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CHPATER-IV

RESULTS AND DISCUSSION


The organic acids (lactic acid and citric acid) were produced through fermentation by utilizing food industrial wastes (sugar cane molasses and corn steep liquor) as a carbon and nitrogen source for the microbial flora. The bacterial culture (Lactobacillus delbrueckii) was used for lactic acid fermentation, whereas fungus (Aspergillus niger) was used for production of citric acid. The cultures Lactobacillus delbrueckii and Aspergillus niger were isolated from their indigenous sources such as fresh milk, yoghurt, cheese, soil, wheat bran and rice bran. Lactobacillus spp. were identified on the basis of different biochemical, enzymatic and sugar fermentation tests whereas Aspergillus niger was identified on the basis of its morphological characteristics during different stages of growth. The fermentation conditions were optimized for the production of organic acids with special reference to temperature, time and various substrate levels. The bacterial viable cells (cfu/mL), fungal biomass (g/100mL), acidity, nitrogen contents and grams of lactic acid or citric acid and sugars in fermented media were estimated at different days of fermentation. The organic acids (lactic acid and citric acid) produced were subsequently used in different foods as acidulants and flavoring agents. The data obtained during the course of this study have been discussed under different headings as given below.

4.1 ISOLATION OF MICROORGANISMS


4.1.1 Lactobacillus spp. 4.1.1.1 Isolation Five yoghurt (Y1-Y5) and five fresh milk (M1-M5) samples were inoculated into nutrient broth from where the growth was shifted to nutrient agar plates. The colonies were morphologically studied after Grams staining and the colonies showing Grams positive, rod shaped morphology were further transferred to the selective medium deMans Rogosa and Sharpe broth (MRS broth). The colony and cellular morphological results of all the isolates are shown in Table 1. Four isolates having Grams +ve and rod

shaped morphology were selected for further isolation and purification. The growth from MRS broth shifted to MRS agar plates was further subjected to purification by sub-culturing technique. The purified growth observed after 24 hours of incubation on MRS agar was white in color, regular, large, spherical, convex, smooth and 2.1-4.9 mm in diameter. The luxuriant growth of Lactobacillus spp., was obtained on MRS agar plates. The results of colony characteristics and cellular morphology of the selected isolates are shown in Table 2 and illustrated in Fig 2. The results are similar to the criterion repoted by Harriagan (1998), Wood and Holzapfel (1995) and Holt et al. (1994) who also recommended that Lactobacillus spp. show better and more growth on the selective and enriched media like MRS with similar cellular and colony morphological characteristics as observed in the study. 4.1.1.2 Identification of the isolates The purified cultures were further analyzed on the basis of biochemical, enzymatic and sugar fermentation tests for the identification of Lactobacillus spp. as follows. i Biochemical and enzymatic tests The biochemical tests were performed to confirm the desired bacterial spp. and the results are shown in Table 3. All the isolated cultures showed positive response to methyl red and negative to catalase. ii Sugar Fermentation Tests The results regarding response of pure culture isolates to sugar fermentation tests are shown in Table 3. It is obvious that the culture isolate Y2 showed negative results for gluconate and maltose. The culture isolates Y3 and Y5 exhibited same pattern of results by fermenting only lactose. Similarly, the isolate Y4 fermented single sugar i.e. sucrose. The growth of these isolates studied at 15C and 45C showed positive results at 45C whereas none of the isolates showed growth at 15C. All the selected isolates were also subjected to glucose fermentation test and they produced acid from glucose but no gas was formed, which indicated that the culture isolates were homofermentative. On the basis of these biochemical, enzymatic and sugar fermentation tests it was concluded that the isolates Y2, Y3, Y4 and Y5 were Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus delbrueckii and Lactobacillus bulgaricus,

respectively. The former three cultures were named as IFST-LBA (Institute of Food Science and Technology: Lactobacillus acidophilus), IFST-LBB (Institute of Food Science and Technology: Lactobacillus bulgaricus) and IFST-LBD (Institute of Food Science and Technology: Lactobacillus delbrueckii), respectively. Harrigan (1998) suggested the same criterion for identification of Lactobacillus spp. These results were also in line with the recommendations of Holt et al. (1994) and Wood and Holzapfel (1995) who gave similar pattern for identification of Lactobacillus genera. During this study sugar cane molasses (sucrose source) were used as substrate for fermentation, therefore, Lactobacillus delbrueckii was used sunsequently for lactic acid production due to its sucrose fermenting ability. 4.1.2 Isolation of fungus (Aspergillus niger) The samples from wheat bran, rice bran, cheese and soil were inoculated on potato dextrose agar (PDA) and colony morphology of different isolates was examined. The colonies from cheese and rice samples were found to be from Aspergillus group. The colonies were off white, green and little bit yellowish in colour whereas few colonies were cottony white covering the whole petriplate with a dark central region which was only apparent from backside of the petri plates (Rhizopus group). The Rhizopus group was separated and the isolates from cheese and rice bran were further transferred on PDA repeatedly till purification. The purified colonies were off white and green after 24-48 hours of growth but turned black and dark black after 72 hours of incubation due to spores formation. The spores as well as mycelia were studied under microscope. The fugal biomass and spores observed during the study are shown in Fig 3 and Fig 4. The mycelia were non-septate with a foot-cells and conidiophore ended in a terminal enlarged ellipsoidal spherical swelling. This spherical vesicle bearded phialides that covered its entire surface and therefore the head of conidia was mop-like. By following the complete morphological characters of the fungus it was confirmed that the isolates from cheese and rice bran were Aspergillus niger (A. niger). The A. niger from cheese source was further used for fermentation process.

4.2 UTILIZATION OF FOOD INDUSTRIAL WASTES


4.2.1 Chemical composition of sugar cane molasses and corn steep liquor The chemical composition of sugar cane molasses and corn steep liquor was determined to assess the nutritional potential of the waste material as substrates. The results regarding composition of sugar cane molasses and corn steep liquor are shown in Table 4 and Table 5, respectively. The sugar cane molasses contained moisture 23.8%, ash 11.3%, dry matter 76.2%, nitrogen 0.62%, total sugars 54.8%, reducing sugars 27.1% and non-reducing sugars 27.7%. The corn steep liquor contained moisture 49.3%, ash 7.9%, dry matter 50.7%, nitrogen 3.8%, total sugars 6.2% and reducing sugars 6.2%. The composition of these food waste materials may vary with respect to soil and climatic conditions, variety, maturity stage and the process conditions in the industry. The results are identical to those of Akhtar et al. (1997) who also found that the corn steep liquor contained 42-56% dry matter, 26-45% protein and 2.5-15% sugars. 4.2.2 LACTIC ACID PRODUCTION 4.2.2.1 Fermentation of sugar cane molasses Different levels of sugar cane molasses (0%, 6%, 12%, 18% and 24%) were subjected to fermentation for the production of lactic acid at different temperatures (34C, 38C and 42C) by using Lactobacillus delbrueckii (IFST-LBD) as the culture. The sugar cane molasses were used as carbon source for the culture while yeast and meat extract were used as nitrogen sources. The initial concentration of total sugars, reducing sugars, non-reducing sugars, nitrogen and lactic acid in the fermentation media is given in Table 6. The parameters studied during fermentation process are discussed below. i Utilization of total, reducing and non-reducing sugars It is obvious from the statistical results in Table 7 that fermentation time and substrate levels showed highly significant effect on utilization of total, reducing and non-reducing sugars during this fermentation process. The effect of temperature on utilization of total and non-reducing sugars was found to be non-significant while the utilization of reducing sugars was significantly influenced by temperature. The first order interactions of all the variables were non-significant except the one between fermentation

time and substrate levels on utilization of total, reducing and non-reducing sugars in the media during fermentation. Utilization of total sugars The total sugar contents in the media gradually decreased with the increase in fermentation time. Significantly high total sugar utilization was observed after 8 days of fermentation followed by 7 days of fermentation with mean values of 8.16 g/100 mL and 7.92 g/100 mL, respectively. However, the utilization of total sugars was significantly lower at the initiation of fermentation (Fig 5). This indicated that utilization of total sugars increased significantly as a function of fermentation period i.e. days. The highest utilization of total sugars was found in 24% (8.04 g/100 mL) substrate level followed by 18% (6.05 g/100 mL) substrate level (Fig 6). However, utilization of total sugars was not observed in the media containing no substrate. The first order interaction between fermentation time and substrate levels revealed that the highest utilization of total sugars occurred after 8 days of fermentation period when 24% sugar cane molasses were added as substrate in the media whereas, low amount of total sugars was utilized at the start of experiment in all the media containing different levels of substrates. However, no total sugars utilization was detected in the media without substrate throughout the fermentation period. The utilization of total sugars ranged from 0.00 to 12.95 g/100 mL as shown in Fig 7 and Table 8. The results further showed that per day utilization of total sugars was the maximum at 3rd day of fermentation whereas in subsequent days its utilization was decreased (Fig 8). Utilization of reducing sugars The utilization of reducing sugars increased gradually with increase in fermentation time. Significantly the highest amount of reducing sugars was utilized after 8 days of fermentation and significantly the lowest amount was used at the beginning of fermentation process. The utilization of reducing sugars ranged from 0.00 to 3.94 g/100 mL during fermentation period of 0 to 8 days (Fig 9). The utilization of reducing sugars also increased with increase in temperature of fermentation. Significantly, higher reducing sugars were utilized at 42C temperature during the fermentation process. The results for utilization of reducing sugars at 38C and 34C temperatures were statistically non-significant. During lactic acid fermentation of

