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Haruto Kumura
Field Science Center for Northern Biosphere
Professor
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Last Updated :2024/05/09

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Affiliation

  • Field Science Center for Northern Biosphere

Job Title

  • Professor

J-Global ID

Research Interests

  • 微生物   食品   

Research Areas

  • Life sciences / Food sciences

Educational Organization

Association Memberships

  • 北海道畜産草地学会   日本畜産学会   日本栄養・食糧学会   日本農芸化学会   

Research Activities

Published Papers

  • Napaporn Chintagavongse, Haruto Kumura, Toru Hayakawa, Jun-Ichi Wakamatsu, Koichi Tamano
    Journal of bioscience and bioengineering 2024/03/01 
    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.
  • Haruka Abe, Yang Zhai, Yu Toba, Hiroki Masumo, Toru Hayakawa, Haruto Kumura, Jun-Ichi Wakamatsu
    Food chemistry 441 138317 - 138317 2024/01/02 
    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.
  • Yang Zhai, Haruka Abe, Hung-Cheng Wang, Toru Hayakawa, Haruto Kumura, Jun-Ichi Wakamatsu
    Food chemistry 427 136755 - 136755 2023/11/30 
    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.
  • Qingyun Huang, Nodoka Miyaki, Zongfei Li, Yutaroh Takahashi, Satoshi Ishizuka, Toru Hayakawa, Jun-Ichi Wakamatsu, Haruto Kumura
    Journal of the science of food and agriculture 103 (8) 4234 - 4241 2023/06 
    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.
  • Toru Hayakawa, Yu Kubono, Shuji Fujii, Jun‐ichi Wakamatsu, Haruto Kumura
    Animal Science Journal 94 (1) 1344-3941 2023/01
  • Yang Zhai, Hung-Cheng Wang, Toru Hayakawa, Haruto Kumura, Jun-Ichi Wakamatsu
    Food chemistry 395 133604 - 133604 2022/11/30 
    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.
  • Napaporn Chintagavongse, Hayate Takiguchi, Chi Ming-Hsuan, Koichi Tamano, Toru Hayakawa, Jun-Ichi Wakamatsu, Tomohiro Mitani, Haruto Kumura
    Journal of the science of food and agriculture 2022/01/23 
    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.
  • Md. Kauser-Ul-Alam, Toru Hayakawa, Haruto Kumura, Jun-ichi Wakamatsu
    Meat Science 176 108467 - 108467 0309-1740 2021/06 [Refereed]
     
    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.
  • Hung-Cheng Wang, Toru Hayakawa, Haruto Kumura, Jun-ichi Wakamatsu
    Food Bioscience 40 100870 - 100870 2212-4292 2021/04 [Refereed]
  • Nihon Chikusan Gakkaiho 91 (4) 389 - 394 1346-907X 2020/11/25 [Refereed]
  • Md. Kauser-Ul-Alam, Yu Toba, Shoji Hioki, Toru Hayakawa, Haruto Kumura, Jun-ichi Wakamatsu
    Foods 9 (11) 1583 - 1583 2020/10/31 [Refereed]
     
    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.
  • Md Asaduzzaman, Momo Ohya, Haruto Kumura, Toru Hayakawa, Jun-ichi Wakamatsu
    Meat Science 165 108109 - 108109 0309-1740 2020/07 [Refereed]
     
    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.
  • Napaporn Chintagavongse, Tomoki Yoneda, Chi Ming-Hsuan, Toru Hayakawa, Jun-Ichi Wakamatsu, Koichi Tamano, Haruto Kumura
    Journal of the science of food and agriculture 100 (13) 4834 - 4839 2020/06/01 [Refereed][Not invited]
     
    BACKGROUND: Some species belonging to the genus Aspergillus have been used in traditional Japanese fermentation foods. A. sojae is the species responsible for high proteolytic activity. Freeze-drying treatments followed by physical disruption enables the pulverisation of mycelia of A. sojae RIB 1045 grown in whey protein base solid media. Through this protocol, intracellular proteases were extracted to compare extracellular protease activity in terms of the reaction pH dependence in the presence or absence of the inhibitors. RESULT: With different sensitivities to inhibitors, intracellular and extracellular proteases showed the highest activity under the acidic region, which was 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 and were allowed to ripen for three 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 were found in CP and FDP experimental cheese, 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: An 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 degree of WSN compared with A. oryzae, as shown in our previous study. This article is protected by copyright. All rights reserved.
  • Wakamatsu JI, Kawazoe H, Ohya M, Hayakawa T, Kumura H
    Meat science 161 107989 - 107989 0309-1740 2019/10 [Refereed][Not invited]
     
    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.
  • Mofassara Akter, Akiko Shiraishi, Haruto Kumura, Toru Hayakawa, Jun‐ichi Wakamatsu
    Animal Science Journal 90 (6) 774 - 780 1344-3941 2019/06 [Refereed]
     
    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.
  • Haruto Kumura, Megumi Satoh, Taiki Machiya, Makoto Hosono, Toru Hayakawa, Jun‐ichi Wakamatsu
    International Journal of Dairy Technology 72 (3) 403 - 408 1364-727X 2019/04/26 [Refereed]
     
    The lipolytic and proteolytic activity of Penicillium camemberti PC TT033 and Penicillium roqueforti PR G3, cultured on the whey solids or simulated cheese media, were compared under several pH reaction conditions. Lipolytic activity was higher when both strains had been cultured on the whey medium than on the simulated cheese medium, whereas proteolytic activity was less influenced by the culture medium. The relationship between the reaction pH and these enzyme activities was dependent on the culture medium, which suggested that the expression level and balance of isozyme rely on the culture substrate.
  • Jun-ichi Wakamatsu, Mofassara Akter, Fumika Honma, Toru Hayakawa, Haruto Kumura, Takanori Nishimura
    LWT 101 599 - 606 0023-6438 2019/03 [Refereed]
  • Application of red pigment producing edible fungi for development of a novel type of functional cheese.
    Kumura, H, Ohtsuyama, T, Matsusaki Y, Taitoh, M, Koyanagi, H, Kobayashi, K, Hayakawa, T, Wakamatsu, J, Ishizuka S
    Journal of Food Processing and Preservation 2018 [Refereed][Not invited]
  • Haruto Kumura, Chiharu Saito, Yuko Taniguchi, Taiki Machiya, Yutaroh Takahashi, Ken Kobayashi, Akira Kimura
    Advances in Dairy Research 05 (03) 2017 [Refereed][Not invited]
  • Ken Kobayashi, Yusaku Tsugami, Kota Matsunaga, Shoko Oyama, Chinatsu Kuki, Haruto Kumura
    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1863 (8) 2006 - 2016 0167-4889 2016/08 [Refereed][Not invited]
     
