GB2612082A - Process of production of xylooligosaccharides from peels of orange fruit - Google Patents

Abstract

The present disclosure relates generally to the synthesis of xylooligosaccharides. More specifically, a process for production of xylooligosaccharides from peels of orange fruit, the process comprising the steps of: (a) extracting and precipitating xylan from the peels; and (b) hydrolysing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides. Preferably, the process comprises the steps of: (a) subjecting the peels to alkali lysis by a basic solution comprising sodium hydroxide; (b) extracting xylan by treating with steam to give alkali-solubilized xylan; (c) precipitating the xylan by adding an organic solvent; and (d) hydrolysing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides. More preferably, the peels of orange fruit are dried peels of orange fruit. The organic solvent may be ethanol or anhydrous methanol. The xylooligosaccharides comprise xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and combinations thereof. The xylooligosaccharides produced by the present process are potential prebiotics. The process of the present disclosure gives high yields with low time requirement thereby improving the efficiency of the production.

Description

PROCESS OF PRODUCTION OF XYLOOLIGOSACCHARIDES FROM PEELS OF ORANGE FRUIT

FIELD OF THE INVENTION

[0001] The present disclosure relates generally to the field of synthesis of vlooligosaccharides. More specifically, the disclosure is directed to a process of production of xylooligosaccharides from peels of orange fruit.

BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Xylooligosaccharides (XOS) are saccharides or sugar oligomers made of two to up to six xylose units linked via 131-4 linkages. They are nutritionally valued as prebiotic compounds that promote growth of gut bacteria and improve the gut microbiota. XOS are not digested by stomach enzymes and are therefore available for consumption by intestinal microbes. Commercially XOS is produced from xylan which is a complex polysaccharide and is a type of hemicellulose. Xylan is the second most abundant hemicellulose material found in corn cobs, corn husks, wheat bran, rice bran, sugarcane bagasse, wheat straw, rice straw, almond shell, peanut shell, and Sehinta Nervostun grass.

[0004] Production of xylooligosaccharides involves two steps: (i) the extraction of xylan from a given substrate and (ii) hydrolysis of the substrate to give the XOS. Recent studies show many excising and commercially available extraction processes for xylan. However, identifying an economically feasible process along with appropriate reaction conditions for xylan extraction suitable for a particular substrate is a challenging process. This must be followed by identifying a suitable process for hydrolyzing the xylan obtained to give maximum yield of XOS. In earlier investigations, xylooligosaccharides have been prepared from various organic waste materials such as corn cobs, corn husks, rice barn, wheat bran, sugarcane bagasse, wheat straw, rice straw, almond shell, peanut shell, and Sehima nervosum grass.

(AK. Samanta, Natasha Jayapal, A.P. Kolte, S. Senani, Manpal Sridhar, K.P. Suresh, K.T. Sampath. "E)izymatic production of xylooligosaccharides from alkali solubilized xylan of natural grass (Sehima nervosum). " Bioresource Technology 112 (2012) 199-205; A.K. Samanta, S. Senani, Atul P. Kolte, Manpal Sridhar, K.T. Sampath, Natasha Jayapal and Anusuya Devi "Production and in vitro evaluation of xylooligosaccharides generated from corn cobs." Food and bioproducts processing 9 0 ( 2 0 1 2): 466-474; and A. K. Samanta,Natasha Jayapal, A. P. Kolte,S. Senani, Manpal Sridhar,Sukriti Mishra,C. S. Prasad & K. P. Suresh. "Application of Pigeon Pea (Cajanus calm)) Stalks as Raw Material for Xylooligosaccharides Production." Appl Biochem Biotechnol (2013) 169:2392-2404). Most of the techniques are completely based upon enzymatic hydrolysis of xylan. Some of the Patent documents demonstrating these methods are provided below: [0005] Patent no. IN209418 (2007) discloses a process for manufacturing xylooligosaccharides, comprising: steaming a corncob meal under conditions of a temperature of 150 to 250°C and a pressure of 20 to 29N1Pa; filtering the steamed corncob meal to obtain a filtrate from which solid has been removed; performing an enzyme treatment by reacting xylanase with soluble xylan in the filtrate to hydrolyze the soluble xylan; removing any suspended matter from a reaction product obtained during the enzyme treatment; and drying the reaction product.