sugar cane molasses, the utilization of reducing sugars ranged from 2.49-2.55 g/100 mL (Fig 10). It is also evident that the reducing sugars were utilized significantly in high amount in the media containing 24% substrate level (3.96 g/100 mL) followed by media with 18% substrate level (3.08 g/100 mL) as shown in Fig 11. The interaction of fermentation time and substrate levels showed almost same trend of reducing sugars utilization with respect to time in all the media (Fig 12). The utilization of total sugars was increased as a function of fermentation time and substrate level. However, per day utilization of reducing sugars was higher at 2nd and 3rd day of fermentation in the media with different substrate levels and then it was leveled off (Fig 13). However, significantly the highest utilization of reducing sugars was observed after 8 days of fermentation in 24% level of substrate (6.200.07 g/100 mL) and significantly no reducing sugars were utilized at the initiation of fermentation process (0.00 g/100 mL) as shown in Table 9. Utilization of non-reducing sugars The non-reducing sugars utilization showed linear relationship with fermentation time. With increasing fermentation time the utilization of non-reducing sugars increased progressively. It ranged from 0.00 to 4.22 g/100 mL during fermentation period of 0 to 8 days. However, after 6 and 7 days of fermentation utilization of non-reducing sugars was statistically non-significant with each other (Fig 14). Significantly the highest utilization of non-reducing sugars was observed in the media which contained 24% (4.07 g/100 mL) substrate level followed by the media containing 18% (2.97 g/100 mL) sugar cane molasses (Fig 15). Non-reducing sugars utilization in the medium with no substrate was found to be zero. The combined effect of fermentation time and substrate levels shown in Fig 16 indicated that the utilization of non-reducing sugars increased as a function of fermentation time in all media, except in the media without substrate, where non-reducing sugars were not found during fermentation. The increase in utilization of non-reducing sugars started after one day of fermentation. However, significantly higher amount of non-reducing sugars was utilized after 8 days of fermentation in the media containing 24% sugar cane molasses (Table 10). Per day utilization of non-reducing

sugars was increased as a function of fermentation time and was the maximum at 3rd day of fermentation and after this it was leveled off (Fig 17). ii Nitrogen utilization, titrable acidity and bacterial viable count The statistical results for utilization nitrogen, titrable acidity and bacterial viable count are shown in Table 11. The results revealed that fermentation time, temperature and substrate levels significantly affected the utilization of nitrogen. While, the effect of all first order interactions between different variables was found non-significant for nitrogen utilization. The effect of fermentation time (days) and substrate levels was found to be highly significant on titrable acidity, while effect of temperature was found to be non-significant. Similarly, the interaction of all the variables showed non-significant effect on titrable acidity, except that of fermentation time and substrate levels. The results further revealed that the effect of all variables was found to be highly significant on bacteria viable count in the media during fermentation, except interactive effect of fermentation time, substrate levels and temperature, for this parameter. Nitrogen utilization The data regarding effect of fermentation time on nitrogen utilization during fermentation period is shown in Fig 18. The nitrogen was utilized significantly higher after 8th day of fermentation of sugar cane molasses for lactic acid production by IFST-LBD culture. The utilization of nitrogen was 0.00 to 3.12 g/100 mL during fermentation period from 0 to 8 days. The nitrogen utilization was significantly higher when fermentation was carried out at 42C with mean value of 1.91 g/100 mL, while lower nitrogen utilization was recorded at 34C with a mean value of 1.80 g/100 mL (Fig 19). The effect of different substrate levels on the utilization of nitrogen is shown in Fig 20 which indicated that maximum nitrogen utilization took place in the media containing 18% substrate levels with a mean value of 1.93 g/100 mL, whereas, less nitrogen utilization was observed in other media. However, the utilization of nitrogen was statistically non-significant among the media having 12%, 18% and 24% substrate levels.

Titrable acidity The titrable acidity was monitored to determine the extent of lactic acid production from sugar cane molasses with the passage of fermentation time. The titrable acidity as percent of lactic acid gradually increased with increase in time of fermentation upto 7 days of fermentation and the difference in titrable acidity estimated at 7 and 8 days of fermentation was found statistically non-significant (Fig 21). The titrable acidity ranged from 0.27 to 5.20% during 0 to 8 days of fermentation. The titrable acidity was significantly higher in the media containing 18% substrate followed by the media containing 24% substrate with mean values of 5.47% and 5.42%, respectively (Fig 22). The difference in titrable acidity for 18% and 24% substrate levels was observed statistically non-significant. The interaction of fermentation time and variation in substrate levels on titrable acidity (Table 12) showed significantly higher titrable acidity after 7 days of fermentation in the media containing 18% substrate, but significantly lower titrable acidity in the media with no substrate, where no acid was produced during fermentation. The results also showed that the titrable acidity increased upto 6 days of fermentation in the media containing 18% and 24% substrate levels and after 6 days of fermentation almost stability in further increase in titrable acidity was found. Similarly, in the media containing 6% and 12% substrates, stability with respect to titrable acidity was found after 4 to 5 days of fermentation and almost no change in titrable acidity was observed in the control experiment during the whole period of fermentation (Fig 23). Bacterial viable count The results for viable count in the fermentation media during lactic acid production from sugar cane molasses indicated an increase in viable count with the increase in fermentation time. The viable count was significantly higher at 6 days of fermentation with a mean value of 6.8x1013cfu/mL (Fig 24). However, after 6 days of fermentation there was a decline in the number of viable counts. The viable count also increased significantly with increase in temperature and it was higher at 42C, and significantly the lower at 34C (Fig 25). The combined effect of fermentation time and temperature on viable count indicated (Fig 26) that the count was significantly the lowest at the start of fermentation, and it was significantly higher after

6 days of fermentation carried out at various temperatures. The maximum viable count was found at 42C after 6 days of fermentation when effect of fermentation time and temperature was pooled. Significantly the highest viable count in the media containing 18% (4.1x1013 cfu/mL) substrate level and the lowest viable count in the media without any substrate (9.1x1010 cfu/mL) was observed (Fig 27). The interaction of fermentation time and substrate levels was significant with regard to viable bacterial count and it was higher after 6 days of fermentation in the media containing 18% substrate level with mean value of 9.7x1013 cfu/mL (Table 13). However, the trend of bacterial viable count as a function of fermentation time and substrate levels was similar. The viable count was significantly higher after 6 days of fermentation in all the media except in the media containing no substrate (Fig 28). The interaction of substrate levels and temperature showed the highest viable count at 42C in the media containing 18% substrate level and the viable count was significantly lower in the media containing no sugar source (Fig 29). iii Lactic acid production The analysis of variance for lactic acid production from sugar cane molasses is shown in Table 14 which indicated that the lactic acid production was affected significantly by the fermentation time, temperature and substrate levels. However, the first order interactions among these variables were found to be non-significant, except the interaction of fermentation time and substrate levels, which was significant for this parameter. The lactic acid production increased with increase in fermentation time till the completion of the experiment i.e. 8th day (5.85 g/100 mL) of fermentation of sugar cane molasses by lactic acid culture IFST-LBD. However, the difference in lactic acid production between 7th and 8th day of fermentation was found statistically non-significant (Fig 30). The lactic acid production from sugar cane molasses was significantly higher at 42C than at other temperatures. The lactic acid production was 3.77 g/100 mL, 3.69 g/100 mL and 3.67g/100 mL at 42C, 38C and 34C temperature, respectively.

However, the difference in lactic acid production between 34C and 38C temperature was found statistically non-significant (Fig 31). The lactic acid production with 18% and 24% substrate levels was found significantly, the highest (4.99 g/100 mL and 4.93 g/100 mL) followed by at 12% substrate level (3.27 g/100 mL) as is evident from Fig 32. However, no lactic acid formed in the medium without substrate due to non-availability of carbon source to the microorganisms. The interaction of fermentation time and substrate levels (Fig 33) showed an increasing trend of lactic acid production with increase in fermentation time and substrate levels. Significantly the highest lactic acid production took place after 7 days of fermentation (7.760.08 g/100 mL) in the media containing 18% sugar cane molasses as carbon substrate (Table 15). Although the maximum lactic acid production was achieved after 7 days of fermentation, however, per day lactic acid production showed higher lactic acid production at 2nd and 3rd day of fermentation as shown in Fig 34. Conclusion On the basis of these results it was concluded that the 7 days of fermentation, 42C temperature and 18% substrate level were the optimum conditions for lactic acid production from sugar cane molasses as carbon substrate by Lactobacillus delbrueckii (IFST-LBD). The maximum yield of lactic acid was 7.760.08 g/100 mL (77.6 g/L) with a mean recovery of 78.30 percent with respect to initial total sugar contents (9.910.20 g/100 mL) of the medium.

