A process for preparing fructo-oligosaccharides with a high degree of polymerization

Abstract

A process for preparing fructo-oligosaccharides with a high degree of polymerization is disclosed. The process comprises treating a substrate comprising fructo-oligosaccharides with a low degree of polymerization with at least one selected from the group consisting of Aureobasidium pullulans, Aspergillus niger, an enzyme preparation having an endoinulinase activity, and combinations thereof at a temperature in the range of 40 to 60°C to obtain the fructo-oligosaccharides with the high degree of polymerization.

Description

[0001] A PROCESS FOR PREPARING FRUCTO-OLIGOSACCHARIDES WITH A HIGH DEGREE OF POLYMERIZATION

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a process for preparing fructo-oligosaccharides (‘FOS’). Particularly it relates to a process for preparing FOS with a high Degree of Polymerization (‘DP’).

[0004] BACKGROUND

[0005] FOS are non-digestible oligosaccharides that occur naturally in a wide variety of foods such as banana, tomato, wheat, and onion. FOS are commercially available as a food ingredient or a nutritional supplement and have Generally Recognized as Safe (‘GRAS’) status. According to a growing body of research, FOS may provide several health benefits, for example, improvements in plasma lipids and lipoproteins, reduction in serum insulin and glucose concentrations, and inhibition of harmful bacteria in gut. Further, FOS have sweetening profile, and water retention properties similar to those of sucrose and sorbitol. They are also known to improve moisture retention, texture, and shelf life of food products. Due to these properties, FOS are widely used as alternative sweetener, and prebiotic or functional food. Shorter-chain FOS having a DP of up to 5 provide quicker gut health benefits, including rapid increases in beneficial bacteria, enhanced immunity, and immediate relief from constipation. They are also sweeter, which makes them more appealing as a dietary sweetener. Higher-chain FOS having a of DP 6 or more offer a more sustained prebiotic effect, with benefits that develop gradually overtime, such as better support for gut flora diversity, reduced gastrointestinal discomfort, and enhanced mineral absorption. These are ideal for long-term gut health management.

[0006] SUMMARY

[0007] The present disclosure relates to a process for preparing FOS with a high DP. The process comprises treating a substrate comprising FOS with a low DP with at least one selected from the group consisting of Aureobasidium pullulans, Aspergillus niger, an enzyme preparation having an endoinulinase activity, and combinationsthereof at a temperature in the range of 40 to 60°C to obtain the FOS with the high DP.

[0008] BRIEF DESCRIPTION OF DRAWINGS

[0009] FIG 1. shows High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (‘HPAEC-PAD’) chromatograms of the FOS with the low DP and the FOS with the high DP obtained in Example 2.

[0010] DETAILED DESCRIPTION

[0011] Reference will now be made in detail to embodiments of the present disclosure. The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner, simply because it is being utilized in conjunction with a detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several features, no single one of which is solely responsible for its desirable attributes, or which is essential to practicing the inventions herein described.

[0012] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.

[0013] The terms “a,” “an,”, and “the” are used to refer to “one or more” (i.e., to at least one) of the grammatical object of the article.

[0014] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion and are not intended to be construed as “consists of only”, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.

[0015] Likewise, the terms “having” and “including”, and their grammatical variants are intended to be non-limiting, such that recitations of said items in a list are not to the exclusion of other items that can be substituted or added to the listed items.

[0016] Also, any numerical range recited herein is intended to include all sub -ranges subsumed therein. For example, a range of "1 to 10" is intended to include any and all subranges between and including the recited minimum value of 1 and the recited maximum value of 10, that is, all subranges beginning with a minimum value equal to or greater than 1 and ending with a maximum value equal to or less than 10, and all subranges in between.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0018] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described.

[0019] The full forms of the abbreviations used herein and not defined elsewhere in the present disclosure are as follows:

[0020] 1. G: Glucose

[0021] 2. F: Fructose

[0022] 3. S: Sucrose4. F3 : Trisaccharide units of fructans

[0023] 5. F4: Tetrasaccharide units of fructans

[0024] 6. F5: Pentasaccharide units of fructans

[0025] 7. F6: Hexasaccharide units of fructans

[0026] 8. GF2: 1-kestose;

[0027] 9. GF3: Nystose,

[0028] 10. GF4: furanosylnystose; and

[0029] 11. GF5: IF-fructofuranosylnystose

[0030] The present disclosure relates to a process for preparing FOS with a high DP. The process comprises treating a substrate comprising FOS with a low DP with at least one selected from the group consisting of Aureobasidium pullulans, Aspergillus niger, an enzyme preparation having an endoinulinase activity, and a combination thereof at a temperature in the range of 40 to 60°C to obtain the FOS with the high DP.

