Food Enzymes Research Articles

Comprehensive Understanding of Laboratory Evolution for Food Enzymes: From Design to Screening Innovations.

In the field of food processing, enzymes play a pivotal role in improving product quality and flavor, and extending shelf life. However, the exposure of traditional food enzymes to high temperatures during processing often leads to a decrease in activity or even inactivation, limiting the effectiveness of their application under high-temperature conditions. Therefore, the modification of thermostability and activity of enzymes to adapt to extreme conditions through protein engineering has become a key way to improve the efficiency and economic benefits of industrial production. Directed evolution and semirational design strategies in the laboratory have proven to be broadly applicable frameworks for biochemical researchers in the food field, including those who are beginners. In this review, we systematically summarize semirational design strategies and high-throughput screening strategies, and introduce some intuitive computer simulation software to improve the thermostability and enzyme activity of food enzymes. The application of these strategies and techniques provides a comprehensive guide for the optimization of food enzymes. In addition, the latest hot topics of genetically engineered food enzymes in the field of application are discussed.

Safety evaluation of the food enzyme α‐amylase from the non‐genetically modified Bacillus amyloliquefaciens strain UN‐01

Abstract The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the non‐genetically modified microorganism Bacillus amyloliquefaciens strain UN‐01 by Nagase (Europa) GmbH. The production strain qualified for the qualified presumption of safety approach and no issues of concern arose from the production process of the food enzyme, therefore, the Panel considered that toxicological studies were unnecessary. It is intended to be used in five food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed during two processes, dietary exposure was calculated only for the remaining three food manufacturing processes. It was estimated to be up to 0.434 mg TOS/kg body weight per day in European populations. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. Known sources of food allergens were used in the food enzyme manufacturing process. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Quality characteristics of Hokkaido brown bear meat sauces prepared with rice koji mold and food enzymes

Quality characteristics of Hokkaido brown bear meat sauces prepared with rice koji mold and food enzymes

Subchronic Toxicity Evaluation of Dietary Administration of a Fungal Biomass From Rhizomucor pusillus.

Fungal-derived food products align with sustainable food supply principles and offer a sustainable and nutritious option for consumers. Rhizomucor pusillus strain CBS 143028 has emerged as a candidate food ingredient. Fermentation of R. pusillus CBS 143028 results in a mycelium biomass mainly comprising fungal proteins, cell wall components, and micronutrients. Although R. pusillus has a history of safe use in the production of food enzymes and in traditional fermented foods, the fungal biomass obtained after fermentation of R. pusillus CBS 143028 is considered a novel food and requires a thorough safety assessment. To this end, a 90-day oral toxicity study was conducted in which Wistar rats (10/sex/group) were provided diets containing 0, 100,000, 200,000, or 300,000 ppm of R. pusillus mycelium. Standard toxicity study parameters as given in OECD Test Guideline 408 were examined. The mean achieved dosages of R. pusillus mycelium were 6398, 12,738, or 19,668 mg/kg body weight/day for males and 7235, 14,949, or 22,461 mg/kg body weight/day for females in the low-, mid-, and high-dose groups, respectively. Although statistically significant differences were reported, these effects were not considered biologically relevant or test article-related due to atypical values in the control groups, the lack of a dose response relationship, or were attributed to normal biological variation. Thus, the no-observed-adverse-effect level (NOAEL) was established at 300,000 ppm (corresponding to 19,668 and 22,461 mg/kg body weight/day for males and females, respectively), the highest concentration tested.

Efficacy evaluation of food enzyme addition in fermented adzuki bean paste production

Efficacy evaluation of food enzyme addition in fermented adzuki bean paste production

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Safety evaluation of the food enzyme carboxypeptidase C from the genetically modified Aspergillus niger strain PEG.

