Journal of Advanced Laboratory Research in Biology

E-ISSN: 0976-7614
Volume 5, Issue 4, October 2014
PP 169-187
https://e-journal.sospublication.co.in

Review Article

Botanical and Protein Sweeteners

Fawibe O.O.1, Ogunyale O.G.1, Ajiboye A.A.2 and Agboola D.A.1*
1
Department of Biological Sciences, Federal University of Agriculture, P.M.B 2240, Abeokuta, Ogun State, Nigeria.
2
Department of Biological Sciences, P.M.B. 4494, Osun State University, Osogbo, Osun State, Nigeria.

Abstract: Plant species with unusual taste properties such as bitterness, sourness or sweetness and others with a
taste- modifying components; have long been known to man, although their exploitation has been limited.
Exponential growth in the number of patients suffering from diseases caused by the consumption of sugar has
become a threat to mankind's health. Artificial low-calorie sweeteners available in the market may have severe side
effects. It takes time to figure out the long-term side effects and by the time these are established, they are replaced
by a new low-calorie sweetener. Saccharine has been used for centuries to sweeten foods and beverages without
calories or carbohydrate. It was also used on a large scale during the sugar shortage of the two world wars but was
abandoned as soon as it was linked with the development of bladder cancer. Naturally occurring sweet and taste
modifying proteins (Thaumatin, Curculin, Miraculin, Brazzein, Pentadin, Monellin, Mabinlin) present in plants
such as Thaumatococcus daniellii (Marantaceae), Curculigo latifolia (Hypoxidaceae), Synsepalum dulcificum
(Sapotaceae), Pentadiplandra brazzeana (Pentadiplandraceae), Dioscoreophyllum cumminsii (Menispermaceae),
Capparis masaikai (Capparaceae) are being seen as potential replacements for the currently available artificial low
calorie sweeteners. Most protein sweetener plants such as S. dulcificum, P. brazzeana, C. masaikai, are shrubs; C.
latifolia, T. daniellii, are perennial herbs while D. Cumminsii is an annual liana.

Keywords: Nutritive and Non-Nutritive Sweeteners, Protein sweetener, Natural Sugar, Artificial Sugar.

1. Introduction per gram. For most sugars, this is about 17 calories per
teaspoon. Sweeteners that do not supply calories are
The role sweeteners play in the diet is constantly referred to as non-nutritive sweeteners. Synthetic
debated. Sweeteners of one kind or another have been (artificial) sweeteners may be nutritive or non-nutritive.
found in human diets since prehistoric times. Terms Synthetic sweeteners must pass the approval of the
such as sugar-free, sugar alcohols, sucrose, corn Food and Drug Administration (FDA) before they can
sweeteners, etc. can be confusing. Each of the be marketed in the United States. The three synthetic
sweeteners available to consumers has specific sweeteners currently approved by the FDA are
applications and certain limitations. A variety of aspartame, saccharin and acesulfame-K. Aspartame is a
sweeteners exist to help consumers satisfy their desire nutritive sweetener. Saccharin and acesulfame-K are
for sweetness. Sweeteners are used in foods for several non-nutritive sweeteners (Halliday, 2008).
reasons, besides adding sweetness. Sugar is used as a
preservative in jams and jellies; it provides body and 1.1 Nutritive Sweeteners
texture in ice cream and baked goods; it aids in
fermentation in bread and pickles (Birch G. Gerard, Sugars: All natural sugars are composed of simple
2000). carbohydrates. Carbohydrates are classified into three
Sweeteners that supply energy (calories) are categories: monosaccharides (one unit of sugar),
referred to as nutritive sweeteners, even though they disaccharides (two units of sugar) and polysaccharides
lack other nutrients essential for growth and health (many units of sugars).
maintenance. Nutritive sweeteners provide four calories

*Corresponding author:
E-mail: [email protected].
Botanical and Protein Sweeteners Agboola et al

The simpler forms of carbohydrates are called sugar, fructose and glucose. Honey is more
sugars, and the more complex forms are either starches concentrated than crystalline table sugar, so it contains
or dietary fibers. The simple carbohydrates follow: more calories per equal measure than table sugar.
Honey is often proclaimed as more nutritious than
Classification of Most Common Carbohydrates sugar. It does provide trace minerals and B vitamins in
Monosaccharides Disaccharides Polysaccharides
very minute amounts, but it is misleading to actually
(Single Sugars) (Double Sugars) (Complex Sugars)
say honey is more nutritious. The contribution of these
Glucose Sucrose Starch nutrients to the overall diet is insignificant.
Fructose Lactose Glycogen
Maple sugar: Maple sugar is produced by boiling off
Galactose Maltose Cellulose (fiber)
the liquid from the syrup derived from the spring flow
Fructose: Fructose is the sweetest tasting of all the of the sap of mature sugar maples. Maple sugar is
natural sugars. It is found in fruits and honey. Fructose mainly sucrose.
is known as the fruit sugar. It also has been referred to In human nutrition, the primary role of
as levulose. Fructose is one and one-half times as sweet carbohydrates is to supply energy. A gram of
as sucrose. carbohydrate contains 4 calories (energy). The body
prefers this source of energy over all other available
Glucose: Glucose is in nearly all plant foods. Glucose sources of energy nutrients.
is known as the "blood sugar" because it is the main Carbohydrates are known as the "energy sparing
sugar circulating in blood and is the main form into nutrient". The energy available from carbohydrates
which the body converts other sugars and spares protein from being used for energy so that
carbohydrates. protein may build and repair body tissue. When
Another name for glucose is dextrose. Glucose is carbohydrates are absent from the diet, protein is used
rapidly absorbed into the bloodstream from the for energy.
intestines. Glucose is unusual in that it can be absorbed Carbohydrates are found in almost all plant foods
into the bloodstream to some degree through the lining and one animal source -- milk. There are nutrient
of the mouth. differences between these sugar sources.
Sucrose is the most abundant sugar in plants. Fruit furnishes the same monosaccharides and the
Sucrose is a disaccharide composed of two simple same calories per monosaccharide gram of weight as
sugars and when sucrose is digested it separates the two refined table sugar and honey. The difference is that in
chemically attached simple sugars, glucose and fruit the sugars are diluted in a large quantity of water
fructose, for the body to absorb and use for energy. that contains vitamins, minerals and fiber.
Sucrose is the most common sugar for household and The significant difference between sugar sources is
industrial use. It is produced by concentrating the sugar not between "natural" and "refined," but between
from sugar cane or sugar beet juice. concentrated sugars (honey, table sugar, concentrated
Traditionally, the word sugar has been used to fruit juices, corn sweeteners) and the diluted, naturally
imply sucrose, but this can cause confusion because occurring sugars in foods such as oranges, corn, milk,
there are so many other sugars currently on the market. and potatoes.
Sucrose is purified and granulated to various stages to There is strong evidence that sugar plays a part in
provide raw, white, brown and powdered sugars. dental cavities. Dental cavities are caused by the acid
During processing, a dark coloured liquid, molasses, is by-product of bacterial growth in the mouth, and
produced. bacteria thrive on carbohydrates. Therefore, sugars are
Lactose: Lactose is the sugar found in milk. Lactose is implicated as the cause of cavities. Any carbohydrate-
primarily used in products to provide bulk. It is seldom containing food, including bread, bananas, milk and
used as a sweetener because it is the least sweet of the concentrated sugars, can support bacterial growth.
sugars. Lactose has two monosaccharide parts, glucose It takes about 24 hours for a large enough build-up
and galactose, which are normally broken apart by the of bacteria to accumulate on a tooth to produce cavity-
enzyme lactase. Some individuals may lose the ability causing acid. Brushing after eating carbohydrates and
to digest lactose and become lactose intolerant. once-a-day, flossing may effectively prevent cavity
Cheese, although a product of milk, has less lactose formation, regardless of the carbohydrate content of the
because the lactose changes during the fermentation diet. Some individuals may never get cavities because
process. they have inherited resistance to them.
Maltose: Maltose is produced during bread making and Corn Syrup: This glucose sweetener was developed in
beer brewing. When you eat or drink a food source of the 1920s by treating cornstarch with acid, heat and/or
maltose, the maltose is split into two glucose units so enzymes. Corn syrup is not as sweet as sucrose but is
they can be absorbed. often used with or in place of sucrose to provide "body"
and "texture" in food.
Honey: Honey is a natural syrup made from plants by
honeybees. The same sugars in honey are found in table

