Identification of cheese rancidity-related lipases in Aspergillus oryzae AHU 7139.

The adjunct product with enzymatic activity from Aspergillus oryzae is beneficial for flavor enrichment in the ripened cheese. However, an excessive lipolytic reaction leads to the release of volatile free fatty acids. Accordingly, a strong off-flavor (i.e., rancidity) has been detected when A. oryzae AHU 7139 is used. To identify the rancidity-related lipase from this strain, we evaluated the substrate specificity and lipase distribution using five mutants cultured on a whey-based solid medium under different initial pH conditions. The results showed a higher diacylglycerol lipase activity than triacylglycerol lipase activity. Moreover, an initial pH of 6.5 for the culture resulted in higher lipolytic activity than a pH of 4.0, and most of the activity was found in the extracellular fraction. Based on the gene expression analysis by real-time polymerase chain reaction and location and substrate specificity, five genes (No. 1, No. 19, mdlB, tglA, and cutL) were selected among 25 annotated lipase genes to identify the respective knockout strains. Because ΔtglA and ΔmdlB showed an outstanding involvement in the release of free fatty acids, these strains were applied to in vitro cheese curd experiments. In conclusion, we posit that triacylglycerol lipase (TglA) plays a key role as the trigger of rancidity and the resulting diglycerides have to be exposed to diacylglycerol lipase (MdlB) to stimulate rancidity in cheese made with A. oryzae AHU 7139. This finding could help screen suitable A.oryzae strains as cheese adjuncts to prevent the generation of the rancid-off flavor.

Water extractability of the zinc protoporphyrin IX-myoglobin complex from Parma ham is pH-dependent.

The bright red color of Parma ham is mainly derived from zinc protoporphyrin IX (ZnPP), which exists in both water-soluble and insoluble states. Water-soluble ZnPP mainly binds to hemoglobin, however, the presence of water-insoluble ZnPP remains unexplained. Therefore, we aimed to elucidate how ZnPP exists in a water-insoluble state by focusing on its binding substance. Depending on the skeletal muscle, water-insoluble ZnPP comprised 30-50% of total ZnPP. The ZnPP water extractability was positively correlated with muscle pH. Water-insoluble ZnPP was extractable with a high-pH solution and existed as a complex with myoglobin or hemoglobin; nevertheless, myoglobin-binding ZnPP was more abundant. Furthermore, the water solubility of the myoglobin globin moiety at pH 5.5-6.0 was reduced by ZnPP binding. These results suggest that water-insoluble ZnPP mainly exists as a ZnPP-Mb complex, with low solubility attributed to the low pH of the ham.

Dissociation of ferriheme from oxidized heme proteins and re-reduction of ferriheme to ferroheme are crucial for the formation of zinc protoporphyrin IX in nitrite/nitrate-free dry-cured meat products.

Zinc protoporphyrin IX (ZnPP) is the dominant red pigment in nitrate/nitrite-free dry-cured meat products such as Parma ham, and it is considered to be a potential alternative to nitrite/nitrate for reddening dry-cured meat products. Ferroheme and ferriheme dissociated from heme proteins in meat were proposed as substrates to form ZnPP. To elucidate their specific formation mechanism, nitric oxide, carbon monoxide, and azide were used to stable heme in heme proteins. The exogenous hemoglobin derivatives bound with these ligands showed lower heme dissociation compared with exogenous oxyhemoglobin and did not contribute to ZnPP formation. Meanwhile, azide inhibited almost all ZnPP formation by binding to ferriheme, indicating ferriheme dissociation from oxidized heme proteins, predominantly for ZnPP formation. Free ferriheme could not be converted to ZnPP unless it was reduced to ferroheme. Overall, ferriheme dissociated from oxidized heme proteins was the dominant substrate for conversion to ZnPP after re-reduction to ferroheme.

L-histidine inhibits the heat-induced gelation of actomyosin in a low ionic strength solution.

The heat-induced gelation of actomyosin plays a key role in meat processing. Our previous study showed that L-histidine could affect the characteristics of a heat-induced gel of myosin on a low ionic strength. To apply the specific effect of L-histidine to meat processing, the heat-induced gel properties of actomyosin in the presence of L-histidine were investigated. Actomyosin in a low ionic strength solution containing L-histidine did not form a gel upon heating. The dynamic rheological properties of actomyosin in low ionic strength solutions were distinct depending on the presence or absence of L-histidine. Electron microscopy showed that, heated at 50°C, actomyosin in a low ionic strength solution containing L-histidine remained a filamentous structure. The surface hydrophobicity of actomyosin was stable up to 50°C in a low ionic strength solution containing L-histidine. In conclusion, L-histidine might suppress the aggregation of actomyosin and inhibit heat-induced gelation in a low ionic strength solution.

