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.

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.

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.

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.

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.

Metabolome analysis of milk fermented by γ-aminobutyric acid-producing Lactococcus lactis.

γ-Aminobutyric acid (GABA) is one of the most important functional components in fermented foods because of its physiological functions, such as neurotransmission and antihypertensive activities. However, little is known about components other than GABA in GABA-rich fermented foods. A metabolomic approach offers an opportunity to discover bioactive and flavor components in fermented food. To find specific components in milk fermented with GABA-producing Lactococcus lactis 01-7, we compared the components found in GABA-rich fermented milk with those found in control milk fermented without GABA production using capillary electrophoresis time-of-flight mass spectrometry. A principal component analysis score plot showed a clear differentiation between the control milk fermented with L. lactis 01-1, which does not produce GABA, and GABA-rich milk fermented with a combination of L. lactis strains 01-1 and 01-7. As expected, the amount of GABA in GABA-rich fermented milk was much higher (1,216-fold) than that of the control milk. Interestingly, the amount of Orn was also much higher (27-fold) than that of the control milk. Peptide analysis showed that levels of 6 putative angiotensin-I-converting enzyme (ACE)-inhibitory peptides were also higher in the GABA-rich fermented milk. Furthermore, ACE-inhibitory activity of GABA-rich fermented milk tended to be higher than that of the control milk. These results indicate that the GABA-producing strain 01-7 provides fermented milk with other functional components in addition to GABA.

IL-5 promotes induction of antigen-specific CD4+CD25+ T regulatory cells that suppress autoimmunity.

Immune responses to foreign and self-Ags can be controlled by regulatory T cells (Tregs) expressing CD4 and IL-2Rα chain (CD25). Defects in Tregs lead to autoimmunity, whereas induction of Ag-specific CD4+CD25+ Tregs restores tolerance. Ag-specific CD4+CD25+ FOXP3+Tregs activated by the T helper type 2 (Th2) cytokine, IL-4, and specific alloantigen promote allograft tolerance. These Tregs expressed the specific IL-5Rα and in the presence of IL-5 proliferate to specific but not third-party Ag. These findings suggest that recombinant IL-5 (rIL-5) therapy may promote Ag-specific Tregs to mediate tolerance. This study showed normal CD4+CD25+ Tregs cultured with IL-4 and an autoantigen expressed Il-5rα. Treatment of experimental autoimmune neuritis with rIL-5 markedly reduced clinical paralysis, weight loss, demyelination, and infiltration of CD4+ (Th1 and Th17) CD8+ T cells and macrophages in nerves. Clinical improvement was associated with expansion of CD4+CD25+FOXP3+ Tregs that expressed Il-5rα and proliferated only to specific autoantigen that was enhanced by rIL-5. Depletion of CD25+ Tregs or blocking of IL-4 abolished the benefits of rIL-5. Thus, rIL-5 promoted Ag-specific Tregs, activated by autoantigen and IL-4, to control autoimmunity. These findings may explain how Th2 responses, especially to parasitic infestation, induce immune tolerance. rIL-5 therapy may be able to induce Ag-specific tolerance in autoimmunity.

Plasminogen activation by lactic acid bacteria.

Plasminogen was incubated with lactic acid bacteria and the plasmin activity in the mixture was measured. Three of 15 strains tested revealed significant plasminogen activation ability. Lactococcus lactis subsp. lactis biovar diacetylactis NIAI C59 showed the highest activity. The strain activated not only human plasminogen but also bovine plasminogen. The activity demonstrated a high level of thermal stability within a range of pH 3.0-9.0. The plasminogen activator activity in strain C59 increased after 15 h of cultivation, and reached a plateau after 21 h. A remarkable amount of activity was transferred to the solution when C59 cells were incubated in buffer solutions at pH 9.0 and above.

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.

CD4+CD25+ T cells alloactivated ex vivo by IL-2 or IL-4 become potent alloantigen-specific inhibitors of rejection with different phenotypes, suggesting separate pathways of activation by Th1 and Th2 responses.

