What is Enzyme Modified Cheese?

Enzyme modified cheese or EMC as they are known are a cost effective alternative to natural cheeses. They are produced using enzymes on the cheese curds or immature cheeses to produce a more intense flavor profile as compared to that of natural cheeses. Hence they may be used at a lower level to impart the same flavor. However, EMC although having a strong flavor profile, do not mimic the textural properties of natural cheeses. EMC have approximately a 15-30 fold more intense flavor and are available as pastes or spray-dried powders. EMC find use in processed foods to give a cheesy flavor or to improve the flavor of a comparatively bland cheese product.

The cheese flavor is a result of the proteolytic, glycolytic and lipolytic pathways. While manufacturing EMC these pathways are only followed, the only difference being the use of enzymes rather than the entire culture micro-organism. The culture technique developed when a certain gentleman mixed curd slurry with NaCl in the ratio 2:1 and blended it. He then incorporated enzymes into it and kept the mixtures at 30 C for 4-5 days with constant agitation. A liquid cheese product with characteristic Cheddar, Brick or Romano flavor could be produced from fresh curd in 4-5 days. Nowadays, either of the following two approaches to manufacturing EMC may be taken. Either the hydrolysis of fat and protein occur simultaneously in one step or each of the hydrolysis is carried out separately and then the end products blended together to give the final EMC product.

EMC flavors available include Cheddar, Mozzarella, Romano, Feta, Parmesan, Blue, Gouda, Swiss, Colby and Brick. These cheese flavors find application in cheese analogues, chips, pasta products, salads, ready to eat foods, frozen foods, canned foods and low fat cheese spreads or cheese substitutes.


Reference:

Kieren N. Kilcawley, Martin G. Wilkinson, Patrick F. Fox, Enzyme Modified Cheese, International Dairy Journal, 8, 1998, 1-10.

Wheat Gluten

Gluten is a storage protein found in cereals like triticale, rye, barley; but perhaps the most well known is wheat gluten. Gluten is made up of the monomeric peptide gliadin and the polymeric peptide  glutenin. Some individuals suffer from Celiac disease- an allergic response to gluten. Hence, the advent of “gluten free” claims on many different food products. Wheat gluten is used in many different ways, for both, food and non-food uses.

Bakery: Perhaps the oldest and best known functional use of gluten is in bread making. Although insoluble in water, gluten can bind approximately twice its weight water giving rise to a hydrated viscoelastic mass. This property of gluten helps  hold together the dough used for making bakery products. In addition, owing to its elasticity, gluten can stretch and expand and help trap in the carbon dioxide bubbles generated during fermentation of bread.  

Meat analogues: When subjected to extrusion processing, gluten proteins align to form microfibrils that in turn form a macroscopic fibrous structure. These microfibrils upon hydration swell and give a fleshy appearance to the texturized wheat gluten. The viscoelastic properties of gluten also enable it to be moulded into a desired shape. This property has been harnessed in making meat or sea food replacements. Gluten may be processed and presented as  high value seafood like crab meat. The extrusion process works on gluten to give it the mouth feel and texture of meat.

Condiment: Although gluten is lacking in some essential amino acids like leucine and threonine, it has a high proportion of glutamine. By chemical methods such as deamidation, this glutamine can be converted to glutamic acid. Wheat gluten has thus been used to produce monosodium glutamate or a liquid similar to soy sauce.

Fortification and breakfast cereals: Wheat gluten is used as a low cost additive to fortify flours having low protein content. Even breakfast cereals make use of wheat gluten to increase their protein content and give a characteristic texture to the product. The most notable amongst these is the Kellog’s K cereal which employs wheat gluten as one of its ingredient. Due to its ability to bind water, gluten has been used as a binding agent for fruit purees used as fillings in nutritional fruit bars. It has also been used in producing synthetic cheese and as replacement for sodium caseinate in imitation cheese products.

Non-food uses: Gluten also has many non-food applications such as its use in adhesive bandages, biodegradable materials and edible coatings. Peptides from gluten have also found use in cosmetics. 

Reference:

Day et al, Wheat-gluten uses and Industry needs, Trends in Food Science & Technology, 17 (2006) 82–90

Randomly Amplified Polymorphic DNA

Randomly amplified polymorphic dna (RAPD) as the name suggests are the products of a PCR  reaction primed by arbitrarily selected primers.

How is it different from conventional PCR?

In conventional PCR, one designs the forward and reverse primers with the aim that they will anneal to the gene of interest. In order to accomplish this, it is necessary that one knows the sequence of the gene of interest. The next few steps follow a cycle wherein the primers first anneal, then a polymerase extends them and again the extended products are denatured making them ready for the next cycle of primer annealing.

In case of RAPD, the knowledge of the gene sequence is not a pre-requisite. One selects random primers which at low temperature display lower fidelity and hence anneal to many arbitrary sites on the DNA template with a variety of mismatches. After a few initial steps of annealing at lower temperature the amplification may then be carried out according to the conventional PCR or may also be continued even at the same temperature. When these amplified products are run on a gel, a pattern of bands is obtained which is unique for a particular species and is dependent upon the primers used.

