Optimization of Xanthan Gum Fermentation Utilizing Food Waste by GRD Journals

GRD Journals- Global Research and Development Journal for Engineering | Volume 6 | Issue 5 | April 2021 ISSN- 2455-5703

Optimization of Xanthan Gum Fermentation Utilizing Food Waste Sakshi Shiram Student Department of Food Biotechnology Ajeenkya DY Patil University, Pune Pranav Venugopal Student Department of Food Biotechnology Ajeenkya DY Patil University, Pune

Amogh Tungare Student Department of Food Biotechnology Ajeenkya DY Patil University, Pune

Neha Gondekar Student Department of Food Biotechnology Ajeenkya DY Patil University, Pune

Biswa Prasun Chatterji Associate Professor Department of Food Biotechnology Ajeenkya DY Patil University, Pune

Abstract Xanthan Gum, a product used widely in the food industry, possesses high commercial value. The problem with Industrial Xanthan fermentation is its prohibitive cost of using pure sucrose as substrate. Food waste such as Carrot and Pumpkin peels contains glucose, sucrose, vitamins and minerals that are utilized by Xanthomonas campestris at a favourable pH. Effect of pH, whey and multivitamins have been studied on the yield. Xanthan was purified using chilled Isopropyl alcohol following a standard protocol. The yields of xanthan gum with Carrot, Pumpkin and MGYP at pH 6.0 were 40.88g/L, 31.4g/L and 14.06, respectively. Altering the pH in the experiment from 6.0 to 6.8 immensely influenced the biopolymer’s yield. Supplementing media with whey and multivitamins rendered positive results. Tackling the problem of food waste in India is difficult and we have recycled food waste to make an economical medium for a high-value food additive i.e., Xanthan gum. Keywords- Xanthan Gum, Xanthomonas Campestris, Exopolysaccharide, Beverage Waste, Low-Cost Production, Rheological Properties

I. OBJECTIVE In the present study, the xanthan gum is produced by emphasizing on food-waste, which holds a great potential to plummet the waste and provides benefits financially as well as to the environment. Production of xanthan gum by food waste entails certain factors: 1) high carbon source such as glucose and sucrose 2) good nitrogen source 3) adequate temperatures and pH.

II. INTRODUCTION Xanthan gum was unearthed in the early 1960s by a team of scientists lead by Allene Rosalind Jeanes at the United States Department of Agriculture. In addition, it is amongst a few of the microbial polysaccharide to be approved by the Food and Drug Administration (FDA) in 1968 as a stabilizer and thickening agent in the food industry. This biopolymer synthesized chemically has a natural origin from the microbes. However, an array of factors including increased market price and demand, higher cost of production using traditional substrates may be a limiting factor for raw material utilized for fermentation. For robust evidence, it has been reported that the price of xanthan gum (US$ 4000-5000/ton) has been gradually increasing because of higher production cost of glucose and sucrose (US$ 400–600/ton) [1]. Hence, many researchers and scientists have set forth their undivided attention to elicit a cost-effective alternative for xanthan gum substrate. Moreover, the researchers do believe that the use of natural polymers using industrial biotechnology is crucial in terms of production volumes. Owing to investigate an environmentally friendly alternative, many researchers used substrates which were mostly agro food-waste such as cheese whey, malt grains, apple pomace, grape juice, sugarcane molasses, sugar beet molasses, passion fruit peels [2] . Therefore, this study includes a similar attribute using fruit peels to economize the fermentation process. This approach must be inexpensive, causing significantly low harm to the environment along with reinforcing the notion of sustainable manufacturing, Furthermore, this strategy of optimizing xanthan gum using sustainable outlook will lead to tremendous safety standards, thereby escalating compliance standards [3].

