Nutritional Evaluation of Milk-, Plant-, and Insect-Based Protein Materials by Protein Digestibility Using the INFOGEST Digestion Method.

The INFOGEST method is a valuable tool for understanding and monitoring food digestion as an alternative to in vivo assays. However, few studies have compared animal and alternative protein sources in terms of protein quality using the INFOGEST method. This study aimed to evaluate the protein quality of milk-, plant-, and insect-based protein materials by in vitro protein digestibility and in vitro digestible indispensable amino acid score (DIAAS), following the INFOGEST method. Milk-based protein materials had the highest protein digestibility (86.1-90.8%), followed by soy (85.1%) and wheat (82.3%). These materials had significantly higher protein digestibility compared with zein (65.1%), cricket (63.6%), and mealworm (69.5%). Additionally, the mean values of in vitro DIAAS of milk-based protein materials (105.0-137.5) were higher than those of plant- and insect-based protein materials (1.9-91.0). Milk-based protein materials have higher protein quality than plant- and insect-based protein materials by the nutritional evaluation following the INFOGEST digestion method.

Plasma Albumin Redox State Is Responsive to the Amino Acid Balance of Dietary Proteins in Rats Fed a Low Protein Diet.

We recently reported that plasma albumin redox state, which correlates with albumin synthesis rate, could be associated with the quality of dietary protein. Aiming to elucidate the association between them, plasma albumin redox state was investigated in rats fed various kinds of AIN-93G-based low protein diets. Plasma albumin redox state was shifted to a more oxidized state in rats fed 3% casein (CN) diet than those fed 3% whey protein or 3% wheat gluten diet, while supplementing 3% CN diet with cystine reversed it to a more reduced state, indicating that cystine would complement the shortage of cysteine in CN, thereby increasing albumin synthesis rate. Supplementation with glutathione, a cysteine-containing antioxidative tripeptide, normalized hepatic glutathione redox state modulated by ingestion of 3% CN diet, but it only reversed the oxidized shift of plasma albumin redox state to an extent similar to cystine alone or the constituting amino acid mixture of glutathione (i.e., glutamic acid, cystine, and glycine), indicating that glutathione would primarily serve as a source of cysteine rather than exert its antioxidative activity. Plasma albumin would thus be influenced by amino acid balance in dietary proteins, and it could be useful as a biomarker that contributes to prevention of protein under-nutriton, caused by not only insufficient protein intake but also ingestion of poor-quality protein.

The Combination of Bifidobacterium breve and Three Prebiotic Oligosaccharides Modifies Gut Immune and Endocrine Functions in Neonatal Mice.

BACKGROUND: Several types of oligosaccharides are used in infant formula to improve the gut microbiota of formula-fed infants. We previously reported that a combination of 3 oligosaccharides (lactulose, raffinose, and galacto-oligosaccharides; LRG) and Bifidobacterium breve effectively increased B. breve numbers, acetate, and the expression of several immune- and gut hormone-related mRNAs in neonatal mice gut. OBJECTIVE: We investigated whether changes in neonatal gut microbiota alter gut immune and endocrine development. METHODS: We first compared postnatal day (PD) 14 with PD21 in C57BL/6J male mouse pups to identify the physiologic immune and endocrine changes during development. In a separate study, we administered phosphate-buffered saline (control group; CON), B. breve M-16V (M-16V), or M-16V + LRG to male mouse pups from PD6 to PD13, and analyzed the gut microbiota and immune and endocrine parameters on PD14 to evaluate whether M-16V + LRG accelerates gut immune and endocrine development. RESULTS: The proportion of regulatory T (Treg) cells in the CD4+ cells of large intestinal lamina propria lymphocytes (LPLs) was significantly increased (63% higher) at PD21 compared with PD14. The serum glucagon-like peptide (GLP)-1 tended to be lower (P = 0.0515) and that of GLP-2 was significantly lower (58% lower) at PD21 than at PD14. M-16V + LRG significantly increased the Treg proportion in large intestinal LPL CD4+ cells (20% and 29% higher compared with CON and M-16V, respectively) at PD14. M-16V + LRG also caused significant changes in expression of large intestinal mRNAs that are consistent with developmental progression, and increased serum concentrations of GLP-1 (207% and 311% higher compared with CON and M-16V, respectively) and GLP-2 (57% and 97% higher compared with CON and M-16V, respectively) at PD14. CONCLUSIONS: Neonatal administration of M-16V + LRG alters the gut microbiota and enhances gut immune and endocrine development in suckling mice.

