This paper presents a novel microfluidic DNA digestion system incorporating a high performance micro-mixer. Through the appropriate control of fixed and periodic switching DC electric fields, electrokinetic forces are established to mix the DNA and restriction enzyme samples and to drive them through the reaction column of the device. The experimental and numerical results show that a mixing performance of 98% can be achieved within a mixing channel of length 1.6 mm when a 150 V/cm driving voltage and a 5 Hz switching frequency are applied. The relationship between the mixing performance, switching frequency, and main applied electric field is derived. It is found that the optimal switching frequency depends upon the magnitude of the main applied electric field. The successful digestion of lambda-DNA using Eco RI restriction enzyme is demonstrated. The DNA-enzyme reaction is completed within 15 min in the proposed microfluidic system, compared to 50 min in a conventional large-scale system. Hence, the current device provides a valuable tool for rapid lambda-DNA digestion, while its mixer system delivers a simple yet effective solution for mixing problems in the micro-total-analysis-systems field.

Cockroaches of the subfamily Panesthiinae (family Blaberidae) are among the few major groups of insects feeding on decayed wood. Despite having independently evolved the ability to thrive on this recalcitrant and nitrogen-limited resource, they are among the least studied of all wood-feeding insect groups. In the pursuit of unraveling their unique digestive strategies, we explored cellulase and xylanase activity in the crop, midgut, and hindgut lumens of Panesthia angustipennis and Salganea taiwanensis. Employing Percoll density gradient centrifugation, we further fractionated luminal fluid to elucidate how the activities in the gut lumen are further partitioned. Our findings challenge conventional wisdom, underscoring the significant contribution of the hindgut, which accounts for approximately one-fifth of cellulase and xylanase activity. Particle-associated enzymes, potentially of bacterial origin, dominate hindgut digestion, akin to symbiotic strategies observed in select termites and passalid beetles. Our study sheds new light on the digestive prowess of panesthiine cockroaches, providing invaluable insights into the evolution of wood-feeding insects and their remarkable adaptability to challenging, nutrient-poor substrates.

In this work, performance and microbial structure of a digestion (food waste-only) and a co-digestion process (mixture of cow manure and food waste) were studied at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The highest methane yield (480 mL/g VS) was observed in the mesophilic digester (MDi) fed with food waste alone. The mesophilic co-digestion of food waste and manure (McoDi) yielded 26% more methane than the sum of individual digestions of manure and food waste. The main volatile fatty acid (VFA) in the mesophilic systems was acetate, averaging 93 and 172 mg/L for McoDi and MDi, respectively. Acetate (2150 mg/L) and propionate (833 mg/L) were the main VFAs in the thermophilic digester (TDi), while propionate (163 mg/L) was the major VFA in the thermophilic co-digester (TcoDi). The dominant bacteria in MDi was Chloroflexi (54%), while Firmicutes was dominant in McoDi (60%). For the mesophilic reactors, the dominant archaea was Methanosaeta in MDi, while Methanobacterium and Methanosaeta had similar abundance in McoDi. In the thermophilic systems, the dominant bacteria were Thermotogae, Firmicutes and Synergistetes in both digesters, however, the relative abundance of these phyla were different. For archaea, the genus Methanothermobacter were entirely dominant in both TDi and TcoDi.

The digestive methods employed by amphioxus (Branchiostoma)-both intracellular phagocytic digestion and extracellular digestion-have been discussed since 1937. Recent studies also show that epithelial cells lining the Branchiostoma digestive tract can express many immune genes. Here, in Branchiostoma belcheri, using a special tissue fixation method, we show that some epithelial cells, especially those lining the large diverticulum protruding from the gut tube, phagocytize food particles directly, and Branchiostoma can rely on this kind of phagocytic intracellular digestion to obtain energy throughout all stages of its life. Gene expression profiles suggest that diverticulum epithelial cells have functional features of both digestive cells and phagocytes. In starved Branchiostoma, these cells accumulate endogenous digestive and hydrolytic enzymes, whereas, when sated, they express many kinds of immune genes in response to stimulation by phagocytized food particles. We also found that the distal hindgut epithelium can phagocytize food particles, but not as many. These results illustrate phagocytic intercellular digestion in Branchiostoma, explain why Branchiostoma digestive tract epithelial cells express typical immune genes and suggest that the main physiological function of the Branchiostoma diverticulum is different from that of the vertebrate liver.

To evaluate the use of genipin in delaying enzymatic digestion of corneal stroma.

Omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential for human health but prone to oxidation. While esterification location is known to influence the stability of omega-3 in triacylglycerols (TAGs) in oxidation trials, their oxidative behavior in the gastrointestinal tract is unknown. Synthesized ABA- and AAB-type TAGs containing DHA and EPA were submitted to static in vitro digestion for the first time. Tridocosahexaenoin and DHA as ethyl esters were similarly digested. Digesta were analyzed by gas chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy. Besides the formation of di- and monoacylglycerols, degradation of hydroperoxides was detected in ABA- and AAB-type TAGs, whereas oxygenated species increased in tridocosahexaenoin. Ethyl esters were mainly unaffected. EPA was expectedly less susceptible to oxidation prior to and during the digestion process, particularly in sn-2. These results are relevant for the production of tailored omega-3 structures to be used as supplements or ingredients.

Microplastics (MPs) are a widely recognized global problem due to their prevalence in natural environments and the food chain. However, the impact of microplastics on human microbiota and their possible biotransformation in the gastrointestinal tract have not been well reported. To evaluate the potential risks of microplastics at the digestive level, completely passing a single dose of polyethylene terephthalate (PET) through the gastrointestinal tract was simulated by combining a harmonized static model and the dynamic gastrointestinal simgi model, which recreates the different regions of the digestive tract in physiological conditions. PET MPs started several biotransformations in the gastrointestinal tract and, at the colon, appeared to be structurally different from the original particles. We report that the feeding with microplastics alters human microbial colonic community composition and hypothesize that some members of the colonic microbiota could adhere to MPs surface promoting the formation of biofilms. The work presented here indicates that microplastics are indeed capable of digestive-level health effects. Considering this evidence and the increasing exposure to microplastics in consumer foods and beverages, the impact of plastics on the functionality of the gut microbiome and their potential biodegradation through digestion and intestinal bacteria merits critical investigation.

A better understanding of how dietary lipids are processed by the human body is necessary to allow for the control of satiation and energy intake by tailored lipid systems. To examine whether rats are a valid model of human dietary lipid processing and therefore useful for further mechanistic studies in this context, we tested in rats three lipid emulsions of different stability, which alter satiety responses in humans. Different sets of 15 adult male Sprague Dawley rats, equipped with gastric catheters alone or combined with hepatic portal vein (HPV) and vena cava (VC) catheters were maintained on a medium-fat diet and adapted to an 8 h deprivation/16 h feeding schedule. Experiments were performed in a randomized cross-over study design. After gastric infusion of the lipid emulsions, we assessed gastric emptying by the paracetamol absorption test and recorded in separate experiments food intake and plasma levels of gastrointestinal hormones and metabolites in the HPV. For an acid stable emulsion, slower gastric emptying and an enhanced release of satiating gastrointestinal (GI) hormones were observed and were associated with lower short-term energy intake in rats and less hunger in humans, respectively. The magnitude of hormonal responses was related to the acid stability and redispersibility of the emulsions and thus seems to depend on the availability of lipids for digestion. Plasma metabolite levels were unaffected by the emulsion induced changes in lipolysis. The results support that structured lipid systems are digested similarly in rats and humans. Thus unstable emulsions undergo the same intragastric destabilization in both species, i.e., increased droplet size and creaming. This work establishes the rat as a viable animal model for in vivo studies on the control of satiation and energy intake by tailored lipid systems.

Effects of two foods with bioactive constituents (black tea brew, BTB and grape seed powder, GSP) on lipid digestibility was studied. Lipolysis inhibitory effect of these foods was examined using two test foods (cream and baked beef) with highly different fatty acid (FA) composition. Digestion simulations were performed either using both gastric and pancreatic lipase, or only with pancreatic lipase according to the Infogest protocol. Lipid digestibility was assessed based on the bioaccessible FAs. Results showed the triacylglycerols containing short- and medium-chain FAs (SCFA and MCFA) are non-preferred substrates for pancreatic lipase; however, this is not characteristic for GL. Our findings suggest that both GSP and BTB primarily affect the lipolysis of SCFAs and MCFAs, because the dispreference of pancreatic lipase towards these substrates was further enhanced as a result of co-digestion. Interestingly, GSP and BTB similarly resulted in significant decrease in lipolysis for cream (containing milk fat having a diverse FA profile), whereas they were ineffective in influencing the digestion of beef fat, having simpler FA profile. It highlights that the characteristics of the dietary fat source of a meal can be a key determinant on the observed extent of lipolysis when co-digested with foods with bioactive constituents.