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Botanical and Protein Sweeteners Agboola et al

High-Fructose Corn Syrup: Made from corn syrup by Disease Control state that some individuals may have
converting glucose into fructose, this unique enzymatic an unusual sensitivity to aspartame. Statistically, some
process, developed in 1970, provides a much sweeter sensitivity to aspartame could be expected with over
product, allowing a reduction in quantity used. 100 million U.S. individuals consuming products
Currently, the soft drink industry uses high-fructose containing aspartame.
corn syrup as its main nutritive sweetener. It may be
listed as corn sweetener or high fructose corn syrup on 1.2 Non-Nutritive Sweeteners
a label. Non-nutritive sweeteners do not provide any
calories when consumed. There are two non-nutritive
Sugar Alcohols: These sweeteners are commercially
produced from glucose, or derived from fruits and sweeteners currently approved by FDA; Saccharin and
vegetables. The most common sugar alcohols are Acesulfame-K.
sorbitol, mannitol, maltitol, and Xylitol. Prunes have Saccharin was discovered in 1879 and has been
marketed for over 80 years. Due to the length of its safe
the largest amount of the naturally occurring sorbitol of
use, saccharin was given GRAS (generally regarded as
any fruit normally eaten in the United States. Apples
and pears also are high in sorbitol. Sugar alcohols are safe) status.
found in dietetic candies, chewing gums, and as a In 1977, FDA proposed removing saccharin from
public use because of a Canadian study of saccharin
coating on the tablets and gums.
and bladder cancer in rats. Public opposition led
Products containing sugar alcohols may carry the
Congress to pass a moratorium on the FDA's action to
label "sugarless," "sugar-free" or "no sugar," but they
take saccharin off the market. This moratorium is still
are still carbohydrates and supply calories. Sugar
alcohols do not promote tooth decay. They are absorbed in effect. Products containing saccharin must carry a
warning label that states: Use of this product may be
more slowly than sugars, which may be the reason they
hazardous to your health. This product contains
can have a laxative effect.
saccharin which has been determined to cause cancer
Aspartame: This artificial sweetener was discovered in in laboratory animals.
1965. In 1981, the FDA approved aspartame as a table- Saccharin has not been shown to cause cancer in
top sweetener and as an ingredient in dry foods, such as human beings. Saccharin is approximately 300 times as
dry bases for beverages and cold cereals. In 1983, sweet as sucrose and is stable within a wide range of
approval was extended to carbonated beverages. temperatures.
Aspartame is marketed in the U.S. under the trade Acesulfame-K was approved by FDA in July 1988
names of NutraSweet and Equal. Aspartame provides as a free-flowing table-top sweetener and for use in dry
the same energy as any protein, four calories per gram base beverage mixes, puddings and desserts, chewing
because aspartame is the methyl ester of two amino gum and dairy product analogues (including toppings).
acids (proteins) -- aspartic acid and phenylalanine. Acesulfame-K is marketed under the names of Sunette
Aspartame is 180-200 times sweeter than sucrose; and Sweet One. Acesulfame-K is about 200 times
it does not contribute a significant amount of calories sweeter than sucrose. Acesulfame-K is heat stable and
due to the small amount needed to sweeten products. can be used in baking. Petitions for its use in soft drinks
Due to a possible excess of phenylalanine for and baked goods have been filed.
phenylketonuric (PKU) children, aspartame must carry Cyclamates were removed from the open market in
a warning label stating: "Phenylketonuric: contains 1969 because of research that linked cyclamates as a
phenylalanine". Phenylketonuria is a genetic disease in possible cancer-causing agent in rats. Petitions have
which the body cannot produce the enzyme necessary been filed to bring back cyclamates as an alternative
for the body to use phenylalanine. sweetener.
The Acceptable Daily Intake set by the FDA is 50 Traditional methods to sweeten foodstuffs, feed,
milligrams of aspartame per kilogram of body weight. and other consumer products have relied on the use of
In terms of aspartame-sweetened soft drink usage, this low-molecular-weight sweetening agents, especially
equates to four to five 12 ounce cans for a 40 pound sucrose. In recent years, however, there has been an
child or about 17 cans for a 150 pound adult. Adequate increasing demand for low-calorie sweeteners. Together
data is not available to establish the safety of aspartame with this trend, there is also an increase in the demand
for children less than two years of age. There are few, if for healthy and natural eating products. Therefore, In
any, reasons to use a sugar substitute for children less order to address this need, there is an intense and
than two years of age. Children need energy to grow. ongoing search for alternative sweeteners. The
Over 200 commercial products have aspartame as alternative sweeteners that have been developed show a
one of their ingredients. The use of aspartame is limited very intense sweetening profile. They are several orders
at high or prolonged temperatures because it breaks of magnitude sweeter than sucrose on both a molar and
down and loses its sweetness. Although there have been weight basis. Therefore, in order to supply the same
over 6,000 unsolicited complaints to the FDA sweetening effect as sucrose, these compounds can be
concerning aspartame, the FDA supports the safety of used in minute amounts. This results in almost
aspartame for the general population. The centres for

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Botanical and Protein Sweeteners Agboola et al

negligible addition to the calorie count, as well as a In humans, the sweet taste is mainly due to the
product that does not contribute to tooth decay. recently discovered T1R2-T1R3 receptor, two of the
Moreover, the alternative and intense sweetening three members of the T1R class of taste-specific
additives also can be used in diabetic foods, since they proteins hypothesized to function in combination as a
do not trigger a demand for insulin in these patients. heterodimer. The human T1R2-T1R3 receptor
Finally, some intense sweeteners also have been found recognizes natural and synthetic sweetness and T1R1-
to be strong flavor enhancers, thus expanding their T1R3 recognizes the umami taste (Nelson, 2002). So
range of applications. far there are seven known sweet and taste-modifying
The prevalence of obesity and diabetes has proteins, namely Brazzein, Thaumatin, Monellin,
increased dramatically in recent years in the United Curculin, Mabinlin, Miraculin and Pentadin (Faus,
States, with similar patterns seen in several other 2000). The key residues on the protein surface
countries, including India as well (Levine et al., 2003). responsible for biological activity have not yet been
Diabetes mellitus is a chronic disease caused by an identified with certainty for any of these proteins.
inherited or acquired deficiency in production of insulin Monellin was found to be 100000 times sweeter than
by the pancreas or by the ineffectiveness of the insulin sucrose on a molar basis (Hung et al., 1999) followed
produced (Pinget and Boullu-Sanchis, 2002). Artificial by Brazzein and Thaumatin which are 500 times (Ming
sweeteners like Saccharin, Aspartame, Cyclamate and et al., 1994) and 3000 times sweeter than sucrose (van
Acesulfame-K are used worldwide as low-calorie der Wel and Loeve, 1972) respectively (the latter two
sweeteners by patients affected by diseases linked to the on a weight basis). All of these proteins have been
consumption of sugar, e.g. diabetes, hyperlipemia, isolated from plants that grow in tropical rainforests.
caries, obesity etc. but they have side effects such as Although most of them share no sequence homology or
psychological problems, mental disorders, bladder structural similarity, Thaumatin shares extensive
cancer, heart failure and brain tumors. Sweet proteins homology with certain non-sweet proteins found in
have the potential to replace these artificial sweeteners, other plants. The potential industrial applications of
by acting as natural, good, low-calorie sweeteners, as these proteins are the low-calorie sweetener industry
we know that proteins do not trigger a demand for and the cola, snacks, food and chocolate industries.
insulin in these patients whereas sucrose does.

