk without compromising microbiological safety.

There are over 400 varieties of cheeses representing fewer than 20 distinct types, and these are grouped

or classified according to texture or moisture content, whether ripened or unripened, and if ripened,

whether by bacteria or molds [34]. Table 9.9 shows the classification of cheeses according to their curing

Handbook of Food Preservation, Second Edition

Cheese Varieties and Their Classification

Principal Curing Characteristics and Examples

No curing—must be made from pasteurized milk

Cottage, Quark, Cream, Mozzarella

Ripened by bacteria and surface microorganisms

Limburger, Brick, Port du Salut

Ripened primarily by bacteria, without eyes

Provolone, Edam, Gouda, Cheddar, Parmesan,

Ripened primarily by bacteria, with eyes

Ripened principally by internal mold growth

Roquefort, Stilton, Gorgonzola, Cheshire, Danish Blue

characteristics. The majority of cheeses, with the exception of

Setting (curdling) the milk

whey cheeses, are made using variations of the same basic

process, as illustrated in Figure 9.1. Slight variations of these

and the use of different milks combine to generate the huge

range of cheeses available today.

In general, the process of manufacture starts with the prepaCooking the curds

ration of milk. Milk generally receives a treatment equivalent to

pasteurization at the start of the processing. The milk is then

Draining whey or dipping curds

cooled to the fermentation temperature, which depends on the

type of cheese to be manufactured, 29°C–31°C for Cheddar,

Stilton, Gouda, Camembert, and Leicester higher temperatures

are employed in the manufacture of high-scalded cheeses such

as Emmental, Gruyère, and Italian cheeses. Milk is inoculated

with an appropriate lactic starter. The starter culture produces Curd transformation (some varieties)

lactic acid, which, with added rennin, gives rise to curd formation. In addition, lactic acid is also responsible for the fresh

acidic flavor of unripened cheeses and plays a major role in the

suppression of pathogenic and some spoilage microorganisms

and in the production of volatile flavor compounds and the synthesis of lipolytic and proteolytic enzymes involved in the ripenFIGURE 9.1 Basic steps in cheese making.

ing process of cheese. The starter organisms most used for

cheese production are mesophilic starters, strains of Lactococcus

lactis and its subspecies. Thermophilic starters such as Lb. helveticus, Lb. casei, Lb. lactis, Lb. delbrueckii

subsp. bulgaricus, and Streptococcus thermophilus are used in the production of cheeses where a higher

incubation temperature is employed. Propionic bacteria, molds such as Penicillium camemberti, P. candidum, P. roqueforti and red- or yellow-smearing cultures such as Bacterium linens are also added, depending on the type of cheese to be manufactured. The time of renneting and the amount added differ with

cheese type. After coagulation of the milk, the curd is cut into small cubes for whey expulsion. The curd is

further shrunk by heating it and then pressed to expel more whey, followed by salting. Finally, the cheese

is ripened under conditions appropriate to the cheese in question.

Cheese ripening involves a complex series of chemical and biochemical reactions. Proteolysis and lipolysis are two primary processes in cheese ripening with a variety of chemical, physical, and microbiological

Fermentation as a Method for Food Preservation

changes occurring under controlled environmental conditions [48,49]. These reactions are of importance to

the flavor and texture development in cheeses [50–52]. Flavor compounds include peptides and amino acids,

free fatty acids, methyl ketones, alkanes, lactones, and aliphatic and aromatic esters.

Although most ripened cheeses are the products of metabolic activities of LAB, several known cheeses

owe their particular character to other related organisms. In the case of Swiss cheese, Propionibacterium

shermanii is added to the lactic bacteria Lb. bulgaricus and Streptococcus thermophilus. Propionibacteria

contribute to the typical flavor and texture of Swiss-type cheese [53]. The lipolytic and proteolytic activities of molds play an important role in the maturation of some cheeses. In blue cheese such as Roquefort

and Stilton, Penicillium roqueforti grows throughout the cheese and imparts the blue-veined appearance

characteristic of this type of cheese. Penicillium camemberti is associated with surface-ripened soft

cheeses such as Camembert and Brie.

