Generally Recognized As Safe Research Articles

Granulation and encapsulation of N-Acetylcysteine (NAC) by internal phase separation

The encapsulation of small hydrophilic molecules offers advantages for the delivery of hydrophilic food supplements and food ingredients. These advantages include: masking off-flavors, reducing astringency , and controlling release profiles. We used an internal phase separation (IPS) method for simultaneous granulation and encapsulation of a small hydrophilic food supplement in corn oil. To prove that IPS is a suitable method for the encapsulation of small hydrophilic molecules with controllable release times, we used the nutritional supplement N -acetylcysteine (NAC) as our model molecule. NAC has a variety of health and pharmaceutical benefits however it suffers from poor organoleptic properties such as a bitter aftertaste, astringency, and a strong sulfur smell. We used IPS to produce free-flowing granules containing 56–78% NAC with diameters ranging from 100 to 1000 μm. All the materials used in this process were either food safe or generally recognized as safe (GRAS). The granulated/encapsulated samples were characterized using FTIR, NMR, SEM, and LC-MS methods to confirm the presence of unreacted NAC in high loading concentrations. The kinetics of NAC release was then monitored using conductometry. The combination of hydrophilic carboxymethyl cellulose sodium salt (CMC) and hydroxypropyl methylcellulose (HPMC) gums with a hydrophobic coating such as ethyl cellulose (EC), provided an effective barrier to ensure a 5 min time window for NAC dispersion into water and drinks before the capsule starts to dissolve in water and loses more than 30 wt % of its total NAC loading. • Internal phase separation was used for NAC granulation and encapsulation. • Using all GRAS materials, NAC was granulated and encapsulated in high loading. • The encapsulated samples, showed delayed NAC release in water. • The spherical granules of encapsulated NAC can be used in ready-to-mix product.

Identificación de especies de Collletotrichum aisladas de fruto de mango y su efecto de control por la aplicación de compuestos GRAS

The fungus Colletotrichum sp. is the causal agent of anthracnose in mango fruit and leads to substantial postharvest losses (>30 %). Efficient control agents continue to be investigated to reduce the incidence of this pathogen, preferably of a biological-organic nature. The objective of this work was to evaluate the in vitro application of commercial-grade generally recognized as safe (GRAS) compounds as chitosan, hydrogen peroxide, acetic acid, and peracetic acid, in the control of Colletotrichum species isolated from mango fruit. The application of commercial chitosan (1.5 % and 2.0 %) confirmed its antifungal effect, with an average inhibition of 34 %, no significant difference was shown with respect to reactive grade chitosan. Hydrogen peroxide, acetic acid, and peracetic acid completely inhibited the development of C. asianum at concentrations > 1.0 %. Each GRAS agent caused morphological damage to the spores, including loss of turgidity, intracellular disorder, effusion of liquid from the cytoplasm, and total loss of integrity. Scanning electron microscopy confirmed the damage to the mycelia, with collapsed and dehydrated structures due to exposure to the control agents. Overall, commercial GRAS agents showed an in vitro control effect on the growth of C. asianum at different stages of their development.

Designing a Highly Efficient Biosynthetic Route for Lacto-N-Neotetraose Production in Escherichia coli.

Recently, the biosynthesis of human milk oligosaccharides (HMOs) has been attracting increasing attention. Lacto-N-neotetraose (LNnT) is one of the most important neutral-core HMOs with promising health effects for infants. It has received Generally Recognized as Safe (GRAS) status and is the second HMO commercially added in infant formula after 2'-fucosyllactose. In previous studies, a series of engineered Escherichia coli strains have been constructed and optimized to produce high titers of precursor lacto-N-triose II. On the basis of these strains, LNnT-producing strains were constructed by overexpressing the β1,4-galactosyltransferase-encoding gene from Aggregatibacter actinomycetemcomitans NUM4039 (Aa-β1,4-GalT). Interestingly, an appreciable LNnT titer was obtained by weakening the metabolic flux of the UDP-GlcNAc pathway and simply overexpressing the essential genes lgtA, galE, and Aa-β1,4-GalT in lacZ-, wecB-, and nagB-deleted E. coli. Subsequently, LNnT synthesis was optimized through balancing the expression of these three biosynthetic enzymes. The optimized strain produced LNnT with an extracellular titer of 12.1 g/L in fed-batch cultivation, with the productivity and specific yield of 0.25 g/L·h and 0.27 g/g dry cell weight, respectively.

