UTILIZATION OF LOCALLY AVAILABLE COST EFFECTIVE SUBSTRATES IN PROBIOTIC MEDIUM FORMULATION
The interest in probiotics has increased considerably in recent years since they have commercial applications in pharmaceutical, biomedical and food processing industries. According to the World Health Organisation (WHO), probiotics are live organisms which when administered in an adequate amount, confers health benefits to the hosts. It is generally considered to promote the balance of intestinal microbiota and increase health benefits ADDIN EN.CITE <EndNote><Cite><Author>Yang</Author><Year>2014</Year><RecNum>2</RecNum><record><rec-number>2</rec-number><foreign-keys><key app=”EN” db-id=”a92w55stz5zafce2rxkvpwebx9r955ewdvs0″ timestamp=”1551250472″>2</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Yang, Shih-Chun</author><author>Lin, Chih-Hung</author><author>Sung, Calvin T</author><author>Fang, Jia-You</author></authors></contributors><titles><title>Antibacterial activities of bacteriocins: application in foods and pharmaceuticals</title><secondary-title>Frontiers in microbiology</secondary-title></titles><periodical><full-title>Frontiers in microbiology</full-title></periodical><pages>241</pages><volume>5</volume><dates><year>2014</year></dates><isbn>1664-302X</isbn><urls></urls></record></Cite><Cite><Author>Yang</Author><Year>2014</Year><RecNum>2</RecNum><record><rec-number>2</rec-number><foreign-keys><key app=”EN” db-id=”a92w55stz5zafce2rxkvpwebx9r955ewdvs0″ timestamp=”1551250472″>2</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Yang, Shih-Chun</author><author>Lin, Chih-Hung</author><author>Sung, Calvin T</author><author>Fang, Jia-You</author></authors></contributors><titles><title>Antibacterial activities of bacteriocins: application in foods and pharmaceuticals</title><secondary-title>Frontiers in microbiology</secondary-title></titles><periodical><full-title>Frontiers in microbiology</full-title></periodical><pages>241</pages><volume>5</volume><dates><year>2014</year></dates><isbn>1664-302X</isbn><urls></urls></record></Cite></EndNote> (Yang et al.
2014). Some of the beneficial aspects of probiotic consumption include improvement of intestinal health by regulation of microbiota, stimulation and development of immune system, synthesizing and enhancing the bioavailability of nutrients, reducing the risk of certain other diseases (Nagpal et al., 2012).In spite of advantages, the growth and utilization of probiotics in the fermentation process have to be economical so that the probiotic products would be affordable by everyone.
Probiotic microorganisms are sold in capsules, tablets, sachets and in the form of food products like fermented milk, ice cream, candies, etc. Growing probiotic microbes in large scale is essential to get the live biomass in higher concentration. Growth medium can represent almost 30% of the cost for microbial fermentation. (L.R. Rodrigues et al.2006). Complex media which are widely employed for the growth of probiotics are not economically attractive due to their high amount of expensive nutrient supplements such as beef extract, glucose, and peptone. These nutrient supplements improve the nutritional quality of the medium, as they contain growth promoting compounds besides organic nitrogen and carbonaceous compounds. However, the use of supplements in large quantities is very expensive and can reach as much as 32% of lactic acid production (Lima et al. 2010). So, there is a need to develop the nutrient supplements which are industrially acceptable processes concerning productivity and economic level. Nevertheless, much effort in process optimization at the engineering and biological levels has been done from several inexpensive wastes thereby decreasing the production costs (L.R. Rodrigues et al.2006).
In recent years, there is an increasing trend towards utilization of agro-industrial wastes. One such largest cellulosic agro industrial wastes is sugarcane bagasse, a fibrous residue of cane stalk which is left over after crushing and extraction of juice from the sugarcane (Pandey et al., 2000). In the context of the sugar industry, approximately 280?kg dry weight of sugarcane crop residues (sugarcane bagasse and cane leaf matter) are generated per tonne of wet cane harvested (Jonker et al. 2015). Bagasse consists of approximately 50% cellulose, 50% ?-cellulose, 30% pentosans, 25% hemicellulose, 25% lignin, and 2.4% ash (Parameswaran et al., 2009). Since it has low ash content, it provides numerous advantages for usage in the bioconversion process using microbial cultures. Sugarcane bagasse has several applications which include paper production, electricity production and products based on fermentation. Several products which have been obtained from the processes involves bagasse includes chemicals and metabolites such as alkaloids, mushrooms, protein enriched animal feed (single cell protein), enzymes, biofuel, etc.
