Descrioción anatómica del caracol

Purpura panza

,empleado Por los tejedores mixtecos

Source: Natural & Synthetic Organic & Inorganic Vegetal, Animal, Microbial 1856. Discovery of mauve by Perkin (purpure of Tyre) 1906. review of 80 commercial products (30 never tested before, 26 with mixed results. Only 7 were approved.

Impact of Biotechnology on the current colorant Industry

Microbial processes

Monascus purpureus

(polyketide pigments)

Phaffia rhodozyma

(Astaxanthin)

Spirulina maxima Dunallella salina

(Phycyanin) ( b -carotene) Extraction from seeds and flowers Marigold flowers (lutein)

Crocus sativus

(saffron; a crocin)

Bixa orellana

(achiote) Extraction from waste products crawfish, red crab, shrimp (Astaxanthin) Enzymatic process resolution of zeaxanthin Tissue culture

Lithospermum eryhrorhizon

(shikonin) Genetic Engineering

Escherichia coli

(indigo)

CAROTENOIDES Uno de los grupos de pigmentos naturales mas distribuidos y diversos en la naturaleza. >600 estructuras conocidas.

Algunas especies los sintetizan

de novo

mientras que otras, particularmente animales, solo capaces de absorberlos y metabolizarlos. Síntesis en la naturaleza de unos 100 millones de toneladas anuales Carotenos: C40 cadenas hidrocarbonadas ( generalmente anaranjados a rojizos) Xantophylls : Sus derivados oxigenados (generalmente amarillos) De los conocidos, un 10% tiene valor como vitamina A . Además del beta caroteno, los más importantes entre ellos son el alfa-caroteno y la b eta criptoxantina. La condición fundamental para que tengan actividad vitamínica es que tengan cerrado y sin oxidar al menos uno de los anillos de los extremos de la estructura.

Licopeno, zeaxantina y luteína no tienen valor como vitamina A, aunque son muy importantes como pigmentos, y pueden tener también actividad como antioxidantes.

Annato (Bixa orellana L)

Bixin (Apocarotenoide) Achiote (Mexico), Urucum (Brasil). también: roucou, onato, bija, orlean, … Extracción tradicional en proceso acuoso a alta temperatura Bixin es soluble en grasa pero no en agua; tratada con alcali, Se hidroliza el ester metílico para producir: norbixin , (dicarboxilic) que es soluble en agua.

Method of removing pigment from annatto seed

United States Patent 4204043

EXAMPLE Bixa seeds (36 seeds, approximately 2.5 g), harvested one day earlier near San Isidro, Costa Rica, Placed in a 250 ml flask with 60 ml of water and 0.1 g of Spark-L (a pectinase produced by Miles Laboratories, Elkhart, Ind., containing polygalacturonase and pectinesterase). The flask was stirred occasionally over a period of 18 hours at ambient temperature (22 °-25° C.). The contents of the flask had a good odor with no indication of fermentation having occurred. The aqueous dispersion of the annatto was decanted from the seeds. As a control, the procedure described above was repeated without addition of enzyme. Removal of Pigment Treatment From Seed (%) : Water & pectinase 70% Water (control) 10%

astaxanthin capsanthin ß-carotene lutein zeaxanthin canthaxanthin Astaxantina (4 5 veces más eficiente) Astaxanthin is produced by microalgae , yeast , salmon , trout , krill , shrimp , crayfish , crustaceans , and the feathers of some birds.

$2000-4000 US/Kg Salmón: 35-75 mg/Kg alimento ( 10-15% costo dieta ) MERCADO DE ALGUNOS CAROTENOIDES

b

-Caroteno: sintético: $750 US/Kg natural: $1800 US/Kg Cenpazuchil Cantaxantina: $1500 US/Kg

Amounts of astaxanthin found in different organisms [7]

Natural Astaxanthin Sources

Salmonids Plankton Krill Arctic shrimp Phaffia Yeast Haematococcus pluvialis

Astaxanthin Concentration(ppm)

~ 5 ~ 60 ~ 120 ~ 1200 ~ 8000 ~40,000

Haematococcus pluvialis

(0.5-2% dry weight)

Phaffia rhodozyma

Adonis aestivalis (200-350 mg/flower)

Brevibacterium lacticola

INDIGO

•

The world largest selling textile dye (by vol): 13, 000 ton/year with a value of $200 million dollars.

•

From Mollusks genus Murex

•

European woad plant: Isatis tinctoria

•

Asian: Indigofera tinctoria

•

1883 Adolf von Baeyer (chemical structure) Nobel prize.

•

1897 BASF---



chemical synthesis

•

Genencor recombinant E. coli with 9 genes cloned.

