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Combination of in vitro intestinal continuous fermentation and intestinal cell models to investigate Salmonella and probiotics interactions in infected gut microbiota
S316
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
indicated that the methanol extracts and the derived phytochemicals from CPTo and CPS are great potential sources as natural antioxidant agents. doi:10.1016/j.jbiotec.2010.09.296 [P-F.53] Biogenic amines degradation by moderate halophile, Brevibacillus sp. SK35 Sornchai Sinsuwan 1,∗ , Aungkawipa 2 Rodtong , Jirawat Yongsawatdigul 1
Montriwong 1 , Sureelak
1
School of Food Technology, Suranaree University of Technology, Thailand 2 School of Microbiology, Suranaree University of Technology, Thailand Keywords: biogenic amine; reduction; Brevibacillus sp.; fish sauce
Biogenic amines (BAs) are widely found in fermented foods and exert to adverse effects on the central nervous and vascular systems of human. The level of BAs in food was normally regulated. We attempted to investigate the potential of bacterial strain isolated from fish sauce fermentation in biogenic amine reduction. In addition, optimum conditions for BAs reduction of the selected strain and partial characterization of the amine oxidase produced by such a strain were elucidated. Among 7 strains, namely Virgibacillus sp. (SK33 and SK37), Staphylococcus sp. (SK1-1-5 and SKM20-7-9), Brevibacillus sp. SK35, Halomonas sp. SKW12, and Corynebacterium sp. SK1-1-6, were tested for histamine reduction capabilities and BAs production. All strains produced BAs, namely typtamine, tyramine, spermine, and spermidine, in the similar level. Brevibacillus sp. SK35 was able to reduce histamine at 99%, whereas histamine degradation of other stains was about 0-26%. The strain reduced simple amines to a greater extent than the polyamines with reduction order of putrescine > cadaverine > tyramine > tryptamine > histidine > spermidine > spermine. Optimum condition of BAs reduction was at 35 ◦ C, pH 8, and 10% NaCl when cultivated in the modified MØller broth. Amine oxidase was localized in intracellular with molecular weight of 45 kDa based on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) activity staining using various BAs as substrates. Optimum conditions of the enzyme were at 35 ◦ C and pH 7-8. doi:10.1016/j.jbiotec.2010.09.297 [P-F.54] Study about the addition of chemically modified starches (crosslinked cornstarches), dextrins, and oats fiber in baked pound cake M.E. Sánchez-Pardo ∗ , E. Jiménez-García, I. González-García Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería Bioquímica. Plan de Ayala y Prolongación de Carpio, Casco de Santo Tomás. C. P. 11340., Mexico Keywords: chemically modified starches; dextrins; pound cake; oats fiber The pound cake is one of the sweet sub-products of breadmaking, the modified starches contribute to the diet the same calories that the native starches, but have advantages in baking, since its functionality is important as well as the addition of the oats fiber. dextrins are used to obtaining a healthy product and good quality. It is so also justified the incorporation of fiber oatmeal due to their functional properties and health benefits.
The objective of this work was to evaluate the influence of these additives in baking products such as pound cake. In this study flour of wheat, chemically modified starches (cross-linked cornstarches), dextrins, and oats fiber were used. The wheat flour used to elaborate the pound cake had ash content 0.3-0.5 g/100 g sample and 12 g protein/100 g sample. The pound cake density was determined by the ratio mass/volume, mass was obtained by weighing pound cakes in an analytical scale an hour after baking. The volume “v” of each cake was obtained by the rape seed displacement method. Sensory evaluation consisted of a hedonic test of seven points using 100 untrained panelists. The results were evaluated with a Friedman bilateral variance rank analysis. Pound cake elaborated with the incorporation of modified starches, dextrin and fiber of oats presented higher volume (12%) and lower density, as well as a distribution of the alveoli most homogenous (a = 0.89). Sensorial judge indicated that the product added with the additive mixture (10 g dextrins/100 g; 25 g chemically modified starches/100 g; and 25 g oats fiber/100 g) presented better characteristics than conventional one. The acceptance level was “likes much”. This study focuses on the effect of a modified starches (crosslinked cornstarches), dextrins, and oats fiber on the properties of pound cake baked, these additives improved several quality parameters. doi:10.1016/j.jbiotec.2010.09.298 [P-F.55] Combination of in vitro intestinal continuous fermentation and intestinal cell models to investigate Salmonella and probiotics interactions in infected gut microbiota A. Zihler 1,∗ , M. Gagnon 1 , C. Chassard 1 , C.P. Braegger 2 , C. Lacroix 1 1
