METHOD FOR THE PRODUCTION OF ERYTHRITOL FROM RENEWABLE RESOURCES

CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY

The present application claims priority from Indian patent application number (202121053279) filed on (19/11/2021 ), incorporated herein by a reference.

FIELD OF THE INVENTION

The invention relates to a method for the preparation of erythritol using yeast strain, it particularly relates to the use of renewable resource as a carbon source and microbial lysate as nitrogen as well as a nutrient source. More particularly it relates to the use of ethanol as a carbon source and microbial lysate as nitrogen as well as a nutrient source for erythritol production by fermenting it with Moniliella pollinis.

BACKGROUND

India is huge producer of sugar. Sugar has vast potential to deliver multiplicity of bio chemicals. Erythritol is one of the functional polyols and sweeteners, commonly used in low sugar and “sugar free” foods. It is a low-calorie tetraol with relative sweetness of 0.65 and energy about 0.24 calories per gram and is safe for diabetics. Erythritol has a heat absorbing effect and therefore gives a cooling sensation as it i dissolves in mouth. Unlike other artificial sweetners, Erythritol does not cause stomach upset. It has low hygroscopicity, good stability towards heat. It is stable at low pH and is non fermentable. Erythritol has a low solubility in water and is easy to crystallize.

In recent years, Erythritol is being widely used in functional foods such as low-sugar and health care products in addition to its uses in medicine and chemical fields. It is believed that in coming years Erythritol will find applications in many diverse fields therefore its synthesis is currently receiving much attention.

There are three main methods for the production of Erythritol 1) Extraction method 2) chemical synthesis method 3) Fermentation method. Erythritol is commercially produced by fermentation of glucose, derived either from sucrose or starch using hypertonic yeast. This method is economical and provides high recovery of erythritol. With the growing interest in production of renewable fuels, most of the molasses and starch & other renewable resources are now being diverted to produce ethanol. Therefore, along with renewable resource like molasses, use of ethanol too as a feedstock for producing erythritol makes an interesting proposal. Such fermentation is novel and has not been reported till date.

The present invention discloses method of production of erythritol by fermentation of ethanol and other renewable resources by yeast and the factors responsible for said fermentation. The invention also discloses a cost-effective media formulation to reduce the cost associated with fermentation media having nutrients like nitrogen, phosphorous, sulphur, trace elements and growth factors like amino acids.

BRIEF DESCRIPTION OF THE INVENTION

A process for the production of erythritol by fermentation comprises a yeast strain capable of producing erythritol and fermentation medium containing renewable resources as a carbohydrate source and microbial cell lysate as a nutrient as well as nitrogen source. Said fermentation media is sterilized at 121 °C for 20 minutes forming a first stream and then fermenting said first stream at desired condition for specific time period forming a fermented stream. Next, yeast is separated from fermented stream and forming a second stream without yeast. Further, said second stream is treated with activated charcoal for about 2 hours forming a third stream forming a fourth stream. Said fourth stream is purified by using ion exchange chromatography forming a final stream which is concentrated in an evaporator to get final product.

DETAILED DESCRIPTION OF THE INVENTION

Present invention discloses a novel process for erythritol production using fermentation medium containing renewable resources as a carbon source and microbial cell lysate (MCL) as a nutrient as well as nitrogen sources. The present invention is more about producing erythritol using inexpensive nitrogen as well as carbon source such as molasses and sugar cane juice. The process also discloses the novel process of using ethanol as a carbon source and MCL as a nitrogen source for erythritol production where other by products like glycerol or ethanol are not produced during fermentation.

In one embodiment of the present invention, said process includes following steps: 1) Yeast culture 2) Preparation of microbial cell lysate (MCL) 3) Preparation of pre-fermentation culture for fermentation 4) Preparation of fermentation media 5) Fermentation 6) down streaming process

Each step has one or more elements for performing specific or optional functions as required for erythritol production using low-cost media. A person skilled in the art may appreciate different variations and/ or combinations of these elements that may be used to perform the objects of the invention disclosed herein.

1 ) Yeast culture

In one of the embodiments of present invention, any yeast strain which can produce erythritol from renewable resources is used in the present invention. The yeast, Moniliella pollinis is used for the present invention. Moniliella is a unique genus of basidiomycetous black yeast that has resistance towards high sugar concentrations, ferments glucose and produces many types of sugar polyols. In embodiment of present invention an osmotolerant basidiomycetous fungi M. pollinis strain CBS 461.67 is procured from CBS Netherland. The yeast peptone dextrose (YPD) medium is used for pre-culturing and maintaining the yeast strains. It consists of about 1% w/v of yeast extract, about 2% w/v of peptone and about 2% w/v of dextrose with initial pH 5 to 5.5. YPD agar contains about 2% agar in addition to other components. The optimal growth temperature of said yeast is about 30 °C and pH is about 5.5 to 7. The colour of M. pollinis strain CBS 461.67 is white to yellow-white during initial incubation. After prolonged incubation, it becomes yellowish dark brown on YPD agar plates.