    Alveolar mammary epithelial cells (MECs) in mammary glands are highly specialized cells that produce milk for suckling infants. Alveolar MECs also form less permeable tight junctions (TJs) to prevent the leakage of milk components after parturition. In the formation process of less permeable TJs, MECs show a selective downregulation of Cldn4 and a localization change of Cldn3. To investigate what induces less permeable TJs through these compositional changes in Cldns, we focused on two lactogenesis-related hormones: prolactin (Prl) and glucocorticoids. Prl caused a downregulation of Cldn3 and Cldn4 with the formation of leaky TJs in MECs in vitro. Prl-treated MECs also showed low beta-casein expression with the activation of STAT5 signaling. By contrast, dexamethasone (Dex), a glucocorticoid analogue, upregulated Cldn3 and Cldn4, concurrent with the formation of less permeable TJs and the activation of glucocorticoid signaling without the expression of beta-casein. Cotreatment with Prl and Dex induced the selective downregulation of Cldn4 and the concentration of Cldn3 in the region of Tjs concurrent with less permeable TJ formation and high beta-casein expression. The inhibition of Prl secretion by bromocriptine in lactating mice induced the upregulation of Cldn3 and Cldn4 concurrent with the downregulation of milk production. These results indicate that the coactivation of Prl and glucocorticoid signaling induces lactation-specific less permeable TJs concurrent with lactogenesis. (C) 2016 Elsevier B.V. All rights reserved.
  • Ken Kobayashi, Chinatsu Kuki, Shoko Oyama, Haruto Kumura
    EXPERIMENTAL CELL RESEARCH 340 (2) 295 - 304 0014-4827 2016/01 [Refereed][Not invited]
     
    Lactose is a milk-specific carbohydrate synthesized by mammary epithelial cells (MECs) in mammary glands during lactation. Lactose synthesis is downregulated under conditions causing inflammation such as mastitis, in which MECs are exposed to high concentrations of inflammatory cytokines. In this study, we investigated whether inflammatory cytokines (TNF-alpha, IL-1 beta, and IL-6) directly influence the lactose synthesis pathway by using two types of murine MEC culture models: the monolayer culture of MECs to induce lactogenesis; and the three-dimensional culture of MECs surrounded by Matrigel to induce reconstitution of the alveolar structure in vitro. TNF-alpha caused severe down-regulation of lactose synthesis related genes concurrently with the degradation of glucose transporter 1 (GLUTI) from the basolateral membranes in MECs. IL-1 beta also caused degradation of GLUTI along with a decrease in the expression level of beta-1,4-galactosylransferase 3. IL-6 caused both up-regulation and down-regulation of the expression levels of lactose synthesis-related genes in MECs. These results indicate that TNF-alpha, IL-1 beta, and IL-6 have different effects on the lactose synthesis pathway in MECs. Furthermore, TNF-alpha triggered activation of NFKB and inactivation of STAT5, suggesting that NF kappa B and STAT5 signaling pathways are involved in the multiple adverse effects of TNF-alpha on the lactose synthesis pathway. (C) 2015 Elsevier Inc. All rights reserved.
  • Takaaki Uejyo, Chinatsu Kuki, Shoko Oyama, Haruto Kumura, Ken Kobayashi
    CELL AND TISSUE RESEARCH 359 (2) 643 - 653 0302-766X 2015/02 [Not refereed][Not invited]
     
    The mammary gland is a highly specialized organ that is able to repeat development and regression (involution) of alveolar structures for milk production. Mammary involution consists in two phases. The first phase is reversible and lasts until approximately 48 h after weaning in mice. Interestingly, an extended milking interval can change the milk-secretory activity of alveolar epithelial cells (AECs) before the first phase of involution begins. In this study, we investigate the changes in the ability of AECs to secrete milk during the involution progression. Careful observation of the number and locations of cleaved caspase-3 positive AECs revealed that the first phase of involution occurred approximately 24 h after weaning and the second phase began between 48 and 72 h after weaning. However, initial changes in the milk production ability of AECs began just 1 h after weaning and milk production gradually ceased within 24 h. In addition, activation of STAT3 and inactivation of STAT5 had occurred in some AECs by 6 h after weaning and more broadly by 24 h. In addition, milk production processes such as nutrient uptake, synthesis, and secretion ceased by 24 h post-weaning. Interestingly, enlarged cytoplasmic lipid droplets were observed in AECs 12 h after weaning even though the expression levels of genes relevant to triglyceride production (Srebp1 and AQP3) were down-regulated. These results indicate that several changes in the milk production ability of AECs occur during expanded suckling intervals and prior to involution.
  • Shota Miyata, Yozo Oda, Chika Matsuo, Haruto Kumura, Ken Kobayashi
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 62 (49) 11854 - 11861 0021-8561 2014/12 [Not refereed][Not invited]
     
    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation.
  • Ken Kobayashi, Shoko Oyama, Takaaki Uejyo, Chinatsu Kuki, Md Morshedur Rahman, Haruto Kumura
    VETERINARY RESEARCH 44 119  0928-4249 2013/12 [Not refereed][Not invited]
     
    Mastitis, the inflammation of mammary glands resulting from bacterial infection, disrupts milk production in lactating mammary glands. In this study, we injected lipopolysaccharide (LPS), one of the endotoxins from Escherichia coli into mouse mammary glands to disrupt milk production, and we investigated the influence of LPS on nutrient uptake, synthesis, and secretion processes for milk component production in alveolar epithelial cells (AEC). The expression of genes relevant to the three-staged milk component production process (nutrient uptake, synthesis, and secretion of milk components) were down-regulated within 12 h after LPS injection in AEC. The internalization of glucose transporter 1 (GLUT-1) from the basolateral membrane to the cytoplasm occurred in accordance with the down-regulation of gene expression 3 h after LPS injection. The abnormal localization of adipophilin and beta-casein was also observed in the LPS-injected mammary glands. SLC7A1, an amino acid transporter, was up-regulated 3 and 6 h after LPS injection. Furthermore, the inactivation of signal transducer and activator of transcription 5 (STAT5) and the activation of STAT3 and nuclear factor-kappa B (NFkappaB) occurred 3 h after LPS injection. These results indicate that the nutrient uptake, synthesis, and secretion of milk components in AEC are rapidly shut down in the lactating mammary glands after LPS injection.
  • Ken Kobayashi, Takaaki Uejyo, Shoko Oyama, Md. Morshedur Rahman, Haruto Kumura
    Cell and Tissue Research 354 (2) 495 - 506 0302-766X 2013/11 [Not refereed][Not invited]
     
