Generally Recognized As Safe Status Research Articles

REGULATION OF FUNCTIONAL FOODS IN UKRAINE AND THE WORLD. PROSPECTS FOR THE USE OF POSTBIOTICS AS FUNCTIONAL INGREDIENTS

Functional food allows to individualize the characteristics and necessities of each person. Over the past few decades, the demand for products that have a positive impact on human health has exploded worldwide. The market for functional foods in developed countries is estimated at USD 300 billion, which makes this sector attractive for investment. Accordingly, the production of functional foods and ingredients needs to be regulated at the state level. There is no harmonization in the definitions of functional foods and their classification at both the global and state levels. Despite the fact that the concept and terminology of functional food and the means of achieving it is well-established, the term “functional food” is often absent in the legislative acts of countries that actively produce functional foods, and the term “health-related food” is used instead. As a rule, health-related foods are subject to quality and safety requirements that are identical to those of conventional food products. Exceptions are regulations that establish rules for labeling FFPs and statements regarding their physiological effects or the content of a particular ingredient that may affect physiological aspects. In Europe and the USA, much attention is paid to the safety of “health-related food” and the novel food products. European Food Safety Authority and the US Food and Drug Administration require sponsors to submit information on the composition of multiple batches of a product to support safety evaluations for novel foods and Generally Recognized as Safe (GRAS) ingredients. In Ukraine, the legal status of the terms “functional food product” and dietary supplement was regulated by Law of Ukraine 771 “On Basic Principles and Requirements for Food Safety and Quality”, but in the version of this law dated 16.01.2020, this term was excluded, the term “dietary supplement” was edited and the term “novel food product or ingredient” was included, which is related to the process of harmonization of Ukrainian and European legislation in the food sector. In 2020, the Ministry of Health of Ukraine approved Order No. 1145 “On Approval of the Requirements for Nutrition Claims and Health Claims for Food Products”, which regulates the following concepts: claims and conditions for their use; health claims allowed to be used in food labeling and advertising, except for claims about reducing the risk of diseases and claims related to the development and health of children; claims about reducing the risk of diseases and claims related to the development and health of children. Unfortunately, Ukrainian regulations do not contain claims and conditions for the use of claims about pro- and prebiotics that have GRAS status worldwide. Particular attention should be paid to the possibility of using postbiotics as functional food ingredients with immunological activity.

FEMA GRAS assessment of natural flavor complexes: Allspice, anise, fennel-derived and related flavoring ingredients.

The FEMA Expert Panel program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavoring ingredients in food has resulted in the publication of an updated constituent-based procedure as well as publications on the safety evaluation of many botanical-derived NFCs. This publication, ninth in the series and related to the ninth publication, describes the affirmation of the generally recognized as safe (GRAS) status for NFCs with propenylhydroxybenzene and allylalkoxybenzene constituents under their conditions of intended use as flavoring ingredients added to food. The Panel's procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology for the NFCs themselves and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s) with suspected genotoxic potential, the estimated intake of the individual constituent is compared to the TTC for compounds with structural alerts for genotoxicity and if exceeded, a margin of exposure is calculated using BMDL10 values derived from benchmark dose analyses using Bayesian model averaging, as presented in the tenth article of the series. Safety evaluations for NFCs derived from allspice, anise seed, star anise, sweet fennel seed and pimento leaves were conducted and their GRAS status was affirmed for use as flavoring ingredients. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food.

VT-1161-A Tetrazole for Management of Mono- and Dual-Species Biofilms.