[0031] In accordance with various embodiments, the FOS with the high DP obtained from the disclosed process has the DP in the range of 6 to 10. In an embodiment, the DP is in the range of 6 to 8.

[0032] In various embodiments, the FOS with the low DP has the DP in the range of 3 to 6. In some embodiments, the low DP is in the range of 3 to 5.

[0033] In some embodiments, the substrate further comprises sucrose. In some embodiments, the substrate comprises the FOS with the low DP and the sucrose in a ratio in the range of 5 to 95% and 95 to 5%.

[0034] The treatment is carried out at pH in the range of 4.5 to 8. In some embodiments, the pH is in the range of 5.5 to 7.The treatment is carried out for a duration of 1 to 24 hours. In some embodiments, the treatment is carried out for a duration of 15 to 20 hours.

[0035] The treatment is carried at stirring rotations in the range of 75 to 200 rpm. In some embodiments, the stirring rotations are in the range of 125 to 150 rpm. In an embodiment, an orbital shaker is used for stirring.

[0036] In an embodiment, the substrate is treated with the enzyme preparation having endoinulinase activity. In accordance with various embodiments, the enzyme preparation having endoinulinase activity may be a pure endoinulinase enzyme, or a crude enzyme preparation having endoinulinase activity. In an embodiment, the enzyme preparation having endoinulinase activity comprises the pure endoinulinase enzyme and is derived from Aspergillus niger. In an embodiment, the pure endoinulinase enzyme is obtained from commercial sources. In another embodiment, the enzyme preparation having endoinulinase activity is the crude enzyme preparation and is derived from Aspergillus niger.

[0037] The amount of the enzyme preparation having endoinulinase activity needed in the disclosed process depends on various factors such as process temperature, amount of various raw materials, pH, allowable process duration, and desired conversion rates. These and other relevant factors may be determined by the person skilled in the art following the generally accepted procedures in this technical field. In accordance with various embodiments, the enzyme preparation is present in an amount of about 1400 to 2800 units per ml of the substrate.

[0038] In accordance with the various embodiments, the substrate is treated with Aureobasidium pullulans and / or Aspergillus niger. In some embodiments, Aureobasidium pullulans and / or Aspergillus niger containing biomass contains 0.4 to 1.5% (wt / vol) microbial cells in a 55 to 60 brix sucrose solution at 55°C.The FOS with the high DP obtained from the process can be recovered by any suitable method. Examples of the suitable methods include chromatography such as Simulated Moving Bed Chromatography (‘SMBC’).

[0039] In accordance with various embodiments, the recovered FOS has purity of 95 to 100%. In various embodiments, the recovered FOS comprises 0.5-3% F3; 0.5-10% F4; 5-29% DP6; 1-11% DP7; 0.5-4% DP8, 0.5-8% GF2; 0.5-24% GF3; 13-43% GF4; and 18-41% GF5.

[0040] The FOS with the low DP may be obtained by any suitable process. It may also be obtained from commercial sources.

[0041] In an embodiment, the FOS with the low DP is obtained by treating a substrate comprising sucrose with at least one selected from the group consisting of Aspergillus sp., Aureobasidium sp., an enzyme preparation having fructosyltransferase activity, and combinations thereof at a temperature in the range of 50 to 60°C and pH is the range of 4.5 to 8.0; and recovering the FOS with a DP. The FOS with the low DP can be recovered by any suitable method, for example, SMBC.

[0042] The enzyme preparation having fructosyltransferase activity can be a pure fructosyltransferase enzyme or a crude enzyme preparation having fructosyltransferase activity. In accordance with an embodiment, the pure fructosyltransferase enzyme is obtained from a commercial source.