    The mammary alveolus is a highly specialized structure that secretes milk for suckling infants during lactation. The secreting alveolus consists in alveolar epithelial cells (AECs) and myoepithelial cells and is surrounded by microvascular endothelial cells, adipocytes and several immune cell types such as macrophages and neutrophils. During normal lactation, these cells play distinct roles needed to maintain the secretory ability of the mammary alveolus. However, inflammation resulting from pathogenic bacterial infections causes structural and functional regression of the secreting alveolus in the lactating mammary gland. We initiated artificial inflammation in the mammary glands of lactating mice by injecting lipopolysaccharide (LPS), as a mammary inflammation model and investigated, by immunohistochemical analysis, the early response of the cells constituting and surrounding the alveolus. Some AECs sloughed away from the alveolar epithelial layer and showed progression of apoptosis detected by immunostaining of cleaved caspase-3 after LPS injection. Adipocytes exhibited transient shrinkage and re-accumulation of lipid droplets, although the numbers of adipocytes did not demonstrate a significant difference. Activation of F4/80-positive cells around the mammary alveolus was observed 3 h after LPS injection. However, the recruitment of CD11b-positive cells into the alveolar lumen was not observed until 12 h after LPS injection. Myoepithelial cells were contracted after LPS injection. LPS injection around the alveolus did not induce any detectable structural changes in capillaries surrounding the alveolus. Thus, cell-specific behavior and tissue remodeling of the alveolus occur after LPS injection in a time-dependent manner. © 2013 Springer-Verlag Berlin Heidelberg.
  • Ken Kobayashi, Shoko Oyama, Atsushi Numata, Md. Morshedur Rahman, Haruto Kumura
    PLoS ONE 8 (4) 1 - 12 1932-6203 2013/04/23 [Not refereed][Not invited]
     
    Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling. © 2013 Kobayashi et al.
  • H. Kumura, T. Sawada, Y. Oda, M. Konno, K. Kobayashi
    JOURNAL OF DAIRY SCIENCE 95 (7) 3629 - 3633 0022-0302 2012/07 [Refereed][Not invited]
     
    Among the lipids in bovine milk, minor components such as conjugated linoleic acids and phospholipids are more attractive than triacylglycerols from the standpoint of biological activity. To explore novel functions of bovine milk polar lipids (MPL), topical application to murine dorsal skin was introduced as an assay system. The acetone-insoluble lipid fraction derived from bovine milk was dispersed in ethanol and applied to 9-wk-old C57BL/6N female mice for 3 wk. In combination with visual assessment of the dorsal pigmentation, the progression of the hair cycle was estimated by calculating the ratio of subcutis to dermis thickness. The administration of MPL led to earlier progression of the hair cycle compared with administration of the vehicle. In some cases, the extent of MPL-induced hair cycle progression was comparable to that in animals treated with minoxidil, the most well-known reagent that initiates anagen. These results indicate that the MPL preparation contains a dermal penetrative component that can regulate the hair cycle and, thus, this preparation possesses potential for cosmetic use.
  • Ken Kobayashi, Haruto Kumura
    HISTOCHEMISTRY AND CELL BIOLOGY 136 (5) 587 - 594 0948-6143 2011/11 [Not refereed][Not invited]
     
    Tight junctions (TJs) are the most apical junctional complexes and restrict the fluid flux through the paracellular pathway. In the mammary glands, the tightness of TJs occurs shortly after parturition to prevent the leakage of milk components from the lumen and the loosening of TJs is induced immediately after weaning. Claudins are transmembrane proteins, and their composition at the apical-most regions determines the permeability of TJs. In this study, we investigated the localization and expression patterns of claudin-3 and -4 in the mammary glands around the lactation period because it is unclear how claudins construct mammary TJs in the apical-most regions. Our results showed that claudin-3 and -4 change not only their level of expression but also their localization in the processes of parturition, lactation, and weaning. Claudin-3 was concentrated in the apical-most regions during lactation, whereas claudin-4 gradually decreased at the beginning of lactation and increased drastically immediately after weaning. The qualitative change of claudin-3 was also identified by western blotting analysis as an additional band around the lactation period. In addition, parts of the mammary epithelial cells showed intensive positive reactions to claudin-4 in the lateral membrane and cytoplasm after weaning, concurrently with the involution mammary glands. These results indicate that claudin-3 in the apical-most regions maintains the impermeable TJs during lactation, and claudin-4 contributes to the permeability changes of TJs immediately after parturition and weaning.
  • H. Kumura, T. Ishido, K. Shimazaki
    JOURNAL OF DAIRY SCIENCE 94 (2) 657 - 667 0022-0302 2011/02 [Refereed][Not invited]
     
    Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese,, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25 degrees C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of beta-lactoglobulin and a-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein.
  • Takuya Ishido, Toki Nishiyama, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaiki, Hidemasa Motoshima, Kazuhiro Kawai
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 66 (1) 76 - 80 0026-3788 2011 [Refereed][Not invited]
     
    Lactoperoxidase is an antibacterial protein found in milk. Its activity forms the basis of the lactoperoxidase system, a solution composed of lactoperoxidase, SCN- and H2O2. In this study, lactose, beta-galactosidase, glucoseoxidase, lactoperoxidase and KSCN were used to produce the antibacterial active chemical, hypothiocyanite ion (OSCN-). Development of antibacterial activity can be attributed to the generation of H2O2 from glucose by glucoseoxidase-mediated lactose hydrolyzation. The stable and continuous production of OSCN- was confirmed and antibacterial activity towards E. coli, P. fluorescens, P. syringae and other microorganisms was estimated with a lactoperoxidase system using lactose as the primary substrate. This novel lactoperoxidase system could be utilized to prevent bacterial spoilage of raw milk, as well as in new applications such as teat dips for the maintenance of bovine udder hygiene.
  • Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki
    INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY 45 (3) 453 - 458 0950-5423 2010/03 [Refereed][Not invited]
     