VT-1161 is a novel tetrazole antifungal agent with high specificity for fungal CYP51 (compared to human CYP enzymes) which has been proven to have fewer adverse effects and drug-drug interaction profiles due to fewer off-target inhibitors. In this study, we evaluated the anti-biofilm potential of VT-1161 against mono- and dual-species biofilms of Candida albicans, Klebsiella pneumoniae and Staphylococcus aureus. VT-1161 inhibited planktonic growth of all three strains, with an MIC value of 2 µg mL-1 for C. albicans and 0.5 µg mL-1 for K. pneumoniae and S. aureus, and killed 99.9% of the microbial populations, indicating a cytocidal action. Additionally, VT-1161 showed an excellent anti-biofilm action, since it inhibited mono-microbial biofilms by 80% at 0.5 µg mL-1, and dual-species biofilms of C. albicans/K. pneumoniae and C. albicans/S. aureus by 90% at the same concentration. Additionally, the eradication of mature biofilms after 24 h of VT-1161 exposure was excellent, reaching 90% at 2 μg mL-1 for both mono- and dual-species biofilms. In such mixed biofilms, the use of VT-1161 was revealed to be an alternative treatment because it was able to reduce the number of cells of each species during both inhibition and eradication. Since long-term therapy is necessary for most fungal biofilm infections due to their recurrence and obstinacy, VT-1161 showed low cytotoxicity against normal human cell lines and also against the invertebrate model Caenorhabditis elegans. Considering the excellent anti-biofilm potential and its GRAS (generally recognized as safe) status, VT-1161 may find use in the prevention or therapeutic treatment of mono- or poly-microbial biofilms.

Study of the persistence and dynamics of recombinant mCherry-producing Yarrowia lipolytica strains in the mouse intestine using fluorescence imaging.

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.

The sweet protein brazzein as a promising natural sweetener

In the modern world, due to the overconsumption of sugar-containing products, the problem of obesity is relevant. Among the many sweeteners that minimize sugar intake, a group of sweet-tasting proteins is up-and-coming. Brazzein is the smallest of the sweet proteins (54 aa, 6473 Da), and it is also safe for obese and diabetic people since it does not affect blood sugar and insulin levels. Brazzein has high thermal stability over a wide pH range: from 2 to 8 [1]. To increase the level of sweetness of brazzein, mutant variants of this protein were created through site-directed mutagenesis, the sweetest of which is triple mutant H31R/E36D/E41A, which is 22,500 times sweeter than sucrose [2]. Since the content of brazzein in the fruits of the natural source (Pentadiplandra brazzeana) is extremely low (0.2%), various methods have been developed to obtain brazzein using heterologous expression systems, which used as producers: bacteria (Escherichia coli, Lactococcus lactis), yeast (Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum) and animals (Mus musculus) [35]. Despite the short peptide sequence, the industrial production of recombinant protein faced several problems, including low protein yield (e.g in mouse milk it was detectable on western blot analysis only) and loss of sweetness. Аn extremely relevant and promising way to obtain recombinant brazzein is the optimization of extracellular expression in bakers yeasts with the GRAS (Generally recognized as safe) status, since the safety of these microorganisms for human health can potentially significantly reduce the number of brazzein purification steps and thereby reduce its cost to consumers.

β-caryophyllene, a cannabinoid receptor 2 agonist, decreases the motivational salience and conditioning place preference for palatable food in female mice.

β-caryophyllene (BCP) is a cannabinoid receptor CB2 agonist plant-derived terpenoid found in different essential oil plants, including rosemary, black pepper, copaiba and cannabis. It has GRAS (generally recognized as safe) status and is approved by the FDA (Food and Drug Administration) for food use. BCP displays agonist activity on the CB2 receptor and is a potential therapeutic target in several neuropsychiatric disorders, including anxiety and drug addiction. Unlike CB1 receptors, activation of the CB2 receptors is devoid of psychotomimetic and addictive properties. In this regard, this study aimed to evaluate the effects of BCP on incentive salience ("wanting") performance and motivational properties elicited by sweetened palatable foods in female Swiss mice. After 9days of training for incentive salience performance for a sweet reward (hazelnut cream with chocolate), food-restricted mice received a systemic injection of BCP (50 and 100 mg/kg) before testing over 3days. Moreover, independent groups of female mice were tested on sweet reward-induced conditioned place preference (CPP) for 22 consecutive days. To evaluate BCP effects on the expression of seeking behaviour for sweetened food, mice received a single intraperitoneal injection of BCP (50 mg/kg) 30min before testing on the CPP task. BCP significantly decreased the incentive performance for a sweet reward compared with the control group in a CB2 receptor-dependent manner. Also, BCP suppressed the expression of sweet reward-CPP. Altogether, these preclinical data demonstrate the potential role of BCP in treating disorders associated with food addiction-like behaviour.

