An Overview of Biotransformation for the Sustainability of Sweet Tasting Proteins as Natural Sugar Replacers

: According to WHO, sugar intake rates should be reduced due to the connection of sugar with diseases. However, reducing sugar in foods is a challenge both for food manufacturers and consumers. Therefore, sweet-tasting proteins may solve the problems with a sweet taste, health ben-efits, and without caloric contents. So far, known natural sweet-tasting proteins are brazzein, curculin, thaumatin, monellin, miraculin, and mabinlin. Nevertheless, natural sources of sweet proteins might be extinct in the future due to overconsumption. Thus, biotransformation studies of sweet proteins are promising with high yield rates, quality, fewer by-products, and more sustainable solutions.


Introduction
Sugar is a crucial compound for food processing with characteristics of texture, stability, mouthfeel, flavour, colour and preservation features [1]. Moreover, sugar is an energy source of our body as a carbohydrate, but excessive sugar consumption is an issue of obesity [2]. According to the World Health Organization [3], less than 10% of total energy should be intaken from free sugars for adults. However, nowadays, over sugar consumption is a challenging issue because of caused disorders in the body such as weakening of immunity [4], diabetes, cardiovascular diseases and cancer [5].
On the other hand, sweet-tasting is a genetically evolutionary survival mechanism for human-being because of psychological necessity [6]. Nevertheless, sweetness causes addiction with tooth decay, weight gain, obesity, type 2 diabetes mellitus, high blood cholesterol, depression and cancer [7][8][9]. Thus, due to the side effects of over sugar consumption, removing sugar has been suggested from the GRAS (Generally Regarded as Safe) list of FDA (U.S. Food and Drug Administration) by Lustig et al. [10].
In conclusion, the review aims to discuss natural and recognized sugar replacers as sweet-tasting proteins with health-promoting activities, and sustainability features by biotransformation. So far, known natural sweet proteins are brazzein, curculin, thaumatin, monellin, miraculin, and mabinlin. Thus, the scope includes identifying sweet-tasting proteins as natural food ingredients.  Brazzein is a derivative of Pentadiplandra brazzeana Baillon, which is found in African tropical forests naturally [11]. Brazzein is the smallest sweet-tasting protein with a 54 amino acid structure [12]. The sweetness of brazzein is 2000 times higher than 5% sucrose solution [13], and the stability of brazzein maintains up to 80 °C [14] which is an important feature for food manufacturing.

Brazzein
Because of original plant location and limited brazzein production cause alternative ways searching, and bioconversion is the best way to manufacture brazzein close to natural. The first brazzein biotransformation study was made via Escherichia coli in 2000. However, following brazzein biotransformation studies in E. coli exhibited a lower sweet taste than the product of the original plant. Later, sweet brazzein manufacturing has been achieved with Pichia. Pischia cells have released about 120 mg/L of brazzein in 6 days. Nevertheless, Kluyveromyces lactis has produced about 104 mg/L of brazzein into the cultured medium in a short period, and recombinant brazzein's sensory characteristics were similar to the original plant product [11]. Recently, Bacillus licheniformis has been applied for brazzein extraction, due to its fast-growing, high secretion, and low cost [12]. Thus, the rBrazzein genes have been expressed and 57 mg/L of brazzein has been produced at 36 h. Ebrazzein and Bbrazzein demonstrated 400 and 266 times more sweetness characteristics than sucrose respectively.
For plant biotransformation studies of brazzein, the most applied mediums are maize, corn, rice and lettuce [15][16]. Moreover, brazzein has been achieved to produce about 400 μg/g in corn seeds, and corn brazzein allows for industrial production which can solve issues related to the sustainability of the original brazzein in the future [11]. Thus, the sweet taste of brazzein starts slower than sucrose and may replace sugar in food processing with novel food applications.

Curculin
Curculin is extracted from Molineria latifolia (Dryand. ex W.T.Aiton) Herb. ex Kurz, which is native to Malaysia [11]. Dried fruits of Molineria are used by local people against the bitter taste of black tea, and sour foods [17]. Therefore, the compound is a promising ingredient for future food production as a novel material.
Indeed, curculin demonstrates 550 times more sweetness than sucrose on a weight basis [18]. Moreover, water solutions of the curculin exhibit a strong sweet taste at low pH [17]. Thus, the feature might be applied for innovative food productions.
So far, gene expression studies of curculin have been made via E. coli, but homodimeric forms of the compound have not exhibited any sweet taste; heterodimeric forms of curculin have demonstrated characteristics of sweet taste [19]. Thus, the natural source of curculin is unsustainable, and biotransformation studies of curculin have exhibited valuable results for flavour enhancing and sweet taste features ( [20]. Besides, the characteristics of curculin are attractive and promising.