    P>In the present study, we investigated the in vitro growth responses of fourteen strains of four Bifidobacterium spp. (Bifidobacterium infantis, B. breve, B. bifidum, B. longum) against bovine lactoferrin (bLf) at various concentrations. Bacterial strains were grown in deMan, Rogosa and Sharpe (MRS) broth with or without bLf, and growth was monitored by measuring absorbance at 660 nm. A dose-dependent and strain-dependent growth response was observed. Bifidobacterium spp. were ranked into high, medium and low according to their calculated relative growth response levels against lactoferrin (Lf). Strains showed better growth responses against holo-type Lf. However, no inhibitory effects at high concentrations (4 mg mL-1) or with apo-type Lf were observed. These results strongly suggest that the growth response of Bifidobacterium spp. against bLf may be a selection criterion for their use in fermented products. In addition, use of holo-type Lf in fermented food products may be more effective for probiotic growth, and could also be used as a source of iron to the host.
  • Seema Kafley, Woan-Sub Kim, Haruto Kumura, Kei-Ichi Shimazaki
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 65 (3) 245 - 248 0026-3788 2010 [Refereed][Not invited]
     
    As whey protein being a precursor of bioactive substances this study was conducted to evaluate the influence of Whey Protein Isolate (WPI) and its peptic or tryptic hydrolysates on the growth of probiotics. The probiotic bacteria used in this study included selected strains of bifidobacteria (B. longum, B. bifidum, B. breve, B. infantis) and Lactobacillus acidophilus. The influence of the supplementation of WPI or its enzymatic hydrolysates on the growth of selected strains during 30 hours of incubation was studied in vitro by measuring the absorbance at 600 nm. Different concentrations of pepsin and trypsin (0.2 and 0.5% (w/w)) were used for hydrolysis of WPI. The growth stimulation of bifidobacteria was demonstrated to be higher with the peptic hydrolysates of WPI in comparison to WPI or its tryptic hydrolysates, whereas both the W PI and its peptic or tryptic hydrolysates stimulated the growth of L. acidophilus compared to the control. This study provides evidence that the functional peptides released from major proteins of hydrolysed WPI, i.e. the peptides of a-lactalbumin or beta-lactoglobulin stimulated the growth of probiotics, depending on the respective strain.
  • Md. Morshedur Rahman, Woan-Sub Kim, Toshiaki Ito, Haruto Kumura, Kei-ichi Shimazaki
    ANAEROBE 15 (4) 133 - 137 1075-9964 2009/08 [Refereed][Not invited]
     
    Lactoferrin, a major whey protein of human milk, is considered as growth promoter for bifidobacteria, the predominant microorganisms of human intestine. In the present study, in vitro growth promotion and cell binding ability of bovine lactoferrin to several strains of Bifidobacterium longum have been demonstrated. A dose-dependent as well as strain-dependent growth promotion effect by lactoferrin was observed. Cell binding ability of lactoferrin was inspected under an inverted confocal laser scanning microscope by incubation bacterial cells with biotinylated bovine lactoferrin and FITC-conjugated avidin. Fluorescence staining showed bovine lactoferrin binding to ail tested strains. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the extracted membrane and cytosolic fraction of each B. longum strain by far-Western blot technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on these results, we suggest that existence of lactoferrin-binding protein could be a common characteristic in bifidobacteria. It can also be hypothesized that lactoferrin-binding protein in bifidobacteria is not only involved in growth stimulation mechanism but also could play different roles. (C) 2009 Elsevier Ltd. All rights reserved.
  • Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki
    BIOTECHNOLOGY LETTERS 31 (6) 863 - 868 0141-5492 2009/06 [Refereed][Not invited]
     
    Bovine lactoferrin promotes bifidobacterial growth. Its binding to bifidobacteria is thought to be responsible for such action. After separating the bovine lactoferrin half molecule and extraction of surface proteins from bifidobacteria, binding profiles were observed by immunoblotting. No binding appeared when lactoferrin C-lobe was reacted with the cell surface proteins on a polyvinylidene difluoride membrane. Conversely, a 50-kDa band appeared when the surface proteins were reacted with either intact or nicked bovine lactoferrin. This result strongly suggests that the binding region could be lactoferrin N-lobe. Interestingly, despite the absence of binding, C-lobe enhanced bifidobacterial growth.
  • Haruto Kumura, Shinya Takii, Ubune Iwase, Kei-ichi Shimazaki
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 64 (1) 10 - 13 0026-3788 2009 [Refereed][Not invited]
     
    BALB/c mice were immunized with the food antigen ovalbumin (OVA) via intraperitoneal injection to prepare OVA-sensitized splenocytes, which were subsequently cultured in the presence of disrupted yeast preparation (DYP) from three different strains of Kluyveromyces lactis. In the absence of DYP, splenocytes from OVA-sensitized mice demonstrated elevated IgE production in response to OVA exposure, whereas addition of K. lactis suppressed IgE production in a dose-dependent manner. In the light of the observation of degranulation study using rat basophilic leukemia RBL-2H3 cells, K. lactis S16 was selected for oral administration to OVA-sensitized BALB/c mice. However, DYP of K. lactis S16 did not appear to modify the concentration of serum IgE. Furthermore, no difference in IgE production in response to OVA stimulation was found between spleen cells isolated from animals fed a diet supplemented with DYP from K. lactis S 16 and those from control animals. Six weeks after OVA-sensitization, the fecal IgA content of mice fed DYP from K. lactis S16 was slightly higher than that of the two other experimental groups. The substance present in the DYP responsible for the suppression of IgE production in vitro should be identified in order to reproduce the observed immunomodulatory effects in vivo more efficiently.
  • Haruto Kumura, Yukie Ikezu, Erika Tajima, Kei-ichi Shimazaki
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 64 (3) 256 - 259 0026-3788 2009 [Refereed][Not invited]
     