Sugarcane bagasse has been widely used for production of industrial enzymes. Many bacteria, yeast and filamentous fungi have been cultivated on this sugarcane bagasse by fermentation process. A key step in the fermentation of bagasse includes pre-treatment method which is done for the conversion of polysaccharides into fermentable sugars. Different pre-treatment methods include acid process, such as auto hydrolysis, steam explosion and acid-catalysed hydrolysis (Batalha et al., 2015; Neves et al., 2016; Putro et al., 2016), or alkaline processes, such as ammonia fibre expansion, AFEX (Krishnan et al., 2010) and alkaline-sulfite treatment (Laurito-Friend et al., 2015). Among several pre-treatment methods done for lignocellulosic bioresources (Hendriks and Zeeman, 2009; Mosier et al., 2005), steam explosion has been proven to be effective for different materials (Saddler et al., 1993), including sugarcane bagasse (Mart?n et al., 2002, 2008; Rocha et al., 2011), and its effectiveness can further be increased if lignin is removed from the pre-treated material (Palonen, 2004).
2. MATERIALS AND METHODS:
Sugarcane Bagasse (SCB) was collected from the local vendor of NIT Campus, Rourkela. It was dried at 60°C, milled in a Hammer mill to a particle size of 16/60 mesh and stored in an air tight polyethylene bag, at room temperature for further use.
2.2 Proximate Composition
2.2.1 Moisture content
The moisture content of sugarcane bagasse was calculated by standard procedure (Association of Official Analytical Chemists (AOAC) method, 2005) by weighing the samples before and after drying at a hot air oven at 105? C and then weight loss is calculated.
2.2.2 Fat content
The fat content of sugarcane bagasse was estimated by standard procedure (AOAC method,2005). Five grams of moisture-free bagasse powder sample was weighed(W) and put inside the cellulose thimble. A pre-weighed beaker(W1) and 90mL petroleum ether were taken in it. The thimble was dipped into the solvent and boiled at 90?C. The fat in the bagasse sample was extracted by steps like rinsing and recovery. In the end, the solvent is evaporated from the beaker and collected in the condensation vessel. Then the beaker is dried in a hot air oven and it was weighed(W2). The fat content was calculated by the equation
2.2.2 Protein content
The protein content of the sugarcane bagasse was estimated by standard method. Initially, the organic nitrogen content was quantified using the Kjeldahl procedure(AOAC,2005). Two grams of moisture free bagasse powder was taken in digestion tube along with 4g of catalyst mixture(cupric sulphate: potassium sulphate= 5:1) and 10mL concentrated H2SO4.These samples are digested in the presence of manifolds(15% sodium hydroxide and 1.5L of distilled water) for 2 hours and cooled. It is then distilled in the presence of mixed indicator(2mL methyl red and 10mL bromocresol green) and boric acid(4%). The obtained distillate was titrated against 0.1N HCl. The protein content was determined by multiplying the organic nitrogen by a factor of 6.25.
2.2.3 Fiber content
The crude fiber content of the sugarcane bagasse was estimated by Weende method(AOAC,2005). Two grams of fat free powdered bagasse sample was taken in a preweighed crucible(W0). It is then washed with 150mL H2SO4(1.25%) for 1 hour(500? C for 15 mins). It is followed by washing with distilled water. Again it is washed with alkali, i.e., 150mL NaOH solution (1.25%) for 1 hour (500? C for 15 mins). The crucible was then dried in a hot air oven at 105?C for three hours and it is weighed(W1) and heated in a muffle furnace for 3 hours at 450? C. After that, it is cooled in a desiccator again and it was weighed(W2). Then the fiber content was calculated
2.2.4 Ash content
2.3 Pre-treatment of Sugarcane Bagasse
Pretreatment of sugarcane bagasse was done by steam explosion method. To prepare sugarcane hydrolysates, different composition of SCB 5%, 7.5%, 10%, 12.5%, 15%, 17.5% and 20% were suspended in 100 ml of water (M-I, M-II, M-III, M-IV, M-V, M-VI, M-VII) respectively. It is then autoclaved (1atm) for 15 mins. The liquid fraction was separated by filtration on Buchner funnel, using Whatman no. 1 paper and the pH was adjusted to 7 using NaOH.
2.4 Microorganism and inoculum
Lactobacillus helveticus MTCC 5463 was used throughout the experiment. It was grown in Man, Rogosa and Sharpe(MRS) medium. The MRS medium has the following composition(g/L): glucose (20.0), peptone (10.0), yeast extract (5.0), meat extract (10.0), sodium acetate (5.0), ammonium citrate (2.0), K2HPO4 (5.0), MgSO4.7H2O (0.1) and MnSO4.4H2O (0.05). Cells of L. plantarum cultivated in MRS Broth were collected by centrifugation (8000 x g, 10 min, 4° C) and suspended in 0.9% saline (pH 7.2) and this seed culture is used as an inoculum.
2.5 Evaluation of Growth of L. helveticus in Sugarcane Bagasse Hydrolysate
The SCB hydrolysate was fermented using L.helveticus. About 100 ml of SCB hydrolysate of different concentrations (5%, 7.5%, 10%, 12.5%, 15%, 17.5% and 20%) were placed in 250 ml Erlenmeyer flask (M-I, M-II, M-III, M-IV, M-V, M-VI, M-VII) and it was supplemented with yeast extract (0.5g), polysorbate (0.1 g), ammonium citrate (0.2 g), sodium acetate (0.5 g), K2HPO4 (0.2 g) and MgSO4.7H2O (0.01 g). These contents were sterilized at 121° C, allowed to cool and then inoculated with 10% seed culture of L. plantarum. The bacteria were then grown at (37° C± 1) for 48 hours and all the experiments were done in triplicates and each replicate contained 10 ml of inoculated medium. Growth in all the experiments were measured by taking optical density (OD at 600nm) for every 6 hours.