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

A. Backbone of Xanthan Xanthan’s main chain is comprised of D-glucose units joined by the b-l position of one unit with the fourth position of the next unit, a linear backbone analogous to the cellulose chemical structure. The basic structure of xanthan comprises of monomer units of pentasaccharide (Fig 1). The currently recognised Xanthan structure consists of units of beta-D-glucopyranose. At the C-3 position, Trisaccharide side-chains are bound to alternative sugar residues on the main chain. Two mannose residues and glucuronic acid form the side-chain. The terminal residue of beta-D-mannopyranose is bound to the residue of beta-D- glucuronic acid, which in tum is bound to the non-terminal residue of alpha-D- mannopyranose. As an acetic acid ester, the 6'-0H group of the nonterminal D- mannopyranose residue is present. On the D-mannopyranosyl end groups of side- chains, pyruvate acetal groups are found. The impacts of different glycosidic (or other) links in any polysaccharide's backbone is a significant aspect in altering the conformation of the polysaccharide chain and its properties. Rheological (flow) properties exhibited by xanthan varies with difference in amount of pyruvate (1 to 6 percent). The standards for xanthan composition are as follows: 37% glucose, 43.4% mannose, 19.5% glucuronic acid along with 4.5% acetate and 4.4% pyruvate [4].

Fig. 1: Chemical structure of Xanthan gum [5]

B. Production of Xanthan Gum Biosynthesis of xanthan involves a complex mechanism incorporating a multi- enzyme process. In the biosynthesis of xanthan, the initial stage is the absorption of carbohydrates that may happen by active transport or facilitated diffusion. This is accompanied by substrate phosphorylation with a hexokinase enzyme which uses adenosine 5'-triphosphate[6]. Biosynthesis involves converting the phosphate-phorylated substrate to the different sugar nucleotides needed by enzymes such as UDP-Glucopyrophosphorylase for the integration of the polysaccharide-repeating cell. For the synthesis of xanthan with an acceptable repeating unit, UDPglucose, GDP-man-nose, and UDP-glucuronic acid are essential. Cabbage supplies the carbohydrate substrates, proteins, and minerals for cell proliferation in the biofilm formation of xanthan on the cabbage plant by Xanthomonas campestris. Carbon sources, nitrogen sources, trace minerals and pH conditions are given under laboratory conditions or industrial fermentation in a fashion that simulates natural conditions [7]. C. Xanthan’s Commercial Production Xanthan gum development on an industrial level is undertaken by utilizing affordable substrates and nutrients. Carbohydrate sources, such as sucrose; whey has been extensively used in the processing medium. Whey also contains nitrogen as well as other growth factors that are sufficient. A high carbon to nitrogen ratio demands an effective conversion of the carbon source to the desired polysaccharide output. Inorganic sources of nitrogen such as ammonium or nitrate salts are sufficient, and a wide range of complex sources of nitrogen such as yeast extract, soy-meal peptone and soybean whey are also useful to produce xanthan. The presence of phosphorus is needed by X. campestris, which is typically applied in the form of a phosphate buffer. Cultivation of the batch growth takes two days, stirred-tank reactor is most frequently used for process of batch fermentation. Normally, processing is carried out in a temperature range of 25°C to 35°C [7]. The transition of oxygen volume is determined both by the speed of agitation and airflow rate. Polysaccharide accumulation commences during the initial growth development and continues after growth. During fermentation, the pH decreases due to the production of organic acids. When the pH drops below 5.0, the production of xanthan declines significantly. Therefore, it is important for the fermentation medium to be monitored at an optimum pH of 7.0 using a buffer or base addition during the phase [7]. To distribute the added air uniformly within the medium, ideal agitation is

