Browsing by Subject "Enzyme"

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Enzymatic hydrolysis of vegetable and insect proteins using technical enzyme preparations
(2021) Großmann, Kora Kassandra; Fischer, Lutz
The present dissertation covers the usage of technical enzyme preparations (TEPs) for vegetable and insect protein hydrolysis, due to the mounting interest in alternative protein sources to cover the increasing demand for food from a growing world population. The TEPs, as defined in this study, are enzyme preparations that include side activities and are used in food processing. Today, TEPs are used by food manufacturers based on the supplier’s information that usually states the main enzyme activity and includes information on side activities only in some cases. However, knowledge about the activity profile is crucial as side activities can contribute to the properties of the protein hydrolysates produced (e.g. degree of hydrolysis [DH], liberation of amino acids) and the final food product quality. In the first study, an automated photometric analyzer (GalleryTM Plus, Thermo Fisher Scientific) was introduced for the comprehensive activity determination of TEPs. The new setup of the analyzer covered 32 synthetic and natural substrates in order to determine aminopeptidase, carboxypeptidase, dipeptidylpeptidase and endopeptidase activities distinguishably. Accordingly, the overall proteolytic activity of TEPs was quantified and detailed information about the substrate spectra and peptidase side activities was generated. Furthermore, several batches of the industrial TEP Flavourzyme1000L were measured. By determining 32 peptidase activities, batch variations were shown. As Flavourzyme1000L is standardized by its supplier Novozymes on only one activity (leucine aminopeptidase), the additional 31 new peptidase activities determined showed differences of the side activities of the batches. In addition, the study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting lupine protein hydrolysates (DH and liberation of amino acids). Due to the determination of 32 peptidase activities (so-called “activity fingerprint”), TEPs were selected specifically to increase, for example, the DH. The two TEPs P278 and DZM were selected due to their complementary peptidase activities, as an example of this study. The combination of these two TEPs resulted in an increase of the DH of 47%. Now, TEPs can be selected targeted more on the basis of their peptidase activities to, for example, increase the hydrolysis efficiency of lupine protein by combining complementary peptidase activities. In the second study, six food-grade TEPs (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K and PeptidaseR) were investigated regarding their influence on the hydrolysis of soy, pea and canola protein. The hydrolysates were investigated analytically concerning their DH and free amino acid profiles and sensorically concerning the taste attributes umami and bitter. By using a random forest model, the taste attributes bitter and umami were connected to specific peptidase activities (exo- and endopeptidase activities). Furthermore, out of the six selected TEPs, the usage of ProteAX-K showed high umami and low bitter taste of the vegetable protein hydrolysates (soy, pea and canola). In line with the first study, the second study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting vegetable protein hydrolysates. Based on these new insights, TEPs can be selected more specifically based on their peptidase activity profiles to direct the formation of desired taste attributes of the protein hydrolysates. In the third study, two TEPs with various peptidase activities (Flavourzyme1000L, ProteaseA “Amano”2SD) were applied for the hydrolysis of insect proteins. This study investigated the potential of cricket and mealworm protein and their hydrolysates regarding their sensory potential. The sensory profiles of the insect proteins were altered by, firstly, applying proteolytic hydrolysis and then a Maillard reaction (30 min, T = 98°C, 1% (w/v) xylose) to the hydrolysates. The initially earthy-like flavor of the insect proteins resulted in modified taste profiles described by e.g., savory-like attributes, due to both processing steps. Furthermore, 38 odor-active molecules (1 alcohol, 5 acids, 11 aldehydes, 5 ketones and 16 heterocyclic compounds) were identified by gas chromatography-olfactometry (GC-O). The identified molecules are also found in meat and edible seafood products. The third study showed that the flavoring profile of insect proteins was modified and can be developed further by the respective processing.
Food-grade Lactobacilli expression systems for recombinant enzymes
(2013) Böhmer, Nico; Fischer, Lutz
Lactobacilli are Gram-positive bacteria used throughout the food industry as traditional starters for various fermented foods. Lactobacilli would be superior for recombinant enzyme production regarding the food safety demands since most of them are Generally Recognised As Safe (GRAS) organisms. The major advantages of Lactobacilli as food-associated microorganisms used for recombinant enzyme production are their safe and sustainable use as overall safety food-grade expression systems. In the work presented, Lactobacilli were studied in detail as food-grade expression systems for recombinant enzyme production. In a first analysis, the two pSIP expression systems, pSIP403 and pSIP409, were investigated to produce a hyper-thermophilic Beta-glycosidase (CelB) from Pyrococcus furiosus in Lactobacillus plantarum NC8 and Lactobacillus casei as hosts, respectively. Both Lactobacilli harbouring the pSIP409-celB vector produced active CelB in batch bioreactor cultivations, while the specific CelB activity of the cell-free extract was about 44% higher with Lb. plantarum (1,590 ± 90 nkatpNPGal/mgprotein) than with Lb. casei (1,070 ± 66 nkatpNPGal/mgprotein). A fed-batch bioreactor cultivation of Lb. plantarum NC8 pSIP409-celB resulted in a specific CelB activity of 2,500 ± 120 nkatpNPGal/mgprotein. A basal whey medium with supplements was developed as an alternative to the cost intensive MRS medium used. About 556 ± 29 nkat pNPGal/mgprotein of CelB activity was achieved in bioreactor cultivations using this medium. It was shown that both Lactobacilli were potential expression hosts for recombinant enzyme production. An additional approach was performed to produce a metagenome-beta-galactosidase using Lb. plantarum NC8 with the pSIP expression system. Using this system, a quite low maximal galactosidase activity of only 0.18 nkatoNPGal/mgprotein was detected. A 13 times higher