Protein-glutaminase improves water-/oil-holding capacity and beany off-flavor profiles of plant-based meat analogs.

An unresolved challenge for plant-based meat analogs (PBMAs) is their lack of juiciness. Saturated fats significantly contribute to the juiciness of PBMAs, but there are concerns about the undesirable health effects related to saturated fats; thus, demand for their replacement with vegetable unsaturated oils has increased. Although many food additives are used to reduce the leakage of unsaturated oils, this solution cannot meet the clean-label requirements that have been trending in recent years. In this study, we aimed to develop better consumer-acceptable methods using protein-glutaminase (PG) to improve the juiciness of PBMA patties to meet clean-label trends. We found no significant difference between the visual surface of control and PG-treated textured vegetable proteins (TVPs). However, the microstructure of PG-treated TVP had a more rounded shape than that of the control TVP as observed under a scanning electron microscope. After grilling process, the PBMA patties composed of PG-treated TVP showed significantly higher liquid-holding capacities (a juiciness indicator) than the control patties. This suggested that PG treatment could potentially produce PBMA patties with increased juiciness. Interestingly, after the PG-treated TVP underwent the wash process, we found that PG treatment of TVP easily reduced the various beany off-flavor compounds by 58-85%. Moreover, the results of the in vitro protein digestion test showed that the amounts of free amino nitrogen released from PBMA patties composed of PG-treated TVP were 1.5- and 1.7-fold higher than those from control patties in the gastric and intestinal phases, respectively. These findings indicate that PG treatment of TVP could enhance the physical, sensory, and nutritional properties of PBMA patties and meet the clean-label requirements.

Decolorization and detoxication of plant-based proteins using hydrogen peroxide and catalase.

The gap between the current supply of meat and its predicted future demand is widening, increasing the need to produce plant-based meat analogs. Despite ongoing technical developments, one of the unresolved challenges of plant-based meat analogs is to safely and effectively decolor plant proteins that originally exhibit yellow-brown or strong brown color. This study aimed to develop an effective and safe decoloring system for soy-based protein products using food-grade hydrogen peroxide and catalase. First, soy-based protein isolate (PI) and textured vegetable protein (TVP) were treated with hydrogen peroxide, and then the residual hydrogen peroxide was degraded using catalase. This process caused notable decolorization of PI and TVP, and residual hydrogen peroxide was not detected in these products. These findings indicate that this process could safely and effectively decolorize soy-based proteins. Interestingly, this decoloring process enhanced the solubility, water- and oil-holding capacities, foaming capacity, and emulsifying stability of decolored soy-based PI. Additionally, cooking loss and juiciness of decolored TVP-based foods were improved compared to those of non-treated foods. These findings indicate that the decoloring process also enhances the physical properties of soy-based protein products.

Cyclodextrins produced by cyclodextrin glucanotransferase mask beany off-flavors in plant-based meat analogs.

The widening gap between the supply and demand for meat products has increased the need to produce plant-based meat analogs as protein sources. Meat analogs are principally composed of soy-based textured vegetable proteins. Despite ongoing technical developments, one of the unresolved challenges for plant-based meat analogs is the off-flavor from soy, which limits their consumer acceptability. Among the various methods developed for overcoming this challenge, masking the beany flavors with cyclodextrins (CDs) is an attractive, cost-effective, and safe strategy. However, the current established CD treatment method does not meet the requirement for a clean-label. This study aimed to develop more acceptable off-flavor-masking technologies for plant-based patties for modern clean-label preferences using enzymatic methods. We used the cyclodextrin glucanotransferase (CGT), "Amano," as a commercially available food-grade CGT. The CGT-catalyzed reaction in plant-based patties yielded 17.1 g/L CD. As CGT could yield sufficient CD in the patties, we investigated whether CDs produced by CGT could mask the off-flavors released from the plant-based patties. The CGT-treated patties had significantly lower volatilization amounts of the known beany off-flavor-generating compounds compared to the non-treated patties. Moreover, CGT treatment improved the texture of the patties and increased their water- and oil-holding capacity. As CGT is rendered inactive after cooking, it would not be considered an additive. These findings indicated that CDs produced by the CGT reaction could effectively mask off-flavors of meat analogs and improve their physical properties while meeting clean-label requirements.

Neobacillus kokaensis sp. nov., isolated from soil.

A Gram-stain-variable, motile, aerobic, spore-forming, rod-shaped bacterium, designated as strain LOB 377T, was isolated from soil sampled in Koka County (now Konan City), in Shiga Prefecture, Japan. To determine its taxonomic position, the bacterium was evaluated by a polyphasic approach based on genomic, phenotypic and chemotaxonomic tests. From phylogenetic analysis based on 16S rRNA gene sequences, strain LOB 377T (LC570960) was revealed to have the highest similarity to the type strain of Neobacillus mesonae FJAT-13985T (9.1 %). The average nucleotide identity and digital DNA-DNA hybridization values between both strains based on whole genome sequences were 84.1 and 42.8 %, respectively, which were below the recommended thresholds. The strain grew at 15-45 °C (optimum, 25-37 °C), at pH 5.5-9.5 (optimum, pH 6.5-8.5) and with 1.0-2.0 % (w/v) NaCl. Cell-wall peptidoglycan of the strain contained meso-diaminopimelic acid. The major menaquinone was menaquinone-7. Predominant fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C14 : 0 and C16 : 1 ω11c. The polar lipids were phosphatidylglycerol, unidentified phospholipid and unidentified aminophospholipid. The DNA G+C content was 40.5 mol%. According to the genomic, phenotypic and chemotaxonomic results, strain LOB 377T represents a novel species in the genus Neobacillus, for which the name Neobacillus kokaensis sp. nov. is proposed. The type strain is LOB 377T (=ATCC 31382T=NBRC 114637T=DSM 113418T).

Synergistic effects of laccase and pectin on the color changes and functional properties of meat analogs containing beet red pigment.

The widening gap between current supply of meat and its future demand has increased the need to produce plant-based meat analogs. Despite ongoing technical developments, one of the unresolved challenges of plant-based meat analogs is to safely and effectively imitate the appearance of raw and cooked animal-based meat, especially the color. This study aimed to develop a more effective and safe browning system for beet red (BR) in plant-based meat analog patties using laccase (LC) and sugar beet pectin (SBP). First, we investigated the synergistic effects of SBP and LC on BR decolorization of meat analog patties. We discovered that the red tones of LC-treated patties containing BR and SBP were remarkably browned after grilling, compared to patties that did not contain SBP. Notably, this color change by LC + SBP was similar to that of beef patties. Additionally, the hardness of LC-treated meat analog patties containing BR was higher than those that did not contain BR. Interestingly, the presence of SBP and LC enhanced the browning reaction and functional properties of meat analogs containing BR. This is the first report on a browning system for meat analogs containing BR using enzymatic methods to the best of our knowledge.

Improved functional properties of meat analogs by laccase catalyzed protein and pectin crosslinks.

The gap between the current supply and future demand of meat has increased the need to produce plant-based meat analogs. Methylcellulose (MC) is used in most commercial products. Consumers and manufacturers require the development of other novel binding systems, as MC is not chemical-free. We aimed to develop a novel chemical-free binding system for meat analogs. First, we found that laccase (LC) synergistically crosslinks proteins and sugar beet pectin (SBP). To investigate the ability of these SBP-protein crosslinks, textured vegetable protein (TVP) was used. The presence of LC and SBP improved the moldability and binding ability of patties, regardless of the type, shape, and size of TVPs. The hardness of LC-treated patties with SBP reached 32.2 N, which was 1.7- and 7.9-fold higher than that of patties with MC and transglutaminase-treated patties. Additionally, the cooking loss and water/oil-holding capacity of LC-treated patties with SBP improved by up to 8.9-9.4% and 5.8-11.3%, compared with patties with MC. Moreover, after gastrointestinal digestion, free amino nitrogen released from LC-treated patties with SBP was 2.3-fold higher than that released from patties with MC. This is the first study to report protein-SBP crosslinks by LC as chemical-free novel binding systems for meat analogs.

Feeding Aspergillus protease preparation combined with adequate protein diet to rats increases levels of cecum gut-protective amino acids, partially linked to Bifidobacterium and Lactobacillus.

Our recent study indicated that dietary Aspergillus oryzae-derived protease preparation (AP), through its enzymatic activity, exerted a bifidogenic effect in rats. We hypothesized that dietary AP links to protein degradation and subsequently elevates gut-protective amino acids (AAs) in rats fed adequate protein diet. In this study, dietary AP markedly increased the relative abundance of Bifidobacterium and Lactobacillus and the levels of free threonine, alanine, proline, taurine, ornithine, phenylalanine, cystine, and γ-aminobutyric acid in the cecum contents of rats fed with an adequate protein diet, but not in those fed with a low-protein diet. The elevated AAs, except ornithine and phenylalanine, potentially have gut-related health benefits. Some of the AP-modulated free AAs appeared to be associated with the relative abundance of Bifidobacterium and Lactobacillus. Thus, AP combined with adequate protein diet is likely to increase the levels of cecum beneficial free AAs, which is partially associated with the relative abundance of the probiotics.

Bayesian penalized-likelihood reconstruction algorithm suppresses edge artifacts in PET reconstruction based on point-spread-function.

PURPOSE: The Bayesian penalized-likelihood reconstruction algorithm (BPL), Q.Clear, uses relative difference penalty as a regularization function to control image noise and the degree of edge-preservation in PET images. The present study aimed to determine the effects of suppression on edge artifacts due to point-spread-function (PSF) correction using a Q.Clear. METHODS: Spheres of a cylindrical phantom contained a background of 5.3 kBq/mL of [18F]FDG and sphere-to-background ratios (SBR) of 16, 8, 4 and 2. The background also contained water and spheres containing 21.2 kBq/mL of [18F]FDG as non-background. All data were acquired using a Discovery PET/CT 710 and were reconstructed using three-dimensional ordered-subset expectation maximization with time-of-flight (TOF) and PSF correction (3D-OSEM), and Q.Clear with TOF (BPL). We investigated ß-values of 200-800 using BPL. The PET images were analyzed using visual assessment and profile curves, edge variability and contrast recovery coefficients were measured. RESULTS: The 38- and 27-mm spheres were surrounded by higher radioactivity concentration when reconstructed with 3D-OSEM as opposed to BPL, which suppressed edge artifacts. Images of 10-mm spheres had sharper overshoot at high SBR and non-background when reconstructed with BPL. Although contrast recovery coefficients of 10-mm spheres in BPL decreased as a function of increasing ß, higher penalty parameter decreased the overshoot. CONCLUSIONS: BPL is a feasible method for the suppression of edge artifacts of PSF correction, although this depends on SBR and sphere size. Overshoot associated with BPL caused overestimation in small spheres at high SBR. Higher penalty parameter in BPL can suppress overshoot more effectively.

Consumption of an acid protease derived from Aspergillus oryzae causes bifidogenic effect in rats.

A marked elevation in the abundance of Bifidobacterium was found in the cecum of rats that were fed a high-fat diet supplemented with an Amano protease preparation (derived from Aspergillus oryzae). The protease preparation contains several digestive enzymes, including acid protease (AcP), alkaline protease, and amylase. We hypothesized that the elevation in the abundance of Bifidobacterium by Amano protease preparation is associated with the digestive enzymes involved in the protease preparation. To test this hypothesis, this study was conducted to investigate if such bifidogenic effect is because of the AcP. Rats were fed a high-fat diet containing purified AcP obtained from the Amano protease preparation for 2 weeks. The numbers of Bifidobacterium in the cecum and feces of rats were markedly elevated by the dietary supplementation of 1 g/kg Amano protease. Bifidobacterium numbers were unaffected by supplementation with purified AcP (0.096 g/kg) at the level equivalent to the AcP amount found in the 1-g/kg Amano protease diet. Bifidobacterium numbers in the cecum and feces, and lactate levels in the cecum were significantly (P<.05) elevated when rats were fed a diet containing 0.384 g/kg AcP (4-fold higher amount of AcP than that used in the 1-g/kg Amano protease diet). Thus, the bifidogenic effect of 1 g/kg Amano protease diet could not be explained by the AcP. However, intriguingly, supplemental AcP was found to cause a significant bifidogenic effect at the dose that is 4-fold higher than that used in the 1-g/kg Amano protease diet.

Nitrogen Dissociation via Reaction with Lithium Alloys.

Lithium alloys are synthesized by reactions between lithium metal and group 14 elements, such as carbon, silicon, germanium, and tin. The nitrogenation and denitrogenation properties are investigated by thermal and structural analyses. All alloys dissociate the nitrogen triple bond of gaseous molecules to form atomic state as nitrides below 500 °C, which is lower than those required for conventional thermochemical and catalytic processes on nitride syntheses. For all alloys except for germanium, it is indicated that nanosized lithium nitride is formed as the product. The denitrogenation (nitrogen desorption) reaction by lithium nitride and metals, which is an ideal opposite reaction of nitrogenation, occurs by heating up to 600 °C to form lithium alloys. Among them, the lithium-tin alloy is a potential material to control the dissociation and recombination of nitrogen below 500 °C by the reversible reaction with the largest amount of utilizable lithium in the alloy phase. The nitrogenation and denitrogenation reactions of the lithium alloys at lower temperature are realized by the high reactivity with nitrogen and mobility of lithium. The above reactions based on lithium alloys are adapted to the ammonia synthesis. As a result, ammonia can be synthesized below 500 °C under 0.5 MPa of pressure. Therefore, the reaction using lithium alloys is recognized as a pseudocatalyst for the ammonia synthesis.

The quest for industrial enzymes from microorganisms.

Satoshi Omura, Professor Emeritus at Kitasato University, was awarded the Nobel Prize for his discovery of a substance of tremendous value to mankind from a microorganism. As a researcher who regularly deals with enzymes produced by microorganisms and a person engaged in microorganism-based business, Professor Omura's Nobel Prize fills me with great pride and joy. It is perhaps not surprising that this Nobel Prize-winning research would emerge from Asia, specifically Japan, where people live in harmony with nature rather than try to conquer it. At Amano Enzyme Inc., we devote ourselves to searching for novel enzymes from microorganisms. While incorporating my own experiences, I will recount the stories of a few discoveries of valuable enzyme-producing microbes in soil and bacterial strain libraries. I will also briefly introduce microbial strain library construction as a tool for facilitating the identification of the desired producing bacteria.

Crystal structure of ß-galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants.

UNLABELLED: ß-Galactosidase (EC 3.2.1.23) from Bacillus circulans ATCC 31382, designated BgaD, exhibits high transglycosylation activity to produce galacto-oligosaccharides. BgaD has been speculated to have a multiple domain architecture including a F5/8-type C domain or a discoidin domain in the C-terminal peptide region from amino acid sequence analysis. Here, we solved the first crystal structure of the C-terminal deletion mutant BgaD-D, consisting of sugar binding, Glyco_hydro, catalytic and bacterial Ig-like domains, at 2.5 Å. In the asymmetric unit, two molecules of BgaD-D were identified and the value of VM was estimated to be 5.0 Å(3) · Da(-1). It has been speculated that BgaD-D consists of four domains. From the structural analysis, however, we clarified that BgaD-D consists of five domains. We identified a new domain structure comprised of ß-sheets in BgaD. The catalytic domain exhibits a TIM barrel structure with a small pocket suited for accommodating the disaccharides. Detailed structural information for the amino acid residues related to activity and substrate specificity was clarified in the catalytic domain. Furthermore, using the structural information, we successfully constructed some thermostable mutants via protein engineering method. DATABASE: Coordinates for the BgaD-D structure have been deposited in the Protein Data Bank under accession code 4YPJ.

Catalytic modification in dehydrogenation properties of KSiH3.

A number of known catalysts, which have been proven to be very effective for several hydrogen species, were studied in order to determine their effects on the hydrogen ab/desorption properties of KSiH3. Among all the catalysts used in this work, mesoporous Nb2O5 is found to be quite effective, with a reduction in activation energy from 142 kJ mol(-1) for pristine KSi to 63 kJ mol(-1) for mesoporous-Nb2O5-added KSi, thus allowing desorption to start at 100-120 °C. Any disproportionation is not observed in the controlled hydrogenation process. The mechanism for this improvement is also proposed in detail. The kinetic modifications on the ab/desorption properties of KSiH3 provide an alternative to the well-known family of heavy BCC alloys which are capable of working in the same temperature range but with a lower gravimetric hydrogen content, almost half of the KSi system.

The discoidin domain of Bacillus circulans ß-galactosidase plays an essential role in repressing galactooligosaccharide production.

The recently cloned ß-galactosidase from Bacillus circulans ATCC 31382, designated BgaD, contains a multiple domain architecture including a F5/8 type C domain or a discoidin (DS) domain in the C-terminal peptide region. Here we report that the DS domain plays an essential role in repressing the production of galactooligosaccharides (GOSs). We prepared deletion mutants and point-mutated forms of rBgaD-A (deletion of the BgaD signal peptide) to compare their reaction behaviors. The yields of GOSs for all of the point-mutated forms as well as the deletion mutants of rBgaD-As increased as compared to rBgaD-A. In particular, W1540A mutant BgaD-A (rBgaD-A_W1540A) produced much more GOSs than rBgaD-A. Surface plasmon resonance experiments indicated that both the wild-type and the W1540A mutant DS domains showed high affinity for galactosyllactose. rBgaD-A, which has a wild-type DS domain, showed high hydrolytic activity toward galactosyllactose, while the hydrolytic activities of rBgaD-D, without a DS domain, and rBgaD-A_W1540A, with a mutant DS domain were extremely low. The findings obtained in this study indicate that the wild-type DS domain of rBgaD-A has a function that aids galactosyllactose molecules to be properly oriented within the active site, so that they can be hydrolyzed efficiently to produce galactose/glucose by inhibiting the accumulation of GOSs.

Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex.

Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 Å resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 Å resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed.

Cloning and expression of a ß-galactosidase gene of Bacillus circulans.

A gene of ß-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(®). Using the cloned gene, recombinant ß-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.

Causes of the production of multiple forms of ß-galactosidase by Bacillus circulans.

The presence of multiple types of ß-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four ß-galactosidases, ß-Gal-A, ß-Gal-B, ß-Gal-C, and ß-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. ß-Gal-B, ß-Gal-C, and ß-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while ß-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for ß-Gal-A, showed biphasic behavior. ß-Gal-A was truncated to yield multiple ß-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of ß-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for ß-Gal-D.

X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 A resolution using a twinned crystal.

Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6(3). Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33-2.3 A. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD.