Optimization of Small Peptide Feed from Milk Thistle Residue by Synergistic Fermentation of Multiple Strains and Proteases

In order to improve the utilization rate of the milk thistle residue, this study used the synergistic fermentation of multiple strains and proteases to increase the small peptide content of the fermented feed produced by the milk thistle residue. Taking the small peptide content of the milk thistle residue fermented feed as an indicator, the optimal fermentation process was obtained by single-factor optimization experiments and the response surface methodology. The optimal fermentation process was as follows: fermentation time of 7 days, inoculum size of 15%, inoculation ratio of aerobic strains: anaerobic strains = 1: 2, solid-state fermentation water content of 66%, fermentation temperature of 36℃, and amount of protease was 0.25% acid protease+0.25% bromelain. Under the above process, the small peptide content of the fermented feed from milk thistle residue was greatly improved to 57.86%. These results inferred that the added proteases were beneficial to the growth of fermentative microorganisms, the secretion of protease and the increase of the small peptide content.

Partial characterization of digestive proteases in Pacific red snapper Lutjanus peru Nichols & Murphy, 1922 (Perciformes: Lutjanidae)

Pacific red snapper (Lutjanus peru) is an important commercial species in Mexico with great aquaculture potential; however, digestive physiology is still unknown. Therefore, the objective of the present work was to characterize the digestive proteases of L. peru juvenile using biochemical and electrophoretic techniques. Results showed a higher acid protease activity than the alkaline proteases, trypsin, chymotrypsin, and leucine aminopeptidase (LAP). The optimum temperature for acid proteases was between 30 to 40°C. Trypsin activity showed two maximum peaks of temperature (30 and 50°C), while alkaline proteases, chymotrypsin, and LAP had optimum temperatures of 50, 50 to 60, and 40°C, respectively. Moreover, the optimum pH of acid proteases was between 2 and 3. Also, alkaline proteases, trypsin, chymotrypsin showed pH optimums at pH 6, 9, and 5, respectively, although LAP showed two optimum pH values at 6 and 9. Acid protease zymogram showed three isoforms, totally inhibited by pepstatin A. Alkaline protease zymogram revealed six bands (125.4, 67.2, 57.9, 48.6, 29.8, and 26.9 kDa), which were inhibited by specific serine-proteases and metalloproteases inhibitors. In conclusion, the main digestion in L. peru depends on stomach proteases, which are characteristic of carnivorous fish, followed by intestinal digestion supported mainly by chymotrypsin.

Production, Optimization, and Characterization of an Acid Protease from a Filamentous Fungus by Solid-State Fermentation

Acid proteases represent an important group of enzymes, extensively used in food and beverage industries. There is an increased demand for acid proteases adapting to the industrial extreme environment, especially lower pH. Thus, this necessitates the search for a better acid protease from fungi that best performs in industrial conditions. The fungal isolates were isolated from grape and dairy farm soil using potato dextrose agar and further screened for protease production based on the hydrolysis of clear zone on skim milk agar. The potential fungi were then subjected to secondary screening under solid-state fermentation (SSF). After the secondary screening, the potential fungus was identified to the genus level by the macroscopic and microscopic methods. The growth conditions and media composition for the potential fungus were further optimized under SSF. The crude enzyme produced by the potential isolate was characterized after partial purification by acetone and ammonium sulfate precipitation. A total of 9 fungal isolates showed protease production in primary and secondary screening; however, one potential isolate (Z1BL1) was selected for further study based on its protease activity. The isolate was identified to the genus Aspergillus based on their morphological features. The maximum acid protease from the isolate Z1BL1 was obtained using fermentation media containing wheat bran as a solid substrate, 1 mL of 3.2 × 106 inoculum size, 50% moisture content, and pH 4.5 upon 120-h incubation at 30°C. The acetone-precipitated enzyme exhibited the maximum activity at 50°C and pH 5 with stability at pH 4–6 and temperature 40–60°C. Thus, the acid protease produced from Aspergillus showed suitable enzyme characteristics required in the industry and could be a candidate for application in the food industry after further purification.

Reducing glucoamylase usage for commercial-scale ethanol production from starch using glucoamylase expressing Saccharomyces cerevisiae

The development of yeast that converts raw corn or cassava starch to ethanol without adding the exogenous α-amylase and/or glucoamylase would reduce the overall ethanol production cost. In this study, two copies of codon-optimized Saccharomycopsis fibuligera glucoamylase genes were integrated into the genome of the industrial Saccharomyces cerevisiae strain CCTCC M94055, and the resulting strain CIBTS1522 showed comparable basic growth characters with the parental strain. We systemically evaluated the fermentation performance of the CIBTS1522 strain using the raw corn or cassava starch at small and commercial-scale, and observed that a reduction of at least 40% of the dose of glucoamylase was possible when using the CIBTS1522 yeast under real ethanol production condition. Next, we measured the effect of the nitrogen source, the phosphorous source, metal ions, and industrial microbial enzymes on the strain’s cell wet weight and ethanol content, the nitrogen source and acid protease showed a positive effect on these parameters. Finally, orthogonal tests for some other factors including urea, acid protease, inoculum size, and glucoamylase addition were conducted to further optimize the ethanol production. Taken together, the CIBTS1522 strain was identified as an ideal candidate for the bioethanol industry and a better fermentation performance could be achieved by modifying the industrial culture media and condition.

Production, Optimization and Characterization of an Acid Protease From Filamentous Fungi by Solid-state Fermentation

Acid proteases represent an important group of enzymes, extensively used in food and beverage industries. There are a diversification of food industries and thus an increasing demand for biocatalysts capable of adapting the industrial extreme environments. These demands can be covered by a plant and animal proteases; however there is a shortage to meet the present industrial demands. This necessitates the search for an alternative acid protease sources from fungi. The fungal isolates were recovered from grape and dairy farm soil using potato-dextrose Agar. The fungi were further screened for protease production based on the hydrolysis of clear-zone on skim-milk agar media. The potential fungi were then subjected to secondary screening under solid-state fermentation. After primary and secondary screening, the potential fungus (isolate Z1BL1) was identified to the genus level by combination of macroscopic and microscopic morphological study. The growth condition and media composition for potential fungal isolate (Z1BL1) was further optimized under solid-state fermentation. The crude enzyme produced from isolate Z1BL1 was characterized after partial purification by acetone and ammonium sulphate precipitation. A total of 9 fungal isolates were showed protease production in primary and secondary screening, however 1 potential isolate (Z1BL1) was selected for further study based on its protease activity. The potential fungus, isolate Z1BL1 was identified to the genus Aspergillus based on their morphological features. The optimization of media composition and growth conditions for acid protease production from Z1BL1 were slightly increased the protease activity. The acetone precipitated enzyme exhibited the maximum activity at 50 0C and pH 5 with stability at pH 4-6 and temperature 40-60 0C. Thus based on the above findings, the acid protease produced from Aspergillus was shown suitable enzyme characteristics required in industry and could be a candidate to be applicable in food industry after further purification by high resolution techniques.