The food enzyme carboxypeptidase C (EC 3.4.16.5) is produced with the genetically modified Aspergillus niger strain PEG by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in nine food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.053 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with a xylanase obtained from A. niger strain XEA. The Panel considered this food enzyme as a suitable substitute for the carboxypeptidase to be used in the toxicological studies, because both strains were derived from the same recipient strain, the location of the inserts was comparable, no partial inserts were present and the production methods were essentially the same. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1850 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 901. A homology search for the amino acid sequence of the food enzyme to known allergens was made and one match with a wheat allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially in wheat-allergic individuals, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme endo-1,3(4)-β-glucanase from the non-genetically modified Talaromyces versatilis strain PF8.

The food enzyme endo-1,3(4)-β-glucanase (3-(1-3;1-4)-β-d-glucan 3(4)-glucanohydrolase; EC 3.2.1.6) is produced with the non-genetically modified Talaromyces versatilis strain PF8 by Erbslöh Geisenheim AG. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was calculated to be up to 0.110 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2229 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure resulted in a margin of exposure of at least 20,264. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory or contact allergens were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the food enzyme endonuclease from the non-genetically modified Penicillium citrinum strain NP 11-15.

The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non-genetically modified Penicillium citrinum strain NP 11-15 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.006 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1010 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 168,333. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of an extension of use of the food enzyme β-glucosidase from the non-genetically modified Penicillium guanacastense strain AE-GLY.

The food enzyme β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) is produced with the non-genetically modified Penicillium guanacastense strain AE-GLY by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in four food manufacturing processes. Subsequently, the applicant has requested to extend its use to include three additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. The dietary exposure was calculated to be up to 0.206 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Using the no observed adverse effect level reported in the previous opinion (943 mg TOS/kg bw per day), the Panel derived a margin of exposure of at least 4578. Based on the previous evaluation, the assessment of the new data and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of an extension of use of a food enzyme containing endo-polygalacturonase, pectinesterase, pectin lyase and non-reducing end α-l-arabinofuranosidase activities from the non-genetically modified Aspergillus niger strain PEC.

The food enzyme has four declared activities: endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase (endo-cleaving); EC 3.2.1.15), pectinesterase (pectin pectylhydrolase; EC 3.1.1.11), pectin lyase ((1-4)-6-O-methyl-α-d-galacturonan lyase; EC 4.2.2.10) and non-reducing end α-l-arabinofuranosidase (α-l-arabinofuranoside non-reducing end α-l-arabinofuranosidase; EC 3.2.1.55). It is produced with the non-genetically modified Aspergillus niger strain PEC by DSM Food Specialties B.V. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant has requested to extend its use to include four additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme-TOS was estimated only for the remaining six processes. The dietary exposure was calculated to be up to 0.612 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (204 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 333. Based on the previous evaluation, the assessment of the new data and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Safety evaluation of the food enzyme triacylglycerol lipase from the non-genetically modified Limtongozyma cylindracea strain AE-LAYH (B).

The food enzyme, a triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3), is produced with the non-genetically modified Limtongozyma cylindracea strain AE-LAYH (B) by Amano Enzyme Inc. It is intended to be used in six food manufacturing processes. Since residual amounts of food enzyme-total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining five food manufacturing processes. It was estimated to be up to 0.315 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualifies for the quality presumption of safety (QPS) approach of safety assessment and no issue of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A homology search for the amino acid sequence of the food enzyme to those of known allergens was made and one match with a honeybee venom allergen was found. The Panel considered that a risk of allergic reactions by dietary exposure, particularly in individuals allergic to honey, cannot be excluded, but is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the food enzyme manufacturing process, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

High-Level Expression of Sucrose Isomerase in Bacillus subtilis Through Expression Element Optimization and Fermentation Optimization.

