The Influence of Sugars on Pressure Induced Starch Gelatinization

AbstractBesides the application of high pressure (HP) as a non-thermal preservation technology, HP could additionally have a deep impact on the material properties of the treated food. Especially the HP induced swelling and gelatinization of starch influences the processing properties of starch-based food systems and differs in comparison to thermal induced gelatinization. The aim of this study was to examine the impact of HP on starches under different conditions and to influence systematically the gelatinization and pasting properties of wheat starch by the addition of various types of sugar. Caused by limitation of conventional methods, this study also includes the development of an appreciate method based on the particle size measurements of the starch granules. Three methods of measuring particle sizes were examined for an application for pressure treated starches. Finally, an image analysis with microscope, camera and image processing software ImageJ was chosen to perform the analysis. Wheat, tapioca and potato starch with concentration of 5 (w/w) and 25 (w/w) were pressurized at 600MPa for ten minutes at 20°C as well as 60°C, to reach treatment conditions which are suitable for HP food pasteurization. The results showed that ultra HP significantly increased the particle size of the starch granules, whereas the degree of swelling was starch type and temperature dependent. High starch concentrations resulted in a limited swelling caused of the limited water content. This effect is enhanced with increasing swelling properties. Besides this, sugar caused a significant decrease of the granules size. A dependence of this effect with the type of sugar was not examined. This work should be a contribution to expand the understanding of the swelling mechanisms of starch granules under HP and should facilitate a future process and product development of HP pasteurized starch based products

Sterilization of liquid foods by pulsed electric fields – an innovative ultra-high temperature process

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm−1), skim milk (0.3% fat; 5.3 mS cm−1) and fresh prepared carrot juice (7.73 mS cm−1). The combination of moderate preheating (70–90°C) and an insulated PEF-chamber, combined with a holding tube (65 cm) and a heat exchanger for cooling, enabled a rapid heat up to 105–140°C (measured above the PEF chamber) within 92.2–368.9 μs. To compare the PEF process with a pure thermal inactivation, each spore suspension was heat treated in thin glass capillaries and D-values from 90 to 130°C and its corresponding z-values were calculated. For a comparison of the inactivation data, F-values for the temperature fields of both processes were calculated by using computational fluid dynamics (CFD). A preheating of saline water to 70°C with a flow rate of 5 l h−1, a frequency of 150 Hz and an energy input of 226.5 kJ kg−1, resulted in a measured outlet temperature of 117°C and a 4.67 log10 inactivation of B. subtilis. The thermal process with identical F-value caused only a 3.71 log10 inactivation. This synergism of moderate preheating and PEF was even more pronounced for G. stearothermophilus spores in saline water. A preheating to 95°C and an energy input of 144 kJ kg−1 resulted in an outlet temperature of 126°C and a 3.28 log10 inactivation, whereas nearly no inactivation (0.2 log10) was achieved during the thermal treatment. Hence, the PEF technology was evaluated as an alternative ultra-high temperature process. However, for an industrial scale application of this process for sterilization, optimization of the treatment chamber design is needed to reduce the occurring inhomogeneous temperature fields

Impact of different water activities (aw) adjusted by solutes on high pressure high temperature inactivation of Bacillus amyloliquefaciens spores

Much research has been conducted to comprehend the mechanisms of high pressure (HP) inactivation of spores in aqueous systems but for food model systems these information are scarce. In these systems spores can interact with ingredients which then could possibly lead to retarded or reduced inactivation, which can cause a problem for the sterilization process. The protective mechanism of a reduced aw-value is still unclear. HP processing might prove valuable to overcome protective effects of solutes and achieve shorter process times for sterilization under HP. To gain insight into the underlying mechanisms five aw-values (0.9, 0.92, 0.94, 0.96, 1) were adjusted with two different solutes (NaCl, sucrose). Solutions were inoculated with spores of Bacillus amyloliquefaciens and treated at 105, 110, and 115°C at 600 MPa. Further a thermal inactivation was conducted at the same temperatures for a comparison with the HP data. Afterward, the influence of HP high temperature treatment on the inactivation, the dipicolinic acid (DPA)-release and membrane constitution was assessed by plate count, HPLC and flow cytometry (FCM). The results show that during HP treatments sucrose and salt both have a protective effect, in which the influence of sucrose on the retarded inactivation is higher. The threshold water activities (aw), which is 0.94, here salt and sucrose have a significant influence on the inactivation. The comparison of thermal (105–115°C) and HP and high temperature (600 MPa, 105–115°C) treated samples showed that the time needed to achieve a 4–5 log10 inactivation is reduced from 45 (aw = 1) to 75 (aw = 0.9) min at 105°C to 3 (aw = 1) to 15 (aw = 0.9) minutes at 600 MPa and 105°C. The release of DPA is the rate limiting step of the inactivation and therefore monitoring the release is of great interest. The DPA-release is slowed down in high concentrated solutions (e.g., sucrose, salt) in comparison to aw 1. Since there is a difference in the way the solutes protect the spore it could be seen as an inner spore membrane effect. Maybe as shown for vegetative microorganism the solutes can interact with membranes, e.g., the inner spore membrane. Flow cytometry (FCM) measurement data show a similar trend