2) Preparation of microbial cell lysate:

In traditional fermentation, the obtained fermentation broth or supernatant is filtered and purified to get desired components like erythritol with other by-products. Said desired components are purified and crystallized to get commercial erythritol.

Microbial sludge generated from above process is directly discharged into environment due to lack effective treatment; this increases the waste disposal cost and environmental pollution load. In prior art, the use of bacterial sludge as nutrient/ nitrogen source in the form of yeast extract and CSL is reported where yeast extract is prepared by fermentation of yeast in sugar rich feedstock. Said production of yeast extract needs various operations such as aeration, agitation, cell lysis and drying. These processes consume energy which increases production cost of cell extract. Requirement of nutrients and feedstock further add on to the production cost.

In the present invention, during fermentation of sugarcane juice, significant fraction of sugars is utilized for generation of M. pollinis cell biomass. MCL is prepared from M. pollinis cells which are recovered as a by-product during erythritol fermentation, and it used as a source of nutrients for the erythritol production all year-round of plant operation. This results savings in operating cost and capital cost required to generate equivalent yeast extract.

The microbial cell lysate is prepared from initial fermentation. Said microbial cells are separated at the end of fermentation by separation techniques like centrifugation, filtration etc. Next, water is added to cells to form a first cell suspension having about 90 to about 100 optical density at 600 nm (ODeoo 90-100). Said first cell suspension is boiled for about 3 to 5 minutes to form a second cell suspension. Then, said second cell suspension is dried at 60 °C until constant weight of dried microbial cell lysate (MCL). Said dried lysate is then analysed for total protein & nitrogen content by Kjeldahl’s method. Elemental composition is analyzed using AOAC official method. Said MCL comprises of total protein about 45 to 48 % by weight, nitrogen about 6 to about 8 % by weight, iron about 65 to about 75 ppm and zinc about 230 to about 270 ppm. Specifically, Zinc concentration in MCL is higher as compared to yeast extract. Said MCL is used at concentration up to 2 g/L in fermentation medium for further multiple fermentation batches.

3) Preparation of pre-fermentation culture for fermentation:

The culture is revived on agar plates containing yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L (YPD) and agar 20 g/L. said plates are incubated for 48 hrs at 30 °C. The pre-pre culture is prepared by inoculating a single isolated colony grown on YPD agar plate in a first reactor containing MCL- glucose (MCL 1.6 g/L, glucose 20 g/L) medium. The reactor is incubated at 30°C and 150 rpm for 24 h. The pre-fermentation culture is prepared by inoculating a second reactor containing MCL- glucose medium with 5% v/v inoculum from the pre- pre culture reactor. Next, the said reactor is incubated at 30°C and 150 rpm for 24 h. After achieving desired cell concentration at 24 h, about 5 % of pre-fermentation culture is used further to inoculate fermentation medium.

4) Preparation of fermentation media:

Fermentation media is prepared using MCL and renewable sources. In fermentation the MCL is used a nutrient as well as a nitrogen source whereas like molasses, sugar cane juice, sucrose, glucose or ethanol or combination thereof is used as a carbon sources. In fermentation medium, up to 2 g/L MCL is used along with molasses or sugar cane syrup and it is diluted with water to achieve concentration of sugar between 15 and 20 % w/w sugars in the fermentation medium. Then, fermenter is sterilized at 120 °C for 20 min and then used for fermentation process.

In another embodiment of the process, up to 2 g/L MCL is mixed with desired quantity of water in a fermenter, and sterilized at 120 °C for 20 min. After sterilization, cool the fermentation media and then add between 1 and 4 % by weight ethanol in it.

5) Fermentation:

Next, said sterilized media is fermented with Moniliella Pollinis at 30 deg. C and 300 rpm for 144 hrs. Aeration inflow to fermenter is maintained at 5 SLPM (1 VVM) to form a fermentation stream. Said fermented stream is analysed for erythritol and other by-products.

