Download Lecture -18 Modification of food plant taste and appearance

Lecture -18
Modification of food plant taste and appearance
Preventing Discoloration
The post harvest discoloration of fruits and vegetables is a considerable problem
for the food industry. A lack of acceptance of discolored foods by consumers has been
dealt with by the food industry through the use of food additives in a wide range of foods.
However, recently, the safety of some of these food additives, in particular sulfites, has
been questioned.
The enzymes that are thought to be responsible for the initial step in the
discoloration of fruits and vegetables, the oxidation of monophenols and o- diphenols to
o-quinones are polyphenol oxidases. These nucleus-encoded enzymes with a molecular
weight of around 59,000 Daltons are localized in chloroplast and mitochondrial
membranes.
The contention that inhibition of the enzyme polyphenol oxidase would decrease
the extent of discoloration has been tested with transgenic potatoes carrying a number of
different polyphenol oxidase cDNA constructs. Vectors were constructed with either the
full-length or partial potato polyphenol oxidase cDNA in either the sense or antisense
orientation under the control of the cauliflower mosaic virus 35S promoter, the granulebound starch synthase promoter, or the patatin type I promoter (Fig. 1). The latter two
promoters are specific for the potato tuber. The two commercial varieties of potato that
were transformed with these constructs are considered to have a good level of resistance
to black spot (enzymatic discoloration), so an increase in black spot resistance by genetic
manipulation would be greater than what could be attained by traditional breeding
techniques Transgenic plants with the polyphenol oxidase cDNA constructs were
deliberately bruised, and then the extent of black spot damage was assessed. Most of the
transgenic potato plants with an antisense version of the polyphenol oxidase gene under
the control of either the cauliflower mosaic virus 35S promoter or the granule bound
starch synthase promoter were significantly more resistant to black spot than the non
transformed potatoes. The patatin promoter, which may not be fully active in potato
tubers, did not prevent polyphenol oxidase accumulation. The sense constructs all
synthesized increased amounts of polyphenol oxidase and showed larger amounts of
black spot than non transformed control plants. Similar anitsense constructs are likely to
reduce enzymatic discoloration in wide range of commercially important plants.
Fig 1.Sense and antisense polyphenol oxidase gene constructs.
Transcription of either the sense or antisense gene is separately under the control of the
cauliflower mosaic virus 35S promoter (P35S), the granule-bound starch synthase
promoter (PGBSS) or the patatin type I promoter (Ppatatin) with the nopaline synthase
transcription terminator region (tNOS). The abbreviations RB and LB represent the right
and left borders of the T-DNA.
Sweetness
Even though a fruit or vegetable may have high nutritional value if it is not tasty,
humans usually will not eat it. Although palatability of food can be achieved by adding
salt, sugar, flavors, or other ingredients during preparation it would be advantageous to
the food industry if certain foods could be made intrinsically more appetizing.
Monellin, a protein that is found in the fruit of an African plant with the unlikely
name of serendipity (Dioscoreophyllum cumminsii Diels), is approximately 3,000 times
sweeter than Sucrose on a weight basis. This feature makes monellin a candidate as a
sugar substitute, with the added bonus that because it is a protein, it would not have the
same metabolic impact as sugar.
Monellin is a dimer with an A chain of 45 amino acid residues and a B chain of
50 residues; the chains are held together by weak noncovalent bonds. Unfortunately, the
fact that monellin is composed of two separate polypeptide chains limits its usefulness as
a sweetener because it is readily dissociated (denatured) and consequently loses its
sweetness when it is either heated during cooking or exposed to acid (e.g., lemon juice or
vinegar). Also, the need to clone and express in a coordinated manner two separate genes
complicates efforts to produce the protein in either transgenic plants or microorganisms.
To circumvent this problem, a monellin gene that encodes both the A and B
chains as a single peptide was chemically synthesized. The fusion protein was produced
in transgenic tomato and lettuce plants. Two different promoters were used to express the
monellin fusion protein gene. In the experiment with tomatoes, expression was directed
by the tomato fruit-specific promoter E8, which is activated at the onset of fruit ripening.
The construct for the lettuce experiment was under the control of the 35S promoter from
cauliflower mosaic virus. Each construct used the transcription terminationpolyadenylation site from a Ti plasmid nopaline synthase gene. In each case, the
synthetic monellin gene was introduced into plant cells by A. tumefaciens infection,
using the Ti plasmid cointegrate vector system. Monellin was detected in ripe and
partially ripe tomatoes and in lettuce leaves but not in green tomatoes. The monellin level
in tomatoes could also be elevated by a burst of the plant hormone ethylene. However, if
taste tests of these genetically sweetened foods are positive, then this strategy for
sweetening plants without sugar or chemical additives would be applicable to a range of
fruits and vegetables.