[0006] Patent no. IN252011 (2012) relates to a process for obtaining xylooligosaccharides from cereals. The invention is aimed at obtaining oligosaccharides from cereals using cereal/cereal malt enzymes. Xylans present in the cereal/cereal malt/ bran cell wall are hydrolyzed with the cereal/cereal malt enzyme extract, particularly rich with xylanase to obtain xylooligosaccharides. This method exploits the cereal/cereal malt xylanase, which is induced during germination to hydrolyze cereal/cereal malt/cereal bran xylans leading to the liberation of xylooligosaccharides.

[0007] In US6942754B2, xylooligosaccharides were produced from a lignocellulose pulp by enzyme-treating a lignocellulose pulp with hemicellulase, filtering the resultant reaction mixture to separate a liquid fraction from the enzyme-treated pulp, subjecting the separated liquid fraction to a permeation treatment through a separation membrane to separate a non-permeated fraction containing xylooligosaccharides lignin complex with an increased concentration from a permeated fraction, collecting the non-permeated fraction, and separating and recovering xylooligosaccharides from the collected non-permeated fraction.

[0008] Orange peels are one of cheapest raw materials which are readily available in almost every season. Orange peel contains soluble sugars and pectin. According to Rivas era!, (Rivas, B.; Torrado, A.; Torre, P. Converti, A.; Dominguez, J.M. (2008), Submerged citric acid.fermeniation on orange peel aitiohydrolysaie. J. Agric, Food Chem. 56, 2380-2387), orange peel constituted highest percentage of soluble sugars with 16.9%wt. followed by starch, 3.75%wt., fiber which includes cellulose, 9.21%wt.; hemicelluloses 10.5%wt., lignin 0.84%wt., and pectins 42.5%wt. It also has high percentage of ashes, 3.50%wt and proteins 6.50%wt, and the least amount of fats, 1.95%wt. (Kapoor, KR.; Chaudhary, K.; Tauro, P. (1982). Prescott and Dunn's Industrial Microbiology, 4 th edn. G. Reed (Ed), AVI Publishing Co, Wesrport, CT). Recent studies showed that due to presence of hemicellulose and pectins, the orange peels can be utilized to produce prebiotics materials such as xylooligosacchrides, fructooligosacchrides (FOS) and pectin.

[0009] Orange peels contain maximum amount of pectic oligosaccharides (POS) but it has low prebiotic potential compared to XOS and even FOS. The cost of producing a prebiotic material such as XOS from orange peels is 45% lower than that of other conventional fruit pulp industries as shown in the earlier research (Olano-Martin, E., G. R. Gibson, and R. A. Rastall. 2002. Comparison of the in vitro hifidogenic properties of pectins and pectic-oligosaccharides. J. Appl, Microbiol, 93:505-511). Thus, orange peels which are considered waste products can be a great substrate for XOS production.

[0010] XOS production from orange peel as the substrate generally uses enzymatic hydrolysis for XOS extraction from xylan. But enzymatic hydrolysis results in production of undesirable materials which significantly lowers the yield of XOS produced. Enzymatic hydrolysis also decreases the edibility of the XOS due to higher toxicity of any residual enzymes. Moreover, enzymatic hydrolysis has strict pH maintenance requirements and takes longer durations for production, both of which increase complexity and costs of production.

[0011] Thus, there is a need in the art to provide a novel method of production of xylooligosaccharides from orange peel with higher yield, better reaction control and lesser time duration.

OBJECTS OF THE INVENTION

[0012] An object of the present disclosure is to provide a process of production of xylooligosaccharides that is economically feasible and ecologically beneficial.

[0013] An object of the present disclosure is to provide a process of production of xylooligosaccharides from peels of orange fruit.

[0014] Another object of the present disclosure is to provide a process of production of xylooligosaccharides from peels of orange fruit that gives higher yields of XOS and requires lesser time.

SUMMARY OF THE INVENTION

[0015] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0016] The present disclosure provides a process for synthesizing xylooligosaccharide from peels of orange fruit which is ordinarily considered a waste product. The process comprises two key steps which are extraction of xylan from the peels of orange fruit and hydrolysis of the xylan to give XOS.

[0017] In an aspect, the present disclosure provides a process for production of xylooligosaccharides from peels of orange fruit, the process comprising the steps of: (a) extracting and precipitating xylan from the peels and (b) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.

