FIELD OF THE INVENTION
The present invention relates to the field of fermentation and more particularly to a composition for bio-fuel fermentation and a process thereof.

BACKGROUND OF THE INVENTION
Production of bio-fuels such as bio-ethanol from organic by-products has acquired significance in recent years. Currently, bio-ethanol fermentation is the major source of fuel ethanol worldwide. Increasing demand of ethanol as a vital source of fuel and in beverages for human consumption, distillers are using high concentration of antimicrobial agents, for instance, antibiotics, biocides and harmful chemicals to control bacterial contamination in fermentation process to improve recovery of bio-ethanol production.

In fermentation process, fermentation ingredients like molasses, microbial nutrients, inorganic/organic salts, raw water and recycling condensate of fermentation process are the major source of bacterial contaminations. In Indian distilleries, molasses is the majority feedstock of bio-ethanol fermentation and contain majority of contaminations such as Lactobacillus spp. (Gram positive rods); Pseudomonas spp. (Gram negative coco-bacilli), Acetobacter spp., Pediococcus spp. and other Gram positive and Gram negative spp.

Other than molasses, raw water is also one of the major sources of contaminations. In the current study of bio-ethanol fermentation process, the filamentous members of the bacteroidetes (Genus: Haliscomenobacter) was recognized as a major contaminant from raw water used in bio-ethanol fermentation and is not manageable with conventional composition containing antibiotics (such as Penicillin G, Ciprofloxacin, Ampicillin, Tetracycline, Streptomycin, Monensin, Maduramicin and Virginiamycin), biocides (Benzalkonium chloride and Dithiocarbamate) and germicide (such as polyhexa-methylenebiguanide).

Conventional compositions and processes for production of the bio-fuel involve chemical process of reacting ethylene with steam, concentrated acid hydrolysis process, dilute acid hydrolysis, enzymatic hydrolysis and wet milling processes, dry milling process and fractional distillation process.

US2003185811A1 discloses process for preparing a herbal extract including the steps of mixing herbal matter with water to produce an aqueous extract solution, adding a nutritive supplement capable of supporting bacterial fermentation to the solution, seeding the resulting mixture with probiotic bacteria and incubating the seeded mixture to effect fermentation of the herbal matter.

US2011097765A1 discloses compositions and methods for enhanced fermentation of molasses using transglucosidase.

US9562239B2 discloses a process of producing a fermentation product, comprising: liquefying a starch-containing material to dextrin with an alpha-amylase in the presence of an asparaginase and/or an amino acid oxidase; saccharifying the dextrins to a sugar with a glucoamylase and fermenting the sugar using a fermenting organism.

The conventional compositions and processes for production of the bio-fuel may provide yields in return of minimal capital investments, however they suffer from plurality of shortcomings. The conventional process such as enzymatic hydrolysis is expensive and is still in its early stage of development. The contaminants in the existing processes not only deteriorate the molasses quality but also play crucial role to reduce the bio-ethanol recovery in yeast based fermentation by utilizing micro-nutrients and macro-nutrients present for bio-ethanol fermentation. Further, it is very difficult to avoid microbial contamination and to prevent antibiotic resistance during conventional bio-fuel fermentation processes. In other words, prolonged applications of antibiotics lead to emergence of antibiotic resistance in bacterial contaminants, ultimately these contaminants become unmanageable and thus, makes difficult to maintain profitable yield and recovery of the end product.

Therefore, there remains a need in the art for compositions and processes for bio-fuel fermentation which do not suffer from above mentioned discrepancies.

OBJECT OF THE INVENTION
It is an object of the present invention to provide a composition for bio-fuel fermentation to reduce unwanted contaminants.

Another object of the present invention is to provide the composition for bio-fuel fermentation with anti-bacterial properties.

Another object of the present invention is to provide the composition for bio-fuel fermentation such that all the constituents conjointly show synergistic effect for increasing bio-fuel yield.

Another object of the present invention is to provide the composition for bio-fuel fermentation to eliminate the use of antibiotics and biocides.

