Production strategies and applications of microbial single cell oils

Polyunsaturated fatty acids (PUFAs )of the ω-3 and ω-6 class (e.g. , α-linolenic acid, linoleic acid) are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used form any valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF) or solid state fermentation (SSF). The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g., medium, pH-value, temperature, aeration, nitrogensource). From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. Ingeneral, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations andoverall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids or derived fatty acids with emphasis on food applications

Assimilation of alternative sulfur sources in fungi

Fungi are well known for their metabolic versatility, whether it is the degradation of complex organic substrates or the biosynthesis of intricate secondary metabolites. The vast majority of studies concerning fungal metabolic pathways for sulfur assimilation have focused on conventional sources of sulfur such as inorganic sulfur ions and sulfur-containing biomolecules. Less is known about the metabolic pathways involved in the assimilation of so-called “alternative” sulfur sources such as sulfides, sulfoxides, sulfones, sulfonates, sulfate esters and sulfamates. This review summarizes our current knowledge regarding the structural diversity of sulfur compounds assimilated by fungi as well as the biochemistry and genetics of metabolic pathways involved in this process. Shared sequence homology between bacterial and fungal sulfur assimilation genes have lead to the identification of several candidate genes in fungi while other enzyme activities and pathways so far appear to be specific to the fungal kingdom. Increased knowledge of how fungi catabolize this group of compounds will ultimately contribute to a more complete understanding of sulfur cycling in nature as well as the environmental fate of sulfur-containing xenobiotics

Enzymatic production and analysis of antioxidative protein hydrolysates

This review provides an overview on the production and analysis techniques of antioxidative peptides from food proteins. Regarding the production of antioxidative peptides, interlinked factors must be considered. Depending on the protein substrate, different peptidases or peptidase systems containing multiple enzymes as well as a specific production process must be chosen. The antioxidative peptides might be produced in a batch process including multiple pre- and post-treatments, besides the hydrolyses with peptidases itself. As an alternative, the potential of continuous production systems is discussed in this review. Furthermore, robust analyses tools are needed to gain control of the process and final product properties. With no standardized methodology available for antioxidative peptide evaluation, pros and cons of various strategies for peptide separation and antioxidative measurement are discussed in this review. Therefore, this review provides a roadmap for antioxidative peptide generation from various sources for research and development as well as for potential industrial use

Purification and characterization of a fungal aspartic peptidase from Trichoderma reesei and its application for food and animal feed protein hydrolyses

BACKGROUND Various neutral and alkaline peptidases are commercially available for use in protein hydrolysis under neutral to alkaline conditions. However, the hydrolysis of proteins under acidic conditions by applying fungal aspartic peptidases (FAPs) has not been investigated in depth so far. The aim of this study, thus, was to purify a FAP from the commercial enzyme preparation, ROHALASE® BXL, determine its biochemical characteristics, and investigate its application for the hydrolysis of food and animal feed proteins under acidic conditions. RESULTS A Trichoderma reesei derived FAP, with an apparent molecular mass of 45.8 kDa (sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SDS-PAGE) was purified 13.8-fold with a yield of 37% from ROHALASE® BXL. The FAP was identified as an aspartate protease (UniProt ID: G0R8T0) by inhibition and nano-LC-ESI-MS/MS studies. The FAP showed the highest activity at 50°C and pH 4.0. Monovalent cations, organic solvents, and reducing agents were tolerated well by the FAP. The FAP underwent an apparent competitive product inhibition by soy protein hydrolysate and whey protein hydrolysate with apparent Ki-values of 1.75 and 30.2 mg*mL−1, respectively. The FAP showed promising results in food (soy protein isolate and whey protein isolate) and animal feed protein hydrolyses. For the latter, an increase in the soluble protein content of 109% was noted after 30 min. CONCLUSION Our results demonstrate the applicability of fungal aspartic endopeptidases in the food and animal feed industry. Efficient protein hydrolysis of industrially relevant substrates such as acidic whey or animal feed proteins could be conducted by applying fungal aspartic peptidases. © 2022 Society of Chemical Industry

Kinetic parameters of PepX¹ using chromogenic <i>p</i>-nitroanilide substrates.

1<p>Protein concentration of the enzyme solution: 2.61 mg mL⁻¹.</p>2<p>Substrate for the standard assay. Triplicate measurements; the standard deviation was <5%.</p

Characterization of the Recombinant Exopeptidases PepX and PepN from <i>Lactobacillus helveticus</i> ATCC 12046 Important for Food Protein Hydrolysis

<div><p>The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from <i>Lactobacillus helveticus</i> ATCC 12046 were produced recombinantly in <i>E. coli</i> BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-<i>p</i>NA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in <i>E. coli</i> at high levels for the first time. The PepN activity reached 1,000 µkat_{H-Ala-<i>p</i>NA} L⁻¹. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn²⁺-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co²⁺>Zn²⁺>Mn²⁺>Ca²⁺>Mg²⁺. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.</p></div

Lineweaver-Burk linearization of PepX inhibition by H-L-Phe-L-Pro-OH (H-Ala-Pro-<i>p</i>NA as substrate; each point represents the average of triplicate measurements; the standard deviation was <5%).

<p>Lineweaver-Burk linearization of PepX inhibition by H-L-Phe-L-Pro-OH (H-Ala-Pro-<i>p</i>NA as substrate; each point represents the average of triplicate measurements; the standard deviation was <5%).</p