Antimicrobial and Odour Qualities of Alkylpyrazines Occurring in Chocolate and Cocoa Products

A total of 40 pyrazine compounds of cocoa and chocolate were discovered through a literature analysis. Data on the alkylpyrazines’ antibacterial properties were gathered. It was discovered that 12 substances were effective against bacteria, fungus and oomycetes. Following calculations using PASS and AntiBac-Pred, 15 substances were identified as potentially having the ability to inhibit the growth of the Picornavirus, Yersinia pestis, Resistant Mycobacterium tuberculosis, Mycobacterium bovis BCG (Bacillus Calmette-Guerin), Micrococcus luteus and Corynebacterium jeikeium. The study of odour qualities led to the identification of the most potent odourants among alkylpyrazines as well as the aroma composition of cocoa and chocolate. Pyrazines have been given GRAS (generally recognized as safe) status by the Flavor and Extract Manufacturers Association (FEMA) for use as flavouring additives in food, demonstrating that this class of chemicals is a secure substitute for managing and combating microbial contamination, which also provides beneficial odour properties to the recipient. A statistical evaluation of the correlation between the odour threshold and the probability of antibacterial activity was performed. A more in-depth investigation of the antimicrobial and olfactory activities of alkylpyrazines is required in the future.

A Novel Co-crystallization Technique to enhance the Physicochemical property of BCS Class-II drugs using Efavirenz as a model drug

Pharmaceutical co-crystallization, a novel technique, provides for alteration and tailoring of physicochemical properties of active pharmaceutical ingredients (API) notwithstanding maintaining the intrinsic activity of the drug molecule. In the present work, co-crystals of efavirenz (EFA) were prepared with selected conformers of GRAS (Generally Recognized as Safe) status wise salicylic acid (SA) and benzoic acid (BA) using solution crystallization method to improve its dissolution. The assembly of crystal structure was evaluated by means of fourier transformation infrared spectroscopy (FTIR), x-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR). Saturation solubility and in vitro dissolution studies were further employed to investigate co-crystals. Equilibrium solubility profile of EFA-SA and EFA-BA exhibits an enhancement of 1.60 and 1.29 folds of solubility of efavirenz co-crystals as compared to the pure drug. Pharmacokinetic study performed in rats showed AUC0-∞ for co-crystals formulation was higher (13.15±0.02µg hmL−1) than the pure drug suspension formulation 7.85±0.03µg hmL−1. Statistically, AUC0-t of the co-crystal formulation was significantly higher (p<0.05) as compared to pure drug suspension formulation. Higher amount of drug concentration in blood indicated better systemic absorption of efavirenz from co-crystals formulation as compared to the pure drug suspension formulation.

The Integrated Probiotic Database: a genomic compendium of bifidobacterial health-promoting strains.

Background: The World Health Organization defines probiotics as "live microorganisms, which when administered in adequate amounts confer a health benefit on the host". In this framework, probiotic strains should be regarded as safe for human and animal consumption, i.e., they should possess the GRAS (generally recognized as safe) status, notified by the local authorities. Consistently, strains of selected Bifidobacterium species are extensively used as probiotic agents to prevent and ameliorate a broad spectrum of human and/or animal gastrointestinal disorders. Even though probiotic properties are often genus- or species-associated, strain-level differences in the genetic features conferring individual probiotic properties to commercialized bifidobacterial strains have not been investigated in detail. Methods: In this study, we built a genomic database named Integrated Probiotic DataBase (IPDB), whose first iteration consists of common bifidobacterial strains used in probiotic products for which public genome sequences were available, such as members of B. longum subsp. longum, B. longum subsp. infantis, B. bifidum, B. breve, and B. animalis subsp. lactis taxa. Furthermore, the IPDB was exploited to perform comparative genome analyses focused on genetic factors conferring structural, functional, and chemical features predicted to be involved in microbe-host and microbe-microbe interactions. Results and conclusion: Our analyses revealed strain-level genetic differences, underlining the importance of inspecting the strain-specific and outcome-specific efficacy of probiotics. In this context, IPDB represents a valuable resource for obtaining genetic information of well-established bifidobacterial probiotic strains.

