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Production optimization, purification and toxicological assessment of extracellular red pigment from monascus purpureus in submerged culture
Journal of Biotechnology 136S (2008) S743–S750
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Abstracts
VIII-2 Genetically modified foods
VIII2-O-001 Traditional fermented foods: A case study of microbiological and biochemical characterization of bhatooru, seera and siddu of Himachal Pradesh, India Tek Chand Bhalla ∗ , Savitri, Navdeep Thakur Department of Biotechnology, H P University, Shimla 171005, Himachal Pradesh, India E-mail address: [email protected] (T.C. Bhalla). India represents great diversity in climate, ethnic populations, natural resources and religions vis-à-vis versatility in the preparation and consumption of fermented foods and beverages. In Himachal Pradesh, a number of traditional fermented foods and beverages are popular which are unique in comparison to other parts of the country. Bhatooru, chilra, seera, siddu, gulgule, marchu, sepubari, pickles (of local fruits and vegetables) and fermented beverages (kinnauri, chhang, sura, behmii, etc.) are some of the indigenous fermented products of Himachal Pradesh (Savitri and Bhalla, 2007). The microbiological and biochemical studies of cereal based fermented foods (bhatooru, seera and siddu) were performed in order to identify micro flora, changes in enzymatic profiles, and nutritional enhancements during fermentation. Saccharomyces cerevisiae, Lactobacillus sp. and Leuconostoc sp. dominated in bhatooru/siddu and seera fermentation. Besides these organisms, Bacillus sp., Kocuria rhizophila, Enterobacter sakazakii, Pseudomonas sp., Staphylococcus sp. etc. were also isolated from the fermented samples. The increase in microbial load during fermentation declined pH of bhatooru/siddu dough and seera from 5.94 to 4.18 and 6.98 to 3.45 respectively. The fermentation in bhatooru/siddu preparation was initiated with the addition of malera (traditional inoculum) to wheat flour dough, which resulted in increase of total proteins from 13.6% to 18.4% (w/w), decrease of total sugars/carbohydrates and starch content from 74.1% to 50.1% (w/w) and 70.2% to 48.3% (w/w), respectively on dry weight basis. The reducing sugar level of the fermented dough increased from 7.8 mg/g to 10.0 mg/g of dry matter. Initial rise in protease and amylase activity in 6 h of incubation was observed which finally declined as fermentation proceeded. By studying the organoleptic characteristics, bhatooru prepared by using 6% (w/w) malera was observed to have high score in taste, aroma and general acceptability. In seera preparation natural fermentation of whole wheat grain was allowed to proceed in tap water resulting in decrease of total protein and sugars contents, however, increase in starch content
0168-1656/$ – see front matter
was observed. Amylase and protease activity increased in initial stages of fermentation but later activities of both the enzymes decreased. Effect of different amount of water used for steeping of wheat was studied and 1:5 ratio of wheat grains to water resulted in maximum yield. A significant increase in vitamins (thiamine, riboflavin, nicotinic acid, riboflavin, and cyanocobalamin), essential amino acids (Met, Phe, Thr, Lys, and Leu) was observed during fermentation of bhatooru/siddu/seera. Best bhatooru (in terms of increased protein content, vitamins, essential amino acids, good taste, aroma, appearance and overall acceptability) was obtained by using costarters i.e. C1 (S. cerevisiae + L. plantarum + L. brevis) and C2 (T. delbrueckii + L. plantarum + L. brevis). The fermented products have higher amount of proteins, vitamins and essential amino acids and are nutritionally better as compared to non-fermented food products. Reference Savitri, Bhalla, T.C., 2007. Traditional foods and beverages of Himachal Pradesh. Ind. J. Trad. Knowl. 6, 17–24.
doi:10.1016/j.jbiotec.2008.07.1769 VIII2-O-005 Production optimization, purification and toxicological assessment of extracellular red pigment from monascus purpureus in submerged culture Sandipan Chatterjee 1 , Sharmistha Maity 1 , Subhasita Roy 1 , Pritam Chattopadhyay 1 , Angshuman Sarkar 2 , Sukanta Kumar Sen 1,∗ 1
Microbiology Division, School of Life Sciences, Department of Botany, Visva-Bharati University, Santiniketan 731 235, India 2 Department of Statistics, Visva-Bharati University, Santiniketan 731 235, India E-mail address: [email protected] (S.K. Sen).