ETH Zurich, Switzerland University Children’s Hospital Zurich, Switzerland Keywords: In vitro continuous fermentation model; HT29-MTX intestinal epithelial cell line; Salmonella; Probiotics 2
Salmonellosis is a major public health concern with a significant cost in many countries. Imposed by an increasing prevalence of multi-drug resistant strains, biological alternatives for the treatment of Salmonella infections are needed. We combined two original in vitro models closely mimicking the complex in vivo intestinal situation to investigate the therapeutic potential and mechanisms of two promising bacteriocinogenic probiotics, Bifidobacterium thermophilum RBL67 (thermophilicin B67) and Escherichia coli L1000 (microcin B17-producer), active against Salmonella. A recently developed continuous three-stage colonic fermentation model of S. Typhimurium infection with immobilized child fecal microbiota was used to simulate the proximal, transverse and distal human colonic sections. Reactor effluents were directly applied to intestinal mucus-secreting HT29-MTX cell-layers to measure first-level host responses after induction of infection and probiotic treatments. Stable pathogen invasion into HT29-MTX cells (1 ± 0.4%) was recorded after infection accompanied by decreased epithelium integrity (45% lower Trans Epithelial Electrical Resistance compared to non-infected control). After adding E. coli L1000, Salmonella invasion only decreased in the distal reactor (89%) while TEER did not change for all samples. In contrast, anti-Salmonella activity of B. thermophilum RBL67 was most pronounced in the proximal reactor with a 76%-decrease of pathogen invasion and TEER recovered close to levels of non-infected samples. Bio-Plex (Bio-Rad) analysis was used to measure cellular
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
S317
immune responses. Our results highlight the benefits to combine suitable cellular and colonic fermentation models to evaluate host protection activity of probiotics during Salmonella infection, providing efficient tools for mechanistic studies and preclinical development of new antimicrobials.
mulation by down-regulation of SREBP1 and FAS via adiponectin receptor AdipoR2 (Fig. 1). Acknowledgement: This work was supported by Grant-in-Aid for Scientific Research (No. 21580142) from the Ministry of Education, Science, Sports, and Culture in Japan.
doi:10.1016/j.jbiotec.2010.09.299
Reference Hosooka, et al., 2008. Nat. Med. 14, 188–193.
[P-F.56]
doi:10.1016/j.jbiotec.2010.09.300
Effect of Secoisolariciresinol on Adipogenesis T. Sugahara ∗ , S. Tominaga, S. Akiyama, S. Nishimoto
Yamauchi, M.
Maruyama, K.
Ehime University, Japan Keywords: Secoisolariciresinol; Flaxseed; Adipogenesis; adipocytes
[P-F.57] Study comparative of custard inoculated with bacteria lactic of Kefir in different kind of soy milk (Glicine max) M.E. Sánchez-Pardo ∗ , E. Jiménez-García, I. Solano-Ortiz
Introduction: Flaxseed (Linum usitatissimum L.) lignan, (+)secoisolariciresinol (SECO), (−)-SECO, and meso-SECO were stereoselectively synthesized and biological functions were evaluated. As a result, (−)-SECO suppressed triglyceride (TG) production in 3T3-L1 adipocytes. In this research, we focused on the effects of (−)-SECO on the regulation of adipogenesis in vitro and in vivo. Methods: Differentiation of 3T3-L1 pre-adipocytes into adipocytes was induced by the addition of insulin, dexamethasone and isobutyl-methylxanthine. Following differentiation, (−)-SECO was added to the medium, and the effects on adipocytes were examined. (−)-SECO was orally administrated to C57BL/6 mice with high-fat diet, and adipogenesis-relevant parameters including body weight and energy intake were examined. Results: (−)-SECO stimulated differentiation of 3T3-L1 preadipocytes by enhancement of PPAR␥ level, and suppressed TG accumulation in the cells. In addition, adiponectin production was facilitated by (−)-SECO. These facts suggest that (−)-SECO stimulates differentiation of 3T3-L1 pre-adipocytes by up-regulation of PPAR␥ expression and adiponectin production. The protein downstream of tyrosine kinases-1 (Dok1) is known to function as a multisite adapter molecule in insulin signalling, and promotes PPAR␥ expression (Hosooka et al., 2008). Dok1 expression was obviously enhanced by (−)-SECO within 6 h in 3T3-L1 preadipocytes. These facts suggest that (−)-SECO accelerated Dok1 expression, and Dok1 released PPAR␥ expression modulated by Ras cascade to facilitate adipocytes differentiation (Fig. 1). Increase in body weight and adipocytes weight of C57BL/6 mice fed with high-fat diet was significantly restricted by oral administration of (−)-SECO. In addition, mRNA expression for PPAR␥ and adiponectin was stimulated by intake of (−)-SECO. (−)-SECO intake enhanced serum adiponectin level, and adiponectin suppressed TG accu-
Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería Bioquímica. Plan de Ayala y Prolongación de Carpio, Casco de Santo Tomás. C. P. 11340. México, D. F., Mexico Keywords: soy milk; kefir granules; acceptance level; inoculated In Mexico there is a tendency to eat more nutritious dessert products because we have accustomed to eating food with sugar; custard has been reformulated as a dessert to get the lactic acid bacteria from kefir, and protein soybeans contribute to tasty into this food that cover the daily nutritional needs and are innovative in using soy milk. The aim of this work was the designing of custard made with commercial soy milk, soy milk powder and compare it with custard of cow’s milk; all inoculated with lactic acid bacteria of kefir in order to elaborate a functional food of high quality. We conducted a formulation that consisted of 65% milk, 24% egg, 9% sugar, 1.3% water, 0.9% corn starch and 0.4% essence of vanilla. Custard was inoculated with kefir granules before and after fermentation, and with the granules (106 UFC) to obtain a functional product through comparative study using different raw materials. A comparative study of these products was experimented with different kind of milks. The acceptance level was higher when the custard was elaborated with soy milk (“I like very much”) and soy milk industrialized (“I like”) prepared from commercial soybean meal, than cow’s milk. As well as the presence of yeasts was lower in the custard elaborated with milk fermented with kefir, regarding the custard inoculated directly with the kefir granules had higher presence of yeasts. Two hedonics scales 7 were used, which consisted of a tasting of the product without inoculate and another inoculated. The first, the taste of the custard didn’t chance; and the second, it was an increase in the taste of the product, and as a result an increase in the level preference. The combination of soy milk and inoculate of kefir increased the level of preference. doi:10.1016/j.jbiotec.2010.09.301
Fig. 1. Effect of (-)-SECO on adipogenesis.
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
indicated that the methanol extracts and the derived phytochemicals from CPTo and CPS are great potential sources as natural antioxidant agents. doi:10.1016/j.jbiotec.2010.09.296 [P-F.53] Biogenic amines degradation by moderate halophile, Brevibacillus sp. SK35 Sornchai Sinsuwan 1,∗ , Aungkawipa 2 Rodtong , Jirawat Yongsawatdigul 1
Montriwong 1 , Sureelak
1
School of Food Technology, Suranaree University of Technology, Thailand 2 School of Microbiology, Suranaree University of Technology, Thailand Keywords: biogenic amine; reduction; Brevibacillus sp.; fish sauce
Biogenic amines (BAs) are widely found in fermented foods and exert to adverse effects on the central nervous and vascular systems of human. The level of BAs in food was normally regulated. We attempted to investigate the potential of bacterial strain isolated from fish sauce fermentation in biogenic amine reduction. In addition, optimum conditions for BAs reduction of the selected strain and partial characterization of the amine oxidase produced by such a strain were elucidated. Among 7 strains, namely Virgibacillus sp. (SK33 and SK37), Staphylococcus sp. (SK1-1-5 and SKM20-7-9), Brevibacillus sp. SK35, Halomonas sp. SKW12, and Corynebacterium sp. SK1-1-6, were tested for histamine reduction capabilities and BAs production. All strains produced BAs, namely typtamine, tyramine, spermine, and spermidine, in the similar level. Brevibacillus sp. SK35 was able to reduce histamine at 99%, whereas histamine degradation of other stains was about 0-26%. The strain reduced simple amines to a greater extent than the polyamines with reduction order of putrescine > cadaverine > tyramine > tryptamine > histidine > spermidine > spermine. Optimum condition of BAs reduction was at 35 ◦ C, pH 8, and 10% NaCl when cultivated in the modified MØller broth. Amine oxidase was localized in intracellular with molecular weight of 45 kDa based on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) activity staining using various BAs as substrates. Optimum conditions of the enzyme were at 35 ◦ C and pH 7-8. doi:10.1016/j.jbiotec.2010.09.297 [P-F.54] Study about the addition of chemically modified starches (crosslinked cornstarches), dextrins, and oats fiber in baked pound cake M.E. Sánchez-Pardo ∗ , E. Jiménez-García, I. González-García Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería Bioquímica. Plan de Ayala y Prolongación de Carpio, Casco de Santo Tomás. C. P. 11340., Mexico Keywords: chemically modified starches; dextrins; pound cake; oats fiber The pound cake is one of the sweet sub-products of breadmaking, the modified starches contribute to the diet the same calories that the native starches, but have advantages in baking, since its functionality is important as well as the addition of the oats fiber. dextrins are used to obtaining a healthy product and good quality. It is so also justified the incorporation of fiber oatmeal due to their functional properties and health benefits.