CD4(+)CD25(+)Foxp3(+) T cells are regulatory/suppressor cells (Tregs) that include non-antigen (Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naive CD4(+)CD25(+) Treg develop into specific Tregs is unknown. Here, we generated adaptive Tregs by culture of naive CD4(+)CD25(+)Foxp3(+) T cells with allo-Ag and either interleukin-2 (IL-2) or IL-4. Within days, IL-2 enhanced interferon-gamma receptor (Ifngammar) and Il-5 mRNA and IL-4 induced a reciprocal profile with de novo IL-5Ralpha and increased IFN-gamma mRNA expression. Both IL-2- and IL-4-alloactivated CD4(+)CD25(+) Tregs within 3 to 4 days of culture had enhanced capacity to induce tolerance to specific donor but not to third-party cardiac allografts. These hosts became tolerant as allografts functioned more than 250 days, with a physiologic ratio of less than 10% CD4(+)CD25(+)Foxp3(+) T cells in the CD4(+) population. CD4(+)CD25(+) T cells from tolerant hosts given IL-2-cultured cells had increased Il-5 and Ifngammar mRNA. Those from hosts given IL-4-cultured cells had enhanced IL-5Ralpha mRNA expression and IL-5 enhanced their proliferation to donor but not third-party allo-Ag. Thus, IL-2 and IL-4 activated allo-Ag-specific Tregs with distinct phenotypes that were retained in vivo. These findings suggested that T-helper 1 (Th1) and Th2 responses activate 2 pathways of adaptive Ag-specific Tregs that mediate tolerance. We propose they be known as T-suppressor 1 (Ts1) and Ts2 cells.

Molecular-based analysis of changes in indigenous milk microflora during the grazing period.

Variations in milk microflora caused by changes in the cow feeding environment (from inside to outside grazing) were analyzed using a molecular-based approach comprising denaturing gradient gel electrophoresis and real-time PCR. After 8 d of outside grazing, changes in milk microflora were observed. Denaturing gradient gel electrophoresis analysis showed that the predominant bacterial group in the milk belonged to the Lactobacillus species during the experimental period, whereas the genus Staphylococcus gained in numbers during the outside grazing period in addition to Lactobacillus. To investigate the quantitative dynamics of staphylococci, real-time PCR was performed using staphylococcal-specific primers. Real-time PCR analysis revealed that the population of staphylococci increased during the outside grazing period. Our combined denaturing gradient gel electrophoresis and real-time PCR approach enables precise monitoring of the dynamics of both total bacteria and specific bacterial species in milk. Our results provide information on grazing management and the manufacture of dairy products.

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.

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.

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.

Immunomodulatory and cytotoxic effects of various Lactococcus strains on the murine macrophage cell line J774.1.

In a series of in vitro culture experiments using the murine macrophage-like cell line, J774.1, we investigated the ability of 46 different Lactococcus lactis strains to induce production of the cytokines interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-alpha. The extent of induction of IL-6, IL-12 and TNF-alpha was strain-specific and was not related to subspecies, biovariety, or the source of the isolate. When incubated with a high concentration of viable cells of some lactococcal strains, J774.1 cells hardly produced cytokines in which case the percentage of J774.1 cells that were double-stained with the apoptosis probe FITC-labeled annexin V and propidium iodide was significantly increased. This finding suggests that perturbation of cytokine induction is due to the cytotoxic effects of these strains. On the other hand, when incubated with living cells of other strains, even at a high concentration, J774.1 cells produced IL-6, IL-12 and TNF-alpha. In these cases, FITC-labeled annexin V interacted with these cells, suggesting that incubation with these strains causes phosphatidylserine to be exposed at the cell surface. The ability of these strains to induce TNF-alpha, but not IL-6 and IL-12, was lost after heat treatment, suggesting that the stimulus required for TNF-alpha induction is heat sensitive and is different from those required for IL-6 and IL-12 induction. The specificity of cytokine induction by different lactococci is discussed in terms of interaction of non-pathogenic bacteria with macrophages, as well as the implications for the use of lactococci as probiotics.

Interference of in vitro and in vivo growth of several intestinal bacteria by Lactococcus strains.

The ability of Lactococcus strains to inhibit the growth of intestinal bacteria was examined. In in vitro cocultures, we observed that among eighteen Lactococcus strains tested, the ability to inhibit growth of Escherichia coli varied, with the L. lactis N7 showing the greatest growth inhibition. Strain N7 (8.94 x 10(10) CFU/day for 7 days) was orally administered to mice, and the viable count of strain N7 in feces appeared at a level of 10 (4-5) CFU/g. After administration, the proportion of Bacteroidaceae to total intestinal bacteria decreased. Lactococci may act as probiotic bacteria by inhibiting the growth of harmful bacteria.

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.