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CHAPTER TWO

2. Protein Sweeteners plant starts fruiting when only 1 ft tall. It produces fruit
practically year around. In native habitat, two large
2.1 Miracle Fruit crops are available yearly, each after a rainy season.
The mature bushes usually have a few fruits hanging
 Scientific names: Synsepalum dulcificum, around all year. Seed to fruit in 2 to 3 years and Flower
Synsepalum subcordatum to fruit in 30 to 45 days.
 Family: Sapotaceae One of the many virtues of miracle fruit is its
 Common name: Miracle Fruit ease of cultivation, although, like with any plant, it
 Origin: Ghana (Tropical West Africa) would be nice if you learn its likes and dislikes before
inviting it into your home or garden. If planted in
It is one of the strangest tropical fruits. The most alkaline limestone-based soils, the plant may die. These
unusual thing about it is the effect it has on one's taste plants seem to live for acid, thriving in it, and then
after this miraculous berry has been consumed. The converting it! They must have rich, well-drained soils
“miracle” is that if lemon or other sour food is eaten that are acid in pH, with lots of peat moss, and require
after the miracle fruit, the sour tastes sweet as if sugar constant supply of micronutrients. On alkaline soils,
has been added. That kind of magical experience is they often are grown in large containers with generous
unforgettable! The interest in this plant is so high that amounts of peat moss for sustained success in fruiting.
anyone who has a plant always finds eager volunteers It makes an excellent container tree, which gives it the
to test its sweetening properties. A natural chemical in added benefit of mobility. Since it is so easily
the fruit masks the tongue’s sour taste buds so that containerized, almost anyone can grow this plant
lemons taste like lemonade or lemon pie, or lemon whether they have an outside planting area or not.
candy. What causes the miracle? The fruit has a unique Miracle fruit can be a rewarding indoor plant. It is not a
taste changing glycoprotein that inhibits the taste buds' fast-growing plant, which is another benefit for those
perception of sour taste. The sweet sensation lasts for who would like to grow it in their house or greenhouse.
half an hour to a few hours (Slater and Joanna, 2007). It thrives under warm temperatures and high humidity.
A 10 inch plant is happy in a one-gallon pot. One that
2.1.1 History size will flower and fruit at least twice a year, probably
The plant was discovered in West Africa, where more frequently.
the native diet revolved around a few basic foods, Miracle Fruit thrives best in partial shade. When
mostly of sour taste. Just imagine the delight of the plants are small they are subject to frost damage, so
people when someone ate a few red berries and later ate they should be container-grown and kept indoors or
a meal of sour foods, to find everything suddenly moved to protected locations when frost or freeze
sweet! The West African natives use the fruit to threatens. Older plants may sustain some leaf and minor
sweeten sour palm wine (beer) Pito, and fermented twig damage but may sustain cooler temperatures
maize bread - Kenkey. without being killed. Although not thought to be frost
This West African wonder was not botanically tolerant, the Synsepalum plants have been observed
identified and named until the middle of the 19th growing in Florida in protected from wind locations as
century as Synsepalum dulcificum, a member of the far north as Tampa.
Sapotaceae family, relative of the Sapodilla (Manilkara When propagating miracle fruit, the seeds are
zapota) (Theerasilp et al., 1989). sown in a rich, well-drained medium, just barely
Bill Whitman was the first to grow the plant covered, and water lightly every other day. Seeds
successfully in the US. He had a seedling all of 7" tall generally come up in about eight to ten weeks, but grow
and a cutting of 4" bearing fruit upon the young twigs. slowly the first year, often being only two to three
inches tall at the end of almost one year of growth. It
2.1.2 The Plant really takes three to four years before the plants reach a
This small, evergreen shrub grows very slowly to height of more than fifteen to twenty inches, and then
a height of 4-6 ft in container, and 10-15 ft in natural they start to grow more rapidly. However, when
habitat. Eventual size depends on where the plant is growing this plant from seed, it takes few years before
grown; a 10 year old plant might be easily only 4-5 feet the first fruit is been enjoyed. If possible, it is always
tall. It forms an oval to pyramidal shaped bush or small recommended to get a mature full specimen, at least 1 ft
tree. Inconspicuous brown-and-white 1/2 inch flowers tall that is ready to bloom.
are followed by bright scarlet, 1 inch football-shaped There are 2 known species of Synsepalum that
fruit, sweet and pleasant tasting. Most of the fruit is carry miracle fruits. Synsepalum dulcificum is a
taken up by a single large seed, but the pulp around it smaller-leaf version (leaves are narrow) and is
can be nibbled off and then for the next hour or so, somewhat slower growing plant. Synsepalum
anything one eats that is sour has a sweet flavor. The subcordatum (Giant Miracle Fruit) is a larger leaf

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variety and grows into a small tree. The fruit is slightly dimer miraculin in its crude state have the taste-
bigger than those of S. dulcificum, and are produced modifying activity of turning sour tastes into sweet
more profusely, especially in the first years. With age, tastes (Igeta et al., 2006).
the fruit crop amounts of these two species become
about the same (Theerasilp et al., 1989). 2.1.5 Sweetness Properties
Miraculin, unlike curculin (another taste-
2.1.3 Miraculin modifying agent), is not sweet by itself, but it can
Miraculin is a natural sugar substitute, a change a sour beverage into a sweet beverage, even for
glycoprotein extracted from the fruit of Synsepalum a long period after consumption. The anti-sweet
dulcificum (Theerasilp and Kurihara, 1988). The berry, compound, Gymnemic acid suppresses the sweet taste
which contains active polyphenols, was first of miraculin, as it does for sucrose. The duration and
documented by explorer Chevalier des Marchais, who intensity of the taste-modifying phenomena depend on
searched for many different fruits during a 1725 various factors: miraculin concentration, duration of
excursion to its native West Africa. The protein is a contact of the miraculin with the tongue, and acid
single polypeptide with 191 amino acid residues concentration. Maximum sweet-induced response has
(Nirasawa et al., 2001). been shown to be equivalent to the sweetness of 17%
Miraculin itself is not sweet. However, after the sucrose solution (Matsuyama et al., 2009).
taste buds are exposed to miraculin, ordinarily sour Glycoprotein is sensitive to heat: when heated
foods, such as citrus, are perceived as sweet. This effect over 100°C, miraculin loses its taste-modifying a
lasts up to an hour. Thus, miraculin has the unusual property. Miraculin activity is inactivated at pH below
property of modifying sour taste into a sweet taste 3 and pH above 12 at room temperature. The sweet
(Temussi, 2002). modifying effect stays at pH 4 (in acetate buffer), for 6
Miraculin works by binding to the sweet months at 5°C.
receptors on the tongue. The Miraculins effect lasts as The detailed mechanism of the taste-inducing
long as the protein is bound to the tongue, which can be behaviour is still unknown. It has been suggested that
up to an hour. It makes most acidic foods taste sweet, the miraculin protein can change the structure of taste
but does not improve the taste of bitter things (Aaron cells on the tongue. As a result, the sweet receptors are
Rowe, 2006). activated by acids, which are sour in general. This
The active substance, isolated by Prof. Kenzo effect remains until the taste buds return to normal. The
Kurihara a Japanese scientist, was named miraculin two histidine residues (i.e. His29 and His59) appear to
after the miracle fruit when Kurihara published his be mainly responsible for the taste-modifying behavior.
work in Science in 1968. One site maintains the attachment of the protein to the
membranes while the other (with attached xylose or
2.1.4 Glycoprotein Structure arabinose) activates the sweet receptor membrane in
Miraculin was first sequenced in 1989 and was acid solutions. Further research is being conducted at
found to be a glycoprotein consisting of 191 amino the University of Tokyo using a system of cultured cells
acids and some carbohydrate chains (Theerasilp et al., that allowed the testing of human taste receptors at
1989). various pH values to uncover the mechanism. As
Miraculin occurs as a tetramer (98.4 kDa), a already known miraculin binds strongly to the sweet
combination of 4 monomers group by dimer. Within taste receptors on our tongues; however, it does not
each dimer 2 miraculin glycoproteins are linked by a activate receptors at neutral pH. Once an acid is
disulfide bridge (Kurihara, 1992). introduced, the miraculin protein changes shape in such
The molecular weight of the glycoprotein is 24.6 a way that it turns on the sweet receptors, it is bound to,
kDa, including 3.4 kDa (13.9% of the weight) of sugar causing an ultra-sweet sensation without affecting other
constituted (on molar ratio) of glucosamine (31%), flavors tasted. Once the acidic food is swallowed,
mannose (30%), fucose (22%), xylose (10%) and miraculin returns to its inactive shape until the next
galactose (7%). acidic food comes along. This can continue for about an
The taste-modifying protein, miraculin, has hour while the miraculin protein is still bound to the
seven cysteine residues in a molecule composed of 191 taste receptor (Matsuyama et al., 2009).
amino acid residues. Both tetramer miraculin and native

Amino acids sequence of glycoprotein miraculin unit adapted from Swiss-Prot biological database of protein sequence.