Field Survey of Dietary Supplements and Foods with Probiotics Added – Annotation of USA Label Compliance

As US regulatory framework lays the foundation of safety aspects and the efficacy of clinical effects of food products, specifically dietary supplement products, the reality of regulatory compliance is not well understood. Especially, live microorganisms as dietary supplements have many challenges in label compliance due to the unclear regulatory framework, but also due to the complexity of such living organisms. Field surveys were conducted in Southern California, USA (specifically in the Orange County and Los Angeles County regions) at local retailers or online from January 2019 to April 2021. Dietary supplements and food categories that included probiotics specifically into formula were assessed for label compliance and ingredient safety through manual annotations and curation of information. The data were observed and documented statistically. Eighty (80) unique products were collected where the number of probiotic strains ranged from 34 to 1 strain. Thirty-three (33) unique statement of identity were recorded, range of violations were maximum 9 to minimum 0. Violations were checked against the US label compliance per Chapter 21 of the Code of Federal Regulations (CFR) in food and dietary supplement label compliance regulations. Eighty-one (81) unique probiotics were observed where 33 were not confirmed of GRAS (generally recognized as safe) status, and 6 had no NDI (new dietary ingredient) status. Most labels were not in compliance with all the regulatory nuances that were found. Although most probiotics were only listed on the species level, it is still a topic worthwhile the discussion of increasing regulatory awareness to the industry. Non-compliance with labels could be mitigated by such educational outreach.

Some Important Metabolites Produced by Lactic Acid Bacteria Originated from Kimchi.

Lactic acid bacteria (LAB) have been used for various food fermentations for thousands of years. Recently, LAB are receiving increased attention due to their great potential as probiotics for man and animals, and also as cell factories for producing enzymes, antibodies, vitamins, exopolysaccharides, and various feedstocks. LAB are safe organisms with GRAS (generally recognized as safe) status and possess relatively simple metabolic pathways easily subjected to modifications. However, relatively few studies have been carried out on LAB inhabiting plants compared to dairy LAB. Kimchi is a Korean traditional fermented vegetable, and its fermentation is carried out by LAB inhabiting plant raw materials of kimchi. Kimchi represents a model food with low pH and is fermented at low temperatures and in anaerobic environments. LAB have been adjusting to kimchi environments, and produce various metabolites such as bacteriocins, γ-aminobutyric acid, ornithine, exopolysaccharides, mannitol, etc. as products of metabolic efforts to adjust to the environments. The metabolites also contribute to the known health-promoting effects of kimchi. Due to the recent progress in multi-omics technologies, identification of genes and gene products responsible for the synthesis of functional metabolites becomes easier than before. With the aid of tools of metabolic engineering and synthetic biology, it can be envisioned that LAB strains producing valuable metabolites in large quantities will be constructed and used as starters for foods and probiotics for improving human health. Such LAB strains can also be useful as production hosts for value-added products for food, feed, and pharmaceutical industries. In this review, recent findings on the selected metabolites produced by kimchi LAB are discussed, and the potentials of metabolites will be mentioned.

The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis

BackgroundBacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP.ResultsAs proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity.ConclusionOur results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level.Graphic abstract

The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins.

Bacillus subtilis is a successful host for producing recombinant proteins. Its GRAS (generally recognized as safe) status and its remarkable innate ability to absorb and incorporate exogenous DNA into its genome make this organism an ideal platform for the heterologous expression of bioactive substances. The factors that corroborate its value can be attributed to the scientific knowledge obtained from decades of study regarding its biology that has fostered the development of several genetic engineering strategies, such as the use of different plasmids, engineering of constitutive or double promoters, chemical inducers, systems of self-inducing expression with or without a secretion system that uses a signal peptide, and so on. Tools that enrich the technological arsenal of this expression platform improve the efficiency and reduce the costs of production of proteins of biotechnological importance. Therefore, this review aims to highlight the major advances involving recombinant expression systems developed in B. subtilis, thus sustaining the generation of knowledge and its application in future research. It was verified that this bacterium is a model in constant demand and studies of the expression of recombinant proteins on a large scale are increasing in number. As such, it represents a powerful bacterial host for academic research and industrial purposes.

