Sweeteners and sweet taste enhancers in the food industry

This review will discuss the current knowledge on the existing alternatives for sugar replacement and studies that are being done towards the identification of substances with enhancement


Introduction
Since the end of the twentieth century, obesity has been one of the greatest public health concerns worldwide (Disse et al., 2010), with consequences beyond esthetic issues, as it is considered a risk factor for many different diseases (Fujiwara et al., 2012;Ribeiro & Santos, 2013), such as cardiac conditions, hypertension and diabetes.To limit the prevalence of diseases related to the excessive consumption of sugar, researchers have worked on the isolation of naturally occurring compounds and development of synthetic sweeteners.These sweeteners are able to replace sugar totally or partially, but are rarely as satisfying as the full sugar alternative because they fail to trigger physiological satiety mechanisms (Raben et al., 2002;Swithers et al., 2010).
Due to the ongoing demand for alternative sugar substitutes, there has been a large research effort over the past several decades aimed at identifying the human genes coding for the sweet taste receptor.The use of high-throughput screening technologies and classical discovery approaches has enabled the discovery of novel sweeteners as well as sweet taste enhancers, known as positive allosteric modulators (PAMs), which are absolutely tasteless at the intended usage levels, but potentially enhance the activity of agonists of the sweet taste receptors and, therefore, sweetness perception.
Onnova Market Onsights published that over 16% of global soft drinks launched in the 2016 used either no added sugar, low sugar or sugar free claims.Along with the interest in sugar reduction, there has also been a continuing focus on clean label formulations, such as stevia and monk fruit.Soft drinks accounted for 20% of recent launches featuring stevia (Gelski, 2016).

Practical Application:
The food industry uses more and more sweeteners to supply the demand for alternative sugar substitutes in products with no added, low or sugar free claims.Along with the interest in sugar reduction, there has also been a continuing industrial focus on clean label formulations, such as in the natural sweeteners added products.To increase this knowledge, the article presents detailed information concerning potency, technological properties and food applications of synthetic and natural sweeteners.Furthermore, this work discusses the challenges and possible application of sweet taste enhancers in the food industry.
properties.Here, we also describe the advantages and possible applications of sweet taste enhancers in the food industry.

Sweet taste receptor
To evaluate the quality of food components, sensory information of taste is needed.On general, humans can detect at least five basic taste qualities: sweet, salty, umami, sour and bitter (Meyers & Brewer, 2008).The mechanism of detection of these taste qualities begins with the taste receptor on the apical membrane of taste receptor cells in the tongue.These receptors bind to taste substances, recognizing them and activate the taste cells which then transmit sensory information to nerve cells and into the brain (Strapasson et al., 2011).
Physiologically, sweet, bitter and umami tastes are mediated by G protein-coupled receptors (GPCRs), called type 1 taste receptors (T1Rs) for sweet and umami and type 2 taste receptors (T2Rs) for bitter taste.Type 1 taste receptor 3 (T1R3) subunit combines with type 1 taste receptor 2 (T1R2) subunit to form a sweet taste receptor, which is expressed in the oral cavity, where it provides input on the caloric and macronutrient contents of ingested food.This receptor recognizes all the compounds (natural or artificial) perceived as sweet by people (Belloir et al., 2017).Each of these has a section characterized by a large extracellular venus flytrap domain (VFT), which is stabilized in the closed conformation when a sweetener binds.Apparently, the sweet taste enhancers are most likely to bind to T1R2 subunit adjacent to the flytrap opening, linked to a transmembrane domain (TDM) via a short cysteine-rich domain (CRD), helping it to stay closed once the sweetener has struck (Nelson et al., 2001;Zhao et al., 2003).

Alternatives to sugar
Early in the 20th century, scientists have identified many synthetic and natural sweet-tasting compounds.Most of these compounds consist of substances with a very intense sweet taste that are used in small amounts to replace the sweetness of a much larger amount of sugar (Gardner et al., 2012) and are referred to as high potency (HP) sweeteners.Sweeteners are chemically diverse including natural sugars, sugar alcohols, natural and synthetic sweeteners, and sweet-tasting proteins.These substances have a continuous great interest as they assist the food industry in developing good-tasting reduced calorie products to meet the consumers' and health professionals' expectations (Klug & Lipinski, 2011).
The number of approved substances has increased substantially in the past three decades, allowing food developers to choose from a variety of sweeteners, as they differ from each other not only in potency, but also in mouthfeel, duration of sweetness, aftertaste, solubility and stability.These current alternatives to sugar enable manufacturers to use an ingredient or a combination of ingredients best suited for a given product, since no sweetener is perfect for all uses (Nabors, 2011).Synthetic or naturally occurring sucrose substitutes are required to exhibit a sucrose-like taste quality with properties such as demonstrated non-toxicity, non-cariogenic and no effects on blood glucose or insulin.Studies suggest that sweetness, when not accompanied by calories, might result in ambiguous psychobiological signals that confuses the body's regulatory mechanisms, leading to a loss of control over appetite and overeating (Bellisle & Drewnowski, 2007).
The usage of sweeteners in food and beverages is regulated according to the accepted daily intake (ADO) values, which define the estimated amount of a determined substance that a person can consume on average every day without risk, assigned by regulatory agencies following review of safety assessment studies (Kroger et al., 2006).
The Unites States Food and Drug Administration published in 2015 an issue regarding additional information about high-intensity sweeteners permitted for use in food in the United States.Saccharin, aspartame, acesulfame potassium, sucralose, neotame, advantame, steviol glycosides and Luo Han Guo fruit extracts were reviewed regarding approved use and safety for human consumption.For example, sucralose has been extensively studied and more than 110 safety studies were reviewed by FDA in approving the use as a general purpose sweetener for food (U.S. Food and Drug Administration, 2015).

