[1]

SOURCE MATERIAL: Do you know Brazzein? Join us and discover the novel,

ideal alternative sweetener, natural, high intensity, and smallest sweet

protein.

INDEX

Title 2

Molecule 2

1.Elevator Pitch 2

2. Summary of test result 2

3.Description 2

Composition, natural origin 2

Safety for human consumption 3

Molecule sweetness, taste profile 3

Profile determination 3

Sweetness effect production 3

Applications or types of beverages and foods can be sweetened with it 4

Aliments or foods and beverages in which it wouldn’t work well 4

Color 4

Water Solubility and limitations 4

Heat and pH stability 4

Light- stability 5

Micro-stability 5

Shelf-life 5

Calories and my history with it 5

Novel, original, different from other sweeteners 5

Taste in beverages and foods in comparison with sucrose 5

Compatibility with other sweeteners 6

Available source 6

4. Why should brazzein be chosen for an award? 6

5.Health regulatory or commercial restrictions for using the Source Material 7

General Recognize As Safe 7

Grandfather clause 8

Recognition Procedures 8

A. Code of Regulations and Scientific Procedures 8

B. Experience based on common use 8

C. Intended Use 9

GRAS Projects and Commercial viability 9

Bio-fermentation/Biotechnology 10

6.Discovery, Development, or Usage of Source Material 11

Discovery 11-12

Extraction method 13

Development 13

Invention 14

Usage of the Source Material and consumer perception 15

Property rights 16

U.S patent documents 17

Foreign patent documents 17

7.References 18-19

8. Annex: Graphic and animation document 20

[2]

Do you know Brazzein? Join us and discover the novel, ideal alternative

sweetener, natural, high intensity, and smallest sweet protein.

Molecule: Brazzein

1. Elevator Pitch: molecule

Sweet natural protein, zero calorie, good taste, harmless for teeth, suitable for diabetes, obesity

2. Summary of test result: Not done.

Robens,

Sweet is currently unavailable to the general public. We have no need at this time

for sales and or distribution. I will keep your information on file and contact you when we

make this product available

Stephen Felderstein, Vice President Sales, North America

Natur Research Foods, Inc.

16133 Ventura Boulevard, 7th Floor

Encino, California 91436.

T. 310.473.5389

F. 310.473.1086

E. stephenf@naturresearch.com

W. www.naturresearch.com

3. Description

Composition, Natural origin

It has a single chain of 54 amino acid residues with eight disulfides bonds:

[3]

pyrE-D-K-C-K-K-V-Y-E-N-Y-P-V-S-K-C-Q-L-A-N-Q-C-N-Y-D-C-K-L-D-K-H-A-R-S-G-E-C-F-Y-D-E-

K-R-N-L-Q-C-I-C-D-Y-C-E-Y.

It folds into a 3-stranded antiparallel β-sheet and one α-helix. It has 8 cysteines, connected

into a network of 4 disulfide bonds and it has a 3D structure. It was isolated from a West

African plant, Oubli.

Safety for human consumption

No concerns are known. It has been consumed in food and beverages for centuries

by natives in West Africa either raw or in cooked form. The body is able to metabolize it

like any other protein.

Molecule sweetness, taste profile

The major form isolated from its natural source contains 80% pyr E at its N-

terminus. The remainder, 20%, is des-pyr E. The pyr E containing brazzein is 500 times

sweetener than a 10% sucrose solution on a weight basis and 200 times sweeter when

compared to 2% sucrose solution and is 19.000 times sweeter than sucrose on a per-

molecule basis.

Brazzein has a good taste similar to sugar. In tests, it was shown to be closer to

sugar with little or no metallic or bitter after taste.

Profile determination

Mammalian sweet taste perception is mediated by the Taste type 1 Receptor (T1R)

family of class CG-Protein Coupled Receptors (GPCRs).

Sweetness effect production

Based on X-ray and NMR, residues 29–33, 36, 39–43 and C-terminus are involving

[4]

in its sweet taste. Brazzein surface charge is important and Arg-43, Lys, Tyr, His, Asp and

Glu are also important for its sweetness.

Applications

➢ Bake goods (breads, cakes, etc.)

➢ Beverages (soft drink, cola, juice, tea, etc.)

➢ Chewing gum

➢ Dairy products (ice cream, yoghurt, etc.)

➢ Confectionaries (chocolates, candies)

➢ Impalatable pharmaceutical products.