Sucrose isomerase is an important food enzyme that catalyzes the isomerization of sucrose into isomaltulose, a functional sugar widely used in food industry, while the production level of sucrose isomerase in food safe host strains was much lower than industrial requirement. Bacillus subtilis is an excellent host strain for recombinant protein expression, which owns the characteristics of powerful secretory capability and generally recognized as safe state. In this study, the expression of sucrose isomerase in B. subtilis was improved through expression element optimization and fermentation optimization. Firstly, the extracellular chaperone PrsA was overexpressed to enhance extracellular folding of sucrose isomerase, which improved the recombinant expression level by 80.02%. Then, the protein synthesis level was optimized through promoter screening, improving the recombinant expression level by 60.40%. On the basis of strain modification, the fermentation conditions including nitrogen source, carbon source, metal ion, pH and temperature were optimized successively in shake-flask. Finally, the 3 L bioreactor cultivation condition was optimized and yielding a sucrose isomerase activity of 862.86 U/mL, the highest level among the food safety strains. This study provides an effective strategy to improve the expression level of food enzymes in B. subtilis.

Molecular insights into the interaction between graphene oxide and β-galactosidase: From multispectral experiment, enzyme kinetics experiment to computational simulations

β-galactosidase has potential applications in various scientific disciplines, including the food industry, genetic engineering, and enzyme engineering. In this study, multi-spectral experiments combined with enzyme kinetics experiments and computational simulation were used to explore the interaction mechanism, structural and functional changes, and effects on the microenvironment of graphene oxide and β-galactosidase. Graphene oxide was synthesized using the improved Hummer method, and the characterization experiments demonstrated that the structure was flat and free of cracks, and the functional groups satisfied the required amount. Zeta potential experiments and molecular docking results jointly confirmed that graphene oxide and β-galactosidase were combined by van der Waals forces, electrostatic forces, and hydrophobic forces. Fluorescence quenching experiments showed that the quenching constant decreased from 2.90 × 1012 mLμg-1s−1 to 1.76 × 1012 mLμg−1 s−1 with the increase of temperature, indicating that the quenching mode was static quenching, accompanied by increased polarity and decreased hydrophilicity of β-galactosidase. Circular Dichroism Spectrum, Fourier Transform infrared spectroscopy, the ONPG experiment, and molecular dynamics simulation proved that the stability and activity of β-galactosidase were improved after binding with graphene oxide. These findings provide support for the potential applications of β-galactosidase in addressing lactose intolerance, optimizing gene expression efficiency, and developing innovative biosensors or bioprocessing systems.

Construction of a Kluyveromyces lactis strain with multi-copy integration for enhanced bovine chymosin production by CRISPR/Cas9 and UV mutagenesis

Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcymD, and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcymD-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.

The study of stabilizing food enzymes using b-cyclodextrins

Background: Today, biologically active additives, which contain hydrolytic enzymes (amylase, lipase, protease), are widely used in the digestive industry. These enzymes are often obtained from animal and vegetable raw materials, -for example, from the pancreas of a reindeer, the Fabricius bag of chickens, and Medusomyces Gisevii Lindau (Kombucha). However, in obtaining enzymes, the activity of amylase and lipase may decrease due to active forms --of proteases. Objective: The main goal of this research work was to evaluate the stabilizing effect of nanostructures–cyclodextrins on the activity of hydrolytic enzymes (protease, amylase, lipase) obtained from animal raw material (chicken bursa of Fabricius) and microorganism cultures (Kombucha). Identify the relationship at which the negative influence of external environmental factors is leveled and, as a result, enzymatic activity is maintained for a certain period of time. Methods: The protein concentration in the solution was determined using the Lowry method. A modified method by Rukhlyadiva was used to determine the amylolytic activity. The definition of lipolytic activity is based on modified Shibabi-Bishop method. To determine the amylolytic activity, a modified method by Rukhlyadiva was used. Results: The relationship between b-cyclodextrin and enzymes was determined, at which enzymatic activity is maintained for a certain period. Conclusion: Thus, cyclodextrins in solution stabilizes the activity of proteases, amylases, and lipases, which is of great practical importance in the food and pharmaceutical industries. Keywords: kombucha, pancreas, enzymes, amylase, lipase, protease, cyclodextrins