Synthetic sweeteners
Many synthetic compounds were identified serendipitously without the understanding of the mechanism for sweetness in a time when taste and smell were key dimensions for evaluation, and this routine testing continued into the 20 th century.With the development of chromatography and spectroscopy methods, human routine sensory assessment of compounds was gradually discontinued.Nevertheless, the unintentional discovery of many sweet taste compounds continued in the years to follow (Schiffmann & Gatlin, 1993;Davies, 2010).
The majority of the currently available sweeteners in the world market are synthetic compounds, but their use depends on the granting of legislative approval, for which individual countries have their own regulatory requirements (Grenby, 1991;U.S. Food and Drug Administration, 2015).Despite the considerable market value of synthetic sweeteners, problems have been perceived with some of these compounds in terms of their safety, stability, cost, and/or quality of taste.As shown in Table 1, ten synthetic non-caloric sweeteners were discussed by diverse authors in terms of potency, technological properties and beverage and food applications.

Natural sweeteners
On recent years, as the incidence of overweight and diabetic people has increased worldwide, there has been a great demand for new alternative low or no-calorie sweeteners for dietetic and diabetic purposes (Kim & Kinghorn, 2002;Freitas et al., 2014).There are many artificial sweeteners, but their use is limited because they are often associated with harmful side effects (Tandel, 2011).As a consequence, the search for sugar substitutes of natural sources has led to the discovery of a number of substances that possess an intensely sweet taste or taste-modifying properties.Dver a hundred plant materials have been found to taste sweet because they contain large amounts of sugars and/or polyols or other sweet constituents.Although these sweeteners may have some caloric value, their contribution to energy intake is negligible in the amounts used (Sardesai & Waldshan, 1991;Bellisle & Drewnowski, 2007).
Several of these highly sweet natural products have either been commercialized as sweeteners or flavoring agents in some countries as pure compounds, compound mixtures and refined extracts or are under development.The potency, technological properties and beverage and food applications of 12 natural sweeteners were discussed by diverse authors and are summarized in Table 2.

Sweetness modulators
Non-caloric HP sweeteners are generally limited in taste quality (Riera et al., 2007;Li et al., 2011) by many negative attributes, such as off-tastes, delay in sweetness onset, low maximal sweetness response, lingering aftertaste and sweet tastes that desensitize the gustatory system.Researchers have studied ways of combining non-caloric substances with one another or with carbohydrate sweeteners with different properties in order to obtain a complimentary taste profile that could reproduce the taste of sugar to its best (Priya et al., 2011).However, they noted that only minimal sweet taste enhancement could be achieved with such blends and apparent synergy in taste tests could only be observed at lower sweetener levels.Even though sweeteners, in general, show small enhancement effects, blends and new sweetener formulations remain very popular among the food and beverage industries.
Recently, by the use of screening technologies and classical discovery approaches, researchers were able to identify completely new flavor ingredients, such as PAMs.These molecules are enhancers, which possess no taste on their own, but potentiate the taste of sugar (Dttinger & Hofmann, 2003;Dttinger et al., 2003;Davies, 2010).