    Five strains of yeast (Saccharomyces cerevisiae G1, Kluyveromyces lactis S25, Debaryomyces hansenii R3, D. occidentalis R1, and Yarrowia lipolytica S12) isolated from blue cheese were selected to prepare cell-free extracts and observe their growth promotion effects on three strains of bifidobacteria (B. breve ATCC1 5700, B. infantis ATCC1 5697 and B. longum ATCC1 5707) cultivated in MRS broth supplemented with cysteine-hydrochloride (0.05%). Optical density observation revealed that addition of these yeast extracts at a concentration of 0.1% was sufficient to stimulate the growth of these bifidobacteria strains. In particular, the yeast extract from K. lactis S25 induced the logarithmic phase earlier for B. infantis ATCC1 5697 and B. longum ATCC15707. The effect of yeast extract from K. lactis S25 was further investigated using five strains of bifidobacteria (B. bifidum ATCC1 5696, B. breve ATCC1 5700, B. infantis ATCC1 5697, B. longum BB536 and ATCC15708) in the skim milk culture system. In the presence of yeast extracts, the viable cell count was higher than for that of the control, in particular for B. bifidum ATCC1 5696 and B. longum ATCC 15708 and BB536. The viability of B. bifidum ATCC15696 and B. longum BB536 during refrigeration was significantly improved when yeast extracts from K. lactis were added.
  • T. Khusniati, W. -S. Kim, S. Yanagisawa, H. Kumura, K. Shimazaki
    JOURNAL OF FOOD SAFETY 28 (4) 601 - 608 0149-6085 2008/11 [Refereed][Not invited]
     
    In order to study the possibility of utilizing aromatic materials for the preservation of milk, the antibacterial effects of various aromatic materials against Pseudomonas fluorescens inoculated in milk were investigated. The effects of aromatic materials were generated using the "vapor contact method" and bacterial growth was measured by the menadione-catalyzed luminol chemiluminescence method. Wasabi (Wasabi japonica) and mustard (Brassica nigra L.) were found to be more effective than garlic (Allium sativum L.), ginger (Zingiber officinale Roscoe), sansho (Zanthoxylum piperitum) and peppermint (Mentha piperita). In addition to these materials, four types of commercially available food preservation sheets were tested for antibacterial activity. Sheets containing mustard extracts showed stronger antibacterial activities. Eucalyptus extract-treated sheets showed a moderate effect.
  • Md. M. Rahman, W. -S. Kim, T. Ito, H. Kumura, K. Shimazaki
    APPLIED BIOCHEMISTRY AND MICROBIOLOGY 44 (5) 478 - 481 0003-6838 2008/09 [Refereed][Not invited]
     
    In the present study, lactoferrin binding to bifidobacteria and detection of lactoferrin-binding protein in membrane fractions of several bifidobacteria have been demonstrated. This is the first report showing the binding of bovine lactoferrin to four Bifidobacterium spp. (B. infantis, B. breve, B. bifidum, and B. longum) incubated with biotinylated lactoferrin and fluorescein-conjugated avidin and observed under an inverted confocal laser scanning microscope. Fluorescence staining showed lactoferrin binding at the pole of the bacterial cells. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the membrane fraction of Bifidobacterium spp. by far-western blotting technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on the results of this and previously reported studies, we suggest that binding of lactoferrin to Bifidobacterium longum is strain dependent.
  • Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki
    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 24 (8) 1593 - 1598 0959-3993 2008/08 [Refereed][Not invited]
     
    Thirteen strains of four different Bifidobacterium spp. were observed for their autoaggregation ability and surface hydrophobicity, and correlation between these two traits was determined. Bifidobacteria were classified into high, medium and low autoaggregation strains according to autoaggregation ratio measured from changes in absorbance of media. High autoaggregation strains showed microscopic clustering of cells, whereas low and medium autoaggregation strains showed no such clustering. Autoaggregation ability decreased in high autoaggregation strains but increased in medium and low autoaggregation strains when the assay was performed at higher temperature (37 degrees C compared with 25 and 10 degrees C). Bacterial strains belonging to the high, medium or low autoaggregation group were correlated in terms of their surface hydrophobicity and evaluated based on changes in absorbance of the bacterial suspension before and after extraction with xylene. These results indicate that autoaggregation ability, together with surface hydrophobicity and microscopic image could be used for evaluating the adhesion ability of potential probiotic bifidobacterial strains. Moreover, a synergistic effect of pH and media may be involved in autoaggregation.
  • Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kel-Lchi Shimazaki
    ANAEROBE 14 (2) 73 - 77 1075-9964 2008/04 [Refereed][Not invited]
     
    Changes in autoaggregation ability and surface hydrophobicity of bifidobacteria with addition of bovine lactoferrin in liquid media were investigated. Lactoferrin addition caused loss of autoaggregation ability, disappearance of microscopic clusters and produced consistent turbidity in the cultured medium compared with control. Similar outcomes with addition of bovine lactoferrin hydrolysates (pepsin), bovine transferrin or ovotransferrin suggested that the effect is not lactoferrin-specific. On the other hand, addition of proteins, except bovine transferrin, did not alter surface hydrophobicity. These results indicate that one or more surface components involved in autoaggregation of bifidobacteria are proteins. (c) 2008 Elsevier Ltd. All rights reserved.
  • Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 62 (1) 6 - 8 0026-3788 2007 [Refereed][Not invited]
     
    Three isoforms (bLf-1, bLf-2 and bLf-3) were purified from bulk bovine lactoferrin using ion exchange chromatography. The purified isoforms were identified by SDS-PAGE and Western blot analysis with anti-lactoferrin antibody. The molecular masses of bLf-1, bLf-2 and bLf-3 were estimated to be 84.6, 83.1 and 83.1 kDa, respectively, by MALDI-TOF mass spectrometry. Lactobacillus acidophilus CH-2 was used to investigate whether these isoforms have any effect on bacterial growth. The bacterial strain was grown in MRS medium with bovine lactoferrin or bovine lactoferrin isoforms, or without lactoferrin (control) using a 96-well microplate. Growth patterns at different times were measured by monitoring the absorbance using a microplate reader at 600 nm. All purified isoforms, as well as bovine lactoferrin, exhibited significant growth stimulation effects on L. acidophilus CH-2 as compared with the control. Furthermore, isoform with the highest molecular mass exhibited the most potent effect. Thus, bovine lactoferrin isoforms apparently exert prebiotic effects.
  • Ran E. Yoshise, M. Matsumoto, H. Chiji, H. Kuwata, K. Shin, K. Yamauchi, Y. Tamura, T. Tanaka, H. Kumura, K. Shimazaki
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 62 (4) 446 - 450 0026-3788 2007 [Refereed][Not invited]
     