ANTIBIOTIC RESISTANCE PROFILE OF THE NEWLY ISOLATED LACTIC ACID BACTERIA STRAINS FROM TRADITIONAL FERMENTED FOODS

The use of antibiotics is a major problem everywhere around the globe and in step with this, there's currently an increased public and scientific interest. Microbial resistance is a vital issue for the organizations like EFSA, WHO, FDA, and FAO because it is developing rapidly and is an increasingly serious health concern within the world. Microbial resistance comes as a result of continuous exposure of microorganisms to antibiotics. Lactic acid bacteria (LAB) constitute a really important group of microorganisms that can inhabit different conditions and environments, as the different parts of the gastrointestinal tract of humans and animals, and are an important element of the microbiota of fermented foods. The FDA and EFSA authorities have given them the status referred to as GRAS (Generally Recognized as Safe) and QPS (Qualified Presumption of Safety). Continuous exposure of LAB to environments with antibiotics may prompt them to become an intrinsic or extrinsic reservoir of genes accountable for antibiotic resistance. Knowledge of antibiotic susceptibility of bacteria with probiotic potential is important. Also, it's essential to define their resistance profile. Ten newly isolated strains from traditional fermented foods were used for determining the antibiotic resistance profile. The resistance to antibiotics varied among the examined strains and a few of the antibiotics resulted in complete resistance. Chromosomal DNA of LAB was analyzed for the antibiotic resistance genes. Only five of eight vancomycin-resistant strains have shown that they contain the resistance gene in chromosomal DNA. None of the genes that determined the resistance to other antibiotics have been detected in chromosomal DNA.

Efficacy of pectin-based caproic acid, caprylic acid, linalool, and cuminaldehyde coatings in reducing Salmonella Heidelberg on chicken eggs

Among the animal derived food products, contamination of poultry eggs, and egg shell surface is one of the major causes for foodborne salmonellosis in the United States. As a means of reducing the pathogen transfer to the internal egg contents, polysaccharide-based coatings containing antimicrobial phytochemicals could potentially serve as a biocontrol strategy for shelled egg products. The current study investigated the efficacy of four GRAS (Generally Recognized as Safe)-status plant-derived compounds, namely, caproic acid (CAO), caprylic acid (CAY), linalool (LIN) and cuminaldehyde (CUM), as pectin-based coating treatments, individually or in combination, for reducing Salmonella Heidelberg (SH) on shell eggs. A three-strain mixture of SH (~8.0 log CFU in 50 μL inoculum) was spot-inoculated on surface sterilized white-shelled eggs. Eggs were evenly coated with either pectin-based treatments of CAO (1%), CAY (1%), LIN (1%) and CUM (1%), individually, or a combination of 4 phytochemicals (COMB- each phytochemical at 0.5% v/v level of inclusion). The treated eggs were stored at 4°C and SH counts were enumerated on days 0, 1, 3, 5, 7, 14, and 21 of storage. The study was replicated thrice, 3 eggs/treatment/day time point, and the data were analyzed using two-way ANOVA with significance tested at p < 0.05. On day 0, pectin-coated control eggs had ~7.6 log CFU of SH/egg. At the end of refrigerated storage (day 21), pectin-based coating of CAO and CAY at 1% level reduced SH by 2.0–2.5 log CFU/egg (P < 0.05) when compared to controls. In addition, the CUM and LIN based coatings produced 3.0 log and 3.9 log reduction, respectively, in SH counts on eggs by day 21 of storage. Among the treatments with phytochemical combinations, COMB1 [pectin (2%) + Caprylic acid, caproic acid and cuminaldehyde (each at 0.5% level)] was found to be most effective, reducing SH counts to 2.5–3.3 log CFU/egg from day 0 through day 14, and by the end of storage period (day 21), a 3.5 log CFU reduction/egg (p < 0.05) compared to untreated controls. Morphological studies of treated eggs using atomic force microscopy (AFM) have shown that the roughness of eggs can be influenced by a combination of various compounds. Results indicate the potential efficacy of the aforesaid phytochemicals in reducing SH on shell eggs; however, further studies investigating their industrial feasibility and effects on sensory attributes of eggs are warranted.