Aliments in which it wouldn’t work well

➢ Caramel

➢ Soy sauce

➢ Milk chocolate

➢ Roasted coffee

➢ Toast

Color: green to red during ripening

Solubility and limitations: Very water-soluble. Its solubility is at least 50 mg/ml. Its

isoelectric point (pI= 5.4) is lower than those of other sweet proteins, with pI > 7.0.

It has its lowest solubility at its pI.

Stability:

Heat and pH stability

[5]

Very heat and pH stable. Through assay it was demonstrated its sweetening power

doesn’t diminish after incubation for 2 hours at 980C at a concentration of 0.5mg/ml, at

different pH levels 2; 4; 6; 8 in different concentration buffers of 50 mM.

Light: red, black increased sweetness; light blue decreased it; dark blue, scored as water.

Micro-stability: Excellent for its low calorie, then its low water activity and unavailable

water for the microorganism growth.

Shelf-life: it has a long shelf-life due to its heat stability.

Calories and my history with it

Since I was informed on August 27, 2017, I was looking for a sugar substitute with

the required criterions and on September 29, I discovered Brazzein. It has zero glycemic

indexes. Like sucrose, it contains 4 calories per gram. It is so sweet that any food portion

will contain zero calories.

Novel, original, different from other sweeteners

In November 1994, it was discovered in the red pulp of Pentadiplandra brazzeana.

Whereas monk fruit has a juicy-fruit type candy state profile and Stevia as a licorice

aftertaste, brazzein has more of a sucrose-type profile than other natural sweetener

proteins such as thraumatin or monellin.

Taste in beverages and foods in comparison with sucrose

➢ It has a taste profile closer to sucrose.

➢ It has a lingering aftertaste.

➢ The onset and duration of its sweetness is lightly different and longer than

sugar.

[6]

Compatibility with other sweeteners

It can be used to reduce the aftertaste of other sweeteners and complete their

flavor. It has a synergistic effect if mixed with Stevia and it is suitable to be mixed with

other artificial sweeteners, acesulfame K that elicits a quick but short sweetness response

and aspartame.

Available Source

➢ Oubli’s berries

➢ Maize

➢ Bacterias

➢ Yeast

➢ Milk

4. Why should brazzein be chosen for an award?

Considering the point of view of its sweetness, heat stability, high water solubility

and minimum molecular weight, brazzein is currently the superior protein sweetener. For

its properties like zero glycemic index, low or zero calories contribution, it can help the

human being to fight against the diabetes, the obesity, the hyperlipemia, heart disease

and other pathologies.

For no known side effects, everybody can use it. So its sweetness, the absence of

bitter after taste and the possibility for mass-production make it a realistic, cost-effective

alternative. The opportunity to explore this protein for potential health application in the

low calorie sweetener industry makes it the best product in a very near future in the

worldwide market.

[7]

5. Health regulatory or commercial restrictions for using the Source Material

Actually, Brazzein is not yet available for commercial distribution due to regulatory

process and lack of Food and Drug Administration (FDA) and European Food Safety

Authority (EFSA) approval. To understand the process, let’s review some concepts.

Generally Recognized As Safe

GRAS, an acronym for the phrase Generally Recognized As Safe is an American

Food and Drug administration (FDA) designation that a chemical or substance added to

food is considered safe by experts and so is exempted from the usual Federal Food, Drug

and Cosmetic Act (FFDCA) food additive tolerance requirements. The concept of food

additives being ¨generally recognized as safe¨ was first described in Food Additives

Amendment of 1958, and all additives introduced after this time had to be evaluated by

new standards.

A GRAS determination can be self-affirmed or the FDA can be notified of a

determination of GRAS by qualified nongovernmental experts.

A. Self-affirmed: The manufacturer of this chemical or substance had performed all

necessary research, including the formation of an expert panel to review safety concerns

and is prepared to use these findings to defend its product’s GRAS status.

B. FDA Response to GRAS notification: The manufacturer has performed all the

aforementioned due diligence and submitted a GRAS notification to inform the FDA of a

determination that the use of a substance is GRAS. Following evaluation, the FDA provides

three possible responses:

1. FDA does not question the basis for the notifier’s GRAS determination

[8]

2. The notification does not provide a sufficient basis for GRAS determination basis.

3. The FDA has, at the notifiers’s request, ceased to evaluate GRAS notice.

Grandfather clause

A grandfather clause or grandfather policy is a provision in which an old rule

continues to apply to some existing situations while a new rule will apply to all future

cases. For substances used in food prior to January 1, 1958, a grandfather clause allows

experience based on common used in asserting they are safe under the conditions of their

intended use.

