Comparison of taste characteristics between koji mold-ripened cheese and Camembert cheese using an electronic tongue system.

Koji mold, classified in the genus Aspergillus, is used to produce traditional Japanese fermented foods such as miso, soy sauce, and sake. In recent years, the application of koji mold to cheese ripening has attracted attention, and cheese surface-ripened with koji mold (koji cheese) has been studied. In this study, to evaluate the taste characteristics of koji cheese, an electronic tongue system was employed to measure the taste values of cheese samples ripened using 5 strains of koji mold in comparison with commercial Camembert cheese. All koji cheese samples exhibited lower sourness and greater bitterness, astringency, saltiness, and umami richness than the Camembert cheese samples. The intensity of each taste characteristic differed depending on the koji mold strain. These results indicate that koji cheese has a different taste value than conventional mold-ripened cheese. Furthermore, the results also indicate that various taste characteristics can be achieved by selecting different koji molds.

Volatile and soluble metabolite profiles in surface-ripened cheeses with Aspergillus oryzae and Aspergillus sojae.

To determine the impact of traditional koji molds on chemical characteristics of soft-type natural cheese, novel surface mold-ripened cheeses with Aspergillus oryzae and Aspergillus sojae were studied by non-targeted metabolite profiling. Comprehensive water-soluble and volatile metabolite profiles of koji cheese were evaluated among five Aspergillus strains and other mold-ripened cheeses. Time-course changes in the metabolite profiles and degrading enzyme activities were also compared with those of an industrial Penicillium candidum starter culture. Koji cheeses differed from Camembert, Brie, and blue cheeses in higher lactic acid, amino acid, and acetoin levels and lower methyl ketone and volatile fatty acid levels. Time-course analysis revealed the associations of rapid accumulations of glutamic, aspartic, and 3-methylbutanoic acids and 3-methylbutanal with higher proteolytic activity, and methyl ketone and fatty acid derivative suppressions with lower lipolytic activity. Ethyl butanoate, diacetyl, and malic acid also characterized koji cheeses as strain-dependent metabolites. This study highlighted the key compositional difference derived from cheese ripening with Aspergillus strains. The findings could help quality improvements of koji cheese product.

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria.

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

The survival impact of preoperative FOLFOX for resectable locally advanced rectal cancer: the R-NAC-01 study.

PURPOSE: In this follow-up of the R-NAC-01 study, we assessed the long-term oncological benefit of four courses of modified leucovorin, 5-fluorouracil (FU), and oxaliplatin (mFOLFOX6) chemotherapy before rectal surgery. METHODS: In this prospective, multicenter study (UMIN 000012559) involving 11 hospitals in Japan, patients with lower rectal cancer underwent four cycles of mFOLFOX6 chemotherapy and subsequent surgery within four to six weeks. The 3-year recurrence-free survival and local recurrence rates were then reported. RESULTS: Of 41 patients (36 males, 5 females; mean age: 60.8 years old) who received 4 courses of chemotherapy, 40 underwent total mesorectal excision, and 1 underwent total pelvic exenteration. R0 resection was achieved in 40 patients, but none showed a pathological complete response. Twenty-nine patients received adjuvant chemotherapy for an average of 4 months. The 3 year recurrence-free survival and local recurrence rates in patients undergoing curable resection were 72.8% and 8.5%, respectively. cStage III patients with adjuvant chemotherapy had a significantly higher 3 year recurrence-free survival than those without adjuvant chemotherapy (76.6 vs. 40.0%, log-rank p = 0.03). CONCLUSION: Four courses of mFOLFOX6 chemotherapy before surgery may be a promising treatment strategy for locally advanced rectal cancer. Adjuvant chemotherapy might be needed for cStage III patients, even after four courses of neoadjuvant mFOLFOX6.

Lactobacillus buchneri subsp. silagei subsp. nov., isolated from rice grain silage.