Pharmaceutical novel solid forms of Milrinone with advanced physicochemical properties

Milrinone (MLN) is a second-generation bipyridine phosphodiesterase (PDE) inhibitor developed by modifying amrinone chemically. It is a PDE-III antagonist that promotes cardiac function and systemic vasodilator in chronic decongested cardiovascular disease by increasing cAMP levels. It is classified under Biopharmaceutics Classification System (BCS) class II, which implies, high permeability and low water solubility. Using mechanochemical and solution crystallization, five novel molecular solids of the cardioprotective drug (MLN) with Generally Recognized As Safe (GRAS) co-formers, namely 2-hydroxybenzoic acid (2-HBA), 2,3-dihydroxybenzoic acid (2,3-DHBA), 2,4-dihydroxybenzoic acid (2,4-DHBA), fumaric acid (FA), and adipic acid (AA) were successfully synthesized. All the solid forms were characterized by PXRD, SCXRD, and thermal analysis (TGA and DSC). The structural analysis of the complexes reveals that the dimeric interaction of MLN itself remains intact in all the structures. Molecular assemblies made with AA and 2,3-DHBA produced channel structures, whereas the remaining formed various forms of sheet structures. A physicochemical property analysis revealed that the synthesized molecular solid exhibits higher solubility than the parent form. Furthermore, the theoretical analyses corroborate well with the solubility of molecular complexes. We believe that the novel solid forms for MLN are promising candidates for addressing poor solubility difficulties while also providing a new avenue for further improving the material's physicochemical properties.

Effect of antimicrobial treatments applied individually and in combination on the growth of Listeria monocytogenes in Queso Fresco at 3 different temperatures

Queso fresco (QF), a fresh soft cheese, is one of the most popular Hispanic cheeses in the United States and is frequently associated with Listeria monocytogenes outbreaks. Listeria monocytogenes can grow and thrive at room temperature as well as refrigeration temperatures. A combination of antimicrobial agents provides a larger spectrum of listeriostatic and listeriocidal activity resulting in a more effective approach toward the control of L. monocytogenes. In this study, we evaluated the efficacy of 3 Food and Drug Administration-approved generally recognized as safe (GRAS) antimicrobials, nisin (NIS), lauric arginate ethyl ester (LAE), and ε-polylysine (EPL), and the endolysin PlyP100 individually and in combination for control of L. monocytogenes in QF at 4°C, 7°C, and 10°C. Additionally, growth curves of L. monocytogenes were obtained in BHI broth and QF at these temperatures. In order for an antimicrobial to be considered a postlethality treatment for L. monocytogenes, it should not allow an increase of more than 2-log over the product's shelf life. Three treatments, PlyP100, PlyP100 + NIS, and EPL + LAE, effectively kept the pathogen below the 2 log growth threshold at 4°C. However, at 7°C and 10°C, none of the antimicrobial treatments could inhibit L. monocytogenes growth (i.e., <2 log). Overall, our results suggest the importance of considering the effect of cold storage temperatures above 4°C on the antilisterial efficacy of antimicrobial treatments in QF.

Bioconversion of Jackfruit Seed Waste to Fungal Biomass Protein by Submerged Fermentation.

The bioconversion of jackfruit seed into a valuable product like Single-Cell Protein (SCP) was carried out using Generally Recognized As Safe (GRAS), five filamentous fungi such as Penicillium expansum, Aspergillus niger, Aspergillus oryzae, Rhizopus microsporus, and Candida intermedia by submerged fermentation. Data obtained from the study showed that supplementation of jackfruit seed extracts with inorganic nitrogen sources and glucose as a carbon source enhanced fungal biomass and SCP production. Among the various fungal strains, A. niger gave the highest biomass and protein yield of 4.01 ± 0.07g/l and 1.82 ± 0.03g/l, respectively, on the 9th day of fermentation followed by P. expansum (3.65 ± 0.04g/l and 1.68 ± 0.03g/l, respectively). C. intermedia growth was not recorded in all the experimental media. The present study revealed that fungal biomass has shown low crude fat, crude fiber, and total genomic DNA content ranged from 1.10 ± 0.20 to 4.95 ± 0.40%. A. niger and P. expansum were the most efficient in the conversion of sugar (55.83 ± 0.3% and 54.71 ± 0.4%, respectively) to yield biomass in sugar supplementation media. P. expansum and A. niger were the most promising fungal strains to produce fungal biomass protein using inexpensive agro-waste materials.