SUMMARY OF THE INVENTION
The present invention is described hereinafter by various embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only, and are not intended to limit the scope of the claims. In addition, a number of materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary, and are not intended to limit the scope of the invention.

According to an embodiment of the present invention, a composition for bio-fuel fermentation comprises, but not limited to, a plurality of plant extracts or plant extract fractions and a plurality of enzymes.

According to an embodiment of the present invention, the plurality of plant extracts or plant extract fractions are selected from a group consisting of, but not limited to, extracts of Gymnema Sylvestre, Senna alata, Clerodendrum infortunatum, Berberis aristata root, Cinnamomum Verum Bark, Cynodon dactylon, Moringa oleifera, Allium sativum and Azadirachta indica.

According to an embodiment of the present invention, the plurality of plant extracts or plant extract fractions are present in a range of, but not limited to, 1 mg/mL to 10 mg/mL.

According to an embodiment of the present invention, the plurality of enzymes are selected from a group consisting of, but not limited to, proteolytic enzymes and amylolytic enzymes.

According to an embodiment of the present invention, the proteolytic enzymes and amylolytic enzymes are selected from a group consisting of, but not limited to, fungal alpha amylase, bacterial alpha amylase, glucoamylase or neutral protease.

According to an embodiment of the present invention, the plurality of enzymes are present in a range of, but not limited to, 1 ppm to 100 ppm.

According to an embodiment of the present invention, the composition shows anti-bacterial activity against, but not limited to, microbial consortia and one or more contaminants.

According to an embodiment of the present invention, the microbial consortia is selected from a group consisting of, but not limited to, Lactobacillus species, Pseudomonas species, Acetobacter species, Haliscomenobacter species, other Gram positive and Gram negative species or a combination thereof.

According to an embodiment of the present invention, the one or more contaminants are selected from a group consisting of, but not limited to, Lactobacillus specie, Pseudomonas species, Acetobacter species, Pediococcus species and other Gram positive and Gram negative species.

According to an embodiment of the present invention, the plurality of plant extracts or plant extracts fractions shows maximum anti-bacterial activity at the concentration in a range of but not limited to, 1 mg/mL to 10 mg/mL.

According to an embodiment of the present invention, the bio-fuel is, but not limited to, bio-ethanol.

According to an embodiment of the present invention, a process for bio-fuel fermentation comprises the steps of but not limited to, mixing molasses with a solvent to form a first mixture, mixing a composition in the first mixture to form a second mixture, incubating the second mixture, adding dried active yeast in the incubated second mixture to obtain a third mixture, incubating the third mixture to obtain the bio-fuel and performing distillation of the obtained bio-fuel. Further, the first mixture obtained is wort.

According to an embodiment of the present invention, the solvent is, but not limited to, water.

According to an embodiment of the present invention, the second mixture is incubated at temperature ranging from, but not limited to, 30°C to 35°C for 0.5 hours.

According to an embodiment of the present invention, the dried active yeast is, but not limited to, Saccharomyces cerevisiae.

According to an embodiment of the present invention, the third mixture is incubated at temperature ranging from, but not limited to, 30°C to 35°C for 24 hours.

According to an embodiment of the present invention, the composition further comprises, but not limited to, a plurality of plant extracts or plant extract fractions and a plurality of enzymes.

According to an embodiment of the present invention, the plurality of plant extracts or plant extract fractions are selected from a group consisting of, but not limited to, extracts of Gymnema Sylvestre, Senna alata, Clerodendrum infortunatum, Berberis aristata root, Cinnamomum Verum Bark, Cynodon dactylon, Moringa oleifera, Allium sativum and Azadirachta indica.

According to an embodiment of the present invention, the plurality of plant extracts or plant extract fractions are present in a range of but not limited to, 1 mg/mL to 10 mg/mL.

According to an embodiment of the present invention, the plurality of enzymes are selected from a group consisting of, but not limited to, proteolytic enzymes and amylolytic enzymes.