Recognition Procedures

The use of a food substance may be GRAS under sections 201 (s) and 409 of the

Act, and FDA’s implementing regulations in 21 CFR 170.3 and 21 CFR 170.30 through

scientific procedures or for a substance use in food before 1958 through experience based

on common use in food under 21 CFR 170.30 (b).

A. Code of Regulations and Scientific Procedures

The code of Federal Regulations, revised as of April 1, 2010 includes title 2117030

(b) that provides General recognition of safety through scientific procedures requires the

same quantity and quality of scientific evidence as is required to obtain approval of the

substance as a food additive. It is based upon published studies which may be

corroborated by the application of unpublished scientific studies, other data and

information of methods.

B. Experience based on common use

Under 21 CFR 170.30 (c) and 170.3 (b), general recognition of safety through

[9]

experience based on common use in foods requires a substantial history of consumption

for food use by a significant number of consumers.

In the brazzein case, only we can use the first method, scientific procedures

because it was after 1994 they were looking for approval for its use due to the possibility

to have production-mass, even though this product was already mentioned by Baillon in

1868 and it was used by many people from Ghana, Gabon, Cameroon, etc., but the

attention is now focused on the product made by bio-fermentation due to the

impossibility to have enough from the natural source berries.

C. Intended used

The substance must be shown to be ¨generally recognized as safe¨ under the

conditions of its intended use. The proponent of the exemption has the burden of proving

that the use of the substance is generally recognized as safe. To establish such

recognition, the proponent must be shown that there is a consensus of expert opinion

regarding the safety of the use of the substance.

GRAS Projects and commercial viability

Efforts to scale up the ingredient for commercial use have initiated. On the

regulatory front, Natur Research Ingredients chose to perfect and optimize the

manufacturing process first, prior to seeking regulatory approval, given this is required for

achieving success in obtaining it because they can’t apply for regulatory approval, make

improvements in the manufacturing process and continue with the same regulatory

process.

Then, the manufacturing process already exists, and manufactures are looking

[10]

for a partner, ideally with expertise in the sweeteners business to handle distribution.

By bio-fermentation technology, using food-grade bacteria commonly used actually for

many foods and beverages, a large quantity of brazzein can grow in large tank vessels in a

matter of days. Potential mass production could provide a valuable opportunity for the

commercial use of brazzein.

Actually Brazzein is not yet approved for use by the FDA and more studies and

experimentation lie ahead before it can be used in any products. A company was formed

to bring it to the market as a sweetener in 2008 and there was promise to start selling the

product by 2010 once it obtained agreement from the FDA that its brazzein produced by

bio-fermentation is generally recognize as safe (GRAS). In addition many leading food and

beverage companies are already evaluating samples of the sweetener and attempt to

prove that the synthetic brazzein can still be listed as brazzein on the ingredient label and

is as natural as the original because the source material for this ingredient is the same

fruit berry of the West African plant Pentadiplandra brazzeana Baillon and several

toxicological and allergological studies are performing to demonstrate there is no

concerns about it like the natural product.

However, in 2017, Brazzein still does not obtain a GRAS waiver from FDA and they

continue to seek partners to help that the product can be on the market. For this reason,

we decide to be part of this project because, based on the existing evidence about

biotechnology, specifically bio-fermentation safety and once all the criterions

aforementioned are fulfilled, we hope in a near future, we can successfully obtain it.

Bio-fermentation

[11]

Bio-fermentation is a kind of biotechnology, which is defined by United Nations

Convention as ¨any technological application that uses biological systems, dead organism,

or derivative thereof to make or modify products or processes for specific use.¨

Biotechnology includes a broad range of applications including bioengineering, bio-

remediation, gene therapy, genetic selection, bio-degradation, cloning, bio-fermentation,

etc. Many products produced by bio-fermentation are since 1960s and 1980s used for

food application.

Like those products, the brazzein made by bio-fermentation of high quality with

high level of standardization will obtain the GRAS based on experts consensus either self-

affirmed by the manufacturer Loren Miles, the owner of Los Angeles based Natur

Research Ingredients Inc and Natur Research Foods Inc, and other companies with

experiences in the use of sweetness products for its distribution or having the response

for GRAS by FDA and will overcome the restrictions to be used extensively because

biotechnology, specifically bio-fermentation, is not new and safe if it is used in a wise and

careful way to ensure production of safe food and to meet the expectation of the society

and once the manufacturer fulfills all required criterions for that.

