Importance of controlling microbes during sugar production from cane and its effect on sugar yield & losses of suga

Importance of controlling microbes during sugar production from cane and its effect on sugar yield & losses of sugar By V. M. Kulkarni 6th International Sugar Conference, November 10 – 13, 2012. Aswan, Egypt WWW.VM BIOTECH.COM

Sugar / Sucrose It is known that sugar is not “manufactured” by us in sugar factory; but it is produced in the plant – Sugarcane cultivated by the farmer in the field. We, in sugar factory, extract, purify and separate sucrose. During this process, we can’t separate all sucrose in to bag as some sucrose is lost due to presence of impurities in sugarcane juice and other parameters. Thus it is impurities that determines fate of sucrose – to be in bags or to be in molasses.

Sugar losses • Sugar losses are of 4 types • Chemical, due to changes in pH and temperature – can be reduced only by strict control on parameter • Microbial, due to direct consumption of sugars for growth • Enzymatic – microbial and invertase present in sugarcane cellsand • Indirect losses due to microbial metabolites. http://www.vmbiotech.com

Philosophy of V.M.Biotech • We have introduced the new thought of giving importance to impurities rather than purity and paper presented in ISSCT 2005 at Guatemala received good response • ‘Prevention is better than Cure’ is a base of our new process • We aim to use technology to prevent impurity formation rather than remove them later www.vmbiotech.com

What is appropriate chemicals • Our philosophy is “prevention is better than cure” and selection on the basis of natural logical principals • We have observed during microbial control studies in more than 100 factories that proper control on microbial activity is essential to control impurity development including color • Let us look at this most crucial factor www.vmbiotech.com

Microorganisms • Are microscopic, can’t be seen by necked eye, requires microscope with 1000 X magnification (oil immersion lens). • Their surface to volume ratio is very high. • They multiply very rapidly, and hence can exert impact on environment. • They are omnipresent, omnipotent and omnivorous …… just like GOD ! www.vmbiotech.com

Microbes and God. • Microbes are present every where – from dry, hot dessert to the cold’s of Antarctica. • They can grow in absence of oxygen. • They can grow at : • 1050 C, Yellowstone National Park Geezers to – 500 C at Antarctica. • pH 0.5, Thiobacillus to many alkalophiles at pH11.5 • Distill water to high salinity of the Dead Sea, They also are known to cause foaming / deterioration of molasses 900 Brix in storage tanks. www.vmbiotech.com

Microbes and God. • They can degrade KCN, and can produce toxin few hundred times toxic than KCN. • They are so versatile that they can degrade all most any thing. • When they are angry – they produce epidemics which kills many people, and when are worshiped, we are blessed with antibiotics. • Without them our daily food requirements are incomplete and improper use can have food poisoning. www.vmbiotech.com

Thus microorganisms are • Funny little entities, they won’t grow if they don’t want to – even if you all dance on table; and they will continue to grow if they want to grow – even if you all dance on the table. • Dr. A. D. Agate, Professor of Microbiology. www.vmbiotech.com

Microorganisms and survival • Microbes do have phenomenal ability to sustain adverse conditions for very long time, and they grow rapidly as soon as favorable conditions returns. • Microorganisms are known to survive under totally abnormal conditions for 7.5 billion years! Desulfovibro. • Microbes form colonies, too difficult to penetrate – called biofilm www.vmbiotech.com

Survival of Microbes • Microbes Protect Themselves in nature by: • 1. growing as biofilms • Resistant to penetration by antimicrobials, important in sugar mills • Modifying or excluding antibacterial agent • 2. Forming spores • Most resistant form of bacteria. Generally found in sugar

Biofilms • Bacteria have the ability to colonize process surfaces • this leads to build up of slime materials or biofilms. • The biofilm can become • an ecosystem with a wide • variety of pathogenic and • spoilage microorganisms • and a penetration barrier for • biocides. Rod-shaped bacterium

Bacterial Biofilms SEM image ofsixday old Pseudomonas aeruginosa biofilm.

Microorganisms in Sugar Industry. • Sugar cane and mill house products are most nutritive for many microbes to grow rapidly and consume sugar to produce various metabolites which hinders in sugar production by causing process difficulties and adversely affects sugar quality. • Many of them are not eliminated by boiling juice for clarification and in fact some grow at clarifier and continue to grow during further process. www.vmbiotech.com

Microorganisms in Sugar Industry. • Microbes gain entry via cut ends of harvested cane and continue their growth till they are eliminated. • Depending on environmental conditions, PJ contains about 106 to 109 cfu per ml. • They vary qualitatively form place to place and season to season, cold climate favors yeast while Lactobacillus predominates in summer. www.vmbiotech.com

Microorganisms in Sugar Industry. • These microbes grow and consume sugar at rapid rate and it is believed that more than 1% on cane sugar is lost during cut – to – mill delay. • Further, microbes produce various metabolites that interferes in the process and affects sugar recovery and sugar quality adversely. www.vmbiotech.com

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Products of microbial metabolism. • Inversion of sucrose to reducing sugars. • Alcohol generation – Yeasts • CO2 gas formation • Production of various acids. • Polymerization of glucose / fructose to form dextran, oligosaccharides, leavan and other polysaccharides.