According to an embodiment of the present invention, the proteolytic enzymes and amylolytic enzymes are selected from a group consisting of, but not limited to, fungal alpha amylase, bacterial alpha amylase, glucoamylase or neutral protease.

According to an embodiment of the present invention, the plurality of enzymes are present in a range of, but not limited to, 1 ppm to 100 ppm.

BRIEF DESCRIPTION OF THE DRAWINGS
So that the manner in which the above recited features of the present invention can be understood in detail, a more particular to the description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawing. It is to be noted, however, that the appended drawing illustrates only typical embodiments of this invention and are therefore not to be considered limiting of its scope, the invention may admit to other equally effective embodiments.

These and other features, benefits and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the views, wherein:

Fig. 1 is a flow chart illustrating a process (100) for bio-fuel fermentation, in accordance with an embodiment of the present invention.

Fig. 2 is a graph showing percent survival of molasses consortia after treatment with a composition C1 for bio-fuel fermentation at various concentrations, in accordance with an embodiment of the present invention.

Fig. 3 is a graph showing percent survival of Lactobacillus spp. after treatment with the composition C1 at various concentrations, in accordance with an embodiment of the present invention.

Fig. 4 is a graph showing percent survival of Haliscomenobacter spp. after treatment with the composition C1 at various concentrations, in accordance with an embodiment of the present invention.

Fig. 5 is a graph showing percent survival of molasses consortia after treatment with the composition C2 at various concentrations, in accordance with an embodiment of the present invention.
Fig. 6 is a graph showing percent survival of Lactobacillus spp. after treatment with the composition C2 at various concentrations, in accordance with an embodiment of the present invention.

Fig. 7 is a graph showing percent survival of Haliscomenobacter spp. after treatment with the composition C2 at various concentrations in accordance with an embodiment of the present invention.

Fig. 8 is a graph showing percent survival of sugar consortia after treatment with the composition C2 at various concentrations, in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS
While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described, and are not intended to represent the scale of the various components. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim. As used throughout this description, the word "may" is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein is solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes.
In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.

The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only, and are not intended to limit the scope of the claims. In addition, a number of materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary, and are not intended to limit the scope of the invention.

In accordance with an embodiment of the present invention, a composition for bio-fuel fermentation and a process thereof is disclosed. The composition comprises a plurality of plant extracts or plant extract fractions and a plurality of enzymes. The constituents of the composition for bio-fuel fermentation show synergistic effect that enhances the yield of bio-fuel.

In accordance with an embodiment of the present invention, the plurality of plant extracts or plant extract fraction are selected from, but not limited to, a group consisting of extracts of Gymnema Sylvestre, Senna alata, Clerodendrum infortunatum, Berberis aristata root, Cinnamomum verum Bark, Cynodon dactylon, Moringa oleifera, Allium sativum and Azadirachta indica. The plurality of the plant extracts or plant extract fraction are present in an amount ranging from, but not limited to, 1 mg/mL to 10 mg/mL.

In accordance with an embodiment of the present invention, the plurality of the enzymes are selected from, but not limited to, a group consisting of proteolytic and amylolytic enzymes. Further the proteolytic and amylolytic enzymes are selected from, but limited to, a group consisting of fungal alpha amylase, bacterial alpha amylase, glucoamylase, acid proteases and neutral protease. The pluralities of enzymes are present in an amount ranging from, but not limited to, 1 ppm to 100 ppm.

In accordance with an embodiment of the present invention, the composition for bio-fuel fermentation exhibits antimicrobial activity against one or more contaminants and microbial consortia selected from, but not limited to, a group consisting of Lactobacillus spp., Pseudomonas spp., Acetobacter spp., Haliscomenobacter spp., (other Gram positive and Gram negative spp.) or a combination thereof. Further, the one or more contaminants are selected from a group consisting of, Lactobacillus species., Pseudomonas species, Acetobacter species, Pediococcus species and other Gram positive and Gram negative species.