6. Discovery, development, or usage of Source Material

Discovery

The initial source for Brazzein is the fruit of Pentadiplandra brazzeana Baillon, a

species described as a Tiliacae by Baillon in 1868, which is presently classified in the

Pentadiplandraceae, a family created by Hutchinson in 1928. It is a small protein with a

molecular mass of 6.4 kDa. By this time, due to the lack of technology to extract the

[12]

protein from the berries, it is not developed until:

➢ November 1994 when the scientists, Ding Ming, Göran Hellekant, discovered

brazzein isolating it in the red pulp which is sweet and containing it. They described

its weigh molecular, its charge.

➢ In July 1996, Izawa H. Ota M, Kohmura M, Ariyoshi reported the amino acids

sequence of brazzein in¨ Synthesis and characterization of the Sweet protein

Brazzein.¨

➢ In 1998, Caldwell et al determined the solution structure 3D of brazzein by nuclear

magnetic resonance (NMR) spectroscopy and described its topology. Gao et al did

the same in 1999.

➢ In 2000, Assadi-Porter, Aceti, Cheng et al described the three sites of its structure.

➢ In 2000, Pfeiffer et al reported its sweet perception is more similar to sucrose than

that of the other sweet proteins.

➢ In April 2000, Assadi Porter FM, Aceti DJ, Markley JL reported the sweetness

determinants sites of brazzein and its sweetness description on a per-molecule

basis in 2003.

➢ In 2003, Jin and collaborators shown that between 25 brazzein mutants tested,

four mutants (D29A, D29K, D29N, and E41K) were significantly sweeter than wild-

type brazzein, while three mutants (A2ins, D2N, Q17A) were as sweet as the wild-

type brazzein.

➢ In June 2004, Tancreti T, Pastore A, Salvadori S, Esposito V, Temussi PA reported

the interaction of sweet proteins with their receptor.

[13]

➢ In 2011, Yoon and coworkers demonstrated the importance of the positive charge

in the 29-33 and 39-43 regions and the role of K30 and R33 to maintain full sweetness,

the side chain of Q17 residues is fundamental for the sweet taste. Assadi Porter and

collaborators suggested also the 9-19 region is involved in sweetness elicitation.

Extraction method

Ding Ming, Göran Hellekant described too the best method to extract it from the

pulp, which consists in the use of 0.1moles/liter (M) phosphate buffer at pH 7.0 containing

5% glycerol, 0.1 milimoles/liter (mM) DTT, 20 micrograms/ml (µg/ml) phenylmethyl

sulfonyl fluoride (PMSF), 0.1 mM ethylenediamine tetra acetic acid (EDTA) and 0.5% weight

volume (w/v) polyvinylpolyrrolidone (PVP) at 4%. The proteins which precipitated between

30 and 85% ammonium sulfate saturation, were separated on a Sephacryl S-100 column in

50 mM phosphate buffer at pH 7.0. Finally, brazzein was purified on a CM- Sepharose CL-6B

column by a Sodium chloride gradient of 0.1 to 0.4 M in 20 mM sodium citrate at pH 3.6.

Development

Brazzein encompasses a group of related sweet proteins extracted and purified

from the fruit of the Africa plant, Pentadiplandra brazzeana. It occurs in three natural

forms, the amino acids sequences of which are known: Type 1 is 54 amino acids in length

and has an N- Terminal pyrrolidone carboxylic acid; Type II is 54 amino acids in length and

has an N-terminal glutamine; and Type III is 53 amino acids in length and has an N-terminal

aspartic acid.

In addition to the natural forms of brazzein, variants of brazzein, i.e. mutated and

recombinant proteins with varied sequences and properties have been created by genetic

[14]

engineering such as those described in US pat. Nos. 5,326,580; 5,346,998; 5,527,555;

5,741,537; 7,153,535; 6,274,707 and in Jin et al., Chem. Senses 28: 491-498; Assadi-Porter,

et al., Arch. Biochem. Biophys.376: 259-265, 2000. Assadi-Porter, et al., Chem. Senses 30

(Suppl.1): i90-i91, 2005.

Using food-grade methodology and yeast fermentation, for enhanced production

and improvement purification of a natural, they obtain high-quality brazzein protein that

enables its commercial production. This unique protein has applications as a sweetener in

the baking, beverage, and table-top product industries.