100181 In an embodiment, the present disclosure provides a process for production of xylooligosaccharides from peels of orange fruit, the process comprising the steps of: (a) subjecting the peels to alkali lysis by a basic solution comprising sodium hydroxide; (b) extracting xylan by treating with steam to give alkali-solubilized xylan; (c) precipitating the xylan by adding an organic solvent; and (d) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.

[0019] In an embodiment, the peels of orange fruit may be dried peels of orange fruit. In a preferred embodiment, the dried peels may be crushed before use.

[0020] In an embodiment, the xylooligosaccharides obtained may be further purified. In an embodiment, purification of xylooligosaccharides may be performed by activated charcoal or by ultrafiltration [0021] In an aspect, the present disclosure provides xylooligosaccharides obtained from peels of orange fruit by a process comprising the steps of (a) subjecting the peels to alkali lysis by a basic solution comprising sodium hydroxide; (b) extracting xylan by treating with steam to give alkali-solubilized xylan; (c) precipitating the xylan by adding an organic solvent; and (d) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.

[0022] Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1 provides the high-performance liquid chromatography (HPLC) chromatogram of xylan produced by the process as per an embodiment of the present disclosure.

Figure 2 provides the high-performance liquid chromatography (HPLC) chromatogram of standard xylan.

Figure 3 provides the high-performance liquid chromatography (HPLC) chromatogram of xylooligosaccharides produced by the process as per an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0025] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0026] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0027] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the

S

numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0028] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0029] As used in the description herein and throughout the claims that follow, the meaning of "a," "an,-and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0030] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to." [0031] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0032] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0033] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

[0034] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

100351 It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0036] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

100371 The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0038] As used herein, the term 'alkali lysis' refers to a pre-treatment step wherein the substrate is degraded by a strong basic solution. This process helps separate out the hemicelluloses and lignin from the substrate. Particularly, the base solubilizes the extracted xylan.

[0039] As used herein, the term 'alkali-solubilized xylan' refers to the xylan which has separated out from the substrate and is dissolved in the basic solution.

[0040] As used herein, the tenn xylooligosaccharides', refers to oligomers comprising di-to penta-131-4 linked D-xylopyranosyl residues.

100411 The terms 'substrate', 'orange peel', 'powdered orange peel', 'peels of orange fruit' and 'dried orange peel' have been used interchangeably.

100421 In an embodiment, the present disclosure provides a process for production of xylooligosaccharides from peels of orange fruit, the process comprising the steps of (a) extracting and precipitating xylan from the peels; and (b) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.

[0043] In an embodiment, the present disclosure provides a process for production of xylooligosaccharides from peels of orange fruit, the process comprising the steps of (a) subjecting the peels to alkali lysis by a basic solution comprising sodium hydroxide; (b) extracting xylan by treating with steam to give alkali-solubilized xylan; (c) precipitating the xylan by adding an organic solvent; and (d) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.

[0044] In a preferred embodiment, the peels of orange fruit may be dried peels of orange fruit. In a preferred embodiment, the dried peels may be crushed to powder before use.

[0045] In an embodiment, the orange fruit used for the present purpose may be selected from fruits of any of the species or varieties of oranges including, but not limited to, Citrus sinensis, Citrus aurantium, Citrus retieulata, Citrus trifoliate, blood orange, Valencia orange, navel orange, or cara cara oranges [0046] In an embodiment, the basic solution is a sodium hydroxide solution in water. Sodium hydroxide is made use of due to its superior alkali nature compared to other bases, especially potassium hydroxide. Alkali lysis is considered to be a significant method to dissolve all impurities (such as lignin based substances) because it dissociates ester linkages between hemicelluloses and lignin and produces maximum concentration of free hemicellulose.

100471 In an embodiment, the basic solution may have a concentration of sodium hydroxide in the range of about 6% to about 218% by weight. In a preferred embodiment, the basic solution may have a concentration of sodium hydroxide in the range of about 11% to about 13% by weight. The increase in the concentration of basic solution results in a consequent increase in the recovery of xylan.

