Improvement of Methods for Safety Control of Microbial Producers and Food Produced Therewith.

The study substantiates the necessity to implement the algorithm of molecular-genetic assessment of biosafety of the genetically modified microorganisms (GMM) and to develop standardized methods to test the genetically modified strains producing enzymes, bioactive substances, and other products of microbial synthesis prior to their use in food industry. Analysis of microbial producers and related food products for the presence of GMM-associated DNA revealed high incidence of the marker genes amp and lacZ in enzyme preparations and in mycelium of industrial genetically modified producer of Aspergillus genus. The procedure of extraction of DNA from mycelium of mold fungi is optimized by including the stage of additional purification of the extracts, assessment of their purity by PCR with universal ITS primers, and determination of effective DNA concentration in the samples prior to conduction of the molecular genetic assay. For identification and genotyping of mold fungi (the biotechnological producers of enzyme preparations), the Sanger sequencing method was adapted. Using this modified method, we determined the species of five equivocally identified strains of Aspergillus genus. To identify the closely-related micromycetes of Ascomycota division, a genotyping algorithm was developed based on amplification of total DNA with expanded panel of primers and DNA sequencing by capillary electrophoresis.

[Assessment of the influence of an enzymal preparation - a complex of glucoamylase and xylanase from Aspergillus awamori Xyl T-15 on the intestinal microbiom and immunological indicators of rats].

The requirements for the safety of food products obtained by microbial synthesis are including as obligation for to conduct toxicological studies - the study of various biochemical and immunological markers of toxic effects. The necessity of these studies is explained by a possible change in the structure of food ingredients produced by a microbial cell and, consequently, a change in their biological properties, as well as the possible presence of living forms and/or DNA of producer strains or of their toxic metabolites in these ingredients. At the same time, it is well known that the nutrient composition of foods has a significant impact on the composition and properties of microorganisms that make up the gut microbiome, which, in turn, determines the immune status. The purpose of the research was to justify the analyses of gut microbiocenosis composition for inclusion in the protocol of safety investigation of foods obtained by microbial synthesis [on the example of an enzyme preparation (EP) - a complex of glucoamylase and xylanase from a genetically modified strain of Aspergillus awamori Xyl T-15]. Material and methods. In experimental studies carried out for 80 days, Wistar rats (males and females) were used. The study of the effect of EP (a complex of glucoamylase and xylanase from a genetically modified Aspergillus awamori Xyl T-15 strain) in dozes 10, 100 and 1000 mg/kg body mass on the cecum microbiome and the immune status (content of cytokines and chemokines: IL-1a, IL-4, IL-6, IL-10, IL-17A, INF-γ, TNF-α, MCP-1, MIP-1a and Regulated on Activation Normal T-cell Expressed and Secreted - RANTES) was carried out. Results. It has been shown that EP - a complex of glucoamylase and xylanase from A. awamori Xyl T-15 at doses of 100 mg/kg or more causes mild disturbances in the composition of gut microbiocenosis. At the same time, these disorders have a significant immunomodulat ory and immunotoxic effect on the body, which manifests itself in a dose-dependent change in the profile of pro-inflammatory cytokines and chemokines in blood and spleen. The adverse effect of EP on the body is probably due to the formation of metabolites that are not formed during usual digestive processes in the gastrointestinal tract. The minimum effective dose (LOAEL) of EP was 100 mg/kg body weight In accordance with established requirements, the activity of the EP should not appear in ready-to-use food. Subject to this requirement, amount of EP entering the body cannot exceed the established LOAEL level. Therefore, a complex of glucoamylase and xylanase can be used in food industry, subject to the establishment of regulations «for technological purposes¼ for A. awamori Xyl T-15 strain. Conclusion. The data obtained on the relationship between the state of the microbiome and the immune status upon the introduction of EP indicate the need to include indicators of the state of gut microbiocenosis in the test protocol of safety.

A comprehensive review of metabolic and genomic aspects of PAH-degradation.