    Lactoferrin is an important bioprotective milk protein and acts to protect infants against bacterial attack in both direct and indirect manners. The former includes antimicrobial effects against pathogenic bacteria, i.e., bacteriostatic and bactericidal activities. The latter includes modulation of the immune system through action against intestinal epidermal cells and Peyer's patch, and through the control of intestinal microflora, as growth of lactic acid bacteria and bifidobacteria populations stimulated by lactoferrin. In this report, the behavior of lactoferrin orally administered to rats was analyzed using ELISA and Western blot analysis with anti-lactoferrin polyclonal antibody and anti-N-lobe and C-lobe monoclonal antibody. The amounts ingested were 900 mg lactoferrin/kg body weight. Thirty minutes after oral administration of lactoferrin solution (10%, 2 ml), lactoferrin and its fragments were detected in the stomach and small intestine. In the cecum, lactoferrin was detected 120 min after administration. Interestingly, the C-lobe but not the N-lobe of lactoferrin was detected by Western blot analysis using anti-N-lobe and anti-C-lobe monoclonal antibodies. These results were confirmed by SELDI affinity mass spectrometric assay.
  • RAHMAN Md. Morshedur, KIM Woan-Sub, ITO Toshiaki, KUMURA Haruto, SHIMAZAKI Kei-ichi
    Bioscience of Microbiota, Food and Health JAPAN BIFIDUS FOUNDATION 26 (3) 75 - 79 1342-1441 2007 [Refereed][Not invited]
     
    In the present study, binding of bovine lactoferrin to bifidobacteria was demonstrated. This is the first report showing the binding of bovine lactoferrin to Bifidobactesrium spp. under a transmission electronic microscope using biotinylated bovine lactoferrin and gold-conjugated streptavidin. In addition, we confirmed that bovine lactoferrin-binding protein exists on the surface of bifidobacteria.
  • Haruto Kumura, Natsuko Minato, Kei-Ichi Shimazaki
    JOURNAL OF DAIRY RESEARCH 73 (4) 449 - 453 0022-0299 2006/11 [Refereed][Not invited]
  • Tetsuya Tanaka, Shin Murakami, Haruto Kumura, Ikuo Igarashi, Kei-ichi Shimazaki
    BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 84 (5) 774 - 779 0829-8211 2006/10 [Not refereed][Not invited]
     
    Toxoplasina gondii is an obligatory intracellular parasitic protozoan transmitted via the ingestion of raw, infected meat that causes congenital infections. In a cell-free environment, virulent Toxoplasnia was strikingly resistant to H2O2. The activity of H2O2 or H2O2 generated by glucose-glucose oxidase against the resistant tachyzoite stage of pathogenic T. gondii was enhanced by adding KI and bovine lactoperoxidase (bLPO), referred to here as the bLPO system. Replacing bLPO (heme content, 90%) with recombinant bLPO (heme content, 6%) did not enhance the parasiticidal activity with KI and H2O2. These results indicated that heme contributed to the enzyme activity and resulted in the killing of tachyzoites of T gondii. Tachyzoites treated with the bLPO system also lost the ability to penetrate the mouse fibroblast cell line (NIH/3T3), and could be killed intracellularly after exposure by bLPO to a mouse macrophage cell line (J774A.1). These findings suggested that toxicity was mediated through small amounts of H2O2 generated by phagocytic events in naive macrophages, and by the peroxidative activity of bLPO. Our observations suggest that the bLPO system could help prevent the development of Toxoplasmosis in humans after ingesting raw, infected meat.
  • K Takase, K Hagiwara, H Onodera, Y Nishizawa, M Ugaki, T Omura, S Numata, K Akutsu, H Kumura, K Shimazaki
    BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 83 (2) 239 - 249 0829-8211 2005/04 [Refereed][Not invited]
     
    The milk protein, lactoferrin, is known to have antibacterial, antiviral, and antifungal activities. To explore the possibility of conferring disease resistance in plants by expressing this protein, the gene for the full-length human lactoferrin (HLF), as well as the N-lobe, the N-terminal half molecule (HLFN), was introduced into rice plants and expressed constitutively under the control of the cauliflower mosaic virus 35S promotor. Western blot analysis of leaves from HLF-transgenic rice plants showed an 80 kDa-band, which was about 1-2 kDa less than human milk lactoferrin. HLFN was expressed as a 45-kDa protein and retained its heparin-binding property. Deglycosylation experiments suggested that both proteins produced by the plants had plant-type oligosaccharide chains. The transgenic rice plants were assessed for resistance against disease-causing bacteria, virus, and fungi. Of the pathogens tested, significant resistance against Burkholderia (Pseudomonas) plantarii, the causative agent of bacterial seedling blight disease, was observed in the transgenic plants expressing HLF or HLFN.
  • KIM Woan-Sub, RAHMAN Md. Morshedur, KUMURA Haruto, SHIMAZAKI Kei-ichi
    Bioscience of Microbiota, Food and Health JAPAN BIFIDUS FOUNDATION 24 (4) 119 - 123 1342-1441 2005 [Refereed][Not invited]
     
    The effect of bovine lactoferrin hydrolysates on the growth of four species of bifidobacteria, B. bifidum, B. longum, B. breve and B. infantis was investigated. It was observed that the growth of 3 species of bifidobacteria (B. bifidum, B. breve and B. infantis) was stimulated by bovine lactoferrin hydrolysates. These results suggest the possibility that lactoferrin, digested in the intestine, acts as a bifidogenic factor for the growth of bifidobacteria.
  • H Kumura, Y Tanoue, M Tsukahara, T Tanaka, K Shimazaki
    JOURNAL OF DAIRY SCIENCE 87 (12) 4050 - 4056 0022-0302 2004/12 [Refereed][Not invited]
     
    To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir. Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Among the 8 species, K. lactis showed higher adhesive ability than K. marxianus, K. lodderae, and D. hansenii. The other 4 species were poorly adhesive. All species other than K. marxianus and C. humilis were resistant to acidic conditions. In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27degreesC; however, it was evident for C. humilis and a strain of D. occidentalis when incubated at 37degreesC. Moreover, the influence of proteinase treatment of living cells of K. lactis and K. lodderae on their adhesion to Caco-2 cells was evaluated. Although a slight reduction was recognized when K. lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible. These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K. lactis and K. lodderae to Caco-2 cells. No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K. lactis. In conclusion, K. lactis was the most attractive to continue study for use as probiotic microorganisms.
  • H Kumura, A Miura, E Sato, T Tanaka, K Shimazaki
    JOURNAL OF DAIRY RESEARCH 71 (4) 500 - 504 0022-0299 2004/11 [Refereed][Not invited]
  • NY Lee, K Kawai, Nakamura, I, T Tanaka, H Kumura, K Shimazaki
    JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (10) 1267 - 1269 0916-7250 2004/10 [Not refereed][Not invited]
     