[0048] Steam extraction is performed to extract the xylan from the peels to generate alkali-solubilized vial'. In an embodiment, the steam extraction may be performed in a pressure vessel. In an embodiment, the steam extraction involves treating the peels with steam at a temperature in the range of about 110°C to 130°C, preferably 120°C and pressure in the range of about 12psi to about 18psi, preferably about 15 psi. In a preferred embodiment, the peels may be exposed to steam having a temperature of 120°C at 15 psi pressure for about 45 minutes.

[0049] In order to obtain maximum extraction of xylan from the peels, the peels to steam ratio must be set appropriately. In an embodiment, the peels to steam ratio for steam extraction may be in the range of about 0.5:5 to about 1.5:15, preferably the peels to steam ratio is about I: 10, [0050] Orange peels comprise hemicelluloses, and lignin among many other constituents. The other constituents are insoluble in alkali and are thereby separated from the hemicellulose-xylan. The insoluble components are precipitated by centrifugation. In an embodiment, the alkali-solubilized xylan produced from step (b) may be centrifuged to remove any lignin extracted from the peels before proceeding for precipitation step. In an embodiment, the centrifugation may be performed at about 4500 rpm to about 5500 rpm for about 20 minutes to about 40 minutes. In a preferred embodiment, the centrifugation may be performed at about 5000 rpm for about 30 minutes.

[0051] In an embodiment, the centrifugation may be followed by separation of the alkali-solubilized xylan from the precipitated lignin. This separation may be performed by decanting or by any filtrating means known in the art, preferably the filtering means is a cellulose filter paper.

[0052] Once the other constituents are removed, next step is the precipitation of xylan. The alkali-solubilized xylan is maintained at a pH range of about 4.7 to about 5.3, preferably about pH 5 for precipitation. In an embodiment, the pH may be maintained by a mild acid, selected from the group comprising of glacial acetic acid, citric acid and malic acid.

[0053] Once the pH is set, the xylan may be precipitated by the organic solvent followed by its separation by centrifugation. In an embodiment, the organic solvent may be selected from ethanol, or anhydrous methanol. In a preferred embodiment, the organic solvent is cold ethanol, preferably with a concentration in the range of about 65% v/v to about 75% v/v, most preferably the organic solvent is cold ethanol with a concentration of about 70% v/v. In a preferred embodiment, the centrifugation of step (c) may be performed at about 7500 rpm to about 8500 rpm for about 8 minutes to about 12 minutes, preferably about 8000 rpm for about 10 minutes at room temperature.

[0054] In an embodiment, the organic solvent may be added in an amount of about 65% to about 75% viv with respect to the amount of alkali-solubilized xylan.

[0055] The centrifugation of xylan gives pellets of xylan that may be dried to be used for XOS production immediately or may be powdered and stored for later use.

[0056] Once the xylan is obtained from the orange peel, the second critical step is the hydrolysis of the xylan. The present disclosure performs the hydrolysis with tartaric acid. In an embodiment, hydrolyzing the precipitated xylan may be performed at a pH range of about 4.5 to about 6.5, preferably at a pH of about 5.5. In an embodiment, hydrolyzing the precipitated xylan may be performed at a temperature range of about 85 °C to about 95 °C, preferably about 90°C.

[0057] In an embodiment, the tartaric acid may be a tartaric acid solution in water having a concentration in the range of about 0.10 vv to about 0.20 v/v.

100581 In an embodiment, the tartaric acid may be employed in an amount of about 0.1% to about 0.3% by weight based on the amount of xylan. After adding the tartaric acid the mixture may be incubated. In a preferred embodiment, the incubation may be performed in a shaking incubator. The incubator may have a shaking speed of about rpm to about 170 rpm, preferably about 150 rpm. In an embodiment, the incubation may be performed for about 2 hours to about 20 hours, preferably about 2 hours to about 6 hours. To obtain the xylooligosaccharides, the hydrolysis reaction is arrested by boiling followed by filtration.

[0059] In an embodiment, the xylooligosaccharides obtained by the process recited above may be further purified. In an embodiment, purification of xylooligosaccharides may be performed by activated charcoal or by ultrafiltration.

[0060] In an embodiment, the xylooligosaccharides comprise di to penta-oligomers of xylose. In an embodiment, the xylooligosaccharides produced by the process recited above comprises xylose, xylobiose, xylomiose, xylotetraose, xylopentaose, and combinations thereof.