The use of additives in food plays a significant role to enhance its quality, hence, acceptability (Mapari et al., 2005). Out of 29 approved food colours, 16 are synthesized from petrochemicals and their longterm intake is a matter of great concern. The rice fermented with Monascus sp. has been in vogue in East Asian countries. Monascus purpureus, a potent red pigment producing fungus was optimized for its growth and pigment yield by varying the physical
S744
Abstracts / Journal of Biotechnology 136S (2008) S743–S750
and nutritional parameters in submerged culture (Carvalho et al., 2003). The pigment complex was extracted (with soxhletor) and purified by column chromatography to obtain the red pigment, monascorubramine. Spectrophotometric analysis showed a consolidated peak at 500 nm (Carvalho et al., 2005). Red pigment was maximally produced at 20 g/l of glucose. Monosodium glutamate at 0.3% as nitrogen source was found optimal for red pigment (72 U/g dry cell mass). Antibacterial activity and toxicity of the purified pigment were tested. It was observed that the purified red fraction was active only against Gram positive bacteria. Feeding trials did not produce any significant symptoms of toxicity or mortality either immediately or in post feeding period. Data were statistically analyzed through a second-degree curve, y = a + b*x + c*x2 where, was the independent variable and y was the dependent variable (pigment yield), using MATLAB 6.1 (The MathWorks, Inc. Massachusetts) and found significant.
Jongrungruang, S., Kittikoop, P., Yongsmith, B., Bavavoda, R., Tanasupawat, S., Boonuclomlap, U., Thebtaranonth, Y., 2004. Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang. Phytochemistry 65, 2569–2575. Kalyarat, K. 2005. Personal Communication. Yongsmith, B., Chitradon, L., Krairak, S., Tabloka, W., Bavavoda, R., 1991. Cassawa fermentation of yellow pigments and amylolytic enzymes of a mutant of Monascus spp. in submerge cultivation. Microb. Util. Renewable Resour. 7, 354–363. Yongsmith, B., Krairak, S., Bavavoda, R., 1994. Production of yellow pigments in submerged culture of a mutant of Monascus spp. J. Ferment. Bioeng. 78, 223–228.
References
doi:10.1016/j.jbiotec.2008.07.1771
Carvalho, J.C., Pandey, A., Babitha, S., Soccol, C.R., 2003. Production of Monascus biopigments: an overview. Agrofood Ind. Hi-tech. 14, 37–42. Carvalho, J.C., Oishi, B.O., Pandey, A., Soccol, C.R., 2005. Biopigments from Monascus: strain selection, citrinin production and color stability. Braz. Arch. Biol. Technol. 48, 885–894. Mapari, S.A.S., Neilson, K.F., Larsen, T.O., Frisvad, J.C., Meyer, A.S., Thane, U., 2005. Exploring fungal biodiversity for the production of water soluble pigments as potential natural food colorants. Curr. Opin.Biotechnol. 16, 231–238.
yellow mutant, produced unique and innovative UV-absorbing yellow compounds with their strong antimutagenic and antioxidant characteristics should lend globally some parts in protecting various kind of market products (food and non food) from deterioration. References
VIII2-Y-002 Evaluation of lactic acid bacteria dynamics during plaa-som fermentation using amplified 16S ribosomal DNA restriction analysis Phikunthong Kopermsub 1,2,∗ , Sirinda Yunchalard 2,3 1
doi:10.1016/j.jbiotec.2008.07.1770 VIII2-O-014 Fermentation of functional monascus yellow pigments on rice solid culture Busaba Yongsmith 1,∗ , Kaew Kangsadalampai 2 1
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 2 Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand E-mail address: [email protected] (B. Yongsmith). This paper communicates our new finding on the secondary Monascus yellow mutants which are capable on producing high yellow pigmentation at single wavelength (max 370 nm) instead of red pigments at multi wavelength (max 420, 500 nm) produced by its parents in 1990 (Yongsmith et al., 1991). One yellow mutant, strain KB20M10.2 could efficiency synthesize yellow pigments in both submerged fermentation using cassava starch with soybean flour (Yongsmith et al., 1994), and in solid state fermentation using rice or cereal products as substrate. In rice solid culture, yellow, tangerine or tan (brown) rice are produced which mainly affected by rice treatment preparation, rice variety, initial pH, and initial moisture content. Maximum yellow pigments of rice solid culture at room temperature for 15 days is 2000 U/gdw. No citrinin or aflatoxin could be detected. Crude ethanol yellow pigment extracts could serve not only as an efficient yellow colourant but also as an effective antimutagenic substance using Ames and fruit fly tests. In addition, two new purified azaphilones pigments (monascusone A and monascusone B) were isolated from the CH2 Cl2 extract of yellow rice solid culture together with known azaphilones, monascin and FK 17-P2 b2 (Jongrungruang et al., 2004). Moreover, either crude ethanol yellow extract or its key purified yellow pigment (monascusone A) could serve as strong antioxidant analyzed by FRAP or DPPH assay (Kalyarat, 2005). Our finding suggested that Thai local strain Monascus kaoliang KB20M10.2, the new potent
