Background information on mannitol formed by Leuconostoc bacteria

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1 The Measurement of Mannitol in Sugar Beet Factories to Monitor Deterioration and Processing Problems Eggleston, G. 1 and Jean-Marc Huet 2 Groupement Echanges Techniques

2 Presentation Outline Background information on mannitol formed by Leuconostoc bacteria Mannitol versus dextran Development of an enzymatic mannitol method for sugar beet factories now Official ICUMSA Method GS8-12 (2010) Uses of mannitol in sugar beet factories Major Conclusions

3 Background Information on Mannitol

4 Major Metabolites of hetero-fermentative Leuconostoc Bacteria of Interest to the Sugar Beet Industry phosphoketolase dextransucrase nsucrose G F H 2 O Dextran (glucose) n + fructose isomaltooligosaccharides (up to 7dp) leucrose, palatinose Leuconstoc mesenteroides Levan + glucose (fructose)n nsucrose levansucrase No O 2 Glucose O 2 mannitol dehydrogenase lactic acid + ethanol lactic acid + acetic acid + CO 2 + CO 2 M it l Fructose Mannitol + lactic + acetic + CO 2 acid acid

5 Hetero-fermentative Lactic Acid Bacteria, Leuconostoc and Lactobacillus Bacteria Mannitol (ppm) Plating Counts (CFU x 10 7 /ml) Acetobacter t pasteurianus < Bacillus coagulans < Bacillus megaterium < Bacillus subtilis < Bacillus stereothermophilus < Lactobacillus buchneri Lactobacillus fermentum Lactobacillus fructosus Lactobacillus vaccinostercus Leuconostoc mesenteroides Eggleston, Basso, Amorim, Lima Paulillo and Basso, Zuckerindustrie

6 ASI Enzymatic Antibody Haze Dextran Dextran Dextran High MW Dextran High MW Dextran High MW Dextran Medium MW Dextran Medium MW Dextran Low MW Dextran Pectins Oligosaccharides Too Long and Complicated Too Expensive and Imprecise Other Micro-particles Not Specific and Difficult to Interpret

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8 Relations between mannitol and metabolite contents Acetic acid y = 5,54x - R 2 = 0, Mannit tol (ppm) D Lactic acid y = 3,54x - R 2 = 0,97 Dextran y = 1.31x R 2 = Metabolite (ppm)

9 Official ICUMSA Method GS8-12 (2010) The Determination of Mannitol in Beet Juices, Thin Juices and Syrups by an Enzymatic Method Eggleston and Harper (2006). Food Chem. Huet (2008). GS8 Report. ICUMSA. Eggleston (2009). AVH Proceedings. Huet (2010). GS8 Report. ICUMSA.

10 Mannitol dehydrogenase Mannitol + NAD + Fructose + NADH + H + Reaction Conditions 340 nm absorbance b Temperature : Room temperature ph : 10.5 (use glycine buffer) Reaction time : 7 min Simple Equipment Required: (usually present at sugar beet factories) Test-tubes Volum. flasks Pipettes Spectrophotometer Disposable cuvettes Timer

11 Linearity y = x R 2 = nm Abs at Mannitol (ppm)

12 Effect of Temperature Delta abs/ /min Temperature ( o C) Mannitol measured after ~7 min at room temp or within 4 min at 40 o C

13 Interference of Other Sugars Determination of mannitol in the presence of other sugarcane juices in simulated 14.0 Brix juices Simulated juice Mannitol conc. Added ppm Mean % recovery of mannitol Total variation CV (%) Sucrose Sucrose + glucose Sucrose + glucose + fructose Sucrose Sucrose + glucose Sucrose + fructose Sucrose + glucose + fructose Sucrose + glucose + fructose + dextran N=

14 Mannito ol (ppm) by enzymatic method y = x R 2 = Accuracy Mannitol (ppm) by IC method No statistical differences between the two methods were found at the 95% probability level for any cane juice studied

15 Sugar Beets Jean Marc Huet Tested, adapted, and improved the enzymatic method for sugar beet cossette and diffuser juices: Absorbance in sample cells sometimes increased before enzyme added adding a 3 min stabilization time before MDH addition overcame this problem Accuracy and precision (precision less at lower mannitol values) were acceptable

16 Recovery Tests in Raw and Thin Juices Mannitol Recovered in Raw Juice Added (ppm) ppm Recovery % CV % Recovered in Thin Juice Mannitol + NAD + Fructose + NADH + H +