Reducing Sugars. • Also present naturally in cane, concentration depends on variety, growing conditions, maturity and degree of freshness. • Microbes produce enzyme invertase (also present in sugarcane cells) which converts to reducing sugars. • Higher reducing sugars indicate that sucrose is lost either after harvesting or during milling. • Usually fresh mature cane have RS % Bx 2.00, more RS is due to stale cane.

Acids. • Present naturally in cane. • They are produced by degradation of reducing sugars by many microbes. • Concentration depends on cane maturity and freshness of cane. • High concentration indicates sugar losses, consequently more sucrose is lost in molasses. • Fresh, mature cane juice has TA 7.5 % Bx.

Coloring matter. • Present naturally in cane. • Concentration depends on cane variety, maturity and freshness of cane. • Trash and tops contains coloring matter in large quantity. • Higher coloring matter are found to be associated with higher acidity, staleness and higher microbial count.

Polysaccharides - Dextran : Origin • Dextran is present in cane / juice as a result of the infection of bacteria Leuconostoc (Lactobacillus group) • Concentration mainly depends on cut – to – mill delay; poor housekeeping also forms dextran. • Hot humid conditions favors dextran formation. • Burnt cane, small billets, damaged cane also favors dextran formation.

Dextran : Processing • Increases viscosity – dextran’s physical property. • Poor clarification – act as protective colloids & hinders aggregation & settling of Ca phosphate. • Decreases crystal growth rate. • Drop in boiling house performance. • Deteriorates molasses exhaustion. • Decreased pan and centrifugal capacity. • Pol increases are possible.

Dextran : some consequences du Boil SASTA 2001 • Assume 85 pty, S 10% >>> Bx. 11.8 • Literature : lose 1 g / L for 250 mg / L dextran made.

Dextran : some yardsticks. • Each 250 ppm in juice represents direct sucrose loss of 1000 mg. • 1000 ppm on Bx inflates molasses purity by 3.15 (eq. 0.75 units per 1000 ppm on Bx in molasses) • For every 300 ppm in syrup a purity increase 1 unit can be expected in final molasses. • 1000 ppm on brix in molasses (250 ppm on brix in mixed juice) gives a loss of exhaustion performance of 1.2 to 1.4 units of purity. • Dextran in sugar is about one tenth of that in syrup. • White sugar with > 150 ppm dextran will produce distorted candy

Oligosaccharides : • These are small molecules of about 2 to 10 monosaccharide (M.Wt. < ca 1600). • They are mainly ketoses and theanderose. • They are formed during cut – to – mill delay and during process. • Concentration in juice depends on climatic conditions and cane burning.

Oligosaccharides : processing. • Dominant crystal habit modifiers. • Reduces rate of crystallization. • Accumulates rapidly in burnt cane. • Hygroscopic – similar to invert. • Some strongly incorporated in crystals. - about 50 X more than invert. • Slight effect on pol. • Effect can be reduced by minimizing cane delays and good mill sanitation.

Other polysaccharides : Levans. • These are high molecular wt., mostly water soluble polysaccharides (B-2-6 linked polyfructosans.) • Exhibit negative optical rotation. • Formed by action of levansucrase on sucrose, produced by bacteria (Bacillus species) • This fructose polymer is of factory origin than field origin and can be controlled by effective mill sanitation.

Rules of Sucrose Degradation.Clarke et al. 1997 • Sucrose degrades in acid more easily than in alkali, and invert is more reactive in alkali than in acid. • In acid, the rate of sucrose hydrolysis is faster than the rate of degradation of its inversion products. • In alkali, the rate of sucrose degradation is much less than the rate of glucose and fructose degradation. • Alkaline degradation (pH<8.5) of sucrose does not result inversion products, hence the loss of sucrose to invert is a consequence of the acid hydrolysis which provides glucose and fructose for further alkaline degradation. www.vmbiotech.com

Microbial degradation of sugars • Sucrose is first converted to glucose and fructose, which are then degraded / utilized by microbes to produce various metabolites. • Glucose is utilized to form dextran, acids, alcohol, gas and other polysaccharides. • Fructose is converted to glucose and also used to form complex polysaccharides. • No rules apply for these conversion and all reaction can occur irrespective of pH and temperature. www.vmbiotech.com

Microbial degradation of sugarsat high temperature • Microbes capable of growing at higher temperature also gain entry via cane, they remain dormant at normal temperature. • These thermophiles grow in clarifier and during further process. • Major end product of their metabolism (80%) is Lactic acid. • Thermal degradation of invert also produces acid www.vmbiotech.com

Evaluation of sucrose loss • Purity Drop from PJ to MJ. • Analysis of Dextran. • Analysis of alcohol. • Microbial count. • Rise in Reducing sugars from PJ to MJ. • Rise in RS as well as acidity from PJ to MJ to Clear juice till Final Molasses www.vmbiotech.com

Purity Drop from PJ to MJ. • There are many optically polar compounds in cane juice like dextran, reducing sugars, amino acids and organic acid. • Amount of these compounds vary from cane to cane and also depend on microbial growth. • Due to this large variation and changes due to microbial growth from PJ to MJ, Purity Drop is never a reliable criteria for estimation of sugar loss.