In vitro study on the antimicrobial activity of eleven essential oils against oral cavity microbiota

Obtained from aromatic plants, essential oils have been utilised as natural food preservatives. Their ability to inhibit microorganism growth results from their varied chemical compositions. Furthermore, due to their classification as generally recognised as safe (GRAS) and low toxicity levels, there has been an increased study of essential oils’ compositions and antimicrobial properties against pathogens living in the human oral cavity. An initial evaluation was conducted with 11 essential oils to verify their antimicrobial activities in vitro against opportunistic pathogenic bacteria Staphylococcus aureus, Streptococcus sanguinis and Streptococcus mutans, also a model yeast Candida albicans. The antimicrobial activity was evaluated by agar disk diffusion assay, while PT kindly provided chemical composition data. Darjeeling Sembrani Aroma. The result showed that tested essential oils exhibited an inhibition zone range of 8.13 to 40.00 mm for bacteria and 7.63 to 44.04 mm for yeast. Chemical compositions of the oils revealed that the major compounds are monoterpenes, followed by sesquiterpenes and phenylpropanoids.

Risk on the Table: Food Production, Health, and the Environment ed. by Angela N. H. Creager and Jean-Paul Gaudillière (review).

Reviewed by: Risk on the Table: Food Production, Health, and the Environment ed. by Angela N. H. Creager and Jean-Paul Gaudillière Jonathan Rees (bio) Risk on the Table: Food Production, Health, and the Environment Edited by Angela N. H. Creager and Jean-Paul Gaudillière. New York: Berghahn, 2021. Pp. 366. The technology of food production both involves machines that make more food faster, as well as various processes which make that food more desirable to consumers. This is a book that focuses on the second of these categories. The editors deliberately invoke the industrialization of food in the introduction to the book, but the subsequent chapters deal more with chemistry than they do with technology in the mechanical sense. The authors are in dialogue with food and food adulteration literature more than with the broader issues related to industrialization. Nonetheless, since food, food technology, and food science all cross international boundaries, the risks associated with its industrialization and mass production eventually became global. For example, lowering the price or increasing the supply of something might bring the possibility of food poisoning or the long-term risk of cancer from ingesting some food additives. As Heiko Stoff explains in his chapter, a coloring agent like Butter Yellow (for margarine) fell out of favor after World War II in Germany because of the risk consumers faced when eating it. On the other hand, as the book's coeditor Angela Creager explains in her chapter, new food testing technology showed natural carcinogens in foods that Americans consumed every day. In another place at another time, these things might have been banned, but risk tolerance was very high in the United States during the 1970s and early 1980s. Even once these risk perceptions hardened during the late 1980s, the American political system had become too industry friendly to effectively regulate these substances. The second half of the book focuses almost exclusively on the United States. In these chapters, you can see the high risk-tolerance of the U.S. regulatory regime for much of the last one hundred years. Hannah Landecker, for example, writes about the use of arsenic-based drugs in American industrial-scale meat production. These drugs moved growth-promoting hormones through cows, thereby increasing supply and lowering price. Despite some early opposition to these substances on health grounds, it took until 2015 for the Food and Drug Administration to withdraw approval of those drugs because of new discoveries regarding the arsenic that remained in the bodies of chickens. All drugs are poisonous to some degree, but even the U.S. government developed limits to how much it wanted eaters to consume. In their chapter, Maricel V. Maffini and Sarah Voge show the limitations of these regulations by focusing on the holes in the framework created by the 1958 Food Additives Amendment to the Food, Drug, and Cosmetics [End Page 899] Act of 1938. Although inspired by low levels of pesticide residues and other chemicals found in food and milk supplies, substances that are generally recognized as safe (GRAS) are still legal in the food supply under the law. While some substances that are potentially harmful simply haven't been tested, others have been grandfathered in despite known risks that they might cause harm. In short, the system set up to regulate food in the United States tolerates risks from various innovations because it favors whatever facilitates production on an industrial scale unless extraordinary proof of harm can be found, and even then action isn't guaranteed. Collectively, the chapters in this collection suggest that the tolerance for risk in the United States has generally been greater than elsewhere for both cultural and political reasons. The book's weak spot is the failure of some of the authors who focus on the United States to make at least a passing attempt at upholding the earlier section's global perspective. Luckily, the editors have brought together enough international work to form a broad picture of changes in the global food system. This is an extremely welcome view of how those changes were received in different places at different times. Jonathan Rees Jonathan Rees is professor of history at Colorado State...