[0043] In an embodiment, the crude enzyme preparation having fructosyltransferase activity is derived from Aspergillus sp. In an embodiment, the enzyme preparation is derived from Aspergillus niger and is present in an amount in the range of 2200-2600 units per ml of the substrate. In an embodiment, Aspergillus sp. is Aspergillus niger MCC0252.In an embodiment, the crude enzyme preparation having fructosyltransferase activity is derived from Aureobasidium sp. In an embodiment, Aureobasidium sp. is Aureobasidium pullulans. In an embodiment, the enzyme preparation is derived from Aureobasidium pullulans and is present in an amount in the range of 1300-1600 units per ml of the substate. In an embodiment, Aureobasidium sp. is Aureobasidium pullulans MCC0127.

[0044] The invention will now be described with respect to the following examples, which do not limit the disclosed method in any way and only exemplify the claimed method. It will be apparent to those skilled in the art that various modifications and variations can be made to the method / process of the present disclosure without departing from the scope of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the method / process disclosed herein.

[0045] EXAMPLES

[0046] Instruments and Materials: One or more of the following instruments and materials were used in this example provided below.

[0047] 1. Microbial cultures: The cultures of Aureobasidium pullulans strain MCC0127 and Aspergillus niger strain MCC0252 were obtained from India.

[0048] 2. Endoinulinase enzyme: Novozym® 960 from Novozymes.

[0049] 3. Media for maintaining Aureobasidium pullulans and Aspergillus niger cultures separately: Glucose Yeast Peptone (‘GYP’) agar slants containing (g / 100 ml): 1 g of glucose, 0.6 g of yeast extract, 1 g of peptone and 20 g of agar.

[0050] 4. Media for seed inoculum and production of biomass: GYP liquid media containing of (g / 100 ml): 1 g of peptone, 5 g of sucrose, 0.5 g of di-potassium hydrogen phosphate, 0.2 g potassium di-hydrogen phosphate, 0.2 g of magnesium sulphate, 0.25 g of sodium chloride, and 0.001 g of polypropylene glycol (‘PPG’) as an antifoam agent. The pH of the media was adjusted to 6.5 using IN NaOH or IN HCL solution.5. pH digital meter: For measuring the pH.

[0051] 6. Fermenter: 50L fermenter (M / s. Scigenics, Chennai) with 35 1 working volume

[0052] Example 1: Production of biomass of Aureobasidiumpullulans strain MCC0127 and Aspergillus niger strain MCC0252

[0053] Two batches of 35 1 of each of the production media were sterilized by heating at 121°C for 20 mins. The two batches of the media were then cooled down to 27°C and transferred to fermenters. Overnight grown inocula of Aureobasidium pullulans and Aspergillus niger strains were transferred separately to each of the batches. The fermenter temperature and stirring rotations were maintained at 27°C and 100 rpm respectively. Samples from both fermenters were taken at different intervals to check biomass concentration. Batches of Aureobasidium pullulans and Aspergillus niger strains were harvested once maximum Packed Cell Volume (‘PCV’) was obtained.

[0054] Example 2: Preparation of the EOS with a high DP using Aureobasidium pullulans MCC0127 and Aspergillus niger MCC0252 in accordance with an embodiment of the present disclosure.

[0055] Biotransformation 1 (‘BT-1’) - Production of EOS with low DP: A 55 brix sucrose solution was prepared in water with a working volume of 400 ml. The sucrose solution so obtained was transferred to a 21 capacity conical flask and preincubated at 55°C. The preincubated sucrose solution was then mixed with 0.75% of cell pellet of Aureobasidium pullulan strain and incubated at 55°C under stirring rotations of 150 rpm. Samples were withdrawn at different hours to analyze pH, the amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0056] HPLC column: Shodex NH2P504E;

[0057] Mobile phase: acetonitrile: water (70:30);

[0058] Flow rate: 1 ml / min;

[0059] Column temperature: 30°C.Purification 1 (‘P-1’): After BT-1, the microbial cells were heated up to 80°C for 30 mins and the heated solution was filtered to obtain the FOS with DP of up to 6 of 59% purity. 55 brix solution of the FOS so obtained was prepared. 150 ml of the 55 brix solution of the FOS was passed through an SMBC separation column filled with 150 ml of Applexion resins (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) with a flow rate of 8 ml / min. The temperature was maintained at 65°C. Different fractions of 15 ml were collected (Pl-Fl to P1-F10). The initial fractions (Pl-Fl to P1-F4) were eluted with high purity FOS solution (95% purity). The FOS and other sugars content was measured using HPLC. The HPLC details are as follows:

[0060] HPLC column: Shodex NH2P504E;

[0061] Mobile phase: acetonitrile: water (70:30);

[0062] Flow rate: 1 ml / min;

[0063] Column temperature: 30°C.