Sweetener receptor positive allosteric modulators
Studies involving chimeric receptors, mutagenesis and molecular modeling have revealed that the GPCRs are very susceptible to allosteric modulation.By screening synthetic chemical libraries, PAMs have been identified for several members of the metabotropic glutamate receptor and GABA B receptor (Koizumi et al., 2011).Every synthetic PAM identified for these receptors binds to the TDM to enhance activity and sometimes  increases affinity of the natural ligand (glutamate, aminobutyric acid or calcium) within the VFT.While sweeteners bind near the hinge region to trigger the initial closure of the VFT, enhancers bind near the opening of the pocket and stabilize the closed conformation by strengthening the hydrophobic interactions between the two lobes and lowering entropic penalties of lobe closure.Upon binding to the receptors, PAMs exhibit little or no intrinsic agonist activity on their own or act both as agonists on their own and as enhancers for the endogenous agonists.
On order to improve the taste of HP sweeteners, PAMs may bind to the sweetener receptor without activating it, but do so in a manner that they cause carbohydrate sweeteners such as sucrose, fructose and glucose to bind with higher affinity.These molecules would be enhancers, or PAMs of the sweetener receptor.As they possess no sweetness activity, formulations associating carbohydrate sweeteners with PAMs should accurately replicate carbohydrate sweetener taste (Servant et al., 2011;DuBois & Prakash, 2012).
The first examples of PAMs for the sweet taste receptor were identified at the company Senomyx Oncorporation (San Diego, CA, USA) in 2007.Dne of these molecules, SE-2 was able to significantly enhance the activity and the potency of sucralose in a cell-based assay, but was not sweet on its own on the intended usage level and had no activity in the absence of sucralose (Gray, 2014).
Tested in a solution, it showed a reduction of four to six folds in the amounts of sugar, while maintaining the sweetness intensity.Further studies have identified other potent PAMs, SE-3 and SE-4, displaying an altered specificity, which allowed a reduction up to 50% in the amount of sugar, preserving the sweetness intensity.Ot was also identified that SE-2 could be used to reduce the off-tastes related to high levels of sucralose in some products, and neither SE-2 or SE-4 were found to impart any bitterness, metallic taste or other temporal effects, allowing its use on different products which could taste identically to the fully sweetened equivalent (Zhang et al., 2010).

Industrial applications and challenges
The definition of flavor enhancer is laid down in point 14 of Annex O of Regulation (EC) No 1333/2008 on food additives: "[…] flavor enhancers are substances which enhance the existing taste and/or odor of a foodstuff […]" (European Flavour Association, 2015).According to the European Commission's Directorate General for Health and Consumers Guidance notes on the classification of flavoring substances with modifying properties and a flavor enhancer: "Substances that mainly enhance sweetness of food through intensifying the taste of added sugars or sweeteners should be considered as flavor enhancers.The intended function of the added substance is to enhance sweet flavor, thus leading to the possibility of reducing the amount of added sweet ingredients.The same approach would apply if the substance is added mainly to enhance the saltiness or sourness of food […]" (European Union, 2014).
PAMs were approved by the Flavor and Extract Manufacturers Association (FEMA) as flavoring substances that have been described to be used in food and beverage applications, such as soft drinks and powdered beverages (Watson, 2014).Ot is apparent that the sweetener PAM technology is an exciting approach to calorie reduction of products with a taste quality which is not possible with HP sweeteners, but the results achieved so far are quite modest.Ot remains to be demonstrated that compounds exhibiting both agonist and enhancement properties are responsible for increasing effects, and not only additive sweetness (DuBois & Prakash, 2012).However, PAMs do not have minimal toxicology data published by, for example, EFSA or WHD.Safety human tests of PAMs in combination with sucrose has not been adequately performed.
Dther challenges in the field of PAMs include: identification of more efficacious PAMs that could enhance the taste of sugar to 20-fold in order to cut beverage calories and enable the labeling of zero-calorie discovery of different enhancers for each sweetener, increasing specificity and therefore efficacy (Servant et al., 2010(Servant et al., , 2011)); isolation of PAMs with a broader spectrum of activity that are able to improve taste qualities of synthetic and natural HP sweeteners; identification of PAMs from natural origin with strong enhancement effects that are cost-effective because of the consumers' search for natural ingredients (Scott-Thomas, 2014).

Conclusions
Led by the increasing consumer desire for reduced calorie products with a more realistic sugar taste, the search for low or no-calorie sweeteners will continue.On recent years, although consumers have shown considerable interest for natural sweeteners as part of a growing trend for healthful products lacking of synthetic ingredients, compromise on taste quality is unlikely.On addition, some of the plant-derived substances are difficult to be isolated and many of them do not grow outside their natural environment, compromising their stability and being costly to produce.
With the discovery of human genes coding for the sweet taste receptor, the identification of novel synthetic and natural HP sweeteners is possible.However, the compounds identified and developed until now fail to mimic the taste of sugar.So far, sweetness enhancement has been reported for sweeteners combinations apparently showing modest synergistic effects and remains very popular in the industry.Nonetheless, measuring the effect of these combinations is difficult, since each of the components present inherent physicochemical properties and flavor characteristics that might be complementary.
On contrast, PAMs are not agonists and do not taste sweet on their own, simplifying the interpretation of the enhancement properties.They are able to enhance sweetness using a low concentration of modulator in a cost-effective manner, maintaining the real taste of sugar without any increase in caloric value.Therefore, the continuous search towards identifying additional selective enhancers for other commercially relevant sweeteners acting within the VFT of the sweet taste receptor could revolutionize the field of flavor development and sweetened products.

Table 1 .
Potency, technological properties and beverage and food applications of synthetic sweeteners.

Table 2 .
Potency, technological properties and beverage and food applications of natural sweeteners.