SIGNAL (29) MKELTMLSLS FFFVSALLAA AANPLLSAA

1-50 DSAPNPVLDI DGEKLRTGTN YYIVPVLRDH GGGLTVSATT PNGTFVCPPR
51-100 VVQTRKEVDH DRPLAFFPEN PKEDVVRVST DLNINFSAFM PNPGPETISS
101-150 WCRWTSSTVW RLDKYDESTG QYFVTIGGVK FKIEEFCGSG FYKLVFCPTV
151-191 CGSCKVKCGD VGIYIDQKGR GRRLALSDKP FAFEFNKTVY F

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Botanical and Protein Sweeteners Agboola et al

2.1.6 As a Sweetener
As miraculin is a readily soluble protein and
relatively heat stable, it is a potential sweetener in
acidic food (e.g. Soft drinks). Japanese researcher’s
more or less successful attempts to mass produce it is
focused on recombinant technology. While attempts to
express it in yeast and tobacco plants have failed,
researchers have succeeded in preparing genetically
modified E. coli bacteria, lettuce and tomatoes that
express miraculin. The scientists' crops resulted in 40
micrograms of miraculin per gram of lettuce leaves,
which was considered a large amount. Two grams of
Plate B: Showing the mesocarp of the fruit.
lettuce leaves produced roughly the same amount of
miraculin as in one miracle fruit berry (Slater and
Joanna, 2007).
Miraculin was never approved for use as a
sweetener by the United States Food and Drug
Administration (FDA). In the 1970s the Miralin
Company planned on bringing miraculin to market and
was founded with investments by Reynolds Metals,
Barclays, and Prudential. Legal advice and contact with
the FDA indicated that miraculin would be approved as
generally recognized as safe as the berries had been
eaten for centuries in Africa with no reports of adverse
reactions (substances used in food prior to January 1, Plate C: Showing the potted shrub.
1958, through either scientific procedures or through
experience based on common use in food can be
designated GRAS). However, on the eve of the
product's launch in 1974 the FDA determined that
miraculin would be considered a food additive and thus
require years more testing. At this point, the company's
investment capital could not sustain them and Miralin
folded. Afterward, Miralin requested the FDA
documents under the freedom of information act. The
documents were nearly completely blacked out, and the
rationale for the sudden change in regulation was not
revealed (Theerasilp and Kurihara, 1988). Plate D: Showing the prolificity of the shrub.
Limitations: Miraculin is a non-heat-stable protein,
subject to denaturation from heating, and thus miracle 2.2 Lemba
berries are not taste-bud active when cooked.
While miraculin changes the perception of taste,  Scientific names: C. latifolia, C. capitulata, C.
it does not change the food's chemistry, leaving the racemosa, C. orchioides
mouth and stomach vulnerable to the high acidity of  Family: Hypoxidaceae
some foods, such as lemon juice, that may cause  Common name: Lemba
irritation if eaten in large quantities.  Origin: Malaysia

The genus Curculigo belongs to the family

Hypoxidacea, which consists of approximately 20
species of exclusively tropical origin (Kocyan, 2007).
In Malaysia, it is locally known as Lemba and the
common species are Curculigo latifolia Dryand which
is a perennial herb with specialized underground stems.
The four other species found are namely C. latifolia, C.
capitulata, C. racemosa and C. orchioides. The plants
are not cultivated but found abundantly distributed in
both Peninsular Malaysia and Borneo. The plants grow
about two meters tall and the petiole up to 1 meter long
Plate A: Showing the Shape of the Fruit. that consist of blade elliptical leaf, 30-100cm x 5-l0cm,

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Botanical and Protein Sweeteners Agboola et al

subglabrous. The leaf stalks are one-third the length of taste-modifying activity of the protein (discussed
the leaves and they overlap one another at their bases to below) remains unchanged when it is incubated at 50°C
form a thick stem. Inflorescence ovoid to flowers for 1 hr between pH 3 and 11. The plant is referred to
cylindrical, compact, 2-6cm x 2-6cm, bracts, 1-6cm locally as 'Lumbah'.
long; green, flower color; yellow; fruit ovoid, 10-
25mm, tiny seeds and sweet. 2.2.2 Protein Structure
The leaves are tough, thin and broad, who makes The active form of curculin is a heterodimer
leaves them very suitable for wrappings (Buchanan, consisting of two monomeric units connected through
1999). It grows well in a hilly area and it is a shade- two disulfide bridges. The mature monomers each
loving plant, thriving partly in shaded or sunless consists of a sequence of 114 amino acids, weighing
conditions, with abundant water supply. It prefers 12.5 kDa (curculin 1) and 12.7 kDa (curculin 2),
fertile, well-drained soils, rich in organic matter. Leaves respectively. While each of the two isoforms is capable
are elliptic with numerous distinct parallel primary of forming a homodimer, these do not possess the sweet
veins and the plants can easily be mistaken for ground taste nor the taste-modifying activity of the
orchids or young palms. Flowers are yellow with the heterodimeric form. To avoid confusion, the
exception of C. orchioides, which have pink flowers. In heterodimeric form is sometimes referred to as
Borneo, traditionally the leaves are used for wrapping "neoculin" (Suzuki et al., 2004). These sweet proteins
foods or pounded and wrapped around the knees to ease are postulated to evoke activities by interacting with the
joint pains. The remarkable feature, however, lies in the T1R2-T1R3 heterodimeric sweet taste receptor similar
fruits. Interest in Lemba has increased owing to their to low molecular mass sweet compounds, such as
roles as alternative sweeteners and the possible sugars and aspartame.
beneficial implications in the prevention of diabetes.
The fruit contained sweet proteins called curculin, 2.2.3 Sweetness Properties
which exhibit taste-modifying activity (Barre et al., Curculin is considered to be a high-intensity
1997). Curculin modifies taste bud sensations by sweetener, with a reported relative sweetness of 430-
changing acidic or tasteless food into sweetness. 2070 times sweeter than sucrose on a weight basis
Curculin extract was first developed into natural (Yamashita et al., 1990).
sweeteners by Yamashita et al., (1990) but A sweet taste, equivalent to a 6.8% or 12%
commercialized products are hindered by fruit sucrose solution, was observed after holding curculin in
productions as these plants are yet to be cultivated. the mouth in combination with clear water or acidified
Preliminary studies on germination rate of Lemba seeds water (citric acid), respectively. The sweet taste lasts
showed that seeds were unable to germinate on for 5 minutes with water and 10 minutes with an acidic
germinating trays, jiffy bags or blotter papers. It was solution. Sweetness was also observed with other acids
suspected that seeds may be recalcitrant or may require such as ascorbic acid (vitamin C) and acetic acid
special germinating media or pre-treatments. In (Yamashita et al., 1990).
addition to this, success of seed germination may be The taste-modifying activity of curculin is
affected by fruit ripeness and maturity, where fruit reduced in the presence of ions with two positive
developmental studies and ripening of this plant was charges (such as Ca2+ and Mg2+) in neutral pH
never reported. The difficulty in using seed as solutions, although these ions have no effect in acidic
propagules has enticed the needs for other means of solutions. In the same way, monovalent ions (such as
propagation. As these plants are monocots, the most Na+ and Cl−) have no effect in solutions with either
suitable vegetative propagules are corms and rhizomes. neutral or acidic pH.
Although the "sweet-inducing" mechanism is
2.2.1 Curculin unknown, it is believed that one active site of curculin
Curculin is a sweet protein that was discovered strongly binds to the taste receptor membranes while a
and isolated in 1990 from the fruit of Curculigo second active site fits into the sweet receptor site. The
latifolia (Hypoxidaceae) (Yamashita et al., 1990) a latter site is thought to be responsible for the induction
plant from Malaysia. Like miraculin, curculin exhibits of sweetness. Presence of Ca2+ and/or Mg2+, water and
taste-modifying activity; however, unlike miraculin, it acids tune the binding of the active site of curculin to
also exhibits a sweet taste by itself. After consumption the receptor site and therefore modify perceived
of curculin, water and sour solutions taste sweet. The sweetness (Yamashita et al., 1990).
Amino acid sequence of sweet protein curculin adapted from Swiss-Prot biological database of protein sequences.

