The development of extruded meat alternatives using Maillard-reacted beef bone hydrolysate and plant proteins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

Figures are re-used with permission.This research thesis aimed to process beef bone extract into a flavoursome protein ingredient to be added to extruded meat analogues to form meat alternatives and study their impact on the structural, textural, and sensory properties of meat alternatives. The thesis consists of three main parts. In the first part, two methods namely enzymatic hydrolysis and Maillard reaction (MR) treatments were evaluated for their suitability of modifying the flavour character of beef bone extract to become flavoursome protein ingredients. The second part studied the effects of soy protein concentrate (SPC) to wheat gluten (WG) ratio as a way of improving the structural and textural properties of current extruded meat analogues. The third part studied the effects of flavoursome protein ingredient (i.e. Maillard-reacted beef bone hydrolysate) with plant proteins on extruded meat alternatives. It also investigated the effects of moisture contents on extruded meat alternatives and their application in sausages. To begin, an experimental study on the effects of enzymatic hydrolysis treatments (i.e. single, simultaneous and sequential) on the physicochemical properties of beef bone extract using Protamex®, bromelain, and Flavourzyme® was conducted. Next, the changes in the physicochemical properties and volatile compounds of beef bone hydrolysates during heat treatment as a result of the MR were investigated. Beef bone hydrolysates were combined with ribose in aqueous solutions and heated at 113°C to produce Maillard reaction products (MRPs). Results showed that Flavourzyme® was the most effective in increasing the proportion of low Mw peptides, reducing viscosity and enhancing the flavour intensity of beef bone extract. Concurrently, the effects of SPC to WG ratio at a constant mass of SPC and WG on the physicochemical properties of extruded meat analogues were studied. Meat analogues containing 30%WG showed the highest degree of texturisation, fibrous structure, hardness and chewiness using instrumental and sensory analysis. For the third part of this research thesis, the effects of flavoursome protein ingredient (i.e. Flavourzyme®-MRP) at different concentrations (0, 10, 20, 30 and 40% wet weight) with plant proteins on extruded meat alternatives were investigated. Meat alternatives containing 20%MRP obtained the highest sensory scores for appearance, meaty aroma, meaty taste, and overall acceptability. Results showed that the addition of MRP with soy protein concentrate and wheat gluten to produce meat alternatives changed the textural, structural, and sensory properties significantly. The effects of moisture content (MC) on the physicochemical properties of extruded meat alternatives made from Flavourzyme®-MRP and plant proteins were studied. Samples were extruded at different dry feed rate of 1.8, 2.2, 2.6 and 3.0 kg/h to obtain MC of 60%MC, 56%MC, 52%MC and 49%MC, respectively. Meat alternatives at 49%MC were the closest in terms of both textural and microstructural properties to reference sample, boiled chicken breast. Results showed that the change in MC as a process parameter played an important role in the formation of fibrous structure in extruded meat alternatives. Lastly, the physicochemical properties of sausages made from extruded meat alternatives at different MC were conducted. Five sausages made from meat alternatives (S49%MC, S52%MC, S56%MC and S60%MC) and chicken breast (SCB) as a reference sample were prepared. Results showed that S49%MC had the highest sensory scores among all sausages made from meat alternatives. However, SCB obtained the highest sensory scores for all attributes except for appearance among all sausages at a 95% confidence level. Overall, the present work demonstrated that a flavoursome protein ingredient (i.e. Flavourzyme®-MRP) from low-value meat by-product (i.e. beef bone extract) can be successfully incorporated into extruded meat analogues to form meat alternatives with high aroma and taste quality while maintaining fibrous structure. However, further work needs to be done to improve the textural and sensory properties of sausages made from extruded meat alternatives

Disrupted Functional Connectivity of Cornu Ammonis Subregions in Amnestic Mild Cognitive Impairment: A Longitudinal Resting-State fMRI Study

Background: The cornu ammonis (CA), as part of the hippocampal formation, represents a primary target region of neural degeneration in amnestic mild cognitive impairment (aMCI). Previous studies have revealed subtle structural deficits of the CA subregions (CA1-CA3, bilateral) in aMCI; however, it is not clear how the network function is impacted by aMCI. The present study examined longitudinal changes in resting state functional connectivity (FC) of each CA subregion and how these changes relate to neuropsychological profiles in aMCI.Methods: Twenty aMCI and 20 healthy control (HC) participants underwent longitudinal cognitive assessment and resting state functional MRI scans at baseline and 15 months afterward. Imaging data were processed with published routines in SPM8 and CONN software. Two-way analysis of covariance was performed with covariates of age, gender, education level, follow up interval, gray matter volume, mean FD, as well as global correlation (GCOR). Pearson’s correlation was conducted to evaluate the relationship between the longitudinal changes in CA subregional FC and neuropsychological performance in aMCI subjects.Results: Resting state FC between the right CA1 and right middle temporal gyrus (MTG) as well as between the left CA2 and bilateral cuneal cortex (CC) were decreased in aMCI subjects as compared to HC. Longitudinal decrease in FC between the right CA1 and right MTG was correlated with reduced capacity of episodic memory in aMCI subjects.Conclusion: The current findings suggest functional alterations in the CA subregions. CA1 connectivity with the middle temporal cortex may represent an important neural marker of memory dysfunction in aMCI

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Building Reconfigurable Circuitry in a Biochemical World

International audienceRealizing complex systems within a biochemical environment is a common pursuit in synthetic biology, an emerging technology with promising potential in biomedicine and other applications. Such systems achieve certain computation through properly designed biochemical reactions. Despite fruitful progress being made, most existing reaction designs have fixed target functionality. Their lack of reconfigurability can be disadvantageous, especially when a system has to adapt to a varying biochemical environment. In this paper, we propose an analog approach to economically construct a reconfigurable logic circuit similar to a silicon based field programmable gate array (FPGA). The effective "logic" and "interconnect" of the circuit can be dynamically reconfigured by controlling the concentrations of certain knob species. We study a potential biomedical application of our reconfigurable circuitry to disease diagnosis and therapy at a molecular level

Reconfigurable Neuromorphic Computation in Biochemical Systems

International audienceImplementing application-specific computation and control tasks within a biochemical system has been an important pursuit in synthetic biology. Most synthetic designs to date have focused on realizing systems of fixed functions using specifically engineered components, thus lacking flexibility to adapt to uncertain and dynamically-changing environments. To remedy this limitation, an analog and modularized approach to realize reconfigurable neuromorphic computation with biochemical reactions is presented. We propose a biochemical neural network consisting of neuronal modules and interconnects that are both reconfigurable through external or internal control over the concentrations of certain molecular species. Case studies on classification and machine learning applications using the DNA strain displacement technology demonstrate the effectiveness of our design in both reconfiguration and autonomous adaptation

Plant proteins for future foods: A roadmap

10.3390/foods10081967Foods108196

Hybrid Simulations of Heterogeneous Biochemical Models in SBML

International audienc