WO2010086841A2

WO2010086841A2 - An enzymatic process to enhance total reducing sugars (fermentable & un-fermentable sugars) in molasses, post production during transport and storage

Info

Publication number: WO2010086841A2
Application number: PCT/IB2010/050506
Authority: WO
Inventors: Sathyanathan Dhinakar Kompala; Sucharita Swati Dash; Subramani Ramachandrappa
Original assignee: Richcore Life Sciences Pvt. Ltd.
Priority date: 2009-02-02
Filing date: 2010-02-03
Publication date: 2010-08-05
Also published as: WO2010086841A3; WO2010086841A4; WO2010086841A8

Abstract

The present invention relates to an enzymatic process which maintains and/or enhances quantity of Total Reducing Sugars (TRS) in stored molasses resulting in enhanced ethanol yield on fermented with yeast.

Description

Title of Invention: AN ENZYMATIC PROCESS TO ENHANCE TOTAL REDUCING SUGARS (FERMENTABLE & UN- FERMENTABLE SUGARS) IN MOLASSES, POST

PRODUCTION DURING TRANSPORT AND STORAGE

Technical Field

[1] The present invention relates to a process facilitated by enzymes that maintains and enhances availability of total reducing and fermentable sugars in molasses by hy- drolyzing various polysaccharides present in molasses, arresting deterioration of molasses and enhancing their shelf life during the long-term storage. Background Art

[2] The term molasses (from Greek word 'meli' meaning honey) is referred specifically to the final viscous byproduct obtained in the preparation of sucrose by repeated evaporation, crystallization and centrifugation of juices from sugar cane or sugar beet. The various sources of molasses are cane sugar, sugar beet, starch including maize and sweet sorghum, citrus and hemicellulosic extract. It is one of the major byproducts of sugar industry and is widely used for the industrial production of ethanol, food additive and animal or poultry feed. The quality of molasses depends on the maturity of the sugar cane, sugar beet, starch, maize, sweet sorghum and citrus juice etc the amount of sugar extracted, and the method of extraction. The flavor and color of molasses varies depending on how early or late in the process the molasses is extracted.

[3] Because of its unusual properties, molasses has several uses. It can be used as the base material for fermentation into rum, and as the carbon source for in situ remediation of chlorinated hydrocarbons. Also, it can be used as a minor component of mortar for brickwork. It can be used as a chelating agent to remove rust where a rusted part stays a few weeks in a mixture of 1 part molasses and 10 parts water. Molasses can also be added to the soil of almost every plant to promote microbial activity. Blackstrap Molasses is often used in horticulture as a flower blooming and fruiting enhancer, particularly in organic hydroponics. Blackstrap Molasses may also be used as an iron supplement for those who cannot tolerate the constipation associated with iron supplementation. Molasses is also used as an additive in livestock grains to increase the protein content and also commonly used in dark brewed beverages like stout and very heavy dark ales.

[4] One of the most important industrial applications of molasses is that it is used to produce ethanol by fermentation of available sugar using various micro-organisms such as yeast. Ethanol fermentation is a complex biological process for the production of alcohol from sugar. Alcohol is widely used for many different purposes. The organisms such as yeast, under anaerobic conditions, convert glucose to ethanol. The stoichiometric equation for the production of alcohol by fermentation is given below:

[5] C₆H₁₂O₆ (Glucose ) > C₂H₅OH (Ethyl Alcohol ) + 2CO₂ (Carbon

Dioxide) -265kcal /kg cal

[6] The level of sugars available in molasses for fermentation is very critical because reduced levels of sugar result in low level of ethanol production. Indian Alcohol Industry mostly consumes sugarcane molasses as the raw material. Various components in molasses and other factors have direct impact on fermentation process such as:

[7] • Low purity cane juice leads to formation of non-sugars in molasses, which creates problem in the fermentation process.

[8] • Molasses produced by double sulphitation process contain sulphur compounds, which are toxic to the living organisms.

[9] • Fresh molasses lowers the fermentation efficiency due to high sulphite content.

[10] • Old molasses also shows poor recovery due to formation of complex compounds by reaction of reducing sugars with nitrogenous bodies. Disclosure of Invention Technical Problem

[11] However one of the most critical issues with molasses is the long term storage.

Molasses are usually stored in the industries for 10 days to 6 months in storage tank of the volume varying from 1 to 5 lakh liters. The sugars in molasses are easily available to the microbial deterioration and other environmental factors causing, the quality of molasses to drop with time in terms of content of Total Reducing Sugars (TRS) such as glucose, fructose, glyceraldehyde, lactose, arabinose and maltose that falls approximately 2 to 3 % if molasses are stored for more than 2 months. Exogenous factors such as high temperature also enhance the process of deterioration or spoilage leading to short shelf life and loss of available sugar, thereby resulting in low ethanol yield. Therefore, 1-2 months stored molasses is ideal for fermentation as during storage sludge settles at bottom & entrapped gases like SO2 escapes. Molasses are properly cooled in the sugar factory before it is pumped to the molasses storage tank and molasses storage tanks are cooled by external surface cooling. Technical Solution

[12] In India, the production of molasses is about 8-12 million tonnes per year, major portion of which serves as a raw material for ethanol production in more than 200 distilleries. Owing to the increasing demand and cost of molasses, it is extremely important to have a process that maintains quality and enables long term storage of molasses without affecting the levels of TRS.