Analysis of Dextran • Haze method analyzes dextran from sugar and is not reliable for juices. • Starch and other polysaccharides interferes in the estimation and cause errors. • Dextran is produced by Leuconostoc, which belongs to lactobacillus family, and thus lactic acid is better indicator & can account for other non dextran forming microbes.

Analysis of alcohol • Yeasts are one of the major contaminant in harvested cane. • Thus alcohol estimation is used to determine cut – to – mill delay. • However, difference in temperature and dynamic flow of juices, it can’t be used reliably for losses from PJ to MJ.

Microbial Count • Samples can’t be preserved, and spot analysis is never representative. • Microbes grow in geometric phase. • They are in very large number more than 107 per ml. • Thus delay in plating by just 1 minute can influence results dramatically. • Thus this criteria can never give true picture.

Rise in Reducing sugars from PJ to MJ. • This is considered to be better criteria. • Reduction in rise in reducing sugars form PJ to MJ can be due to two reasons : i) Prevention of inversion of sucrose and ii) Destruction of reducing sugar, which is very harmful to the process. • Thus this criteria alone is insufficient to know sugar losses. www.vmbiotech.com

Rise in RS and acidity from PJ till Molasses • Since degradation product of reducing sugar is acid, its estimation by titration along with the analysis of reducing sugar can reliably evaluate sugar losses. • When biocide capable of killing microbes is used, it must show downstream effects. • Molasses being stable and can truly represent large amount of cane, thus is most reliable sample and results are reproducible and accurate. www.vmbiotech.com

Proper analysis only can throw better light on process. www.vmbiotech.com

Control of microbial sugar loss • Sugarcane juice is most nutritive and contains organic matter, suspended and dissolved solids in large quantities, which acts as protective agents and do not allow many chemicals to kill microbes. • However, chemicals used at mills are called as mill sanitizers and they are :

Control of microbial sugar loss • Halogen based biocides. • Other oxidizing biocides. • Quaternary ammonium compounds. • Dithiocarbamate based biocides.

Halogen biocides : Limitations. • They react with reducing and organic matter which is in plenty in cane juice, thus are ineffective in sugarcane juice condition. • Further, they form many carcinogenic compounds by reacting with amino acids and can remain in sugar. Hence are not recommended for mill sanitation.

Other oxidizing biocides • Include formalin, H2O2, ClO2, peroxyacetic acid, and Ozone. • Reducing matter in cane juice and organic matter is very high, which consumes these compounds and protects microbes. • Although, these are safe to use, will require at very high dose to make them uneconomical. • They are difficult to handle and are very corrosive.

Quaternary ammonium compounds : mode of action. • Bacterial cell walls contains n-peptidoglycan, this gives negative charge to the bacterial surface. • Quats is attracted to these negatively charged sites of the bacteria. Then biocide penetrates the cell wall (structure) to reach protein material on the cytoplasmic membrane, interacts with suitably charged sites in proteins like carboxyl groups. • This disorganizes membrane, thus resulting in denaturizing & precipitation of proteins thus disturbs normal functioning of cell nutrition causing death.

Quaternary ammonium compounds • Comparative bactericidal activity (BS: 6471) of Home Quats • ------------------------------------------------------------------------------------------- • cetrimide 266.6 PPM • Alkyl benzyl dimethyl amm chloride 200 PPM • Alkyl benzyl trimethyl amm chloride 200 PPM • Dodecyl dimethyl amm chloride 200 PPM • Dodecyl ethyl methyl anthosulfate 333.3 PPM • ------------------------------------------------------------------------------------------

Quaternary ammonium compounds • Quats, especially benzalkonium,are preferred in food industries for sanitation. • Biocide activity is reduced drastically in presence of 250 ppm salts of calcium and magnesium, which are more than 800 ppm in cane juice. • At low dose (less than 20 ppm) they are not biocidal and can induce resistance in microbes and some bacteria can use quats as food for their growth.

Performance of QUATS in Hard Water* • ____________________________________________________________________ • microorganisms • BCK Diluted Pseudomonas E. coli Bacterium proteus Salmonella typhi • in aeroginosa 6749 B- 196 4635 3390 • ____________________________________________________________________Distilled Water 100 ppm 50 ppm 50 ppm 50 ppm • Hard Water 1500 ppm 250 ppm 250 ppm 250 ppm • * ( 300 ppm ) • ___________________________________________________________________

Importance of controlling microbes during sugar production from cane and its effect on sugar yield & losses of suga