In situ fortification of vitamin B12 in grain materials by fermentation with Propionibacterium freudenreichii

Vitamin B12 is a micronutrient that is predominantly present in food of animal origin. Therefore, developing plant-origin food with a nutritionally relevant content of vitamin B12 can increase the dietary intake of this vitamin among people with limited consumption of animal products. Since its chemical synthesis is overly complicated and expensive, the commercial vitamin B12 used for food fortification is exclusively produced via a biotechnological process. As compared to fortification with this commercial form of vitamin B12, in situ fortification via fermentation can be a more cost-effective alternative. As a commonly consumed staple food, grains are excellent vehicles for enrichment with micronutrients. Propionibacterium freudenreichii is the only food-grade microorganism with the ability to produce vitamin B12. Because P. freudenreichii has a low growth rate and is sensitive to acidic conditions, sterilized grain materials have mostly been used so far to produce a high vitamin B12 content. The sterilization process, however, alters the technological properties of grain-based raw materials and decreases the feasibility of the process. The present thesis focuses on in situ fortification of vitamin B12 in native grain materials by fermentation with P. freudenreichii. This study has demonstrated that fermentation of wheat flour, whole-wheat flour and wheat bran with P. freudenreichii resulted in a physiologically significant level of vitamin B12 (up to 155 ng/g dw) after 7 days. Whole-grain wheat flour and wheat bran had a higher content of vitamin B12 than refined wheat flour. However, the propagation of Enterobacteriaceae indicated that monoculture fermentation with P. freudenreichii cannot dominate the microflora, to guarantee microbial safety and control endogenous microbiota present in grain materials. Thus, an effective co-culture of Lactobacillus brevis ATCC 14869 and P. freudenreichii was established through a pre-screening to ensure microbial safety. During co-fermentation in wheat bran, P. freudenreichii produced a high level of vitamin B12 (ca. 183 ng/g dw on day 3). Moreover, controlling pH during fermentation could greatly enhance the vitamin B12 production (up to 332 ng/g dw on day 3). Meanwhile, L. brevis showed a strong inhibition on the propagation of Enterobacteriaceae during fermentation, as expected. The wider applicability of the established co-culture was demonstrated by fermenting 11 types of grain materials, including cereals, pseudocereals and legumes, with P. freudenreichii and L. brevis. P. freudenreichii produced a nutritionally significant level of vitamin B12 in most of the grain materials. The highest production was found in the rice bran (ca. 742 ng/g dw), followed by the buckwheat bran (ca. 631 ng/g dw), after fermentation. Meanwhile, the addition of L. brevis was able to dominate indigenous microbes during fermentation and thus greatly improve microbial safety during the fermentation of different grain materials. Overall, this thesis demonstrates that the fermentation of grain materials with P. freudenreichii and an appropriate co-culture, such as L. brevis, is a promising way to provide vitamin B12 in non-sterilized grain-based materials, without compromising microbial safety. Meanwhile, selecting raw materials that provide optimal conditions for P. freudenreichii can significantly improve the efficacy of vitamin B12 synthesis.B12-vitamiinia esiintyy pääasiassa vain eläinperäisissä elintarvikkeissa. Tämän vuoksi on tärkeää kehittää B12-vitamiinia sisältäviä kasviperäisiä elintarvikkeita kuluttajille, jotka syövät vähän tai eivät lainkaan eläinperäisiä tuotteita. Koska B12-vitamiinin syntetisointi kemiallisesti on erittäin monivaiheinen ja kallis prosessi, elintarvikkeiden täydentämiseen käytetty vitamiini tuotetaan yksinomaan teollisesti mikrobien avulla. B12-vitamiinin tuottaminen suoraan elintarvikkeeseen in situ voisi kuitenkin olla taloudellisempi vaihtoehto. Yleisesti käytettävät viljamateriaalit ovat hyviä kasvatusalustoja mikrobeille, joiden tiedetään tuottavan tiettyjä bioaktiivisia yhdisteitä, kuten vitamiineja. Propionibacterium freudenreichii on ainoa elintarvikekäyttöön hyväksytty mikro-organismi, jolla on kyky tuottaa B12-vitamiinia. Tämän väitöskirjatyön tavoite oli tutkia B12-vitamiinin tuottamista in situ viljapohjaisiin alustoihin P. freudenreichii -bakteerin avulla. Tutkimuksessa osoitettiin, että valkoisten ydinvehnäjauhojen, kokojyvävehnäjauhojen ja vehnäleseen fermentointi P. freudenreichii –bakteerilla tuotti merkittävän määrän B12-vitamiinia seitsemän päivän aikana. Fermentointi yksinomaan propionibakteerilla ei kuitenkaan pystynyt estämään täysin joidenkin haitallisten bakteereiden kasvua ja siten takaamaan tuotteen mikrobiologista turvallisuutta. Kun vehnäleseen fermentointiin käytettiin samanaikaisesti sekä P. freudenreichii -bakteeria että Lactobacillus brevis –maitohappobakteeria, havaittiin L. breviksen estävän tehokkaasti haitallisten bakteerien kasvua. Kehitetyn P. freudenreichiin ja L. breviksen yhteisviljelyn vaikutusta B12-vitamiinin tuottoon ja mikrobiologiseen laatuun tutkittiin vehnän lisäksi 11 erilaisella vilja-, pseudovilja- ja palkokasvimateriaalilla. P. freudenreichii tuotti B12-vitamiinia useimmissa materiaaleissa ravitsemuksellisesti merkittäviä määriä. Eniten P. freudenreichii tuotti B12-vitamiinia riisileseessä ja seuraavaksi eniten tattarileseessä kolmen päivän fermentoinnin aikana. Lisäksi todettiin, että L. breviksen vaikutuksesta tuotteiden mikrobiologinen laatu säilyi hyvänä. Tämä väitöskirjatyö osoitti, että fermentointi käyttäen P. freudenreichii -bakteeria yhdessä L. brevis -maitohappobakteerin kanssa on lupaava menetelmä tuottaa B12-vitamiinia viljapohjaisiin materiaaleihin ilman, että tuotteen mikrobiologinen laatu heikkenisi. Lisäksi tässä tutkimuksessa todettiin, että B12-vitamiinin optimaaliseen tuottoon vaikuttaa suuresti lähtömateriaalin koostumus