Impact of surface structure and feed gas composition on Bacillus subtilis endospore inactivation during direct plasma treatment

This study investigated the inactivation efficiency of cold atmospheric pressure plasma treatment on Bacillus subtilis endospores dependent on the used feed gas composition and on the surface, the endospores were attached on. Glass petri-dishes, glass beads, and peppercorns were inoculated with the same endospore density and treated with a radio frequency plasma jet. Generated reactive species were detected using optical emission spectroscopy. A quantitative polymerase chain reaction (qPCR) based ratio detection system was established to monitor the DNA damage during the plasma treatment. Argon + 0.135% vol. oxygen + 0.2% vol. nitrogen as feed gas emitted the highest amounts of UV-C photons and considerable amount of reactive oxygen and nitrogen species. Plasma generated with argon + 0.135% vol. oxygen was characterized by the highest emission of reactive oxygen species (ROS), whereas the UV-C emission was negligible. The use of pure argon showed a negligible emission of UV photons and atomic oxygen, however, the emission of vacuum (V)UV photons was assumed. Similar maximum inactivation results were achieved for the three feed gas compositions. The surface structure had a significant impact on the inactivation efficiency of the plasma treatment. The maximum inactivation achieved was between 2.4 and 2.8 log10 on glass petri-dishes and 3.9 to 4.6 log10 on glass beads. The treatment of peppercorns resulted in an inactivation lower than 1.0 log10. qPCR results showed a significant DNA damage for all gas compositions. Pure argon showed the highest results for the DNA damage ratio values, followed by argon + 0.135% vol. oxygen + 0.2% vol. nitrogen. In case of argon + 0.135% vol. oxygen the inactivation seems to be dominated by the action of ROS. These findings indicate the significant role of VUV and UV photons in the inactivation process of B. subtilis endospores

Challenges and advanced concepts for the assessment of learning and memory function in mice

The mechanisms underlying the formation and retrieval of memories are still an active area of research and discussion. Manifold models have been proposed and refined over the years, with most assuming a dichotomy between memory processes involving non-conscious and conscious mechanisms. Despite our incomplete understanding of the underlying mechanisms, tests of memory and learning count among the most performed behavioral experiments. Here, we will discuss available protocols for testing learning and memory using the example of the most prevalent animal species in research, the laboratory mouse. A wide range of protocols has been developed in mice to test, e.g., object recognition, spatial learning, procedural memory, sequential problem solving, operant- and fear conditioning, and social recognition. Those assays are carried out with individual subjects in apparatuses such as arenas and mazes, which allow for a high degree of standardization across laboratories and straightforward data interpretation but are not without caveats and limitations. In animal research, there is growing concern about the translatability of study results and animal welfare, leading to novel approaches beyond established protocols. Here, we present some of the more recent developments and more advanced concepts in learning and memory testing, such as multi-step sequential lockboxes, assays involving groups of animals, as well as home cage-based assays supported by automated tracking solutions; and weight their potential and limitations against those of established paradigms. Shifting the focus of learning tests from the classical experimental chamber to settings which are more natural for rodents comes with a new set of challenges for behavioral researchers, but also offers the opportunity to understand memory formation and retrieval in a more conclusive way than has been attainable with conventional test protocols. We predict and embrace an increase in studies relying on methods involving a higher degree of automatization, more naturalistic- and home cage-based experimental setting as well as more integrated learning tasks in the future. We are confident these trends are suited to alleviate the burden on animal subjects and improve study designs in memory research

Challenges and advanced concepts for the assessment of learning and memory function in mice