New Insights into the Virulence Traits and Antibiotic Resistance of Enterococci Isolated from Diverse Probiotic Products.

The GRAS (generally recognized as safe) status of Enterococcus has not yet been authenticated, but enterococci, as probiotics, have been increasingly applied in human healthcare and animal husbandry, for instance as a dietary supplement, feed additive, or growth promotor. The food chain is the important route for introducing enterococci into the human gut. The pathogenicity of Enterococcus from probiotic products requires investigation. In the study, 110 commercial probiotic products used for human, animal, aquaculture, and plants were examined, among which 36 enterococci were identified, including 31 from Enterococcus faecium, 2 from E. faecalis, 2 from E. casseliflavus, and 1 from E. gallinarum. Strikingly, 28 of the 36 enterococci isolated from probiotics here did not mention the presence of Enterococcus in the labeled ingredients, and no Enterococcus isolates were found from 5 animal probiotics that were labeled with the genus. In total, 35 of the 110 products exhibited hemolysis, including 5 (10.6%) human probiotics, 14 (41.2%) animal probiotics, 8 (57.1%) aquaculture probiotics, and 8 (53.3%) plant probiotics. The detection rates of virulence factors associated with adhesion, antiphagocytosis, exoenzyme, biofilm, and other putative virulence markers (PVM) in 36 enterococci were 94.4%, 91.7%, 5.6%, 94.4% and 8.3%. Twenty-six of the 36 isolated strains exhibited biofilm formation ability, where 25 strains (69.4%) and one (2.8%) were strong and weak biofilm producers, respectively. We analyzed the resistance rates against erythromycin (97%), vancomycin and ciprofloxacin (8%), tetracycline (3%), and high-level aminoglycosides (0%), respectively. High detection rates of msrC/lsaA (86%) and aac(6′)-Ii (86%) were observed, followed by vanC (8%), tetM (3%). The Tn5801-tetM-like integrative conjugative element (ICE) was identified in E. gallinarum, exhibiting resistance to tetracycline (64 μg/mL). Seven probiotic E. faecalis and E. faecium, as active ingredients in human probiotics, shared the same STs (sequence types) and were distinct from the STs of other contaminated or mislabeled enterococci, indicating that two particular STs belonged to native probiotic isolates. These findings advocate appropriate assessments of enterococci when used in probiotics.

Milhocina como fonte proteica para produção de bacteriocina por Enterococcus faecium

A produção de bacteriocina por Bactérias Ácido Lácticas tem atraído grande atenção por causa do seu status GRAS (Generally Recognized as Safe), e seu uso potencial como aditivo para a conservação de alimentos. A utilização de substrato com características que forneçam nutrientes necessários porem com reduzido custo, está entre os itens essenciais para produção e uso comercial de bacteriocinas. Portanto, o objetivo deste trabalho foi avaliar a viabilidade da produção de enterocinas, utilizando soro de leite e água de maceração de milho (milhocina) como substrato em 18 e 24 horas de cultivo. Enterococcus faecium (Efm20, Efm22, Efm24, Efm25) e Enterococcus faecalis (Efs27) foram caracterizados quanto à atividade antimicrobiana de enterocinas presentes no sobrenadante de cultivo livre de células (CFS) contra Listeria innocua CLIP 12612. O caráter proteico da substância antimicrobiana foi confirmado, sendo sua atividade inibitória perdida após o tratamento dos CFS com proteases. Em contrapartida a atividade antimicrobiana dos CFS se manteve termoestável após tratamento com calor (80°C e 100°C) e também quando tratado com catalase, o que confirma que essa atividade do CFS não está relacionada com a ação de H2O2 e ácido láctico (CFS neutralizado com NaOH). Pela contagem de células bacterianas (UFC/mL) dos isolados cultivados nos diferentes meios, observou que houve maior quantidade de UFC em MRS com substratos. A atividade antimicrobiana dos CFS obtido dos meios MRS com substrato em comparação com o MRS foram similares quando em difusão em ágar, porém ao quantificar essa atividade observou maiores valores de unidade arbitrária para os CFS obtidos em meio MRS com milhocina em ambos os tempos avaliados 18 e 24 horas. Em conclusão, constatou-se que os substratos testados apresentam grande potencial para serem aplicados na fabricação de bioconservação de produtos alimentícios.