2.6 Analytical methods
The quantification of lactic acid concentration was determined using high-performance liquid chromatography (HPLC) with a refractive index detector(Shimadzu, Japan). Agilent Hiplex-H column was used with 5mM H2SO4 as a mobile phase, with flow rate 0.7mL/min and column temperature at 60?C. Amount of lactic acid was identified and quantified from standards purchased form HiMedia, India and Sigma, USA.
2.7 Experimental Design:
The response surface methodology was applied to understand the interaction of various variables and then used to find the optimum production that affects the response.
2.7.1 Central Composite Design:
The experimental CCD was carried out to optimize the production of biomass and lactic acid. Thus a central composite circumscribed randomized experimental design was used, with two variables (incubation time and pH), two-star points (±? =1.41421) and two blocks and five replicates at the center point resulting in a total of 14 runs. The independent variables, experimental range, and levels investigated for the CCD is given in table 1
26098507620Range and levels
00Range and levels
Table 1: Experimental range and levels of independent variables used in the central composite design
Batalha, Larisse Aparecida Ribas, Qiang Han, Hasan Jameel, Hou-min Chang, Jorge Luiz Colodette, and Fernando Jos? Borges Gomes. “Production of fermentable sugars from sugarcane bagasse by enzymatic hydrolysis after autohydrolysis and mechanical refining.” Bioresource technology 180 (2015): 97-105.
Hendriks, A. T. W. M., and G. Zeeman. “Pretreatments to enhance the digestibility of lignocellulosic biomass.” Bioresource technology 100, no. 1 (2009): 10-18.
Krishnan, Chandraraj, Leonardo da Costa Sousa, Mingjie Jin, Linpei Chang, Bruce E. Dale, and Venkatesh Balan. “Alkali?based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol.” Biotechnology and Bioengineering 107, no. 3 (2010): 441-450.
Laurito-Friend, D. F., F. M. Mendes, F. M. Reinoso, A. Ferraz, and A. M. F. Milagres. “Sugarcane hybrids with original low lignin contents and high field productivity are useful to reach high glucose yields from bagasse.” Biomass and Bioenergy 75 (2015): 65-74.
Maiorella, B. L. “Ethanol.” Comprehensive biotechnology. 3 (1985): 861-914.
Medina, Carlos Orestes Mart?n, Marcelo Marcet, and Anne Belinda Thomsen. “Comparison of wet oxidation and steam explosion as pretreatment methods for bioethanol production from sugarcane bagasse.” BioResources 3, no. 3 (2008): 670-683.
Mosier, Nathan, Charles Wyman, Bruce Dale, Richard Elander, Y. Y. Lee, Mark Holtzapple, and Michael Ladisch. “Features of promising technologies for pretreatment of lignocellulosic biomass.” Bioresource technology 96, no. 6 (2005): 673-686.
Neves, P. V., A. P. Pitarelo, and L. P. Ramos. “Production of cellulosic ethanol from sugarcane bagasse by steam explosion: Effect of extractives content, acid catalysis and different fermentation technologies.” Bioresource technology208 (2016): 184-194.
Palonen, Hetti, Folke Tjerneld, Guido Zacchi, and Maija Tenkanen. “Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin.” Journal of Biotechnology 107, no. 1 (2004): 65-72.
Pandey, Ashok, Carlos R. Soccol, Poonam Nigam, and Vanete T. Soccol. “Biotechnological potential of agro-industrial residues. I: sugarcane bagasse.” Bioresource technology 74, no. 1 (2000): 69-80.
Parameswaran, Binod. “Sugarcane bagasse.” In Biotechnology for Agro-Industrial Residues Utilisation, pp. 239-252. Springer, Dordrecht, 2009.
Putro, Jindrayani Nyoo, Felycia Edi Soetaredjo, Shi-Yow Lin, Yi-Hsu Ju, and Suryadi Ismadji. “Pretreatment and conversion of lignocellulose biomass into valuable chemicals.” RSC Advances 6, no. 52 (2016): 46834-46852.
Rocha, GJ D. M., Adilson Roberto Gon?alves, B. R. Oliveira, E. G. Olivares, and C. E. V. Rossell. “Steam explosion pretreatment reproduction and alkaline delignification reactions performed on a pilot scale with sugarcane bagasse for bioethanol production.” Industrial Crops and Products 35, no. 1 (2012): 274-279.
Saddler, J. N., L. P. Ramos, and C. Breuil. “Steam pretreatment of lignocellulosic residues.” BIOTECHNOLOGY IN AGRICULTURE (1993): 73-73.