Optimization of apple pomace based medium and fermentation conditions for pigment production by Sarcina sp.

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1 Indian Journal of Natural Products and Resources Vol. 2(4), December 2011, pp Optimization of apple pomace based medium and fermentation conditions for pigment production by Sarcina sp. V K Joshi*, Devender Attri and Neerja S Rana 1 Department of Postharvest Technology, 1 Department of Basic Sciences Dr. Y.S.P. University of Horticulture and Forestry, Nauni, Solan , Himachal Pradesh, India Received 19 July 2010; Accepted 2 August 2011 The growth of Sarcina sp. (isolated from water) as a source of microbial pigment on apple pomace based medium was studied. 20 g/l of apple pomace gave maximum yield of biomass and carotenoids in the apple pomace based medium. The use of glucose (0.1%) in the apple pomace based medium gave the highest yield of biomass (8.897g/l) and carotenoids (12.87mg/100g). The effect of carbon and nitrogen sources on yield of biomass and carotenoids production by Sarcina sp. (isolated from water) a dark yellow colour producing bacteria was studied in apple pomace based medium. Among the different nitrogen sources tried, potassium nitrate (0.3%) gave the maximum production of biomass (12.30 g/l) and carotenoids (12.80mg/100g). Beyond 0.3% concentration however, there was decrease both in yield and carotenoids contents. Among different ph values (from 3.0 to 6.0), a ph of 5.5 was optimized whereas, a temperature of 35 o C with an incubation period of 72 h produced the highest quantity of biomass and carotenoids. Keywords: Apple pomace, Fermentation, Microbial pigments, Carotenoids, Sarcina sp. IPC code; Int. cl. ( ) A61K 36/00 Introduction Colour is the most important attribute of any article especially food. Natural colours were added from the earlier times in our food, but with the increase in demand synthetic colour like coaltar dyes were developed that dominated the natural colours like turmeric. However, the synthetic colours being used in food are mutagenic, carcinogenic and cause other health related problems. Metanil yellow caused testicular damage in gametogenic elements to arrest spermatogenesis in guinea pigs, rats and mice 1. whereas, reproductive and neurobehavioural toxicity by tartrazine in mice 2. The toxicity of tomato red, a popular food dye blend on male swiss albino rats was determined 3. The food dye (Apple green) was also found moderately mutagenic at high doses 4. Besides imparting colour to the products, the natural colours have been found to possess anti-cancer activity. The presence of food colour in the human diet is being considered beneficial because of their action as pro-vitamin 5,6, antioxidant 7 or possibly as tumour inhibiting agents 8. In addition to plants, a number of microorganisms are also known to produce pigment, viz. and. Rhodotorula, Sarcina, *Correspondent author, vkjoshipht@rediffmail.com; Phone: Cryptococcus, Monascus purpureus, Phaffia rhodozyma, Bacillus sp Thus, the food industry has become increasingly interested in the use of microbial technology to produce colours for use in foods. It can also help to overcome the growing public concern over the adverse health effects of addition of synthetic colours in various food products 12. However, to produce the microbial pigment, the medium is too complex and requires a number of expensive chemicals 13. There is a need to develop a suitable medium which is cheap and easy to prepare for the cultivation of pigments producing microorganisms. Apple pomace is a by-product of apple juice processing industry and its disposal creates environmental problem. It contains peel, seed and remaining solid parts and is estimated to be 20-30% of the total processed crop It is a rich source of carbohydrates, minerals and vitamin-c and thus, has potential to support the growth of microorganisms. In our earlier efforts, the apple pomace based media have been used to produce pigment from Rhodotorula, Micrococcus and Chromobactor sp. 10,11,18. The food products need colour of various shades and different microorganisms have ability to produce colour of different shades. In an attempt to produce biocolour of different shades, another