Invention

An expression vector for the production of a brazzein protein by a yeast cell

comprising at least two cassettes, each cassette comprising a promoter sequence, a

secretion signal sequence, a brazzein protein reading frame sequence, and a termination

sequence, wherein the at least two cassettes are present in an expression vector

comprising an integration sequence, for integration of the cassettes into the yeast genome

at a specific site.

A recombinant P. pastoris cell comprising at least two copies of a gene construct

comprising a p GAP promoter sequence Type II or Type III protein reading a frame

sequence and a termination sequence, wherein the copies of the gene construct are

present at a preselected integration site within the P. pastoris genome. A method for

producing enhanced levels of brazzein protein is presented, comprising the steps of:

A) Transforming one or more cells in a fermentation medium under aerobic

condition, wherein the transformed cells produce enhanced levels of a brazzein

[15]

protein compare with cells containing only one of the brazzein protein gene.

B) A method for enhancing production of a foreign protein in recombinant Pichia is

presented, comprising the steps of

C) Initiating a culture of the recombinant Pichia, under aerobic conditions.

D) Reducing oxygen levels in the culture at less than 5% and

E) Maintaining oxygen levels in the culture at less than 5%.

A method for purifying a protein from a yeast fermentation culture is also

presented, comprising the ordered steps of:

A) Adjusting the pH of the fermentation medium above or below the isoelectric point

of the protein to be purified

B) Removing the cells from the pH adjusted fermentation medium.

C) Subjecting the cell-free fermentation medium to ion- exchange chromatography

and eluting the protein as a solution

D) Passing the eluted protein solution through a first membrane to remove unwanted

high molecular weight solutes and

E) Concentrating and diafiltering the protein permeate on a second membrane to

remove low molecular weight solutes.

Finally, it is important to mention there is another brazzein source production:

maize. Currently, Nektar worldwide and Prodigene a spin-off of Pioneer Hi- Bred

International, the world’s largest seed company, have genetically engineered maize

that produces large amounts of brazzein.

Usage of the source material and consumer perception

[16]

The berries of the plant Pentadiplandra brazzeana Baillon contains 3 to 5 reniform

seeds surrounded by a thick soft layer of pulp, which turns from green to red during

ripening. Its Geographical distribution is from Nigeria east to the Central African Republic

and south to DR Congo and Angola. The oubli plant from which the protein was isolated

grows specifically in Gabon, Ghana, Congo, Nigeria and Cameroon, where its fruit has been

consumed by the apes and local people for a long time. P. brazzeana fruits are so sweet

that African locals call them ¨Oubli¨ or ¨Forgetting¨ in their vernacular language because

their taste is said to encourage nursing infants to forget their mother’s milk, as once they

eat them. They are said to forget to come back to the village to see their mother due to its

sweetness property.

7. Any person or entity having any intellectual property rights or potential claim

of intellectual property rights in the source material.

Property rights

The Wisconsin Alumni Research Foundation is the independent nonprofit

technology transfer organization serving the University of Wisconsin Madison and

Morgridge Institute for Research. Brazzein came to the attention of industry after a US

researcher observed people and animals eating the berries in West Africa. Researchers at

the University of Wisconsin have been granted US patents 5, 326, 58; 5,346,998; 5,527,555

and 5,741,531 and European patent 684995 for brazzein, the genetic sequence coding for it

and the transgenic organisms where it has been added. Subsequent work has focused on

making transgenic organisms that produce brazzein in the laboratory. In the followed list,

all the existing patents about brazzein are:

[17]

U.S patent documents:

➢ Patent No.: US 8,741,621B2- Date of patent: Jun.3,2014- Inventors:

Alfred Carlson, Terre Haute IN (US); Richard W. Armentrout, Decatour,

IL (US)

Timothy Peter Ellis, Boxborough, MA (US).

➢ 4,495A 1/1985 Brujard et al 435/6.14

➢ 5,326,580A 7/1994 Hellkant et al

➢ 5,346,998A 9/1994 Hellkant et al

➢ 5,527,555A 6/1996 Hellkant et al

➢ 5,741,537A 4/1998 Hellkant et al

➢ 5,759,802A 6/1998 Maki et al 435/69.1

➢ 6,274,707B1 8/2001 Markley et al

➢ 7,153,535B2 12/2006 Jim

➢ 2007/0122895A1 5/2007 Raymond et al

➢ 2009/0221078A1 9/2009 Caimi et al 435/471

➢ 2009/0311749A1 12/2009 Takagi et al 435/69.1

➢ 2010/0076176A1 3/2010 Miles et al 530/370

➢ 2011/0014650A1 1/2011 Young et al 435/69.1

➢ 2011/0021843A1 1/2011 Bailey et al 568/367

Foreign Patent Documents

➢ WO WO94/24280 10/1994

➢ WO WO99/25835A1 5/1999.