Maraş Et Sucuğunda Ekzopolisakkarit Üreten Mikroorganizmaların Tespiti

Ekzopolisakkaritler (EPS) de laktik asit bakterilerinin (LAB) fermantasyon sonucu hücre dışında oluşturdukları polisakkarit yapıda maddelerdir. EPS’ler prebiyotik özellik gösterdikleri gibi gıda endüstrisinde jelleştirici, emülsifiye edici, stabilizatör olarak da kullanılmaktadırlar. Ayrıca EPS’nin sağlık üzerine bağışıklık sistemini geliştirici, kolesterolü düşürücü, antiülser ve antitümör gibi olumlu etkilerinin olduğu belirtilmiştir. Genel olarak güvenilir kabul edilen (GRAS) özellikte olmaları da ekzopolisakkaritlerin önemini artırmaktadır. Bu çalışmada, fermente sucuk üretiminde hakim flora olan laktik asit bakterilerinden EPS üreten bakterileri türleri ve ürettikleri EPS miktarı tespit edilmiştir. Bu amaçla Kahramanmaraş piyasasındaki kasaplarda üretilen 10 farklı kasaptan elde edilen sucuk örneklerinden LAB izole edilmiş ve izole edilen laktik asit bakterilerinin ürettikleri ekzopolisakkarit miktarı fenol sülfürik asit yöntemi ile belirlenmiştir. Daha sonra izole edilen bakterilerin karakterizasyonu PCR ile yapılmıştır. Sonuç olarak 10 farklı kasaptan temin edilen sucuklarda Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus ve Bacillus subtilis bakterileri tespit edilmiştir. Bu sucuklardan izole edilen toplam 56 suşun ürettiği EPS miktarının 20,19 ile 109,10 ppm arasında değiştiği tespit edilmiştir. Kullanılan örnekler içerisinde hem en düşük hem de en yüksek EPS üreten suşun L. plantarum olduğu belirlenmiştir.

Antimicrobial effect of essential oils in content of edible films (review)

The globalization of food trade, the growing demand for minimally processed, easy-to-prepare, ready-to-eat food, and the proliferation of centralized food processing pose serious problems for food safety. Outbreaks of infectious diseases of alimentary origin stimulate the search for innovative ways to increase the safety and shelf life of ready-to-eat foods. No less important is the problem of solid waste disposal, a significant share of which is food packaging. Therefore, in the last decade, interest in edible films and packaging has increased significantly. Essential oils are natural antibacterial agents that can be successfully used in edible films as a substitute for synthetic compounds. Numerous studies have shown that antimicrobial films and coatings with the inclusion of essential oils are effective in reducing the level of pathogenic microorganisms such as Listeria monocytogenes, Salmonella Typhi, Staphylococcus aureus, Escherichia coli O157: H7 and others. Essential oils have the status of GRAS (Generally Recognized as Safe) registered by the European Commission and the United States. They contain a wide variety of secondary metabolites that are capable of inhibiting or slowing the growth of bacteria, yeasts and moulds. In addition to external application on the surface of food systems, the components of essential oils can diffuse into food to neutralize the target microorganisms. The article summarizes current information on the sources, properties, limitations and potential application of volatile essential oils and their compounds in edible films and packaging to ensure food safety. Despite the many advantages, the inclusion of these compounds in the composition of edible films requires additional research, in particular to determine the optimal inhibitory concentrations, mechanical and functional properties of the films and so on. Key words: essential oils, edible films, antibacterial additives, biodegradation, food safety.

Lactococcus lactis NCDO2118 exerts visceral antinociceptive properties in rat via GABA production in the gastro-intestinal tract.