In addition to monellin, several other sweet-tasting proteins have been reported
(Table 1). The genes for some of these proteins have been isolated and characterized.
Fructans are naturally occurring polymers of fructose that are not usually
degraded in the human digestive tract. However, some beneficial bacteria in the human
intestinal tract can utilize fructans. Small fructans, i.e., up to five monosaccharide units,
have a sweet taste and can be used as a natural low-calorie sweetener instead of sucrose.
Fructans are generally produced on a large scale in an expensive process from sucrose by
fungal invertases, or they are extracted from the roots of chicory plants or Jerusalem
artichoke tubers. To develop an inexpensive means of producing fructans, researchers
engineered sugar beet plants to convert the sucrose that it normally stores in the vacuole
of taproot parenchyma cells into fructans. This was done by transforming sugar beets
with a genetic construct that contained the 1-sucrose: sucrose fructosyl transferase cDNA
from jerusalem artichokes under the transcriptional control of the3SS promoter. To
transform sugar beets, the genetic construct was introduced into protoplasts in the
presence of polyethylene glycol. The transgenic sugar beets accumulated fructan up to
40% of the taproot dry weight. The mixture of fructans that was produced by the
transgenic sugar beet plants is essentially the same as the fructan that is produced
enzymatically.
Starch
The starch that is found in most crop plants such as potato consists of 20 to 30%
(straight-chain) amylose and of 70 to 80% (branched-chain) amylopectin. The ratio of
amylose to amylopectin has a large influence on the physical and chemical properties of
the starch. For many industrial applications it would be useful to have starch that is
highly enriched in either amylose or amylopectin. Starch is normally synthesized in a
stepwise process (Fig 2).The enzyme ADP-glucose pyrophosphorylase catalyzes the
transfer of ADP (from ATP) to glucose-I-phosphate to form ADP-glucose. The enzyme
starch synthase catalyzes the transfer of glucose from ADP-glucose to the nonreducing
end of a preexisting glucan chain, which is a short version of an amylose chain, by means
of an α-1,4 linkage. Branching can occur, catalyzed by starch branching enzyme, when
two glu can chains are joined by an α-1,6 linkage. To generate potatoes with a high
percentage of amylose and a corresponding low percentage of amylopectin, plants were
transformed using A. tumefaciens with antisense versions of the starch branching enzyme
under the transcriptional control of the 35S promoter. In transgenic lines that exhibited
only approximately 1% of the normal amount of the starch branching enzyme activity the
fraction of amylose increased from around 28% to 60 to 89% of the starch content. This
is an initial step toward developing potatoes with unique starch compositions that may be
used either for food or as a source of unusual starches with unique industrial properties.
Figure 2. The major reactions in the biosynthesis of starch
Since starch is both inexpensive and very abundant, it is used in a number of
industrial processes as a thickener, a gelling agent, or an adhesive. In addition, it is a
substrate for the production of high-fructose syrups and ethanol. The production of highfructose syrups requires the starch to be degraded, usually at high temperature by the
enzyme α-amylase, and then treated, also at high temperature, with glucose isomerase,
which converts the glucose (released from the starch that was degraded by α-amylase)
into fructose. The enzymes that catalyze these reactions account for a major portion of
the cost of the industrial conversion of starch into fructose. Therefore, reducing the cost
of the enzymes makes the overall process more attractive. With this in mind, researchers
constructed a bifunctional enzyme containing the essential regions of α-amylase and
glucose isomerase by fusing together, the genes for these enzymes using polymerase
chain reaction (PCR) (Fig. 3). Of course care was taken to ensure that the chimeric gene
retained the correct reading frames of the two genes. When the bifunctional enzyme was
synthesized in E. coil, both α-amylase and glucose isomerase activities were found, and
both activities were identical to found that in the thermo tolerant micro organisms. The
chimeric gene was introduced into potato plants under the transcriptional control of the
granule bound starch synthase promoter. Biochemical analysis revealed considerable
increase in glucose and fructose concentration compared to non transformed plants.
Figure 3. Construction of an α-amylase-glucose isomerase fusion gene.
The α--amylase and glucose isomerase genes were from Bacillus stearothermophilus and
Thermus themophilus , respectively. The portions of the PCR primers that do not match
the target genes are designed to retain both genes in the same reading frame and to
include appropriate restriction endonuclease sites.