2 422 INDIAN J NAT PROD RESOUR, DECEMBER 2011 microorganism Sarcina was isolated and evaluated for pigment production. Use of apple pomace for the production of pigments by Sarcina sp. has been described. Since the microbial growth and pigment production are known to be affected by various factors, this aspect was also looked into and the results have been discussed here. Materials and Methods Apple pomace The partially dried apple pomace (10-11% moisture) was procured from Himachal Horticultural Produce, Processing and Marketing Corporation, Processing unit at Parwanoo, Himachal Pradesh (India). It was further mechanically dehydrated at 60±1 o C till its moisture content reached 4±1% which generally took 3-4 h. The dried apple pomace was then grounded into fine powder and packed in polyethylene pouches for further studies. Isolation of microorganisms The pigment producing microorganisms were isolated from different sources like soil, water, juices, decomposed bread and apple pomace using serial dilution plate techniques 19. These were plated on nutrient agar medium as per the standard practices 20. The colonies from the plates showing different coloured shades were picked up, isolated by streak plating and purified. The organisms were identified by morphological and biochemical tests, viz. catalase test, oxidase test, Vogas-Proskauer test, citrate utilization test, Simmon s citrate test, etc. as per the standard practice. Medium optimization Media preparation The ground apple pomace varying in concentration was supplemented with the other ingredients as detailed in Table 1. The concentration of apple pomace was gradually increased and the quantities of other ingredients, viz. yeast extract, peptone and dextrose were decreased accordingly. Each medium was sterilized at 121 C for 20 min. The ph (4.5) of the media was kept constant. After sterilization, the medium was poured in petri plates. After solidification, inoculation was done by pouring 80 μl cell suspension in sterilized water on the surface of solid medium, ascetically. Inoculum was spread by a glass spreader and the plates were kept in an incubator at 35ºC±1ºC in the inverted position as per the practices. Each experiment was replicated thrice. Effect of nitrogen sources Ferrous ammonium sulphate, ammonium sulphate, sodium nitrate, peptone and potassium nitrate were added separately to the respective optimized apple pomace based media in different concentrations (0.1 to 0.5%) and the media were sterilized at 121 o C for 15 min. The yield of biomass and carotenoids were recorded in each plate. Effect of carbon sources Maltose, lactose, glucose and fructose each at the rate of 0.1 to 0.5% were added to the respective optimized apple pomace based media that was sterilized at 121 o C for 15 minutes. Observations were recorded for biomass and carotenoids as stated earlier. Effect of ph and temperature Effect of ph, incubation temperature and incubation period on the growth of Sarcina sp. was also studied. The ph of the each apple pomace based medium was adjusted to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 using 0.1 N NaOH solution or 1N HCl. Rest of the conditions remained the same. To find out the optimum temperature for the maximum pigment production by the microorganism, a range of temperature (25, 30 and 35 o C) was employed. Effect of different incubation periods ranging from 24 to 96 h on the growth and pigmentation by the microorganism was determined. While studying the effect of these factors on yield and pigmentation one factor at a time was considered while the other remained constant. The incubation period that showed maximum biomass production was selected for each organism. Growth estimation Extent of growth was measured by taking into the extent of surface of the media covered by the pigment producing microorganism in all the experiments and Table 1 Details of the ingredients (g/l) used in apple pomace based media preparation for microbial pigments Ingredient (g/l) M 1 M 2 M 3 M 4 M 5 M 6 M 7 M 8 M 9 M 10 M 11 M 12 Peptone Yeast extract Glucose Apple pomace

3 JOSHI et al.: OPTIMIZATION OF APPLE POMACE FOR PIGMENT PRODUCTION BY SARCINA SP. 423 was expressed as per cent of the total area of the petriplate. To measure the yield quantitatively, the growth was removed from surface of the media by scratching with a metallic sterilized spatula. The biomass was dried at 60±1 o C and weighed 20. Pigment extraction The dried biomass of pigment producing microorganism was ground to fine powder and dissolved in 60 ml of respective solvent (acetone, petroleum ether, ethanol, and hexane) till the biomass became colourless. After extraction, the solvent was evaporated and the pigment was recovered and packed in polyethylene pouches. 