Selection of primers

A single random primer will seldom be informative. When we use many such primers and then score them; will the RAPD profile make some sense in terms of polymorphism. Polymorphism maybe detected when the fingerprint of one sample shows an amplified band while the other sample does not even when the same primer is used with both the samples.

RAPD may be generated due to mutation in template (insertions/deletions) or presence of alleles in heterozygous individuals. However, these cannot be pinpointed as such because RAPD markers are dominant i.e they amplify many loci in a single go with a single primer.     

Some of the amplified bands are easily recognizable while some others may be difficult to interpret. The ambiguity arises due to the fact that every primer will possess different power to distinguish between different sites of amplification. Also there will exist competition between primers and different sites. Certain amplified fragments may interfere in the amplification or separation of other segments. Reproducibility of the RAPD pattern becomes problematic below a certain concentration of genomic DNA and may produce smears. On the other hand, there maybe poor resolution if the genomic concentration is very high                   

Reference:

Molecular Biomethods Handbook, second edition, John M Walker, Ralph Rapley,  chpt 10, pg 132-147                                                                                                                                               

What is Sitosterolemia?

Cholesterol synthesized by the human body plays an important role as part of the cell membrane. It is a type of sterol. However, plants do not synthesize cholesterol. Instead they have phytosterols of which sitosterol is a comparatively abundant phytosterol. When we eat plant derived foods, phytosterols are ingested into the body but they are not absorbed into the blood. Sitosterolemia is a rare recessive autosomal disease in which individuals absorb large quantities of plant sterols and this is stored in their blood and tissues. It is also referred to as phytosterolemia or plant sterol storage disease.

The clinical signs may include small yellowish outgrowths on various parts of the body, called xanthomas. These may also occur within the body such as in the tendons.  These are made up of accumulated lipids.  Joint stiffness and haemolytic anaemia may also be present.

The underlying cause of this genetic disorder is mutations in the genes ABCG5 or ABCG8. These genes code for a transport protein sterolin (sterolin 1 and sterolin 2 respectively) The sterolin 1 and sterolin 2 then form a heterodimer which acts as a transporter protein. Sterolin is the protein involved in transport of sterols out of the apical cells of the intestines. When there exists a mutation in any one of these genes, this transport protein malfunctions resulting in sitosterolemia. An interesting observation is that, mutations have always been seen in both the alleles of any 1 of the genes never in both of them together.

People diagnosed with sitosterolemia need to consume foods lower in plant and shellfish sterols. Patients may be given ezitimibe which acts as a sterol absorbtion inhibitor. If the individual is not responding to this therapy then use of cholestryramine and/or partial ileal bypass surgery may be recommended.

References:

http://ghr.nlm.nih.gov/condition/sitosterolemia

http://www.ncbi.nlm.nih.gov/books/NBK131810/

What is a Biofilm?

A biofilm can be said to be the community house of micro-organisms or maybe a microbial city. The term is generally used with respect to bacteria but biofilms can also be made my protozoans or yeast.Many free floating bacteria (planktonic forms) give up their nomadic existence and tend to aggregate in a place and live a sedentary lifestyle within a biofilm.

Biofilms can be formed on any surface as long as the bacteria sense that nutrients would be available to them. Initially it may attach to the surface with its pili. Later on, other bacteria may also come and attach themselves to the same surface. In this way a microcolony would be formed. Now as the number of inhabitants start increasing, the bacteria decide to fortify their dwelling place and hence secrete an exopolysaccharide (EPS). This exopolysaccharide builds up the matrix of the biofilm within which many bacteria may live.

A biofilm may comprise of the same species of bacteria or related species which may benefit each other in a symbiotic relationship. Also, the bacteria living together in this manner communicate with each other via quorum sensing. This involves small chemical messengers sent by one bacterium that affects the behaviour of the other bacteria around it. Also the bacteria utilize a different repertoire of genes when they are in a biofilm as compared to when they are free floating. Due to proximity there is also a high rate of horizontal gene transfer within the different species in a biofilm.

Since most biofilms are complex colonies made of many different micro-organisms even the structure of the polysaccharides that form the extracellular matrix of the biofilm tends to differ, sometimes even within the same biofilm. The biofilms maybe neutral in nature or polyanionic or polycationic. It has also been found that the polysaccharide which comprises the biofilm is very similar to what the planktonic cells secrete as an exopolysaccharide. Also, mutant cells which do not secrete EPS are also not capable of forming biofilms by themselves. However, in a mixed microbe setting this does not matter much because if one of the species cannot contribute to the matrix of the biofilm, the other species may do that work.

Living together in such a manner has its benefits. The EPS biofilm provides protection against antimicrobials. Due to this, biofilms are difficult to destroy by medicines.  Also, since the antimicrobials cannot reach them, the bacteria also tend to be more resistant and as a consequence, more virulent.
It also is a strong protective measure from adverse environmental conditions.

However, even though biofilm formation most of the times is undesirable, it does have its uses. Biofilms have been harnessed in waste water treatment and bio remediation.