Supplementary effect of whey components on the monascin productivity of Monascus sp.

BACKGROUND: Monascus sp. has been used in fermented foods for centuries. It can synthesize yellow, red, and orange pigments as secondary metabolites. Here, we focused on yellow pigment monascin, responsible for anti-inflammation and antidiabetic effects, and investigated whether whey could be a suitable substrate with or without rice powder for monascin production using M. purpureus AHU 9085, M. pilosus NBRC 4520 and M. ruber NBRC 32318. RESULTS: The growth and monascin production of the three Monascus strains were dependent on three liquid media consisting of whey and/or rice. All strains showed the best growth in a rice and whey mixed medium, in which M. ruber NBRC 32318 exhibited the highest total monascin production. Subsequent investigation of the effects of whey components indicated that a mineral cocktail in whey was particularly effective in stimulating the monascin production efficiency of M. ruber NBRC 32318. However, this recipe exhibited less stimulation, or even inhibition, for M. pilosus NBRC 4520 and M. purpureus AHU 9085, respectively. In terms of total monascin production, rice with whey provided the highest amount due to growth promotion along with relatively high production efficiency. CONCLUSION: The effect of whey on growth and monascin production was strongly dependent on the Monascus strains. Even a mineral cocktail in whey could regulate monascin productivity in a strain-specific manner. Further studies are needed to elucidate the mechanism behind the diverse responses by the minerals in the production of monascin from Monascus. © 2023 Society of Chemical Industry.

Zinc protoporphyrin IX predominantly exists as a complex non-enzymatically bound to apo-hemoglobin in Parma ham.

Most of the water-soluble zinc protoporphyrin IX (ZnPP) in Parma ham mainly exists as complexes with hemoglobin and myoglobin (ZnPP-Hb and ZnPP-Mb). To elucidate the formation mechanism of these complexes, a new experimental model to produce higher amount of water-soluble ZnPP complexes was established. ZnPP-Hb was detected as the main water-soluble ZnPP complex in this model, which is the same as that in Parma ham. Adding exogenous Hb into this model promoted higher ZnPP formation than with Mb added, indicating that Hb was the superior substrate for generating ZnPP compared to Mb. The increase in non-heme iron content with ZnPP formation in both the Hb- and Mb-added groups indicated that the release of iron ion from heme was a crucial step in ZnPP formation. ZnPP-Hb was formed when ZnPP non-enzymatically bound with apo-Hb. These results revealed the mechanism of why ZnPP-Hb is more dominant in Parma ham than to ZnPP-Mb.

Clinical evaluation of a new rapid immunochromatographic test for detection of Bordetella pertussis antigen.

A more rapid and less complicated test to diagnose pertussis is required in clinical settings. We need to detect Bordetella pertussis, which mainly causes pertussis, as early as possible, because pertussis is more likely to become severe in infants, and people around them can easily become a source of infection due to its strong infectivity. Nevertheless, methods that can detect B. pertussis rapidly and efficiently are lacking. Therefore, we developed a new immunochromatographic antigen kit (ICkit) for the early diagnosis of pertussis. The ICkit detects B. pertussis antigens in a nasopharyngeal swab without equipment and provides the result in about 15 min with a simple procedure. Additionally, a prospective study to evaluate the ICkit was conducted in 11 medical institutions, involving 195 cases with suspected pertussis. Compared with the real-time polymerase chain reaction (rPCR), the sensitivity and specificity of the ICkit were 86.4% (19/22) and 97.1% (168/173), respectively. The ICkit detected the antigen in both children and adults. Furthermore, the ICkit detected the antigen until the 25th day from the onset of cough, when rPCR detected the antigen. Thus, the ICkit demonstrated a high correlation with rPCR and would help diagnose pertussis more rapidly and efficiently.

A study of lipolysis induced by adjuncts from edible Aspergillus sp. solid culture products on ripened semi-hard cheese.