Polyaromatic hydrocarbons (PAHs) are considered as hazardous organic priority pollutants. PAHs have immense public concern and critical environmental challenge around the globe due to their toxic, carcinogenic, and mutagenic properties, and their ubiquitous distribution, recalcitrance as well as persistence in environment. The knowledge about harmful effects of PAHs on ecosystem along with human health has resulted in an interest of researchers on degradation of these compounds. Whereas physico-chemical treatment of PAHs is cost and energy prohibitive, bioremediation i.e. degradation of PAHs using microbes is becoming an efficient and sustainable approach. Broad range of microbes including bacteria, fungi, and algae have been found to have capability to use PAHs as carbon and energy source under both aerobic and anaerobic conditions resulting in their transformation/degradation. Microbial genetic makeup containing genes encoding catabolic enzymes is responsible for PAH-degradation mechanism. The degradation capacity of microbes may be induced by exposing them to higher PAH-concentration, resulting in genetic adaptation or changes responsible for high efficiency towards removal/degradation. In last few decades, mechanism of PAH-biodegradation, catabolic gene system encoding catabolic enzymes, and genetic adaptation and regulation have been investigated in detail. This review is an attempt to overview current knowledge of microbial degradation mechanism of PAHs, its genetic regulation with application of genetic engineering to construct genetically engineered microorganisms, specific catabolic enzyme activity, and application of bioremediation for reclamation of PAH-contaminated sites. In addition, advanced molecular techniques i.e. genomic, proteomic, and metabolomic techniques are also discussed as powerful tools for elucidation of PAH-biodegradation/biotransformation mechanism in an environmental matrix.

[Chemical food safety: development of methodological and regulatory base].

The review presents the results of studies carried out in the Federal Research Centre of Nutrition and Biotechnology in the direction of food toxicology, the purpose of which was to improve the risk assessment methodology, substantiate hygienic regulations for the content of chemical contaminants in foodstuffs and develop methods for their detection and quantitative determination. New challenges and problems associated with the control and regulation of chemical contaminants in foods are associated, firstly, with the identification of previously unrecognized chemical factors harmful to human health, and, secondly, are caused by the progress of technologies, accompanied by the emergence of new sources of nutrients and methods of processing foodstuffs, which, along with many benefits and advantages, creates new potential risks to consumer health. Among the priority chemical pollutants, which should be mentioned currently as objects of improved regulation and control methods, are toxic elements (organic and inorganic forms of arsenic, mercury, nickel), veterinary drugs, phycotoxins, phytotoxins, new mycotoxins, various forms of polychlorinated biphenyls and polycyclic aromatic hydrocarbons, biologically active substances of plant origin, concentrated during the production of extracts, as well as so-called technological contaminants, food additives, residual amounts of technological aids. An independent problem is the assessment of risks from nanoparticles and nanomaterials used in the production of foodstuffs, as well as enzyme preparations and food ingredients produced with the help of genetically modified microorganisms. The system of toxicological and hygienic assessment and control of chemical contaminants in foodstuffs operating in Russia is constantly being improved on the basis of new scientific data to substantiate the permissible levels of their content in products and new methods of analysis. The results obtained are reflected in the regulatory documents of the Russian Federation and the Eurasian Economic Union.

[Study on the development of measures to manage the risks associated with the food produced using microbial synthesis].

In order to increase the efficiency of food production, micro-organisms are used whose genetic material has been modified by directed mutagenesis or by transgenesis. Such products belong to a new type of products, the mandatory condition for the use of which is to confirm its safety when used in food. The aim of the article - justification of the need to improve the system for assessing the safety of foods obtained by microbial synthesis. Material and methods. The analysis and generalization of current scientific researches published in the databases Scopus, Web of Science, PubMed, RSCI, as well as national and international regulatory and legislative documents have been carried out. Results and discussion. The analysis of scientific data, legislative and regulatory documents of international legislation, the European Union, as well as other economically developed countries, has shown that a mandatory element of assessing the possibility of safe use of food produced by microbial synthesis is the study of the sequence of transgenic insert nucleotides in the producer strain in order to analyze the presence of pathogenicity determinants, antibiotic resistance, and the ability to produce toxic metabolites. The data obtained in vitro on the absence of risks of using both producer strains and the enzyme preparations and other ingredients synthesized by them in the food industry should be confirmed in experiments in vivo. Currently, the need to comply with these requirements, as well as the main criteria for assessing the risks of such food, are mainly provided by legislation and regulations of the Russian Federation. At the same time, the system of sanitary and hygienic assessment of the safety of producer strains and food ingredients produced by them needs to be updated.