[0064] Biotransformation 2 (‘BT-2’) - Production of the FOS with high DP: The FOS solution (95% purity) obtained above was concentrated to a 60 brix solution. 400 ml of said 60 brix of FOS solution was transferred to a 2 1 capacity conical flask and preincubated at 55°C. The preincubated solution was then mixed with 0.75% of cell pellet of Aspergillus niger strain and incubated at 55°C under stirring rotations of 150 RPM. Samples were withdrawn at different hour interval to analyze pH and amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0065] HPLC column: Shodex NH2P504E;

[0066] Mobile phase: acetonitrile: water (70:30);

[0067] Flow rate: 1 ml / min;

[0068] Column temperature: 30°C.

[0069] Purification 2 (‘P-2’): After BT-2, the microbial cells were heated up to 85°C for 30 mins. The heated solution so obtained was filtered to obtain the FOS with the DP of up to 8 of >80% purity. 60 brix solution of the FOS so obtained was prepared. 150 ml of said 60 brix solution of the FOS was passed through SMBC separation columnfilled with 150 ml of Applexion resins (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) with a flow rate of 8 ml / min. The temperature was maintained at 65°C. Different fractions of 15 ml each were collected (P2-F1 to P2-F10). The initial pooled fractions (P2-F1 to P2-F4) were eluted with high purity FOS (>99% purity). The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0070] HPLC column: Shodex NH2P504E;

[0071] Mobile phase: acetonitrile: water (70:30);

[0072] Flow rate: 1 ml / min;

[0073] Column temperature: 30°C.

[0074] Results: Table 1 provides the content of the low and high FOS solutions obtained after BT-1, P-1 and BT-2, and P-2 respectively. Table 2 provides the contents of P2-F1 to P2-F4.Table 1: Content of the FOS Solutions Obtained at Various Stages

[0075]

[0076] Table 2: Contents of Fractions P2-F1 to P2-F4

[0077]

[0078] Observations: During BT-2, further transfructosylation reaction happened and the short chain FOS (GF2 & GF3) composition was further extended and produced FOS with higher DP (DP8). During the P-2, glucose was removed completely, and purity of the high DP FOS was increased to >95% with increasing content of DP8. Glucose, fructose and sucrose were removed completely and the high DP FOS solution with 100% purity was obtained in P2-F1 and P2-F2.

[0079] Example 3: Preparation of the FOS with high DP using Aureobasidiumpullulans MCC0127 in accordance with an embodiment of the present disclosure.

[0080] BT-1 - Production of FOS with low DP: 55 brix sucrose solution was prepared in water. 400 ml of said solution was transferred to a 2 1 capacity conical flask, and preincubated at 55°C. The preincubated solution was mixed with 0.75% of cell pellet of Aureobasidium pullulan strain and incubated at 55°C under stirring rotations of 150 rpm. Samples were withdrawn at different hour intervals to analyze pH, the amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0081] HPLC column: Shodex NH2P504E;

[0082] Mobile phase: acetonitrile: water (70:30);

[0083] Flow rate: 1 ml / min;

[0084] Column temperature: 30°C.

[0085] P-1: After BT-1, the microbial cells were heated up to 80°C for 30 mins. The heated solution was filtered to obtain the FOS with the DP of up to 6 of 59% purity. 55 brix solution of the FOS so obtained was prepared. 150 ml of the 55-brix solution of the FOS was passed through an SMBC separation column filled with 150 ml of Applexion resins (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) with a flow rate of 8 ml / min. The temperature was maintained at 65°C. Different fractions of 15 ml were collected (Pl -Fl to P1-F10). The initial fractions (Pl -Fl to P1-F4) were eluted with high purity FOS solution (95% purity). The FOS and other sugars content was measured using HPLC. The HPLC details are as follows:HPLC column: Shodex NH2P504E;

[0086] Mobile phase: acetonitrile: water (70:30);

[0087] Flow rate: 1 ml / min;

[0088] Column temperature: 30°C.