The contaminants deteriorate molasses quality and reduce the bio-fuel recovery during the fermentation process by utilizing the macronutrients and micronutrients from the fermentation process. Moreover, the composition eliminates the need of antibiotics such as Penicillin, Ciprofloxacin, Ampicillin, Tetracycline, Streptomycin, Monensin, Maduramicin and Verdiniamycin. Further, the use of biocides such as Benzalkonium chloride (BKC), Diethyldithiocarbamate and sodium (DTC) is also eliminated.

In accordance with an exemplary embodiment of the present invention, the bio-fuel is, but not limited to, an alcohol. Further, the alcohol is, but not limited to, bio-ethanol. Bio-ethanol can be used as a high-performance fuel component thereby reducing the use of cancer-causing gasoline compounds such as ethylbenzene, xylene, toluene and benzene.

Figure 1 is a flow chart illustrating the process (100) for bio-fuel fermentation in accordance with an embodiment of the present invention.

At step 102, as shown in figure 1, molasses is mixed in a solvent to form a first mixture. Fermentation of molasses is a biological process in which sucrose from molasses is converted into cellular energy that eventually produces ethanol and carbon dioxide. Molasses is byproduct obtained during sugar refining in sugar industries and is broadly used in the process of the fermentation.

Molasses contains a number of essential minerals such as calcium, magnesium, manganese, potassium, copper, iron, phosphorus, chromium, cobalt, and sodium. Further, the solvent is, but not limited to, water. Consequently, the use of water as a solvent makes the process ecofriendly and cost-effective as well. The first mixture obtained is, but not limited to, wort.

At step 104, a composition having a plurality of plant extracts or plant extract fraction and a plurality of enzymes is added in the first mixture to form a second mixture. Further, the plurality of plant extracts or plant extract fraction are selected from, but not limited to, a group consisting of extracts of root Gymnema Sylvestre, Senna alata, Clerodendrum infortunatum, Berberis aristata root, Cinnamomum Verum Bark, Cynodon dactylon, Moringa oleifera, Allium sativum and Azadirachta indica. The plurality of the plant extracts or plant extract fraction are present in an amount ranging from, but not limited to, 1 mg/mL to 10 mg/mL.

In accordance with an embodiment of the present invention, the plurality of the enzymes are selected from, but not limited to, a group consisting of proteolytic and amylolytic enzymes. The proteolytic enzymes are capable of digesting or hydrolyzing proteins that are present in the molasses obtained from the sugar industries. Also, the amylolytic enzymes convert the starch from the molasses into dextrin which is further converted to glucose. Further, the proteolytic and amylolytic enzymes are selected from, but limited to, a group consisting of fungal alpha amylase, bacterial alpha amylase, glucoamylase, acid proteases and neutral protease. The pluralities of enzymes are present in an amount ranging from, but not limited to, 1 ppm to 100 ppm.

At step 106, the second mixture is incubated at a temperature ranging from, but not limited to, 30ºC to 35ºC for, but not limited to, 0.5 hours.

At step 108, dried active yeast is added in the second mixture after the incubation forming a third mixture. Further, the yeast can be, but not limited to, Saccharomyces cerevisiae. The dries active yeast is instant yeast that further metabolizes sugar in the absence of oxygen and facilitates the yield of ethanol and carbon dioxide.

At step 110, the third mixture is incubated at a temperature ranging from, but not limited to, 30ºC to 35ºC for 24 hours to obtain the bio-fuel. The provided temperature during incubation helps in facilitating the necessary activity of the yeast for the process of fermentation.

In accordance with an embodiment of the present invention, the bio-fuel distillation is performed after the 24 hours incubation period at 30ºC to 35ºC and the amount of bio-fuel is measured.

Hereinafter, non-limiting examples of the present invention are provided for more detailed explanation which is not meant to limit the scope of the invention in any manner.