New developments in biotechnology applied to microorganisms.

EFSA was requested by the European Commission (in accordance with Article 29 of Regulation (EC) No 178/2002) to provide a scientific opinion on the application of new developments in biotechnology (new genomic techniques, NGTs) to viable microorganisms and products of category 4 to be released into the environment or placed on the market as or in food and feed, and to non-viable products of category 3 to be placed on the market as or in food and feed. A horizon scanning exercise identified a variety of products containing microorganisms obtained with NGTs (NGT-Ms), falling within the remit of EFSA, that are expected to be placed on the (EU) market in the next 10 years. No novel potential hazards/risks from NGT-Ms were identified as compared to those obtained by established genomic techniques (EGTs), or by conventional mutagenesis. Due to the higher efficiency, specificity and predictability of NGTs, the hazards related to the changes in the genome are likely to be less frequent in NGT-Ms than those modified by EGTs and conventional mutagenesis. It is concluded that EFSA guidances are 'partially applicable', therefore on a case-by-case basis for specific NGT-Ms, fewer requirements may be needed. Some of the EFSA guidances are 'not sufficient' and updates are recommended. Because possible hazards relate to genotypic and phenotypic changes introduced and not to the method used for the modification, it is recommended that any new guidance should take a consistent risk assessment approach for strains/products derived from or produced with microorganisms obtained with conventional mutagenesis, EGTs or NGTs.

Rapid Monitoring of Viable Genetically Modified Escherichia coli Using a Cell-Direct Quantitative PCR Method Combined with Propidium Monoazide Treatment.

The commercialization of industrial genetically modified microorganisms (GMMs) has highlighted their impact on public health and the environment. Rapid and effective monitoring methods detecting live GMMs are essential to enhance current safety management protocols. This study aims to develop a novel cell-direct quantitative polymerase chain reaction (qPCR) method targeting two antibiotic-resistant genes, KmR and nptII, conferring resistance against kanamycin and neomycin, along with propidium monoazide, to precisely detect viable Escherichia coli. The E. coli single-copy taxon-specific gene of D-1-deoxyxylulose 5-phosphate synthase (dxs) was used as the internal control. The qPCR assays demonstrated good performance, with dual-plex primer/probe combinations exhibiting specificity, absence of matrix effects, linear dynamic ranges with acceptable amplification efficiencies, and repeatability for DNA, cells, and PMA-treated cells targeting KmR/dxs and nptII/dxs. Following the PMA-qPCR assays, the viable cell counts for KmR-resistant and nptII-resistant E. coli strains exhibited a bias% of 24.09% and 0.49%, respectively, which were within the acceptable limit of ±25%, as specified by the European Network of GMO Laboratories. This method successfully established detection limits of 69 and 67 viable genetically modified E. coli cells targeting KmR and nptII, respectively. This provides a feasible monitoring approach as an alternative to DNA processing techniques to detect viable GMMs.

Propidium Monoazide-Treated, Cell-Direct, Quantitative PCR for Detecting Viable Chloramphenicol-Resistant Escherichia coli and Corynebacterium glutamicum Cells.

With the rapid development and commercialization of industrial genetically modified microorganisms (GMMs), public concerns regarding their potential effects are on the rise. It is imperative to promptly monitor the unintended release of viable GMMs into wastewater, the air, and the surrounding ecosystems to prevent the risk of horizontal gene transfer to native microorganisms. In this study, we have developed a method that combines propidium monoazide (PMA) with a dual-plex quantitative PCR (qPCR) approach based on TaqMan probes. This method targets the chloramphenicol-resistant gene (CmR) along with the endogenous genes D-1-deoxyxylulose 5-phosphate synthase (dxs) and chromosomal replication initiator protein (dnaA). It allows for the direct quantitative detection of viable genetically modified Escherichia coli and Corynebacterium glutamicum cells, eliminating the requirement for DNA isolation. The dual-plex qPCR targeting CmR/dxs and CmR/dnaA demonstrated excellent performance across various templates, including DNA, cultured cells, and PMA-treated cells. Repeatability and precision, defined as RSDr% and bias%, respectively, were calculated and found to fall within the acceptable limits specified by the European Network of GMO Laboratories (ENGL). Through PMA-qPCR assays, we determined the detection limits for viable chloramphenicol-resistant E. coli and C. glutamicum strains to be 20 and 51 cells, respectively, at a 95% confidence level. Notably, this method demonstrated superior sensitivity compared to Enzyme-Linked Immunosorbent Assay (ELISA), which has a detection limit exceeding 1000 viable cells for both GM bacterial strains. This approach offers the potential to accurately and efficiently detect viable cells of GMMs, providing a time-saving and cost-effective solution.

Metagenomic Characterization of Multiple Genetically Modified Bacillus Contaminations in Commercial Microbial Fermentation Products.

Genetically modified microorganisms (GMM) are frequently employed for manufacturing microbial fermentation products such as food enzymes or vitamins. Although the fermentation product is required to be pure, GMM contaminations have repeatedly been reported in numerous commercial microbial fermentation produce types, leading to several rapid alerts at the European level. The aim of this study was to investigate the added value of shotgun metagenomic high-throughput sequencing to confirm and extend the results of classical analysis methods for the genomic characterization of unauthorized GMM. By combining short- and long-read metagenomic sequencing, two transgenic constructs were characterized, with insertions of alpha-amylase genes originating from B. amyloliquefaciens and B. licheniformis, respectively, and a transgenic construct with a protease gene insertion originating from B. velezensis, which were all present in all four investigated samples. Additionally, the samples were contaminated with up to three unculturable Bacillus strains, carrying genetic modifications that may hamper their ability to sporulate. Moreover, several samples contained viable Bacillus strains. Altogether these contaminations constitute a considerable load of antimicrobial resistance genes, that may represent a potential public health risk. In conclusion, our study showcases the added value of metagenomics to investigate the quality and safety of complex commercial microbial fermentation products.

Biodiversity risk assessment of genetically modified Chaetoceros gracilis for outdoor cultivation.

A genetically modified (GM) strain of the diatom Chaetoceros gracilis expressing the phosphite dehydrogenase gene (ptxD), which is a useful gene both for the biological containment and the avoidance of microbial contamination, was characterized to estimate the risk against the biodiversity by laboratory experiments. GM strain could grow in the medium containing phosphite as a sole source of phosphorus, while its general characteristics such as growth, salt tolerance, heat and dehydration resistance in the normal phosphate-containing medium were equivalent to those of wild type (WT) strain. The increase in potential toxicity of GM strain against plant, crustacean, fish and mammal was also disproved. The dispersal ability of WT strain cultured in an outdoor raceway pond was investigated for 28 days by detecting the psb31 gene in vessels, settled at variable distances (between 5 and 60 m) from the pond. The diatom was detected only in one vessel placed 5 m apart. To estimate the influence on the environment, WT and GM strains were inoculated into freshwater, seawater and soil. The influence on the microbiome in those samples was assessed by 16S rRNA gene amplicon sequencing, in addition to the analysis of the survivability of those strains in the freshwater and the seawater. The results indicated that the effect to the microbiome and the survivability were comparable between WT and GM strains. All results showed that the introduction of the ptxD gene into the diatom had a low risk on biodiversity.

DNA walking strategy to identify unauthorized genetically modified bacteria in microbial fermentation products.

Recently, unexpected contaminations of unauthorized genetically modified microorganisms (GMM) carrying antimicrobial resistance (AMR) genes were reported in microbial fermentation products commercialized on the food and feed chain. To guarantee the traceability and safety of the food and feed chain, whole-genome sequencing (WGS) has played a key role to prove GMM contaminations via the characterization of unnatural associations of sequences. However, WGS requires a prior microbial isolation of the GMM strain, which can be difficult to successfully achieve. Therefore, in order to avoid such bottleneck, a culture-independent approach was proposed in this study. First, the screening for the aadD gene, an AMR gene conferring a resistance to kanamycin, and for the pUB110 shuttle vector, carrying the aadD gene and commonly used to produce GMM, is performed. In case of a positive signal, DNA walking methods anchored on the two borders of the detected pUB110 shuttle vector are applied to characterize unknown flanking regions. Following to the sequencing of the generated amplicons, unnatural associations of sequences can be identified, allowing to demonstrate the presence of unauthorized GMM. The developed culture-independent strategy was successfully applied on commercialized microbial fermentation products, allowing to prove the presence of GMM contaminations in the food and feed chain.