85 per cent along with the highest total carotenoids (12.17 mg/100g). However, in other media the yield and carotenoid content were significantly decreased. Increase in the quantity of apple pomace to 30 % and subsequently to 60% (M7 to M12) and reducing the dextrose level to 0.25% decreased all the parameters examined. Going by the results, it is also apparent that concentration of apple pomace of more than 20 g/l is supporting neither biomass nor pigment production. Therefore, it seems that one or more ingredients of apple pomace might be inhibitory in the medium where apple pomace was used at a concentration higher than 20 g/l. Therefore, a concentration of 20 g/l of apple pomace powder was considered optimum for Carotenoids Carotenoid contents were determined as per the procedure given by Ranganna 21. One gram of the sample was dissolved in the petroleum ether and ground till whole of the colour was extracted, followed by the addition of sodium sulphate (3%). The separated coloured portion of the solvent was collected and the final volume was made as specified. Optical density of the extract was taken at 449 nm and the quantity of carotenoids was calculated from the standard curve. Concentration x final volume x dilution μg of carotenoids = x 100 Weight of sample Results and Discussion Isolation and identification of microorganisms from agar plates The yellow colour producing colonies from water samples were isolated. Different morphological and biochemical tests were performed on the isolates. The isolates that was Gram+ve, dark yellow, without motility, catalase and oxidase positive, negative for acid production, without utilization of glycerol, mannose, lactose, capable of hydrolysis of gelatin and nitrate, lacked in arginine dehydrolase, produce Simmon s citrate, no growth with 7.5% NaCl was identified as Sarcina as per Bergy s 22. Optimization of apple pomace based media It is clear from Figure 1 that the media M1 to M4 and M6 showed better biomass and carotenoids production by Sarcina than the others. It is also evident that as the quantity of apple pomace was increased, the growth of pigment producing microorganism declined. The highest growth was supported by M6 medium where the quantity of apple pomace was 20 g/l and the surface area covered on petri plate on this media was Fig.1 Optimization of apple pomace based media for the pigment production using Sarcina sp.

4 424 INDIAN J NAT PROD RESOUR, DECEMBER 2011 incorporation to the basic media for the highest growth and pigment production by Sarcina. Reducing the glucose content from 1% to 0.5% did not affect the pigment production but lowering the glucose content to 0.25% also reduced the biomass as well as carotenoid production. In our earlier work on Chromobacter and Micrococcus also a concentration of apple pomace of 20 g/l was found to be optimum for growth and pigment production. The variation can be attributed to the ability of different microorganisms to use different sources of carbon, depending upon the biochemical pathway and the enzyme system of specific microorganism. Clearly, the use of apple pomace has reduced the use of other ingredients to make the medium cheaper that has supported the growth of pigments producing microorganisms. Effect of different carbon sources and their concentrations There were significant differences among various carbon sources for yield and carotenoids production (Table 2). The maximum yield of biomass was recorded in medium with glucose (8.02 g/l) followed by fructose (7.23 g/l). However, the carotenoid yield was statistically at par in glucose and fructose having mg/100 g, and mg/100 g, respectively. The use of maltose produced the lowest quantity of biomass. Among various concentrations of glucose (range 0.1 to 0.5%), the highest yield (8.02 g/l) was observed in 0.1% concentration of glucose with carotenoid content of mg/100 g (Figure 2). But increase in the concentration of the carbon source continuously reduced both the yield of biomass and pigment. An appraisal of the results on the type of sugar used in the media showed that although the yield of biomass was drastically different the carotenoid content remained almost similar though statistically different. This shows that the type of sugar influenced the growth of Sarcina than the pigment production. In our earlier work on pigment production by Micrococcus highest yield of biomass was recorded in medium with fructose (0.1%) and it increased up to 0.2% of fructose, then declined 10. Different microorganisms have different ability to use specific carbon source within certain concentration range and this has exactly observed in this study also 22. Effect of different nitrogen sources and their concentration Effect of different nitrogen sources on the yield and carotenoids (Table 3) revealed that the highest yield of biomass (12.93 g/l) and carotenoids production (12.80 mg/100 g) took place where potassium nitrate was used Table 2 Effect of carbon source on the yield and carotenoid production by Sarcina sp. Carbon source (0.1%) Yield of biomass (g/l) Carotenoid (mg/100 g) dwb Glucose Fructose Lactose Maltose CD (P<0.05) dwb = Dry weight basis Fig. 2 Effect of different glucose concentrations on the yield and carotenoids production by Sarcina sp.

5 JOSHI et al.: OPTIMIZATION OF APPLE POMACE FOR PIGMENT PRODUCTION BY SARCINA SP. 425 followed by sodium nitrate (12.23 mg/l) and ammonium sulphate (11.97 g/l). To produce biomass and pigment using apple pomace in solid state fermentation, sodium nitrate was found to be the best for Micrococcus 10 while for Rhodotorula it was ferrous ammonium sulphate 11. The differences are attributed to the ability of microorganism to use different nitrogen sources, using specific pathway. Among various concentrations of potassium nitrate, there was a significant increase in the yield (from mg/l to mg/100ml) and carotenoids up to 0.3%, after which both the yield and carotenoids production decreased. The lowest yield was recorded in the medium with 0.5% concentration of potassium nitrate. Decrease in yield at higher nitrogen levels could be attributed to the toxicity of nitrogen source at higher concentration. So 0.3% concentration of potassium nitrate has been found optimum for the growth of Sarcina sp. (Figure 3). Table 3 Effect of nitrogen source on the yield and carotenoid production by Sarcina sp. Nitrogen source (0.1%) Yield of biomass (g/l) Carotenoid (mg/100 g) dwb Ferrous ammonium sulphate Ammonium sulphate Sodium nitrate Peptone Potassium nitrate Control CD (P<0.05) dwb = Dry weight basis Effect of temperature and ph on growth and pigment production The results (Table 4) shown that the maximum biomass yield (6.14 g/l) and quantity of pigment produced by (12.61 g/l) Sarcina occurred at a temperature of 35 o C while the lowest growth took place at a temperature of 25 o C (1.45 g/l). Since the optimum temperature for growth and development of Sarcina has been reported as 35 o C 22, it could be a reason for better growth and pigmentation at 35 C in our study. For production of pigment by Micrococcus in solid state fermentation on the apple pomace medium a temperature of 35 C was found to be optimum. It is apparently related to the specific type of microorganism involved wherein the specific microorganism is having specific temperature of growth. Significant differences were found both in yield and carotenoid production by Sarcina at different ph values of the media. Perusal of data (Table 4) further revealed that at ph 3 and 3.5, there was no growth of Sarcina at all and hence, no pigment production took place. The highest growth and pigmentation was observed at ph 5.5 where the yield was recorded as 6.50 g/l and carotenoid quantity as mg/100 g. Both the yield and carotenoid were statistically at par with that produce in the medium with ph 6.0. At ph 4.0 and below, the growth of bacteria except acid tolerant bacteria is very less or negligible. So no growth or pigment production by Sarcina sp. at ph value 3.0 and 3.5 could be attributed to unfavourable ph value. It was 6.0 ph that was found optimum for Fig. 3 Effect of different concentrations of potassium nitrate on the yield and carotenoids production by Sarcina sp.