SIGNAL (22) MAAKFLLTIL VTFAAVASLG MA

1-50 DNVLLSGQTL HADHSLQAGA YTLTIQNKCN LVKYQNGRQI WASNTDRRGS
51-100 GCRLTLLSDG NLVIYDHNNN DVWGSACWGD NGKYALVLQK DGRFVIYGPV
101-114 LWSLGPNGCR RVNG
PROPEP (22) GITVAKDSTE PQHEDIKMVI NN

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Botanical and Protein Sweeteners Agboola et al

2.2.4 As a Sweetener
Like most proteins, curculin is susceptible to
heat. At a temperature of 50°C (122°F) the protein
starts to degrade and lose its "sweet-tasting" and "taste-
modifying" properties, so it is not a good candidate for
use in hot or processed foods. However, below this
temperature both properties of curculin are unaffected
in basic and acidic solutions, so it has potential for use
in fresh foods and as a table-top sweetener.
Because curculin is not widely found in nature,
efforts are underway to produce a recombinant form of
the protein. In 1997, curculin was expressed in E. coli Plate H: Showing the fruit of the plant containing Curculin.
and yeast, but the recombinant protein did not exhibit
"sweet-tasting" or "taste-modifying" activity (Kurihara In addition to the challenges related to
et al., 1997). However, a 2004 study obtained a commercial production of the protein, there are many
recombinant curculin, expressed in E. coli, exhibiting regulatory and legal issues remaining to be resolved
"taste-modifying" and "sweet-tasting" properties before it can be marketed as a sweetener. Curculin
(Suzuki et al., 2004). currently has no legal status in the European Union and
the United States. However, it is approved in Japan as a
harmless additive, according to the List of Existing
Food Additives established by the Ministry of Health
and Welfare (English publication by JETRO).

2.3 Oubli

 Scientific name: Pentadiplandra brazzeana

 Family: Pentadiplandraceae
 Common name: Oubli
 Origin: Gabon and Cameroon

The plant grows in Gabon and Cameroon, where

Plate E: Showing the perennial monocotyledonous plant.
the fruit has been consumed by the apes and local
people for a long time. The berries of the plant are
incredibly sweet. African locals call them "Oubli"
(French for "forgot") in their vernacular language
because their taste helps nursing infants forget their
mother's milk, as once they eat them they forget to
come back to the village to see their mother (Stein,
2002)

2.3.1 Brazzein
Brazzein is smaller, more heat-stable and pH-
stable member of the set of proteins known to have
intrinsic sweetness. The protein, consisting of 54 amino
Plate F: Showing the arrangement of the flowers. acid residues, is reported to be between 500 and 2000
times sweeter than sucrose and represents an excellent
alternative to available low-calorie sweetener. It was
originally isolated from the fruit of an African plant
Pentadiplandra brazzeana Baillon (Jin et al., 2003).
Heat and pH stability of the protein makes it an ideal
system for investigating the chemical and structural
requirements of a sweet-tasting protein. It was first
isolated as an enzyme by the University of Wisconsin–
Madison in 1994 (Ming and Hellekant, 1994).
Brazzein is found in the extracellular region. The
tissue it's found in is the pulp surrounding the seeds.
With pentadin, discovered in 1989, brazzein is
Plate G: Showing the arrangement of the flower in acropetal the second sweet-tasting protein discovered in this
succession. African fruit (Van der Wel, 1989).

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Botanical and Protein Sweeteners Agboola et al

Like the other natural sweet-proteins such as Increasing interest of brazzein makes it difficult
monellin and thaumatin, it is highly sweet. to source naturally from Gabon, but it can also be
synthesized by a solid-phase method. Recombinant
2.3.2 Protein Structure proteins were successfully produced via E. coli.
The monomer protein, consisting of 54 amino The Texas companies Prodigene and Nectar
acid residues, is the smallest of the sweet proteins with Worldwide were among the licensees to use Wisconsin
a molecular weight of 6.5 kDa (Hellekant and Danilova, Alumni Research Foundation patents on brazzein, and
2005). The amino acid sequence of brazzein, adapted genetically engineer the enzyme into maize. Brazzein
from the Swiss-Prot biological database of protein, is as then can be commercially extracted from maize through
follows: QDKCKKVYEN YPVSKCQLAN ordinary milling. Approximately one ton of maize
QCNYDCKLDK HARSGECFYD EKRNLQCICD yields 1-2 kilograms of Brazzein. It can also be
YCEY. engineered into plants like wheat to make pre-
The structure of brazzein was determined by sweetened grains, e.g. for cereals (Halliday, 2008).
proton nuclear magnetic resonance (NMR) at a pH 5.2
and 22 degrees C. Brazzein has four evenly spaced 2.3.5 Application
disulfide bonds and no sulfhydryl groups. Sweet carbohydrates have several problems
3D analysis of brazzein showed one alpha-helix associated with their use, such as high caloric content,
and three strands of anti-parallel beta sheet. It is not tooth decay and diabetes mellitus. This leads to demand
similar to either of the other two sweet-tasting proteins, for non-sugar alternatives. However, non-sugar
monellin and thaumatin (Izawa et al., 2000). alternatives show also deficiencies such as being
However, a recent 3D study shows that these unsuitable in most cooking or baking applications.
three proteins possess similar "sweet fingers" believed Their organoleptic qualities are also inferior and
to elicit the sweet taste concern from a toxicological point of view can be
Residues 29–33 and 39–43, plus residue 36, as raised for sweeteners, such as synthetic sweeteners,
well as the C-terminus, were found to be involved in which lack a history of human consumption.
the sweet taste of the protein. The charge of the protein Brazzein combines a long history of human
plays also an important role in its interaction with the consumption, small size with high sweet potency,
sweet taste receptor. solubility and exceptional thermostability. It tastes
Based on this knowledge a synthesized improved purely sweet with no sourness, saltiness or bitterness.
brazzein, called pGlu-1-brazzein, was reported to be These qualities make it a very good alternative.
twice sweet as the natural counterpart (Jin et al., 2003). However, as is a characteristic of many high-intensity
sweeteners, the sweetness of brazzein grows slightly
2.3.3 Sweetness Properties slower than that of sucrose. The sweetness of brazzein
On a weight basis, brazzein is 500 to 2000 times is readily washed from the tongue and neither mouth
sweeter than sugar, compared to 10% sugar and 2% cooling nor extensively lingering occurs. It often
sugar solution respectively (Izawa et al., 2000). Its improves the mouthfeel of beverages when blended
sweet perception is more similar to sucrose than that of with other sweeteners and works well in both citric acid
thaumatin with a clean, sweet taste with lingering and phosphate beverage systems.
aftertaste and with a slight delay longer than aspartame Brazzein combines well with most high-intensity
in an equi-sweet solution (Pfeiffer et al., 2000). sweeteners such as acesulfame-K and aspartame,
Brazzein is stable over a broad pH range from providing both quantitative and qualitative synergy.
2.5 to 8 and heat stable at 98°C for 2 hours (Hellekant Also, it improves stability, flavor and mouthfeel when
et al., 2005). blended with acesulfame-K and aspartame, either alone
or blended. It typically reduces the side taste of other
2.3.4 As a Sweetener sweeteners; for example, a blend of stevioside and
Brazzein represents an alternative to available brazzein is superior in taste quality to stevioside alone.
low-calorie sweeteners. As a protein, it is safe for Brazzein has been expressed in yeast (Guan et al.,
diabetics and very soluble in water (> 50mg/mL) (Birch 1995), fruits and vegetables to increase their sweetness
G. Gerard, 2000). and in grains to be economically extracted and used as
When blended with other sweeteners, sweeteners sweetened flour (Faus, 2000).
such as aspartame and stevia, brazzein reduces side
aftertaste and complements their flavor (Halliday, 2.3.6 Brazzein Controversy
2008). Despite the fact that the sweet taste of the berries
Unlike other natural sweeteners, apart from was well known in West Africa, the University claims
thaumatin, its sweet profile is closer to sucrose. Unlike that the sweet compound (brazzein) is its own invention
other sweet-tasting proteins, it can withstand heat, and admit to no connection with Gabon.
which makes it suitable for any industrial food This fact, which involved the appropriation of
manufacture (Hellekant et al., 2005). legal rights by means of patents over indigenous