Genetically Modified Micro-Organisms for Industrial Food Enzyme Production: An Overview.

The use of food enzymes (FE) by the industrial food industry is continuously increasing. These FE are mainly obtained by microbial fermentation, for which both wild-type (WT) and genetically modified (GM) strains are used. The FE production yield can be increased by optimizing the fermentation process, either by using genetically modified micro-organism (GMM) strains or by producing recombinant enzymes. This review provides a general overview of the different methods used to produce FE preparations and how the use of GMM can increase the production yield. Additionally, information regarding the construction of these GMM strains is provided. Thereafter, an overview of the different European regulations concerning the authorization of FE preparations on the European market and the use of GMM strains is given. Potential issues related to the authorization and control of FE preparations sold on the European market are then identified and illustrated by a case study. This process highlighted the importance for control of FE preparations and the consequent need for appropriate detection methods targeting the presence of GMM, which is used in fermentation products.

Production of Vitamin B2 (Riboflavin) by Microorganisms: An Overview.

Riboflavin is a crucial micronutrient that is a precursor to coenzymes flavin mononucleotide and flavin adenine dinucleotide, and it is required for biochemical reactions in all living cells. For decades, one of the most important applications of riboflavin has been its global use as an animal and human nutritional supplement. Being well-informed of the latest research on riboflavin production via the fermentation process is necessary for the development of new and improved microbial strains using biotechnology and metabolic engineering techniques to increase vitamin B2 yield. In this review, we describe well-known industrial microbial producers, namely, Ashbya gossypii, Bacillus subtilis, and Candida spp. and summarize their biosynthetic pathway optimizations through genetic and metabolic engineering, combined with random chemical mutagenesis and rational medium components to increase riboflavin production.

Characterisation of microorganisms used for the production of food enzymes.

This document is intended to assist the applicant in the preparation and the presentation of an application, as foreseen in Article 17.3 of Regulation (EC) No 1332/2008, for the authorisation of food enzymes. It specifically covers the characterisation of microorganisms used as production organisms.

Safety and efficacy of l-valine produced using Corynebacterium glutamicum CGMCC 11675 for all animal species.

The product subject of this assessment is l-valine produced by fermentation with a strain of Corynebacterium glutamicum (CGMCC 11675). It is intended to be used in feed and water for drinking for all animal species and categories. Owing to the uncertainties regarding the possible genetic modification of the original production strain, the FEEDAP Panel cannot conclude on the safety of the additive l-valine produced with C. glutamicum CGMCC 11675 for the target species, the consumers, the users and the environment. The FEEDAP Panel has concerns on the safety for the target animals of the simultaneous oral administration of valine-containing additives via feed and water for drinking. In the absence of data, the FEEDAP Panel cannot conclude on the potential of l-valine produced with C. glutamicum CGMCC 11675 to be toxic by inhalation, irritant to skin or eyes, or on its potential to be a dermal sensitiser. The product is considered an efficacious source of the amino acid l-valine for all animal species. The supplemental l-valine requires protection against rumen degradations in order to be as efficacious in ruminant as in non-ruminant species.

Guidance on the characterisation of microorganisms used as feed additives or as production organisms.

[Table: see text]. ABSTRACT: This guidance document is intended to assist the applicant in the preparation and the presentation of an application, as foreseen in Article 7.6 of Regulation (EC) No 1831/2003, for the authorisation of additives for use in animal nutrition. It specifically covers the characterisation of microorganisms used as feed additives or as production organisms.

Synthetic Phosphorus Metabolic Pathway for Biosafety and Contamination Management of Cyanobacterial Cultivation.

Recent progress in genetic engineering and synthetic biology have greatly expanded the production capabilities of cyanobacteria, but concerns regarding biosafety issues and the risk of contamination of cultures in outdoor culture conditions remain to be resolved. With this dual goal in mind, we applied the recently established biological containment strategy based on phosphite (H3PO3, Pt) dependency to the model cyanobacterium Synechococcus elongatus PCC 7942 ( Syn 7942). Pt assimilation capability was conferred on Syn 7942 by the introduction of Pt dehydrogenase (PtxD) and hypophosphite transporter (HtxBCDE) genes that allow the uptake of Pt, but not phosphate (H3PO4, Pi). We then identified and disrupted the two indigenous Pi transporters, pst (Synpcc7942_2441 to 2445) and pit (Synpcc7942_0184). The resultant strain failed to grow on any media containing various types of P compounds other than Pt. The strain did not yield any escape mutants for at least 28 days with a detection limit of 3.6 × 10-11 per colony forming unit, and rapidly lost viability in the absence of Pt. Moreover, growth competition of the Pt-dependent strain with wild-type cyanobacteria revealed that the Pt-dependent strain could dominate in cultures containing Pt as the sole P source. Because Pt is rarely available in aquatic environments this strategy can contribute to both biosafety and contamination management of genetically engineered cyanobacteria.