Elephant's breast milk contains large amounts of glucosamine.

Hand-reared elephant calves that are nursed with milk substitutes sometimes suffer bone fractures, probably due to problems associated with nutrition, exercise, sunshine levels and/or genetic factors. As we were expecting the birth of an Asian elephant (Elephas maximus), we analyzed elephant's breast milk to improve the milk substitutes for elephant calves. Although there were few nutritional differences between conventional substitutes and elephant's breast milk, we found a large unknown peak in the breast milk during high-performance liquid chromatography-based amino acid analysis and determined that it was glucosamine (GlcN) using liquid chromatography/mass spectrometry. We detected the following GlcN concentrations [mean ± SD] (mg/100 g) in milk hydrolysates produced by treating samples with 6M HCl for 24 hr at 110°C: four elephant's breast milk samples: 516 ± 42, three cow's milk mixtures: 4.0 ± 2.2, three mare's milk samples: 12 ± 1.2 and two human milk samples: 38. The GlcN content of the elephant's milk was 128, 43 and 14 times greater than those of the cow's, mare's and human milk, respectively. Then, we examined the degradation of GlcN during 0-24 hr hydrolyzation with HCl. We estimated that elephant's milk contains >880 mg/100 g GlcN, which is similar to the levels of major amino acids in elephant's milk. We concluded that a novel GlcN-containing milk substitute should be developed for elephant calves. The efficacy of GlcN supplements is disputed, and free GlcN is rare in bodily fluids; thus, the optimal molecular form of GlcN requires a further study.

Combinational effects of prebiotic oligosaccharides on bifidobacterial growth and host gene expression in a simplified mixed culture model and neonatal mice.

It is important to provide formula-fed infants with a bifidobacteria-enriched gut microbiota similar to those of breastfed infants to ensure intestinal health. Prebiotics, such as certain oligosaccharides, are a useful solution to this problem, but the combinational benefits of these oligosaccharides have not been evaluated. This study investigated the benefits of oligosaccharide combinations and screened for an optimal combination of oligosaccharides to promote healthy gut microbiota of formula-fed infants. In vitro and in vivo experiments were performed to assess the bifidogenic effects of lactulose (LAC) alone and LAC combined with raffinose (RAF) and/or galacto-oligosaccharide (GOS), using a mixed culture model and neonatal mice orally administered with these oligosaccharides and Bifidobacterium breve. In the in vitro culture model, the combination of the three oligosaccharides (LAC-RAF-GOS) significantly increased cell numbers of B. breve and Bifidobacterium longum (P<0·05) compared with either LAC alone or the combination of two oligosaccharides, and resulted in the production of SCFA under anaerobic conditions. In the in vivo experiment, the LAC-RAF-GOS combination significantly increased cell numbers of B. breve and Bacteroidetes in the large intestinal content (P<0·05) and increased acetate concentrations in the caecal content and serum of neonatal mice. Genes related to metabolism and immune responses were differentially expressed in the liver and large intestine of mice administered with LAC-RAF-GOS. These results indicate a synergistic effect of the LAC-RAF-GOS combination on the growth of bifidobacteria and reveal possible benefits of this combination to the gut microbiota and health of infants.

Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages.