Dynamic simulation of a peristaltic micropump considering coupled fluid flow and structural motion

This paper presents lumped-parameter simulation of dynamic characteristics of peristaltic micropumps. The pump consists of three pumping cells connected in series, each of which is equipped with a compliant diaphragm that is electrostatically actuated in a peristaltic sequence to mobilize the fluid. Diaphragm motion in each pumping cell is first represented by an effective spring subjected to hydrodynamic and electrostatic forces. These cell representations are then used to construct a system-level model for the entire pump, which accounts for both cell- and pump-level interactions of fluid flow and diaphragm vibration. As the model is based on first principles, it can be evaluated directly from the device's geometry, material properties and operating parameters without using any experimentally identified parameters. Applied to an existing pump, the model correctly predicts trends observed in experiments. The model is then used to perform a systematic analysis of the impact of geometry, materials and pump loading on device performance, demonstrating its utility as an efficient tool for peristaltic micropump design

Surface micromachined electrostatically actuated micro peristaltic pump

An electrostatically actuated micro peristaltic pump is reported. The micro pump is entirely surface micromachined using a multilayer parylene technology. Taking advantage of the multilayer technology, the micro pump design enables the pumped fluid to be isolated from the electric field. Electrostatic actuation of the parylene membrane using both DC and AC voltages was demonstrated and applied to fluid pumping based on a 3-phase peristaltic sequence. A maximum flow rate of 1.7 nL min^–1 and an estimated pumping pressure of 1.6 kPa were achieved at 20 Hz phase frequency. A dynamic analysis was also performed with a lumped-parameter model for the peristaltic pump. The analysis results allow a quantitative understanding of the peristaltic pumping operation, and correctly predict the trends exhibited by the experimental data. The small footprint of the micro pump is well suited for large-scale integration of microfluidics. Moreover, because the same platform technology has also been used to fabricate other devices (e.g. valves, electrospray ionization nozzles, filters and flow sensors), the integration of these different devices can potentially lead to versatile and functional micro total analysis systems (µTAS)

Induced Stem Cells as a Novel Multiple Sclerosis Therapy.