Gut disorders associated to irritable bowel syndrome (IBS) are combined with anxiety and depression. Evidence suggests that microbially produced neuroactive molecules, like γ-aminobutyric acid (GABA), can modulate the gut-brain axis. Two natural strains of Lactococcus lactis and one mutant were characterized in vitro for their GABA production and tested in vivo in rat by oral gavage for their antinociceptive properties. L. lactis NCDO2118 significantly reduced visceral hypersensitivity induced by stress due to its glutamate decarboxylase (GAD) activity. L. lactis NCDO2727 with similar genes for GABA metabolism but no detectable GAD activity had no in vivo effect, as well as the NCDO2118 ΔgadB mutant. The antinociceptive effect observed for the NCDO2118 strain was mediated by the production of GABA in the gastro-intestinal tract and blocked by GABAB receptor antagonist. Only minor changes in the faecal microbiota composition were observed after the L. lactis NCDO2118 treatment. These findings reveal the crucial role of the microbial GAD activity of L. lactis NCDO2118 to deliver GABA into the gastro-intestinal tract for exerting antinociceptive properties in vivo and open avenues for this GRAS (Generally Recognized As safe) bacterium in the management of visceral pain and anxious profile of IBS patients.

Deproteinization of Chitin Extracted with the Help of Ionic Liquids.

The isolation of chitin utilizing ionic liquid 1-ethyl-3-methylimidazolium acetate has been determined to result in polymer contaminated with proteins. For the first time, the proteins in chitin extracted with ionic liquid have been quantified; the protein content was found to vary from 1.3 to 1.9% of the total weight. These proteins were identified and include allergenic proteins such as tropomyosin. In order to avoid ‘traditional’ hydroxide-based deproteinization of chitin, which could reduce the molecular weight of the final product, alternative deproteinization strategies were attempted. Testing of the previously reported deproteinization method using aqueous K3PO4 resulted in protein reduction by factors varying from 2 to 10, but resulted in significant phosphate salt contamination of the final product. Contrarily, the incorporation of GRAS (Generally Recognized as Safe) compound Polysorbate 80 into the polymer washing step provided the polymer of comparable purity with no contaminants. This study presents new options for the deproteinization of chitin that can replace traditional approaches with methods that are environmentally friendly and can produce high purity polymer.

The ameliorative effect of Lactobacillus paracasei BEJ01 against FB1 induced spermatogenesis disturbance, testicular oxidative stress and histopathological damage

Fumonisin B1 (FB1) is a possible carcinogenic molecule for humans as classified by the International Agency for Research on Cancer (IARC) in 2B group. In livestock, it is responsible for several mycotoxicoses and economic losses. Lactobacillus strains, inhabitants of a wide range of foodstuffs and the gastrointestinal tract, are generally recognized as safe (GRAS). Thus, the aim of this work was to evaluate the protective effect of Lactobacillus paracasei (LP) against FB1-induced reprotoxicities including testicular histopathology, sperm quality disturbance, and testosterone level reduction. Pubescent mice were divided randomly into four groups and treated for 10 days. Group 1: Control; Group 2: FB1 (100 μg/kg b.w); Group 3: LP (2 × 109 CFU/kg b.w); Group 4: LP (2 × 109 CFU/kg b.w) and FB1 (100 μg/kg b.w). After the end of the treatment, animals were sacrificed. Plasma, epididymis, and testis were collected for reproductive system studies. Our results showed that FB1 altered epididymal sperm quality, generated oxidative stress, and induced histological alterations. Interestingly, these deleterious effects have been counteracted by the LP administration in mice. In conclusion, LP was able to prevent FB1-reproductive system damage in BALB/c mice and could be validated as an anti-caking agent in an animal FB1-contaminated diet.

Recent Advances in Directed Yeast Genome Evolution.

Saccharomyces cerevisiae, as a Generally Recognized as Safe (GRAS) fungus, has become one of the most widely used chassis cells for industrial applications and basic research. However, owing to its complex genetic background and intertwined metabolic networks, there are still many obstacles that need to be overcome in order to improve desired traits and to successfully link genotypes to phenotypes. In this context, genome editing and evolutionary technology have rapidly progressed over the last few decades to facilitate the rapid generation of tailor-made properties as well as for the precise determination of relevant gene targets that regulate physiological functions, including stress resistance, metabolic-pathway optimization and organismal adaptation. Directed genome evolution has emerged as a versatile tool to enable researchers to access desired traits and to study increasingly complicated phenomena. Here, the development of directed genome evolutions in S. cerevisiae is reviewed, with a focus on different techniques driving evolutionary engineering.

Carotenoid Recovery from Tomato Processing By-Products through Green Chemistry.

