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.

Zinc Protoporphyrin-Rich Pork Liver Homogenates as Coloring Ingredients in Nitrite-Free Liver Pâtés.

This study aimed to investigate the coloring ingredient potential of liver homogenates that form Zn protoporphyrin (ZnPP), a natural red pigment, after anaerobic incubation. Liver homogenates were used to develop nitrite-free sterile pork liver pâtés. These homogenates were applied in the formulation of pâtés directly or after centrifugation to obtain a pellet that was highly concentrated in ZnPP. Both the whole homogenate and its insoluble fraction were adjusted to pH 7.5 before their use in the formulation of pâtés with and without antioxidant (0.5% ascorbate plus 0.1% tocopherol) addition. Pâtés formulated with the whole homogenate showed color and texture characteristics that were similar to those of the positive control with nitrite. However, high levels of the insoluble fraction also led to pâtés with improved color characteristics but with a two-fold softened texture. Therefore, the form and amount of ZnPP added played roles in the final appearance of the product. The ZnPP pigment was more stable than heme in the sterilization treatment, and antioxidant addition proved to be unnecessary. The ZnPP-rich ingredients allowed for the preparation of nitrite-free cooked liver pâtés with a stable red color and could thus be potentially applied in other uncured cooked meat products.

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.

Evidence of the mechanism underlying zinc protoporphyrin IX formation in nitrite/nitrate-free dry-cured Parma ham.

A large amount of zinc protoporphyrin IX (ZnPP) is found in nitrite/nitrate-free dry-cured meat products, such as Parma ham, and is known to contribute to the favorable bright red color of the latter. ZnPP is a metalloporphyrin, in which zinc is coordinated, instead of iron, in the porphyrin ring. ZnPP proved to be more stable than heme, and its formation should be favored in dried meat products to improve color without the addition of nitrites or nitrates. Toward that, understanding the mechanisms of formation of ZnPP in nitrite/nitrate-free dry-cured ham would be important. In this lecture, I introduce some of our research group's findings regarding the endogenous and exogenous factors contributing to the formation and distribution of ZnPP in Parma ham and why ZnPP formation is suppressed in common cured meat products.

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.

Enhancement of sperm motility and viability by turmeric by-product dietary supplementation in roosters.

Improving sperm motility and viability are major goals to improve efficiency in the poultry industry. In this study, the effects of supplemental dietary turmeric by-product (TBP) from commercial turmeric production on sperm motility, viability, and antioxidative status were examined in domestic fowl. Mature Rhode Island Red roosters were divided into two groups - controls (groupC) without TBP administration and test subjects (groupT) fed a basal diet supplemented with 0.8g of TBP/day in a temperature-controlled rearing facility (Experiment 1) and 1.6g/day under heat stress (Experiment 2) for 4 weeks. In Experiment 1, TBP dietary supplementation increased the sperm motility variables straight-line velocity, curvilinear velocity, and linearity based on a computer-assisted semen analysis, 2 weeks following TBP supplementation. In Experiment 2, using flow cytometry, sperm viability at 3 and 4 weeks following TBP supplementation was greater in Group T than C, and this increase was consistent with a reduction in reactive oxygen species (ROS) production at 2 and 4 weeks. The results of both experiments clearly demonstrate that dietary supplementation with TBP enhanced sperm motility in the controlled-temperature conditions as well as sperm viability, and reduced ROS generation when heat stress prevailed. Considering its potential application in a range of environments, TBP may serve as an economical and potent antioxidant to improve rooster fertility.

Myosin substitution rate is affected by the amount of cytosolic myosin in cultured muscle cells.

In striated muscles, approximately 300 myosin molecules form a single thick filament in myofibrils. Each myosin is continuously displaced by another myosin to maintain the thick filament structure. Our previous study using a fluorescence recovery after photobleaching (FRAP) technique showed that the myosin replacement rate is decreased by inhibition of protein synthesis, but myosin is still exchangeable. This result prompted us to examine whether myosin in the cytoplasm is involved in myosin replacement in myofibrils. To address this, FRAP was measured in green fluorescent protein (GFP)-tagged myosin heavy chain 3 (Myh3) expressing myotubes that were treated with streptolysin-O (SLO), which forms pores specifically in the plasma membrane to induce leakage of cytoplasmic proteins. Our biochemical data demonstrated that the cytoplasmic myosin content was reduced in SLO-permeabilized semi-intact myotubes. Furthermore, FRAP experiments showed a sluggish substitution rate of GFP-Myh3 in SLO-permeabilized myotubes. Taken together, these results demonstrate that the myosin substitution rate is significantly reduced by a decreased amount of myosin in the cytoplasm and that cytoplasmic myosin contributes to myosin replacement in myofibrils.

