Locust bean or guar? Molecular methods for detecting additions of guar gum to locust bean gum

1. 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 INEC annual meeting Granada, May 25-26, 2000

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 E 412 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.

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

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) LBG enzyme LBG guar lectin GUAR support bound guar is detected with the same lectin labeled with an enzyme immobilized lectin lectin binds guar but not LBG color readings

11. 1.2 1.0 0.8 0.6 0.4 0.2 0 Enzyme-Linked Lectin Assay (ELLA) Optical density E 410 commercial samples LBG Guar Tara 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. G U A R G U A R L B G L B G Marker 1 Marker 2 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

20. G U A R G U A R L B G L B G Marker 2 Marker 1 Unveiling the Markers sequences • Isolation of markers from gel • Sequencing

21. G U A R G U A R G U A R L B G L B G L B G BcnI ClaI HaeIII Marker 1 restriction analysis PCR amplification and digestion

22. G U A R G U A R G U A R L B G L B G L B G Marker 2 restriction analysis SmaI XhoI HaeIII

23. G U A R G U A R L B G L B G Marker 2 Marker 1 Markers sequences • Isolation of markers from gel • Sequencing

24. Carob vs. guar sequences (Marker 2) different identical P3 GUAR 1 1 23413241244244323423 2444123 2 414311221212441123424 50 CAROB 1 1234132412442443234232444123 4 414311221212441123424 50 GUAR 51 413 33 2324433 1 13 2 42131112443 2 3442112 3 233324423341 1 4 100 CAROB 51 413 11 2124433 4 13 4 42131112443 4 3442112 1 233324423341 3 4 100 PG22 GUAR 101 2 2332422234323312331222 3 213 2 3 2 3 213 2112333 14 43 1 3 2 2 - 149 CAROB 101 4 2332442234321312331222 1 213 4 3 4 3 431 2132333 33 43 3 3 3 2 1 150 GUAR 150 -4 3 2 1313 411 122 12 132 11 24 2 122 - 3 24 41 21 1223 1 1 3 33 43 2423 197 CAROB 151 22 3 3 1313 232 122 31 132 42 24 4 122 2 3 42 41 14 1223 3 1 1 33 21 2423 200 GUAR 198 2 1 1 2331 3 4122 1 122 112 444 113 2421 3 32 1 22122 4 2 24 12334324 247 CAROB 201 1 1 3 2331 1 4122 4 122 333 444 424 2421 1 32 3 22122 3 2 43 12334324 250 PG21 GUAR 248 11241 4 1 22122 1 1242 1 44 11 4 2 313 2 42433 34 13212 42 3 2 43 13 -3 296 CAROB 251 3--23 4 3 22122 4 1242 3 44 42 4 - 313 4 42433 42 13212 344 2 12 13 41 297 PG21 GUAR 297 4 1 3 11 2 11 112 24 313 111 24333 nnnn4 341 2423 4 13 133 2- 4123 13 341 CAROB 298 4 3 3 32 2 24 112 32 313 224 24333 11342 341 3232 4 22 123 41 4123 31 347 P4 GUAR 342 211 432 4 2433 13 4221342 4 32222 1 143 3 3231242111442341413 391 CAROB 348 324 432 2 2433 31 4421342 2 32222 3 143 1 3231242111442341413 397 P4 GUAR 392 4414423224224 405 CAROB 398 4414423224224 410

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

26. Investigation of DNA extraction from locust bean and guargums (E 410 and E 412)

27. L B G G UAR 30% 20% 10% 12% 6% 2% guar+LBG mixture 1 mixture 2 Specific amplification of guar DNA from mixtures of LBG and known additions of guar gum 300 200 100

28. LBG Specific detection of guar DNA in commercial samples labeled as E 410 300 Guar 200 100 Commercial samples labeled as E 410

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

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