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Botanical and Protein Sweeteners Agboola et al

biomedical knowledge without compensation to the known to exist in the Princes Islands, Angola, the
indigenous groups, is considered an act of Biopiracy by Central African Republic, Uganda and Indonesia.
GRAIN and Green Peace (The European Patent Katemfe is a rhizomatous, perennial and
Directive, 2008). monocotyledonous herb, propagating itself by rhizomes
(Onwueme et al., 1979). About 2 or 2.5m long petioles
arise from the rhizomes depending on the age and the
environment of the plant. At the end of these long
petioles are large, broad and oval papery, tough,
versatile leaves that are about 45cm long and 30cm
broad. The leaves are ovate-elliptic rounded, truncate at
the base, and shortly acuminate at the apex.
The inflorescence of T. daniellii usually arises
from the lowest node and may be simple or forked with
spikes about 8 to 10cm in length and bracts, usually
umbricate, about 3 to 4cm in length (Tomlinson, 1961).
The flowers that may be as long as the bracts form in
Plate I: Showing the oval shape of the fruit. short spikes close to the ground at the base of a swollen
petiole. Sepals are broadly linear and about 1cm in
length. Corolla tubes are short and lobes are oblong and
about 2.5 to 3cm long. As many as 10 to 12 purplish-
pink flowers may form on each inflorescence, but
usually only 2, 3 or 4, rarely more than 4, of these form
matured fruits. The plant flowers most of the year, but
is most prolific from July until late October, followed
by fruit formation, maturing and ripening from January
until mid-April (Onwueme et al., 1979).
The fruit grows on short stalks close to the ground
and may be covered with plant debris as it clusters on
the soil surface within the reach of insects and rodents.
Plate J: showing the arrangement of the fruit. It is pyramidal or trigonal in shape, maturing from a
dark-green through brown to crimson or bright-red
colour when fully ripe and may weigh between 6 and
30g depending on whether it has one, two or three
seeds. Within the fruit are the black hard seeds that are
covered by a thin layer of sticky, transparent gel. The
seeds look more like stones when dried, obviously
showing its hardness and impervious nature. It also has
a soft, fleshy and juicy cap called an aril, which
contains sweeter substances (Onwueme et al., 1979).
Local uses are multipurpose, ranging from
cultivation as a fetish plant in Gabon to collecting
leaves for wrapping and boiling food in Ghana
Plate K: showing the fruit mesocarp containing the sweet protein. (Facciola, 1998) (Fig. 2). Large quantities of the fruit
are consumed by the local people to sweeten over
2.4 Sweet Prayer Plant fermented palm wine and sour foods. From the aril of T.
daniellii, an intensely sweet, non-toxic and heat stable
 Scientific name: Thaumatococcus daniellii protein – thaumatin - is extracted, used as a sweetener
Benth. or a taste modifier in beverages, desserts, chewing gum
 Family: Marantaceae and pet foods.
 Common name: Sweet prayer plant, Katemfe
 Origin: West Africa 2.4.1 Thaumatin
Thaumatin is a low-calorie sweetener and
The sweet prayer plant or katemfe flavour modifier. The substance, a natural protein, is
(Thaumatococcus daniellii Benth.) grows throughout often used primarily for its flavour-modifying
the hot, humid tropical rainforest and coastal zone of properties and not exclusively as a sweetener (Green,
West Africa. Its natural habitat is the undergrowth of 2001).
forest trees. T. daniellii is particularly found in southern The thaumatins were first found as a mixture of
parts of Ghana, Cote d’Ivoire and Nigeria. It is also proteins isolated from the katemfe fruit

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Botanical and Protein Sweeteners Agboola et al

(Thaumatococcus daniellii Bennett) of West Africa. kilodalton protein from olive pulp, which was purified
Some of the proteins in the thaumatin family are natural and confirmed to cause an allergic reaction in a skin
sweeteners roughly 2000 times more potent than sugar. prick test. The protein had amino acid sequence
Although very sweet, thaumatins taste is markedly ATFXIVNQXTYTVXAAASP, which is similar
different from sugars. The sweetness of thaumatin (homologous) to that of thaumatin-like proteins
builds very slowly. Perception lasts a long time, leaving (Palomares et al., 2008).
a liquorice-like aftertaste at high usage levels. It
consists of 207 amino acid residues with eight 2.4.3 Production
intramolecular disulfide bonds and contains no free Within West Africa, the katemfe fruit has been
cysteine residues. It aggregates upon heating at pH 7.0 locally cultivated and used to flavor foods and
above 70oC, whereupon its sweetness disappears beverages for some time. The fruit's seeds are encased
(Kaneko and Kitabatake, 1999). Thaumatin is highly in a membranous sac, or aril, that is the source of
water soluble, stable to heating, and stable under acidic thaumatin. In the 1970s, Tate and Lyle began extracting
conditions. thaumatin from the fruit. In 1990, researchers at
Unilever reported the isolation and sequencing of the
2.4.2 Biological Role two principal proteins found in thaumatin, which they
Thaumatin production is induced in katemfe in dubbed thaumatin I and thaumatin II. These researchers
response to an attack upon the plant by viroid were also able to express thaumatin in genetically
pathogens. Several members of the thaumatin protein engineered bacteria.
family display significant in vitro inhibition of hyphal Thaumatin has been approved as a sweetener in
growth and sporulation by various fungi. The thaumatin the European Union (E957), Israel, and Japan. In the
protein is considered a prototype for a pathogen- United States, it is a Generally Recognized as Safe
response protein domain. This thaumatin domain has flavoring agent (FEMA GRAS 3732).
been found in species as diverse as Oryza sativa and Thaumatin is a pertinacious substance with a
Caenorhabditis elegans. Thaumatins are pathogenesis- very high sweetening capacity than sugar (3000 times).
related (PR) proteins, which are induced by various The gene to produce Thaumatin has been genetically
agents ranging from ethylene to pathogens, and are engineered into Escherichia coli and other micro-
structurally diverse and ubiquitous in plants (Palomares organization so that it can be produced on a large scale.
et al., 2008). They include thaumatin, osmotin, tobacco
major and minor PR proteins, alpha-amylase/trypsin 2.4.4 Uses of the Fruit
inhibitor, and P21 and PWIR2 soybean and wheat leaf The fruit of T. daniellii has diverse uses because
proteins. The proteins are involved in systematically of the presence of the protein, thaumatin which is
acquired resistance and stress response in plants, utilized for various uses. Industrially, thaumatin which
although their precise role is unknown. Thaumatin is an is present in the mesocarp of the fruit has a high
intensely sweet-tasting protein (on a molar basis about pharmaceutical potential in drugs, confectioneries and
100,000 times as sweet as sucrose) found in the West mineral drink manufacturing as a sweetener.
African shrub Thaumatococcus daniellii: it is induced They indicated it to be 100,000 times sweeter
by an attack by viroids, which are single-stranded than sugar; hence, it is used as a sweetener and flavor
unencapsulated RNA molecules that do not code for enhancer. Thaumatin contains many amino acids, some
protein. The thaumatin protein I consist of a single of which give it a lingering aftertaste that limits its
polypeptide chain of 207 residues. human use to products such as medicines. It is also used
Like other PR proteins, thaumatin is predicted to to enhance flavor of pet foods and to flavor beverages
have a mainly beta structure, with a high content of (Higginbotham, 1986).
beta-turns and little helix. Tobacco cells exposed to The thaumatin gene can be engineered directly
gradually increase salt concentrations develop a greatly into selected fruits and vegetable crops to improve their
increased tolerance to salt, due to the expression of flavor and sweetness (Zemanek and Wasserman, 1995).
osmotin, a member of the PR protein family (Singh et Generally, natural sweeteners may be both nutritive and
al., 1999). Wheat plants attacked by barley powdery flavourable and thus popular both as food and
mildew express a PR protein (PWIR2), which results in flavouring. However, because common sugar and other
resistance against that infection. The similarity between nutritive sweeteners such as honey and corn syrup are
this PR protein and other PR proteins to the maize associated with health problems (such as obesity and
alpha-amylase/trypsin inhibitor has suggested that PR tooth decay) or even a principal cause of this life
proteins may act as some form of the inhibitor (Mauch threatening sickness (diabetes), efforts have been made
et al., 2004). from 19th century to produce non-nutritive sweetness
Thaumatin-like protein has been found in olive which may be natural or synthetic includes saccharin,
fruit, and it can cause allergic reactions. A worker in an aspartame, cyclamates and thaumatin. It combines well
olive-oil mill in Jaen, Spain had respiratory symptoms with monosodium glutamate and is used in typical
at work. His blood serum was used to extract a 23-

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Botanical and Protein Sweeteners Agboola et al

Japanese seasonings as well as in chewing gum The outer bark of the stalk is used mainly for
(www.Britannica.com). ornamental bags. The inner bark is used as a sponge
and pulp while the stalk is used to wave fish trap. The
2.4.5 Uses of Stalk and Roots roots are used for medicinal purpose to cure ailments
The stalk Thaumatococcus daniellii is used for such as stomach ache and typhoid fever (Van Der Wel
weaving mats, fish trap and ornamental bags and is also and Loeve, 1972).
used as a sponge for pulping roll.
2.5 Serendipity Berry

 Scientific name: Dioscoreophyllum cumminsii

 Family: Menispermaceae
 Common name: Serendipity berry
 Origin: West Africa

The search for natural sweeteners has prompted

intensive research on plants with sweetening properties.
Dioscoreophyllum cumminsii (Stapf) Diels is a
dioecious, semelparous annual Liana found as a late
Plate L: Showing the aril of the fruit. successional, understorey species in West African semi-
deciduous forests. The fruits and subterranean tubers
are intensely sweet and are both edible. The active
principle in Dioscoreophyllum cumminsii Diels fruits
(commonly known as serendipity berries) as a low
molecular weight protein named monellin (after the
Monell Chemical Senses Centre in Pennsylvania,
U.S.A). The sweetening substance in D. cumminsii is a
protein (monellin), which is 3000 times as sweet as
sugar. As a non-carbohydrate sweetener, monellin has
potentially been used in combating tooth decay, in low-
calorie diets for diabetics and dieters and as a sweetener
in the food industry. Unfortunately, it is one of the
Plate M: Showing the trigonal shape of the fruit. threatened plant species in the country because of
massive habitat loss and fragmentation. Conservation is
now needed as a salvage program. Population size is a
major determinant of extinction risk. This has prompted
the application of growth and population dynamics
models to viability analysis of wild species. The aim of
population viability analysis (PVA) is to determine the
minimum viable population size (MVP) or area (MVA)
of a particular species. In this paper, the MVP was
estimated for D. cumminsii uses genetic models. The
models simulated minimum effective population size of
6040 individuals’ ha−1 in the ratio of 5032 males to
1008 females. This suggests that to retain evolutionary
Plate N: Showing the long petiole of the leaf. potential, D. cumminsii requires an effective population
size of more than the 500–5000 range considered
adequate for many other species.

2.5.1 Monellin
Monellin, a sweet protein, consists of two non
covalently associated polypeptide chains, an A chain of
44 amino acid residues and a B chain of 50 amino acid
residues. The protein can be purified from the fruit of
Dioscoreophyllum cumminsii grown in West Africa and
is approximately 100,000 times sweeter than sugar on a
molar basis and several thousand times sweeter on a
weight basis (Kohmura, 1998). Single-chain monellin
Plate O: Showing the leaf shape.
(SCM), which is an engineered 94-residue polypeptide,

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Botanical and Protein Sweeteners Agboola et al

has proven to be as sweet as native two-chain monellin Capparis masaikai, known as Mabinlang, grows in
and is more stable than the native monellin at high the subtropical region of the Yunnan province of China
temperature and in acidic environments. Native and bear fruits of tennis-ball size. The mature seeds are
monellin is relatively sensitive to heat or acid treatment, used in traditional Chinese medicine. Capparis is a
which may cause separation of the sub-units and flowering plant genus in the family Capparaceae. These
denaturation of the protein. Despite misgivings about plants are shrubs or lianas and are collectively known
the stability of the protein to heat and acid, downstream as caper shrubs or caper bushes. Capparis species occur
processes have been established. Its D-enantiomer has over a wide range of habitat in the subtropical and
been crystallized and analyzed by X-ray tropical zones.
crystallography at 1.8 Å resolutions. Two crystal forms Shrubs or climbers, to 7.5m tall. New branches
(I and II) were found under crystallization conditions reddish, slightly flat, with vertical ridges and stipular
similar, but not identical, to the crystallization stripes, densely shortly rust-colored tomentose. Stipular
conditions of natural L-monellin (Lee et al., 1999). One spines to 5mm but often absent on flowering twigs,
NMR study of a non-sweet analog in which the AspB7 stout, recurved, sulcate, hollow, base inflated, apex
of protein was replaced by AbuB7 (L-2-Aminobutylic sharp. Petiole 1.2-2.1cm, ca. 2mm in diam., trichomes
acid), showed similar 3-dimensional structures of these like those on branches; leaf blade elliptic, oblong, or
two proteins, indicating that the lack of the beta- sometimes elliptic-obovate, 7-20 × 3.5-9cm, nearly
carboxyl group in the AbuB7 analog is responsible for leathery, often dark reddish brown when dry, abaxially
the loss of sweetness. Recent research on identifying densely rust-colored shortly tomentose but glabrescent,
binding sites on the receptor by means of structure-taste adaxially almost glabrous, midvein slightly broad,
relationships, found that four monellin analogues, abaxially lavender and raised, and adaxially impressed,
[AsnA16]-, [AsnA22]-, [GlnA25]-, and [AsnA26]- secondary veins 6-10 on each side of midvein and
monellin were 7500, 750, 2500, and 5500 times as abaxially lavender and slightly raised, reticulate veins
sweet as sucrose on a weight basis, respectively. Thus, not obvious, base rounded to broadly cuneate, apex
among them, [AsnA22]-monellin and [GlnA25]- rounded to obtuse or sometimes acute to acuminate.
monellin were less sweet than the native monellin Inflorescences axillary subumbels or axillary and
(Tancredi et al., 2004). terminal together forming a 10-20cm panicle, 3-8-
flowered, densely rust-colored shortly tomentose, often
with abortive leaflets; peduncle 1-5cm. Pedicel 3-4cm.
Sepals 8-12 × 5-8mm, outside densely rust-colored
shortly tomentose, inside glabrous; sepals of outer
whorl inwardly concave to hemispheric, leathery;
sepals of inner whorl slightly inwardly concave, thin.
They are also used as sweets; when chewed the
seeds elicit a sweet taste (Liu, 1993). The origin of the
sweet taste was identified as sweet-tasting proteins
named Mabinlins. They are highly sweet, 100-400
times sweeter than sucrose on a weight basis (Nirasawa,
Plate P: Showing the mode of stem (liana). 2001). Caper bushes are mainly used by humans for
their fruit, which are rich in micronutrients. The fruit of
other species, such as Karir (C. decidua), are also used
for cooking; C. mitchellii and the Wild passionfruit (the
local subspecies of C. spinosa) are well-known bush
tucker in Australia. Mabinlang seeds (C. masaikai) are
eaten as sweets.
Mabinlang is also used in Traditional Chinese
Medicine. Aspalathos, the root of a shrub contained for
example in the sacred Ancient Egyptian incense
(kyphi), is sometimes considered to be C. spinosa.
Other species have also recorded uses in herbalism and
folk medicine; dedicated research is largely lacking,
Plate Q: Showing the type of fruit. however. Mabinlins are sweet-tasting proteins found in
Mabinlang seed (and possibly in other Capparis
2.6 Mabinlang species); at least one of them is highly resistant to heat.
The market for mabinlins is presently not large, but this
 Scientific name: Capparis masaikai is mainly due to insufficient supply rather than to lack
 Family: Capparaceae of demand.
 Common name: Mabinlang
 Origin: China

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Botanical and Protein Sweeteners Agboola et al

Caper bushes from arid regions - chiefly C.

decidua - are highly useful in landscape gardening,
afforestation and reforestation. They can stop soil
erosion and preserve agricultural land. Any large-
flowered species can be used to attract butterflies. The
Crimson Rose (Atrophaneura hector), a spectacular
swallowtail butterfly of South Asia, likes to visit
flowers of C. spinosa in the winter.

2.6.1 Mabinlin
Mabinlin is a sweet protein with the highest
known thermostability. It is derived from Capparis
Plate R: Showing the type of flower.
masaikai and its sweetness was estimated to be around
400 times that of sucrose on weight basis. It consists of
an A chain with 33 amino acid residues and a B chain
composed of 72 residues. The B chain contains two
intramolecular disulfide bonds and is connected to the
A chain through two intermolecular disulfide bridges
(Guan et al., 2000). Its heat stability is due to the
presence of these four disulfide bridges. The sweetness
of Mabinlin-2 is unchanged after 48 hr incubation at
boiling point and of Mabinlin-3 and -4 is unchanged
after 1 hr at 80°C (Kohmura and Ariyoshi, 1992).

Plate S: Showing seeds which contain the sweet protein.

Plate T(b): Showing the berry.

Plate T(a): Showing the berry.

Table 1. Showing the comparison between the sweet proteins.

Protein Miraculin Curculin Brazzein and Pentadin Thaumatin Monellin Mabinlin

Family of Plant Sapotaceae Hypoxidaceae Pentadiplandraceae Marantaceae Menispermaceae Capparaceae
Synsepalum Curculigo Pentadiplandra Thaumatococcus Dioscoreophyllum
Scientific Name Capparis masaikai
dulcificum latifolia brazzeana daniellii cumminsii
Common Name Miracle fruit Lemba Oubli Sweet prayer plant Serendipity berry Mabinlang
Origin West Africa Malaysia Gabon and Cameroon West Africa West Africa China
Mode of Stem Shrub Perennial herb Shrub Perennial herb Annual liana Shrub
Degree of Sweetness in
2000 430-2070 500-2000 3000 100000 400
Relation to Sucrose
o o o o o o
Thermostability 100 C for 1hr 50 C for 1hr 98 C for 2hrs 70 C for 1 hr 50 C for 1hr 100 C for 48hrs

Ph Range 3-12 3-11 2.5-8 1-6 2-9 4-7.5

Number of Amino Acid
191 228 54 207 94 105
(Residue)

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CHAPTER THREE

3. Interaction of Sweet Proteins and their sweeteners and sweet proteins interact with the same
Receptor receptor, the human T1R2-T1R3 receptor. Studies have
shown that the T1R3 receptor protein is encoded by the
Humans detect taste with taste receptor cells. These Tas1r3 gene involved in transduction of sweet taste
are clustered in the taste buds. Each taste bud has a pore (Inoue et al., 2004).
that opens out to the surface of the tongue enabling Recently, it has been found that T1R3-independent
molecules and ions taken into the mouth to reach the sweet- and umami-responsive receptors and/or
receptor cells inside. There are five primary taste pathways also exist in taste cells (Damak et al., 2003).
sensations salty, sour, sweet, bitter and umami. Sweet
and umami (the taste of monosodium glutamate) are the
main pleasant tastes in humans. T1Rs are mammalian
taste receptors that assemble two heteromeric G-
protein-coupled receptor complexes T1R1+T1R3, an
umami sensor, and T1R2+T1R3, a sweet receptor
(Zhao et al., 2003).
Sweet and taste-modifying proteins interact with
the T1R2-T1R3 receptor with a different mechanism
compared to small molecular weight compounds
(Tancredi et al., 2004). Recently, it has been shown that
the T1R2-T1R3 receptor has many characteristics
similar to the mGluR, apart from some minor
differences in the active site region.
The major work by Kunishima et al., solving the
crystal structure of the N-terminal active site region of
the subtype 1 of mGluR both free and complexed with
glutamate has helped a lot in understanding the
mechanism of interaction between ligand and T1R2-
T1R3 receptor. Their structural work on mGluR and its
N-terminal domain showing considerable
conformational change induced by the glutamate
complexation (Kunishima et al., 2000). The 'Active'
and 'resting' conformations of m1-LBR, an extracellular
ligand binding region of mGluR, is modulated by the
dimer interface. The protomer can form 'open' or
'closed' confirmations and is made up of two domains
namely LB1 and LB2. The population of active
conformers depends on ligand binding, i.e. the so called
'closed-open_A'. The ligand-free receptor exists as two
different structures, free form I (open-open_R), the
'resting' conformation with two open protomers and free
form II (closed-open A), nearly identical to the
complexed form. The mechanism suggested by these
structures is that the receptor is in dynamic equilibrium,
and that ligand binding stabilizes the 'active' dimer.
There are thus two ways, in principle, to activate the
receptor: first, to complexate form I with the proper
ligand (glutamate for the mGluR, aspartame or any
other small molecular weight sweetener for the T1R2-
T1R3 receptor) and second, by shift the equilibrium
between free form I and free form II in favor of free
form II.
The exact mechanism of interaction of sweet
proteins with the T1R2-T1R3 sweet taste receptor has
not yet been elucidated. Low molecular mass

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Botanical and Protein Sweeteners Agboola et al

CHAPTER FOUR

4. List of Sugar Substitutes and their Equivalent such as "no sugar added" ice cream. Erythritol is
Sweetness to Sugar gaining momentum as a replacement for these other
sugar alcohols in foods as it is much less likely to
The three primary compounds used as sugar produce gastrointestinal distress when consumed in
substitutes in the United States are saccharin, large amounts. In many other countries, xylitol,
aspartame, and sucralose. Maltitol and sorbitol are often cyclamate and the herbal sweetener stevia are used
used, frequently in toothpaste, mouthwash, and in foods extensively.

4.1 Natural Sugar Substitutes

Table 2. Showing the sweetness and energy densities of natural sugar substitutes in comparison to those of sucrose.

Sweetness Sweetness by Energy

Name Notes
by weight food energy density
Brazzein 800 Protein
Curculin 550 Protein
Erythritol 0.7 14 0.05
Glycyrrhizin 50
Glycerol 0.6 0.55 1.075 E422
Hydrogenated starch
0.4–0.9 0.5×–1.2 0.75
hydrolysates
Inulin
Isomalt 0.45–0.65 0.9–1.3 0.5 E953
Lactitol 0.4 0.8 0.5 E966
Luo Han Guo 300
Mabinlin 100 Protein
Maltitol 0.9 1.7 0.525 E965
Malto-
oligosaccharide
Mannitol 0.5 1.2 0.4 E421
A protein that does not taste sweet by itself, but modifies
Miraculin
taste receptors to make sour things taste sweet temporarily
Naturally-occurring sweetener isolated from the plant
Monatin
Sclerochiton ilicifolius
Monellin 3,000 Protein; the sweetening ingredient in serendipity berries
Osladin
Pentadin 500 Protein
Sorbitol 0.6 0.9 0.65 E420
Extracts known as rebiana, Truvia, PureVia; mainly
Stevia 250
containing rebaudioside A, a steviol glycoside
Tagatose 0.92 2.4 0.38
Thaumatin 2,000 Protein; E957
Xylitol 1.0 1.7 0.6 E967

4.2 Artificial Sugar Substitutes

Table 3. Showing the sweetness and energy densities of artificial sugar substitutes in comparison to those of sucrose.

Sweetness
Name Trade name FDA approval Notes
(by weight)
Acesulfame potassium 200 Nutrinova 1988 E950
Alitame 2,000 (Withdrawn) Pfizer
Aspartame 160–200 NutraSweet, Equal 1981 E951
Salt of aspartame-acesulfame 350 Twin sweet E962
Cyclamate 30 (Banned, 1969) E952, Abbott
Dulcin 250 (Banned, 1950)
Glucin 300
Neohesperidin dihydrochalcone 1,500 E959
Neotame 8,000 NutraSweet 2002 E961
P-4000 4,000 (Banned, 1950)
Saccharin 300 Sweet'N Low 1958 E954
Sucralose 600 Kaltame, Splenda 1998 E955, Tate & Lyle

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CHAPTER FIVE

5. Conclusion [11]. Hellekant, G. and Danilova, V. (2005). Brazzein a

Small, Sweet Protein: Discovery and
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