4.2.2.2 Fermentation of corn steep liquor The microorganisms require a suitable source of carbon and nitrogen for their proper growth and maintenance. The selection of these sources for fermentation is very important for efficient growth of microorganisms. In the present study the attempts were made to determine the suitability of corn steep liquor (CSL) as nitrogen source instead of yeast and meat/beef extract for lactic acid production by Lactobacillus delbrueckii (IFST-LBD) strain. Different levels of corn steep liquor (0%, 6%, 12%, 18% and 24%) were added into the fermentation media to find out the optimum level for maximum production of lactic acid from glucose as a carbon source. The fermentation was carried out at different temperatures i.e. 34C, 38C and 42C. The initial concentration of total sugars, reducing sugars, non-reducing sugars, nitrogen and lactic acid contents in the fermentation media is given in Table 16 and results obtained are discussed here inafter. i Utilization of total sugars The statistical results given in Table 17 showed that fermentation time (days) and substrate levels had significant effect on utilization of total sugars, whereas, the effect of temperature was found to be non-significant for this parameter. The first order interactions of all the variables were non-significant, except the one between fermentation time and substrate levels. The utilization of total sugars during lactic acid production increased progressively with increase in fermentation time (0.00 to 7.04 g/100 mL). Significantly the highest amount of total sugars was utilized at 8th day of fermentation, while the lowest amount was utilized at the beginning of fermentation process (Fig 35). The results in Fig 36 showed that higher amount of total sugars was utilized in the media containing 18% (6.85 g/100 mL) substrate level, whereas lower amount (1.91 g/100 mL) of total sugars utilization in the media with no substrate was found in this study (p<0.05). The interaction of fermentation time and substrate levels (Fig 37) indicated a linear increase in utilization of total sugars after 1 day of fermentation in almost all the media. However, significantly the highest amount of total sugars was utilized at 8th day of fermentation in the media containing 18% substrate with a mean value of 9.760.16 g/100 mL. However, no total sugars utilization was observed at the initiation of fermentation in all the media (Table 18). Utilization of total sugars evaluated on daily

basis showed higher utilization at 3rd day of fermentation in the media containing 12%, 18% and 24% substrate levels while at 1st and 4th day of fermentation in the media having 6% and 0% corn steep liquor, respectively (Fig 38) ii Utilization of reducing and non-reducing sugars Due to the addition of glucose as the only sugar source for fermentation, the non-reducing sugars were absent in the media, and hence the total sugars were actually reducing sugars (glucose). Therefore, the results for reducing and non-reducing sugars are not interpreted in this section of the study. iii Utilization of nitrogen The statistical results in Table 19 indicated that effect of fermentation time, substrate levels and temperature on nitrogen utilization was highly significant. However, first order interaction between substrate levels and fermentation time was also found to be significant while interactive effect of all other variables was non-significant. The utilization of nitrogen during lactic acid production monitored at different intervals of fermentation time increased as a function of fermentation time (Fig 39). Significantly the highest amount of nitrogen was utilized after 8 days of fermentation whereas at zero day of the fermentation it was not consumed at all. The temperature also exhibited highly significant effect on nitrogen utilization during lactic acid production from corn steep liquor. The nitrogen utilization was found the highest at temperature 42C. It was noticed that 0.35 g/100 mL, 0.33 g/100 mL and 0.32 g/100 mL of nitrogen were utilized when fermentation was carried out at temperatures of 42C, 38C and 34C, respectively (Fig 40). The effect of substrate levels on nitrogen utilization showed that the utilization of nitrogen was significantly higher in the media containing 24% (0.47 g/100 mL) substrate followed by the media containing 18% (0.42 g/100 mL) corn steep liquor (Fig 41). However, no nitrogen was utilized in the media without any substrate throughout the experimental period of fermentation process. The interaction between fermentation time and substrate levels showed that significantly the highest nitrogen utilization was observed at 7-8th day of fermentation in the media containing 24% corn steep liquor (Table 20). The results further revealed that with the increase in substrate level and fermentation time the utilization of nitrogen was also increased and the stability stage in further utilization of nitrogen was delayed due to

availability of high levels of nitrogen (Fig 42). However, per day utilization of nitrogen in the media containing different substrate levels was randomly changed. This may be due to the production of other nitrogenous metabolites like enzymes and amino acids by the culture (Fig 43). iv Titrable acidity and bacterial viable count The total titrable acidity (percent lactic acid production) and viable bacterial count (cfu/mL) in the fermentation media was also estimated during lactic acid production under various fermentation conditions while using corn steep liquor as a nitrogen source. The statistical results (Table 21) indicated significant effect of fermentation time and substrate levels on the titrable acidity, whereas the effect of temperature was found to be non-significant. The effect of first-order interactions of all other variables was found to be non-significant (p>0.05) on titrable acidity except the interactive effect of fermentation time and substrate levels. However, in case of viable count the effect of all the variables and their first-order interactions was found to be significant, except the interaction among fermentation time, substrate levels and temperature. Titrable acidity The titrable acidity was significantly lower at the start of fermentation but increased with the increase in fermentation time. It increased from 0.29% to 6.75% during 0 to 8 days of fermentation. However, the titrable acidity measured at 8th and 7th day of fermentation was statistically identical (Fig 44). The results shown in Fig 45 indicated significantly higher titrable acidity in the media containing 18% substrate (6.64%) while significantly lower value in zero percent substrate (1.84%) was recorded. The differences in titrable acidity in 12% and 24% substrate levels were statistically non-significant. The interaction of fermentation time and substrate levels on titrable acidity revealed that the titrable acidity also increased with increase in substrate level and fermentation time (Fig 46). Maximum titrable acidity was observed in the media containing 18% substrate and at 8th day of fermentation (Table 22). The results further revealed that after 24 hours (1 day) of fermentation there was a linear increase in titrable acidity.

Viable bacterial count The results for viable count at different time intervals during fermentation shown in Fig 47 indicated that the viable count of bacteria increased with the increase in fermentation time, but it declined after 6 days of fermentation. Significantly higher viable count (6.7 x 1013 cfu/mL) was found at 6th day of fermentation. The viable count also increased with increase in temperature and significantly higher number observed at 42C (3.1 x 1013 cfu/mL), while, significantly lower viable count (2.5 x 1013 cfu/mL) was found at 34C (Fig 48). The combined effect of fermentation time and temperature on viable count (Fig 49) was significantly higher at 42C and after 6 days of fermentation, while significantly lower viable count was found at the start of fermentation at all the temperatures. Significantly the highest viable count with mean value of 4.3 x 1013 was found in the media containing 18% substrate followed by 12% substrate (4.1 x 1013 cfu/mL) and significantly lower count was observed in the media lacking any substrate (Fig 50). The interaction between fermentation time and substrate levels showed that with increasing substrate level and fermentation time the viable count was also increased (Fig 51). Significantly higher viable count was observed after 6 days of fermentation in the media containing 18% substrate with a mean value of 1.0x1014 cfu/mL. The viable count was significantly lower in the control experiment throughout fermentation, which reflects the importance of nitrogen source for microbial growth (Table 23). Similarly, the interaction between substrate levels and temperature showed that higher viable count was observed at 42C and in 12% and 18% substrates with mean values of 4.5x1013 and 4.6x1013 cfu/mL, respectively (Table 24). v Lactic acid production The analysis of variance for lactic acid production is given in Table 25 which indicated that fermentation time and substrate levels showed significant effect on lactic acid production, while temperature had non-significant effect. Similarly, the first order interactions of all other variables were found to be non-significant except the interaction between fermentation time and substrate levels. The lactic acid production was found significantly the lowest at the initiation of fermentation but it gradually increased as a function of fermentation time. It was

significantly higher after 5 days of fermentation. The lactic acid production ranged from 0.00 to 5.55 g/100 mL during 0 to 8 days of fermentation. However, the increase in lactic acid production after 5, 6, 7 and 8 days of fermentation was statistically non-significant (Fig 52). The lactic acid production was found significantly the highest in the media containing 18% substrate (5.95 g/100 mL), and it was found significantly lower in the media lacking nitrogen source i.e. 0% substrate (1.48 g/100 mL). The differences in lactic acid production between 12% and 24% substrates were found statistically non-significant (Fig 53). The interaction between fermentation time and substrate levels was significant, and the lactic acid production increased with increment in fermentation time and substrate level (Fig 54). High amount of lactic acid was achieved at 18% substrate level after 5th day of fermentation time (Table 26). There was a linear increase in lactic acid production in all the treatments after 24 hours of fermentation and the maximum production level was achieved after 4th day of fermentation in the media containing 0%, 6% and 24% substrate levels, and after 5th day of fermentation in case of 12% and 18% substrates. The lactic acid production ranged from 0.00 to 8.140.17 g/100 mL during the whole fermentation process. However, it was further observed that the higher rate of lactic acid production was achieved at 3rd day of fermentation in the media containing 12%, 18% and 24% substrate levels, while at 1st and 2nd day of fermentation in case of 6% and 0% substrate levels, respectively as shown in Fig 55. Conclusion On the basis of these results it was concluded that significantly the highest lactic acid yield was obtained after 5 days of fermentation in the media containing 18% substrate level during lactic acid production by Lactobacillus delbrueckii (IFST-LBD) using corn steep liquor as nitrogenous substrate. The maximum yield of lactic acid was 8.140.17 g/100 mL (81.4 g/L) with a mean recovery of 73.49 percent with respect to initial total sugars (11.080.24 g/100 mL) in the medium.