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

indispensable. Medium agitation is useful for increasing the rate of nutrient transfer across the cell membrane, which stimulates the growth rate of the microorganism. Following the decades of commencing several carbon sources to xanthan fermentation industry now these feedstocks could be labelled into five categories: unmixed simple sugars; Agro-industrial by-products incorporated directly consumable ingredients such as whey and molasses; agro-industrial wastes involving previous hydrolytic processes like acid hydrolysates of fruit pulps; pulps in solid state fermentation including apple pomace, and potato peels; and lower grade juices from fruits such as date [8]. D. Characteristics of Xanthan Gum In aqueous solutions, xanthan manifests pseudoplasticity (shear-reversible property). Based on its helical shape, this characteristic solution property of the xanthan gum can be described. As the shear rate increases, the viscosity of xanthan in solution declines. As the temperature is elevated, most aqueous solutions of other polysaccharides show a sharp decline in viscosity [9]. In a native, ordered conformation, Xanthan occurs in solution at a moderate temperature. As the temperature of an aqueous solution of xanthan increases, the viscosity unexpectedly tends to increase, signaling a difference in the conformation. A xanthan solution will retain its ordered form and viscosity up to 100 ° C in the presence of salts such as sodium or potassium chloride. Xanthan gum powder is soluble in hot and icy water, but insoluble in most organic solvents. In contrast with other gum solutions, Xanthan gum solution demonstrate a high degree of viscosity in comparison to other alternative polysaccharide solutions this property makes it more competitive as stabilizer and thickener. The solution of Xanthan gum is strongly pseudoplastic, lacks thixotropy [10]. Xanthan gum pseudoplasticity enhances sensory qualities in finished goods, eases production and ensures proper pourability [11]. Xanthan Gum solution can tolerate pH variations, i.e., under both acidic and alkaline environments, they are effective. Furthermore, it has thermal stability that makes it preferable to most other polysaccharides that are water soluble. Xanthan gum is flavourless and therefore has no influence on the flavour of other ingredients in food. E. Application of Xanthan Gum Xanthan gum is extensively used in drinks and other liquid beverages. In case of fruit pulp beverage, the gum helps to maintain a good suspension with ends up giving a better appearance to the product. In dairy industry, a cocktail of xanthan gum along with guar gum and/or locust bean gum is highly preferred as a stabilizer for cold beverages such as ice creams, sorbets, frozen yogurt, puddings etc. It enhances the firmness of the product as well as providing good syneresis control. In sauce and dressings, thanking to its rheological properties, xanthan gum provides stability to the emulsion. It also gives an exceptional mouth feel to the salad dressing along with its pseudoplasticity which helps the dressing to stay on the salad. However, in bakery industry, xanthan gum is considered as an egg substitute without compromising its appearance, taste, and texture. In paint industry. At a low shear rate, xanthan is highly viscous which allows the paint to stick to the brush without dripping. Pharmaceutical sector makes a wide usage of xanthan gum, where it acts as a carrier that rescues the drug to the desired location while not wasting the natural ingredients. It has broad scope of utility in standard drug delivery also because of its biodegradability, inexpensive and ease of accessibility. In Agriculture, xanthan gum enhances the flow-ability of these chemicals by equally suspending the solid particles. It has also been reported to be a sole reason for the chemical grasp and perpetuity [12]. F. Literature Review Table 1: Previous research work of various authors using different substrate Time Author Name Substrate Temperature(°C) pH Yield(g/L) (Hrs.) Dr. Biswa Prasun Mango 24 30 6 20.84 Chatterji [13] Sapodilla 24 24 7.4 11.83 Tahereh Grape Juice 72 28 6.5-7 14.35 Ghashghaei [14] Sugar beet Murat Ozdal [15] 60 30 7 20.5 molasses N. Pineapple 72 30 7 8.48 Amenaghawon [16] Peels ShrimpShell Larissa Alves deSousa Costa [17] 120 28 7 4.64 extract Zichao Wang [18] Glycerol 60 28-30 7 33.9 Palm Riadh ben salah [19] date 30 7 43.35 Juice Cheesewhey Niknezhad [20] 48 16.4 lactose Sugar beet Kalogiannis S [21] 24 7 53 Molasses Passion 28 48 7 6.7±1.9 Fruit peel Fabiana P.dos Santosa [22] Green 24 7 5.5±3.8