[13] The present invention relates to a process facilitated by enzymes that maintains and enhances availability of TRS and fermentable sugars in molasses by hydrolyzing various polysaccharides present in molasses, compensating and minimizing losses due to deterioration of molasses and enhancing their shelf life during the long-term storage. The invention has a great commercial value as molasses has various industrial application and hence maintaining sugar- level, quality and shelf-life is commercially very valuable.

[14] The object of the present invention is to maintains and enhance the availability of

TRS and fermentable sugars in molasses during long time storage by hydrolyzing various polysaccharides present in molasses, arresting deterioration of molasses and enhancing their shelf life.

[15] The present invention provides a process facilitated by enzymes that enhances availability of TRS and fermentable sugars in molasses during long term storage and the said process comprises of following steps:

[16] 1. Molasses sample were obtained from various distilleries across India and molasses producing countries across the world.

2. A mixture of enzymes such as xylanase, amylase, beta glucanase, cellulase, alpha galactosidase and xylose isomerase in various combinations and ratio was added to molasses along with the antibiotic virginiamycin during the period of storage.

3. Total reducing sugar was estimated by Fehling's titrimetric method after the interval of 10 days to study the change in reducing sugar content.

[17] Molasses are the final byproduct obtained in the preparation of sucrose by repeated evaporation, crystallization and centrifugation of juices from sugar cane and from sugar beets. Molasses are usually stored in the industries for 10 days to 6 months in storage tank of the volume varying from 1 to 5 lakh liters. The biggest problem with storage of molasses is that since sugars in molasses are easily available to the microbial deterioration, the quality of molasses drops in terms of content of Total Reducing Sugar (TRS) that falls approximately 2 to 3 % if molasses are stored for more than 2 months. Exogenous factors such as high temperature also enhance the process of deterioration or spoilage leading to short shelf life and loss of available sugar, thereby resulting in low ethanol yield. Advantageous Effects

[18] The present invention relates to a process facilitated by microbial enzymes that maintains and enhances availability of TRS and fermentable sugars in molasses by hydrolyzing various polysaccharides present in molasses, arresting deterioration of molasses and enhancing their shelf life during the long-term storage. Description of Drawings [19] Figure 1 : Increase in Total reducing Sugar ( % TRS) in molasses samples treated with enzyme combination during 60 days storage period at 25 - 30 ⁰C as compared to control sample without enzymes stored under similar conditions for the same duration. [20] Figure 2 : Concentrations of sucrose in molasses sample with enzymatic treatment

(test) and without enzymatic treatment (control) during 60 days storage period at 25 -

30 ⁰C. [21] Figure 3 : Concentrations of glucose in molasses sample with enzymatic treatment

30 ⁰C. [22] Figure 4 : Concentrations of fructose in molasses sample with enzymatic treatment

30 ⁰C. [23] Figure 5 : Concentrations of xylose in molasses sample with enzymatic treatment

30 ⁰C.

Best Mode [24] More especially, the present invention describes a process facilitated by microbial enzymes that maintains and enhances availability of TRS and fermentable sugars in molasses by hydrolyzing various polysaccharides present in molasses. The process also arrests deterioration of molasses, enhances sugar content and shelf life of molasses during the long-term storage enabling high ethanol yields from the molasses. [25] The molasses are rich in minerals like calcium, phosphorous, potassium, magnesium, sodium, chlorine and sulfur as depicted in Table 1. [26] Table 1: Composition of molasses

[Table 1] [Table ]

[27] The composition of molasses varies depending upon the factors such as components of soil, climatic conditions, harvesting practices, manufacturing practices, handling and storage conditions etc. Apart from fermentable saccharides such as sucrose, fructose, glucose, and maltose, the molasses also contain unfermentable polysaccharides such as amylose, amylopectin, hemicellulose, cellulose, xylans, pectins etc. which can be hy- drolyzed to fermentable saccharides by specific enzymes addition of which to molasses during long-term storage conditions enables conversion of unfermentable polysaccharides into fermentable saccharides leading to enhanced level of available sugars.

[28] In the present invention, specific enzymes such as xylanase, amylase, beta glucanase, cellulase, alpha galactosidase and xylose isomerase in specific ratio and quantity have been added to molasses at sugar mill post production and/or in transport systems/ vehicle and/or while filling into storage tanks from transport system/vehicle and/or in storage tanks and/or in transfer system between storage tank and fermentors. The enzymes increase the amount of available total reducing sugars (fermentable sugars & unfermentable sugars) by hydrolyzing the polysaccharides present in molasses and additionally also arrest the decrease in TRS levels. During storage of molasses, these enzymes:

• Break down the oligosaccharides to easily fermented monosaccharides, and;

• Isomerize the unfermented xylose to a readily fermentable xylulose.

[29] The combination of enzymes working in tandem also increases availability of fermentable sugars that may be converted to ethanol by Saccharomyces cerevisiae, thereby ensuring higher yields of ethanol from molasses.

