# Introduction actobacillus microorganisms are known as the favorite residents of digestive tract. Lactic acid is the only or main product of the microorganisms, by which they can reinforce an acidic environment. These conditions are not suitable for many pathogenic bacteria in digestive tract and thus destroy the bacteria (Kandler & Weiss, 1985). The bacteria obtain their required energy through metabolism of sugars during the fermentation process, and produce lactic acid as the main and final product (Champagne et al., 2005). Lactic acid is widely used as a flavoring and preservative in food, pharmaceutical, leather and textile industries. The acid is also polymerized into a biodegradable poly lactic acid (PLA) that is used in the manufacture of sutures to close wounds as well as prostheses in the pharmaceutical industry. In addition, it is applied to make products with chemical base (Chooklin et al., 2011). In today's industry, this acid is produced both as fermentative and chemical form. With today's improvement in fermentative processes, fermentative production of lactic acid is 90% and its chemical production is only 10% (Chiarini et al., 1996). Lactobacillus casei is a strain of lactic acid bacteria with a remarkable phenotype and variabile genotype having a variety of bacterial colonies, some of which are present in the human digestive duct. Homofermentative L. casei can produce L(+)-lactic acid. Lactic acid, which is found in both optically active form of D(-) and L(+), is produced by a chemical reaction through hydrolysis of lactonitriles and microbial fermentation processes (Altiok et al., 2006). One of the features of the industrial microorganisms to produce lactic acid is their ability of quick and complete fermentation of cheap raw materials, the need for a minimum amount of nitrogen material and high yield of production (Narayanan et al., 2004). Nabi Bidhendi and Bani Ardalan (2004) studied continuous and discontinuous production of lactic acid from whey using immobilized lactobacillus, compared the use of wooden laminates, the use of brick and glass with adsorption and eggshell with glutaraldehyde with covalent bonding and observed that wood can have the maximum amount of adsorption and is the best stabilizer for production. Parmjit et al. (2010) explored whey fermentation and production of L(+)-lactic acid by Lactobacillus casei. The effects of various parameters such as pH, medium, temperature, inoculation, age of inoculation, mixing and incubation time were examined on the increase of lactose into lactic acid. Optimization of vaporization conditions resulted in the reduced fermentation time and increased conversion rate of lactose into lactic acid. Chooklin et al. (2011) investigated the palm syrup fermentation by Lactobacillus casei TISTR 1500 as the microorganism producing lactic acid. They observed that physical and chemical properties determine the amount of lactic acid production, and total palm syrup concentrate, dry cell weight and lactic acid increase by increasing sugar. Lactobacillus have complex food requirements because they are microorganisms that can have a limited bioproductivity and cannot make the growth factors needed by their body. They cannot just grow on the carbon source and mineral salts of nitrogen. Therefore, they need a different set of amino acids and vitamins. The growth factors are usually provided by nitrogen sources such as yeast extract, peptone and ammonium sulfate. In particular, yeast extract has the greatest effect due to the presence of purines, pyrimidine and vitamins B (Hujanen et al., 2001;Narayanan et al., 2004;Yu et al., 2008). According to all the scientific resources available, this study tried to optimize suitable culture medium to produce a high concentration of the bacterium and the resultant metabolites (Typically, lactic acid) by considering the minimum facilities at a laboratory scale so that the results could be used in the food and pharmaceutical systems. # II. # Materials and Methods Microstructures: Lactobacillus casei donated by the Laboratory of Pharmacy faculty, Tehran University was used in this study. Medium: in order to select a suitable sugar source, 100cc ingredients of the medium MRS (except for the sugar source) were first prepared in a 250cc Erlenmeyer flask in six separate flasks, then the studied sugars (sucrose, and then sugar beet molasses) were added to