Two Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, catalase-negative bacteria, designated strains SG162T and NK01, were isolated from Japanese rice grain silage and total mixed ration silage, respectively. They were initially identified as Lactobacillus buchneri based on the 16S rRNA gene sequence similarities. However, the two strains were separated into a distinct clade from L. buchneri DSM 20057T (=JCM 1115T) through whole-genome sequence-based characterization, forming an infraspecific subgroup together with strains CD034 and S42, whose genomic sequences were available in the public sequence database. Strains within the subgroup shared 99.4-99.7 % average nucleotide identity (ANI) and 97.5-99.0 % digital DNA-DNA hybridization (dDDH) with each other, albeit 96.9-97.0 % ANI and 76.0-76.6 % dDDH against DSM 20057T. Strains SG162T and NK01 could utilize more substrates as sole carbon sources than DSM 20057T, potentially owing to the abundance of genes involved in carbon metabolism, especially the Entner-Doudoroff pathway. The inability of γ-aminobutyric acid (GABA) production was evidenced by the lack of glutamate decarboxylase and glutamate/GABA antiporter genes in the new subgroup strains. Strain SG162T grew at 10-45 °C (optimum, 30 °C), pH 3.5-8.0, and 0-8 % (w/v) NaCl. Its genomic DNA G+C content was 44.1 mol%. The predominant fatty acids were C16 : 0, C19 : 0 cyclo ω8c, and summed feature 8. On the basis of the polyphasic characterization findings, strains SG162T and NK01 represent a novel subspecies of L. buchneri, for which the name Lactobacillus buchneri subsp. silagei subsp. nov. is proposed. The type strain is SG162T (=JCM 32599T=DSM 107969T), and strains CD034 and S42 are also transferred to L. buchneri subsp. silagei.

Autoantigen specific IL-2 activated CD4+CD25+T regulatory cells inhibit induction of experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4+CD25+T cells both from sensitized hosts or from naïve hosts after ex-vivo activation by PNM and rIL-2. Transfer of naïve CD4+CD25+T cells has no effect on EAN, nor did naïve CD4+CD25+T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4+CD25+Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naïve CD4+CD25+T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential.

Alloactivation of Naïve CD4+CD8-CD25+T Regulatory Cells: Expression of CD8α Identifies Potent Suppressor Cells That Can Promote Transplant Tolerance Induction.

Therapy with alloantigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rß2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8-CD25+T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10-20% and CD8ß on <5% of these cells. These cells expressed ifngr and Il12rb2. CD8α+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8α+CD25+Treg consistent with the CD8α+ cells expressing IL-12R. In MLC, the CD8α+ fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4+CD8-CD25+T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α+ cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4+CD8-CD25+Foxp3+T cells was a marker of activated and potent Treg that included alloantigen-specific Treg.

Characterization of unique metabolites in γ-aminobutyric acid-rich cheese by metabolome analysis using liquid chromatography-mass spectrometry.

Fermented dairy products comprise many functional components. Our previous study using fermented milk showed that the γ-aminobutyric acid (GABA)-producing Lactococcus lactis 01-7 strain can produce unique metabolites such as antihypertensive peptides, whereas this study was designed to find the unique metabolites in GABA-rich cheese using the 01-7 strain. Metabolites between cheese ripening with the non-GABA-producing L. lactis 01-1 strain (control) and GABA-rich cheese ripening with a mixture of 01-1 and 01-7 strains were compared. GABA and ornithine were detected in GABA-rich cheese using an amino acid analyzer and citrate was detected in the control cheese using HPLC. Metabolome analysis using LC-MS showed that peptides with unknown function and those with antihypertensive activity were higher in the GABA-rich cheese than in the control cheese. Further analysis of the amount of the YLGY derivatives showed that the amount of YL in the GABA-rich cheese was lower than that in the control. PRACTICAL APPLICATIONS: Clarification of metabolites in cheese contributes to the improvement of cheese ripening, thereby providing consumers with unique cheese with good nutritional and functional characteristics. The use of the 01-7 strain as a cheese starter might provide a functional cheese with antihypertensive-, antioxidative-, and anxiolytic-like activities.

Preoperative FOLFOX in resectable locally advanced rectal cancer can be a safe and promising strategy: the R-NAC-01 study.

PURPOSE: The aim of this study was to assess the safety of rectal surgery after 5-fluorouracil-leucovorin-oxaliplatin chemotherapy (FOLFOX6). METHODS: This was a prospective, multicenter study in 11 Japanese hospitals. We included patients with rectal cancer who received 4 courses of modified FOLFOX6 (mFOLFOX6) before rectal surgery and examined the postoperative complication rate, the clinicopathological response, and the rate of chemotherapy-related adverse events (UMIN 000012559). RESULTS: The study population included 36 men and 5 women. The average age of the patients was 60.8 years and the average body mass index was 23.1 kg/m2. After 4 courses of chemotherapy, grade 2 peripheral nerve disorder and other grade 3 adverse events were seen in 3 patients each (7.3%). Twenty-eight (73.7%) and 8 (21.1%) patients underwent low anterior resection and abdominoperineal resection, respectively. The pelvic nerves were preserved in 35 patients. Surgical morbidity (grade ≥ 3) occurred in 4 patients (10.5%). Anastomotic leakage occurred after surgery in 2 patients (7.1%). No patients achieved pathologically complete remission. However, downstaging of the clinical stage and N stage was seen in 17 (41.5%) and 22 (53.7%) patients, respectively. CONCLUSIONS: Surgery after four courses of mFOLFOX6 chemotherapy can be a safe and promising strategy for patients with locally advanced rectal cancer.