We reported previously that microRNA (miRNA) are present in whey fractions of human breast milk, bovine milk, and rat milk. Moreover, we also confirmed that so many mRNA species are present in rat milk whey. These RNA were resistant to acidic conditions and to RNase, but were degraded by detergent. Thus, these RNA are likely packaged in membrane vesicles such as exosomes. However, functional extracellular circulating RNA in bodily fluids, such as blood miRNA, are present in various forms. In the current study, we used bovine raw milk and total RNA purified from exosomes (prepared by ultracentrifugation) and ultracentrifuged supernatants, and analyzed them using miRNA and mRNA microarrays to clarify which miRNA and mRNA species are present in exosomes, and which species exist in other forms. Microarray analyses revealed that most mRNA in milk whey were present in exosomes, whereas miRNA in milk whey were present in supernatant as well as exosomes. The RNA in exosomes might exert functional effects because of their stability. Therefore, we also investigated whether bovine milk-derived exosomes could affect human cells using THP-1 cells. Flow cytometry and fluorescent microscopy studies revealed that bovine milk exosomes were incorporated into differentiated THP-1 cells. These results suggest that bovine milk exosomes might have effects in human cells by containing RNA.

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation.

In the typical isoprenoid-biosynthesis pathway, condensation of the universal C(5)-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C(10)-C(20)). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

Crystallization and preliminary X-ray crystallographic study of a methyltransferase involved in 2-methylisoborneol biosynthesis in Streptomyces lasaliensis.

The biosynthetic pathway of the off-flavour terpenoid alcohol 2-methylisoborneol (2-MIB) requires geranyl pyrophosphate methyltransferase (GPPMT) to methylate GPP before the cyclization reaction. GPPMT is the first example of an S-adenosyl-L-methionine-dependent methyltransferase that acts on general intermediates such as geranyl pyrophosphate and farnesyl pyrophosphate in isoprenoid biosynthetic pathways. In this study, recombinant GPPMT was overproduced, purified and crystallized in the absence and presence of cofactor, cofactor analogue and substrate. Well diffracting crystals of apo GPPMT containing one molecule in the asymmetric unit were obtained and the structure of this form was solved by the molecular-replacement method. Two crystal forms of the tertiary complex with GPP and sinefungin were also obtained. Structure analysis of these crystals is currently under way in order to understand the enzyme reaction mechanism.

Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol.

To identify the genes for biosynthesis of the off-flavor terpenoid alcohol, 2-methylisoborneol (2-MIB), the key genes encoding monoterpene cyclase were located in bacterial genome databases by using a combination of hidden Markov models, protein-family search, and the sequence alignment of their gene products. Predicted terpene cyclases were classified into three groups: sesquiterpene, diterpene, and other terpene cyclases. Genes of the terpene cyclase group that form an operon with a gene encoding S-adenosyl-l-methionine (SAM)-dependent methyltransferase were found in genome data of seven microorganisms belonging to actinomycetes, Streptomyces ambofaciens ISP5053, Streptomyces coelicolor A3(2), Streptomyces griseus IFO13350, Streptomyces lasaliensis NRRL3382R, Streptomyces scabies 87.22, Saccharopolyspora erythraea NRRL2338, and Micromonospora olivasterospora KY11048. Among six microorganisms tested, S. ambofaciens, S. coelicolor A3(2), S. griseus, and S. lasaliensis produced 2-MIB but M. olivasterospora produced 2-methylenebornane (2-MB) instead. The regions containing monoterpene cyclase and methyltransferase genes were amplified by PCR from S. ambofaciens, S. lasaliensis, and Saccharopolyspora erythraea, respectively, and their genes were heterologously expressed in Streptomyces avermitilis, which was naturally deficient of 2-MIB biosynthesis by insertion and deletion. All exoconjugants of S. avermitilis produced 2-MIB. Full-length recombinant proteins, monoterpene cyclase and methyltransferase of S. lasaliensis were expressed at high level in Escherichia coli. The recombinant methyltransferase catalyzed methylation at the C2 position of geranyl diphosphate (GPP) in the presence of SAM. 2-MIB was generated by incubation with GPP, SAM, recombinant methyltransferase, and terpene cyclase. We concluded that the biosynthetic pathway involves the methylation of GPP by GPP methyltransferase and its subsequent cyclization by monoterpene cyclase to 2-MIB.