DISCUSSION Microorganisms are not only harmful but also make crucial contributions to the welfare of humanity. These are widely used in food industry for production of a large number of fermented food products and at the same time these are also very helpful for conversion of food industrial wastes into value added products. The ability of microorganisms to convert large and complex molecules into the simplest one depends upon the type of culture and the growth requirements which include both intrinsic and extrinsic environmental conditions. The selection of a suitable culture to convert the specific type of substrate into useful products thus plays a vital role in fermentation technology. For the production of these useful products at industrial level, attempts are being made by the researchers to identify the cheapest substrates to reduce the cost of production. In this study the similar attempt was made to observe the suitability of food industrial wastes like sugar cane molasses and corn steep liquor for lactic acid production by Lactobacillus delbrueckii. The suitability of sugar cane molasses as carbon source instead of glucose and the suitability of corn steep liquor as nitrogen source instead of yeast extract and meat/beef extract were examined for lactic acid production, using Lactobacillus delbrueckii (IFST-LBD) strain. During fermentation of sugar cane molasses as a carbon source the yeast and meat extract was used as nitrogen sources whereas during fermentation of corn steep liquor as nitrogen source, glucose was used as a carbon source. The fermentation conditions were optimized with different fermentation times, temperatures and substrate levels. The results for lactic acid production revealed that 12.95 g/100 mL (97.30%) and 9.760.16 g/100 mL (88.09%) total sugars were utilized during fermentation of sugar cane molasses and corn steep liquor, respectively. These results are related to the findings of El-Sherbiny et al. (1986), Mintian et al. (2005) and Richter and Berthold (1998) who observed 80.7%, 71% and 89% total sugar utilization during lactic acid production from different substrates, respectively. The comparatively more utilization of total sugars during fermentation of sugar cane molasses than the findings of other researchers might be due to the proper selection of culture and substrate. The culture Lactobacillus delbrueckii has the ability to ferment sucrose being the basic carbon source in sugar cane

molasses. The results obtained are also in accordance with Nancib et al. (2001) who reported 78 to 89% sugar utilization during lactic acid production from date juice. In another study Yuji et al. (1997) who worked on lactic acid production from bread wastes, achieved 47.2 % of total sugars conversion to D-lactic acid in 72 h under static incubation conditions. The results obtained by Yuji et al. (1997) as 47.2% utilization of total sugars were comparatively lower when compared to the findings of this study and other researchers. This low conversion rate might be due to the use of different types of substrates. Moreover, relatively lower sugar conversion rate during fermentation of corn steep liquor than for sugar cane molasses might be due to the growth inhibition of cells by impurities remaining in CSL as suggested by Underwood et al. (2004). In the present study nitrogen content was affected significantly due to variation in fermentation time, substrate levels and temperature during fermentation. The utilization of nitrogen was increased with increase in substrate level, fermentation time and the temperature of fermentation. The maximum nitrogen utilization during lactic acid production was 3.25 g/100 mL (32.5 g/L) and 0.82 g/100 mL (8.2 g/L) from sugar cane molasses and corn steep liquor, respectively. These results are in line to the findings of Nancib et al. (2001) who observed 20 g/L to 30 g/L maximum utilization of nitrogen during lactic acid production from date juice. The titrable acidity and bacterial viable count was monitored during fermentation to observe the completion of fermentation. The stability or decline in titrable acidity and bacterial viable count reflect the termination of fermentation process. In the present study the titrable acidity of the media was stable after 7 to 8 days of fermentation while the viable bacterial count was significantly higher at 6th day of fermentation of both substrates and thereafter the viable count declined indicating the completion of fermentation. A logarithmic increase (log phase) in bacterial viable count was started within 24 hours of fermentation in both the cases. With increasing substrate level the log phase of bacterial growth was also prolonged indicating a direct relationship between viable count and available sugars in the medium. However, the log phase was shorter in the media containing lower substrate level (low concentration of sugars) due to rapid depletion of nutrients. These results are in agreement with the findings of Bustos et al. (2004), Luis et al. (2003a) and El-Sherbiny et al. (1986) who also reported the

completion of fermentation during 4 to 8 days for lactic acid production from different substrates. The variation in time of fermentation might be due the difference in initial sugar contents of the media i.e. lower the sugar contents, lower was the time of fermentation. The lactic acid production was significantly influenced by fermentation time, temperature and substrate levels. In case of sugar cane molasses the maximum lactic acid was produced after 7 days of fermentation in the media containing 18% substrate level with a mean value of 7.76 g/100 mL (77.6 g/L) at 42C was found to be the suitable temperature for optimum production of lactic acid by Lactobacillus delbrueckii. The maximum recovery of lactic acid when calculated with respect to initial total sugar contents of the media was 78.30 percent. Without carbon source no lactic acid was produced due to unavailability of fermentable sugars for the microorganisms. While in case of corn steep liquor the best results for lactic acid production were obtained in the media containing 18% substrate level and after 5 days (120 hours) of fermentation time with an average yield of 8.14g/100 mL (81.4 g/L) and 73.49% average recovery with respect to initial total sugar contents. Without addition of any nitrogenous substrate the lactic acid yield was decreased. These results fall with in the limits reported by Luis et al. (2003a) who obtained 11.27 to 85.03% lactic acid recovery after 96 hours (4 days) of fermentation by Lactobacillus delbrueckii, using CSL as nitrogen source. The results are also supported by the findings of Kim et al. (2003) who achieved 91% overall yield of lactic acid at 42C with initial pH 6.0 of the media during lactic acid production from food wastes through fermentation with Lactobacillus delbrueckii and observed that without supplementation of nitrogen-containing nutrients, the lactic acid yield was decreased. The results further showed that the corn steep liquor was found to be the effective nitrogenous substrate instead of yeast and meat extract. These findings are also in accordance to that of Lee (2005) who suggested that the replacement of yeast extract with corn steep liquor could be an effective substitute for lactic acid production. The results further support the findings of Luis et al. (2003b) who conducted lactic acid fermentation at 41.5C with Lactobacillus delbrueckii and recovered upto

65.2 g/L lactic acid (72% recovery) from 90 g/L of glucose when 20 g/L corn steep liquor was added in the media. The results could be justified by the findings of a number of scientists who optimized fermentation conditions for lactic acid production from various substrates while using different cultures. Bustos et al. (2004) predicted a maximum lactic acid concentration (58.9 g/L) after 96 hour fermentation. Narita et al. (2004) got 14.7 g/L lactic acid (73.5% recovery) from 20 g/L raw starch with an initial pH 6.0. Aksu and Kutsal (1986), El-Sherbiny et al. (1986) found 70% and 62.6 % lactic acid recovery by Lactobacillus delbrueckii, respectively after the optimum fermentation period of 8 days. Similarly, the results obtained by Garde et al. ( 2002), Bai et al. (2003), Lee et al. (2004), Mintian et al (2005), Bulut et al (2004) and Richter and Berthold (1998) also support the findings of the present study who reported 51 to 88%, 97 %, 74 %, 68.8%, 60% and 94% recovery of lactic acid, respectively. In, the present study during lactic acid production from sugar cane molasses, with increasing substrate level (initial total sugars) the lactic acid production was also increased. The results are supported to the findings of Shirai et al. (2001) who also found that the high initial glucose (sugars) increase the amount of lactic acid production. The differences in lactic acid recovery during this study with that of other researchers might be due to the variation in temperature, substrate levels, types of substrates and the selection of suitable culture for fermentation. It may be concluded from the present results that maximum lactic acid may be produced by Lactobacillus delbrueckii at 42C, in 7-8 days of fermentation while using sugar cane molasses and corn steep liquor as carbon and nitrogen sources at the rate of 18 percent and would be the cheaper sources fo industrial production of lactic acid. Moreover, it is also concluded that the food industrial wastes are not actually the wastes instead these would be the cheaper and suitable substrates for production of different valuable products.

4.2.3 CITRIC ACID PRODUCTION 4.2.3.1 Fermentation of sugar cane molasses The production of citric acid through fermentation by Aspergillus niger was carried out at different temperatures (20C, 24C and 28C) and different levels of sugar cane molasses (0%, 6%, 12%, 18% and 24%) using as carbon/sugar substrate. The initial concentration of total sugars, reducing sugars, non-reducing sugars, nitrogen and citric acid in the fermentation media is given in Table 27 and the results obtained during the course of this study are described here in. i Utilization of total, reducing and non-reducing sugars The statistical results in Table 28 indicated that fermentation time, substrate levels and fermentation time x substrate levels significantly affected the utilization of total, reducing and non-reducing sugars during fermentation. The effect of temperature on utilization of total and reducing sugars was also found significant while the utilization of non-reducing sugars was not significantly influenced by temperature. The first order interaction of all other variables showed non-significant influence on utilization of total, reducing and non-reducing sugars except interaction between fermentation time and substrate levels. Utilization of total sugars The total sugars utilization was progressively higher with increase in fermentation time during citric acid fermentation of sugar cane molasses. The results showed that the highest total sugars utilization took place at 8th day of fermentation with a mean value of 7.02 g/100 mL while significantly the lowest utilization of total sugars was found at the start of fermentation. However, the results for total sugars utilization at 8th and 7th day of fermentation as well as at 6th and 7th day of fermentation for utilization were found statistically not different (Fig 56). The total sugars were utilized significantly higher at 28C (4.67 g/100 mL), whereas, significantly lower utilization of total sugars was recorded at 20C (4.58 g/100 mL) as is evident from Fig 57. The difference in utilization of total sugars between temperature 28C and 24C was observed non-significant. The utilization of total sugars was significantly higher in the media containing 24% (6.50 g/100 mL substrate level) followed by the media having 18% (5.80 g/100 mL)