[0061] The above recited process provides a higher yield of xylooligosaccharides than conventionally known processes of extracting xylooligosaccharides from orange peel. In an embodiment, the process may provide xylooligosaccharides with a yield as high as 18%. The yields of 18% are better than the older experiments done with XOS production via enzymatic methods like in Dr. Samanta et at [Samanta, AK., Senani, S., Kolte, A.P., Sridhar, M., Jayapal, Natasha, Sharath, B., 2011. Enzymatic production of xylooligosaccharides from corn husks. In: Proceedings of National Symposium on Innovative and Modem Technologies for Agricultural Productivity, Food Security and Environmental Management, Mangalore, p. 92.], where a lower yield of (1.618 mg/m1) of XOS was obtained. The XOS produced by the present disclosure has a higher scale of purity of up to 95%.

100621 The time taken for conventional processes to convert xylan to XOS, such as ones employing enzyme hydrolysis may be over 6 hours. In some embodiments, the time taken for the conversion of xylan to xylooligosaccharides by the process of the present disclosure may be under 90 minutes. Thus the present disclosure provides a process with shorter time duration for XOS production from xylan making it more efficient. The process of the present disclosure provides greater control over pH of hydrolysis step by providing a wider working pH range, unlike enzymatic hydrolysis conventionally performed on orange peels which necessarily require a pH of 5.

[0063] The process is industrially scalable and provides a means of consuming orange peels which are generally considered waste products. Thus, the process reduces ecological burden by using waste organic sources that are bio-renewable and readily available.

[0064] The process is cost-effective compared to enzymatic hydrolysis of xylan produced in Gupta, P.K., Agrawal, P. and Hegde, P., 2017. Value addition of orange fruit wastes in the enzymatic production of xylooligosaccharides. African Journal of Biotechnology, 16(24), pp.1324-1330; as has been shown below in Table 1 and Table 2: Table 1: Cost analysis for the XOS production from Gupta et al. S. Particulars Total quantity present Actual Cost (1NR) mentioned Quantity used Estimated No. cost of used quantity ([NR) 1 Orange peels waste sample 5kg <5 5 kg dried orange peels 20 (obtained from to produce xylan.

fmit shops and homes) 2 Xylanase 10 gm 8841 4U = 10.6mg 9.371 enzyme 3 Sodium 500 gm 412 For 12%= 30gm/50mL 24.2 Hydroxide 4 Citric Acid 500 gm 630 2.1077 gm in 200 m L 2.66 monolwdrate Di sodium 500 gm 510 1.4339 gin in 200 m L 1.46 hydrogen phosphate 6 Ethanol 500mL 470 To produce 100 mg of 197.4 (C211501) XOS 3 volumes of 70% 95% ethanol is required hence = 300 ml (210 ml ethanol + 90 mL water) 7 Glacial 500 ml 630 To maintain pH 5 37.8 Acetic approximate 30 mL Acid (approximate) 8 Activated 500 mg 266 To purify 100 mL of 10.64 Carbon unpurified XOS solution requires nearly 100 mg activated charcoal 9 Ethanol 100 ml 47 For 100 mL of XOS 23.5 required for purification by purification activated charcoa150% (95%) of ethanol is required hence= 100 ml (50m1 ethanol + 50 ml water) Electricity 100 units 900 50 450 consumption (approximate)* for uses in all lab equipments Total 00 gm 777.013/ 1 approxim ate of XOS Table 2: Cost analysis for the XOS production from present invention Sr. Particulars Total quantity present Actual Quantity used Estimated No. Cost cost of used (INR) quantity mentioned (INR) 1 Orange peels 5kg <5 5 g dried orange peels to 20.00 waste (obtained produce xylan.

sample from fruit shops and homes) 2 Tartaric Acid 1 kg 200 1 M ( 3.75g in 25m1) 0.75 3 Sodium 1 kg 80 For 12%= 30gm/50m1 2.5 Hydroxide 4 Ethanol 1 L 100 To produce 1000 mg of 2.1 (C2H5014) XOS 3 volume of 70% 95% ethanol required hence = 300 ml (210 in! ethanol + 90 ml water) Also rcsusable for 10 times (21/10=2.1) Glacial 1 L 100 To maintain pH 5 3 Acetic approximate 30 ml Acid (approximate) 6 Electricity 100 units ( 480 2 units 7 consumption approximate)* for uses in all lab equipments Total gm 35.35/ 1 approximate of XOS *power in BESCOM in Bangalore, India is slab wise 0 units to 30 units is charged INR 3.75, then 31 units to 100 units is charged JINX 5.20.