Exploring the connection between food and midgut digestive enzymes to improve honey bee (Apis mellifera) nutrition

Honey bee malnutrition is the leading challenge in beekeeping. Finding a high-quality and cheap alternative to polyfloral pollen is essential. To find a quick and cheap analysis that can be used to evaluate different substitutes for pollen in bee nutrition, caged bees were fed with four types of patties based on sugar, yeast, pollen, and Tenebrio molitor flour (SG, YG, PG, and TG group, respectively). Midgut and hindgut weight, as well as amylase and protease activity in the midgut, were measured to evaluate the effect of patties. Midgut weight was lowest in SG except on days 21 and 28 when it was similar to PG, and highest in YG on day 14 and 21. In SG and TG midgut weight didn’t change over time, while in YG it decreased from day 14 to day 28 and PG from day 7 to day 21 and 28. Specific amylase activity was the highest in YG, and lowest in SG, while it decreased in the YG and PG with time. Specific protease activity decreased in all groups comparing day 7 to day 28. PG and TG bees had a higher number of enzyme isoforms in comparison to YG bees, while SG bees had the lowest number of isoforms in contrast to all other groups. Mealworm patty fed bees had the highest protein concentration in the midgut at day 14 and 28, and similar to PG bees at day 21, induced similar enzyme profiles as pollen patty indicating its superiority over YG and SG.

Safety evaluation of the food enzyme β-galactosidase from the genetically modified Bacillus licheniformis strain DSM 34099.

The food enzyme β-galactosidase (β-d-galactoside galactohydrolase; EC 3.2.1.23) is produced with the genetically modified Bacillus licheniformis strain DSM 34099 by Kerry Group Services International, Ltd. (KGSI). The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in two food manufacturing processes. Dietary exposure was estimated to be up to 7.263 mg total organic solids/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests, other than an assessment of allergenicity, were considered unnecessary by the Panel. A search for the identity of the amino acid sequence of the food enzyme to known allergens was made and one match with a food allergen from kiwi fruit was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to kiwi fruit, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the food enzyme glucan 1,4-α-maltohydrolase from the genetically modified Saccharomyces cerevisiae strain LALL-MA.

The food enzyme glucan 1,4-α-maltohydrolase (4-α-d-glucan α-maltohydrolase; EC 3.2.1.133) is produced with the genetically modified Saccharomyces cerevisiae strain LALL-MA+ by Danstar Ferment AG. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the processing of cereals and other grains for production of baked products. Dietary exposure was estimated to be up to 0.014 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the identity of the amino acid sequence of the food enzyme to known allergens was made and four matches were found, three with respiratory allergens and one with an allergen from mosquito (injected). The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety assessment of the process Lietpak, based on the EREMA MPR technology, used to recycle post-consumer PET into food contact materials.

The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of the recycling process Lietpak (EU register number RECYC327), which uses the EREMA MPR technology. The input material is hot caustic washed and dried poly(ethylene terephthalate) (PET) flakes originating from collected post-consumer PET containers, including no more than 5% PET from non-food consumer applications. The flakes are heated in a ■■■■■ reactor under vacuum. Having examined the challenge test provided, the Panel concluded that the ■■■■■ decontamination (step 2), for which a challenge test was provided, is critical in determining the decontamination efficiency of the process. The operating parameters to control the performance of this step are temperature, pressure and residence time. It was demonstrated that this recycling process is able to ensure a level of migration of potential unknown contaminants into food below the conservatively modelled migration of 0.1 μg/kg food, derived from the exposure scenario for infants, when such recycled PET is used at up to 100%. Therefore, the Panel concluded that the recycled PET obtained from this process is not of safety concern when used at up to 100% for the manufacture of materials and articles for contact with all types of foodstuffs, including drinking water, for long-term storage at room temperature or below, with or without hotfill. Articles made of this recycled PET are not intended to be used in microwave or conventional ovens and such uses are not covered by this evaluation.