Stem cell replacement is providing hope for many degenerative diseases that lack effective therapeutic methods including multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. Transplantation of neural stem cells or mesenchymal stem cells is a potential therapy for MS thanks to their capacity for cell repopulation as well as for their immunomodulatory and neurotrophic properties. Induced pluripotent stem cell (iPSC), an emerging cell source in regenerative medicine, is also being tested for the treatment of MS. Remarkable improvement in mobility and robust remyelination have been observed after transplantation of iPSC-derived neural cells into demyelinated models. Direct reprogramming of somatic cells into induced neural cells, such as induced neural stem cells (iNSCs) and induced oligodendrocyte progenitor cells (iOPCs), without passing through the pluripotency stage, is an alternative for transplantation that has been proved effective in the congenital hypomyelination model. iPSC technology is rapidly progressing as efforts are being made to increase the efficiency of iPSC therapy and reduce its potential side effects. In this review, we discuss the recent advances in application of stem cells, with particular focus on induced stem/progenitor cells (iPSCs, iNSC, iOPCs), which are promising in the treatment of MS

Study of Ni Metallization in Macroporous Si Using Wet Chemistry for Radio Frequency Cross-Talk Isolation in Mixed Signal Integrated Circuits.

A highly conductive moat or Faraday cage of through-the-wafer thickness in Si substrate was proposed to be effective in shielding electromagnetic interference thereby reducing radio frequency (RF) cross-talk in high performance mixed signal integrated circuits. Such a structure was realized by metallization of selected ultra-high-aspect-ratio macroporous regions that were electrochemically etched in p- Si substrates. The metallization process was conducted by means of wet chemistry in an alkaline aqueous solution containing Ni2+ without reducing agent. It is found that at elevated temperature during immersion, Ni2+ was rapidly reduced and deposited into macroporous Si and a conformal metallization of the macropore sidewalls was obtained in a way that the entire porous Si framework was converted to Ni. A conductive moat was as a result incorporated into p- Si substrate. The experimentally measured reduction of crosstalk in this structure is 5~18 dB at frequencies up to 35 GHz

Integrated surface-micromachined mass flow controller

An integrated surface-micromachined mass flow controller (MFC) that consists of an electrostatically actuated microvalve and a thermal flow sensor is presented here. With a unique design and utilizing a multilayer Parylene process, the active microvalve and the flow sensor are integrated onto a single chip to perform closed-loop flow control. Sensitivity of the flow sensor is 55 μV/(μ/L/min) for airflow and 12.2 μV/(nL/min) for water. The valve is actuated with a 10 kHz AC signal and an applied pressure of 21 kPa can be sealed with an actuation voltage of 200 V_peak (±200 V). For flow control, both Pulse Width Modulation (PWM) and actuation voltage adjustment are demonstrated. PWM shows better performance in terms of controllability and linearity

Surface micromachined and integrated capacitive sensors for microfluidic applications

We have demonstrated an entire series of capacitive sensors using a multi-layer parylene/photoresist surface micromachining technology. The developed sensors are designed for total integration into parylene-based microfluidic systems for real-time system monitoring. Sensors have been demonstrated for the following applications: in-line pressure sensing (range: 0-35 kPa, resolution: 0.03 kPa); liquid front position and/or volumetric measurements (range: 0->50 pL, resolution: <5 pL); and dielectric measurements, which can be used to deduce fluid properties such as liquid composition. The reported sensor technology demonstrates versatility, high sensitivity, small footprints, and easy integration

Fracture mode control: a bio-inspired strategy to combat catastrophic damage

The excellent mechanical properties of natural biomaterials have attracted intense attention from researchers with focus on the strengthening and toughening mechanisms. Nevertheless, no material is unconquerable under sufficiently high load. If fracture is unavoidable, constraining the damage scope turns to be a practical way to preserve the integrity of the whole structure. Recent studies on biomaterials have revealed that many structural biomaterials tend to be fractured, under sufficiently high indentation load, through ring cracking which is more localized and hence less destructive compared to the radial one. Inspired by this observation, here we explore the factors affecting the fracture mode of structural biomaterials idealized as laminated materials. Our results suggest that fracture mode of laminated materials depends on the coating/substrate modulus mismatch and the indenter size. A map of fracture mode is developed, showing a critical modulus mismatch (CMM), below which ring cracking dominates irrespective of the indenter size. Many structural biomaterials in nature are found to have modulus mismatch close to the CMM. Our results not only shed light on the mechanics of inclination to ring cracking exhibited by structural biomaterials but are of great value to the design of laminated structures with better persistence of structural integrity.Research Grants Council (Hong Kong, China). Early Career Scheme (Grant 533312)Hong Kong Polytechnic University. Departmental General Research Funds (Internal Competitive Research Grants 4-ZZA8)Hong Kong Polytechnic University. Departmental General Research Funds (Internal Competitive Research Grants A-PM24)Hong Kong Polytechnic University. Departmental General Research Funds (Internal Competitive Research Grants G-UA20