[0065] In an embodiment, the present disclosure provides xylooligosaccharides obtained from peels of orange fruit by a process comprising the steps of: (a) subjecting the peels to alkali lysis by a basic solution comprising sodium hydroxide; (b) extracting xylan by treating with steam to give alkali-solubilized vim; (c) precipitating the xylan by adding an organic solvent; and (d) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.

[0066] In an embodiment, the present disclosure provides xylooligosaccharides for use as prebiotics. In an embodiment, the present disclosure provides a medicament, composition or formulation comprising the xylooligosaccharides obtained as recited herein.

[0067] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

EXAMPLES

[0068] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

100691 MATERIALS: The orange peels were obtained from orange farms located in the outskirts of Bangalore. All the other chemicals used for the purposes of the following examples were procured from Sigma Aldrich, Bangalore, India.

EXAMPLE 1: Production of xylooligosaccharides [0070] The first key step of the production involved the extraction of xylan from powdered orange peels based on the alkaline lysis method using Sodium hydroxide (NaOH) followed by steam extraction in order to produce maximum possible xylan extraction. 5gm of powdered orange peels was taken and treated with 12% of NaOH prepared in distilled water. This was followed by steam extraction at 120°C, 15 psi pressure for 45 minutes to give alkali-solubilized xylan. For the steam extraction, the ratio of peels to steam, i.e., the solid to liquid ratio was kept at 1:10. The xylan from the peels after getting extracted into the alkali gives alkali-solubilized xylan which was centrifuged at 5,000 rpm for 20 minutes. The solution was then filtered first by zero filter paper followed by Whatman® filter paper 40. Glacial acetic acid (20mL) at pH 5.0 was added to the filtered solution to maintain the acidified condition of the supernatant. On addition of 3 volumes (200 mL of xylan solution added with 600 inL) of ice-cold 70 % ethanol xylan was extracted. The solution was centrifuged at 8,000 rpm for 10 minutes at room temperature to precipitate xylan. The precipitated xylan was then dried in hot air oven at 60 °C till a persistent weight was reached, which was 1.3g. The dried pellets were assessed and changed to powder form and stored at room temperature for further analyses.

[0071] The obtained xylan was characterized by using HPLC. The system used was Waters Analytical System with Waters 515 series HPLC pump and Waters 2996 series Photodiode Array Detection systems. The buffer used was acetonitrile and water in the ratio of 63:37. The column used was C-18 ACE Column having dimensions of 250mm x 4.6mm with silica particle size of 3.5pm and pore size of 300A. The run was performed for 20 minutes and the peaks provided in Figure 1 showed the presence of xylan which was compared with chromatogram of standard xylan (refer Figure 2). The peak ranges (wavelength 200-250 nm range) were observed from 6 to 12 minutes of the run, with 204 sample injection, and flow rate of lmL per minute.

[0072] The second key step involved the hydrolysis of the obtained xylan from powdered orange peels to give xylooligosaccharides. The extracted xylan pellets were subjected to chemical hydrolysis using the commercially available Tartaric Acid. The pH was set at 5.5, temperature at 90 °C and chemical concentration of tartaric acid was kept 1M (0.15w/v). After adding about 5 mL of the tartaric acid, the mixture was kept for an incubation period of 2 hours in a shaking incubator at high temperature which was kept constant at 90°C and XOS was extracted at regular intervals by placing the extracted mixture in a hot water bath to arrest the reaction. The extracted XOS was filtered with the help of a 0.45 filter paper. Confirmation and analysis of obtained XOS was done by FIPLC. 5g of orange peel powder gave 1.3 g of Xylan and that 1.3g of Xylan gave 0.93 g of XOS. Total yield % of XOS was 28% and the purity was equivalent to 90%.