Mabinlin
Mabinlin is found in the seeds of Capparis masaikai Levl. from Yunnan Chinese region [11]. Mabinlin possesses four isoforms, which are mabinlin I-1, mabinlin II, mabinlin III, and mabinlin IV [21]. Mabinlin II is the only compound that is heat stable and the sweetness maintains following 48 hours of incubation at 80 °C. Therefore, the sweetness of mabinlin is 400 times higher than sucrose on a molar basis [22].
Mabinlin ll is difficult to extract from the Capparis but biotransformation studies via E. coli and Lactococcus lactis provide availabilities to produce mabinlin in wide spectrums for food applications [23]. Moreover, biotransformation studies of mabinlin in plants have been made into potato, and the mabinlin ll had an astringent-sweet taste and the amount was 1 mg/ml [19]. Therefore, the sweetness characteristics of mabinlin provide possibilities to apply for vegan foods to mask the bitterness of plant-based ingredients.

Miraculin
Miraculin is found in Richardella (Synsepalum) dulcifica (Schumach. & Thonn.) Baehni, and demonstrates an unsweet feature but can transform a sour taste into a sweet feeling. Miraculin consists of 191 amino acids and N-linked oligosaccharide [24[, which is solely extracted from the Richardella fruit after 6 weeks of pollination, following fruit colour change from green to dark red [25]. The miraculin provides abilities to use for taste enhancing of acids [26]. Thus, miraculin solutions may enhance the flavour characteristics of acids in food products for more than 1 hour [27].
The first biotransformation study of miraculin was made via E. coli [28] without sweet taste characteristics after recombinant miraculin has been produced in transgenic lettuce, and the amount of miraculin was between 33.7 and 43.5 μg/g fresh weight with sweet taste feeling characteristics [29]. Following miraculin has been produced in transgenic tomato and strawberry as well [30]. Thus, biotransformation of miraculin supplies low cost, genetic stabilities, and production via transgenic plants [25].

Monellin
Monellin contains 44 amino acids in one chain and 50 amino acids in another chain as polypeptide bonds [19]. Monellin is a sweet-tasting protein of Dioscoreophyllum cumminsii Diels, and the plant grows naturally in African forests [11]. The sweetness of monellin is 4000 times higher than sucrose on a weight basis [31].
Cultivation studies of Dioscoreophyllum have not been achieved except in natural habitats to obtain stabile monellin [32]. For this reason, biotransformation studies have been implemented, and a specific form of monellin provides flexibility for biotransformation. For instance, the transformation of monellin via E. coli supports sweet flavour during heating with pH stability better than the original compound [19]. Moreover, biotransformation of monellin via S. cerevisiae yielded about 54 g of purified monellin [33].
Transgenic plant studies of monellin have been made into transgenic tomato and lettuce [34]. Therefore, ethylene applied transgenic tomato provided about 23.9 μg/g fresh weight of monellin with high heat stability and elevated sweet taste [35].
To conclude, biotransformation studies of monellin will be carried on to find sustainable solutions for broad applications of the component in food manufacturing. As monellin possesses zero glycemic index which can be applied to diets of diabetic people [36]. Besides, any adverse effects of monellin have not been reported for food applications so far [37]. Thus, the compound may find varied applications in food processing forthcoming.

Thaumatin
The arils of African species Thaumatococcus daniellii Bennett include the sweet-tasting thaumatin proteins, the amount of thaumatin in a ripe fruit is about 30-55 mg/g of fresh weight [38]. The sweetness level of thaumatin is 3000 times more than sucrose without caloric values [39].
The sweetness characteristics of thaumatin attract researchers to find alternative production ways for the sustainability issues. Therefore, biotransformation studies of thaumatin exhibit promising results for future implementations. Thus far, thaumatin gene expressions were made in rice [40], strawberry, barley, tomatoes, potatoes [41], cucumber, and pear to enhance the taste of fruit and vegetables [19]. Hence, plant gene expression studies of thaumatin demonstrate advantages such as low toxicity and a rise in economical incomes.
On the other hand, biotransformation of thaumatin by bacteria and fungi provides much fast growth, control the pathway and high yield of the thaumatin [42]. For instance, E. coli is the most used bacteria for protein expressions, due to well-understood genomics. However, the production of thaumatin via E. coli has supplied low amounts of total thaumatin [43]. Faus et al. [44] have applied synthetic genes of E. coli to express thaumatin proteins, and the study has provided a similar molecular weight with original thaumatin. Following those studies, in 2000 Daniell et al. [45] achieved to produce about 40 mg pure thaumatin with similar sweetness characteristics of the original compound. Nevertheless, the disadvantage of E. coli is toxic with by-products [41]. For this reason, Lactococcus lactis, which has been recommended for gene expression of thaumatin, and has been approved as GRAS [46].
Moreover, thaumatin has been produced also by yeast, and the yield was about 100 mg/L [19], and Pichia pastoris is a good example for commercial thaumatin production without toxins [41]. Hence, thaumatin might be utilized for varied food products in forthcoming with sustainability features due to biotransformation studies.

Conclusion
Natural sugar substitutes promote activities against obesity, type ll diabetes, and cardiovascular diseases. Forthcoming, we may see much more applications of natural sugar replacers with wide utilities. However, huge interest in natural sugar replacers may create extinctions of the sources of sugar substitutes. Therefore, biotransformation studies may bring solutions for issues related to sources of natural sugar substitutes, and with extra advantages such as biotransformation creates fewer environmental issues and is more sustainable for the production [47].

Conflicts of Interest:
The author declare no conflict of interest.