[0089] BT-2 - Production of the FOS with high DP: The FOS solution (95% purity) obtained above was concentrated to a 60 brix solution. 400 ml of said 60 brix of FOS solution was transferred to a 21 capacity conical flask and preincubated at 55°C. The preincubated solution was then mixed with 0.75% of cell pellet of Aureobasium pullulans strain and incubated at 55°C under stirring rotations of 150 rpm. Samples were withdrawn at different hour interval to analyze pH and amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0090] HPLC column: Shodex NH2P504E;

[0091] Mobile phase: acetonitrile: water (70:30);

[0092] Flow rate: 1 ml / min;

[0093] Column temperature: 30°C.

[0094] P-2: After BT-2, the microbial cells were heated up to 80°C for 30 mins. The heated solution so obtained was filtered to obtain the FOS with the DP up to 6 of >85% purity. 60 brix solution of the FOS so obtained was prepared. 150 ml of said 60 brix solution of the FOS was passed through SMBC column filled with 150 ml of Applexion resins (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) with a flow rate of 8 ml / min. The temperature was maintained at 65°C. Different fractions of 15 ml each were collected (P2-F1 to P2-F10). The initial fractions (P2-F1 to P2-F4) were eluted with high purity FOS (>96% purity). The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0095] HPLC column: Shodex NH2P504E;

[0096] Mobile phase: acetonitrile: water (70:30);

[0097] Flow rate: 1 ml / min;

[0098] Column temperature: 30°C.Results: Table 3 provides content of the low and high FOS solutions obtained after BT-1, P-1 and BT-2, and P-2 respectively. Table 4 provides the contents of Fractions P2-F1 to P2-F4.Table 3: Content of the FOS Solutions Obtained at Various Stages

[0099] >

[0100]

[0101] Table 4: Contents of Fractions P2-F1 to P2-F4

[0102]

[0103] Observations: After BT-2 with Aureobasium pullulans strain, further transfructosylation reaction happened and the short chain FOS (GF2 & GF3) composition was further extended and produced FOS with higher DP (DP6). During P-2, glucose was removed completely, and purity of the obtained high DP FOS was increased to >95% with increasing content of DP6. Glucose, fructose and sucrose were removed completely and FOS with high DP with 100% purity was obtained in the first two fractions.

[0104] Example 4: Preparation of the FOS with high DP using Aureobasidium pullulans MCC0127 and commercial endoinulinase in accordance with an embodiment of the present disclosure.

[0105] BT-1 - Production of FOS with low DP: 55 brix sucrose solution was prepared in water. 400 ml of said solution was transferred to a 2 1 capacity conical flask, and preincubated at 55°C. The preincubated solution was mixed with 0.75% of cell pellet of Aureobasidium pullulan strain and incubated at 55°C under stirring rotations of 150 rpm. Samples were withdrawn at different hour intervals to analyze pH, the amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0106] HPLC column: Shodex NH2P504E;

[0107] Mobile phase: acetonitrile: water (70:30);

[0108] Flow rate: 1 ml / min;

[0109] Column temperature: 30°C.

[0110] P-1: After BT-1, the microbial cells were heated up to 80°C for 30 mins. The heated solution was filtered to obtain the FOS with the DP of up to 5 of 59% purity. 55 brix solution of the FOS so obtained was prepared. 150 ml of the 55 brix solution of the FOS was passed through an SMBC separation column filled with 150 ml of Applexion resins (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) with a flow rate of 8 ml / min. The temperature was maintained at 65°C. Different fractions of 15 ml were collected (Fraction Pl -Fl to Pl -Fl 0). The initial fractions (Pl -Fl toP1-F4) were eluted with high purity FOS solution (95% purity). The FOS and other sugars content was measured using HPLC. The HPLC details are as follows:

[0111] HPLC column: Shodex NH2P504E;

[0112] Mobile phase: acetonitrile: water (70:30);

[0113] Flow rate: 1 ml / min;

[0114] Column temperature: 30°C.