[0073] In order to determine the composition and types of XOS oligosaccharides obtained, the mixture of XOS was chromatographed on a HT'LC system (Agilent, USA) equipped with refractive index detector. The XOS were eluted with a mobile phase comprising acetonitrile and water in the proportion of 63:37 at a flow rate of 0.5 mIhnin in ZORBAX carbohydrate column (Agilent, USA). The column temperature was fixed at 25° C. The 20[11 filtered sample was injected through a manual injector. The complete analysis was achieved in 30 minutes. The XOS in the enzymatic hydrolyzate were quantified using average peak areas compared with mixture of standards XOS (X1-X5= (xylose, xylobiose, xylotriose, xylotetrose and xylopentose). The retention time of the major peak was observed at 3 minutes followed by the sub-products of XOS. The HPLC chromatograph of the obtained XOS is provided in Figure 3. The range was seen till 0.185 AU with wavelength 254 EXAMPLE 2: OPTIMIZATION OF PROCESS [0074] Parameters, namely substrate concentration, pH, concentration of tartaric acid, alkali concentration, incubation period and temperature were identified for maximum XOS production in present investigation.

2.1 Effect of amount of substrate on XOS production 100751 The effect of varying substrate, from 1 gm to 5 gm dried orange peels powder, was evaluated on maximum XOS production by using I M Tartaric Acid reaction on xylan pellets. All other conditions were kept same as in Example I. The results obtained are provided in Table No. 1 below: Table No. 1: Effect of amount of substrate on XOS production S. Substrate Weight Obtained Xyl an (gm) Obtained XOS No. (gm) (gm) 1 1 0.210 ± .002 0.07304 +.009 2 2.72 + .003 0.30154+ .004 3 5 1.335 + .001 0.93.009 [0076] The maximum XOS production of 0.93±.009 gm was observed for 5 gm of dried orange peels. The high production was noted due to high rate of hydrolysis. The substrate concentration of 1 gm showed lowest XOS production of 0.07304±.009gm.

2.2 Effect of concentration of tartaric acid on XOS production [0077] The concentration of tartaric acid was varied (0.8M, 1M and 1.2M) to evaluate its effect on production of XOS (X1-X5-xylobiose-xylopentose) from the xylan pellets. For these studies, xylan obtained by Example 1 was extracted with varying concentrations of tartaric acid at pH 5.5, temperature of 901) C, and incubation period of 2h. Results are provided in Table No. 2 below.

Table No. 2: Effect of concentration of tartaric acid on XOS production S. No, Substrate (gm) Tartaric acid (Molar) Obtained Xylan Obtained XOS (gm) (gm) 1 5.0 0.8 0.531 0.387 2 5.0 1.0 1.335 0.930 3 5.0 1.2 0.425 0.296 100781 As observed, the amount of XOS increased with increasing concentration of tartaric acid, which is due to higher activity with increase in concentration. After a point the increase in concentration decreases the amount of XOS. This may be due to negative feedback inhibition in chemicals with continuous increase in concentration.

2.3 Effect of concentration of basic solution on XOS production [0079] 5 gm of orange peels powder was chosen to extract xylan using 2%, 4%, 8% and 12% of sodium hydroxide solution. These solutions were prepared in 250 mL by dissolving 5 gm, 10 gm, 20 gm, 30 gm and 37.5 gm respectively of sodium hydroxide in distilled water. Rest of the procedure was followed as provided in Example I. The results for the amount of XOS produced are provided in Table No. 3.

Table No. 3: Effect of concentration of basic solution on XOS production S. Substrate (gm) Basic solution Xylan (gm) XOS (gm) No. concentration 1 5.0 2% (5 gm in 0.325 0 0619 250 ml) 2 5.0 4% (10 gm in 0.250 0 0573 250 nil) 3 5.0 8% (20 gm in 0.500 0 2132 250 nil) 4 5.0 12% (30 gm in 0.976 0.6304 250 ml) 5.0 15% (37.5 gm in 0.601 0 0731 250 ml) [0080] The maximum XOS production of 0.6304 gm was observed at a concentration of 12% (30 gm in 250 ml) of the basic solution. Higher production of xylan was mostly due to breakage of maximum ester bonds and dissolution of lignin-based impurities with higher concentration of NaOH. The lowest XOS production of.057 gm was observed at an alkali concentration of 4%.