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

Janice Druzian [23] Ali Moshsin [1]

coconut shell Corn cobs Corn Straw Apple Juice Residue Orange Peels

24 48 120

7 7

2.7±0.1 1.0±0.04

30.4

30.19

Larissa Alves de Sousa Costa et.al exhibited their work with an aim to synthesis xanthan gum using three native strains of the bacteria Xanthomonas campestris. They used the aqueous shrimp shell extract as the sole source of nitrogen and carbon for the gum yield and its apparent viscosity. The shrimp cell extract prepared contained enough carbohydrate, protein, lipids, ash, moisture. Furthermore, the media containing different concentrations of Shrimp shell extract filtrate were fortified with 0.01% urea and 0.1% phosphate. After apt fermentation procedure, the media containing 10% (w/v) seafood waste extract along with Xanthomonas strain 1182 produced the highest yield i.e., 4.64g/L xanthan reflecting the highest viscosity amongst all. Hence, the yield obtained was superior to the yield obtained from the standard control media containing sucrose as a carbon source making shrimp shell extract a low-cost alternative in making a valuable end product [24]. A team of researchers used Xanthomonas campestris XG30-18 mutant strain, MW-42-NTG-6 showing promising results to produce high-viscosity as well as acid-resistant xanthan gum. Corn starch + sucrose and soybean protein isolate were used as carbon and nitrogen sources, respectively. Under the optimal fermentation conditions (pH 7.0, 28℃ temperature, 240 r/min rotation speed), fermentation took place manifesting a yield of 3.92% [25].

III. FACTORS AFFECTING OPTIMIZATION OF XANTHAN GUM Optimal factors are required in the production of a high value-added product, Xanthan gum. Therefore, the superlative conditions including carbon source in form of beverage waste, fortification using nitrogen source, an ideal temperature, an effective pH along with the type of fermentation process play a significant role in the growth of a bacterial culture of Xanthomonas campestris as well as Xanthan gum. The following elaborates on the individual roles of the given critical conditions and the speculation of various authors given their results. A. Carbon Source Industries have been searching for a cost-effective alternative for the carbon source in the optimization of xanthan gum as it has a direct impact on the synthesis of UDP- glucose, UDP- glucuronate, and GDP- mannose that primarily initiates the building blocks of the pentasaccharide repeating unit [2]. Hence, this study continues to experiment using fruit peels, an agriculture industrial waste, as a major source of carbon. The traditional means of carbon source are reported to be sucrose and glucose being the dominant ones, whereas pyruvate and fructose set forth low efficiency in xanthan synthesis. According to De Vuyst and Vermeire, the initial concentration of 4-5% sucrose concluded flourishing results of xanthan synthesis. Moreover, many studies concluded that a higher C/N ratio has an astonishing effect on xanthan production [2]. B. Nitrogen Source Nitrogen being an imperative nutrient, can be supplied in the form of organic as well as an inorganic compound which directly influences the growth of Xanthomonas [26]. Several authors studied the outcome of Xanthan synthesis when supplied with different nitrogen and amino acid sources [27]. Firstly, F Letisse et al. monitored that ammonium salts gave an exceptional yield, especially the nitrates which were more suitable for the growth experiment [28]. Secondly, over the differential studies lead by Palaniraj et al., 2011, Kumara Swamy M et al., 2012, and Gomashe et al., 2013, a coherent conclusion rose that yeast extract is an ideal source of nitrogen in the production media which gave thriving xanthan yield [13]. C. Effect of pH The pH abundantly affects and simultaneously controls the growth action and xanthan synthesis [27]. Therefore, through research, it was predicted that a neutral pH, 7.0-7.5, works best for the growth of the Xanthomonas bacterial culture. However, during the fermentation process, the pH drops significantly to 5.0 due to the biopolymer’s acid groups, widely affecting the xanthan gum production. To conclude, various authors contemplated to control the pH with the help of alkalizes such as NaOH or KOH and keeping a stable pH to 7.0-8.0 for higher yield of the biopolymer [29]. D. Effect of Temperature Assorted studies evaluated the optimum temperature range being directly proportional to the xanthan yield and its rheological peculiarity. Moreover, many studies agreed to a temperature of 28°C giving the best xanthan yield. In addition to it, however, as the temperature was spiked to 32°C, the xanthan gum obtained had a low average molecular weight, resulting in a low viscous aqueous solution [29]. According to one study, a higher temperature also triggered thermal degradation of the bacterial exopolysaccharide. Nevertheless, this thermal degradation continued with a long time of fermentation treatment when the temperature reached around 60°C [30]. Hence, at a temperature with proximity to 28°C, the biopolymer obtained had distinct rheological properties i.e., high molecular weight and high viscosity. All rights reserved by www.grdjournals.com