17 Method GS8-12 (2010): The Determination of Mannitol in Beet Juices, Thin Juices and Syrups by an Enzymatic Method ICUMSA 27 th Session GS8 Report

18 Cost of Mannitol Enzymatic Method ~ 60 U.S. cents per analysis (45 US cents NAD,12.47 US cents MDH) Megazyme TM kit is US $4 per analysis Biosentec TM kit is US $4.65 per analysis

19 Uses of Mannitol in Sugar Beet Factories

20 Carbonation Clarification In 1998, Steimetz et al, Zuckerindustrie, reported that mannitol, compared to D and L lactic acids, acetic acid, glucose, fructose, leucrose, and ethanol correlated best with First Carbonation Filtration Coefficient Greater than 60 ppm mannitol predicts problems with First Carbonation Filtration Plant (Huet, ICUMSA, 2010)

21 mg / l Mannitol in the Inlet and Outlet Juices of the Raw Juice Heaters Mannit - content measurement / inlet and outlet of raw juice heater campaign Euskirchen factory, Germany; 2008 (Pfeifer & Langen) % at treated with NaOH 25/09/ /10/ /10/ /10/ at treated with NaOH 23/10/ /10/ /11/ at treated with NaOH 13/11/ /11/ at treated with at treated NaOH with NaOH 27/11/ /12/ /12/ /12/ /12/ /01/ inlet heater mg/l outlet heater mg/l valve position for juice flow

22 Raffinerie Tirlemontoise Company, Belgium, 2008 Mannitol in Raw Juice (ppm) Longhchamps Hollogne Wanze (Central Factory) 22 Oct Oct Oct Oct Oct Steam disinfection 31 Oct Nov Nov Nov Nov Nov Steam disinfection 26 Nov

23 Mannitol ppm French Sugar Beet Factories Sant Louis Sucre Factories Etrepagny Eppeville Cagny Roye Diffuser Juice <DL* 27 <DL <DL Diffuser Juice <DL Preheated Juice <DL 18 <DL Heated Juice JT JC <DL 41 Primary mud 18 <DL JT JC <DL <DL Secondary mud <DL 5 37 JAE <DL Sugar de-dusting d <DL <DL <DL Pressed water <DL 9 18 <DL 10 7 * <DL= lower than detection limits of the enzymatic method

24 French Sugar Beet Factories Groupement Echanges Techniques 2009 GET Factories studies: Mannitol is mainly produced within juice/cossettes heat exchangers: into a range of 5 to 40 g / t beets >95% of mannitol produced or contained in cossettes is extracted with raw juices, the rest with pressed pulp Stillage of raw juice distilleries recycled to the extractor feed water tank: Concentrations up to 200 ppm of mannitol, which may represent about 40 g of mannitol recycled/t beets In this case, ~20% of the mannitol brought or produced is extracted with pressed pulp, the rest with raw juices.

25 Cossettes and Raw Juices (RJ) Mannitol Contents (ppm) Cossettes Cell Juice Extractor RJ Cold RJ RJ after heat exchangers 40 Mannnito ol (ppm) FLD STE PTV TOU 1 TOU 2 NAN SOU French Factory

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28 Mannitol Levels Measured by the Eggleston Enzymatic Method in Sugar Beet Juice and Press Water Products Amalgamated Twin Falls Sugar Beet factory, Idaho, U.S. Date Twin Falls Factory Product Brix Mannitol ppm Mannitol ppm/brix 4/11/2009 Raw juice Diffuser juice; 6:00 pm Diffuser mid tower juice; 6:00 pm Press water from pulp pexiting the diffuser; 6:00 pm Re circulated juice in diffuser*; 6:00 pm Re circulated juice in diffuser*; 8:00 pm Re circulated juice in diffuser*; 10: pm 12/21/2009 Diffuser juice; 12:00 am Diffuser mid tower juice; 12:00 am Press water from pulp exiting the diffuser; 12:00 am Re circulated juice in diffuser*; 12: am Re circulated juice in diffuser*; 2:00 am Re circulated juice in diffuser*; 4:00 am

29 Major Conclusions Mannitol is a relevent tracer of Leuconostoc bacteria An enzymatic mannitol method is now an Official ICUMSA method GS8-12 (2009): The Determination of Mannitol in Beet Juices, Thin Juices and Syrups by an Enzymatic Method Mannitol measurements allow: Predicting and controlling processing problems (infections, filterability, cleaning) Adapting anti-bacterial treatments during processing Undertaking process improvements (dead end pipes elimination etc.) Routine mannitol determinations are necessary to define critical thresholds at each factory