2.4 Effect of pH and temperature on XOS production 2.4.a) The effect of pit: [0081] The effect of different pH, 4.5, 5.5 and 6.5 during hydrolysis step, on XOS production was observed for an incubation period of 6h. All other experimental parameters were kept the same as in Example No. 1. Results are presented in Table No. 4 and it can be noted that pH 5.5 was found to be the most significant for XOS production Table No. 4: Effect of pH on XOS production S. No. Substrate (gm) pH Xylan (gm) XOS (gm) 1 5.0 4.5 0.852 0.6839 gm 2 5.0 5.5 1.335 0.9303 gm 3 5.0 6.5 0.974 0.7221 gm 2.4. b) The effect of temperature: [0082] For the comparison, the temperature was set at 85°C, 90°C and 95°C, and for each test 6 h incubation period, pH 5.5, 12% alkaline concentration and 1M Tartaric Acid were maintained. The results are provided in Table No. 5, it can be seen that highest yield was obtained for 90°C.

Table No. 5: Effect of temperature on XOS production S. No. Substrate (gm) Temperature (°C) Xylan (gm) XOS (gm) 1 5.0 85 0.842 0.6883 gm 2 5.0 90 1.335 0.9103 gm 3 5.0 95 0.954 0.7912 gm 2.5 Effect of incubation period on XOS production [0083] The effect of incubation period was based on fixed amount of orange peels powder (5 gm) treated with 12 % sodium hydroxide solution to produce xylan pellets.

Further the pellets were treated with 11V1 Tartaric Acid at pH 5.5 and temperature 90° C. The incubation periods were set as 60 min, 80 min, 90 min, 100 min and 120 min. All other processes and parameters were set as in Example No. 1. The results are provided in Table No. 6 and suggest that 2 hour incubation period gave maximum yield of XOS.

Table No. 6: Effect of incubation period on XOS production S. No. Substrate (gm) Incubation period Xylan (gm) XOS (gm) (minutes) 1 5.0 60 0.612 0.28 2 5.0 80 0.901 0.41 3 5.0 90 1.335 0.93 4 5.0 100 0.836 0.39 5.0 120 0.728 0.32 [0084] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein merely for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention and should not be construed so as to limit the scope of the invention or the appended claims in any way.

ADVANTAGES OF THE PRESENT INVENTION

100851 The present disclosure provides a process of production of xylooligosaccharides from peels of orange fruit that is economically feasible and ecologically beneficial.

100861 The present disclosure provides a process of production of xylooligosaccharides that gives higher yields of XOS, better pH control and requires lesser time than conventional processes.

Claims (12)

1. CLAIMSWe Claim: I. A process for production of xylooligosaccharides from peels of orange fruit, the process comprising the steps of: (a) extracting and precipitating xylan from the peels; and (b) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.
2. 2. The process as claimed in claim I, wherein the process comprises the steps of: (a) subjecting the peels to alkali lysis by a basic solution comprising sodium hydroxide; (b) extracting xylan by treating with steam to give alkali-solubilized xylan; (c) precipitating the xylan by adding an organic solvent; and (d) hydrolyzing the precipitated xylan using tartaric acid and incubating to give the xylooligosaccharides.
3. 3. The process as claimed in claim 1, wherein the peels of orange fruit are dried peels of orange fruit.
4. 4. The process as claimed in claim 2, wherein the basic solution is a sodium hydroxide solution in water with concentration of sodium hydroxide in the range of 6% to 218% by weight.
5. 5. The process as claimed in claim 2, wherein the step (b) involves treating the peels with steam at a temperature in the range of 110°C to 130°C and pressure in the range of 12psi to 18psi.
6. 6. The process as claimed in claim 2, wherein the peels to steam ratio is in the range of 0.5:5 to 1.5:15.
7. 7. The process as claimed in claim 2, wherein the organic solvent is selected from ethanol, or anhydrous methanol.
8. 8. The process as claimed in claim 2, wherein the organic solvent is added in an amount of 65% to 75% VA" with respect to the alkali-solubilized xylan.
9. 9. The process as claimed in claim 2, wherein the precipitated xylan is hydrolyzed at a pH range of 4.5 to 6.5 and a temperature range of 85 °C to 95 °C.
10. 10. The process as claimed in claim 2, wherein the incubation is performed for 2 hours to 20 hours.
11. 11. The process as claimed in claim 2, wherein the tartaric acid is a tartaric acid solution in water having a concentration in the range of 0.10 to 0.20 wk.
12. 12. The process as claimed in claim 1, wherein the xylooligosaccharides comprise xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and combinations thereof.