Complete Genome Sequence of Carotenoid-Producing Enterococcus gilvus CR1, Isolated from Raw Cow's Milk.

Enterococcus gilvus CR1, isolated from raw cow's milk, can produce carotenoids. The complete genome sequence of this strain was determined using the PacBio RS II platform. The assembly was found to contain a circular chromosome, including carotenoid biosynthesis genes, and comprises 2,863,043 bp, with a G+C content of 41.86% and three plasmids.

Complete Genome Sequence of Lactobacillus paracasei EG9, a Strain Accelerating Free Amino Acid Production during Cheese Ripening.

Lactobacillus paracasei EG9 is a strain isolated from well-ripened cheese and accelerates free amino acid production during cheese ripening. Its complete genome sequence was determined using the PacBio RS II platform, revealing a single circular chromosome of 2,927,257 bp, a G+C content of 46.59%, and three plasmids.

Complete Genome Sequence of Lactobacillus plantarum Strain LQ80, Selected for Preparation of Fermented Liquid Feed for Pigs.

Lactobacillus plantarum LQ80 is a strain isolated from liquid feed for pigs. We determined the complete genome sequence of this strain using the PacBio RS II platform. LQ80 contained a single circular chromosome of 3,230,192 bp, with 44.66% G+C content and seven plasmids.

Complete Genome Sequence of Lactococcus lactis subsp. lactis G50 with Immunostimulating Activity, Isolated from Napier Grass.

Lactococcus lactis subsp. lactis G50 is a strain with immunostimulating activity, isolated from Napier grass (Pennisetum purpureum). We determined the complete genome sequence of this strain using the PacBio RS II platform. The single circular chromosome consists of 2,346,663 bp, with 35.03% G+C content and no plasmids.

Whole-transcriptome analysis of oxidative stress response genes in carotenoid-producing Enterococcus gilvus.

Whole-transcriptome analysis of aerobic stress response gene in Enterococcus gilvus was performed using RNA-sequencing to identify carotenoid-based stress response genes in lactic acid bacteria. The expression of gene responsible for pyruvate dehydrogenase complex synthesis was highly upregulated after aerobic treatment. In addition, the expression of transcriptional regulator spx and genes encoding UvrABC system protein was also upregulated.

Changes in Reactivity In Vitro of CD4+CD25+ and CD4+CD25- T Cell Subsets in Transplant Tolerance.

Transplant tolerance induced in adult animals is mediated by alloantigen-specific CD4+CD25+ T cells, yet in many models, proliferation of CD4+ T cells from hosts tolerant to specific-alloantigen in vitro is not impaired. To identify changes that may diagnose tolerance, changes in the patterns of proliferation of CD4+, CD4+CD25+, and CD4+CD25- T cells from DA rats tolerant to Piebald Virol Glaxo rat strain (PVG) cardiac allografts and from naïve DA rats were examined. Proliferation of CD4+ T cells from both naïve and tolerant hosts was similar to both PVG and Lewis stimulator cells. In mixed lymphocyte culture to PVG, proliferation of naïve CD4+CD25- T cells was greater than naïve CD4+ T cells. In contrast, proliferation of CD4+CD25- T cells from tolerant hosts to specific-donor PVG was not greater than CD4+ T cells, whereas their response to Lewis and self-DA was greater than CD4+ T cells. Paradoxically, CD4+CD25+ T cells from tolerant hosts did not proliferate to PVG, but did to Lewis, whereas naïve CD4+CD25+ T cells proliferate to both PVG and Lewis but not to self-DA. CD4+CD25+ T cells from tolerant, but not naïve hosts, expressed receptors for interferon (IFN)-γ and IL-5 and these cytokines promoted their proliferation to specific-alloantigen PVG but not to Lewis or self-DA. We identified several differences in the patterns of proliferation to specific-donor alloantigen between cells from tolerant and naïve hosts. Most relevant is that CD4+CD25+ T cells from tolerant hosts failed to proliferate or suppress to specific donor in the absence of either IFN-γ or IL-5. The proliferation to third-party and self of each cell population from tolerant and naïve hosts was similar and not affected by IFN-γ or IL-5. Our findings suggest CD4+CD25+ T cells that mediate transplant tolerance depend on IFN-γ or IL-5 from alloactivated Th1 and Th2 cells.