BACKGROUND: Aspergillus sp. has been used in traditional Japanese fermented foods. Protease-containing culture products of A. oryzae have been applied as the adjunct enzyme source to enrich the flavor in ripened cheese. Although proteolysis was stimulated, the increase of free fatty acids (FFA) was recognized in some products. Since an excess amount of FFA accumulation can cause rancidity in cheese products, the assessment of lipase activity was considered to be essential for the cheese adjunct preparation. RESULTS: Although an equal lipase activity from the adjunct materials of A. kawachii NBRC 4308, A. luchuensis RIB 2604 and A. oryzae AHU 7139 was applied to semi-hard cheese, the FFA level was significantly higher in A. oryzae cheese than in the others. Furthermore, the profiles of volatile components were different in experimental cheeses. An in vitro study with experimental curds demonstrated that the high FFA might not depend on the lipase retainability on curds. On the contrary, the pronounced activation of the lipases occurred in A. oryzae after incubation with the curds. Moreover, incubation of the insoluble lipase that had been attached to the cells with skim milk curd extracts allowed the release of lipases from the cells into the medium with remarkable activation. CONCLUSION: A. oryzae AHU 7139 possessed a complex lipolytic system comprising extracellular and cell-binding lipases that were attributed to the increase in FFA in A. oryzae cheese. © 2022 Society of Chemical Industry.

High ZnPP-forming food-grade lactic acid bacteria as a potential substitute for nitrite/nitrate to improve the color of meat products.

Zinc protoporphyrin IX (ZnPP)-forming food-grade lactic acid bacteria (LAB) were screened from various sources for their ability to improve the color of meat products. The effects of salt and nitrite on the ZnPP-forming ability of these bacteria were also investigated. Finally, these bacteria were applied in salt-added minced meat to assess their ability to improve the color. Twenty-five LAB were screened for their ZnPP-forming ability in pork. Most of the strains exhibited maximum growth anaerobically in 3% salt at 30 °C and grew well at pH 5.5 and 6.5. Moreover, 3% salt slightly retarded ZnPP formation; however, nitrite completely inhibited ZnPP formation in all the ZnPP-forming LAB. Thirteen LAB (avoiding duplication and non-food-grade) could form ZnPP in salt-added minced meat, resulting in improvement of the bright red color, high ZnPP autofluorescence, and increased fluorescence intensity. Finally, considering the safety, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris, and Leuconostoc lactis were suggested as promising candidates to improve the color of meat products.

Lactococcus lactis Subsp. Cremoris Produces Zinc Protoporphyrin IX Both Aerobically and Anaerobically and Improves the Bright Red Color of Fermented Meat Products.

This study assessed the color improvement via zinc protoporphyrin IX (ZnPP) formation in nitrite-free, dry-cured sausages processed using five varieties of ZnPP-forming lactic acid bacteria (LAB). The ZnPP contents and color intensity of the sausages and other technological properties were analyzed during the processing of sausages. LAB count and acidity significantly increased in the LAB-inoculated sausages compared to the control group. The bright red color was observed both inside and outside the sausages inoculated with Lactococcus lactis subsp. cremoris and Leuconostoc lactis. However, a brown color was observed on the surface of the sausage inoculated with Lactobacillus spp. The redness of Lactococcus lactis subsp. cremoris-inoculated sausages was close to that of the nitrite-added group. Moreover, the external bright red color was improved by Lactococcus lactis subsp. cremoris due to the aerobic formation of ZnPP. Therefore, Lactococcus lactis subsp. cremoris can be used to improve the color of fermented meat products.

Adjunctive application of solid-state culture products and its freeze-dried powder from Aspergillus sojae for semi-hard cheese.

BACKGROUND: Species belonging to the genus Aspergillus have been used in traditional Japanese fermented foods. Aspergillus sojae is a species responsible for strong proteolytic activity. Freeze-drying treatments followed by physical disruption enables the pulverization of the mycelia of A. sojae RIB 1045 grown in whey protein-base solid media. Intracellular proteases were extracted using this protocol to compare extracellular protease activity in terms of the reaction's pH dependence in the presence or absence of inhibitors. RESULT: With different sensitivities to inhibitors, intracellular and extracellular proteases showed the strongest activity under acidic conditions, which were considered suitable for cheese application. The raw culture product (CP) and its freeze-dried product (FDP) were mixed with cheese curds, prepared according to Gouda-type cheese-making methods, and were allowed to ripen for 3 months. Chemical analysis of the products showed 13.3% water-soluble nitrogen (WSN) in the control, which had received noncultured media, whereas 20.0% and 21.1% WSN was found in the CP and FDP experimental cheeses, respectively. Although these adjuncts significantly increased WSN, an insignificant difference was found between CP and FDP. Free fatty acids in all experimental cheeses were similar, showing that CP and FDP caused no rancid defects. CONCLUSION: The introduction of freeze-drying treatments accompanied by cell disruption resulted in a negligible effect in terms of WSN. However, the application of A. sojae can be beneficial when it comes to increasing the level of WSN compared with A. oryzae, as shown in our previous study. © 2020 Society of Chemical Industry.