[0115] BT-2 - Production of the FOS with high DP: The FOS solution (95% purity) obtained above was concentrated to a 60-brix solution. 400 ml of said 60 brix of FOS solution was transferred to a 21 capacity conical flask and preincubated at 55°C. The preincubated solution was then mixed with 0.1% of Novozym® 960 and incubated at 55°C under stirring rotations of 150 rpm. Samples were withdrawn at different hour interval to analyze pH and amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows: HPLC column: Shodex NH2P504E;

[0116] Mobile phase: acetonitrile: water (70:30);

[0117] Flow rate: 1 ml / min;

[0118] Column temperature: 30°C.

[0119] P-2: After BT-2, the reaction mixture was heated up to 80°C for 30 mins. The heated solution so obtained was filtered using SMBC (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) to obtain the FOS with the DP up to 7 of >84% purity. 60 brix solution of the FOS so obtained was prepared. The temperature was maintained at 65°C. Different fractions of 15 ml each were collected (P2-F1 to P2-F10). The initial fractions (P2-F1 to P2-F4) were eluted with high purity FOS (>93% purity). The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0120] HPLC column: Shodex NH2P504E;

[0121] Mobile phase: acetonitrile: water (70:30);

[0122] Flow rate: 1 ml / min;

[0123] Column temperature: 30°C.Results: Table 5 provides the content of the low and high FOS solutions obtained after BT-1, P-1 and BT-2, and P-2 respectively. Table 6 provides the contents of P2-F1 to P2-F4.Table 5: Content of the FOS Solutions Obtained at Various Stages

[0124]

[0125] Table 6: Contents of Fractions P2-F1 to P2-F4

[0126]

[0127] Observations: After BT-2 withNovozym® 960, further transfructosylati on reaction happened and the short chain FOS (GF2 & GF3) composition was further extended and produced FOS with higher DP (DP7). During P-2, glucose was removed completely, and purity of the obtained high DP FOS was increased to >95% with increasing content of DP8. Glucose, fructose and sucrose were removed completely and FOS with high DP with 100% purity was obtained in the first two fractions.

[0128] Example 5: Preparation of the FOS with high DP using Aureobasidiumpullulans MCC0127 and crude enzyme extracted of Aspergillus niger MCC0252 in accordance with an embodiment of the present disclosure.

[0129] BT-1 - Production of FOS with low DP: 55 brix sucrose solution was prepared in water. 400 ml of said solution was transferred to a 2 1 capacity conical flask, and preincubated at 55°C. The preincubated solution was mixed with 0.75% of cell pellet of Aureobasidium pullulan strain and incubated at 55°C under stirring rotations of 150 rpm. Samples were withdrawn at different hour intervals to analyze pH, the amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0130] HPLC column: Shodex NH2P504E;

[0131] Mobile phase: acetonitrile: water (70:30);

[0132] Flow rate: 1 ml / min;

[0133] Column temperature: 30°C.

[0134] P-1: After BT-1, the microbial cells were heated up to 80°C for 30 mins. The heated solution was filtered to obtain the FOS with the DP of up to 5 of 59% purity. 55 brix solution of the FOS so obtained was prepared. 150 ml of the 55 brix solution of the FOS was passed through an SMBC separation column filled with 150 ml of Applexion resins (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) with a flow rate of 8 ml / min. The temperature was maintained at 65°C. Different fractions of 15 ml were collected (Pl -Fl to P1-F10). The initial fractions (Pl -Fl to P1-F4)were eluted with high purity FOS solution (95% purity). The FOS and other sugars content was measured using HPLC. The HPLC details are as follows:

[0135] HPLC column: Shodex NH2P504E;

[0136] Mobile phase: acetonitrile: water (70:30);

[0137] Flow rate: 1 ml / min;

[0138] Column temperature: 30°C.

[0139] Preparation of crude enzyme extract from Aspergillus niger MCC0252: 15% biomass of Aspergillus niger MCC0252 was mixed with phospho-citrate buffer containing 0.1 M disodium hydrogen phosphate, and 0.1 M HC1 (pH 5.6) and kept in an overhead stirrer for mixing to get a homogenized solution. The biomass cells were disintegrated using GE A Lab Homogenizer PandaPLUS 2000. The homogenizer pressure was maintained between 300, 600, 900, 1200, 1400 mPa and 2-10 cycle were carried out to disintegrate the cells. The temperature was maintained at 5°C. The homogenate biomass was then centrifuged at 8000 rpm for 30 min at 4°C to get a cell free lysate. The cell free lysate so obtained was concentrated up to 10 times and stored in -40°C for further use.

