Stability of serum-free and purified baculovirus stocks under various storage conditions

In a context of large-scale production of baculoviruses in serum-free media for use as gene delivery vectors, the stability of these viruses has become an important factor. The development of robust processes heavily relies on baculovirus stock stability. In the present work, we studied over a period of 300 days the stability of baculovirus vectors produced in serum-free media stored at 4, -20, or -80 degrees C or in liquid nitrogen. The viral stocks investigated were either crude baculovirus supernatant, baculovirus supernatant concentrated 10 times and diafiltered against fresh serum-free media by tangential flow filtration, or baculovirus purified by size exclusion chromatography. The results showed that baculovirus supernatant and diafiltered concentrate stored at 4 degrees C underwent a progressive loss of infectivity after a period of 100 and 50 days of storage, respectively. Aggregation has been recognized as the probable mechanism for the loss of infectivity. Baculovirus stocks were unstable at -20 degrees C, whereas in liquid nitrogen they retained infectivity after successive freeze thaw cycles. Concentration and diafiltration of baculovirus supernatant prior to storing at -80 degrees C contributed to improving viral stock stability over time. Glycerol as well as DMSO and sucrose have proven to be equally effective as additives to maintain the purified baculovirus stability after storage at -80 degrees C or in liquid nitrogen.     

Reducing infectivity of HIV upon exposure to surfaces coated with N,N‐dodecyl,methyl‐polyethylenimine

The infectivity of high‐titer, cell‐free HIV in culture media and human milk is rapidly reduced upon exposure to polyethylene slides painted with the linear hydrophobic polycation N,N‐dodecyl,methyl‐polyethylenimine (DMPEI). Accompanying viral p24 protein and free viral RNA analysis of solutions exposed to DMPEI‐coated surfaces suggests that virion attachment to the polycationic surface and its subsequent inactivation are the likely mechanism of this phenomenon. Biotechnol. Bioeng. 2013; 110: 2058–2062. © 2013 Wiley Periodicals, Inc.     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016     

Serum‐free,chemically defined medium with TGF‐β3 enhances functional properties of nucleus pulposus cell‐laden carboxymethylcellulose hydrogel constructs

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum‐containing medium formulation was compared to a chemically defined, serum‐free medium to determine the effect on matrix elaboration and functional properties of NP cell‐laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor‐beta 3 (TGF‐β₃). Glycosaminoglycan (GAG) content increased in both TGF‐β₃‐treated groups and was highest for treated, serum‐free constructs (9.46 ± 1.51 µg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum‐containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum‐free constructs. Type II collagen content was greater in both serum‐free groups and highest in treated, serum‐free constructs. The equilibrium Young's modulus was highest in serum‐free samples supplemented with TGF‐β₃ (18.54 ± 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 ± 0.46 vs. 19.94 ± 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum‐free, TGF‐β₃ supplemented medium, indicating the importance of medium formulation in NP construct development. Biotechnol. Bioeng. 2010; 105: 384–395. © 2009 Wiley Periodicals, Inc.     

A Thin‐Layer Liquid Culture Technique for the Growth of Helicobacter pylori

Background and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori. Methods: A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD₆₀₀ with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD₆₀₀ contained 1.3 ± 0.1 × 10⁹ CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions: Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.     

Enhancement of the antimicrobial properties of bacteriophage‐K via stabilization using oil‐in‐water nano‐emulsions

Bacteriophage therapy is a promising new treatment that may help overcome the threat posed by antibiotic‐resistant pathogenic bacteria, which are increasingly identified in hospitalized patients. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a wound dressing is a promising alternative. This article evaluates the antimicrobial efficacy of Bacteriophage K against Staphylococcus aureus over time, when stabilized and delivered via an oil‐in‐water nano‐emulsion. Nano‐emulsions were formulated via thermal phase inversion emulsification, and then bacterial growth was challenged with either native emulsion, or emulsion combined with Bacteriophage K. Bacteriophage infectivity, and the influence of storage time of the preparation, were assessed by turbidity measurements of bacterial samples. Newly prepared Bacteriophage K/nano‐emulsion formulations have greater antimicrobial activity than freely suspended bacteriophage. The phage‐loaded emulsions caused rapid and complete bacterial death of three different strains of S. aureus. The same effect was observed for preparations that were either stored at room temperature (18–20°C), or chilled at 4°C, for up to 10 days of storage. A response surface design of experiments was used to gain insight on the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. More diluted emulsions had a less significant effect on bacterial growth, and diluted bacteriophage‐emulsion preparations yielded greater antibacterial activity. The enhancement of bacteriophage activity when delivered via nano‐emulsions is yet to be reported. This prompts further investigation into the use of these formulations for the development of novel anti‐microbial wound management strategies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:932–944, 2014     