A large number of vegetables are preserved by lactic acid fermentation around the world. The most

important commercially fermented vegetables in the west are cabbage (sauerkraut), cucumbers, and

olives. Others include carrots, cauliflower, celery, okra, onions, and peppers. Typically, these fermentations do not involve the use of starter cultures and rely on the natural flora. Brine solutions are prepared in the fermentation of sauerkraut, pickles, and olives. The concentration of salt in the brine

ranges from 2.25% for sauerkraut to 10% for olives. The fermentation yields lactic acid as the major

product. The salt extracts liquid from the vegetable, which serves as a substrate for the growth of LAB.

Growth of undesirable spoilage microorganisms is restricted by the salt. Aerobic conditions should be

maintained during fermentation to allow naturally occurring microorganisms to grow and produce

enough lactic acid, and to prevent growth of spoilage microorganisms. Olives receive a special treatment before brining in that green olives are treated with a 1.25%–2% lye solution (sodium hydroxide),

usually at 21°C–25°C for 4–7 h. This treatment is necessary to remove some of the oleuropein, a bitter compound in olives. In some countries, the fermentation of cucumbers is controlled by the addition of acetic acid to prevent growth of spoilage microorganisms, buffered with sodium acetate or

sodium hydroxide, and inoculated with Lb. plantarum alone or in association with Pediococcus cerevisiae. The controlled fermentation reduces economic losses and leads to a more uniform product over

a shorter period of time. Many researches have shown a sequential involvement for different species

of LAB [1,54–56]. For sauerkraut production, Leuconostoc mesenteroides grows first, producing lactic acid, acetic acid, and CO2, followed by Lb. brevis and finally Lb. plantarum grows producing more

acid and lowering the pH to below 4.0, allowing the cabbage to be preserved for long periods of time

under anaerobic conditions. The LAB chiefly responsible for production of high-salt pickles are initially Pediococcus cerevisiae followed by the more acid-tolerant Lb. plantarum and Lb. brevis.

Leuconostoc mesenteroides makes little contribution in the high-salt pickles but is active in the lowsalt pickles [57]. The microbiology of the olive lactic acid fermentation is complex with a number of

microbial strains being involved. Vaughn et al., [58] have divided the normal olive fermentation into

three stages. The initial stage is the most important from the standpoint of potential spoilage if the

brines are not acidified. Acidification eliminates the original contaminating population of dangerous

Gram-negative and Gram-positive spoilage bacteria and, at the same time, provides an optimum pH

for activity of LAB [59]. The natural flora of green olives, consisting of a variety of bacteria, yeasts,

and molds, carries out the fermentation with LAB becoming prominent during the intermediate stage.

Leuconostoc mesenteroides and Pediococcus cerevisiae are the first lactics to predominate, followed

by lactobacilli, mainly Lb. plantarum and Lb. brevis [60].

Fermented Animal Products

The primary reason for developing methods to ferment meats and fish was to extend the shelf life of these

highly prized, perishable foods. Gram-positive micrococci have an important role in these fermentations

[61]. Several products became popular, including fermented sausages, fish sauces, and fish pastes. Many

of the traditional fermentation methods are still used although the primary reason for their use is no

longer preservation, but because the products are popular for their enhanced flavors.

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Yeast, Enzymes and Yoghurt NEET Notes | EduRev



"Biotechnology may be defined as use of micro-organism, animals, or plant cells or their products to generate different products at industrial scale and services useful to human beings."

A powerful industry based on microbes has been developed in recent time. A careful selection of microbial strains, improved method of extraction and purification of the product, have resulted in enormous yields.

The use of living organisms in systems or process for the manufacturer of useful products, It may involve algae, bacteria, fungi, yeast, cells of Higher plants & animals or subsystems of any of these or Isolated components from living matter.

Old biotechnology are based on the natural capabilities of micro organisms. e.g. formation of Citric acid, production of penicillin by Penicillium notatum New biotechnology is based on Recombinant DNA technology. e.g. Human gene producing Insulin has been transferred and expressed in bacteria like E.coli.

In,modern biotechnology, different types of valuable products are produced with help of microbiology, biochemistry, tissue culture, chemical engineering and genetic engineering, molecular biology and immunology.