As the demand for utilizing environment-friendly and sustainable energy sources is increasing, the adoption of waste-to-energy technologies has started gaining attention. Producing biogas via anaerobic digestion (AD) is promising and well-established; however, this process in many circumstances is unable to be cost competitive with natural gas. In this research, we provide a technical assessment of current process challenges and compare the cost of biogas production via the AD process from the literature, Aspen Plus process modeling, and CapdetWorks software. We also provide insights on critical factors affecting the AD process and recommendations on optimizing the process. We utilize four types of wet wastes, including wastewater sludge, food waste, swine manure, and fat, oil, and grease, to provide a quantitative assessment of theoretical energy yields of biogas production and its economic potential at different plant scales. Our results show that the cost of biogas production from process and economic models are in line with the literature with a potential to go even lower for small-scale plants with technological advancements. This research illuminates potential cost savings for biogas production using different wastes and guide investors to make informed decisions, while achieving waste management goals.

Anaerobic digestion is widely used to process and recover value from food waste. Commercial food waste anaerobic digestion facilities seek improvements in process efficiency to enable higher throughput. There is limited information on the composition of microbial communities in food waste prior to digestion, limiting rational exploitation of the catalytic potential of microorganisms in pretreatment processes. To address this knowledge gap, bacterial and fungal communities in food waste samples from a commercial anaerobic digestion facility were characterised over 3 months. The abundance of 16S rRNA bacterial genes was approximately five orders of magnitude higher than the abundance of the fungal intergenic spacer (ITS) sequence, suggesting the numerical dominance of bacteria over fungi in food waste before anaerobic digestion. Evidence for the mass proliferation of bacteria in food waste during storage prior to anaerobic digestion is presented. The composition of the bacterial community shows variation over time, but lineages within the Lactobacillaceae family are consistently dominant. Nitrogen content and pH are correlated to community variation. These findings form a foundation for understanding the microbial ecology of food waste and provide opportunities to further improve the throughput of anaerobic digestion.

In addition to evolving eusocial lifestyles, two equally fascinating aspects of termite biology are their mutualistic relationships with gut symbionts and their use of lignocellulose as a primary nutrition source. Termites are also considered excellent model systems for studying the production of bioethanol and renewable bioenergy from 2nd generation (non-food) feedstocks. While the idea that gut symbionts are the sole contributors to termite lignocellulose digestion has remained popular and compelling, in recent years host contributions to the digestion process have become increasingly apparent. However, the degree to which host and symbiont, and host enzymes, collaborate in lignocellulose digestion remain poorly understood. Also, how digestive enzymes specifically collaborate (i.e., in additive or synergistic ways) is largely unknown. In the present study we undertook translational-genomic studies to gain unprecedented insights into digestion by the lower termite Reticulitermes flavipes and its symbiotic gut flora. We used a combination of native gut tissue preparations and recombinant enzymes derived from the host gut transcriptome to identify synergistic collaborations between host and symbiont, and also among enzymes produced exclusively by the host termite. Our findings provide important new evidence of synergistic collaboration among enzymes in the release of fermentable monosaccharides from wood lignocellulose. These monosaccharides (glucose and pentoses) are highly relevant to 2(nd)-generation bioethanol production. We also show that, although significant digestion capabilities occur in host termite tissues, catalytic tradeoffs exist that apparently favor mutualism with symbiotic lignocellulose-digesting microbes. These findings contribute important new insights towards the development of termite-derived biofuel processing biotechnologies and shed new light on selective forces that likely favored symbiosis and, subsequently, group living in primitive termites and their cockroach ancestors.

Artichoke is a relevant source of health-promoting compounds such as polyphenols and sesquiterpene lactones. In this study, the bioaccessibility and gut bioavailability of artichoke constituents were evaluated by combining in vitro digestion and large intestine fermentation, metabolomics, and Caco-2 human intestinal cells model. Moreover, the ability of artichoke polyphenols to modulate the in vitro starch digestibility was also explored. An untargeted metabolomic approach based on liquid chromatography quadrupole-time-of-flight (UHPLC/QTOF) mass spectrometry coupled with multivariate statistics was used to comprehensively screen the phytochemical composition of raw, digested, and fermented artichoke. Overall, a large abundance of phenolic acids and sesquiterpene lactones was detected, being 13.77 and 11.99 mg·g-1, respectively. After 20 h of in vitro large intestine fermentation, a decrease in polyphenols and sesquiterpene lactones content was observed. The most abundant compounds characterizing the raw material (i.e., chlorogenic acid and cynaropicrin equivalents) showed an average % bioaccessibility of 1.6%. The highest % bioaccessibility values were recorded for flavonoids such as anthocyanin and flavone equivalents (on average, 13.6%). However, the relatively high bioavailability values recorded for flavonols, phenolic acids, and sesquiterpene lactones (from 71.6% up to 82.4%) demonstrated that these compounds are able to be transported through the Caco-2 monolayer. The phenolic compounds having the highest permeation rates through the Caco-2 model included low molecular weight phenolics such as tyrosol and 4-ethylcatechol; the isoflavonoids 3'-O-methylviolanone, equol 4'-O-glucuronide, and hydroxyisoflavone; together with the methyl and acetyl derivatives of glycosylated anthocyanins. Therefore, although human in vivo confirmatory trials are deemed possible, current findings provide insights into the mechanistic effects underlying artichoke polyphenols and sesquiterpenoids bioavailability following gastrointestinal and large intestine processes.