Searching for high ZnPP-forming edible bacteria to improve the color of fermented meat products without nitrite/nitrate.

In order to improve the color of meat products by producing zinc protoporphyrin IX (ZnPP) in meat, we searched for edible bacteria with high ZnPP-forming ability. Eleven bacteria used in different animal products and 126 bacteria isolated from environmental and probiotic sources were assessed for their ability to form ZnPP. Many bacteria from both sources showed a high ZnPP-forming ability. Only three edible bacteria were identified from the 44 high ZnPP-forming isolates with 16S rRNA gene sequencing. High ZnPP-forming bacteria from both sources were inoculated in aseptic salt-added minced meat, and their ZnPP-forming abilities were evaluated. Lactococcus lactis, Leuconostoc mesenteroides, and Enterococcus faecium from environmental isolates produced a brighter red color, higher ZnPP autofluorescence and fluorescence intensity in salt-added minced meat than control. Furthermore, after heating, the color and ZnPP autofluorescence of the inoculated minced meat persisted to a degree. Therefore, it is possible to improve the color of meat products without nitrite/nitrate by using these promising ZnPP-forming edible bacteria.

Improving the color of meat products without adding nitrite/nitrate using high zinc protoporphyrin IX-forming microorganisms.

Zinc protoporphyrin IX (ZnPP) mainly contributes to the red color of dry cured ham without nitrites/nitrates. Here, we examined the effects of acids used for pH adjustment, pH, and microorganisms on ZnPP formation. The results showed that ZnPP formation and optimal pH were dependent upon the acid type. In the presence of microorganisms, the optimal pH for ZnPP formation shifted to higher values, with the amount of formed ZnPP markedly increased at the shifted optimal pH. Additionally, two bacterial strains isolated from incubated pork homogenate exhibited an enhanced ability to form ZnPP. Although the two isolated bacteria are not edible, inoculation with one bacterium into minced meat resulted in formation of large amounts of ZnPP and color closer to that of nitrite-added meat. These results suggest that appropriate food-grade bacterial strains can improve the color of various fermented meat products in the absence of nitrites/nitrates.

Investigation of contributors to zinc protoporphyrin IX formation at optimum pH 5.5 in pork.

We investigated zinc protoporphyrin (ZnPP) formation in pork at pH 5.5, identified the contributors to ZnPP formation, and verified the involvement of myoglobin in this process. When pork homogenate was separated into two water-soluble fractions (>10 and <10 kDa) and an insoluble fraction, ZnPP formation was suppressed. ZnPP formation was rescued after mixing of all three fractions. Heating of the soluble <10 kDa fraction did not suppress the formation of ZnPP as opposed to heating of the soluble >10 kDa fraction, suggesting that protein(s) presents in the >10 kDa fraction contributed to ZnPP formation. Components of the soluble 10-30 kDa fractions separated by ultrafiltration were important in ZnPP formation. Exogenous myoglobin was not essential for ZnPP formation. A gel filtration study showed that soluble protein(s) with molecular weight higher than that of myoglobin was involved. Therefore, it was suggested that the soluble <10 kDa fraction, the insoluble fraction, and the soluble 10-30 kDa fraction (excluding myoglobin) are essential for ZnPP formation in pork at pH 5.5.

Angiotensin II Receptor Blockers Inhibit the Generation of Epoxyeicosatrienoic Acid from Arachidonic Acid in Recombinant CYP2C9, CYP2J2 and Human Liver Microsomes.

Cytochrome P450 (CYP) 2C9, CYP2C8 and CYP2J2 enzymes, which metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids, have cardioprotective effects including anti-inflammation and vasodilation. We have recently shown that some angiotensin II receptor blockers (ARBs) may inhibit AA metabolism via CYP2C8. Using recombinant CYP2C9, CYP2J2 and human liver microsomes (HLMs), the aim was now to compare the ability of six different clinically used ARBs to inhibit AA metabolism in vitro. The rank order of the ARBs for the 50% inhibitory concentration (IC50 ) of AA metabolism was losartan

Range of Effective Corticosteroid Doses for Alleviating Dyspnea in Terminal Cancer Patients: A Retrospective Review.