The mechanisms underlying the formation and retrieval of memories are still an active area of research and discussion. Manifold models have been proposed and refined over the years, with most assuming a dichotomy between memory processes involving non-conscious and conscious mechanisms. Despite our incomplete understanding of the underlying mechanisms, tests of memory and learning count among the most performed behavioral experiments. Here, we will discuss available protocols for testing learning and memory using the example of the most prevalent animal species in research, the laboratory mouse. A wide range of protocols has been developed in mice to test, e.g., object recognition, spatial learning, procedural memory, sequential problem solving, operant- and fear conditioning, and social recognition. Those assays are carried out with individual subjects in apparatuses such as arenas and mazes, which allow for a high degree of standardization across laboratories and straightforward data interpretation but are not without caveats and limitations. In animal research, there is growing concern about the translatability of study results and animal welfare, leading to novel approaches beyond established protocols. Here, we present some of the more recent developments and more advanced concepts in learning and memory testing, such as multi-step sequential lockboxes, assays involving groups of animals, as well as home cage-based assays supported by automated tracking solutions; and weight their potential and limitations against those of established paradigms. Shifting the focus of learning tests from the classical experimental chamber to settings which are more natural for rodents comes with a new set of challenges for behavioral researchers, but also offers the opportunity to understand memory formation and retrieval in a more conclusive way than has been attainable with conventional test protocols. We predict and embrace an increase in studies relying on methods involving a higher degree of automatization, more naturalistic- and home cage-based experimental setting as well as more integrated learning tasks in the future. We are confident these trends are suited to alleviate the burden on animal subjects and improve study designs in memory research

A systematic review of the development and application of home cage monitoring in laboratory mice and rats

Funding Information: Open Access funding enabled and organized by Projekt DEAL. VV is supported by Jane and Aatos Erkko Foundation. Funding Information: The Vienna BioCenter Core Facilities (VBCF) Preclinical Phenotyping Facility acknowledges funding from the Austrian Federal Ministry of Education, Science & Research; and the City of Vienna. Funding Information: This article is based upon work from COST Action “Improving biomedical research by automated behaviour monitoring in the animal home-cage” (TEATIME; CA20135; cost-teatime.org) supported by COST (European Cooperation in Science and Technology). Funding Information: PK, PM, AJ, BL, CTR, LL, and KH were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC 2002/1 “Science of Intelligence” —project number 390523135. Funding Information: This article is based upon work from COST Action “Improving biomedical research by automated behaviour monitoring in the animal home-cage” (TEATIME; CA20135; cost-teatime.org) supported by COST (European Cooperation in Science and Technology). Publisher Copyright: © 2023, The Author(s).Background: Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside of their home cages at selected time points. However, the outcome of such tests can be influenced by various factors and valuable information may be missed when the animals are only monitored for short periods. These issues can be overcome by longitudinally monitoring mice and rats in their home cages. To shed light on the development of home cage monitoring (HCM) and the current state-of-the-art, a systematic review was carried out on 521 publications retrieved through PubMed and Web of Science. Results: Both the absolute (~ × 26) and relative (~ × 7) number of HCM-related publications increased from 1974 to 2020. There was a clear bias towards males and individually housed animals, but during the past decade (2011–2020), an increasing number of studies used both sexes and group housing. In most studies, animals were kept for short (up to 4 weeks) time periods in the HCM systems; intermediate time periods (4–12 weeks) increased in frequency in the years between 2011 and 2020. Before the 2000s, HCM techniques were predominantly applied for less than 12 h, while 24-h measurements have been more frequent since the 2000s. The systematic review demonstrated that manual monitoring is decreasing in relation to automatic techniques but still relevant. Until (and including) the 1990s, most techniques were applied manually but have been progressively replaced by automation since the 2000s. Independent of the year of publication, the main behavioral parameters measured were locomotor activity, feeding, and social behaviors; the main physiological parameters were heart rate and electrocardiography. External appearance-related parameters were rarely examined in the home cages. Due to technological progress and application of artificial intelligence, more refined and detailed behavioral parameters have been investigated in the home cage more recently. Conclusions: Over the period covered in this study, techniques for HCM of mice and rats have improved considerably. This development is ongoing and further progress as well as validation of HCM systems will extend the applications to allow for continuous, longitudinal, non-invasive monitoring of an increasing range of parameters in group-housed small rodents in their home cages.publishersversionpublishe

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Mechanismen der Bacillus Sporen Keimung und Inaktivierung während der Hochdruckbehandlung