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining.

Severe burns are often treated by means of autologous skin grafts, preferably following early excision of the burnt tissue. In the case of, for example, a large surface trauma, autologous skin cells can be expanded in vitro prior to transplantation to facilitate the treatment when insufficient uninjured skin is a limitation. In this study we have analyzed the impact of the enzyme (trypsin or accutase) used for cell dissociation and the incubation time on cell viability and expansion potential, as well as expression of cell surface markers indicative of stemness. Skin was collected from five individuals undergoing abdominal reduction surgery and the epidermal compartment was digested in either trypsin or accutase. Trypsin generally generated more cells than accutase and with higher viability; however, after 7 days of subsequent culture, accutase-digested samples tended to have a higher cell count than trypsin, although the differences were not significant. No significant difference was found between the enzymes in median fluorescence intensity of the analyzed stem cell markers; however, accutase digestion generated significantly higher levels of CD117- and CD49f-positive cells, but only in the 5 h digestion group. In conclusion, digestion time appeared to affect the isolated cells more than the choice of enzyme.

Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL-1 trypsin is electrosonically sprayed (-3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development.

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.

Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source in the biosphere. Turnover of woody biomass is a critical component of the global carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil fuels to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon cycling by processing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal's gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics, and microscopy, we show that the digestive proteome of the shipworm Lyrodus pedicellatus is mostly composed of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that functions in the hydrolysis of β-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass.

Five experiments were conducted to examine effects of lysophospholipids (LPL) on live weight gain, nutrient digestibility, ruminal fermentation parameters, serum biochemical parameters and rumen bacterial community profile in fattening lambs. Two dietary treatments (pelleted complete feed supplemented without (control diet; CON) or with 0.05% LPL on dry matter basis) were tested in these experiments. Feed and water were provided ad libitum to lambs. The results showed that average daily gain (ADG) tended to increase or was not affected by LPL supplementation. Compared with CON, the supplementation of LPL resulted in an increase in dry matter, crude protein and organic matter digestibilities, and a decrease in neutral detergent fiber and acid detergent fiber digestibilities. Ruminal pH values did not change with LPL supplementation, but the concentrations of ammonia and total short chain fatty acids (SCFAs) were increased. The molar proportion of major individual SCFAs and the ratio of acetate to propionate were not affected by LPL supplementation. While the activity of lipase was decreased with LPL supplementation, all other serum biochemical parameters did not change. Rumen bacterial community was altered by LPL supplementation with the relative abundance of fibrolytic bacteria in the total bacterial population, such as Prevotella, decreased. In conclusion, LPL supplementation can alter feed digestion, but may not result in consistent positive responses in animal growth performance.

Anaerobic digestion is important for the management of livestock manure with high ammonia level. Although ammonia effects on anaerobic digestion have been comprehensively studied, the molecular mechanism underlying ammonia inhibition still remains elusive. In this study, based on metatranscriptomic analysis, the transcriptional profile of microbial community in anaerobic digestion under low (1500mgL-1) and high NH4+ (5000mgL-1) concentrations, respectively, were revealed. The results showed that high NH4+ concentrations significantly inhibited methane production but facilitated the accumulations of volatile fatty acids. The expression of methanogenic pathway was significantly inhibited by high NH4+ concentration but most of the other pathways were not significantly affected. Furthermore, the expressions of methanogenic genes which encode acetyl-CoA decarbonylase and methyl-coenzyme M reductase were significantly inhibited by high NH4+ concentration. The inhibition of the co-expressions of the genes which encode acetyl-CoA decarbonylase was observed. Some genes involved in the pathways of aminoacyl-tRNA biosynthesis and ribosome were highly expressed under high NH4+ concentration. Consequently, the ammonia inhibition on anaerobic digestion mainly focused on methanogenic process by suppressing the expressions of genes which encode acetyl-CoA decarbonylase and methyl-coenzyme M reductase. This study improved the accuracy and depth of understanding ammonia inhibition on anaerobic digestion.