[0140] BT-2 - Production of the FOS with high DP: The FOS solution (95% purity) obtained above was concentrated to a 60 brix solution. 400 ml of said 60 brix of FOS solution was transferred to a 21 capacity conical flask and preincubated at 55°C. The preincubated solution was then mixed with 0.1% of crude enzyme extract obtained above and incubated at 55°C under stirring rotations of 150 rpm. Samples were withdrawn at different hour interval to analyze pH and amounts of FOS and other sugars. The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows:

[0141] HPLC column: Shodex NH2P504E;

[0142] Mobile phase: acetonitrile: water (70:30);

[0143] Flow rate: 1 ml / min;

[0144] Column temperature: 30°C.P-2: After BT-2, the reaction mixture was heated up to 85°C for 30 mins. The heated solution so obtained was filtered to obtain the FOS with the DP up to 6 of >85% purity. 60 brix solution of the FOS so obtained was prepared. 150 ml of said 60 brix solution of the FOS was passed through SMBC separation column filled with 150 ml of Applexion resins (APPLEXION XA 2004 / 32 K Strong Acid Cation Resin) with a flow rate of 8 ml / min. The temperature was maintained at 65°C. Different fractions of 15 ml each were collected (P2-F1 to P2-F10). The initial fractions (P2-F1 to P2-F4) were eluted with high purity FOS (>94% purity). The amounts of the FOS and other sugars were measured by HPLC. The HPLC details are as follows: HPLC column: Shodex NH2P504E;

[0145] Mobile phase: acetonitrile: water (70:30);

[0146] Flow rate: 1 ml / min;

[0147] Column temperature: 30°C.

[0148] Results: Table 7 provides the content of the low and high FOS solutions obtained after BT-1, P-1 and BT-2, and P-2 respectively. Table 8 provides the contents of Fractions P2-F1 to P2-F4.Table 7: Content of the FOS Solutions Obtained at Various Stages

[0149] >

[0150]

[0151] Table 8: Contents of Fractions P2-F1 to P2-F4

[0152]

[0153] Observations: After BT-2 with the crude enzyme extract, further transfructosylati on reaction happened and short chain FOS (GF2 and GF3) composition was further extended and produced FOS with higher DP (DP7). During P-2, glucose was removed completely, and purity of the obtained high DP FOS was increased to >95% with increasing content of DP6. Glucose, fructose and sucrose were removed completely and FOS with high DP with up to 100% purity was obtained in the first two fractions.

[0154] Example 6: Signature profiling using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) of input raw material after P-1 stage (FOS with low DP) of Example 2 and product obtained after P-2 stage (FOS with a high DP) of Example 2.

[0155] After P-1 of Example 2: FOS and other sugars were qualitatively checked using Thermo Fisher Scientific HPAEC-PAD, the conditions of which were as follows: Mob Phase A: 100 mM sodium hydroxide

[0156] Mob Phase B: IM sodium acetate and 100 mM Sodium hydroxide

[0157] Flow: 0.5 ml / min

[0158] Column: CarboPac PA-100 Analytical (4 x 250 mm) and guard (4 x 50 mm) Mode: Integrated amperometry

[0159] Waveform Name: Gold standard PAD

[0160] Waveform Description: Carbohydrates (Std. quad)

[0161] Column TC Temperature Set: 30°C

[0162] Compartment TC Temperature Set: 30°C

[0163] pH upper limit: 13.00

[0164] pH lower limit: 10.00

[0165] Run time: 40 min

[0166] Injection Volume: 20 pl

[0167] After P-2 of Example 2: FOS and other sugars were qualitatively checked using Thermo Fisher Scientific HPAEC-PAD, the conditions of which are as follows:Mob Phase A: 100 mM sodium hydroxide

[0168] Mob Phase B: IM sodium acetate and 100 mM Sodium hydroxide

[0169] Flow: 0.5 ml / min

[0170] Column: CarboPac PA-100 Analytical (4 x 250 mm) and guard (4 x 50 mm) Mode: Integrated amperometry