•

Genencor indigo: more expensive than synthetic product

Cochineal: the best natural pigment (carminic acid)

Insects of the families: Coccoides Aphidoides Porphyropera hameli: (Armenian Re) grows in grasses of Azerbaijan Kermococcus vermilis: (Kermes: grows in oaks) Margarodes polonius: Dactylopius coccus: (roots of a grass in various trees in India & Malaysia) (cacti: Opuntia)

80,000 – 100,000 insects/Kg of raw dried cochineal (22% of their dry weight) Today’s production only a small fraction of thet in XVI-XIX centuries (eg. Canary islands alone produced 3000 ton in 1875).

Extracted with hot water Treatment with protease -----



“carmines of cochineal”.

Strawberry



----



blackcurrent.

• According to the World Health Organization, dietary vitamin A deficiency (VAD) causes some 250,000 to 500,000 children to go blind each year. More than half those who lose their sight die within a year. VAD compromises the immune systems of approximately 40 percent of children under five in the developing world, greatly increasing the risk of severe illnesses from common childhood infections. VAD is most severe in Southeast Asia and Africa.

• • has become a staple food in many African countries. Globally, rice grain is the world's most important source of human food-feeding more than half of the world's population. Rice is a good provider of calories and protein, but rice scientists have long recognized its micronutrient deficiencies. Milled white rice contains essentially no beta-carotene and unmilled brown rice contains a very small amount. Public rice research institutions in the Philippines, Vietnam, India, Bangladesh, China and Indonesia are in various stages of leading efforts to develop locally adapted Golden Rice varieties.*

LOS PARADIGMAS En el arroz no se expresa la Fitoeno sintasa. La primera variedad tenía el gene del narciso y la fitoeno desaturasa de la bacteria

Erwinia pseudonarcissus

.

La nueva variedad tiene la sintasa del maíz y contiene mas de 30 arroz. m g de b caroteno por g de

lycopene

More than 250 million sub-Saharan Africans rely on the cassava, a starchy tuber native to South and Central America, as their staple food. Cassava supplies 38.6% of the caloric requirements in some parts of Africa, where hunger and nutrient deficiencies grip the populace and more than 40% of global cassava production takes place. Launched in July 2005 with $7.5 million from the Bill and Melinda Gates Foundation’s Grand Challenges in Global Health Initiative, the program’s overarching goal is to develop what essentially amounts to a super charged cassava plant variety —one with increased levels of iron, zinc, protein, vitamins, and resistance to the cassava mosaic and brown streak viruses plaguing African farmers.

Cassava

ß-Carotene is a dietary precursor of vitamin A that is synthesized by the methylerythritol phosphate (MEP) pathway in plastids of some plant cells. Conventional cassava roots lack some of the essential enzymes necessary to produce ß-carotene. The initial step in the pathway is controlled by deoxyxylulose-5-phosphate synthase (DXS), which is added to Cahoon’s cassava via the gene

dxs

, originally sourced from a different plant species. Additional steps generate the C5 isopentenyl diphosphate (IPP) that is used as the building block for the synthesis of the C20 geranylgeranyl diphosphate (GGDP). Phytoene synthase (PSY), the product of an introduced gene (

psy

) from a bacterial source, combines two molecules of GGDP to form phytoene, which is converted to ß-carotene via lycopene through a series of desaturation, isomerization, and cyclization reactions. The end result is a noticeably more orangey cassava root.

Arabidopsis

gene,

1-deoxy-d-xylulose 5-phosphate synthase

(

dxs

), which regulates the isoprenoid pathway, a set of biochemical reactions further upstream from the biosynthetic step in which

psy

is involved. Inserting

dxs

, which increases the amount of chemical precursors to beta carotene, was “like turning up the whole isoprenoid pathway,” Cahoon says. He found that inserting both the

psy

and

dxs

genes resulted in a cassava even more orange than the roots with only the

psy

modification —and with 30 times more beta-carotene than normal roots. The BioCassava Plus program has also recently seen significant progress in its goal to introduce biofortified foods into the developing world. Director Richard Sayre says that the program’s

pro-vitamin A cassava plants have been approved for field trials in Nigeria, the world’s number one consumer of the food. In July, the country planted between 4000 and 8000 m2 with

Cahoon’s two-gene GM cassava, the first GM product Nigeria has field tested. “We are quite proud of that,” Sayre says. To advance the BioCassava Plus program to the next stage, Sayre says that more donor money will be needed. He says that the program is “planning on approaching other donors,” but declined to name them.

Capsicum flour

0 96% Ethanol extraction

Capsaicinoids (62%) Carotenoids (1.4%)

30% Ethanol extraction 96% Ethanol extraction

Capsaicinoids (15%) Carotenoids (0%) Capsaicinoids (44%) Carotenoids (2%) Aqueous enzymatic treatment

30% Ethanol extraction 96% Ethanol extraction

Capsaicinoids (80%) Carotenoids (73%) Residual flour Capsaicinoids (26%) Carotenoids (70%) Residual flour Capsaicinoids (27%) Carotenoids (83%) Residual flour

Capsaicinoides



Compuestos causantes de la pungencia en los chiles

(Capsicum annuum).