A common example is the production of curd from milk. Micro-organisms such as Lactobacillus and others commonly called lactic acid bacteria (LAB) grow in milk and convert it to curd. During growth, the LAB produce acids that coagulate and partially digest the milk proteins. A small amount of curd added to the fresh milk as inoculum or starter contain millions of LAB, which at suitable temperatures multiply, thus converting milk to curd, which also improves its nutritional quality by increasing vitamin B12. In our stomach too, the LAB play very beneficial role in checking disease causing microbes.

The dough , which is used for making foods such as dosa and idli is also fermented by bacteria. The puffed-up appearance of dough is due to the production of CO2 gas. Similarly the dough, which is used for making bread, is fermented using baker's yeast (Saccharomyces cerevisiae). A number of traditional drinks (e.g. ''Todi' prepared from sap of palms) and foods are also made by fermentation by the microbes.Microbes are also used to ferment fish, soyabean and bamboo shoots to make foods. Cheese, is one of the oldest food items in which microbes were used. Different varieties of cheese are known by their characteristic textur flavour and taste, the specificity coming from the microbes used. For example, th large holes in 'Swiss cheese' are due to production of a large amount of CO2 by a bacterium named Propionibacterium sharmanii. The 'Roquefort cheese' are ripened by growing a specific fungi on them, which gives them a particular flavour.

Louis Pasteur showed in the middle of nineteenth centuary that beer and butter milk are product of fermentation brought about by "yeast". It is a microscopic single celled organism –Saccharomyces cerevisiae.

Presently however yeast product for human and animal consumption are produced on commercial scale. "Alcoholwas the first product of ancient biotechnology"

There are basically two types of yeasts (i) Baker's yeast (ii) Alcohol yeast or Brewer's yeast Baker's yeast generally utilize during the preparation of food materials to increase the taste of food, flavour in food and nutrients in food. It is also utilized as "leavening agent".

By the incomplete degradation of complex organic compounds [sucrose] by yeast fermentation, alcohol is formed.

Some other common products of yeast fermentation are –

[i] Beer – It is produced from Hordeum Vulgare[Barely] malt and alcohol content is 4-8%

[ii] Wine – Produced from grapes, alcohol content is 10-20%.

[iii] Brandy – Produced by distilation of wine and alcohol content is 43-57%

[iv] Gin – Produced from European Rye-Scale cereal.

[v] Rum – Produced from Molasses of Sugarcane and alcohol contents is 40%

Note – Another yeast which supplies nutritional rich food for Man and animals is Torulopsis utilis.

Industrial utilization of biotechnology involve three steps –

[i] Laboratory scale process

[iii] Manufacturing unit The development from laboratory scale to manufacturing unit is "Scaling up to industrial production"

[i] Laboratory Scale – In this process for the production of desirable product, proper micro organism searched and then suitable strain is selected and multiplied. Proper medium also find out on which selected strain, produce best and more amount of product.

Many number of experiments performed in lab for the analysis and selection of strains and medium. All the equipment are utilized in lab i.e., glass apparatus. All the parameters of the process worked out and precaution are also not down for the smooth running of process such as – proper sterlization of nutrient and microbesstrain, required - pH, suitable aerator, disposal of CO2 if evovled, temperature, by product or product inhibition or stimulation, time of optimum production, separation of product and its purification etc. Ultimately, the laboratory scale process finalized and transfer at pilot plant scale.

[ii] Pilot plant Scale – It is the intermediate stage where working of laboratory scale process is tested. At this stage cost and quality of product throughly checked. Glass apparatus are replaced by stainless steel equipment/containers is called "bio reactor".

To produce in large quantities, the development of bioreactors. where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., useing microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).

The most commonly used bioreacters are of stirring type

A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.

The stirrer faciliates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling parts so that small volumes of the culture can be withdrawn periodically.

Micro organisms can be grown in bioreactors in two ways :

(a) Support growth system – In this method microorganisms are growing as a thin layer or film in the solid medium.

(b) Suspended growth system – By suspending cells or mycelia in the liquid medium is called suspended growth system.

[iii] Manufacturing unit – During the designing of bioreactor for the process often very large size so that it accomodate huge amount of medium.