the two mixes. Three flasks were set to pH 7, the remaining flasks were set to pH 5 by adding some hydrochloric acid (HCl), and then an equal amount of the intended bacterium (Lactobacillus casei) was added to each. Flasks were incubated at temperatures 37 Cº, 40 Cº and 42 Cº with the stirring speed of 150 rpm. To determine the effect of different nitrogen sources, 100cc media were prepared in the 250cc flask with the exception that the two nitrogen sources of yeast extract (YE) and corn steep liquor (CSL) (0.2% yeast extract and the remaining corn steep liquor were added to the medium) replaced with nitrogen sources of MRS medium. The provided media were sampled at intervals of 8, 12 and 24 hours. Measurement of final lactic acid: the exact amount of lactic acid was measured based on the chromatography methods using HPLC. Thus, it was necessary to obtain a standard curve of lactic acid after defining the method properties. Therefore, various concentration samples were prepared from pure lactic acid and 20 microliters of it were injected into the device. Of course, three injections of each concentration were conducted on the intra day and between three different days in order to consider the coefficient of intra day and between days variations. After obtaining the standard curve of lactic acid, test samples were injected that included injecting 20 ml of the freshly prepared supernatant (normal test), the supernatant plus volumetric 1% of lactic acid (test 1%) and the supernatant plus volumetric 2% of lactic acid (test 2%). Each of the test samples were injected three times into the device. Lactic acid was added to the supernatant with the aim of the preparation of internal standard so that the existence of lactic acid could be ensured by increasing the height and area under the intended peak curve in the test sample. The concentration of lactic acid in the supernatant was obtained after obtaining areas under the curve and height of the test sample and data compliance with the standard curve. Experimental design: repeated measures design was used to evaluate the effects of time, source, temperature and pH on lactic acid with the within group factor of time and between group factors of source, temperature and pH in this study. Bonferroni post hoc test was used to detect significant differences for mutually between different levels of time. As well as to detect significant differences between different levels. Moreover, Tukey post hoc test was used to determine significant differences between different levels of source and temperature. SPSS 16 was used to analyze data, and statistical significance was considered at 0.05. # III. # Discussion and Conclusion a) Source of nitrogen The nitrogen source is the most important factor affecting for Lactobacillus growth (Wood & Holzapfel, 1995). However, high levels of nitrogen in the extract can cause cell death (De Lima et al., 2009). Examining Figures 1, 2 and 3, it can be seen that maximum production of lactic acid in three samplings was obtained by adding raw material of yeast extract. The results indicate that the maximum production of lactic acid is 60/90g/L in the comparison between the two nitrogen sources of yeast extract and corn steep liquor that is related to yeast extract at a temperature of 37 °C and pH = 5. As seen, yeast extract had the highest production of lactic acid after 8 hours, 12 hours and 24 hours of incubation of media. Due to the high yield production of lactic acid by Lactobacillus casei using the raw material of the yeast extract compared to corn steep liquor, in all media conditions studied and with regard to the fact that the better treatment with the production of 60.90g/L lactic acid was associated with an increase in the raw material, it could be found that since yeast extract is one of the raw materials necessary for the growth of the microorganism and the best source of nitrogen for growth and lactic acid production, decrease in the use of the yeast extract and replacement of the nitrogen source with corn steep liquor would result in a significant reduction in lactic acid production. indicated that production of lactic acid by Lactobacillus casei in the same medium conditions using the nitrogen source of yeast extract was more than that of the condition when corn steep liquor was provided as nitrogen source with the bacterium. Our results is consitent with these findings. Furthermore, Lactobacillus casei production was increased from the beginnig of the fermentation to 24 after wars by both nitrogen sources of corn steep liquor and yeast extract that is compatible with the results obtained in our study (Hujanen et al., 2001). # b) Carbohydrate source As a sugar source, molasses was compared with sucrose and the effect of each on the lactic acid produced by Lactobacillus casei were studied. Table 1 shows the results. As seen, maximum production of lactic acid is 35/53g/L that is related to the sugar sucrose in the medium after 24 hours of incubation at 37 C° and initial pH 5. The process of increasing the amount of lactic acid produced by sucrose compared to molasses was observed in three samplings (8 hours, 12 hours and 24 hours) and in all conditions ranging from pH = 7 and pH = 5 as well as at temperatures of 37, 40, 42 C°. After studying and observing the results obtained in the test medium conditions, it can be found that lactic acid production in media containing sucrose was significantly higher than that of when molasses was used by Lactobacillus casei. Since sucrose is simpler than molasses, it could be concluded that the more simpler the sugar available the bacteria, the more ability the microorganism will have to use it and produce lactic acid. Chooklin et al. (2011) studied the level of lactic acid using the raw material of palm produced by Lactobacillus casei and observed that lactic acid production increases within 24 hours of the onset of the activity of bacteria. Moreover, they could observe that Lactobacillus casei using glucose had the maximum production of lactic acid. The researchers concluded that maximum production of lactic acid is related to the use of glucose, fructose and sucrose, respectively. The results indicated that the microorganism used simple sugars easier and produced more lactic acid. This result is compatible with our results (Chooklin et al., 2011). # c) Temperature and pH After analyzing the results from the lactic acid produced by Lactobacillus casei at temperatures of 37, 40 and 42 C°, it was seen that the maximum production of lactic acid was at 37 C° and acid production decreases with increasing temperature. As seen in Figures 4, 5 and 6, and this decreased process with the raw material of corn steep liquor is 58/93g/L, 54/03g/L and 43/36g/L after 24 hours at pH = 5 and temperatures of 37, 40 and 42 C°. Among the two pHs used in this study, maximum production of lactic acid was at pH = 5. For example, the production of lactic acid using the nitrogen source of yeast extract at 42 C°, pH = 7 and pH = 5 and equals 48/16g/L and 48/70g/L, respectively. In the case of better treatments related to the use of the raw material of yeast extract and temperature of 37 C°, the production of lactic acid with pH = 7 and pH = 5 equals 59/43g/L and 60/90g/L, respectively. # Conclusion This research findings showed that the best source of sugar from the two sources tested for the growth of the bacterium Lactobacillus casei and lactic acid produced by the bacterium is the sugar source of a better use of sugar sources as our study, it could be concluded that Lactobacillus casei consumed the simple sugars better. The bacteria using yeast extract could produce more Lactic acid compared to the existence of nitrogen source of corn steep liquor in the medium that results from the raw materials necessary for the growth of microorganism in yeast extract. Lactobacillus casei at temperature 37 C ? and pH = 5 could produce the highest lactic acid. It should be noted that although there was no significant difference in the amount of lactic acid produced by Lactobacillus casei when using high levels of yeast extract compared to its decrease and replacement with corn steep liquor, with regard to the significant price difference between the two nitrogen sources and given that more lactic acid was produced by corn steep liquor, economic production could be expected by increasing the amount of corn steep liquor and declining the amount of yeast extract. Furthermore, in the case of sugar sources used in the study, although there was a significant difference in the amount of lactic acid produced by Lactobacillus casei after using sucrose and molasses, replacement of molasses with sucrose could be justified with regard to appropriate production of lactic acid by molasses and a lower price than sucrose. # Volume Issue II Version I ![Hujanen et al. (2001) also provided yeast extract and corn steep liquor as nitrogen sources with Lactobacillus casei and observed that the bacterium could use both nitrogen sources and produce lactic acid. However, evaluation of the results reported in these researchers research Volume XVI Issue II Version I](image-2.png "") 12![Figure 1: Comparison of lactic acid produced by Lactobacillus casei in nitrogen sources of yeast extract and corn steep liquor and sugar sources of sucrose and molasses after 8 hours of bacterial growth.](image-3.png "Figure 1 :Figure 2 :") 3![Figure 3: Comparison of lactic acid produced by Lactobacillus casei in nitrogen sources of yeast extract and corn steep liquor and sugar sources of sucrose and molasses after 24 hours of bacterial growth.](image-4.png "Figure 3 :") 4756![Hujanen et al. (2001) also provided yeast extract and corn steep liquor as nitrogen sources with bacteria. and observed that the bacterium could use both nitrogen sources and produce lactic acid. Using statistical method of surface response, they found optimum temperature around 35 C° as the optimum temperature to produce the greatest amount of acid produced by the above-mentioned bacteria. Moreover, Qi and Yao (2007) reported the optimum temperature of 37 C°(Hujanen et al., 2001;Qi and Yao., 2007).Rincon et al. (1993) also reported the optimum pH of 4.5 that indicates acidophility of the microorganism. This is very close to the optimized pH obtained in our study(Rincon et al., 1993).Volume XVI Issue II Version I The lactic acid produced by Lactobacillus casei after 24 hours of incubation at 37 C? and pH 5 and The lactic acid produced by Lactobacillus casei after 24 hours of incubation at 40C? and pH 5 and 7Volume XVI Issue II Version I © 2016 Global Journals Inc. (US) Lactic acid produced by Lactobacillus casei after 24 hours of incubation at 42C? and pH 5 and 7IV.](image-5.png "Table 4 : 7 Table 5 :Table 6 :") ![](image-6.png "") ![](image-7.png "") © 2016 Global Journals Inc. (US) © 2016 Global Journals Inc. (US) sucrose. Since other studies in this area also represents * Kinetic modeling of lactic acid production from whey by Lactobacillus casei (NRRLB-441) DAltiok FTokatli SHarsa Journal of Chemical Technology & Biotechnology 81 2006 * Challenges in the addition of probiotic cultures to foods CPChampagne NJGardner RoyD Critical Reviews in Food Science and Nutrition 45 13 2005 * Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus LChiarini LMara Tabacchi S Appl Microbial Biotechnol 36 1996 * Potential Use of Lactobacillus casei TISTR 1500 for the Bioconversion of Palmyra Sap to Lactic acid SChooklin Kaewsichan JKaewsrichan Journal of Sustainable Energy & Environment 2 2011 * Response surface optimization of D(-)-lactic acid production by Lactobacillus SMI8 using corn steep liquor and yeast autolysate as an alternative nitrogen source CJ BDe Lima LFCoelho KCBlanco JContiero Afr. J. Biotechnol 8 2009 * Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441 MHujanen SLinko Linko MLeisola Appl Microbiol Biotechnol 2001 * Bergey's Manual of Systematic Bacteriology Kandler NWeiss William UsaWilkins 1985 * Batch And Continuous Production Of Lac. Ticacid From Effect of Various Parameters of Carbon and Nitrogen Sources and Environmental Conditions on the Growth of Lactobacillus Casei in the Production of Lactic Acid Whey By Immobilized Lactobacillus NabiRBidhendi Gh Bani Ardalan Journal Of Environmental Studies 30 34 2004 * L(+) lactic acid fermentation and its product polymerization NNarayanan PKRoychoudhury ASrivastava Electronic Journal of Biotechnology 7 2004 * Production of L(+) Lactic Acid using Lactobacillus casei from Whey SParmjit JohnFPanesar CharlesJKennedy MariaKnill Kosseva Brazilian Archives Of Biology And Technology 2010 * L-Lactic acid production from Lactobacillus casei by solid state ermentation using rice straw BQi RYao BioResources 2 2007 * Optimization of the fermentation of whey by Lactobacillus casei JRincon JFuertes AMoya JMMontegudo LRodriguez Acta Biotechnol 13 1993 * Response surface optimization of L-(+)-lactic acid production using corn steep liquor as an alternative nitrogen source by Lactobacillus rhamnosus GMCC 1466 LYu TLei XRen XPei YFeng Biochemical Engineering Journal 39 2008 * The genera of lactic acid bacteria BJ BWood WHHolzapfel 1995 Blackie Academic and Professional Glasgow