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Bear bile powder is a valuable medicinal material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a certain ratio to taurochenodeoxycholic acid (TCDCA). We had created an engineered E. coli harboring two-step bidirectional oxidative and reductive enzyme-catalyzing pathway that could rapidly convert TCDCA to TUDCA at a specific percentage in shake flasks.
This study provided a practical and environment friendly industrialized process for producing artificial substitute of bear bile powder from cheap and readily available chicken bile powder using engineered E. coli microbial cell factory. It also put forward an interesting deep-tank static process to promote the enzyme-catalyzing reactions toward target compounds in synthetic biology-based fermentation.
Bacterial 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) have both oxidative and reductive activities and can interconvert TCDCA and TUDCA coupling with NADPH or NADH as co-factors [20, 21] (Fig. 1). Utilizing the property of 7α-HSDH and 7β-HSDH, we had created an engineered Escherichia coli with 7α-HSDH and 7β-HSDH genes which could convert a certain proportion of TCDCA to TUDCA via tauro-7-keto lithocholic acid (T-7-KLCA) intermediate using chicken bile powder as substrates . The oxidative and reductive activities of 7α-HSDH and 7β-HSDH are affected by temperature, available oxygen, pH value of the media, and the fermentation time in shake flasks [22,23,24]. Here, in order to make the engineered E. coli work feasible for industrial application, we performed process optimization to balance the bidirectional reactions converting TCDCA to TUDCA and to improve the production as well. Medium composition was optimized with response surface methodology (RSM), a useful tool reducing the number of experiments without neglecting the interactions among the parameters [25, 26], and a special deep-tank static process was developed.
Employing the optimized condition, we carried out fermentation in stirred-tank fermenter using refined chicken powder and crude chicken bile powder as substrates and prepared the fermentation broth into powder products consisting of nearly equal content of TUDCA to natural bear bile acid powder. To our knowledge, this is the first report applying engineered whole-cell factory to make products in large scale containing TUDCA at a certain ratio to TCDCA as in the rare bear bile powder medicinal material.
Results of deep-tank static process optimization. a Duration optimization of agitation and static processes. b Temperature optimization using refined chicken bile powder as substrate. c Temperature optimization using crude chicken bile powder as substrate
Considering the economic costs, we conducted temperature optimization for static process by setting 3.5 h for agitation fermentation coupled with 12 h static process (3.5 h-12 h combination) when refined chicken bile powder was used as substrate and 5 h agitation fermentation with 12 h static process (5 h-12 h combination) when crude chicken bile powder was used as substrate. As shown, when using refined chicken bile powder, keeping static process at 25 C resulted in the highest TUDCA titer and conversion efficiency, 1.12 g/L and 43.13%, respectively. The ratio of TUDCA to TCDCA was 1.12. Keeping at 4 C, 16 C, and 37 C led to less TUDCA and lower conversion efficiency, below 1.95 g/L and 28.62%, respectively (Fig. 5b). The ratios of TUDCA to TCDCA in these groups were less than 0.44. This result suggested 25 C was the optimal temperature for static process using refined chicken bile powder as substrate. When using crude chicken bile powder and holding static process at 4 C, 16 C, 25 C, and 37 C, the TUDCA concentration was respective 1.47 g/L, 1.50 g/L, 1.39 g/L, and 1.46 g/L and TUDCA conversion efficiency was 50.77%, 51.80%, 51.65%, and 50.20%, respectively (Fig. 5c). The ratios of TUDCA to TCDCA ranged from 1.01 to 1.17, all above 1.0. This result indicated that the temperature of static process had little effect on TUDCA formation for crude chicken bile powder substrate and 25 C might be the best choice in view of convenience.
Taken together, it suggested that coupling with 12 h-static process at 25 C would help convert TCDCA to ideal ratio of TUDCA through fermentation of the engineered E. coli BL-pα1β2 strain, either using refined chicken bile powder or using crude chicken bile powder as substrates.
Amino acids in the prepared products were also monitored. Except for taurine, the other five amino acids from the prepared products, leucine, valine, proline, alanine, and alanine-d4, were present at a much higher level than that from drainage bear bile powder (Fig. 8). Besides, all products had a considerable amount of phenylalanine. The level of these amino acids in products produced from refined chicken bile powder was higher than the corresponding one produced from crude chicken bile powder, possibly due to amino acids were enriched in refined chicken bile powder. Interestingly, when the coupled static process was set at 37 C, there was nearly equal amount of taurine in the products from refined chicken bile powder to that in drainage bear bile powder (Fig. 8). This result indicated that keeping the static process at 37 C was not conductive to the production of TUDCA but benefit for the formation of taurine which was the main amino acid component in drainage bear bile powder.
Typical mass chromatogram of amino acids in prepared products from chicken bile powder. a Amino acids in products from refined chicken bile powder. b Amino acids in products from crude chicken bile powder. c Amino acids in products from RCBP when static process at 37 C. d Amino acids in natural bear bile powder sample
An efficient Large-scale fermentation process is important for industrial application of synthetic biology approach to produce value-added natural products. In previous work, we recreated a two-enzyme catalyzing biosynthetic pathway of TUDCA in E. coli which has the potential to produce bear bile powder substitute from cheap and readily available chicken bile powder contributing to the bidirectional oxidative and reductive reactions of 7α-HSDH and 7β-HSDH . In that work, we used a phosphate buffer solution modified M9 minimal medium (PBS-M9) and a two-step fermentation procedure to concentrate bacteria to high density and promote the TUDCA production in shake flasks . Replacing two-step fermentation with one-step method is essential to decrease the cost for chemical production in industrial scale . The challenges of one-step fermentation is to improve the cell growth and the production of target compounds as well [27, 28]. In order to increase the lactic acid production in one-step fed-batch fermentation, Fu et al. used as high as 100 g/L of glucose in the initial media and fed glucose as feedstock during the fermentation process as well . Since first fermentation using PBS-M9 medium in the fermenter got low cell density (OD600 equal to 2.15) and low TUDCA conversion efficiency (1.70%), we then tried several rich media including LB, 2-YT, and SOC but failed to get appropriate TUDCA conversion (Data not shown).
It should be mentioned that the chicken bile powder used in this study is abundant in TCDCA but not in pure TCDCA. There are other compositions like TCA which was also transformed to tauroursocholic acid (TUCA) during the fermentation. The pharmacological evaluation of these products is in progress.
Crude chicken bile powder and refined chicken bile powder were respectively used as substrates for large-scale fermentation each in triplicates. After fermentation, the broth was collected and centrifuged at 8000 rpm for 20 min to get the supernatant which was passed through pre-treated isometric resin D101 . The impurities on D101 resin were firstly removed by deionized water until the eluent was colorless. Then the resin was washed with 95% ethanol to get the eluent until colorless. The eluent was concentrated through rotary evaporation at 50 C and then filtered through 0.45 μm organic filters. The filtrate was evaporated by keeping in water bath at 50 C to form extractum and then dried to constant weight in vacuum drying oven at 50 C. After that, the dried solid was taken out and crushed into powder. The bile acid recovery yield in this study indicated to the main five bile acid yield calculated according to the Eq. (5) as follows: 041b061a72