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

IV. MATERIALS AND METHODOLOGY A. Materials Different glasswares were used throughout the entire experiment were from Borosil®. The chemicals and media used in the experiment were of apt analytical standards and were carefully used without compromising any of the safety standards. Additionally, MuscleBlaze® fuel one whey protein powder and Mega rich® multivitamin capsules were used as a supplement to add novelty to the experiments. B. Microorganisms A purchase was made from the National Chemical Laboratory (NCL), Pune of a pure bacterial culture of Xanthomonas campestris 260 which was supported by an orderly planned approach. NCL, Pune also provided a couple of sets of instructions along with the MYGP media composition. The maintenance made up of sub-culturing the original master culture of Xanthomonas on a solidified MGYP agar plate and liquified MGYP broth. (Fig.2.)

Fig. 2: MGYP liquid broth (on the left) and MGYP agar plate (on the right)

C. Fermentation Process Two culture media were prepared from autoclaved MGYP extract (Malt, Glucose, Yeast, Peptone). One of the MGYP extracts was transferred to a conical flask (MGYP broth) and Agar was added to another MGYP medium and subsequently transferred to a petri dish (MGYP agar). Streaking of the pure culture of Xanthomonas campestris 260 was done on the petri dish and was subjected to incubation. To start the experiment, Borosil glassware was used. All the ingredients of MGYP broth were weighed according to the NCL instructions to prepare 200ml broth. Initially, the pH was adjusted in the range of 6.4-6.8 and later made changes according to the variations. A sample from the streaked petri dish was taken and added to the MYGP broth and placed in the incubator at 30°C at 200rpm for 24 hours. D. Raw Material for Xanthan Gum Preparation Carrot and Pumpkin’s waste peels were selected as raw materials. The nutritional content has been provided below. 1) Carrot (Daucus Carota Subsp. Sativus) Carrots are nutritionally dense root vegetables with 9% carbohydrate-containing prominent levels of dietary fiber (2.8%) and free sugars which mostly include sucrose, glucose, and fructose. They impart their characteristic bright orange colour primarily from β-carotene which is further metabolized as Vitamin A. They also constitute Vitamin K, Vitamin B6, and other useful minerals. 2) Pumpkin (Cucurbita Pepo) Pumpkins are packed with good levels of provitamin A and a moderate amount of vitamin C as well. It also contains 6.5% carbohydrate amongst which 2.76g is the sugar content. Along with vitamins, pumpkin composes minerals such as calcium, magnesium, potassium, etc. The sugar content present in each of the vegetables are demonstrated in Table 2. 3) Preparation of Extracts Leftover peels of carrot and pumpkin were collected from a local vegetable vendor. They were chopped into small equal pieces and weighed up to 100 grams. The weighed pieces were placed in a mixer grinder and about 100ml of distilled water was added to the pieces to soften them. The waste was ground in a mixer grinder and filtered under muslin cloth to remove unwanted waste particulate present in it. This refined mixture of vegetables was used in the entire experiment. Both the extracts were transferred

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

in a flask and the flask was labelled by the initials of their peel waste. Further, the extracts were sterilized using autoclave to eliminate all the unwanted contamination. Fruit

Glucose

Sucrose

Carrot

24.6%

56.9%

Pumpkin

0.823% to 4.49%

0.090% to 5.19%

Table 2: Amount of sugar present in Carrot and Pumpkin Total sugar (per Fructose Galactose 100g) 4.7g 18.5% 0% https://ucanr.edu/d atastoreFiles/608- 463.pdf 1.05% 5g http://www.spkx.n et.cn/EN/abstract/a bstract10955.shtml #:~:text=Results% to 0% 3A%20The%20lin ear%20ranges%20 of,2.60%20%CE% BCg%2FL%2C%2 3.80% 0respectively.