6) Downstream process:

Said fermented broth is subjected to centrifugation to separate yeast thereby forming a second stream which is free of yeast. Said second stream is treated further with desired quantity of activated charcoal with continuous stirring for about 2 hrs forming a third stream. Said third stream is passed through anion exchange resin pre-activated by NaOH. The generated elute is further introduced into cation exchange resin, pre-activated with 5% HCI to get an eluant. Said eluant is then subjected to rotary vacuum evaporator to get crude erythritol crystals. Said crystals are then washed with acetone to obtain pure erythritol crystals having about 96% purity. In yet another embodiment of present invention, the zinc concentration in MCL is upto 250 ppm. Zinc ions helps in increasing activity of erythrose reductase enzyme which catalyzes erythrose to erythritol conversion in erythritol production pathway. Zinc concentration in MCL is higher as compared to yeast extract,

The process has several advantages over the known methods as listed below:

• Use of MCL helps in improving erythritol yield and titre during fermentation.

• The fermentation media is simple and efficient as MCL provides nitrogen and other essential nutrients required for the fermentation process.

• The disclosed process is substantially economical as compared with earlier process. It saves the media cost of fermentation.

• Ethanol utilization is achieved only in presence of MCL which is not observed in presence of other nitrogen sources like yeast extract.

• In Disclosed process, the erythritol is converted from ethanol at late stages of fermentation.

•

Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various experimental results is given in the examples, which demonstrate the advantageous and novel aspects of the process of using a renewable source and MCL in optimal concentration for more yield of erythritol.

Example 1 ) MCL preparation

M. pollinis cells were harvested at the end of initial fermentation. Said harvested cells were separated by centrifugation at 7000 rpm for 10 min and then washed with water. About 70-80 g wet cells were suspended in 1 L water to form a first cell suspension having optical density up to 100. Said first cell suspension was boiled for 3-5 minutes to form a second cell suspension. Then, said second cell suspension is dried at 60 °C until constant weight to obtain dried microbial cell lysate (MCL). Said dried lysate is then analysed for total protein & nitrogen content by Kjeldahl’s method, elemental composition is analyzed using AOAC official method. Typical composition of MCL and yeast extract is given in Table 1 .

TABLE 1 : MCL and Yeast extract analysis and composition: io

As depicted in table 1 , total protein analysed in MCL was lower than that of commercial yeast extract but it has a sufficient nitrogen and other essential nutrients (macro and micronutrients) for M.pollinis cells growth and erythritol production. The elemental analysis of MCL revealed that it contained more sodium, calcium, manganese, iron, and zinc than yeast extract, while it contained less potassium, phosphorous, and sulphur.

Example 2: Analysis of erythrose reductase activity The erythritol production pathway was investigated to identify key enzymes and cofactors essential for erythritol production. Erythrose reductase is responsible for conversion of erythrose to erythritol, Hence the cells grown in presence of MCL and yeast extract along with sugarcane juice as carbon source and said cells were analysed for Erythrose reductase activity.

For analysing erythrose reductase activity, crude enzyme was prepared by lysing the equal quantity of cells from MCL as well as yeast extract media (0.4 g of cells suspended in 50 mM sodium phosphate buffer pH 6 and 1 mM phenyl methyl sulphonyl fluoride) using bead basher for 15 min. The activity of the erythrose reductase enzyme was determined by observing a decrease in absorbance at 340 nm caused by cofactor oxidation (NADPH), as reported by Cheng et al (2018). The assay mixture (1.0 ml) contained 600 microliter (pL) sodium phosphate buffer pH 6 (50mM), 100 pL NADPH (2 mM), 200 pL MCL (1.6 g/L) or yeast extract (10 g/L) and 100 pL D(-) erythrose (100mM). To avoid endogenous oxidation of cofactor, the reaction mixture was allowed to stand for one minute before adding substrate. Erythrose reductase activity of 4 U/mg of protein and 0.39 U/mg of protein was observed in M. pollinis cells grown in presence of MCL and yeast extract respectively.

Example 3: Use of microbial cell lysate and sugarcane juice

In 7.5 L of bioreactor, about 5L fermentation media was prepared using 1 .6 g/L microbial cell lysate (having about 7% total nitrogen) in 4750 ml sugarcane juice which is having about 176 g/L total sugar. Said fermenter was sterilized at about 121 °C for about 20 minutes. After sterilization, said fermenter was inoculated with 5% v/v of prefermented yeast culture. The fermentation was carried out at about 30 °C and 300 rpm for about 144 hrs. Then, said fermented broth was subjected to centrifugation to separate yeast forming a second stream. Said second stream comprises about 51 .2 g/L erythritol, about 12.5 g/L ethanol and about 3.3 g/L glycerol. Said second stream was treated further with 20 g/ L activated charcoal with continuous stirring for about 2 hrs forming a third stream. Said third stream was passed through anion exchange resin, which was pre-activated by using 50 g/L NaOH. The generated elute was further introduced into cation exchange resin pre-activated with 5% HCI to get a 2.835 L of eluant. Said eluant was then subjected to rotary vacuum evaporator at 15 mbar of vacuum and at temperature about 30°C. to produce about 62.08 g crystals of crude erythritol. Said crystals were washed with acetone to obtain about 53 g of pure Erythritol crystals having about 96% purity.