This study aimed to determine the range of mean cumulative corticosteroid doses that could effectively palliate dyspnea in opioid-treated patients with terminal cancer and to investigate the demographic or biochemical factors predictive of corticosteroid responsiveness. To this end, responders and nonresponders were compared with regard to corticosteroid dose and whether they had initiated opioid use before or concomitantly with corticosteroid use. A logistic regression analysis was conducted to assess the impacts of demographic and biochemical factors on corticosteroid effectiveness. The final sample comprised 20 patients who satisfied the selection criteria. The responders accounted for 70% of the total sample (n = 14) and experienced the strongest effect with regard to dyspnea palliation at a mean cumulative dose equivalent to 64.4 mg prednisolone. However, no factors predictive of response were identified. In summary, this retrospective study identified effective corticosteroid doses for dyspnea alleviation in terminal cancer patients. Although our study sample was limited in size, the results support further prospective research.

Effectiveness of Corticosteroid Monotherapy for Dyspnea Relief in Patients with Terminal Cancer.

Dyspnea is a common symptom in patients with cancer, particularly those with late-stage terminal disease. It markedly affects terminal cancer patients, reducing their quality of life. Reduced quality of life also affects survival; therefore, dyspnea is a prognostic factor. However, the role of corticosteroids, which often are used to alleviate dyspnea, has not been sufficiently validated. In this study, we retrospectively investigated whether corticosteroid monotherapy was effective for dyspnea palliation. The effectiveness rate of corticosteroid therapy was 45% in nine male and two female study subjects (mean age: 74.5 years; range: 64-86 years). No significant differences were found between responders and nonresponders in the first-day corticosteroid doses (25.5 ± 10.86 vs. 36.1 ± 16.39 mg, P = .29) or doses administered on 2 days (47.7 ± 25.99 vs. 72.2 ± 32.78 mg, P = .25). The mean ± standard error assessment score changed significantly from 2.7 ± 0.14 at the beginning of corticosteroid administration to 1.5 ± 0.37 at the time of maximum effect (P = .028); however, the decrease to 2.1 ± 0.25 at the final administration was not significant (P = .068). This indicates that corticosteroid therapy relieved dyspnea and could provide an early-stage treatment option.

Combined Effect of Opioids and Corticosteroids for Alleviating Dyspnea in Terminal Cancer Patients: A Retrospective Review.

Dyspnea is a prognostic factor that affects the quality of life of terminal cancer patients, and many reports have described opioid treatment for dyspnea alleviation. Here, we retrospectively evaluated differences in the effects of various opioids administered concomitantly with corticosteroids on dyspnea in 20 terminal-stage cancer patients (13 men, 7 women; mean age [range]: 71 [49-94] years) who received opioids concomitantly with corticosteroids. Effectiveness was assessed throughout administration using the Support Team Assessment Schedule, Japanese version (STAS-J), particularly the subscale indicating how strongly a patient is affected by symptoms. The effectiveness of combined opioid and corticosteroid therapy against dyspnea and the opioid dose comprised the primary and secondary foci, respectively. Among concomitantly treated patients, STAS-J scores at initiation (mean ± SD: 3.1 ± 0.24) and lowest recorded STAS-J scores (1.4 ± 0.22) differed significantly (P = .0034) among those receiving morphine, but not among those receiving oxycodone (P = .068) or fentanyl (P = .18). Concomitant opioid and corticosteroid treatment was associated with a ≥2-point STAS-J score improvement in 14/20 patients (effectiveness: 70%). The opioid dose did not significantly affect dyspnea alleviation. We conclude that concomitant opioid and corticosteroid treatment can effectively alleviate dyspnea in terminal cancer patients.

[Introduction and Evaluation of an Integrated Program to Develop Pharmacotherapeutic Management Skills].

Pharmacists must be able to select and collect important information for patient care, to accurately assess and monitor the status of patients, to develop care plans, propose optimal formulations, and provide advice to patients about pharmacotherapy. The core curriculum of Japan's pharmacy education model was revised in 2013 based on the "Basic competencies required of a pharmacist" that should be developed by the time of a student's graduation. Specific behavioral objectives to be acquired include competency in "The ability to implement pharmacotherapeutic management", which involves gaining the ability to understand patient information, to design and propose prescriptions, and to conduct pharmacotherapeutic assessments. We thus introduced an integrated program for fifth-year students at Hokkaido Pharmaceutical University using problem-based learning and role-playing with simulated patients to teach clinical communication skills and the ability to design pharmaceutical care plans for patients. A survey of students who completed the program after pharmacy practical training revealed that most of them realized the value of the program: they were able to develop precise care plans for medical problems and learned good communication skills to collect information about patients.