[0171] Waveform Name: Gold standard PAD

[0172] Waveform Description: Carbohydrates (Std. quad)

[0173] Column TC Temperature Set: 30°C

[0174] Compartment TC Temperature Set: 30°C

[0175] pH upper limit: 13.00

[0176] pH lower limit: 10.00

[0177] Run time: 40 min

[0178] Injection Volume: 20 pl

[0179] Results: HPAEC-PAD chromatograms of the FOS with the low DP and the FOS with the high DP obtained after P-1 and P-2 stage respectively are depicted in FIG. 1.

[0180] Observations: HPAEC-PAD profiling showed that FOS sample after P-1 stage comprises of low DP FOS ranging from DP3 to DP6 (GF2 to GF5) due to removal of glucose, fructose and sucrose during the chromatography purification stage. After P2 stage, the obtained product (pooled fractions Fl to F4) comprises of the FOS with high DP ranging from DP3 to DP 10 with altered composition.

[0181] INDUSTRIAL APPLICABILITY

[0182] The disclosed process provides FOS with a high DP. The FOS with high DP is known to provide several benefits such as sustained prebiotic effect, support for gut flora diversity, reduced gastrointestinal discomfort, improved nutrient absorption, enhanced mineral uptake, and potential anti-inflammatory effects. The disclosed process is simple and efficient. The product obtained from the disclosed process has a unique high value composition.

Claims

We Claim:

1. A process for preparing fructo-oligosaccharides with a high degree of polymerization, the process comprising treating a substrate comprising fructooligosaccharides with a low degree of polymerization with at least one selected from the group consisting of Aureobasidium pullulans, Aspergillus niger, an enzyme preparation having an endoinulinase activity, and combinations thereof at a temperature in the range of 40 to 60°C to obtain the fructooligosaccharides with the high degree of polymerization.

2. The process as claimed in claim 1, wherein the substrate comprises 50-60 brix solution of the fructo-oligosaccharides with the low degree of polymerization.

3. The process as claimed in any of the preceding claims, wherein the low degree of polymerization is in the range of 3 to 6.

4. The process as claimed in any of the preceding claims, wherein the substrate comprises the fructo-oligosaccharides with the low degree of polymerization and sucrose in a ratio in the range of 5 to 95% and 95 to 5%.

5. The process as claimed in claim 1, wherein the high degree of polymerization is in the range of 6 to 10.

6. The process as claimed in any of the preceding claims, wherein the treatment is carried out at a pH in the range of 4.5 to 8.0.

7. The process as claimed in any of the preceding claims, wherein the treatment is carried out for a duration of 1 to 24 hours.

8. The process as claimed in any of the proceeding claims, wherein the treatment is carried at stirring rotations in the range of 75 to 200 rpm.

9. The process as claimed in any of the preceding claims, wherein the enzyme preparation is present in an amount of at least 1400 to 2800 units per ml of the substrate.

10. The process as claimed in any of the proceeding claims, wherein the enzyme preparation is a pure enzyme or a crude enzyme preparation.

11. The process as claimed in claim 10, wherein the pure enzyme or the crude enzyme preparation is derived from Aspergillus niger.

12. The process as claimed in any of the preceding claims, comprising recovering the fructo-oligosaccharides with the high degree of polymerization by simulated moving bed chromatography.

13. The process as claimed in claim 12, wherein the recovered fructooligosaccharides has purity of 95 to 100% and comprises:- 0.5-3% F3;- 0.5-10% F4;- 5-29% DP6;- 1-11% DP7;- 0.5-4% DP8,- 0.5-8% GF2;- 0.5-24% GF3;- 13-43% GF4; and- 18-41% GF5.

14. The process as claimed in any of the preceding claims, wherein the fructooligosaccharides with the low degree of polymerization is obtained by:- treating a substrate comprising sucrose with at least one selected from the group consisting of Aspergillus sp., Aureobasidium sp. an enzyme preparation having fructosyltransferase activity, and combinations thereofat a temperature in the range of 50 to 60° C and pH in the range of 4.5 to 8.0; and- recovering the fructo-oligosaccharides with the low degree of polymerization.

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