Example 4: Use of yeast extract and sugarcane juice for fermentation.

In 7.5 L of bioreactor, about 5L fermentation media was prepared having 10 g/L yeast extract (having 11% total nitrogen) in 4750 ml sugarcane juice with 156 g/L total sugar. Said fermenter was sterilized at about 121 deg C for about 20 minutes. After sterilization, said fermenter was inoculated with 5% v/v of pre-fermented yeast culture. The Fermentation was carried out at about 30 °C and 300 rpm for about 144 hrs. Then, said fermented broth was subjected to centrifugation to remove yeast forming second stream free of yeast. Said second stream comprises about 13.3 g/L erythritol, about 54.3 g/L ethanol and about 4.4 g/L glycerol.

Example 5: Use of Yeast extract and ethanol

In 7.5 L bioreactor, fermentation media having 10 g/L yeast extract (having 11% total nitrogen)was prepared in 4550 ml water. Said fermenter was sterilized at about 121 °C for about 20 minutes. After finishing the sterilization, said fermenter was inoculated with 5% v/v of pre-fermented culture. After inoculation about 200 ml 99% pure ethanol was added. Fermentation was carried out at about 30 °C and 300 rpm for about 144 hrs. Then, said fermented broth was subjected to centrifugation to separate yeast from second stream. Said second stream was analysed. Analysis revealed absence of erythritol, ethanol and glycerol in the second stream.

Example 6: Use of microbial cell lysate and ethanol

In 7.5 L of bioreactor, fermentation media was prepared having 1 .6 g/L MCL (having 7% total nitrogen) in 4550 ml water. Said fermenter was sterilized at about 121 deg C for about 20 minutes. After sterilization, said fermenter was inoculated with 5% v/v of pre-fermented yeast culture. After inoculation about 200 ml 99% pure ethanol was added and said fermentation was carried out at about 30 °C and 300 rpm for about 144 hrs. next, the fermented broth was subjected to centrifugation to separate yeast, thus forming a second stream which was free of yeast. Said second stream contained only about 6.6 g/L erythritol and no other by-products.

Example 7: Use of MCL media and molasses

In 7.5 L of bioreactor, fermentation media was prepared having 1 .6 g/L MCL (having 7% total nitrogen) in 2250 ml water along with 2.5 Kg of molasses containing 400-500 g/L total sugar. Said fermenter was sterilized at about 121 deg C for about 20 minutes. After sterilization, said fermenter was inoculated with 5% v/v of pre-fermented culture. The Fermentation was carried out at about 30 °C and 300 rpm for about 144 hrs. The fermented broth was then subjected to centrifugation to separate yeast forming second stream which is free from yeast. Second stream was analyzed and found to contain 25.7 g/L erythritol, 6.1 g/L ethanol and 7.1 g/L glycerol.

Example 8: Use of Yeast extract and molasses

In 7.5 L of bioreactor, fermentation media was prepared having 10 g/L yeast extract (having 11% total nitrogen) in 2250 ml water along with 2.5 Kg of molasses containing 400-500 g/L total sugar. Said fermenter was sterilized at about 121 deg C for about 20 minutes. After sterilization, said fermenter was inoculated with 5% v/v of prefermented culture. The Fermentation was carried out at about 30 °C and 300 rpm for about 144 hrs. The fermented broth was then subjected to centrifugation to separate yeast forming a second stream free from yeast. The second stream was analyzed and found to contain 16.2 g/L erythritol, 41 .2 g/L ethanol and 8.3 g/L glycerol.

Example 9: To check the effect of MCL on fermentation and on erythritol production, process of example 3 and 4 were repeated where the sugarcane juice is a carbon source. . Samples were analysed after 72 hours. Table 2 depicts the analysis of fermented samples where efficiency of erythritol production was more in MCL containing fermented media.

Table 2 A) Use of MCL in fermentation media

Table 2 B) Use of yeast extract in fermentation media While the invention has been particularly shown and described with reference to embodiments listed in examples, it will be appreciated that several of the above disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen and unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Although the invention has been described with reference to specific preferred embodiments, it is not intended to be limited thereto, rather those having ordinary skill in the art will recognize that variations and modifications may be made therein which are within the spirit of the invention and within the scope of the claims.