EXAMPLES
Example 1: Method to preparation of water extract of plants Berberis aristate, Cynodon dactylon, Moringa oleifera, Allium sativum, Cinnamomum verum, Azadirachta indica, Gymnema sylvestre, Senna alata and Clerodendrum infortunatum.
For preparing the plant extracts used in the composition C1 for bio-fuel fermentation, the plants (Berberis aristate, Cynodon dactylon, Moringa oleifera, Allium sativum, Cinnamomum verum, Azadirachta indica, Gymnema sylvestre, Senna alata and Clerodendrum infortunatum) were dried and grinded to obtain powder. Then 50 grams of the powdered plant parts (leaves/roots/bark) were added in 500 mL distilled water and the mixture is then kept at rest for 24 hours. The watery layer thus formed is kept collected by filtration with cotton pad followed by Whatman paper based filtration to obtain the filtrate. The filtrate thus obtained was kept in water bath at 60°C to facilitate the evaporation of water and to get moist extracts. The slurry was then dried and measured. The details of plant extracts and plant extract fractions (obtained from plant extracts) were used in composition C1 and C2 for bio-fuel fermentation are shown in table 1 below:

Sr. No. Name of plant Part used for plant extracts Name of plant extracts Organic solvent fraction of plant extracts
1 Moringa oleifera Leaves Dried water extract (powder) Chloroform
2 Azadirachta indica Leaves Water extract (viscous) Chloroform
3 Allium sativum Bulb Dried water extract (powder) Ethanol
4 Cynodon dactylon Stem with Leaves Water extract (viscous) Methanol
5 Gymnema sylvestre Leaves Dried water extract (powder) Ethanol
6 Senna alata Leaves Dried water extract (powder) Ethyl acetate
7 Clerodendrum infortunatum Leaves Water extract (viscous) Acetone
8 Berberis aristata Root Dried water extract (powder) Methanol
9 Cinnamomum verum Bark Dried water extract (powder) Methanol
Table 1: Details of plant extracts and organic solvent fractions

Liquid Chromatography Analysis of Plant Extracts
Liquid chromatography performed to separate active component from active plant extracts such as Berberis aristate, Cynodon dactylon, Moringa oleifera, Allium sativum, Cinnamomum verum, Azadirachta indica, Gymnema sylvestre, Senna alata and Clerodendrum infortunatum by using various organic solvents. One gram of water extract powder of Allium sativum extract mixed with 5 mL of ethanol, vortex properly, then the ethanol fraction separated through separating funnel, collected and dried. Further this fraction tested against fermentation contaminants. Similarly, above method used for water extract powder of Gymnema sylvestre, ethanol fraction collected and tested against fermentation contaminants.

One gram of water extract powder of Moringa oleifera extract mixed with 5 mL of chloroform, vortex properly, then the chloroform fraction separated through separating funnel, collected and dried. Further this fraction tested against fermentation contaminants. Similarly, above method used for water extract powder of Azadirachta indica, chloroform fraction collected and tested against fermentation contaminants.

One gram of water extract powder of Cynodon dactylon extract mixed with 5 mL of methanol, vortex properly, then the methanol fraction separated through separating funnel, collected and dried. Further, this fraction tested against fermentation contaminants. Similarly, above method used for water extract powder of Berberis aristate and Cinnamomum verum then methanol fraction collected separately and tested against fermentation contaminants.

One gram of water extract powder of Senna alata extract mixed with 5 mL of ethyl acetate, vortex properly, then the ethyl acetate fraction separated through separating funnel, collected and dried. Further this fraction tested against fermentation contaminants.

One gram of water extract powder of Clerodendrum infortunatum extract mixed with 5 mL of acetone, vortex properly, then the acetone fraction separated through separating funnel, collected and dried. Further this fraction tested against fermentation contaminants.

Example 2
In an embodiment of the present invention, the composition C1 for bio-fuel fermentation was prepared by using the plurality of plant extracts (all extracts in composition based water) such as Berberis aristate, Cynodon dactylon, Moringa oleifera, Allium sativum, Cinnamomum verum, Azadirachta indica, Gymnema sylvestre, Senna alata, and Clerodendrum infortunatum and the plurality of enzymes such as fungal alpha amylase, bacterial alpha amylase, glucoamylase and neutral protease. The prepared composition C1 for bio-fuel fermentation was subjected to determine antibacterial effect on molasses consortia after 24 hours incubation as illustrated in Table 2 below.