References:

http://mic.sgmjournals.org/content/147/1/3.full.pdf

http://jb.asm.org/content/182/10/2675.full.pdf+html


More reading:
http://www.hypertextbookshop.com/biofilmbook/v004/r003/contents/chapters/chapter001/section005/green/page001.html

What is resistant starch?

Initially, when the word "resistant starch" was coined, it was for the purpose of naming that fraction of starch which did not undergo hydrolyzation with alpha amylase and pullulanase even after 20 min of incubation in vitro. However, now it is more commonly used to refer to that fraction of starch which cannot be digested by the small intestine and is hence not available for absorption by the body.

There exist 4 different types of resistant starch (RS) fractions.

RS1- These starches have a physical coat or covering and are hence protected from the attack of enzymes. Eg: Whole grains, seeds, legumes. These can be made available by processes that interfere with their protection, like milling( in case of grains). Chemically, it can be measured as the difference between the glucose released due to enzyme digestion when the food sample is homogenized and when it is not homogenized. Due to physical protection, these foods are generally heat stable.

RS2- These structures have a relatively compact granular form thus allowing little water or enzymes to penetrate their insides and therefore resistant to breakdown. Eg: raw potatoes, green bananas. However, food processing can soften these starches. Chemically, their content is calculated as the difference between the glucose released upon enzymatic digestion from a boiled, homogenized sample to that of a non boiled non homogenized sample.

RS3- It is the most resistant starch fraction and is not at all susceptible to digestion by pancreatic amylase. It is measured as that fraction which is dispersed by Potassium hydroxide and dimethyl sulfoxide.

RS4- This starch contains novel chemical bonds that are formed because of interaction between the food and the chemical agents or additives used in its processing.

Certain activities like autoclaving of the food, parboiling, baking may increase the RS content of certain foods. On the other hand, microwave cooking, germination of legumes, fermentation may help reduce the RS content.

RS has a small particle size, bland in taste and low water holding capacity. They can be used as a dietary fiber or added to other foods to improve its fiber content or lower its calorific value. It is digested over a long period of time and is thus beneficial to be used by diabetics. It can also function as a laxative.




Reference:

http://onlinelibrary.wiley.com/doi/10.1111/j.1541-4337.2006.tb00076.x/pdf

Why can Deinococcus radiodurans survive high radiation dose?

Deinococcus radiodurans is one of the bacterium that is resistant to ionizing radiation. To say, that they evolved this capacity by natural selection or mutation is difficult, because the background radiation on earth is very low as compared to what this organism can endure for it to have evolved in this manner. However, it has been observed that dessication also causes double stranded DNA breaks similar to that which occur after radiation exposure. Hence,  it can be rationalized that Deinococcus actually developed a mechanism to combat dessication or dehydration which is now being proved useful to combat high doses of radiation.

This is not the only bacterium that shows radiation resistance . There are some other bacteria (Kineococcus radiotolerans, Rubrobacter xylanophilus) as well as archae (Thermococcus gammatolerans) that show radiation resistance. However this particular guy has received much attention. How come these apparently unrelated species show resistance to high doses of radiation? There have been 2 postulates about this. One states that radiation resistance was a widespread phenomenon in the beginning of the world and slowly has been lost by the species. The other says it is a result of convergent evolution or horizontal gene transfer.

It has been found that Deinococcus radiotolerans can survive a radiation dose of 5000 Gy without any adverse effect. What helps it withstand this high stress level? The factors responsible for this ability as well as the exact mechanisms have still not been confirmed but the possibilities have been enumerated.

The Deinococcus has a segmented genome with two chromosomes 2.64 Mb (Chromosome I) and 0.41 Mb (Chromosome II). Apart from this it also has a 0.18 Mb megaplasmid and a 0.045 Mb plasmid. It has between 4 to 10 genome copies per cell depending upon the bacterial growth phase which are stacked one over the other . The more the number of genome copies, more is the resistance to radiation stress. More the number of genome copies means there is more probability of survival of crucial genetic information. This information from the surviving copies may then be used to repair the other damaged strands. The condensed genome is also said to play a part in radiation resistance. Since, it is tightly packed together, even though the strands may be fragmented they cannot diffuse away from each other and hence repair may become somewhat easier.

It has been experimentally found that in presence of a lot of Mn (II) the bacterium can withstand radiation stress better than when it is depleted of them. However in both the instances, the impact is the same, its only that the survival is better in presence of Mn (II). It is not that the bacterium does not undergo damage but it has a very efficient  and error free repair mechanism to repair the damage that has taken place. In fact, the cell growth is halted till all the damaged DNA is repaired and then only the bacterium continues with its life cycle.

 Also, new loci have been found in the genome suggesting newer enzymes or at least novel mechanisms of DNA repair that need to be further looked into. However a lot of work still needs to be carried out so that the mysteries of this "strange" bacterium are revealed.

Reference:
http://www.biochem.wisc.edu/faculty/cox/lab/pdfs/38.pdf

More reading:

http://www.genomenewsnetwork.org/articles/07_02/deinococcus.shtml