Hochdruck in Kombination mit hohen Prozesstemperaturen ermöglicht es, hochwertige sterile Lebensmittel herzustellen. Da unter anderem die Inaktivierungsmechanismen bakterieller Sporen nicht vollständig geklärt sind, wird diese Technologie bisher noch nicht industriell verwendet. Ziel der Arbeit war es, das Keimungs- und Inaktivierungsverhalten von Bacillus subtilis Sporen sowie von isogenen mutierten Stämmen, denen ein Teil des Keimungsmechanismus fehlt, in einem großen Druck-Temperatur-Zeitbereich zu untersuchen. Für reproduzierbare Prozessbedingungen wurden alle Kinetiken unter isothermen und isobaren Bedingungen während der Druckhaltezeit erstellt. Zur Vermeidung systematischer Fehler durch Veränderungen im Medium wurde ein pH-Messsystem für bis zu 130 °C und 1 MPa zur Messung in flüssigen und pastösen Lebensmitteln entwickelt. Es zeigte sich nahezu keine Verschiebung im Dissoziationsgleichgewicht bis 130 °C für Phosphat-Puffer, weshalb dieser für thermische Inaktivierungen verwendet wurde. Ein relativ stabiler pKa-Wert unter Hochdruck konnte durch die Verwendung von ACES-Puffer gewährleistet werden. Nach den Behandlungen wurde der physiologische Zustand der Sporen durch Keimzahlbestimmungen, durchflusszytomtetrische Analysen und eine Quantifizierung der freigesetzten Menge an Dipicolinsäure (DPA) mittels HPLC bestimmt. Für elektronen-mikroskopische Untersuchungen erfolgte die Entwicklung einer Ionenstrahl-Schnittmethode, die es ermöglichte Strukturveränderungen im Inneren der Spore zu untersuchen. Durch diese Methoden konnte ein Inaktivierungsmechanismus für 150 MPa und 37 °C abgeleitet werden, welcher durch eine physiologische DPA-Ausschleusung ausgelöst wird, über eine Cortex-Hydrolyse und den Abbau der kleinen säurelöslichen Proteine (SASP) zu einer Inaktivierung führt. Eine Druckerhöhung auf 550 MPa verlangsamt die Keimung und ein SASP-Abbau ist nicht mehr möglich. Wird dieser Druck mit Temperaturen über 60 °C kombiniert, induziert dies eine schnelle DPA-Ausschleusung sowie eine Inaktivierung. Daraus lässt sich schlussfolgern, dass unter diesen Bedingungen die empfindlichste Struktur die innere Sporenmembran ist. Unter Verwendung eines Differentialgleichungsmodells konnten Isokinetik-Druck-Temperatur-Diagramme für die DPA-Ausschleusung, die Bildung temperaturempfindlicher sowie inaktivierter Sporen berechnet werden. Es wurde bestätigt, dass der erste Schritt der Inaktivierung unter Hochdruck die Freisetzung von DPA ist. Ferner bestimmt die Prozesstemperatur oberhalb eines Grenzdruckes von 600 MPa die Keimungs- und Inaktivierungsgeschwindigkeit. Darüber hinaus ermöglichte das verwendete Berechnungsmodell, Kinetikdaten aus zwei verschiedenen Hochdruckanlagen miteinander zu vergleichen. Die Daten dieser Arbeit und die abgeleiteten Inaktivierungsmechanismen können dazu beitragen, diese vielversprechende Technologie zu kommerzialisieren und somit die Lebensmittelsicherheit zu erhöhen.High pressure combined with elevated temperatures can produce low acid, commercially sterile and shelf-stable food. However, this promising sterilization technology has not yet been applied in industrial settings, which is largely due to the unknown inactivation mechanism of highly resistant bacterial spores. This study aimed to clarify the germination and inactivation behavior of Bacillus subtilis spores and isogenic mutant strains lacking part of the germination apparatus, within a wide pressure–temperature–time domain. To ensure reliable process conditions, all kinetic data were derived under isothermal and isobaric treatment conditions during pressure dwell time. To avoid any biasing errors in the experimental results caused by varying matrix conditions, a pH-measurement system for liquid and semi-solid foods up to 130 °C and 1 MPa was constructed. The use of this system showed that PBS buffer had nearly no shift of dissociation equilibrium up to 130 °C, and was therefore used for all thermal inactivation experiments. However, under high pressure ACES buffer was used, due to its relatively stable pKa value. After the treatment, the physiological state of each spore sample was analyzed using the plate count method and flow cytometry, and the amount of dipicolinic acid (DPA) released was quantified by HPLC. In order to optimize scanning electron microscopic evaluations, a focused ion beam section method was developed to investigate the internal structural changes in spores. Based on this set of methods, a mechanism was proposed in which 150 MPa at 37 °C induces a physiological-like DPA release, followed by cortex and small acid soluble protein (SASP) degradation, resulting in a subsequent inactivation. A pressure increase to 550 MPa retarded the completion of germination and disabled SASP degradation. High pressures in combination with high temperatures (≥ 60 °C) caused a rapid DPA release and spore inactivation. This suggests that the most sensitive structure under these conditions is the inner spore membrane. By using a multiresponse kinetic model, isorate pressure–temperature diagrams for the release of DPA, the formation of heat-sensitive and inactivated spores were calculated. It was confirmed that the first step of inactivation under high pressure is the release of DPA. Above a threshold pressure of 600 MPa the treatment temperature dominates germination and inactivation rate. The model used enabled the comparison of kinetic data derived in two different high pressure units with different compression rates. The kinetic data from this study and the suggested mechanisms can aid in optimizing this promising sterilization technology and will increase food safety

Endospore Inactivation by Emerging Technologies: A Review of Target Structures and Inactivation Mechanisms

ISSN:1941-1413ISSN:1941-142