Composition (mg/ml) Molasses Consortia Titer (CFU/ml) Percent Survival
0 hr 24 hr
Composition C1 0.0 (Control) 2.4 x 105 4.9 x 108 100.0
10.0 7.0 x 104 9.9 x 107 20.2
5.0 1.2 x 105 1.6 x 108 32.7
2.5 2.4 x 105 2.7 x 108 55.1
1.25 2.6 x 105 3.7 x 108 75.5
Table 2: Composition C1 for bio-fuel fermentation showing antibacterial effect on molasses consortia

Similarly, antibacterial effect of the composition C1 for bio-fuel fermentation on the Lactobacillus species isolated from molasses after 48 hours incubation as illustrated in Table 3 below.

Composition (mg/ml) Lactobacillus Percent Survival
0 hr 48 hr
0.0 (Control) 1.6 x 104 1.6 x 107 0
10 1.5 x 104 6.4 x 106 40.0
5 1.2 x 104 8.3 x 106 51.9
2.5 1.4 x 104 9.4 x 106 58.8
1.25 1.5 x 104 1.2 x 107 75.0
Table 3: Composition C1 for bio-fuel fermentation showing antibacterial effect on Lactobacillus species

Anti-bacterial effect of the composition C1 for bio-fuel fermentation on the Haliscomenobacter species i.e. Haliscomenobacter hydrossis isolated from molasses by calculating Optical Density (O.D.) at different concentrations at the wavelength after 24 hours incubation as illustrated in table 4 below.

Composition C1 (mg/ml) Optical Density (OD) at 570 nm
0 hr 24 hr Final OD Percent Survival
0.0 (Control) 0.009 1.886 1.877 100.0
10 1.044 1.046 0.002 0.1
5 0.412 0.415 0.003 0.2
2.5 0.095 0.101 0.006 0.3
1.25 0.054 0.165 0.111 5.9
Table 4: Composition C1 for bio-fuel fermentation showing antibacterial effect on Haliscomenobacter hydrossis

The composition C1 for bio-fuel fermentation showed enhanced alcohol recovery in molasses based bio-ethanol fermentation after 24 hours incubation as illustrated in Table 5 below.

Samples Ingredients Quantity Recovered Alcohol (%)
Control Molasses 110 g 7.05

Water Volume make up to 300 mL
Dried Yeast 3 g
Composition
(10 ppm) Molasses 110 g 7.25

Water Volume make up to 300 mL
Dried Yeast 3 g
Composition 3 g
Table 5: Composition C1 for bio-fuel fermentation showing enhanced alcohol recovery in molasses based fermentation

Example 3
In an embodiment of the present invention, the composition C2 for bio-fuel fermentation was prepared by using the plurality organic solvent fractions of water plant extracts (all fractions of water plant extracts based on organic solvents separation such as methanol fraction of Berberis aristate, methanol fraction of Cynodon dactylon, chloroform fraction of Moringa oleifera, ethanol fraction of Allium sativum, methanol fraction of Cinnamomum verum, chloroform fraction of Azadirachta indica, ethanol fraction of Gymnema sylvestre, ethyl acetate fraction of Senna alata and acetone fraction of Clerodendrum infortunatum) and the plurality of enzymes such as fungal alpha amylase, bacterial alpha amylase, glucoamylase and neutral protease. The prepared composition C2 for bio-fuel fermentation was used to determine the antibacterial effect on molasses consortia after 24 hours incubation as illustrated in Table 6 below.

Extract (water based) Concentration (mg/ml) Molasses Consortia Titer (CFU/ml) Percent Survival -Molasses consortia
0 hr 24 hr 24 hr
Composition 2 0.0 (Control) 3.5 x 107 9.8 x 1013 100.00
10.0 3.3 x 107 1.9 x 1013 19.3
5.0 3.4 x 107 2.7 x 1013 27.5
2.5 3.4 x 107 4.1 x 1013 41.8
1.25 3.4 x 107 6.7 x 1013 68.3
Table 6: Composition C2 for bio-fuel fermentation showing antibacterial effect on molasses consortia

Similarly, antibacterial effect of the composition C2 for bio-fuel fermentation on the Lactobacillus species, Haliscomenobacter hydrossis, and sugar consortia and isolated from molasses after 24 hours incubation as illustrated in table 7, 8 and table 9.

Extract (water based) Concentration (mg/ml) Lactobacillus Titer (CFU/ml) Percent Survival - Lactobacillus
0 hr 48 hr 48 hr
Composition 2 0.0 (Control) 1.0 x 105 3.7 x 1012 100.00
10.0 1.0 x 105 2.1 x 1012 0.58
5.0 1.0 x 105 2.6 x 1012 71.12
2.5 1.0 x 105 2.9 x 1012 78.34
1.25 1.0 x 105 3.4 x 1012 93.31
Table 7: Composition C2 for bio-fuel fermentation showing antibacterial effect on Lactobacillus species

Composition 2 Optical Density (OD) at 570 nm
0 hr 24 hr Final OD Percent Survival - H. hydrossis
0.0 (Control) 0.009 1.886 1.877 100.00
10 1.044 1.046 0.002 0.11
5.0 0.412 0.415 0.003 0.16
2.5 0.095 0.101 0.006 0.32
1.25 0.054 0.165 0.111 5.90
Table 8: Composition C2 for bio-fuel fermentation showing antibacterial effect on Haliscomenobacter hydrossis

Extract (water based) Concentration (mg/ml) Sugar Consortia Titer (CFU/ml) Percent Survival - Sugar consortia
0 hr 24 hr 24 hr
Composition 2 0.0 mg/ml (Control) 1.3 x 105 2.7 x 109 100.00
10.0 1.3 x 105 3.6 x 107 12.99
5.0 1.3 x 105 7.0 x 107 25.27
2.50 1.4 x 105 1.2 x 108 44.77
1.25 1.6 x 105 1.5 x 108 57.03
Table 9: Composition C2 for bio-fuel fermentation showing antibacterial effect on Sugar Consortia

The composition C2 for bio-fuel fermentation showed enhanced alcohol recovery in molasses based bio-ethanol fermentation after 36 hours incubation as illustrated in Table 10 below.

Particular 0 hr 36 hr Alcohol %
pH Gravity pH Gravity
Control 4.56 1.06 4.56 1.00 7.3
Composition 2 (10ppm) 4.53 1.06 4.53 1.05 7.9
Table 10: Composition C2 for bio-fuel fermentation showing enhanced alcohol recovery in molasses-based fermentation

The composition C1 and C2 showed no significant lethal effect on the growth of Saccharomyces cerevisiae after 24 hours incubation as shown in table 11.

Compounds FT Yeast Saccharomyces cerevisiae Percent Survival
Concentration 0 hr 24 hr 24 hr
Control 0.0 mg/ml 4.9 x 106 1.6 x 1011 100.0
Composition 1 10.0 4.4 x 106 1.5 x 1011 97.00
5.00 4.8 x 106 1.6 x 1011 100.0
Composition 2 10.0 4.8 x 106 1.5 x 1011 97.00
5.00 4.5 x 106 1.6 x 1011 100.0
Table 11: Composition C1 and C2 for bio-fuel fermentation showing lethal effect on Saccharomyces cerevisiae

Results
It is concluded from the examples 2 and 3 and figures 2 to 8, that with an increase in the concentration of the composition, the percent survival rate of contaminant decreases. Further, the composition was employed for bio-ethanol recovery in molasses based bio-ethanol fermentation, as illustrated in table 5 and table 10, wherein the composition at the concentration of 10 ppm showed 0.2% and 0.6% enhanced bio-ethanol recovery respectively, as compared to the control (without the composition).