Cytokines affecting CD4+T regulatory cells in transplant tolerance. III. Interleukin-5 (IL-5) promotes survival of alloantigen-specific CD4+ T regulatory cells.

CD4+T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4+CD25+FOXP3+Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg. This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4+, especially CD4+CD25+T cells. CD4+T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4+T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4+CD25+T cells' response to rIL-5 and expression of IL-5Rα was also assessed. rIL-5 was sufficient to promote transplant tolerance mediating CD4+T cells' survival in culture with specific-donor alloantigen. Tolerant CD4+T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4+T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4+CD25+T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4+CD25+T cells expressed IL-5Rα. This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4+CD25+T cells that mediate transplant tolerance.

Cytokines affecting CD4+T regulatory cells in transplant tolerance. II. Interferon gamma (IFN-γ) promotes survival of alloantigen-specific CD4+T regulatory cells.

CD4+T cells that transfer alloantigen-specific transplant tolerance are short lived in culture unless stimulated with specific-donor alloantigen and lymphocyte derived cytokines. Here, we examined if IFN-γ maintained survival of tolerance transferring CD4+T cells. Alloantigen-specific transplant tolerance was induced in DA rats with heterotopic adult PVG heart allografts by a short course of immunosuppression and these grafts functioned for >100days with no further immunosuppression. In previous studies, we found the CD4+T cells from tolerant rats that transfer tolerance to an irradiated DA host grafted with a PVG heart, lose their tolerance transferring ability after 3days of culture, either with or without donor alloantigen, and effect rejection of specific-donor grafts. If cultures with specific-donor alloantigen are supplemented by supernatant from ConA activated lymphocytes the tolerance transferring cells survive, suggesting these cells depend on cytokines for their survival. In this study, we found addition of rIFN-γ to MLC with specific-donor alloantigen maintained the capacity of tolerant CD4+T cells to transfer alloantigen-specific tolerance and their ability to suppress PVG allograft rejection mediated by co-administered naïve CD4+T cells. IFN-γ suppressed the in vitro proliferation of tolerant CD4+T cells. Tolerant CD4+CD25+T cells did not proliferate in MLC to PVG stimulator cells with no cytokine added, but did when IFN-γ was present. IFN-γ did not alter proliferation of tolerant CD4+CD25+T cells to third-party Lewis. Tolerant CD4+CD25+T cells' expression of IFN-γ receptor (IFNGR) was maintained in culture when IFN-γ was present. This study suggested that IFN-γ maintained tolerance mediating alloantigen-specific CD4+CD25+T cells.

Classification and characterization of Japanese consumers' beef preferences by external preference mapping.

BACKGROUND: Over the past few decades, beef producers in Japan have improved marbling in their beef products. It was recently reported that marbling is not well correlated with palatability as rated by Japanese consumers. This study sought to identify the consumer segments in Japan that prefer sensory characteristics of beef other than high marbling. RESULTS: Three Wagyu beef, one Holstein beef and two lean imported beef longissimus samples were subjected to a descriptive sensory test, physicochemical analysis and a consumer (n = 307) preference test. According to consumer classification and external preference mapping, four consumer segments were identified as 'gradual high-fat likers', 'moderate-fat and distinctive taste likers', 'Wagyu likers' and 'distinctive texture likers'. Although the major trend of Japanese consumers' beef preference was 'marbling liking', 16.9% of the consumers preferred beef samples that had moderate marbling and distinctive taste. The consumers' attitudes expressed in a questionnaire survey were in good agreement with the preference for marbling among the 'moderate-fat and distinctive taste likers'. CONCLUSION: These results indicate that moderately marbled beef is a potent category in the Japanese beef market. © 2017 Society of Chemical Industry.

[A Case of Giant Malignant Phyllodes Tumor Associated with Severe Anemia].

A 72-year-old woman noted a mass in the left breast about 5 years ago, but she did not consult a medical institution. She was taken in the ambulance and hospitalized to our department due to severe anemia and malnutrition. A computed tomography( CT)scan indicated an 18×12 cm tumor in her left breast. A fiborsarcoma protuberance was suspected based on needle core biopsy results. Simple mastectomy was performed to control hemorrhage and infection. The resected tumor weighed 2.6 kg. The pathological diagnosis was a malignant phyllodes tumor. We report a patient with giant malignant phyllodes tumor associated with severe anemia.

First Complete Genome Sequence of the Skin-Improving Lactobacillus curvatus Strain FBA2, Isolated from Fermented Vegetables, Determined by PacBio Single-Molecule Real-Time Technology.

The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected.