Food Bioengineering最新文献

Mannosyl compounds have been widely used in nutrition, fodder, and vaccine adjuvant industries. In our previous study, the engineered strains for the biosynthesis of three mannosyl compounds including β-1,2-mannobiose (M2-β-1,2), β-1,2-mannotriose (M3-β-1,2), and mannosylglycerate (MG) have been developed. However, their biological activities have not been reported. Here, those three compounds were successfully purified after fermentation of the engineered strains, and their potential immunomodulatory activities on RAW264.7 macrophages were investigated with commercialized β-1,4-mannotriose (M3-β-1,4) as control. Our results showed that M3-β-1,2 and MG promoted the viability and phagocytic function of RAW264.7. Meanwhile, the cytokine TNF-α and interleukin-6 (IL-6) level of RAW264.7 macrophages were significantly enhanced upon the stimulation of M3-β-1,2 and MG compared with M3-β-1,4. Moreover, MG significantly stimulated macrophages to secrete IL-10 compared with other mannan oligosaccharides. Finally, this study proved that the immunomodulatory activity of M3-β-1,2 and MG on RAW 264.7 cells was mainly through mitogen-activated protein kinases and myeloid differentiation protein 88 (MyD88)-dependent signaling pathways. All these findings suggested that M3-β-1,2 and MG exhibited immunomodulatory activities in the innate and adaptive immune systems, thus facilitating the application potential in developing of mannosyl compounds as an immunomodulator available for food and pharmaceutical area.

Vitamin B₆ is an essential nutrient, which is widely used in food products, feed additives, pharmaceuticals, disease diagnosis, and other fields. Pyridoxal-5′-phosphate (PLP), the active form of vitamin B₆, is an important coenzyme participating in a variety of enzyme reactions. At present, the oxazole method is mainly used for the production of vitamin B₆, but toxic and corrosive intermediates produced in the synthesis process, which does not conform to the green manufacturing. Therefore, there is considerable interest in shifting from chemical processes to sustainable fermentation techniques and research on PLP metabolism of other valuable compounds. In this review, we will briefly describe the enzymes that PLP participated and focus on the advances in vitamin B₆ biosynthesis and discuss its application to engineering bacteria that overproduce other commercially valuable substances including cadaverine, 3,4-dihydroxyphenylacetate-l-alanine, γ-aminobutyric acid, and l-phosphinothricin. It will provide a reference for the biosynthesis of vitamin B₆ or other valuable compounds in which PLP participates in the reaction, and we also look forward to the future development prospect of PLP.

The introduction of heterologous pathways into microorganisms for high-level synthesis of valuable compounds would be extremely meaningful. However, the endogenous heterologous pathway conversion point frequently serves as a bottleneck that limits product synthesis. (+)-Nootkatone is an important functional sesquiterpenoid with a unique grapefruit flavor, which is frequently used as a fragrance. The low catalytic efficiency of the valencene synthase CnVS, the first heterologous enzyme in the biosynthesis of (+)-nootkatone, is the major challenge that restricts the production of (+)-nootkatone in Saccharomyces cerevisiae. In this study, we modified CnVS on the basis of the catalytic environment and mechanism of the enzyme. The residue M560 involving in alkyl transfer during substrate cyclization was found to affect activity. By tuning the residue, mutant M560L exhibited a 60% increase in the unit cell yield of (+)-valencene, which is the precursor of (+)-nootkatone. Furthermore, multistep strategies of metabolic engineering were employed that transferred the metabolic flux to the target product synthesis pathway and displayed the superimposed effect of metabolic and protein engineering on (+)-nootkatone yield. Finally, multicopy integration of tHMG1 and ERG20ERG20-GSG-CnVS^M560L boosted the production of (+)-nootkatone to 85.43 mg/L in flask and 804.96 mg/L (5.29 mg/[L h]) in bioreactor. In conclusion, this study presents a paradigm for constructing microbial cell factories using a combination of protein and metabolic engineering in S. cerevisiae.

Camel milk isolates of lactic acid bacteria were characterized to develop defined starter cultures that could be used for the production of standardized Egyptian Rayeb fermented milk. The isolates have shown a wide diversity of Technological characteristics and antimicrobial activity. Interesting isolates were genetically identified at the species level. Two mixed culture starters composed of Lactiplantibacillus plantarum (FFNL146 and FFNL159) and Lactococcus lactis (FFNL1926 and FFNL2005) along with Leuconostoc pseudomesenteroides FFNL2374 were used in the production of Rayeb from camel milk, cow milk, and their mixture and stored for 14 days under refrigeration. Equally mixed camel and cow milk fermented with both starters had the best results of organoleptic attributes and physicochemical properties compared to samples made of camel milk. This is the first study to present two mixed starter cultures for Rayeb manufacturing. The success of these defined starter cultures will potentially enhance the safety and quality of the Rayeb industry in Egypt.

Non/semi-rational engineering strategies can improve the performance of microbial cell factories under the situations of indistinct metabolic regulations and interactions. However, generating positive variants through these strategies is remarkably low. Using traditional screening methods to evaluate the performance of mutants, such as mass spectrometry and chromatography, is low-throughput, slow-detection, and labor-intensive. Specifically, the efficiency of high-throughput screening (HTS) for strain screening could reach even more 10³−10⁶ strains/day, bridging the gap between non/semi-rational engineering strategies and microbial cell factories construction. This review highlights transcription factor (TFs)-based biosensors in the advances in developing synthetic biosensors for HTS. We hope that this review will help take full advantage of valuable TFs to guide the HTS technology and promote its development.

Recently, a novel two-domain alginate lyase (AlyAL01) was cloned from Algibacter sp. and successfully overexpressed in Escherichia coli BL21(DE3). Biochemical analysis showed that the N-terminal carbohydrate-binding module (CBM) of AlyAL01 had no effect on the enzyme activity and product distributions. Therefore, the N-terminal CBM was substituted with CBM3 to confer the designed chimeric protein with the ability to specifically bind to regenerated amorphous cellulose. As anticipated, the designed chimeric protein (CBM3-L1) exhibited a similar enzyme activity. Moreover, it was found that the CBM3-L1 combined the purification and immobilization in one step with high immobilized efficiency of 65.8%. The immobilized enzyme exhibited good storage stability and moderate reusability. The immobilized enzyme could keep 85% activity when incubated at 4°C for 60 days and 70% activity when incubated at 25°C for 30 days. Furthermore, the immobilized CBM3-L1 kept about 50% of its initial activity after being reused five times. Finally, immobilized CBM3-L1 successively produced well-defined alginate oligosaccharides (AOS) with DPs of 2–6 by controlling reaction time. In sum, our current study provided a feasible strategy for well-defined AOS production.

A xyloglucanase gene (RmXEG12B) was cloned from Rhizomucor miehei CAU432 and successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 26,200 U/mL was achieved after 168 h high-cell-density fermentation. The optimal pH and temperature of RmXEG12B were 5.0 and 55°C, respectively. RmXEG12B showed good stability within pH of 3.0–10.0 and was stable up to 50°C. It exhibited the highest specific activity (5989.3 U/mg) towards tamarind xyloglucan. RmXEG12B hydrolyzed apple pomace xyloglucan to produce the partially hydrolyzed apple pomace xyloglucan, which demonstrated a weight average molecular weight of 1.6 × 10⁴ Da and contained three major oligosaccharides with a degree of polymerization of 7–10. This is a strategy for the comprehensive utilization of apple pomace to produce the partially hydrolyzed apple pomace xyloglucan.

A secreted 50-kDa metalloprotease from Serratia marcescens D15 was identified as rice protein hydrolase (SeMPase), which reached the highest proteolytic activity (477.3 U/ml) in basal medium with 1.5% rice protein (RP) at 35°C and 150 rpm for 48 h. SeMPase exhibited a highest catalytic number for RP (275.8 min⁻¹, K_cat), which contained a highly conserved Zn²⁺-binding (HEIGH) domain, Met-turn (SLMSY), and Ca²⁺ binding domain (GGAGND). RP hydrolysates that prepared by SeMPase showed the highest content of oligopeptides at 72.3%, compared to 64.1% from neutrase, 63.0% from alcalase, 50.2% from trypsin, 47.1% from flavourzyme, and 2.41% from pepsin. Our results showed that cleavage sites of SeMPase exposed in RP are more than those of other proteases because both SeMPase cleavage sites and the amino acid composition of RP are rich in Leu, Val, Phe, Glu, and Ala. SeMPase has significant specificity for the preparation of oligopeptide-enriched RP hydrolysates, which provides a new and highly valued option for industrial preparation of oligopeptide-enriched protein hydrolysates.

Microalgae are widely used in the field of food bioengineering. There are relatively few studies on the synthesis of intracellular electron carriers in microalgae and their related metabolic effects. In this study, overexpression of Geranyl diphosphate: 4-hydroxybenzoate geranyltransferase (lepgt), and the addition of 4-hydroxybenzoic acid (4-HB) as quinone ring precursor were adopted to regulate intracellular plastoquinone (PQ) and ubiquinone (UQ) levels in model algae Chlamydomonas reinhardtii. Small changes in PQ and UQ contents could regulate cell growth and metabolism. The expression of lepgt in C. reinhardtii significantly reduced the intracellular contents of PQ and UQ by 49.14% and 98.30%, respectively. The expression of lepgt observably reduced the photosystem activity and respiration rate and increased the contents of neutral lipid and starch by 63.68% and 19.42%, respectively. Exogenous low concentration of 4-HB (1 mM) can effectively improve the contents of intracellular electron carriers, promote respiration, slightly reduce photosystem efficiency, and increase the maximum specific growth rate by 16.93%. It decreased the protein and oil contents by 15.48% and 15.10%, as well as changed the composition of long-chain fatty acids. This paper provided a novel strategy for developing microalgal cell factories for future food production by perturbation on electron carriers of green algae.

Cells cultivated in bioreactors offer many possibilities for the production of novel and nutritious food products. Scientific and technological advances in cellular agriculture and processing technologies have allowed for the development of new techniques to utilize in vitro animal cells, plant cells, and microorganisms to mimic the organoleptic and nutritional properties of traditional foods as well as to potentially develop entirely new product classes. This review compiles and discusses the state-of-the-art cellular production and processing systems including 3D printing of customizable cell-cultivated food products. In addition to the technological state-of-the art, this article reviews the nutritional characteristics of cell-cultivated foods, introduces examples of new food products, and compiles economic characteristics and environmental impacts of each production system as assessed through technoeconomic analyses and lifecycle assessments. The factors influencing consumer acceptance of cell-cultivated foods are articulated and the potential implications of these new technologies on traditional agricultural industries and food chains are discussed. Lastly, future research and development trajectories are introduced with suggestions for continued development.