Taste-modifying proteins are a natural alternative to artificial sweeteners and flavour enhancers and

have been in use in some cultures for centuries. Use has been limited by the stability and availability
of these proteins, but recently advances in biotechnology have been made that will increase their
availability. These include production in transgenic organisms and protein engineering to increase
stability. Taste-modifying proteins will be available for much wider use in the food industry, and will
reduce dependence on synthetic alternatives. # 1998 Elsevier Science Ltd. All rights reserved

Most taste-active substances are of low molecular weight and are capable of rapidly travelling the
small distance from the surface of food, through salivary mucilage, to the taste receptor. Though this
distance is very small, diffusion is presumably an important parameter in the process and confining
perception to low molecular mass substances allows for a more rapid perception of food. It was
therefore surprising when, in the 1960s, higher molecular mass taste-modifying proteins (TMPs)
were discovered that interact with the taste receptors in a potent and specific manner (Table 1).

TMPs are found in the fruit or seed of several Old World plants and show no primary sequence
similarity.

They are a biochemically heterogeneous group which arose separately but have converged to
perform a common natural function of attraction to fruit and to aid seed dispersal. In the past,
emphasis has been on the sweet flavour of some of these proteins, but sweetness is only one of many
traits these proteins possess. A complex range of taste attributes, such as flavour-enhancing,
bitterness suppression and Umami are commonly cited.

Group 1 proteins are sweet tasting at concentrations as low as 8–10 M. Group 2 proteins modify
perception of taste and cause sour substances to be perceived as sweet.

It may be argued that TMPs are of value to science, particularly as probes in the study of gustation,
but that they have no practical value to the food industry, especially when they are compared with
simpler chemical ingredients that perform similar functions. The inclusion of the most important
TMPs—thaumatin, monellinand miraculin—into research programs for new sweeteners was a
response to the ban on cyclamate use in the USA in 1969 which caused public disillusion with
substances perceived as synthetic. TMPs are natural and have been in cultural use for centuries and
are more acceptable to the public than synthetic ingredients.

These proteins have been the subject of interest to the food industry for many years. However, they
have not been widely used because of their limited availability and, in fact, most traded protein is
still harvested from populations of plants distant from their main markets in Japan, Europe and USA
[1].

Many groups have used biotechnology to try to solve the problem of supply and the first efforts were
published as early as 1982 [2]. However, limited success was achieved until recently. New methods
have been developed that have the potential to remove limits on availability and allow TMPs to
become a practical proposition for the food industry and for food scientists.

TMPs have long been in traditional use by West Africans for flavour improvement and bitterness
suppression of food and drink. They are used to improve the flavour of maize dishes such as agidi,
and in beverages such as palm wine or tea. In modern times they have found uses in the food
processing industry as sweetening agents, flavour enhancers, and animal fodder supplements. These
proteins can act at extremely low concentrations, and because of this low effective dose they are
effectively non-cariogenic and acceptable for diabetics in flavour and sweetening formulations. At
present, thaumatin is the only TMP which is harvested on a large scale and is one of the few intense
sweeteners that has undergone extensive safety evaluation [3].

The use of TMPs depends on their isolation in a stable form and separation from materials in the fruit
that would rapidly cause their breakdown. Proteins vary greatly in their properties. However, the
large number of laboratory studies of TMPs have revealed synthesis and separation methods that
can be adapted to larger-scale methods (Table 2). For example, cation-exchange methods have
shown that TMPs are generally positively charged, and this property has been exploited several times
in downstream processes by binding to suitable negatively charged resins. The most successful
methods will be described in succeeding sections.

Thaumatin purification

Thaumatin is a component of the fruit of Thaumatococcus daniellii which grows in forest areas of
Western Africa. The intense sweetness and greatly desirable taste properties of Thaumatococcus
fruit came to the attention of Western science in 1855 when it was studied by the amateur botanist
William Daniell [4]. However, it remained a curiosity until the 1960s when the cyclamate ban
stimulated surveys for natural alternatives to synthetic sweeteners and work on its chemical nature
began in earnest.

The starting material for thaumatin extraction is Thaumatococcus fruit, which consists of a leathery
pericarp which contains up to three seeds. Each seed is coated with a dense polysaccharide gel, and
tipped with a soft white aril that contains the thaumatin. To date the most successful process is that
developed by Tate and Lyle [5] (see Fig. 1). Whole fruits are mechanically disrupted to expose the aril
to an aqueous solution containing aluminium sulphate and sodium metabisulphate (an antibrowning
substance). Extraction with aluminium salts reduces swelling of the polysaccharide gel, which may
absorb thaumatin. The pH of the liquor containing aluminium sulphate is approximately 3.6, which
also results in precipitation of contaminating material. While aluminium is associated with human
disease [6], because thaumatin is used at extremely low concentrations, the amount of metal added
to food is insignificant. Laboratory-based experiments have shown that cation-exchange methods
are e€ective for the purification of thaumatin, and this is also true in larger-scale methods. Thaumatin
may be bound to cation-exchange media such as sulphopropyl resins either in batch methods or in
large columns. An advantage of binding to the resin is that separation from materials that cause
browning of the extract may be optimized. The extract is preserved by ultrafiltration followed by
freeze drying to form an intensely active powder for use in food formulations.

More recent work on downstream processing of thaumatin has focused on use of aqueous two-phase
partition systems (ATPSs). These systems take advantage of the property that a solution containing
a polymer, such as PEG, and a solution containing a relatively high concentration of salts are not
completely miscible and will form two distinct aqueous phases.
This method may be used in protein purification as proteins distribute themselves unequally between
the two aqueous phases. ATPSs are similar in principle to organic phase partitioning, but the aqueous
phases are much less likely to denature proteins. The method can be used as a simple first stage of
purification for crude cell extracts as an alternative to expensive centrifugation processes for removal
of cell debris and also as a first stage of purification. ATPSs have been used in the purification of
thaumatin from crude bacterial cell lysates [7, 8] (Fig. 1).

Though they have not yet been used in any commercial process for extraction of TMPs they will
provide a simple but powerful tool for purification of TMPs from transgenic sources.

Purification of monellin

Monellin also came to the attention of Western science in 1960s in response to the cyclamate ban
[9]. Monellin is a heterodimer that is relatively sensitive to heat or acid treatment which may cause
separation of the subunits and denaturation of the protein. Despite misgivings about the stability of
monellin to heat or acid, downstream processes have been established (Fig. 2). Monellin may be
purified from the fruit of Dioscoreophyllum cumminsii grown in West Africa, or in plants grown in the
USA [4] (see Fig. 2). Berries are picked and removed from stems before mechanical separation of the
seed from the berry pulp. This is an important process as the seed contains the very bitter substances
colombin and isocolombin [10]. The sweet liquor is then dialysed at 4–10 C to remove low molecular
weight contaminants then UV irradiated. This process has been empirically determined to separate
protein and contaminating carbohydrates. Contaminating protein is precipitated by addition of
polyvinyl alcohol to 20% followed by continuous flow centrifugation (Fig. 2). Natural monellin has not
been marketed on a large scale in the food industry, largely due to the challenge of large scale
purification of a protein vulnerable to heat or acid.

Purification on miraculin

Miracle fruit or Synsepalum dulcificum fruit are ellipsoid, red and have a palatable pulp containing
the seed.

William Daniell [11] first described their property of altering the perception of taste such that sour
foods like lemon or vinegar taste sweet. This alteration of taste perception persists for many minutes
after miracle fruits are consumed.

While fresh berries of S. dulcificum are very potent, damaged fruit or crude extracts become quickly
denatured, and so the aim of protein purification is to separate miraculin from factors in a crude
extract that cause protein breakdown, followed by preservation of miraculin. For purification, the
fruits of S. dulcificum are homogenized in 1% PEG 20,000 in water.

Dissolution of miraculin is achieved by adjusting the pH to 7 with saturated sodium carbonate

solution. The suspension is filtered through glass and added to an equal volume of acetone with
stirring. The precipitate is harvested by centrifugation at 1200 g for 10 min and is washed with 2
volumes of 50% v/v acetone. The washed precipitate is dissolved in 0.1 M potassium phosphate
buffer pH 7.0 (see Fig. 2). Miraculin may be used as a food additive to alter the taste of food, such as
by masking sourness in some foods, or may be taken separately, before consuming a meal.
New technologies

The processes described above are adequate to produce TMPs for the food industry, but they are
limited by the expense of protein purification and by the limited availability of fruit to process. An
obvious solution to these limits is suggested by the protein nature of these ingredients and by their
amenability to genetic engineering and biotechnology. Many attempts have been initiated and there
are several examples of biotechnologies at various stages of success (Table 3). However, there are
two examples of biotechnologies that are on the verge of success and which will greatly increase the
amount of TMPs available to the food industry.

Many groups have used cDNAs isolated from the natural sources of TMPs, though with limited
success [12]. Important breakthroughs have only come with the use of synthetic DNAs adapted to
function within defined host organisms. Synthetic DNA has been used [13] to generate strains of
Saccharomyces cerevisiae that secrete thaumatin to a level of 300 mg/1 of medium.

This protein is relatively easy to purify and the method is scalable. Genetic engineering has been used
to solve problems of stability in monellin, which has prevented adequate exploitation of this protein
in the past, by development of a single chain monellin [14]. This consists of a synthetic DNA coding
sequence for the A and B heterodimer peptides fused with a linker to create a single polypeptide that
cannot dissociate when heated or exposed to acid treatment. The improved gene for single-chain
monellin combined with use of the food yeast Candida utilis can be used to produce monellin to
greater that 50% of total protein. Because the singlechain monellin is much more stable at high
temperatures and at low pH, a simpler method of purification may be used. Yeast cells are disrupted
and then treated at 60 C for 10 min followed by treatment at pH 4.5 for 1 h at 4 C. Centrifugation
produces single-chain monellin, which is sweet and pure. Pilot fermentation experiments have so far
obtained 50 g of monellin from 7 kg of cells.

Future trends

Bulk sweeteners and flavoursome ingredients such as sugars are essential in foods and food
processing and contribute many benefits to foods. However, foods can be substantially improved by
the use of intense sweeteners and flavour enhancers and so there is considerable public demand for
foods that contain them. Unfortunately many artificial substances such as chemical intense
sweeteners and flavour enhancers have attracted public dissatisfaction. For example cyclamate was
suspected of causing health problems [15] and was the subject of a ban in the UK and USA [16].
Artificial sweetners may be replaced in speciality applications by TMPs that confer similar properties
to food and are perceived by the public as a more natural alternative, which has been enjoyed in
West African cultures for centuries [17].

Recent new advances are removing the barrier of scarcity that has limited the use of TMPs, and
further new advances are expected which will exploit structural and biochemical studies of TMPs.
Because of their protein nature, TMPs are amenable to molecular biology and genetic engineering
and this means they have a special potential for improvement. Knowledge of the structure of
monellin found by X-ray crystallography has been used to produce a single-chain monellin with
improved stability at high temperatures and acid conditions; these features greatly improve large-
scale purification. Modification of the coding sequence of thaumatin has also greatly improved yields
in transgenic yeast fermentations.

Molecular biology will also be used in the future to manipulate the quality rather than the quantity
of TMPs available. Properties that may be manipulated in the future are taste intensity, aftertaste
and taste profile which have already been modulated in biochemical experiments. Once the
molecular basis of these changes in taste have been elucidated, then protein engineering may be
used to alter the taste properties of TMPs and allow the production of tailor-made molecules on an
industrial scale.