Department of Biotechnology, Khon Kaen University, Khon Kaen 40002, Thailand 2 Graduate School, Khon Kaen University, Khon Kaen 40002,Thailand 3 Fermentation Research Center for Value Added Agricultural Products (FerVAAP), Khon Kaen University, Khon Kaen 40002, Thailand E-mail addresses: choco [email protected] (P. Kopermsub), [email protected] (S. Yunchalard). Predominant lactic acid bacteria (LAB) species and their dynamics during the fermentation process of plaa-som were determined. Samples were taken during the production process at the following steps and times: instantaneously after mixing (sample 1); 6 h after mixing (sample 2); after freezing at −18 ◦ C and packing (sample 3); and at 24 h intervals during a fermentation at 30 ◦ C for 144 h (samples 4–9). The LAB isolates were recovered from MRS-CaCO3 agar plate inhabiting from these samples and subsequently identified using amplified 16S ribosomal DNA restriction analysis and sequence analysis. Six groups (A–F) of the restriction digestion pattern were revealed among 762 LAB isolates. It was not possible to conclude which LAB strain was the predominant species in sample 1. Thereafter, from samples 2 to 3, Lactococcus garvieae, Streptococcus bovis, and Weissella cibaria were recovered of which Lc. garvieae was revealed as the predominant species. From samples 4 to 7 Lc. garvieae continued to be recovered but in declining numbers, while S. bovis could no longer be recovered. In contrast, W. cibaria was recovered at a higher number and became the predominant species in sample 4. The number of W. cibaria started to decline continually from samples 6 to 9. Sample 5 exhibited mixed LAB species with no significant difference in numbers between the main species recovered including W. cibaria, Pediococcus pentosaceus, and Lactobacillus plantarum. The mixed LAB species found in sample 5 continued to be recovered in samples 6–9 however, Lb. plantarum was found to be the predominant species. Additionally, low numbers of Lb. fermentum were recovered from samples 6–8. The results obtained indicated dynamics of predominant LAB species during plaa-som fermentation including Lc. garvieae, S. bovis, W. cibaria, P. pentosaceus, and Lb. plantarum. This provides useful information for further development of plaa-som starters.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Abstracts
VIII-2 Genetically modified foods
VIII2-O-001 Traditional fermented foods: A case study of microbiological and biochemical characterization of bhatooru, seera and siddu of Himachal Pradesh, India Tek Chand Bhalla ∗ , Savitri, Navdeep Thakur Department of Biotechnology, H P University, Shimla 171005, Himachal Pradesh, India E-mail address: [email protected] (T.C. Bhalla). India represents great diversity in climate, ethnic populations, natural resources and religions vis-à-vis versatility in the preparation and consumption of fermented foods and beverages. In Himachal Pradesh, a number of traditional fermented foods and beverages are popular which are unique in comparison to other parts of the country. Bhatooru, chilra, seera, siddu, gulgule, marchu, sepubari, pickles (of local fruits and vegetables) and fermented beverages (kinnauri, chhang, sura, behmii, etc.) are some of the indigenous fermented products of Himachal Pradesh (Savitri and Bhalla, 2007). The microbiological and biochemical studies of cereal based fermented foods (bhatooru, seera and siddu) were performed in order to identify micro flora, changes in enzymatic profiles, and nutritional enhancements during fermentation. Saccharomyces cerevisiae, Lactobacillus sp. and Leuconostoc sp. dominated in bhatooru/siddu and seera fermentation. Besides these organisms, Bacillus sp., Kocuria rhizophila, Enterobacter sakazakii, Pseudomonas sp., Staphylococcus sp. etc. were also isolated from the fermented samples. The increase in microbial load during fermentation declined pH of bhatooru/siddu dough and seera from 5.94 to 4.18 and 6.98 to 3.45 respectively. The fermentation in bhatooru/siddu preparation was initiated with the addition of malera (traditional inoculum) to wheat flour dough, which resulted in increase of total proteins from 13.6% to 18.4% (w/w), decrease of total sugars/carbohydrates and starch content from 74.1% to 50.1% (w/w) and 70.2% to 48.3% (w/w), respectively on dry weight basis. The reducing sugar level of the fermented dough increased from 7.8 mg/g to 10.0 mg/g of dry matter. Initial rise in protease and amylase activity in 6 h of incubation was observed which finally declined as fermentation proceeded. By studying the organoleptic characteristics, bhatooru prepared by using 6% (w/w) malera was observed to have high score in taste, aroma and general acceptability. In seera preparation natural fermentation of whole wheat grain was allowed to proceed in tap water resulting in decrease of total protein and sugars contents, however, increase in starch content
0168-1656/$ – see front matter
was observed. Amylase and protease activity increased in initial stages of fermentation but later activities of both the enzymes decreased. Effect of different amount of water used for steeping of wheat was studied and 1:5 ratio of wheat grains to water resulted in maximum yield. A significant increase in vitamins (thiamine, riboflavin, nicotinic acid, riboflavin, and cyanocobalamin), essential amino acids (Met, Phe, Thr, Lys, and Leu) was observed during fermentation of bhatooru/siddu/seera. Best bhatooru (in terms of increased protein content, vitamins, essential amino acids, good taste, aroma, appearance and overall acceptability) was obtained by using costarters i.e. C1 (S. cerevisiae + L. plantarum + L. brevis) and C2 (T. delbrueckii + L. plantarum + L. brevis). The fermented products have higher amount of proteins, vitamins and essential amino acids and are nutritionally better as compared to non-fermented food products. Reference Savitri, Bhalla, T.C., 2007. Traditional foods and beverages of Himachal Pradesh. Ind. J. Trad. Knowl. 6, 17–24.