[18]

References

1. Ming, D., Hellekant, G., November 21, 1994. Brazzein, a new high-potency thermostable

sweet protein from Pentadiplandra brazzeana B. 355 (1):106-8. US National library of

Medicine. National Institute of Health.

2. Hellekant, G., Danilova, V., January 1, 2005. Brazzein a small, sweet protein: discovery

and physiological overview chemical senses, Volume 30, Issue supp_1, Pagesi88-i89. USA.

3. Mack, E., August 17, 2016. Brazzein boost could pave way for next super sweetener.

American Chemical Society. USA.

4. Caldwell, J., Abildgaard, F., Dzakula, Z., et al, 1998. Solution structure of the

thermostable sweet- tasting protein brazzein. Nat. Struct. Biol. 5: 427- 431, Pub Med,

Europe PMC.

5. Nakata, K., Hongo, N., Kameda, Y., et al, March 27, 2013. Crystal structure of brazzein.

Pub Med, USA. Sect. D 69: 642-647.

6. Watson, E., February 11, 2014. Brazzein entrepreneur seeks partner to take next-

generation natural sweetener to market. William Reed Business Media Ltd, UK.

7. Walters DE, Hellekant G., December 27, 2006. Interactions of the sweet protein

brazzein with the sweet taste receptor. J. Agric Food Chem. US National Library of

Medicine. National Institute of Health.

8. Scattergood, G., August 31, 2016. Natural sweetener brazzein more ´´commercially

viable¨ thanks to Korean Research. Journal of Agricultural and Food Chemistry. USA.

9. Izawa, K., Kuroda, M., 2016. Brazzein. Comprehensive Natural Products II. Chemical

Ecology. USA.

[19]

10. Dittli, S., Rao, H., Tonelli, M., et al, November 1, 2011. Structural Role of the Terminal

Disulfide Bond in the sweetness of Brazzein. Chemical Senses, Volume 36, Issue 9, Pages

821-830. Oxford University Pres. USA

11. Turner, J., September 8, 2016. Scientists make a sweet discovery: lab-synthesized

brazzein. Food Dive, USA.

12. Hellekant, G., Ming, D., Sept 1, 1994. Brazzein sweetener WO 19940191467A1, USA.

13. Lee, J., Kong, J., Do, H., et al, March 31, 2010. Design and efficient soluble expression

of a sweet protein, brazzein and minor- form mutant. Laboratory of Biomolecular

Chemistry, College of Natural Sciences, Chung- Ang University, Seul 156-756, Korea.

14. Jin, Z., Danilova, V., Asadi- Porter, F., et al, June 5, 2003. Critical regions for the

sweetness of brazzein. Volume 544, issues 1-3, Pages 33-37. Elsevier. Federation of

European Biochemical Societies.

15. Watson, E., October 22, 2012. Beyond Stevia: will brazzein make the natural

sweeteners premier league? William Reed Business Media Ltd, United Kingdom.

16. Generally Recognized As Safe, Febrero 2, 2017. U.S Food and Drug. U.S. Department of

Health and Human Services.

17. Carlson, A., Armentrout, R., Ellis, T., June 3, 2014. Enhanced Production and

Purification of a Natural High intensity sweetener. United States.

18. GRAIN, July 25, 2000. Patents on food CROPS of Patents and Pirates. Barcelona, Spain.

19. Rajan, V., Howard, J.A., February 14, 2017. Brazzein: A natural sweetener. Springer.

20. Jones, J., Kern, M., Clemens, R. and collaborators, March 31, 2016. Functionality of

Sugar in Foods and Health. Comprehensive Reviews in food Science and Food Safety.

[20]

Annex: Graphic of Brazzein structure

Solution structure of brazzein with sweetness-determining sites labeled. Side chains for sweetness-determining sites 1 and 2 are shown. Dashed lines indicate distances, in Ångstroms, between key residues of sites 1 and 2. This figure appears in color in the online version of Chemical Senses.

.

Dr. Robens Molaire, PhD.