Downstream Processing – After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product. The processes include separation and purification, which are collectively referred to as downstream processing. The product has to be formulated with suitable preservatives.

Such formulation has to undergo through clinical trials as in case of drugs. Strict quality control testing for each product is also required. The downstream processing quality control testing vary from product to product.

Some important biotechnological products which are produced with the help of organisms as follows –

Total known enzymes 2,200 and only 1–1.5% are used

(i) Rennet – Manufacturing "Cheese"

Old days cheese had been prepared either using the layer of stomach of Goat or Sheep OR the sap of Fig. tree, containing special enzyme–Ficin. In 1874 a Danish Chemist – Christian Hansen extracted pure rennet enzyme from Calf's stomach for industrial production of cheese. First of all diastase enzyme was identify by payen and persoz (1933) Cheese is mainly two different types.

I. Unripened cheese – Ripened from out side–soft

II. Ripened cheese– It is hard and ripened externally as well as internally.

Manufacturing cheese involve following steps.

(i) Milk is inoculated with starter culture of bacteria – Streptococcus lactis or S.cremoris and warmed at 380C. If higher temperature [500C or more] then S.thermophilus combined with Lactobaccilus lactis, L.bulgaricus or L.helveticus.

(ii) When a certain acidity reached in milk by the activity of species of bacteria then rennet enzyme is added. Curdling of milk occurs within half an hour to one hour.

(iii) The curd is removed and liquid separates out which is called whey [contain 93% water and 5% Lactose].

Lactose of whey is used for the manufacture of Lactic acid – First fermented acid.

If the cheese is used at this stage is calledcottage cheese(unripened stage).

(iv) The salts mixed with cottage cheese and put into the frames and pressed so as to allow removal of whey.

Salts hastens the removal moisture and prevent the growth of undesriable microbes. The frames are removed as soon as the cheese has set sufficiently to maintin its shape.

The ripening period varies from 1–16 months but which is very tasty and nutritious. This is hard and ripened cheese contains about 20-30% fats, 20-35% proteins and small amount of minerals and vitamins. [Cheese which prepared at homes with the help of lemon juice is called Raw cheese] Nearly 400 varieties of cheese available which can be classified into following type –

Type of Cheese

Micro Organisms used

Penicillium camemberti , Brevibacterium ,Streptococcus liquifaciens ,Brevibacterium

Ripend by action of microorganisms on the surface of curd

Combination of surface and interior growths

Propionibacterium sp Geotrichum

Inoculating the organisms throughout the curd

(ii) Proteases – This enzyme obtained from Aspergillus orizae and Bacillus subtilis, Bacillus licheniformis and utilized from the formation of detergents in detergent industry [For removing proteinous strains on clothes]. The bottlejuices are clarified by the use of pectinases and protease.

(iii) Amylases – It works on starch and used in Beer, Bread and Textiles industries.

(iv) Amylase, Gluco amylase and Gluco isomerase – By the action of all these enzymes corn (maize) starch transformed into fructose corn syrup. This syrup is more seeter than sucrose and used in beverage industry to flavoursoft drinks and in baking industry to sweeten biscuits and cakes.

(5) Tissue Plasminogen Activator [TPA] or Streptokinase – This enzyme utilized in medicinal field.

Streptokinase produced by the bacterium Streptococcus and modified by genetic engineering is used as a clot buster for removing clots from the blood vessels of patients who have undergone myocardial infraction leading to heart attack.

Uses Of Enzymes :

(1) Detergents (i) Proteases (ii) a -Amylase (iii) Cellulases (iv) Lipases

(2) Leather Industry

(3) Wool Industries

(4) Glucose from Cellulose

(5) Food, Dairy, Juice and Beverages Industries

(6) Production Of Glucose Syrup Bioactive molecule, cyclosporinA, that is used as an immunosuppressive agent in organ-transplant patients, is produced by the fungus Trichoderma polysporum.

Statins produced by the yeast Monascus purpureus have been commercialised as blood -cholesterol lowering agents. It acts by competitively inhibiting the enzyme responsible for synthesis of cholesterol.

Watch the video: Lactobacillus bulgaricus - Streptococcus thermophilus (May 2022).