substrate concentration (Fig 58). However, utilization of total sugars was not observed in the media without substrate. The interactive effect of fermentation time and substrate levels indicated a linear trend in utilization of total sugars after about 24 hours (1 day) of fermentation and reached to a constant level after 2, 3, 4 and 5 days of fermentation in the media containing 6%, 12%, 18% and 24% substrate levels, respectively (Fig 59). However, significantly higher utilization of total sugars was observed after 8 days followed by 7 days of fermentation in the media containing 24% level of substrate with mean values of 10.410.07 g/100 mL and 10.340.03 g/100 mL, respectively (Table 29). Moreover, the results further showed that the per day utilization of total sugars increased with increase in substrate level of 18% and 24% upto 3 to 4 days of fermentation and then it was leveled off. However, the lower substrate levels (6% and 12%) showed maximum utilization of total sugars at 3rd and 2nd days of fermentation as is shown in Fig 60. Utilization of reducing sugars The results shown in Fig 61 revealed that significantly the highest utilization of reducing sugars was observed at 8th day of fermentation with a mean value of 3.29 g/100 mL, whereas, significantly the lowest amount was utilized at the initiation of fermentation. The utilization of reducing sugars obtained at 7th and 8th days of fermentation and at 6th and 7th days of fermentation was statistically identical with each other. The amount of reducing sugars utilization was 2.15 g/100 mL, 2.17 g/100 mL and 2.19 g/100 mL when fermentation was carried out at temperatures of 20C, 24C and 28C, respectively. However, differences in the amount of reducing sugars utilization at 20C and 24C were statistically non-significant (Fig 62). Different levels of sugar cane molasses also affected the utilization of reducing sugars during citric acid fermentation. Significantly, the highest amount of reducing sugars was utilized in the media containing 24% (3.05 g/100 mL) substrate followed by 18% (2.72 g/100 mL) substrate (Fig 63). The interaction of fermentation time and substrate levels showed maximum utilization of reducing sugars in the media containing 24% sugar cane molasses after 7 and 8 days of fermentation with mean values of 4.850.04 g/100 mL and 4.880.04 g/100 mL, respectively (Table 30). The results further showed that the progressively utilization of

reducing sugars was started within 24 hours of fermentation and it became almost stable after 2nd, 3rd, 4th and 5th day of fermentation in the media containing 6%, 12%, 18% and 24% substrate levels, respectively (Fig 64). This indicated that with increasing substrate level (sugar concentration) in the media the time required for stability of fermentation process was also increased. The results further revealed that the maximum daily utilization of reducing sugars was observed at 2nd, 3rd and 4th day of fermentation in the media containing 6%, 12% and 18-24% substrate levels, respectively (Fig 65). Utilization of non-reducing sugars During fermentation of sugar cane molasses the utilization of non-reducing sugars for production of citric acid was monitored and the results are presented in Fig 66. Significantly, the highest amount of non-reducing sugars was utilized after 8 days of fermentation with a mean value of 3.73 g/100 mL, while significantly the lowest amount was utilized at initiation of fermentation. The difference in the amounts of non-reducing sugars after 6, 7 and 8 days of fermentation as well as after 5 and 6 days of fermentation was statistically at par for this variable. Significantly, the highest amount of non-reducing sugars was utilized in the media with 24% substrate (3.45 g/100 mL) followed by the media containing 18% substrate (3.08 g/100 mL). The results are elaborated in Fig 67. The utilization of non-reducing sugars increased with increasing substrate level and fermentation time. The interaction between fermentation time and substrate levels showed that the maximum utilization of non-reducing sugars occurred in the media containing 24% substrate level (5.320.11 to 5.530.15 g/100 mL) after 5 to 8 days of fermentation time (Table 31). The results further revealed that there was an increase in utilization of non-reducing sugars within 24 hours of fermentation and stability in utilization of non-reducing sugars was observed after 2nd, 3rd, 4th and 5th day of fermentation in the media containing 6%, 12%, 18% and 24% substrate levels, respectively. This indicated that the time required for fermentation of sugar cane molasses was shortened due to rapid depletion of nutrients in the media containing lower substrate levels whereas with increasing substrate level the stability in utilization of non-reducing sugars was delayed (Fig 68). The results shown in Fig 69 indicate stepwise

utilization of non-reducing sugars during the course of fermentation process for the production of citric acid by Aspergillus niger from sugar cane molasses. ii Nitrogen, titrable acidity and fungal biomass The statistical results for utilization of nitrogen, titrable acidity of the media and fungal biomass production during fermentation are shown in Table 32. It indicated that fermentation time and substrate levels had significant effect on nitrogen utilization while the effect of temperature on nitrogen utilization was found to be non-significant. Moreover, the effect of all first order interactions of different variables was non-significant on nitrogen utilization except that of fermentation time and substrate levels. The total titrable acidity in the media during fermentation under different conditions was estimated in terms of percent citric acid and fungal biomass production on dry weight basis. The analysis of variance for titrable acidity showed that the fermentation time, temperature, substrate levels and first order interaction between fermentation time and substrate levels significantly influenced the titrable acidity of the media while the interactions of all other variables showed non-significant effect on the titrable acidity. In case of fungal biomass only the combined effect of fermentation time, temperature and substrate levels (days x temperature x substrate levels) was non-significant while all other variables and their interactions showed highly significant influence on fungal biomass production. Utilization of nitrogen The utilization of nitrogen ranged from 0.00 to 0.16 g/100 mL during fermentation period of 0-8 days. The results after 5, 6, 7 and 8 days of fermentation were statistically found non-significant (Fig 70). Similarly higher utilization of nitrogen was estimated in 24% substrate level followed by 18% substrate level with mean values of 0.14 g/100 mL and 0.13 g/100 mL, respectively. There was a direct relationship between substrate level and nitrogen utilization i.e. with increasing substrate level, the utilization of nitrogen was also increased (Fig 71). The highest nitrogen utilization was observed in the media containing 24% sugar cane molasses after 6-8 days of fermentation with mean values of 0.230.01 g/100 mL as shown in Table 33 and clearly elaborated in Fig 72. The results further revealed that there

was an irregular pattern in daily utilization of nitrogen (Fig 73). This irregularity might be due to the production of enzymes and other nitrogenous metabolites by Aspergillus niger at different intervals of fermentation time. Titrable acidity The titrable acidity gradually increased with the passage of fermentation time and was significantly higher after 6, 7 and 8 days of fermentation followed by 5th day of fermentation with mean values of 4.95%, 4.98%, 4.99% and 4.86%, respectively. Results for titrable acidity after 8, 7 and 6 days of fermentation as well as after 5, 6 and 7 days of fermentation were statistically at par (Fig 74). Significantly, the highest titrable acidity was observed at 28C with a mean value of 3.48 percent during fermentation of sugar cane molasses by Aspergillus niger. Moreover, the results for titrable acidity at 28C and 24C as well as at 24C and 20C were statistically non-significant (Fig 75). Significantly higher titrable acidity was recorded in the media containing 24% substrate followed by 18% substrate while significantly the lowest titrable acidity was found in the media without any level of substrate with mean values of 5.79%, 5.20% and 0.43%, respectively (Fig 76). The interaction between fermentation time and substrate levels showed significantly higher titrable acidity after 5 to 8 days of fermentation in the media containing 24% substrate with a maximum mean value of 8.970.13 percent citric acid (Table 34). Almost the stability in titrable acidity was found after 2, 3, 4 and 5 days of fermentation in the media containing 6%, 12%, 18% and 24% substrate levels, respectively. This showed a direct correlation between substrate level and fermentation time i.e. with increasing substrate level, the fermentation time was prolonged (Fig 77). Fungal biomass Significantly the highest fungal biomass was produced after 6, 7 and 8 days followed by 5 days of fermentation with mean values of 1.73 g/100 mL, 1.71 g/100 mL, 1.68 g/100 mL and 1.62 g/100 mL, respectively. The results after 6, 7 and 8 days of fermentation were statistically non-significant for fungal biomass (Fig 78). The fungal biomass produced by Aspergillus niger at 28C (1.30 g/100 mL) was maximum followed by at 24C and 20C with mean values of 1.26 g/100 mL and 1.18 g/100 mL, respectively (Fig 79). The fungal biomass production was relatively more

at 28C after 8 days of fermentation when effect of fermentation time and temperature was pooled (Fig 80). Significantly, the highest fungal biomass was produced in 24% substrate followed by 18% substrate level with mean values of 1.64 g/100mL and 1.58 g/100 mL, respectively (Fig 81). The first order interaction between fermentation time and substrate levels in Fig 82 showed that there was an increase in fungal biomass production with increasing substrate level and fermentation time and after 3-4 days of fermentation it was leveled off. Significantly, the highest fungal biomass was produced after 8 days of fermentation in the media containing 24% substrate level with a mean value of 2.250.04 g/100 mL (Table 35). Significantly, the highest production of fungal biomass was observed at 28C and in the media containing 24% substrate level followed by 18% and 24% substrate levels at 28C and 24C temperatures with mean values of 1.73 g/100 mL, 1.65 g/100 mL and 1.63 g/100 mL, accordingly (Fig 83). iii Citric acid production The citric acid produced during fermentation was estimated by HPLC and the analysis of variance given in Table 36 indicated that the effect of fermentation time, substrate level and that of first-order interaction between fermentation time and substrate levels was highly significant on citric acid production from sugar cane molasses while interaction of all other variables showed non-significant effect on citric acid production. With, the increments in fermentation time the citric acid production increased until 6 days of fermentation with 4.47 g/100 mL maximum citric acid production. However, the amount of citric acid production decreased to 4.44 g/100 mL during subsequent days. It was observed that the citric acid production at 5th, 6th, 7th and 8th day of fermentation was statistically non-significant (Fig 84). Significantly, the highest production of citric acid was observed in the media containing 24% substrate level with mean value of 4.35 g/100 mL followed by the media containing 18% substrate with a mean value of 3.84 g/100 mL (Fig 85). The interaction of fermentation time and substrate levels (Fig 86) indicated that with increasing time of fermentation and substrate level the citric acid production was increased. The citric acid production after 2, 3, 4 and 5 days of fermentation in the media