Safety and efficacy of l-threonine produced by fermentation using Escherichia coli CGMCC 7.232 for all animal species.

The product subject of this assessment is l-threonine produced by fermentation with a genetically modified strain of Escherichia coli (CGMCC 7.232). It is intended to be used in feed and water for drinking for all animal species and categories. The production strain and its recombinant DNA were not detected in the additive. The product l-threonine, manufactured by fermentation with E. coli CGMCC 7.232, does not raise any safety concern with regard to the genetic modification of the production strain. l-Threonine produced using E. coli CGMCC 7.232 is considered safe for the target species. The FEEDAP Panel has concerns regarding the safety of the simultaneous administration of l-threonine via water for drinking and feed. l-Threonine produced using E. coli CGMCC 7.232 is safe for the consumer. In absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin and eyes or to be a skin sensitiser. There is a risk from the inhalation exposure to endotoxins for persons handling the additive. l-Threonine produced using E. coli CGMCC 7.232 is safe for the environment. The product under assessment is considered an efficacious source of the amino acid l-threonine for all animal species. For l-threonine to be as efficacious in ruminants as in non-ruminant species, it requires protection against degradation in the rumen.

Safety of l-tryptophan technically pure, produced by fermentation with Escherichia coli DSM 25084, KCCM 11132P and SARI12091203 for all animal species based on a dossier submitted by FEFANA Asbl.

In 2015, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) issued an opinion on the safety and efficacy of l-tryptophan produced by fermentation using three strains of Escherichia coli, when used as a nutritional additive for all animal species. The Panel concluded that the safety of l-tryptophan produced by E. coli SARI12091203 for target animals, consumers, users and the environment could not be assessed because the data submitted did not permit the identity and safety of the strain, and the purity of the additive, to be determined. During the current assessment, the applicant withdrew the application for l-tryptophan produced by E. coli SARI12091203. l-Tryptophan produced by E. coli DSM 25084 or KCCM 11132P was considered safe for non-ruminant target species, the consumer and the environment. For both products, the level of endotoxins and the possible dusting potential indicated a risk by inhalation for the user. In the absence of data, a potential for dermal sensitisation could not be excluded. The Commission gave the applicant the possibility of submitting complementary information to allow the FEEDAP Panel to complete its assessment. The additional data on the characterisation of the additives and on their potential for inhalation toxicity and as skin sensitisers are the subject of the current opinion. Due to improvements in the manufacturing process, the level of endotoxins present in the l-tryptophan produced by E. coli KCCM 11132P has been markedly reduced; consequently, the endotoxin content does not represent a health risk for the user. The additive has a low acute toxicity by inhalation and is not considered as a potential skin sensitiser. l-Tryptophan produced by E. coli DSM 25084 is not considered a skin sensitiser. The level of endotoxins in this product, however, represents a risk by inhalation for the user handling the additive.

Safety of l-tryptophan technically pure, produced by Escherichia coli CGMCC 3667, for all animal species based on a dossier submitted by GBT Europe GmbH.

l-Tryptophan, technically pure, is a feed additive produced by fermentation with a genetically modified strain of Escherichia coli. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) of EFSA, issued two opinions on the safety and efficacy of the product, in which it could not conclude on the safety of this additive for target animals, consumer, user and the environment, due to the insufficient characterisation of the genetic modification. The European Commission asked the Authority to deliver an opinion on the safety of l-tryptophan, technically pure, as a nutritional additive for all animal species based on additional data submitted by the applicant. Based on new information provided on the genetic modification, including the presence/absence of antibiotic resistance genes in the production strain, the FEEDAP Panel concludes that the l-tryptophan is safe for target animals, consumers, users and the environment.