The objective of this work was to evaluate the influence of these additives in baking products such as pound cake. In this study flour of wheat, chemically modified starches (cross-linked cornstarches), dextrins, and oats fiber were used. The wheat flour used to elaborate the pound cake had ash content 0.3-0.5 g/100 g sample and 12 g protein/100 g sample. The pound cake density was determined by the ratio mass/volume, mass was obtained by weighing pound cakes in an analytical scale an hour after baking. The volume “v” of each cake was obtained by the rape seed displacement method. Sensory evaluation consisted of a hedonic test of seven points using 100 untrained panelists. The results were evaluated with a Friedman bilateral variance rank analysis. Pound cake elaborated with the incorporation of modified starches, dextrin and fiber of oats presented higher volume (12%) and lower density, as well as a distribution of the alveoli most homogenous (a = 0.89). Sensorial judge indicated that the product added with the additive mixture (10 g dextrins/100 g; 25 g chemically modified starches/100 g; and 25 g oats fiber/100 g) presented better characteristics than conventional one. The acceptance level was “likes much”. This study focuses on the effect of a modified starches (crosslinked cornstarches), dextrins, and oats fiber on the properties of pound cake baked, these additives improved several quality parameters. doi:10.1016/j.jbiotec.2010.09.298 [P-F.55] Combination of in vitro intestinal continuous fermentation and intestinal cell models to investigate Salmonella and probiotics interactions in infected gut microbiota A. Zihler 1,∗ , M. Gagnon 1 , C. Chassard 1 , C.P. Braegger 2 , C. Lacroix 1 1
ETH Zurich, Switzerland University Children’s Hospital Zurich, Switzerland Keywords: In vitro continuous fermentation model; HT29-MTX intestinal epithelial cell line; Salmonella; Probiotics 2
Salmonellosis is a major public health concern with a significant cost in many countries. Imposed by an increasing prevalence of multi-drug resistant strains, biological alternatives for the treatment of Salmonella infections are needed. We combined two original in vitro models closely mimicking the complex in vivo intestinal situation to investigate the therapeutic potential and mechanisms of two promising bacteriocinogenic probiotics, Bifidobacterium thermophilum RBL67 (thermophilicin B67) and Escherichia coli L1000 (microcin B17-producer), active against Salmonella. A recently developed continuous three-stage colonic fermentation model of S. Typhimurium infection with immobilized child fecal microbiota was used to simulate the proximal, transverse and distal human colonic sections. Reactor effluents were directly applied to intestinal mucus-secreting HT29-MTX cell-layers to measure first-level host responses after induction of infection and probiotic treatments. Stable pathogen invasion into HT29-MTX cells (1 ± 0.4%) was recorded after infection accompanied by decreased epithelium integrity (45% lower Trans Epithelial Electrical Resistance compared to non-infected control). After adding E. coli L1000, Salmonella invasion only decreased in the distal reactor (89%) while TEER did not change for all samples. In contrast, anti-Salmonella activity of B. thermophilum RBL67 was most pronounced in the proximal reactor with a 76%-decrease of pathogen invasion and TEER recovered close to levels of non-infected samples. Bio-Plex (Bio-Rad) analysis was used to measure cellular
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
S317
immune responses. Our results highlight the benefits to combine suitable cellular and colonic fermentation models to evaluate host protection activity of probiotics during Salmonella infection, providing efficient tools for mechanistic studies and preclinical development of new antimicrobials.
mulation by down-regulation of SREBP1 and FAS via adiponectin receptor AdipoR2 (Fig. 1). Acknowledgement: This work was supported by Grant-in-Aid for Scientific Research (No. 21580142) from the Ministry of Education, Science, Sports, and Culture in Japan.
doi:10.1016/j.jbiotec.2010.09.299
Reference Hosooka, et al., 2008. Nat. Med. 14, 188–193.
