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INEC annual meeting Granada, May 25-26, 2000 Universitat de les Illes Balears and IMEDEA (CSIC-UIB) Area de Microbiología Departamento de Biología y Departamento.

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Presentation on theme: "INEC annual meeting Granada, May 25-26, 2000 Universitat de les Illes Balears and IMEDEA (CSIC-UIB) Area de Microbiología Departamento de Biología y Departamento."— Presentation transcript:

1 INEC annual meeting Granada, May 25-26, 2000 Universitat de les Illes Balears and IMEDEA (CSIC-UIB) Area de Microbiología Departamento de Biología y Departamento de Recursos Naturales Vicente J. Benedí Locust bean or guar? Molecular methods for detecting additions of guar gum to locust bean gum

2 E 410 vs. E 412: what the EC says (I. Definitions) Off. J. of the E.C. L334, E 410 Locust bean gum is the ground endosperm of the seeds of the natural strains of carob tree, Cerationia siliqua (L.) Taub. (family Leguminosae). Consists mainly of a high molecular weight hydrocolloidal polysaccharide, composed of galactopyranose and mannopyranose units combined through glycosidic linkages, which may be described chemically as galactomannans E 412 Guar gum is the ground endosperm of the seeds of the natural strains of guar plant, Cyamopsis tetragonlobus (L.) Taub. (family Leguminosae). Consists mainly of a high molecular weight hydrocolloidal polysaccharide, composed of galactopyranose and mannopyranose units combined through glycosidic linkages, which may be described chemically as galactomannans

3 E 410 vs. E 412: what the EC says (II. Identification) Off. J. of the E.C. L334, E 410 A. Positive tests for galactose mannose B. Microscopic examination (see next slide) C. Solubility: soluble in hot water, insoluble in ethanol E 412 A. Positive tests for galactose mannose B. Solubility: soluble in cold water

4 E 410 vs. E 412: what the EC says (II. Identification) B. Microscopical identification (Place some…containing 0.5% iodine and…and examine under the microscope.) Locust bean gum contains long stretched tubiform cells, separated or highly interspaced. Their brown contents are much less regularly formed in guar gum. Guar gum shows close groups of round to pear shaped cells. Their contents are yellow to brown. E 410 E 412

5 E 410 vs. E 412: what the EC says (II. Identification) Control LBG Control Guar gum A B

6 Other methods from literature Methods based on the polysaccharide composition Galactose to mannose ratios Methods based on the polysaccharide composition and structure Lectin assays

7 Mannose to galactose ratios (I) EC says nothing in E 410 and E 412 descriptions But when describes E 417 says that mannose:galactose ratios are –3:1 for E 417 –4:1 for E 410 –2:1 for E 412

8 Mannose to galactose ratios (II) 4:1 for E 410; 2:1 for E 412 But, different ratios have been described: –3.0:1 and 1.5:1 (Preuss and Their, Z Lebensm Unters Forsch, 175:93, 1982) –2.7:1 and 1.4:1 (Angelini et al, Riv Soc Ital Alim, 13:479, 1984) –3.7:1 and 2.3:1 (Cheetham et al, Carbohyd Pol 6:257, 1986) –3.7 to 7.7:1 depending on the solubilization temperature of E 410 (Lopes da Silva and Gonzales, Foof Hydrocol. 4:277, 1990)

9 Mannose to galactose ratios (III) Variations difficult demonstration of E 412 presence in E 410/E 412 mixtures Furthermore, man:gal ratios will be affected if other food additives (e.g., man from E 415) Also, man and gal can be present in processed foods, thus affecting the man:gal ratios Techniques are cumbersome and require complex extractions

10 Lectin assays Lectins are plant proteins which bind polysaccharides Different lectins bind different polysaccharides Patel et al. (Leatherhead Food RA) immobilized lectin lectin binds guar but not LBG bound guar is detected with the same lectin labeled with an enzyme LBG GUAR color readings lectin guar LBG enzyme support

11 Enzyme-Linked Lectin Assay (ELLA) LBGGuarTara E 410 commercial samples Optical density buffer

12 Development of new (DNA-based) methods E 410 Locust bean gum is the ground endosperm of the seeds of the natural strains of carob tree, Cerationia siliqua (L.) Taub. (family Leguminosae). Consists mainly of a high molecular weight hydrocolloidal polysaccharide, composed of galactopyranose and mannopyranose units combined through glycosidic linkages, which may be described chemically as galactomannans E 412 Guar gum is the ground endosperm of the seeds of the natural strains of guar plant, Cyamopsis tetragonlobus (L.) Taub. (family Leguminosae). Consists mainly of a high molecular weight hydrocolloidal polysaccharide, composed of galactopyranose and mannopyranose units combined through glycosidic linkages, which may be described chemically as galactomannans

13 DNA and its building blocks

14 Examples of languages (messages) English Musical Score Morse code Chinese DNA

15 DNA vs. amino acid information

16 DNA information is conserved

17 The Polymerase Chain Reaction

18 Amplification step of the PCR

19 Seeds as sources of DNA Germination of seeds (carob and guar) Extraction of DNA from fresh tissues PCR amplification of extracted DNA Electrophoretic analysis of amplification products Marker 1 Marker 2 LBGLBG LBGLBG GUARGUAR GUARGUAR

20 Unveiling the Markers sequences Marker 1 Marker 2 LBGLBG LBGLBG GUARGUAR GUARGUAR Isolation of markers from gel Sequencing

21 Marker 1 restriction analysis BcnI ClaI HaeIII LBGLBG GUARGUAR LBGLBG GUARGUAR LBGLBG GUARGUAR PCR amplification and digestion

22 Marker 2 restriction analysis SmaI XhoI HaeIII LBGLBG GUARGUAR LBGLBG GUARGUAR LBGLBG GUARGUAR

23 Markers sequences Marker 1 Marker 2 LBGLBG LBGLBG GUARGUAR GUARGUAR Isolation of markers from gel Sequencing

24 Carob vs. guar sequences (Marker 2) identical different P3 GUAR CAROB GUAR CAROB PG22 GUAR CAROB GUAR CAROB GUAR CAROB PG21 GUAR CAROB PG21 GUAR nnnn CAROB P4 GUAR CAROB P4 GUAR CAROB

25 Specific amplification of guar DNA from seeds Guar seeds Carob tree seeds

26 Investigation of DNA extraction from locust bean and guar gums (E 410 and E 412)

27 Specific amplification of guar DNA from mixtures of LBG and known additions of guar gum % 20% 10% 12% 6% 2% guar+LBG mixture 1 mixture 2 LBGLBG GUARGUAR

28 Specific detection of guar DNA in commercial samples labeled as E Guar LBG Commercial samples labeled as E 410

29 Specificity of the amplified products uncut T a q I X h o I restrictions Amplification products were cutted with a restrictase specific of the guar sequence analyzed by gel electrophoresis

30 Molecular methods for detecting additions of guar gum to locust bean gum Work developed by: S. Albertí, A. Doménech-Sánchez, V.J. Benedí S. Albertí, A. Doménech-Sánchez, V.J. Benedí M.L. Hernández, J.A. Rosselló M.L. Hernández, J.A. Rosselló Funded by: Carob S.A. Carob S.A. With the collaboration of: A. Juan, J. Sansegundo (Carob S.A., lab) A. Juan, J. Sansegundo (Carob S.A., lab) D. Álvarez, M. Urdiaín (UIB) D. Álvarez, M. Urdiaín (UIB) C. Murillo and R. Jiménez-Flores (CalPoly, USA) C. Murillo and R. Jiménez-Flores (CalPoly, USA)


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