Milk Protein-Stabilized Emulsion Delivery System and Its Application to Foods

Milk proteins, such as casein and whey protein, exhibit significant potential as natural emulsifiers for the preparation and stabilization of emulsion-based delivery systems. This can be attributed to their unique functional properties, such as the amphiphilic nature, GRAS (generally recognized as safe) status, high nutritional value, and viscoelastic film-forming ability around oil droplets. In addition, milk protein has been used as a coating material in emulsion-based delivery systems to protect bioactive compounds during food processing and storage owing to its unique functional properties. These properties include the ability to bind lipophilic bioactive compounds and antioxidant activity. In this review, we present the use of milk proteins as emulsifiers for the formation of emulsions and food applications of milk protein-stabilized emulsion delivery systems.

Making life look and taste better

Making life look and taste better

Application of MS bacteriophages on contaminated trimmings reduces Escherichia coli O157 and non-O157 in ground beef

According to the United States Food and Drug Administration (FDA) agency, bacteriophage solutions targeting the serotype O157:H7 are Generally Recognized as Safe (GRAS) to control STEC during beef processing. However, outbreaks involving the “Big Six” STEC increased the industry concern about those serotypes. The objective of this study was to test the efficacy of MS bacteriophages to reduce the “Big Six” non-O157 STEC in beef. The lysing efficacy of phages isolated for each specific serotype varied from 96.2% to 99.9% in vitro. When applied to contaminated trim, reductions ranging from 0.7 to 1.3 Log of all STEC were observed in ground beef. Bacteriophages may provide an additional barrier against the “Big Six” STEC in ground beef. Results of this research provide support documentation to the FDA to extend GRAS status for bacteriophages as processing aids against all adulterant STEC.

Protein-based nanoparticles for drug delivery purposes.

Proteins represent a group of biopolymers with interesting properties to be employed as raw materials in the preparation of nanoparticles for drug delivery purposes. Due to the inherent properties of proteins (i.e., biodegradability, amphiphilic properties, etc.) the resulting nanoparticles can be considered as versatility platforms for a variety of applications. Moreover, some proteins possess a GRAS (Generally Recognized as Safe) status or are considered as excipients by different Regulatory Agencies. As result of this, the resulting nanoparticles and potential translation to clinic would be facilitated, compared to other materials (i.e., polymers). This review is focused on the main proteins employed in the preparation of nanoparticles as well as the procedures permitting their transformation into nanoparticles able of accommodating a high variety of bioactive compounds and drugs. Moreover, the review also provides examples of application of nanoparticles prepared from albumins, globulins, prolamins or macromolecules derived from proteins.

Natural and engineered promoters for gene expression in Lactobacillus species

Lactobacillus species are attractive hosts for the expression of heterologous proteins, antigens, vaccines, and drugs due to their GRAS (generally recognized as safe) status. The bioengineering techniques open new possibilities of improving Lactobacillus strains. In this regard, the control of the gene expression in Lactobacillus strains through the adequate native or engineered promoters acquires a key role in the development of biotechnological applications and for their function as probiotic bacteria. Depending on the objective sought, the protein produced and the strain used, inducible or constitutive promoters can be chosen. Whereas, when a fine-tuning of gene expression is required, the development of synthetic promoter libraries could be the best approach. In this work, we revise the main constitutive and inducible natural promoters from Lactobacillus strains or from other genus that have been applied in Lactobacillus, as well as the few engineered promoters developed for these bacteria.