E. Preparation of Xanthan Gum A 5ml bacterial solution was taken from the MGYP broth which was added to the conical flask comprising of carrot extract and in the case of MGYP and Pumpkin, a streaking loop was beneficial to collect the colonies from the bacterial plate. After inoculating the vegetable extracts with the host bacterium, they were placed in an incubator. Incubator provided favourable temperature (30°C) and aeration (200rpm) to Xanthomonas which was necessary for getting a noteworthy quantity of Xanthan gum. F. Recovery of Xanthan Gum The conical flask containing the inoculated vegetable peel extract was allowed to ferment for 24 hours. After a 24-hour duration, about 100ml of isopropanol was added to the media. The solution prepared was stored further at 4°C for an hour. About 1 hour later, the xanthan gum was observed precipitated in the supernatant portion of the flasks. With the help of a muslin cloth, all the gum was separated from the broth and placed in a petri dish. The dishes containing the xanthan gum obtained were kept in a hot air oven at 65°C for 15 minutes. The dried gum was then weighed and the records were acquired.

3. A 3. B Fig. 3: 3.A. Xanthan gum precipitated at the top of the fermented MGYP liquid broth; 3.B. Dried Xanthan gum

V. RESULTS AND DISCUSSION A. Yield of Xanthan gum from MGYP, Carrot and Pumpkin Xanthan gum obtained post drying from MGYP, Carrot and Pumpkin were weighed and recorded. MGYP, Carrot, and Pumpkin’s broth were maintained at a pH of 6.0. The average yield of xanthan gum collected from Carrot, Pumpkin and MGYP were 40.88g/L, 31.4g/L, and 15.42g/L, respectively. At pH 6.0, it can be deduced that Carrot showed significant yield of Xanthan gum to that of MGYP and Pumpkin.

Fig. 4: Xanthan gum yield from different substrates

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

Fig. 5: Acquired xanthan gum from MGYP, Carrot(C), and Pumpkin (P) media at pH6.0

B. Optimization of pH Conventional media utilized for Xanthomonas Campestris (MGYP) performs well at pH 6.4. Nevertheless, the study on the effect of pH was conducted to find out which pH would stimulate Xanthomonas campestris to produce significant xanthan gum. The control pH was set at 6.0 and the experimental pH was set at 6.8. From figure.4., it can be seen that the yield of biopolymer obtained from MGYP, Pumpkin and Carrot were 15.42g/L, 31.4g/L and 40.88g/L, respectively. Carrot showed maximum productivity of the gum at pH 6.0. However, changing pH from 6.0 to 6.8 drastically influenced the results as yield of the biopolymer obtained at pH 6.8 from MGYP and Pumpkin was reduced to half of their yield observed at pH 6.0 i.e., 6g/L and 15.86g/L, respectively. Besides, yield obtained from Carrot was affected the most as it showed no yield at pH 6.0. It is reported that optimum pH for Xanthomonas campestris to produce xanthan gum is pH 6 but for biomass it is 6.5 [31], this explains fluctuation of xanthan gum obtained at pH 6.0 and 6.8.

Fig. 6: Effect of pH on Xanthan gum

C. Addition of Whey After a plethora of literature review, we comprehended that introduction of whey could profoundly affect the yield of Xanthan gum. We used “MuscleBlaze fuel one” whey protein powder. For every 50mL broth, 0.15g of protein powder was taken, giving 0.11g of protein. Whey was added to each of the three conical flasks containing carrot, pumpkin, and MGYP broth which were maintained at pH 6.0. In addition, all the flasks were kept in the incubator for 24 hours. Carrot, Pumpkin and MGYP broth at pH 6.0 when enriched with whey gave 48g/L, 58g/L and 24.62g/l of Xanthan gum, respectively. Whereas, broths without whey supplementation at the same pH gave 40.88g/L, 31.4g/L and 15.42g/L, respectively.