containing 6%, 12%, 18% and 24% substrate levels, respectively was stabilized. However, the effect of substrate levels and fermentation time on the rate of citric acid production is shown in Fig 87. It is obvious that the citric acid production in the media containing 6%, 12%, 18% and 24% was maximum at 2nd, 3rd and 4th day of fermentation, accordingly. The results further indicated that the production of citric acid was the maximum after 6 days of fermentation in the media containing 24% substrate level with a mean value of 6.870.12 g/100 mL (Table 37). Conclusion: The optimum citric acid production was achieved after 6 days of fermentation in 24% substrate level using Aspergillus niger as a culture. The maximum citric acid yield was 6.870.12 g/100 mL (68.7 g/L) with a mean recovery of 51.62 percent with respect to initial total sugar contents (13.310.15 g/100 mL) of the media. Although, the maximum citric acid was produced after 6 days of fermentation in the media containing 24% substrate level however, the recovery of citric acid was higher after 6 days of fermentation in the media containing 18% substrate level. The citric acid production at 6th day of fermentation in 18% substrate level was 5.91 g/100 mL with a recovery of 59.64% with respect to initial total sugar contents (9.910.20 g/100 mL). Therefore, it was concluded that the 6 days of fermentation and 18% substrate level were the optimum conditions for citric acid recovery from sugar cane molasses.

4.2.3.2 Fermentation of corn steep liquor The citric acid was also produced through fermentation of corn steep liquor by Aspergillus niger at 20C, 24C and 28C temperatures with 0%, 6%, 12%, 18% and 24% substrate levels. The suitability of corn steep liquor as nitrogen source for citric acid production was studied using glucose as a carbon source. The initial concentration of total sugars, reducing sugars, non-reducing sugars, nitrogen and citric acid in the fermentation media is given in Table 38 and the results during the study were as follow: i Utilization of total sugars The utilization of total sugars during citric acid production from corn steep liquor was significantly affected by fermentation time, temperature and substrate levels. Similarly, first order interactions of all other variable except interaction between fermentation time and substrate levels showed non-significant effect on total sugar utilization (Table 39). The utilization of total sugar contents increased with increment in fermentation time and significantly higher amount of total sugars was utilized after 6, 7 and 8 days of fermentation with non-significant difference followed by 5 days of fermentation with mean values of 7.42 g/100 mL, 7.47 g/100 mL, 7.53 g/100 mL and 6.50 g/100 mL, respectively (Fig 88). The utilization of total sugars was 4.80, 4.69 and 4.61 g/100 mL at 28C, 24C and 20C, respectively. The results for utilization of total sugars at 28C and 24C as well as at 24C and 20C were statistically at par (Fig 89). The highest total sugar utilization was observed in media containing 24% corn steep liquor followed by 18% and 12% with mean values of 5.54 g/100mL, 5.24 g/100 mL and 5.16 g/100 mL, respectively. The difference in utilization of total sugars in the media containing 12% and 18% substrate levels was statistically non-significant with one another (Fig 90). There was a linear increase in utilization of total sugars upto 6 days of fermentation and thereafter it was leveled off i.e. constancy in total sugars utilization was observed in the media containing 6%, 12%, 18% and 24% corn steep liquor as substrate (Fig 91 and Fig 92). The utilization of total sugars was the minimum in the media lacking corn steep liquor. The significantly high utilization of total sugars was observed in the

media containing 24% substrate after fermentation of 6-8 days with a maximum mean value of 8.760.17 g/100 mL (Table 40). ii Utilization of reducing and non-reducing sugars Due to the addition of glucose as the only sugar source for fermentation, the nonreducing sugars were absent in the media, and hence the total sugars were actually reducing sugars (glucose). Therefore, the results for reducing and non-reducing sugars are not interpreted in this part of the study. iii Utilization of nitrogen The effect of fermentation time, temperature and substrate level as well as that of fermentation time x substrate levels was found to be highly significant on nitrogen utilization during citric acid production while using corn steep liquor as nitrogenous source. The effect of first order interaction of all other variables was non-significant on utilization of nitrogen (Table 41). It is evident from the Figure 93 that significantly higher utilization of nitrogen occurred after 7 and 8 days of fermentation followed by 6 days of fermentation with mean values of 0.52 g/100 mL, 0.52 g/100 mL and 0.50 g/100 mL, respectively. However, the results after 7 and 8 days of fermentation were statistically at par for nitrogen utilization. Significantly, the highest nitrogen utilization was observed at 28C followed by 24C and 20C. Moreover, the nitrogen utilization ranged from 0.32 g/100 mL to 0.33g/100 mL under different temperature conditions (Fig 94). Similarly, significantly higher nitrogen utilization was recorded in the media containing 24% substrate (0.46 g/100 mL) followed by 18% substrate (0.41 g/100 mL) as is shown in Fig 95. The pooled data on fermentation time and substrate levels indicated that utilization of nitrogen ranged from 0.00 to 0.830.02 g/100 mL during 0 to 8 days fermentation of corn steep liquor (Table 42). A similar trend for nitrogen utilization was noted in all the media except the one containing no substrate (nitrogen source) in which no utilization of nitrogen was observed (Fig 96). Similarly, although per day utilization of nitrogen in all the media containing different levels of substrate was not linear yet its utilization was increased upto 4 days of fermentation and then it was leveled off (Fig 97).

iv Titrable acidity and fungal biomass The statistical results on titrable acidity and fungal biomass production in Table 43 indicated that fermentation time, temperature and substrate levels significantly affected the total titrable acidity of the media during fermentation. The first order interactions of all these variables were found to be non-significant except the one between fermentation time and substrate levels. However, except the interaction of fermentation time, temperature and substrate levels, all other interactions and variables showed highly significant effect on fungal biomass production during citric acid fermentation of corn steep liquor. Titrable acidity The titrable acidity as a measure of percent citric acid production gradually increased from 0.36% to 6.07% with 0 to 8 days increase in fermentation time during the fermentation process (Fig 98). The results after 6, 7 and 8 days of fermentation were statistically non-significant for titrable acidity. The titrable acidity was significantly higher at 28C and 24C with mean values of 3.98% and 3.93%, respectively. However, the difference in titrable acidity at 28C and 24C was statistically non-significant (Fig 99). The effect of substrate levels on titrable acidity (Fig 100) revealed that the titrable acidity was significantly higher in the media containing 12% (4.47%) and 24% (4.40%) substrates followed by 18% (4.29%) substrate level. The results for titrable acidity of 12% and 24% substrate levels were statistically non-significant (p>0.05). The effect of fermentation time and substrate levels interaction showed that with increasing fermentation time and substrate levels the titrable acidity also increased until it became almost stable after 6 days of fermentation (Fig 101). However, the maximum titrable acidity was obtained after 8 days of fermentation in the media containing 12% substrate with a mean value of 6.820.15% acidity (Table 44). Fungal biomass With the increments in fermentation time the fungal biomass production also increased and it was significantly the highest after 7 and 8 days of fermentation followed by 6 days of fermentation with mean values of 1.96 g/100 mL, 1.99 g/100 mL and 1.94 g/100 mL, respectively. The results for 7th and 8th day as well as for 6th and 7th day

of fermentation showed non-significant differences regarding fungal biomass production (Fig 102). Similarly, with increasing temperature of fermentation the fungal biomass production also increased and it was 1.54 g/100 mL, 1.51 g/100 mL and 1.42 g/100 mL at 22C, 24C and 28C, respectively (Fig 103). The combined effect of fermentation time and temperature showed that fungal biomass production at 28C and 24C was almost similar upto 3 days of fermentation. However, after 3rd day of fermentation slight difference was observed and relatively more biomass production occurred at 28C (Fig 104). The fungal biomass production also increased with increasing substrate level and it was significantly the highest in the media containing 24% substrate and the lowest in the media containing 0% substrate level with mean values of 2.22 g/100 mL and 0.12 g/100 mL, respectively (Fig 105). The interaction of fermentation time and substrate level is shown in Fig 106. It showed that the trend for biomass production was almost same in all the media. However, very little fungal biomass produced in the media containing no nitrogen source. In all other media there was exponential increase in biomass production within 24 hours of fermentation and after about 5-6 days of fermentation a constant stage appeared. The significantly higher fungal biomass was produced after 6 days of fermentation in the media containing 24% substrate with a maximum mean value of 2.82 g/100 mL (Table 45). The fungal biomass production was higher at 28C in the media containing 24% substrate when effect of substrate levels and temperature on biomass production was pooled. Moreover, the results at various temperatures were almost similar in the media containing 0% and 6% substrate levels (Fig 107). v Citric acid production The analysis of variance indicated that effect of fermentation time, temperature, substrate levels and fermentation days x substrate level was found to be highly significant on citric acid production while the effect of first order interaction of all other variables was non-significant on citric acid production from corn steep liquor as nitrogen source (Table 46).

