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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, 09.12.98 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, 09.12.98 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 1.21.2 0.8 0.6 0.4 1.0 0.2 0 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 1 12341324124424432342324441232414311221212441123424 50 CAROB 1 12341324124424432342324441234414311221212441123424 50 GUAR 51 41333232443311324213111244323442112323332442334114 100 CAROB 51 41311212443341344213111244343442112123332442334134 100 PG22 GUAR 101 2233242223432331233122232132323 211233314431322- 149 CAROB 101 42332442234321312331222121343434312132333334333321 150 GUAR 150 -43213134111221213211242122-3244121122311333432423 197 CAROB 151 22331313232122311324224412223424114122331133212423 200 GUAR 198 21123313412211221124441132421332122122422412334324 247 CAROB 201 11323311412241223334444242421132322122324312334324 250 PG21 GUAR 248 112414122122112421441142313242433341321242324313-3 296 CAROB 251 3--23432212241242344424-31344243342132123442121341 297 PG21 GUAR 297 413112 1122431311124333nnnn434124234131332-412313 341 CAROB 298 43332224112323132242433311342341323242212341412331 347 P4 GUAR 342 21143242433134221342432222114333231242111442341413 391 CAROB 348 32443222433314421342232222314313231242111442341413 397 P4 GUAR 392 4414423224224 405 CAROB 398 4414423224224 410

25 Specific amplification of guar DNA from seeds Guar seeds Carob tree seeds 100 200 300 400

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 100 200 300 30% 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 410 300 200 100 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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