Estimulantes en el tracto digestivo, bloqueadores de la transmisión del dolor y promueven el metabolismo energético.



Aplicaciones: Condimentos alimenticios, industria del tabaco, repelentes de protección personal y en compuestos para la agricultura, pinturas marinas, medicamentos contra el dolor.

MeO O CH 3 H O N H

Capsaicina

CH 3

Santamaría R., Reyes Duarte M.D., Barzana E., Fernando D., Gama F.M., Mota M. & Lopez- Munguía A., Journal of Agricultural and Food Chemistry, 48, 7, 3063-3067, 2000.

Generalidades Estructura de los capsaicinoides y su



pungencia Análisis sensorial medido en unidades Scoville



Cuantificación en ppm por HPLC



Modelo de Jancsó-Gabor

H 3 CO N H   CH 3 CH 3 

Receptor de capsaicinoides (Caterina y col. Nature, 1997)

Resultados Conversión de capsaicina en agua utilizando diferentes enzimas

Enzimas Conversión a (%)

Lipasas (b)

Novozym 677 BG Lipasa F Esterasa Diversa 001 Novozym 435

Proteasas (c)

Tripsina Penicilino acilasa 0.3

1 8 30 0.05

0.1

a Conversión de hidrólisis de capsaicina en agua, cuantificando la vainillinamina producida después 24 h de reacción a

(b)

45 ºC y

(c)

37 ºC.

Reacci ón de hidrólisis de la capsaicina

MeO O H 2 O H O N H CH 3 CH 3

Enzima hidrolítica

Capsaicina

MeO H O NH 2

Vainillinamina

O + CH 3 H O CH 3

Acido 8-metil-6-trans-nonanenoico

Reyes- Duarte D., Castillo E. Bárzana E. & López-Munguía A. Biotechnology Letters. 22 :1811-1814, 2000.

Síntesis de N-vainillíl oleamida :

Efecto de la amina terciaria

H 3 HO CO Acido oleico Lipasa B de

Candida antarctica

NH 2 HCl + H 3 HO CO N N H 3 CO + HO NH 2 Vainillinamina DIPEA O N H

N-

V ainillíl oleamida (olvanil)

Reyes D.D., Castillo E., Martinez R. & López Munguía A. Biotechnology Letters 24, 2057-2061. 2002.

Síntesis enzimática de capsiato en un solo sistema



Alcoh ólisis

H 3 CO

Vainillinalcohol



Hidrólisis y síntesis

HO OH H 3 CO HO O N H

Capsaicina Lipasa B de

Candida antarctica

H 3 CO HO O O

Capsiato +

H 3 CO HO

Vainillinamina

NH 2

A -90 mV 80 mV -90 mV B 600 400 C 200 Vm (mV)

Efecto de la capsaicina y sus derivados en el

-80 40 80 50 ms

canal de calcio T de células espermatogénicas y

50 m M Capsaicina 50 m

la relaci ón con su pungencia

Lavado 50 ms 100 50 m M Capsaicina 1.0

D 80 0.8

60 0.6

40 0.4

20 I Ca 0.2

0.0

0 2 4 6 Tiempo (min) 8 10 0 C ap sa (5 ic 0 in m a M) V an illi na (5 m 0 m in a M) C ap si (5 at 0 m o M) 

Sensorialmente, la capsaicina es pungente, su hidrolizado no, ni tampoco el capsiato.

Síntesis de Análogos de la Capsaicina

Síntesis de amidas con altos rendimientos (>80%) en 2M2B (solvente polar) independientemente de la estructura del análogo.

2 análogos con actividad pungente

Análogos de capsaicina : inhibidores de canales Ca2+ T-type

Síntesis de Olvanil

H 3 C O H O N H 2 • H C l + N N • H C l H 3 C O + H O Vanillylamine salt derivative Oleic acid 10 Lipase B from

Candida antarctic a

11 12 9 8 13 14 7 DIPEA 6 15 16 5 4 17 18 3 H 2 1

N

7' 1' 6' 5' Olvanil

O

2' 4' 3' O C H 3 O H Vanillylamine N H 2

Síntesis de Capsiato

H 3 C O H O O N H + Capsaicin E -OH Lipase B from

Candida antarctica

H 3 C O H O Vanilly lamine N H 2 E O O Acyl-Enzyme Complexe H 3 C O H O Vanilly lalcohol O H H 3 C O 3' H O 4' 5' 6' 2' 1' 7' O O 3 1 2 4 Capsiate 5 6 7 8 9 10

Evaluación farmacológica

(Colaboración con Alberto Darszon e Ignacio López)

inhibidores de canales Ca2+ T-type

S íntesis quimoenzimatica del Fenil-acetil-rinvanil

O OCH 3 HO O DCC, DMAP O OCH 3 OH Ricinoleato de metilo O O OMe H 2 N OH 2M2B, Novozym 435 O N H OMe OH O O Fenilacetilrinvanil