The recovery of bioactive compounds from agro-industry-derived by-products sustains circular economy principles by encouraging maximized recycling and minimized waste. Tomato processing by-products are abundant in carotenoids, which have several health-promoting properties, and their reintegration into functional food products represents a major interest for scientists and manufacturers. In the present study, carotenoids were recovered from tomato processing by-products based on the principles of green chemistry by using generally recognized as safe (GRAS) solvents, freeze-drying as pretreatment, and ultrasound in the recovery procedure. Spectrophotometric measurements and HPLC were used to identify and quantify total and individual carotenoids from the extracts. The highest values for lycopene (1324.89 µg/g dw) were obtained when ethyl lactate was applied as a solvent, followed by ethyl acetate with slightly smaller differences (1313.54 µg/g dw). The extracts obtained from freeze-dried samples presented significantly lower amounts of lycopene, indicating that carotenoids are highly susceptible to degradation during lyophilization. Flaxseed, grape seed, and hempseed oils were enriched with carotenoids and their rheological measurements showed favorable viscoelastic properties, especially hempseed and flaxseed oil, with viscosity under 50 mPa·s. Considering the results and the economic perspective of carotenoid recovery from tomato processing by-products, ethyl acetate is suitable, sustainable, and environmentally friendly for carotenoid extraction.

De novo synthesis of 2'-fucosyllactose in engineered Bacillus subtilis ATCC 6051a

Microbial synthesis of 2'-fucosyllactose (2'-FL) has received much attention in recent years. In this study, Bacillus subtilis ATCC 6051a, a nonpathogenic GRAS (generally recognized as safe) organism, was engineered to produce 2'-FL. After a synthetic pathway comprising six genes was incorporated into the host cell genome, only a low level of 2'-FL (120 mg/L) was initially detected in recombinant cell culture. Comparison of heterologous lactose transporters confirmed the superior role of LacY from Escherichia coli for efficient production of 2'-FL in B. subtilis. There are two β-galactosidases, GanA and YesZ, in B. subtilis. Deletion of ganA and yesZ caused by insertion of the lacY cassette led to 2'-FL accumulation in shaking-flask culture at concentrations of 3.13 g/L and 5.56 g/L, respectively, which increased to 6.13 g/L in the double-deletion strain 164FL-GY. In addition, enhanced xylose metabolism in 164FL-GY further increased the concentration of 2'-FL to 7.14 g/L. In fed-batch fermentation, the highest productivity of 0.56 g/L·h was achieved in glycerol and xylose containing medium using engineered B. subtilis, and the accumulated 2'-FL reached 31.2 g/L. This is the first report of recombinant B. subtilis for high-level production of 2'-FL via a de novo synthesis pathway.

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism

BackgroundThe methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production.ResultsIn this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain).ConclusionsThe GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

Engineered Bacillus subtilis for the de novo production of 2′-fucosyllactose

BackgroundThe most abundant human milk oligosaccharide in breast milk, 2′-fucosyllactose (2′-FL), has been approved as an additive to infant formula due to its multifarious nutraceutical and pharmaceutical functions in promoting neonate health. However, the low efficiency of de novo synthesis limits the cost-efficient bioproduction of 2′-FL.ResultsThis study achieved 2′-FL de novo synthesis in a generally recognized as safe (GRAS) strain Bacillus subtilis. First, a de novo biosynthetic pathway for 2′-FL was introduced by expressing the manB, manC, gmd, wcaG, and futC genes from Escherichia coli and Helicobacter pylori in B. subtilis, resulting in 2′-FL production of 1.12 g/L. Subsequently, a 2′-FL titer of 2.57 g/L was obtained by reducing the competitive lactose consumption, increasing the regeneration of the cofactor guanosine-5′-triphosphate (GTP), and enhancing the supply of the precursor mannose-6-phosphate (M6P). By replacing the native promoter of endogenous manA gene (encoding M6P isomerase) with a constitutive promoter P7, the 2′-FL titer in shake flask reached 18.27 g/L. The finally engineered strain BS21 could produce 88.3 g/L 2′-FL with a yield of 0.61 g/g lactose in a 3-L bioreactor, without the addition of antibiotics and chemical inducers.ConclusionsThe efficient de novo synthesis of 2′-FL can be achieved by the engineered B. subtilis, paving the way for the large-scale bioproduction of 2′-FL titer in the future.