6 426 INDIAN J NAT PROD RESOUR, DECEMBER 2011 Table 4 Effect of temperature, ph and incubation period on the yield and carotenoid production by Sarcina sp. Treatments Yield of biomass (g/l) Carotenoid (mg/100 g) dwb Temperature ( o C) CD (P<0.05) ph CD (P<0.05) Incubation period (hrs) CD (P<0.05) dwb = Dry weight basis Micrococcus for pigment and biomass production 10 thus, Sarcina can be best grown at ph 5.5. A study on the incubation period ranging from 24 h to 96 h for Sarcina sp. revealed that the maximum yield and pigmentation of organism occurred at 72 h of incubation period (5.70 g/l). The lowest yield was recorded at 24 h of incubation period (4.80 g/l). However, the yield and carotenoid remained at par at 72 and 96 h of incubation periods. Therefore, 72 h of incubation period was considered optimum both for the growth and pigment production by Sarcina sp. In our early work with pigment production by Rhodototrula and Micrococcus, an incubation period of 72 h was found optimum. The maximum growth was also recorded at this incubation period 11,13,18. However, for Chromobacter maximum incubation period was found 48 h only 10. Conclusion The growth of Sarcina sp. as a source of microbial pigment on apple pomace based medium was found to be promising which in addition to producing value added product (microbial colours) could also reduce environmental pollution as apple pomace would be utilized and not thrown out as per the present practice. The optimum quantity of apple pomace for use in the medium was found to be 20 g/l, supplemented with urea 0.1% glucose as a carbon sources and 0.3% potassium nitrate as nitrogen source for the highest yield of biomass and carotenoids. An incubation period of 72 h and ph of 5.5 were optimum for yield of yellow colour pigment by Sarcina sp. The results clearly show the potential of utilizing apple pomace using Sarcina sp. in solid state fermentation with the conditions optimized. References 1 Khanna SK and Dass M, Toxicity, carcinogenic potential and clinical epidemiological studies on dyes intermediates, J Sci. Industr Res, 1991, 50, Tanaka T, Responsive and neurobehavioral toxicity study of tartrazine administered to mice with diet, Food Chem, Toxicol, 2008, 44, Sharma A, Goyal RP, Chakravarty G and Sharma S, Homotoxic effect of chocolate brown, a commonly used blend of permitted food colours on Swiss Albino mice. Asian Exp Med, 2008, 19, Kaur M, Arora S and Katnoria JK, Evaluation of mutagenic potential of food dye (Apple green), Indian J Sci Technol, 2010, 3(12), Olson JH, Provitamin-A function of carotenoids: The conversion of Beta-carotene into vitamin A, J Nutr, 1989, 119, Johnson EA and Schroeder WA, Microbial carotenoids Adv Biochem Engg Biotechnol, 1995, 53, Burton G W, Antioxidant action of carotenoids, J Nutr, 1991, 119, Bendich A, β-carotene and the immune response, Proceedings of the Nutrition Society, 1991, 50, Joshi VK, Attri D, Anju Bala and Shashi Bhushan, Microbial pigments, Indian J Biotechnol, 2003, 2, Attri D and Joshi VK, Optimization of apple pomace based medium and fermentation conditions for pigment production by Micrococcus species, J Sci Industr Res, 2005, 64, Joshi VK and Attri D, Optimization of apple pomace based medium and fermentation conditions for pigment production by Rhodotorula species, Proc Nat Acad Sci India, 2006, 76 (B) II, Lin TF and Demain AL, Effect of nutrition of Monascus species on formation of red pigment, Applied Microbiol Biotechnol, 1991, 36, Sandhu DK and Joshi VK, Development of apple pomace based medium, optimizing pigment production by Rhodotorula and its characterization, Adv Food Sci (CMTL), 1997, 19, Sargent SA, Steffe JF and Pierson TR, The economic feasibility in plant combustion of apple processing wastes, Agric Wastes, 1986, 15(2), Wang HJ and Thomas RL, Direct use of apple pomace in baking products, J Food Sci, 1989, 54(3), Joshi VK, Apple pomace utilization-present status and future strategies, In: Advances in Biotechnology, Ashok Pandey

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