doi:10.1016/j.jbiotec.2008.07.1769 VIII2-O-005 Production optimization, purification and toxicological assessment of extracellular red pigment from monascus purpureus in submerged culture Sandipan Chatterjee 1 , Sharmistha Maity 1 , Subhasita Roy 1 , Pritam Chattopadhyay 1 , Angshuman Sarkar 2 , Sukanta Kumar Sen 1,∗ 1
Microbiology Division, School of Life Sciences, Department of Botany, Visva-Bharati University, Santiniketan 731 235, India 2 Department of Statistics, Visva-Bharati University, Santiniketan 731 235, India E-mail address: [email protected] (S.K. Sen).
The use of additives in food plays a significant role to enhance its quality, hence, acceptability (Mapari et al., 2005). Out of 29 approved food colours, 16 are synthesized from petrochemicals and their longterm intake is a matter of great concern. The rice fermented with Monascus sp. has been in vogue in East Asian countries. Monascus purpureus, a potent red pigment producing fungus was optimized for its growth and pigment yield by varying the physical
S744
Abstracts / Journal of Biotechnology 136S (2008) S743–S750
and nutritional parameters in submerged culture (Carvalho et al., 2003). The pigment complex was extracted (with soxhletor) and purified by column chromatography to obtain the red pigment, monascorubramine. Spectrophotometric analysis showed a consolidated peak at 500 nm (Carvalho et al., 2005). Red pigment was maximally produced at 20 g/l of glucose. Monosodium glutamate at 0.3% as nitrogen source was found optimal for red pigment (72 U/g dry cell mass). Antibacterial activity and toxicity of the purified pigment were tested. It was observed that the purified red fraction was active only against Gram positive bacteria. Feeding trials did not produce any significant symptoms of toxicity or mortality either immediately or in post feeding period. Data were statistically analyzed through a second-degree curve, y = a + b*x + c*x2 where, was the independent variable and y was the dependent variable (pigment yield), using MATLAB 6.1 (The MathWorks, Inc. Massachusetts) and found significant.
Jongrungruang, S., Kittikoop, P., Yongsmith, B., Bavavoda, R., Tanasupawat, S., Boonuclomlap, U., Thebtaranonth, Y., 2004. Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang. Phytochemistry 65, 2569–2575. Kalyarat, K. 2005. Personal Communication. Yongsmith, B., Chitradon, L., Krairak, S., Tabloka, W., Bavavoda, R., 1991. Cassawa fermentation of yellow pigments and amylolytic enzymes of a mutant of Monascus spp. in submerge cultivation. Microb. Util. Renewable Resour. 7, 354–363. Yongsmith, B., Krairak, S., Bavavoda, R., 1994. Production of yellow pigments in submerged culture of a mutant of Monascus spp. J. Ferment. Bioeng. 78, 223–228.