[30] TRS in molasses after storage with and without enzymes was studied in the present invention. As indicated in Figure 1, the TRS level was enhanced from 49% to 51% in samples treated with enzymes whereas the reducing sugar level in control sample dropped down from 49% to 46% within the 60 days study period under similar storage conditions. This increase in reducing sugar level can be therefore attributed to the enzymes added to the test sample during the molasses storage period.

[31] Since, the enzymes act on complex carbohydrates in the molasses such as starch, xylan, hemicelluloses and cellulose and convert them to simpler monosaccharides resulting in increase in TRS level, the commonly observed drop in reducing sugar level in molasses due to storage thus gets compensated or counteracted with a significantly larger increase in TRS. This maintains and increases the quality of molasses as measured by the TRS level for subsequent fermentation.

[32] Analysis of molasses sample by High Performance Liquid Chromatography (HPLC) shows a distinct pattern of sugar levels in enzyme treated and control samples. Sucrose concentration was reduced by 1% in case of control sample whereas a 0.2% increase was observed in case of the enzyme treated molasses sample after 60 days of storage (Figure 2) which corresponds to a total of 1.2% compensation by use of enzymes. The increase in concentrations of glucose and fructose was however more pronounced than sucrose since these are the end products of hydrolysis of most of the complex polysaccharides such as starch, cellulose and pectin (Figure 3 & 4). The decrease of fructose was greater than that of glucose in the untreated molasses sample. Similarly, the xylose content was found to increase in the samples treated with enzymes as compared to the untreated sample but in this case the decrease in not much pronounced (Figure 5).

[33] The enzymatic treatment was very effective in maintaining or enhancing the TRS levels in molasses sample for up to 60 days of storage while a pronounced reduction of the same was noticed in case of control samples. The reducing sugars released by the action of enzymes helped in restoring the lost sugars on account of various factors such as contamination, climatic conditions and other intrinsic factors in the molasses acquired during sugar processing such as chemicals and proteases. This in turn not only maintained the reducing sugar level but also provided additional substrate for yeast fermentation subsequently resulting in higher alcohol yield. Mode for Invention

[34] In order that this invention be more fully understood, the following preparative and testing examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

[35] Example 1

[36] Sugar estimation after enzymatic action on molasses

[37] Molasses samples were obtained from various distilleries across India and other molasses producing countries. The enzymes such as alpha amylase, glucoamylase, cellulase, xylanase, alpha galactosidase and glucose isomerase were procured from commercially available sources and were of industrial grade. The qualities of individual enzymes were assayed by routine biochemistry before the start of the experiment. Sugar standards for High Performance Liquid Chromatography (HPLC) were procured from Sigma. All other chemicals were of reagent grade and procured locally.

[38] A mixture of enzymes such as xylanase, amylase, beta glucanase, cellulase, alpha galactosidase and xylose isomerase in various combinations (0.5% of the enzyme blend) was added to 500 g of molasses sample followed by proper mixing of the enzyme blend with the molasses. The samples were covered and stored at room temperature (25 - 30⁰C) for a period of 60 days. No enzyme was added to the control samples. Samples were taken at 10 days interval starting from the day of set up (0 day) and analyzed for total reducing sugars and complete sugar profile.

[39] Total reducing sugar was estimated by Fehling's titrimetric method after the interval of 10 days to study the change in reducing sugar content. Complete profiling of soluble sugars was done by HPLC on Zorbax carbohydrate column using acetonitrile: water 75: 25 as mobile phase at flow rate of 1.2 ml/min at 28° C. Detection by was Refractive Index detector at 28° C.

Claims

[Claim 1] A process to efficiently maintain and enhance quantity of total reducing sugars as well as fermentable sugars in molasses during storage, the said process is catalyzed by a mixture of enzymes used in various combinations and ratio, resulting in hydrolysis of polysaccharides into simple sugars.

[Claim 2] The process of claim 1 wherein the enzymes used to catalyze the process are xylanase, amylase, beta glucanase, cellulase, alpha galac- tosidase and xylose isomerase.

[Claim 3] The process of claim 1 wherein the enzymes are added to molasses at the sugar mill, post production or in storage tanks or during transfer of molasses from storage tank to fermentor or during transport of molasses.

[Claim 4] The process of claim 1 wherein enzymatic reaction is carried out at ambient temperature.

[Claim 5] The process of claim 1 wherein the said process results in substantial enhancement in level of total reducing sugar and fermentable sugar as compared to the control samples during molasses storage.

[Claim 6] The process of claim 1 wherein the said process efficiently compensates drop in total reducing sugar and fermentable sugar level in stored molasses and maintains sufficient sugar level for subsequent fermentation.

[Claim 7] The process of claim 1 wherein the said process also increases shelf-life of molasses significantly.

[Claim 8] The process of claim 1 wherein the enhanced level of total reducing sugar and fermentable sugar result in high ethanol yield from molasses fermentation.

[Claim 9] The process of claim 1 wherein the enhanced level of total reducing sugar and fermentable sugar result in increase in quantity of available carbon source for all industrial uses of molasses.