Further, as illustrated in table 2 to 4 and table 6 to 9 that the compositions C1 and C2 show potential antibacterial effect against molasses consortia, Lactobacillus species, Haliscomenobacter hydrossis and sugar consortia at different concentration of the plant extracts. It was observed that maximum antibacterial property was showed at the concentration of 10 mg/mL.

On the other hand, there was no significant lethal effect of plant extracts or plant extract fractions at the concentration range of 5.0mg/mL to 10.0mg/mL were observed against Saccharomyces cerevisiae as shown in table 11.

The composition for bio-fuel fermentation and the process thereof offer a plurality of advantages including, but not limited to, reduction of contamination problems during bio-fuel production and remote emergence of antibiotic oriented resistance in contaminant bacteria. In other words, the composition eliminates the need of antibiotics and biocides for removal of the contaminant bacteria grown during bio-fuel preparation process. Further, the composition for bio-fuel fermentation enhances the yield of the bio-fuel thereby increasing the efficacy.

Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the embodiments shown along with the accompanying drawings but is to be provided broadest scope consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and appended claims.

We Claim:
1. A composition for bio-fuel fermentation, comprising:
a plurality of plant extracts or plant extract fractions; and
a plurality of enzymes.
2. The composition as claimed in claim 1, wherein the plurality of plant extracts or plant extract fractions are selected from a group consisting of, extracts of Gymnema sylvestre, Senna alata, Clerodendrum infortunatum, Berberis aristata root, Cinnamomum verum Bark, Cynodon dactylon, Moringa oleifera, Allium sativum and Azadirachta indica.
3. The composition as claimed in claim 2, wherein the plurality of plant extracts or plant extract fractions are present in a range of 1 mg/mL to 10 mg/mL.
4. The composition as claimed in claim 1, wherein the plurality of enzymes are selected from a group consisting of, proteolytic enzymes and amylolytic enzymes.
5. The composition as claimed in claim 4, wherein the proteolytic enzymes and amylolytic enzymes are selected from a group consisting of, fungal alpha amylase, bacterial alpha amylase, glucoamylase or neutral protease.
6. The composition as claimed in claim 5, wherein the plurality of enzymes are present in a range of 1 ppm to 100 ppm.
7. The composition as claimed in claim 1, wherein the composition shows anti-bacterial activity against microbial consortia and one or more contaminants.
8. The composition as claimed in claim 7, wherein the microbial consortia is selected from a group consisting of, Lactobacillus species, Pseudomonas species, Acetobacter species, Haliscomenobacter species, other Gram positive and Gram negative species or a combination thereof.
9. The composition as claimed in claim 7, wherein the one or more contaminants are selected from a group consisting of, Lactobacillus species., Pseudomonas species, Acetobacter species, Pediococcus species and other Gram positive and Gram negative species.
10. The composition as claimed in claim 1, wherein the plurality of plant extracts or plant extract fractions showed maximum anti-bacterial activity at the concentration in a range of 1 mg/mL to 10 mg/mL.
11. The composition as claimed in claim 1, wherein the bio-fuel is bio-ethanol.
12. A process (100) for bio-fuel fermentation, comprising the steps of;
mixing (102) molasses with a solvent to form a first mixture;
mixing (104) a composition in the first mixture to form a second mixture;
incubating (106) the second mixture;
adding (108) dried active yeast in the incubated second mixture to obtain a third mixture; and
incubating (110) the third mixture to obtain the bio-fuel;
wherein the first mixture obtained is wort.
13. The process as claimed in claim 12, wherein the solvent is water.
14. The process as claimed in claim 12, wherein the second mixture is incubated at temperature ranging from 30°C to 35°C for 0.5 hours.
15. The process as claimed in claim 12, wherein the dried active yeast is Saccharomyces cerevisiae.
16. The process as claimed in claim 12, wherein the third mixture is incubated at temperature ranging from 30°C to 35°C for 24 hours.