References
doi:10.1016/j.jbiotec.2008.07.1771
Carvalho, J.C., Pandey, A., Babitha, S., Soccol, C.R., 2003. Production of Monascus biopigments: an overview. Agrofood Ind. Hi-tech. 14, 37–42. Carvalho, J.C., Oishi, B.O., Pandey, A., Soccol, C.R., 2005. Biopigments from Monascus: strain selection, citrinin production and color stability. Braz. Arch. Biol. Technol. 48, 885–894. Mapari, S.A.S., Neilson, K.F., Larsen, T.O., Frisvad, J.C., Meyer, A.S., Thane, U., 2005. Exploring fungal biodiversity for the production of water soluble pigments as potential natural food colorants. Curr. Opin.Biotechnol. 16, 231–238.
yellow mutant, produced unique and innovative UV-absorbing yellow compounds with their strong antimutagenic and antioxidant characteristics should lend globally some parts in protecting various kind of market products (food and non food) from deterioration. References
VIII2-Y-002 Evaluation of lactic acid bacteria dynamics during plaa-som fermentation using amplified 16S ribosomal DNA restriction analysis Phikunthong Kopermsub 1,2,∗ , Sirinda Yunchalard 2,3 1
doi:10.1016/j.jbiotec.2008.07.1770 VIII2-O-014 Fermentation of functional monascus yellow pigments on rice solid culture Busaba Yongsmith 1,∗ , Kaew Kangsadalampai 2 1
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 2 Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand E-mail address: [email protected] (B. Yongsmith). This paper communicates our new finding on the secondary Monascus yellow mutants which are capable on producing high yellow pigmentation at single wavelength (max 370 nm) instead of red pigments at multi wavelength (max 420, 500 nm) produced by its parents in 1990 (Yongsmith et al., 1991). One yellow mutant, strain KB20M10.2 could efficiency synthesize yellow pigments in both submerged fermentation using cassava starch with soybean flour (Yongsmith et al., 1994), and in solid state fermentation using rice or cereal products as substrate. In rice solid culture, yellow, tangerine or tan (brown) rice are produced which mainly affected by rice treatment preparation, rice variety, initial pH, and initial moisture content. Maximum yellow pigments of rice solid culture at room temperature for 15 days is 2000 U/gdw. No citrinin or aflatoxin could be detected. Crude ethanol yellow pigment extracts could serve not only as an efficient yellow colourant but also as an effective antimutagenic substance using Ames and fruit fly tests. In addition, two new purified azaphilones pigments (monascusone A and monascusone B) were isolated from the CH2 Cl2 extract of yellow rice solid culture together with known azaphilones, monascin and FK 17-P2 b2 (Jongrungruang et al., 2004). Moreover, either crude ethanol yellow extract or its key purified yellow pigment (monascusone A) could serve as strong antioxidant analyzed by FRAP or DPPH assay (Kalyarat, 2005). Our finding suggested that Thai local strain Monascus kaoliang KB20M10.2, the new potent
Department of Biotechnology, Khon Kaen University, Khon Kaen 40002, Thailand 2 Graduate School, Khon Kaen University, Khon Kaen 40002,Thailand 3 Fermentation Research Center for Value Added Agricultural Products (FerVAAP), Khon Kaen University, Khon Kaen 40002, Thailand E-mail addresses: choco [email protected] (P. Kopermsub), [email protected] (S. Yunchalard). Predominant lactic acid bacteria (LAB) species and their dynamics during the fermentation process of plaa-som were determined. Samples were taken during the production process at the following steps and times: instantaneously after mixing (sample 1); 6 h after mixing (sample 2); after freezing at −18 ◦ C and packing (sample 3); and at 24 h intervals during a fermentation at 30 ◦ C for 144 h (samples 4–9). The LAB isolates were recovered from MRS-CaCO3 agar plate inhabiting from these samples and subsequently identified using amplified 16S ribosomal DNA restriction analysis and sequence analysis. Six groups (A–F) of the restriction digestion pattern were revealed among 762 LAB isolates. It was not possible to conclude which LAB strain was the predominant species in sample 1. Thereafter, from samples 2 to 3, Lactococcus garvieae, Streptococcus bovis, and Weissella cibaria were recovered of which Lc. garvieae was revealed as the predominant species. From samples 4 to 7 Lc. garvieae continued to be recovered but in declining numbers, while S. bovis could no longer be recovered. In contrast, W. cibaria was recovered at a higher number and became the predominant species in sample 4. The number of W. cibaria started to decline continually from samples 6 to 9. Sample 5 exhibited mixed LAB species with no significant difference in numbers between the main species recovered including W. cibaria, Pediococcus pentosaceus, and Lactobacillus plantarum. The mixed LAB species found in sample 5 continued to be recovered in samples 6–9 however, Lb. plantarum was found to be the predominant species. Additionally, low numbers of Lb. fermentum were recovered from samples 6–8. The results obtained indicated dynamics of predominant LAB species during plaa-som fermentation including Lc. garvieae, S. bovis, W. cibaria, P. pentosaceus, and Lb. plantarum. This provides useful information for further development of plaa-som starters.