Fig. 8: Effect of Whey on different substrates at pH 6.0

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

Fig. 9: Xanthan gum procured from different liquid broths fortified with whey protein

D. Addition of Vitamin Multivitamins are essential as they act as cofactors in abundant metabolic reactions. A multivitamin capsule “Megarich®” was used to understand the effect of multivitamins on the productivity of Xanthan gum. The compositions of the tablet are enlisted in Table 3. Table 3: The compositions of MegaRich® multivitamin capsule Composition Quantity per capsule Elemental Calcium 75mg Elemental Copper 0.5mcg Elemental Manganese 0.5mg Ferrous Fumarate 30mg Folic acid 0.15mg Ginseng 42.5mg Iodine 0.1mg Magnesium sulphate 3mg Niacinamide 10mg Potassium 2mg Vitamin A 1000 IU Vitamin B1 1mg Vitamin B12 1mcg Vitamin B2 105mg Vitamin B6 1mg Vitamin C 50mg Vitamin D3 200 IU Vitamin E 5mg Zinc oxide 10mg

After autoclaving carrot, pumpkin, and MGYP media, they were allowed to cool down before inoculating with Xanthomonas culture. During inoculation, about half a capsule of MegaRich® multivitamin capsule content was added to each of the flasks. After a fermentation period of 24 hours, there was a substantial growth present in the respective media at pH 6.0. At pH 6, vitamins containing Pumpkin and MGYP media gave around 16.6g/L and 12g/L xanthan yield respectively as shown in Figure10. Whereas, carrot media comprising of vitamins did not respond well to the given fermentation condition accounting for 0g/L yield of the biopolymer. The possible explanation for the unsuccessful outcomes may be the drastic depletion in pH level which could have activated the antioxidant activity of vitamins (vitamin C and vitamin E) which inhibits the bacterial growth causing no biopolymer production.

Fig. 10: Effect of vitamin on the yield of Xanthan gum at pH 6.0

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

However, when pH was slightly increased to 6.8, Carrot media (fortified with vitamins) reported to produce an exceptional xanthan yield of 17.4g/L. On the other hand, Pumpkin media enriched with vitamins gave a slight rise to xanthan’s yield(20g/L) whereas MGYP media (with added vitamins) results diminished to about 9.82g/L yield of xanthan gum as represented in Figure 11.

Fig. 11: Effect of vitamin on the yield of Xanthan gum at pH 6.8

E. Formulation of Results in a Tabular Format Table 3.A: Illustrates yield of Xanthan gum at varying parameters (pH 6.0 constant) Sr no. Raw Material pH Average yield (g/L) 1 MGYP 6 15.42 2 MGYP + Whey 6 24.62 3 MGYP + Vitamin 6 12 4 Carrot 6 40.88 5 Carrot + Whey 6 48 6 Carrot + Vitamin 6 NA 7 Pumpkin 6 31.4 8 Pumpkin + Whey 6 58 9 Pumpkin + Vitamin 6 16.6 Table 3.B: Illustrates yield of Xanthan gum at varying parameters (pH 6.8 constant) Sr no. Raw Material pH Average yield (g/L) 1 MGYP 6.8 6 2 MGYP + Whey 6.8 8.4 3 MGYP + Vitamin 6.8 9.826 4 Carrot 6.8 NA 5 Carrot + Whey 6.8 32.8 6 Carrot + Vitamin 6.8 17.4 7 Pumpkin 6.8 15.86 8 Pumpkin + Whey 6.8 21.8 9 Pumpkin + Vitamin 6.8 20

VI. CONCLUSION AND DISCUSSION In the present study, it was observed that at pH 6 carrot and pumpkin peel waste gave an optimum yield of xanthan gum around 40.88g/L and 31.4g/L respectively in comparison to the control media (MGYP). As a novelty to the entitled study, Vitamins and Whey protein were added separately to study their effect on the yield of this biopolymer. Whey is obtained in India from the manufacturing of chhana and paneer. The annual production of whey is projected to be about 5 million tons. In Europe, the consumption rate is 75%, and in the rest of the world, it was possibly less than 50%, resulting in the waste of a vast amount of material that could be used as food or feed. Besides, environmentalists and technologists have been anxious about its polluting potential as whey is a significant pollutant because it has a high BOD of 30,000-50,000 mg/lit and a chemical oxygen demand of 60,000-80,000 mg/lit. Whey’s wastage represents a major loss of available nutrients and resources as the protein present in whey is classified into primary whey proteins which includes Beta-lactoglobulin (65%), Alpha-lactalbumin (25%), and serum albumin (8%), and small proteins/peptides comprising of Glycomacropeptide (GMP), Bovine serum albumin, lactoferrin, Immunoglobulin, and Phospholipoproteins. Moreover, whey proteins have a Biological Value (BV) of 110, which is