Plant‐made E2 glycoprotein single‐dose vaccine protects pigs against classical swine fever

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at‐risk animals, often at very high cost. Current CSFV‐modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first‐generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide‐spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium‐mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil‐in‐water emulsion adjuvants. We report the manufacturing of adjuvanted, plant‐made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single‐dose vaccination, which was accompanied by strong virus neutralization antibody responses.     

Food deprivation and nicotine correct akinesia and freezing in Na+‐leak current channel (NALCN)‐deficient strains of Caenorhabditis elegans

Mutations in various genes adversely affect locomotion in model organisms, and thus provide valuable clues about the complex processes that control movement. In Caenorhabditis elegans, loss‐of‐function mutations in the Na⁺ leak current channel (NALCN) and associated proteins (UNC‐79 and UNC‐80) cause akinesia and fainting (abrupt freezing of movement during escape from touch). It is not known how defects in the NALCN induce these phenotypes or if they are chronic and irreversible. Here, we report that akinesia and freezing are state‐dependent and reversible in NALCN‐deficient mutants (nca‐1;nca‐2, unc‐79 and unc‐80) when additional cation channels substitute for this protein. Two main measures of locomotion were evaluated: spontaneous movement (traversal of >2 head lengths during a 5 second observation period) and the touch‐freeze response (movement greater than three body bends in response to tail touch). Food deprivation for as little as 3 min stimulated spontaneous movement and corrected the touch‐freeze response. Conversely, food‐deprived animals that moved normally in the absence of bacteria rapidly reverted to uncoordinated movement when re‐exposed to food. The effects of food deprivation were mimicked by nicotine, which suggested that acetylcholine mediated the response. Nicotine appeared to act on interneurons or motor neurons rather than directly at the neuromuscular junction because levamisole, which stimulates muscle contraction, did not correct movement. Neural circuits have been proposed to account for the effects of food deprivation and nicotine on spontaneous movement and freezing. The NALCN may play an unrecognized role in human movement disorders characterized by akinesia and freezing gait.     

Methods for detection of Anticarsia gemmatalis nucleopolyhedrovirus DNA in soil.

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0. 25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.     

Growth of v‐src‐transformed cells in serum‐free medium through the induction of growth factors

The v‐src oncogene is one of only two oncogenes capable of transforming mouse embryo fibroblasts (MEFs) lacking the IGF‐IR gene (R‐cells). R‐/v‐src cells grow robustly in the absence of serum, suggesting the hypothesis that they may produce one or more growth factors that would sustain their ability to proliferate in serum‐free condition. Using proteomic approaches on serum‐free conditioned media derived from v‐src‐transformed cells, we have identified two growth promoting factors: ostepontin and proliferin. Subsequent experiments have indicated that osteopontin plays a prevalent role in promoting growth of v‐src‐transformed cells in serum‐deprived condition. J. Cell. Physiol. 228: 1482–1486, 2013. © 2012 Wiley Periodicals, Inc.     

Insulin‐like growth factor‐I prevents caspase‐mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999     

Serum‐free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential

There have been many clinical trials recently using ex vivo‐expanded human mesenchymal stem cells (MSCs) to treat several disease states such as graft‐versus‐host disease, acute myocardial infarction, Crohn's disease, and multiple sclerosis. The use of MSCs for therapy is expected to become more prevalent as clinical progress is demonstrated. However, the conventional 2‐dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the MSC‐based indications further exacerbates this manufacturing challenge. In contrast, expanding MSCs as spheroids does not require a cell attachment surface and is amenable to large‐scale suspension cell culture techniques, such as stirred‐tank bioreactors. In the present study, we developed and optimized serum‐free media for culturing MSC spheroids. We used Design of Experiment (DoE)‐based strategies to systematically evaluate media mixtures and a panel of different components for effects on cell proliferation. The optimization yielded two prototype serum‐free media that enabled MSCs to form aggregates and proliferate in both static and dynamic cultures. MSCs from spheroid cultures exhibited the expected immunophenotype (CD73, CD90, and CD105) and demonstrated similar or enhanced differentiation potential toward all three lineages (osteogenic, chondrogenic, adipogenic) as compared with serum‐containing adherent MSC cultures. Our results suggest that serum‐free media for MSC spheroids may pave the way for scale‐up production of MSCs in clinically relevant manufacturing platforms such as stirred tank bioreactors. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:974–983, 2014     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Ultrastructure of bovine embryos developed from in vitro–matured and –fertilized oocytes: Comparative morphological evaluation of embryos cultured either in serum‐free medium or in serum‐supplemented medium