[P-F.56]
doi:10.1016/j.jbiotec.2010.09.300
Effect of Secoisolariciresinol on Adipogenesis T. Sugahara ∗ , S. Tominaga, S. Akiyama, S. Nishimoto
Yamauchi, M.
Maruyama, K.
Ehime University, Japan Keywords: Secoisolariciresinol; Flaxseed; Adipogenesis; adipocytes
[P-F.57] Study comparative of custard inoculated with bacteria lactic of Kefir in different kind of soy milk (Glicine max) M.E. Sánchez-Pardo ∗ , E. Jiménez-García, I. Solano-Ortiz
Introduction: Flaxseed (Linum usitatissimum L.) lignan, (+)secoisolariciresinol (SECO), (−)-SECO, and meso-SECO were stereoselectively synthesized and biological functions were evaluated. As a result, (−)-SECO suppressed triglyceride (TG) production in 3T3-L1 adipocytes. In this research, we focused on the effects of (−)-SECO on the regulation of adipogenesis in vitro and in vivo. Methods: Differentiation of 3T3-L1 pre-adipocytes into adipocytes was induced by the addition of insulin, dexamethasone and isobutyl-methylxanthine. Following differentiation, (−)-SECO was added to the medium, and the effects on adipocytes were examined. (−)-SECO was orally administrated to C57BL/6 mice with high-fat diet, and adipogenesis-relevant parameters including body weight and energy intake were examined. Results: (−)-SECO stimulated differentiation of 3T3-L1 preadipocytes by enhancement of PPAR␥ level, and suppressed TG accumulation in the cells. In addition, adiponectin production was facilitated by (−)-SECO. These facts suggest that (−)-SECO stimulates differentiation of 3T3-L1 pre-adipocytes by up-regulation of PPAR␥ expression and adiponectin production. The protein downstream of tyrosine kinases-1 (Dok1) is known to function as a multisite adapter molecule in insulin signalling, and promotes PPAR␥ expression (Hosooka et al., 2008). Dok1 expression was obviously enhanced by (−)-SECO within 6 h in 3T3-L1 preadipocytes. These facts suggest that (−)-SECO accelerated Dok1 expression, and Dok1 released PPAR␥ expression modulated by Ras cascade to facilitate adipocytes differentiation (Fig. 1). Increase in body weight and adipocytes weight of C57BL/6 mice fed with high-fat diet was significantly restricted by oral administration of (−)-SECO. In addition, mRNA expression for PPAR␥ and adiponectin was stimulated by intake of (−)-SECO. (−)-SECO intake enhanced serum adiponectin level, and adiponectin suppressed TG accu-
Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería Bioquímica. Plan de Ayala y Prolongación de Carpio, Casco de Santo Tomás. C. P. 11340. México, D. F., Mexico Keywords: soy milk; kefir granules; acceptance level; inoculated In Mexico there is a tendency to eat more nutritious dessert products because we have accustomed to eating food with sugar; custard has been reformulated as a dessert to get the lactic acid bacteria from kefir, and protein soybeans contribute to tasty into this food that cover the daily nutritional needs and are innovative in using soy milk. The aim of this work was the designing of custard made with commercial soy milk, soy milk powder and compare it with custard of cow’s milk; all inoculated with lactic acid bacteria of kefir in order to elaborate a functional food of high quality. We conducted a formulation that consisted of 65% milk, 24% egg, 9% sugar, 1.3% water, 0.9% corn starch and 0.4% essence of vanilla. Custard was inoculated with kefir granules before and after fermentation, and with the granules (106 UFC) to obtain a functional product through comparative study using different raw materials. A comparative study of these products was experimented with different kind of milks. The acceptance level was higher when the custard was elaborated with soy milk (“I like very much”) and soy milk industrialized (“I like”) prepared from commercial soybean meal, than cow’s milk. As well as the presence of yeasts was lower in the custard elaborated with milk fermented with kefir, regarding the custard inoculated directly with the kefir granules had higher presence of yeasts. Two hedonics scales 7 were used, which consisted of a tasting of the product without inoculate and another inoculated. The first, the taste of the custard didn’t chance; and the second, it was an increase in the taste of the product, and as a result an increase in the level preference. The combination of soy milk and inoculate of kefir increased the level of preference. doi:10.1016/j.jbiotec.2010.09.301
Fig. 1. Effect of (-)-SECO on adipogenesis.