Optimization of Xanthan Gum Fermentation Utilizing Food Waste (GRDJE/ Volume 6 / Issue 5 / 003)

greater than casein, soy protein, meat, or wheat gluten, they also contain a large number of sulphur-containing amino acids including cysteine and methionine. On top of that, the dairy sector faces a financial setback because of many maintenance costs associated with proper whey disposal. In India, for every kilogram of cheese produced, about 8.7 kilograms of whey is generated which is seen as a waste. This whey has an exceptional amino acid profile and therefore it can be utilised for manufacturing xanthan gum, not only this would be cost- effective, it would also address the issue of whey disposal and would lead into the development of high-quality xanthan gum which could be used by various industries. The results were astonishing in case of pumpkin when fortified with whey protein at pH 6, which represented a one-fold increase in xanthan gum’s yield in contrast with normal pumpkin media at same pH. The reason being, whey is an organic nitrogen source that helps bacteria to synthesize amino acids, nucleic acids, and proteins subsequently helping the bacteria to proliferate. The Whey used in the experiment was taken from “Muscleblaze Fuel one Whey protein". The whey was not only providing nitrogen to Xanthomonas Campestris but also supplied essential amino acids like Isoleucine, Leucine, Valine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Histidine as well as semi essential amino acids Arginine, Cysteine, Glycine, Proline, and Tyrosine. These amino acids stimulated the growth of exopolysaccharide. While performing the literature review, it was reported that the most effective amino acid that induced the yield of xanthan gum is Cysteine [32]. This explains the increase in xanthan gum’s yield as the whey used in the experimental broth was containing adequate amount of cysteine in contrast to the control broth. Therefore, increase in xanthan gum’s yield by 8g/L (40.88g/L to 48g/L) was witnessed at pH 6.0. On the other hand, the essential vitals of life (vitamins) gave superlative yield at a pH 6.8 for both the food wastes. The right clarification for an escalated yield could be the role of vitamins in the biochemical pathway of xanthan gum synthesis. The backbone of the biopolymer synthesized is moulded by successive addition of D-glucose, D-glucose-1-phosphate, and UDP-Dglucose. The mannose and glucuronic acid are added to the side chains. Later, the acetyl group from acetyl CoA is transferred to mannose residue. Therefore, the above chemical groups are the precursors of the Entner-Doudoroff pathway, a pathway used by the gram-negative Xanthomonas campestris bacteria as the main pathway for supplying energy. However, after extensive research, it has been concluded that the Entner-Doudoroff pathway works in conjugation with the TCA cycle as a cardinal mechanism for the glucose catabolic process [33]. In the TCA cycle, one or more B complex vitamins are vital to produce energy within the cells. On relevance, vitamins such as Riboflavin (B2), Niacin (B3), and Pantothenic acid (B5) play an essential role as co-enzymes/factors such as FAD, NAD, as one of the components of CoA, or Co-enzyme Q10, respectively. It also concludes that all the vitamins (especially the B complex vitamins) that are essential for the Citric acid cycle are indirectly the requisites for the biosynthesis of the bacterial heteropolysaccharide. Therefore, pumpkin and carrot waste exhibit a yield of 20g/L and 17.4g/L which is much higher than the control media at pH 6.8. To conclude, the present research was performed to obtain a cost-effective, biocompatible, and sustainable polymer using frugal food waste. All the experiments were performed once to get the single results due to the pandemic situation. Covid-19 virus created a huge hurdle in the present research which limited our access to the laboratory. Hence, multiple results were impossible to be acquired for every experiment to reckon the average results by plotting a Standard Deviation graph. Additionally, characterization techniques (e.g., Mass spectroscopy and NMR) to analyze the strength and purity of the xanthan gum synthesized can be used further. An advanced down streaming purification approach is required as well to obtain a pure xanthan gum without any impurities.

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