The citric acid production was significantly the highest after 6, 7 and 8 days of fermentation period followed by 5 days of fermentation with mean values of 4.21 g/100 mL, 4.19 g/100 mL, 4.17 g/100 mL and 3.96 g/100 mL, respectively. However, results after 6, 7 and 8 days of fermentation were found to be non-significant for citric acid production (Fig 108). The citric acid production was significantly, the highest at 28C followed by at 24C with mean values of 2.82 g/100 mL and 2.76 g/100 mL, respectively as is evident from Fig 109. Significantly, the highest citric acid production was observed in 12% substrate level (3.62 g/100 mL) followed by 6%, 18% and 24% percent substrate levels (3.12 g/100 mL, 3.09 g/100 mL and 3.08 g/100 mL, respectively) as shown in Fig 110. The interaction of fermentation time and substrate levels showed that the maximum citric acid was produced in the media containing 12% substrate level after 6-8 days of fermentation with a maximum value of 5.65 g/100 mL (Table 47). Furthermore, there was progressive increase in citric acid production in all the media before 24 hours (1st day) of fermentation and consistency in production rate occurred after 5 days of fermentation in the media containing 0%, 6% and 12% substrate levels while after 6 days of fermentation in 18% and 24% substrate levels. It indicated that with increasing the substrate level, the time required for completion of fermentation was prolonged (Fig 111). Daily production of citric acid by Aspergillus niger showed increased citric acid production upto 2 to 5 days of fermentation and after this it was leveled off (Fig 112). Conclusion On the basis of these results it may be concluded that optimum citric acid production and its percent recovery was achieved after 6 days of fermentation at 28C from 12% corn steep liquor as nitrogenous substrate by Aspergillus niger. The maximum citric acid yield was 5.650.11 g/100 mL (56.5 g/L) with a mean recovery of 52.66% when calculated on the basis of initial total sugar contents (10.730.33 g/100 mL) of the medium.

DISCUSSION: Presently most of the citric acid is produced by fungus through submerged fermentation. The cell growth and citrate production during fermentation occurs at different times and for economic reasons, the substrate sugars must be fully utilized. The selection of suitable substrate and the culture is therefore, very important for the production of citric acid at least cost. The Aspergillus niger has the ability to grow on substrates containing carbon and nitrogen source. The nature, quantity and chemical composition of carbon and nitrogen sources and suitable environmental conditions are very important for successful citric acid production. In the present study the suitability of sugar cane molasses and corn steep liquor as carbon and nitrogen sources, respectively for successful citric acid production by Aspergillus niger was assessed. The citric acid was produced at different temperatures (20C, 24C and 28C), using different substrate levels (0%, 6%, 12%, 18% and 24%) of both sugar cane molasses and corn steep liquor. The total sugar contents gradually decreased from 8.28 g/100 mL to 1.26 g/100 mL with the fermentation time up to 8 days. Maximum utilization of total sugars from sugar cane molasses during 0 to 8 days of fermentation was 7.02 g/100 mL. However, on the whole 10.41 g/100 mL (78.21%) total sugars were utilized during the course of the study. In case of corn steep liquor the overall maximum utilization of total sugars was 8.76 g/100 mL (76.37%), under different fermentation conditions. The results are related to the findings of Jianlong et al. (2000) who reported upto 94.8% utilization of sugars during citric acid fermentation. The results are also within the limits of Ali et al. (2002) who observed 18.5 to 96.55 g/L (1.85 to 9.66 g/100 mL) utilization of sugars during fermentation period of six days. Moreover, the results are closely related to those of Haq et al. (2004), who reported 71.33% utilization of sugars during a fermentation period of 7 days. The consumption of nitrogen during fermentation increased with the increase in fermentation time, temperature and substrate. The maximum utilization of nitrogen was 0.23 g/100 mL and 0.83 g/100 mL during fermentation of sugar cane molasses and corn steep liquor, respectively. However, there was an adverse effect on the production of citric acid by increasing corn steep liquor upto 18% or above as more fungal biomass was

produced with low amount of citric acid production. There was an inverse relationship between citric acid and fungal biomass production, while a direct relationship between nitrogen content of the medium and fungal biomass production was observed. These results are supported by the findings of Ali et al. (2002) who also used 2 g/L (0.2%) nitrogen for optimum citric acid production by fungus. Although the nitrogen utilization during corn steep liquor was relative more in the media containing 18% and 24% substrate level but the best results for citric acid production was achieved from 12% substrate with an average 2.9 g/L (0.29 g/100 mL) utilization of nitrogen which is almost at reliance with the findings of Ali et al. (2002). The fungal biomass production was also affected by the fermentation time, temperature and substrate levels. The fungal biomass production ranged from 0.001 g/100 mL to 2.25 g/100 mL and 0.001 g/100 mL to 2.82 g/100 mL during fermentation of sugar cane molasses and corn steep liquor, respectively. In fermentation of corn steep liquor, the fungal biomass production was more and citric acid recovery was less than that from sugar cane molasses due to high level of nitrogen content. The results are supported by the findings of Alben and Erkmen (2004) who reported increased production of biomass with the addition of ammonium nitrate (nitrogen source). These results are also in accordance with the findings of Haq et al. (2004) and El-Holi and Al-Dalaimy (2003) who reported 16.5 g/L (1.65 g/100 mL) and 33.9 g/L (3.39 g/100 mL) fungal biomass production, respectively during citric acid production from different substrates. The variation in the present results may be due to the use of different substrates and other fermentation conditions like fermentation time, temperature and the strain of Aspergillus niger. The titrable acidity monitored during the fermentation studies is an indicator for the quantity of acid produced and the status of fermentation process. During the fermentation of sugar cane molasses the titrable acidity was higher after 5 to 8 days of fermentation with an overall maximum mean value of 8.97%. The stability in titrable acidity was achieved after 2 to 5 days of fermentation in the media containing 6 to 24% sugar cane molasses as the substrate. The stability in titrable acidity was delayed with the increase in the level of sugar cane molasses. It indicated that with increase in substrate the time of fermentation was increased due to more availability of fermentable sugars.

However, in case of corn steep liquor, the titrable acidity ranged from 0.32 to 6.82% and was higher in the media containing 12% substrate. This indicated that optimum citric acid production was achieved from 12% substrate level with 0.29 g/100 mL average nitrogen utilization. The citric acid production was significantly low without addition of nitrogen source in the media which indicated the importance of nitrogen substrate for optimum production of citric acid. The results are also supported by the findings of Alben and Erkmen (2004) who observed more citric acid production with the addition of nitrogen source (ammonium nitrate) in the media. The optimum conditions observed for citric acid production were 6 days of fermentation with 24% substrate level, from sugar cane molasses as carbon substrate for Aspergillus niger. The maximum citric acid yield from sugar cane molasses was 6.870.12 g/100 mL (68.7 g/L) with a mean recovery of 51.62 percent. However, the maximum citric acid recovery with respect to initial total sugar contents of the mediun was achieved after 6 days of fermentation in the media containing 18% substrate level with a mean recovery of 59.64 percent. The optimum citric acid production was obtained after 6 days of fermentation at 28C from 12% corn steep liquor used as nitrogen substrate. The maximum citric acid yield was 5.650.11 g/100 mL (56.5 g/L) with a mean recovery of 52.66 percent. These results are supported by the findings of Ali et al. (2002) who reported maximum citric acid production after 6 days of fermentation from 15% sugars molasses by Aspergillus niger. They obtained 99.56 3.5 g/L of citric acid from 150 gram total initial sugars with a recovery percentage of about 66%. The results are also in consistent to the findings of Ali et al. (2002) who investigated the kinetics of submerged citric acid fermentation by Aspergillus niger using blackstrap molasses as the basal fermentation media and found that 28C was the optimum temperature for fermentation. Similarly, the results also fall within the ranges reported by Alben and Erkmen (2004) and Jianlong et al. (2000) who got 21.9 % and 82.2% citric acid yield, respectively from different substrates. The results are also supported by the findings of Haq et al. (2004) who reported 31.1 to 96.1 g/L citric acid production from 15% (w/v) sugar cane molasses after 7 days of fermentation with 20.7 to 64.0% recovery. They also observed that 28C temperature and initial pH 6.0 were the optimum conditions for fermentation. In another study they

achieved 71.0% citric acid recovery with respect to initial total sugar contents during citric acid fermentation of sugar cane molasses by Aspergillus niger. The results also agree with the findings of El-Holi and Al-Dalaimy (2003) who observed 106.5 g/L (10.65 g/100 mL) citric acid production from whey supplemented with 15% sucrose after a fermentation period of 16 days by Aspergillus niger. The difference in fermentation time and citric acid production might be due to more initial total sugar contents in the fermentation media. Due to higher initial level of total sugars the time of completion of fermentation was also increased and ultimately more citric acid production was obtained. The results are also supported by the findings of Watanabe et al. (1998) who got 779 g/L and 987 g/L and 922 g/L and 1023 g/L citric acid production within 3 to 9 days of fermentation from the concentrated hydrolysate containing 150 g/L of reducing sugars. They achieved maximally 682% yield of citric acid based on the reducing sugars supplied. A number of other scientists including Chaudhary et al. (1978), Eikmeier and Rehm (1984), Hang and Woodmans (1987), Lee et al. (1989), Gutierrez et al. (1996), Lu et al. (1997), Pintado et al. (1998), (Pera and Callieri (1997) and Fedoseev (1970) also made efforts to optimize the fermentation conditions for citric acid production from various substrates. The present results are almost similar to the findings of these researchers. Minor differences in the results might be due to the selection of culture, type of fermentation technique, type and quantity of substrate and other fermentation conditions. The results for fermentation of sugar cane molasses and corn steep liquor obtained during the course of present study reflect the suitability of these food industrial wastes as carbon and nitrogen sources, respectively for citric acid production by the fungus Aspergillus niger.