The ultrastructure of bovine embryos developed from in vitro‐matured and ‐fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum‐free medium (IVMD101) or in a serum‐containing medium (TCM199+CS) was compared. Embryos up to the eight‐cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight‐cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well‐developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome‐like vesicles were partially filled with electron‐dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome‐like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome‐like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)‐cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome‐like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM‐cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum‐free and serum‐supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos. Mol. Reprod. Dev. 53:325–335, 1999. © 1999 Wiley‐Liss, Inc.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

A Simple and Rapid Cryopreservation Technique for Ciliates: A Long‐Term Storage Procedure Used for Marine Scuticociliates

Pseudocohnilembus persalinus is a free‐living marine scuticociliate that, as a new model organism, has been used in a wide variety of studies. However, long‐term laboratory maintenance for this species is mainly achieved by subculture that requires rigorous culture environments and, too often, cultures of the organism die out for a variety of reasons. Successful transport of viable cultures also poses problems for researchers. This study describes a simple and rapid protocol for long‐term cryopreservation of P. persalinus. The effects of physiological states of individuals before freezing, the type and concentration of cryoprotectant, and optimal temperatures for freezing and thawing were assessed. A cryopreservation protocol, using a mixture of 30% glycerol and 70% concentrated P. persalinus cell culture, incorporating rate‐controlled freezing at ?80 °C before liquid nitrogen storage, maintained a high recovery efficiency after 8 wk of storage. These results suggest that broader application of this protocol to build a cryopreserved marine protozoa culture bank for biological studies may be possible.     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.     

Implications of modifying membrane fatty acid composition on membrane oxidation,integrity, and storage viability of freeze‐dried probiotic,Lactobacillus acidophilus La‐5

The aim of this study was to investigate the effect of altering the fatty acid profile of the lipid membrane on storage survival of freeze‐dried probiotic, Lactobacillus acidophilus La‐5, as well as study the membrane integrity and lipid oxidation. The fatty acid composition of the lipid membrane of L. acidophilus La‐5 was significantly different upon growth in MRS (containing Tween 80, an oleic acid source), or in MRS with Tween 20 (containing C12:0 and C14:0), linoleic, or linolenic acid supplemented. Bacteria grown in MRS showed the highest storage survival rates. No indications of loss of membrane integrity could be found, and membrane integrity could therefore not be connected with loss of viability. Survival of bacteria grown with linoleic or linolenic acid was more negatively affected by the presence of oxygen, than bacteria grown in MRS or with Tween 20 supplemented. A small, but significant, loss of linolenic acid during storage could be identified, and an increase of volatile secondary oxidation products during storage was found for bacteria grown in MRS, or with linoleic, or linolenic acid supplemented, but not for bacteria grown with Tween 20. Overall, the results indicate that lipid oxidation and loss of membrane integrity are not the only or most important detrimental reactions which can occur during storage. By altering the fatty acid composition, it was also found that properties of oleic acid gave rise to more robust bacteria than more saturated or unsaturated fatty acids did. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:799–807, 2015     

Oxyma‐based phosphates for racemization‐free peptide segment couplings

Glyceroacetonide–Oxyma [(2,2‐dimethyl‐1,3‐dioxolan‐4‐yl)methyl 2‐cyano‐2‐(hydroxyimino)acetate ( 1 )] displayed remarkable physico‐chemical properties as an additive for peptide‐forming reactions. Although racemization‐free amide‐forming reactions have been established for N‐urethane‐protected α‐amino acids with EDCI, 1 , and NaHCO₃ in water or DMF‐water media, amide‐forming reactions of N‐acyl‐protected α‐amino acids and segment couplings of oligopeptides still require further development. Diethylphosphoryl–glyceroacetonide–oxyma (DPGOx 3 ) exhibits relative stability in aprotic solvents and is an effective coupling reagent for N‐acyl‐protected α‐amino acids and oligo peptide segments. The conditions reported here is also effective in lactam‐forming reactions. Unlike most of the reported coupling reagents, simple aqueous work‐up procedures can remove the reagents and by‐products generated in the reactions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.