Utilization of Agricultural Waste for the Production of Xylooligosaccharides Using Response Surface Methodology and Their In Vitro Prebiotic Ecacy

Air pollution is a prominent problem recently faced in various parts of India due to the burning of stubbles 43 (coconut husk, corn cob, paddy stubbles, sugarcane bagasse, etc.) which are rich in a lignocellulosic component 44 that can be converted into a prebiotic known as Xylooliogsaccaride (XOS). They can be produced by 45 autohydrolysis, acid hydrolysis and enzymatic hydrolysis of xylan. In the present study, Xylan was extracted from 46 sugarcane bagasse using two alkalis (NaOH and KOH) and the yield was compared. Xylooligosaccharide 47 produced by enzymatic hydrolysis and their factors influencing the yield were optimized using Response Surface 48 Methodology. Xylan and Xylooligosaccharide was characterized by FTIR, NMR, XRD, TGA and ESI-MS. 49 Xylooligosaccharides was investigated for their prebiotic potential by in vitro study. The maximum (Relative 50 yield of 86%) yield of xylan was observed in 20% of NaOH. Xylan peaks at 3762cm -1 , 3347 cm -1 , 2917cm -1 51 represents the OH and CH stretching of xylan. The main signals at 4.26 (H-1), 3.19 (H-2), 3.59 (H-3), 3.63 (H- 52 4) and 3.98 (H-5) ppm determines the existence of xylan. The higher amount of XOS is pH 4.75, temperature 53 45°C, enzyme 4U/ml and for time of 16h. The spectrum of 5.0-5.40ppm and 4.30-4.60ppm represents the α 54 anomeric and β anomeric protons in XOS. They are resistant digested and the reaching percentage to the intestine 55 is 95% unhydrolyzed. The maximum prebiotic index was noted in L.plantarum (1.92) and L.fermentum (1.61). The highest prebiotic index and score was observed in L.plantarum (1.9) and L.fermentum (17). The maximum bacteriocin production of Enterococcus faecium against E.fecalis (13mm) and Streptococcus pyogenes (11mm). Therefore, utilization of agricultural residues for a value-added product not only shows a great impact on environmental issues but also could double the farmer’s income The optimization of XOS production was carried out by Response Surface Model (RSM) -Central Composite Design (CCD). The extracted sugarcane bagasse xylan was subjected to enzymatic hydrolysis using commercial xylanase enzyme extracted from T. viridae (Sigma, Bangalore). The experiments were carried out in triplicates with 29 runs by varying pH (4 to 5.5), temperature (40°C to 55°C), enzyme (4U to 20U) and incubation time (8 154 to 24 hours). Two percent of substrates were added to 10ml of sodium citrate buffer and 1ml of enzyme and 155 incubated in shaking waterbath at x150g for appropriate temperature and time. The aliquots were drawn from the 156 enzymatic hydrolysis and the mixture was heated to 100°C to inactivate the enzyme and the hydrolysate was 157 filtered with Whatman No1 filter paper. Three volumes of ice-cold ethanol were added to the filtrate to precipitate the traces of unhydrolyzed xylan (Samanta et al. 2014) and it was vacuum filtered using G3 sintered crucible filter 159 and the filtrate was analyzed for its non-reducing sugar (XOS) by Lane and Eyon chemical method The optimization study was designed using by Design expert software version 11. to liquid

That could be a promising initiator for the production of biofuel and bioenergy along with value-added 84 biomolecule (Prebiotic). Prebiotics, as the name, implies "Prebefore; bio-life" it is evolved before life evolved.

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But it came to light in 1995 with the definition given by Gibson and Roberfroid (1995) as "Nondigestible food 86 ingredients that beneficially affect the host by selectively stimulating the growth and or activity of one of the 87 limited number of bacteria in the colon" (Samanta et al. 2014).

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XOS is the synthesized prebiotics from various agricultural residues viz., corncob sugarcane bagasse, stalks of 89 cotton, tobacco and shells of pistachios, walnut and groundnut, etc., they are hydrolyzed products of xylan a 90 polysaccharide which is synthesized by various methods. These agricultural wastes are dumped in the field or 91 burned after harvesting (Agrupis and Markawa 1999). By utilizing these wastes will protect the environment from 92 pollution as well as increases the economic status of farmers and generates employment (Akpinar et al. 2009).

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From a nutritional point of view, XOS is known as nondigestable (ND) as they are not degraded in the stomach 94 and reaches the lower bowel, to be utilized by the microbiota residing there (Okazaki et al. 1990;Roberfroid 1999; 95 Collins and Gibson 1999;Vazquez et al.2000). They have also helped in reducing cholesterol and maintains gut 96 health. They are moderately sweet, inhibit the retrogradation of starch, and improves the sensory and nutritional 97 properties of food and are stable for a wide range of pH and temperature (Vorgen 1998). XOS is noncarcinogenic 98 and regulates insulin secretion from the pancreas, besides increasing mineral absorption from the large intestine.

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The Prebiotic potential is attributed by the utilization and nourishment of probiotic via fermentation thrive to 107 maintain the gut microflora diversity by eliminating the harmful pathogen (Gibson et al. 2004). The prebiotic 108 index and score can be calculated by comparing the stimulated growth by prebiotic on beneficial microbial 109 diversity and other intestinal pathogens (Huebner et al. 2007).
Bacteriocins can be defined as extracellularly released peptides or protein molecules which have low molecular 111 weight with a bacteriostatic mode of action of closely related species. Bacteriocins are classified into three classes 112 based on their structure and function (Klaenhammer 1993). The action or effectiveness of the probiotics depends 113 mainly on the type of strain and the amount consumed. Prevention of growth of the pathogenic organisms by 114 occupying all the adhesion sites as pathogenic organisms also need to adhere and attach to the epithelial cells of 115 the intestine. The action might also be due to the synthesis of the acids and generating the acidic environment and 116 prevents the growth of pathogens. The immunological benefits conferred by probiotics are by prevention of 117 allergies due to activation of macrophages and thereby increasing antigen presentation and increases secretion of 118 immunoglobulin A.

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In this research, the xylan was extracted from the sugarcane bagasse by alkali extraction and xylooligosaccharides 120 has been produced by enzymatic hydrolysis and studied for its in vitro prebiotic potential and their bacteriocin

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The dried sample was powdered with a mechanical blender and stored in an airtight container until further usage.         Table 2). The effect of the model was analyzed by regression coefficient, Analysis of variance (ANOVA) and 272 response surface plots (Fig 2).

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The coefficient of the factors was determined by the R 2 value, this value has to be above 0.80 to good fit a model

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In the TGA profile of XOS, the gradient temperature increase elucidates the difference in sample weight due to 348 the presence of volatile groups. For XOS, a weight loss of 17.11% was observed between 100°C -300°C due to 349 the evaporation of water vapors in the sample. After this, the sample may undergo a pyrolysis process where the 350 sample is partially decomposed into ash and the sample loses its weight of 17.90% between 300°C -400°C and 351 the sample would have completely decomposed (14.04%) between 500°C to 600° C into ash by the combustion 352 process (Fig 7). Differential Thermogravimetric analysis (DTG) represents the maximum degradation (Td) at a 353 temperature that determines the stability of the sample. The maximum degradation of the sample was observed at 354 147°C.               Prebiotic e cacy of XOS produced from sugarcane bagasse and Inulin (Commercial prebiotic) Figure 10 Bacteriocin activity from probiotic bacteria grown in prebiotic substituted medium against human pathogens.