Diet-Induced Thermogenesis and Expression Levels of Thyroid Hormone Target Genes and Their Products in Rats Differ between Meat Proteins.

We compared the effects of purified meat proteins on postprandial thermogenesis and on the secretion of and responsiveness to thyroid hormones (THs) in rats. Body temperatures at 2 h after feeding were significantly higher in the chicken and mutton protein groups than in the other groups, and these proteins seem to have a strong thermogenic effect. There were no significant differences in plasma TH concentrations among the groups, but levels of TH-responsive Spot 14 protein in the liver and brown adipose tissue were significantly higher in the chicken and mutton protein groups than in the other groups. Levels of malic enzyme 1 protein in the liver and brown adipose tissue were significantly higher in the chicken protein group than in the other groups except for the mutton protein group. Furthermore, levels of uncoupling protein 1 were higher in the chicken and mutton protein groups than in the other groups. The results suggest that the difference in postprandial thermogenesis of meat is strongly dependent on meat proteins; chicken and mutton proteins are strong promoters of postprandial thermogenesis, and THs may contribute to this effect. Since strong postprandial thermogenesis and high expression levels of TH target genes and their products were not observed in the amino acid group, chicken and mutton proteins or their digested peptides might contribute to these effects.

Dynamics of myosin replacement in skeletal muscle cells.

Highly organized thick filaments in skeletal muscle cells are formed from ~300 myosin molecules. Each thick-filament-associated myosin molecule is thought to be constantly exchanged. However, the mechanism of myosin replacement remains unclear, as does the source of myosin for substitution. Here, we investigated the dynamics of myosin exchange in the myofibrils of cultured myotubes by fluorescent recovery after photobleaching and found that myofibrillar myosin is actively replaced with an exchange half-life of ~3 h. Myosin replacement was not disrupted by the absence of the microtubule system or by actomyosin interactions, suggesting that known cytoskeletal systems are dispensable for myosin substitution. Intriguingly, myosin replacement was independent of myosin binding protein C, which links myosin molecules together to form thick filaments. This implies that an individual myosin molecule rather than a thick filament functions as an exchange unit. Furthermore, the myosin substitution rate was decreased by the inhibition of protein synthesis, suggesting that newly synthesized myosin, as well as preexisting cytosolic myosin, contributes to myosin replacement in myofibrils. Notably, incorporation and release of myosin occurred simultaneously in myofibrils, but rapid myosin release from myofibrils was observed without protein synthesis. Collectively, our results indicate that myosin shuttles between myofibrils and the nonmyofibrillar cytosol to maintain a dynamic equilibrium in skeletal muscle cells.

Comparative study of muscle regeneration following cardiotoxin and glycerol injury.

In the present study, we examined muscle regeneration following two types of chemical injuries, cardiotoxin (CTX) and glycerol, in order to compare their effect on the morphological characteristics during muscle regeneration, in addition we studied the structural changes of the intramuscular connective tissue (IMCT) during the regeneration process, by scanning electron microscopy (SEM) after digestion of the cellular elements of the muscle with sodium hydroxide. Tibialis anterior (TA) muscles of adult male mice were injected either with CTX or glycerol. Muscle degeneration was greater in the CTX-injured model than in the glycerol-injured model at day 4 post injection. Muscle regeneration started at day 7 in both the CTX and glycerol models. However, the CTX-injured model showed a higher myotube density and larger myotube diameter than the glycerol-injured model at days 10 and 14 post injection. On other hand, adipocyte infiltration was detected in the glycerol-injured model. In contrast, no adipocytes could be detected in the CTX-injured model. Furthermore, ultrastructural analysis showed a significant difference in myofiber damage and regeneration between the two models. SEM of the IMCT showed a transient increase in endomysial collagen deposition at early stages of regeneration in the CTX-injured model. In contrast, glycerol-injured model showed slight endomysial collagen deposition. Our results suggest that changes in IMCT affect the efficiency of muscle regeneration. Studying the three dimensional structure of IMCT may help clinical therapies to reduce skeletal muscle fibrosis. To our knowledge this is the first time the changes in IMCT following CTX and glycerol injury using SEM-cell maceration technique have been compared.