4.3 UTILIZATION OF CITRIC AND LACTIC ACIDS IN FOOD PRODUCTS 4.3.1 Utilization of citric acid in apple jam The citric acid produced through fermentation was used in the preparation of apple jam and apple juice (soft drink) and compared with commercially available citric acid. Apple jam and juice were stored and evaluated for chemical and sensory attributes during storage. The results obtained are discussed as under: i Chemical analysis of apple jam The statistical results on chemical characteristics of apple jam given in Table 48 indicated that the effect of storage time and treatments was found to be highly significant on total titrable acidity of the jam; however, all other parameters did not significantly differ due to storage and treatments. The first-order interaction of the variables was also found to be non-significantly different on chemical attributes of product. The titrable acidity (% citric acid) of the apple jam decreased as a function of storage period. The titrable acidity was decreased from 0.77% to 0.71% during a storage period of 0 to 60 days (two months). The results for titrable acidity observed after 15 and 30 days of storage as well as after 45 and 60 were statistically identical. The titrable acidity was also higher in the apple jam prepared by commercial citric acid than in the apple jam prepared by citric acid produced in laboratory with mean values of 0.75% and 0.72%, respectively (Table 49). Although the effect of storage period on all other parameters of apple jam including pH, brix, reducing sugars, non-reducing sugars and total sugars was statistically non-significant however, the brix and reducing sugars of apple jam increased while non-reducing sugars were decreased during storage. The increase in reducing sugars may be due to the hydrolysis on non-reducing sugars which were slightly decreased during storage. The results are support by the findings of El-Ashwah et al. (1985) who reported increase in total soluble solids and reducing sugars, while decrease non-reducing sugars in fruit juices. The results are also supported by the findings of Babsky et al. (1986) and Ahmad et al. (1986) who also reported increase in reducing sugars while decrease in non-reducing sugars during storage of fruit concentrates for 111 days and 12 months, respectively. The results are also related to the findings of Paris (1976) who reported increase in titrable acidity of the jam while

negligible changes in pH, brix, reducing sugars, non-reducing sugars and total sugars upto a storage period of 185 days. ii Sensory evaluation of apple jam The statistical results on sensory attributes of the apple jam showed that all the sensory parameters including color, flavor, taste, texture and overall acceptability were not found statistically different as a function of storage period and treatments (Table 50). The sensory attributes of the jam was not influenced during a storage period of 2 month (Table 51). The results are in line with the studies of Bindra et al. (1974) and Paris (1976) who reported negligible changes in color, flavor, taste and texture of the jam during a storage period of two months. The results on chemical and sensory attributes of the apple jam indicated that the citric acid prepared in laboratory was compatible with that of commercial citric acid for the production of apple jam. 4.3.2 Utilization of citric acid in apple juice i Chemical analysis of apple juice The effect of treatment was non-significant on all the parameters while the effect of storage was highly significant on total titrable acidity, brix and reducing sugar contents of the apple juice whereas all other parameters were not affected by the storage period. Similarly, the interaction between storage days and treatments showed statistically non-significant difference on chemical characteristics (pH, acidity, brix, reducing sugars, non-reducing sugars and total sugars) of apple juice (Table 52). The titrable acidity of the apple juice decreased with storage period and it was significantly the highest at 0 days of the storage period followed by that of 15 days of storage with mean values of 0.30% and 0.26%, respectively which was further decreased to 0.21% after 60 days of storage. The degree of brix and reducing sugar contents of the apple juice increased during storage. Brix was significantly higher after 60 days of storage followed by 45 days of storage with mean values of 15.89 and 15.41, respectively (Table 53). The reducing sugar contents of the apple juice increased from 1.55% to 2.04% during storage of 0 to 60 days. Although, the non-reducing and total sugars were not significantly changed during storage, however there was a decrease in non-reducing

sugars and increase in total sugars during storage of the juice. The differences in chemical characteristics of apple juice (pH, titrable acidity, brix, reducing sugars, non-reducing sugars and total sugars) prepared by commercial and the citric acid prepared in laboratory were found non-significant. The increase in reducing sugars and total sugars during storage may be due to the hydrolysis on non-reducing sugars and polysaccharides, respectively. The decrease in titrable acidity might be due to the increase in reducing sugar contents of the juice. The results are supported by the findings of Sandhu et al. (1985) who observed decrease in titrable acidity of the fruit juice during storage. The results are also supported by the findings of Stein et al. (1986) who reported increase in reducing sugar contents while decrease in non-reducing sugars in grape juices. The results are also related to the findings of Sarolia and Mukherjee (2002), Ghodara and pareek (1985), Ewaidah (1988), Saito et al. (1974), Bindra et al. (1974), Mohy-ud-Din et al. (1973) and Foda et al. (1970) who also reported similar results as in the present results. ii Sensory evaluation of apple juice The statistical results given in Table 54 indicated that the sensory attributes of the apple juice were not significantly affected by storage and treatments. Similarly, the first order interaction between storage and treatments also showed non-significant influence on sensory attributes of the apple juice including color, flavor, taste and overall acceptability. The mean scores for sensory attributes of apple juice assigned by the judges are shown in Table 55 which indicated negligible differences as a function of storage and treatments. The results for sensory attributes (color, flavor, taste and overall acceptability) are related to the findings of Sandhu and Singh (2001), Ahmed and Chaudhry (1993), Ahmad et al. (1993), Rizk et al. (1985), Pino et al. (1987), Crandall and Graumlich (1982) who found no appreciable changes in organoleptic acceptance of soft drinks after 4 to 6 month of storage. The results of the present study indicated that the citric acid produced in the laboratory was equally good for the production of apple jam and apple soft drink as was citric acid available commercially.

4.3.3 Utilization of lactic acid in soft cottage cheese The lactic acid produced through fermentation was used in the preparation of soft cheese and was compared with commercially available lactic acid. The soft cheese thus prepared was stored and evaluated for chemical and sensory attributes during storage. The results obtained are discussed as under: i Chemical analysis of soft cottage cheese The effect of storage days was found to be highly significant on protein contents and total titrable acidity of the cheese while the protein content of the cheese was also significantly affected due to variation in treatments. The effect of storage period, treatment and storage days x treatment on all other chemical parameters (fat, moisture, pH, total solids and lactose) of the cheese was statistically non-significant (Table 56). The protein content of soft cheese was significantly higher at the start of storage and significantly the lower after 7 days of storage. The mean decrease in protein content of the soft cheese was from 21.55% to 19.09% during storage of 0 to 7 days. (Table 57). The titrable acidity (% lactic acid) of the cheese increased with increase in storage period and was found significantly the highest after 6 and 7 days of the storage followed by 5 days with mean values of 0.36%, 0.37% and 0.33%, respectively (Table 57a). Similarly, the titrable acidity of cheese prepared from commercial lactic acid was higher than that of the cheese coagulated by lactic acid prepared in laboratory. The results are related to the findings of Van Hekken et al. (2005) who found that soft cheeses contained 58.41.5% moisture, 15.61.4% protein, 22.91.6% fat, and 2.10.4% salt and to the studies of Martin-Hernandez et al. (1990) who found that soft cheese contains 58% moisture, 15% protein, and 21% fat. The change in protein content and titrable acidity in the present study might be due to the hydrolysis of protein into amino acids as reported by Jin and Park (1996) who concluded the hydrolysis of proteins with storage time and conditions. ii Sensory attributes of soft cottage cheese The sensory attributes of the soft cottage cheese showed that the effect of storage days was highly significant on taste, texture and overall acceptability of the cheese while the effect of treatments on taste and texture of the cheese was also found significant.

However, the sensory attributes of the cheese were not influenced by the first-order interaction between storage and treatments (Table 58). The results shown in Table 59 indicated that the scores assigned by judges to the taste, texture and overall acceptability of the soft cottage cheese gradually decreased during storage. The scores given to taste, texture and overall acceptability of the soft cheese decreased from 6.96 to 6.46, 7.21 to 6.38 and 7.13 to 6.38, accordingly. Furthermore the cheese made by lactic acid prepared in laboratory got higher scores for taste while cheese made by commercial lactic acid for texture. The taste of the cheese coagulated by the lactic acid prepared in laboratory might be due to the presence of different flavouring and other compounds contributed by the bacterial culture during fermentation as suggested by Adda et al. (1982). The results could be justified by the findings of Luck (1977) who observed variable changes in appearance (texture) and reported miner changes in sensory attributes of soft cheese during cold storage for period of one week. Conclusion The lactic and citric acids produced in laboratory were comparable with that of commercially available acids when utilized as food additives in food products including apple jam, apple juice/soft drink and soft cottage cheese.

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