activity of 2.42 nkatoNPGal/mgprotein was produced after the knock out of the interfering native Kluyveromyces lactis Beta-galactosidase in the well-known food-grade K. lactis pKLAC2 expression system. Nevertheless, the best performing expression system for the recombinant production of the metagenome-derived enzyme was the Escherichia coli BL21 strain with a pET vector, resulting in the highest Beta-galactosidase of 82.01 nkatoNPGal/mgprotein. Beside the use of the pSIP expression system, a novel expression system for Lb. plantarum was developed. This system is based on the manganese starvation-inducible promoter from the specific manganese transporter of Lb. plantarum NC8 which was cloned for the first time. The expression of CelB was achieved by cultivating Lb. plantarum NC8 at low manganese concentrations with MRS medium and the pmntH2-celB expression vector. A CelB activity of 8.52 µkatoNPGal/L was produced in a bioreactor. The advantages of the novel expression system are that no addition of an external inducing agent was required, and additionally, no further introduction of regulatory genes was necessary. The new promoter meets the general demands of food-grade expression systems. The glutamic acid racemase of Lb. plantarum NC8 was cloned and characterized in this work for the first time as a possible target for a food-grade selection system for this species. Glutamic acid racemases (MurI, E.C. 5.1.1.3) catalyse the racemisation of L- and D-glutamic acid. MurIs are essential enzymes for bacterial cell wall synthesis, which requires D-glutamic acid as an indispensable building block. Therefore, these enzymes are suitable targets for antimicrobial drugs as well as for the potential design of auxotrophic selection markers. A high expression system in E. coli BL21 was constructed to produce and characterize the biochemical properties of the MurI from Lb. plantarum NC8. The recombinant, tag-free Murl was purified by an innovative affinity chromatography method using L-glutamic acid as the relevant docking group, followed by an anion exchange chromatography step (purification factor 9.2, yield 11%). This two-step purification strategy resulted in a Murl sample with a specific activity of 34.06 µkatD-Glu/mgprotein, comprising a single protein band in SDS-PAGE. The purified Murl was used for biochemical characterization to gain in-depth knowledge about this enzyme. Only D- and L-glutamic acid were recognised as substrates for the Murl with similar kcat/Km ratios of 3.6 sec-1/mM for each enantiomer. The findings in this study may contribute to further development and implementation of food-grade Lactobacilli expression systems for recombinant enzyme production. Furthermore, the results obtained may help to optimise and select hosts and expression systems for industrial enzyme production for the needs of the food industry.
Microplastics interactions with soil organisms
(2022) Schöpfer, Lion; Kandeler, Ellen
Microplastics (MP) are plastic particles from 100 nm to 5 mm with different shapes and chemical compositions. In aquatic ecosystems, MP have proven to affect the biological fitness of aquatic organisms, enter the food web, and act as vectors of pollutants. Agricultural soils are sinks for MP due to inputs via sewage sludges, plastic mulches, and organic fertilizers. However, ecological consequences of MP in agricultural soils are unknown. This doctoral thesis aimed to evaluate the risk of conventional and biodegradable MP for soil organisms in agricultural soils. A microcosm study was combined with a field study and a nematode study to investigate background concentrations, the persistence, and the biodegradation of MP in the soil, and effects of MP on soil microorganisms and nematodes. In the microcosm study, the influence of plastic type, particle size, and soil moisture on the biodegradation of MP in the soil and on effects on soil microorganisms were examined under controlled conditions (25 °C, 230 days). The abundance and composition of the main soil microbial groups was analyzed via phospholipid fatty acids (PLFAs) as biomarkers; activities of C cycling enzymes driving the decomposition of differently complex substances were analyzed as proxies for C turnover. To understand better the role of MP as an interface for specific microbial processes in the soil, e.g. the enzymatic hydrolysis of MP, enzyme activities of individual MP particles extracted from the soil were measured. The site of the field study was a conventionally managed agricultural soil (silt-loam Luvisol) of the Heidfeldhof, University of Hohenheim. No practices associated with significant inputs of MP have been conducted at the site in the past (sewage sludge, organic fertilizers, plastic mulch). In a randomized complete block design, the effects of MP, organic fertilizers (digestate and compost), and their interactions on soil microbiological indicators (microbial biomass, soil enzymes) were studied. Before the setup of the field study, MP background concentrations (particle-based) in the soil were analyzed. The persistence of added MP in the soil was evaluated by comparing MP concentrations in the soil after 1 month and 17 months with initial MP concentrations after addition. In the nematode study, the soil-dwelling nematode Caenorhabditis elegans was exposed to MP feed suspensions on agar plates. The uptake of MP through nematodes and the influence of plastic type and concentration on MP effects on nematode reproduction and body length were examined. In all studies, artificially fragmented MP from a conventional polymer (low-density polyethylene, LDPE) and a biodegradable polymer blend (poly(lactic acid) and poly(butylene adipate-co-terephtalate), PLA/PBAT) were used. The occurrence of both LDPE- and PLA/PBAT-MP is likely in agricultural soils because these are used for plastic mulches and compost bags. Results from this thesis suggest that (1) agricultural soils, including those without management practices related to significant MP entry, contain various MP, indicating diffuse MP inputs via atmospheric deposition, littering, and the abrasion of machinery coatings (a possible newly identified pathway), (2) also biodegradable MP persist and are slowly biodegraded in the soil implying a long term exposure risk for soil organisms to MP, (3) MP have no acute negative effects on microorganisms and C turnover, (4) MP form a specific habitat in the soil, the plastisphere, where MP-specific processes take place, e.g. the enzymatic hydrolysis of PLA/PBAT, (5) MP can enter the soil food web via nematodal uptake and affect nematode reproduction, which could destabilize the soil food web.