    Antibacterial effects of bovine lactoferrin were studied in vitro against microorganisms isolated from mastitic milk in Tokachi area, Hokkaido, Japan. Microorganisms isolated were Eschelichia coli (11 isolates), Klebsiella pneumoniae (5 isolates), enterococci (8 isolates), Staphylococcus aureus (10 isolates), coagulase negative staphylococci (CNS, 13 isolates), streptococci (11 isolates), Prototheca zopfii (7 isolates) and yeast-like fungi (9 isolates). Lactoferrin has been known as a multifunctional protein and its antimicrobial effect is one of the most essential function of it. In order to compare their susceptibilities against lactoferrin, the minimal inhibitory concentration values were estimated by a microplate assay method using 96-well microplate, which involved measuring the optical density of the cultures. Prototheca zopfii was highly sensitive to bovine lactoferrin and complete inhibition of this microorganism was observed even at the low concentration of 7 mug/ml. On the other hand, E. coli and enterococci showed resistance against lactoferrin action and staphylococci showed strain-dependent resistance.
  • T Tanaka, H Morita, YC Yoo, WS Kim, H Kumura, KI Shimazaki
    JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (7) 865 - 869 0916-7250 2004/07 [Not refereed][Not invited]
     
    Lactoferrin (Lf), a member of the transferrin family protein, is an iron-binding protein that is known to interact with mammalian cells through a specific receptor. We examined binding of Lf to Jurkat human lymphoblastic T cell line (Jurkat cells) by far Western blotting, and found that bovine Lf and human Lf bound to the same protein components of Jurkat cells, and that pepsin digestion of Lf disrupts the sites responsible for binding to cellular proteins. We also found that the sugar chains of bovine Lf are not involved in binding between bovine Lf and Jurkat cells. Bovine Lf, bovine transferrin and ovotransferrin bound to the same proteins of Jurkat cells, which had molecular weights of about 35 kDa.
  • WS Kim, M Ohashi, T Tanaka, H Kumura, GY Kim, IK Kwon, JS Goh, K Shimazaki
    BIOMETALS 17 (3) 279 - 283 0966-0844 2004/06 [Refereed][Not invited]
     
    We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.
  • Y Wang, A Suzuki, T Tanaka, H Kumura, K Shimazaki
    JOURNAL OF DAIRY SCIENCE 87 (6) 1627 - 1633 0022-0302 2004/06 [Refereed][Not invited]
     
    Degradation of dextran beads was observed when the water-soluble fraction of a blue cheese extract was applied to the top of a Sephadex G-150 or G-200 column. This phenomenon suggests the presence of a specific enzyme that can hydrolyze dextran. After removal of casein components from the blue cheese fraction, ammonium sulfate treatment and gel filtration chromatography were performed to isolate the enzyme fraction. The enzymatic products were analyzed by thin-layer chromatography and gel filtration chromatography and identified as isomaltooligosaccharides. The isoelectric point of this enzyme fraction was approximately 4.9, as determined by isoelectric focusing using Rotofor, and the molecular weight of the fraction was 65 kDa, as estimated by sodium dodecyl sulfate (SDS)-PAGE. Optimum pH for enzymatic activity was 5.0 to 5.3. A partial N-terminal amino acid sequence of 20 residues was determined to be ATPDEWRSRSIYFMLTDRGA from an enzyme fraction further purified by ion-exchange chromatography and native PAGE. This sequence showed a maximum homology of 80% with alpha-amylase or Taka amylase that originated from various microorganisms.
  • T Tanaka, Y Abe, N Inoue, WS Kim, H Kumura, H Nagasawa, Igarashi, I, K Shimazaki
    JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (6) 619 - 625 0916-7250 2004/06 [Not refereed][Not invited]
     
    Trypanosoma brucei, the causative agent of sleeping sickness in humans, requires transferrin (TF) for growth. Therefore, T. brucei has a TF receptor that allows it to obtain iron from TF. Lactoferrin (LF), a member of the TF family protein, is an iron-binding protein that is found in most biological fluids of mammals. LF has been shown to interact with some bacteria species by specific receptor-ligand binding. We examined the ability of T. brucei to bind bovine LF (bLF) by using a fluorescence test, streptavidin-biotin (SAB) microplate analysis, and far Western blotting using a biotin-streptavidin system. We found that bLF bound to components of T brucei, and that bLF hydrolysate disrupted the sites responsible for binding to parasite proteins. Furthermore, bLF, human LF, bovine TF, and ovotransferrin bound same proteins of T brucei, which exhibited molecular masses of 40 and 43 kDa. The N-terminal amino acid sequence of the 40 kDa bLF binding protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
  • T Tanaka, Y Abe, WS Kim, Xuan, X, H Nagasawa, Igarashi, I, H Kumura, K Shimazaki
    JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (12) 1377 - 1380 0916-7250 2003/12 [Refereed][Not invited]
     
    Lactoferrin (LF), a member of the transferrin (TF) protein family, is an iron-binding protein that is known to interact with bacteria through a specific receptor. We examined the binding of bovine LF (bLF), bovine TF (bTF), and ovotransferrin (OTF) by Toxoplasma gondii using a fluorescence test and the streptavidin-biotin (SAB) method using biotin-streptavidin, and found that bLF, bTF, and OTF bound to the protein components of T. gondii. Furthermore, we confirmed that bLF, bTF, and OTF bound a 42 kDa soluble protein of T. gondii by far Western blot method. These results demonstrated that bLF binding proteins are present on T gondii.
  • T Tanaka, S Nakatani, XN Xuan, H Kumura, Igarashi, I, K Shimazaki
    ANTIVIRAL RESEARCH 60 (3) 193 - 199 0166-3542 2003/11 [Not refereed][Not invited]
     
    Lactoferrin (LF) is an iron-binding protein that is found in milk and other mammalian secretions. We found that bovine lactoferrin (bLF) inhibited both the in vitro infection and replication of canine herpesvirus (CHV) in Madin-Darby canine kidney (MDCK) cells. Incubation of CHV with bLF prevented subsequent infection of MDCK cells. Furthermore, proteins from CHV-infected MDCK cells were resolved by SDS-PAGE, and then bLF CHV-binding proteins were identified by far Western blotting. We demonstrated that the anti-CHV activity of bLF was due to its interaction with CHV as well as with MDCK cells. Both the apo- and holo-forms of bLF inhibited virus multiplication independently of their iron-withholding properties. We also demonstrated that human LF had anti-CHV activity. Our findings suggest that LF could be effective in dogs to provide protection against CHV infection. (C) 2003 Elsevier B.V. All rights reserved.
  • T Tanaka, Nakamura, I, NY Lee, H Kumura, K Shimazaki
    BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 81 (5) 349 - 354 0829-8211 2003/10 [Not refereed][Not invited]
     
    Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.
  • T Tanaka, S Sato, H Kumura, K Shimazaki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 (10) 2254 - 2261 0916-8451 2003/10 [Refereed][Not invited]
     
    Lactoperoxidase (LPO) is a heme-containing oxidation-reduction enzyme present in milk. In this study, the gene encoding bovine lactoperoxidase (bLPO) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLPO was isolated. A bLPO-related recombinant baculovirus-expressed protein of 78 kDa was detected using anti-bLPO antibodies. After digestion with N-glycosidase F, the molecular weight of the recombinant bLPO (rbLPO) decreased. In addition, rbLPO reacted with lectin, indicating that the protein was glycosylated. The rbLPO activity and heme content in the culture supernatants increased upon addition of delta-aminolevulinic acid, which is a heme precursor. Differences in the delta-aminolevulinic acid-dependent circular dichroism spectrum and rbLPO pepsin hydrolysis were observed. These results suggest that the secondary structure and structural stability of rbLPO depends on the heme environment. Our data suggest that this bLPO expression system is useful for studying structure, catalytic mechanisms, and biological function.
  • K Shimazaki, S Maki, Y Wang, M Sato, T Tanaka, H Kumura
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 58 (7-8) 379 - 382 0026-3788 2003 [Refereed][Not invited]
     
    Degradation of dextran beads was observed when the water-soluble fraction of a blue cheese extract was applied to the top of a Sephadex G-150 or G-200 column. This phenomenon suggested the presence of a specific enzyme that can hydrolyze dextran. No degradation of Sephadex G-10, G-25 or G-100, Sephacryl S-300 or S-400, Sepharose CL-2B or CL-4B, agar, agarose or Toyopearl were observed. Three varieties of commercially available blue cheese were studied and the dextran hydrolysis activity of each was investigated by Sephadex-test tube method and colorimetric method. The blue cheese varieties each exhibited unique pH- and temperature-dependent behaviors. One sample showed maximal activity at pH 4.7-4.8 and the others showed a broad pH range between 4.5 and 6.5 for higher activity. Two samples lost their activities at 50degreesC but the third one kept its activity at 60degreesC. These phenomena may be due to differences in microorganisms used during the cheese production process.
  • A Kimura, H Kumura, S Ishizuka, K Mikawa, K Shimazaki, Z Saito
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 58 (11-12) 609 - 611 0026-3788 2003 [Refereed][Not invited]
     
    The lipolytic activity of Lactococcus lactis ssp. cremoris was analyzed using gas chromatography and an agar plate method. A cell free extract obtained by sonication of the living cells was inactive toward milk fat triglycerides. However, production of free fatty acids by the cell free extract was detected when the substrate had been pretreated with indigenous bovine milk lipase. In the cell free extract, monoglyceride lipase activity was detected, independent of the number of carbons in the fatty acids. Thus, it was concluded that the bovine milk lipase degraded milk fat triglycerides to monoglycerides, which were then ready to be hydrolyzed by a monoglyceride lipase in the cell free extract to release free fatty acids. It is likely that the lipolytic contribution of this bacterial strain contributes to flavour development during cheese ripening, due mainly to the hydrolytic activity toward monoglycerides in cheese curd.
  • MS Nam, M Kamio, KI Shimazaki, S Harakawa, T Tanaka, Y Omata, A Saito, H Kumura, Igarashi, I, N Suzuki
    FOOD AND AGRICULTURAL IMMUNOLOGY 14 (2) 139 - 146 0954-0105 2002/06 [Refereed][Not invited]
     
    The reactivity towards the monoclonal antibody (MAb) against bovine lactoferrin C-lobe was compared with six members of the transferrin family of proteins by ELISA and dot-blotting methods. This MAb recognizes WNIPMGL (467-473) of the bovine lactoferrin sequence. Human lactoferrin, Korean native goat lactoferrin and bovine transferrin showed weak reactivity with anti-C-lobe MAb. Human transferrin and ovotransferrin did not react with this antibody. As human lactoferrin in the denatured state showed reactivity with the antibody by ELISA, the configurational features of bovine lactoferrin and human lactoferrin were compared. It is observed that human lactoferrin epitopic site is covered with the bulky group and becomes exposed after denaturation.
  • H Kumura, K Takagaki, T Sone, M Tsukahara, T Tanaka, K Shimazaki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 (6) 1370 - 1373 0916-8451 2002/06 [Not refereed][Not invited]
     
    To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein. Growth and casein degradation were recognized at the cheese-ripening temperature. Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on beta-casein. The cytosol contained more than 90% of the total proteolytic activity which was responsible for the degradation of both alpha(s)- and beta-casein. These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.
  • WS Kim, T Tanaka, H Kumura, K Shimazaki
    BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 80 (1) 91 - 94 0829-8211 2002/02 [Refereed][Not invited]
     
    Lactoferrin is an iron-binding glycoprotein and its bacteriostatic and bactericidal effects on gram-positive and gram-negative bacteria are well known. On the other hand, it is known that certain kinds of lactic acid bacteria are resistant to its antibacterial effects. Moreover, it is reported that lactoferrin promotes the growth of bifidobacteria in in vitro and in vivo experiments. In our experiments, lactoferrin-binding protein was found both in the membrane and cytosolic fractions of Bifidobacterium bifidum Bb-11. The bifidobacteria were grown in anaerobic conditions with lactobacilli MRS broth containing cysteine, harvested by centrifugation, and processed by sonication. The lactoferrin-binding proteins on the PVDF-membrane transferred after SDS-PAGE were detected by far-Western (western-Western) method using biotinylated lactoferrin and streptavidin-labelled horse radish peroxidase. The molecular weights of the lactoferrin binding protein detected in the membrane fraction were estimated to be 69 kDa and those in cytosolic fractions were 20, 35, 50, and 66 kDa.
  • H Kumura, A Tanaka, Y Abo, S Yui, K Shimazaki, E Kobayashi, K Sayama
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 (9) 2098 - 2101 0916-8451 2001/09 [Not refereed][Not invited]
     
    Porcine mammary epithelial cells were isolated to culture on collagen gel followed by gel floating treatment to evaluate differentiation under the culture conditions of serum-free medium, supplemented with combinations of insulin, hydrocortisone, and prolactin. After the culture period, the mammary cells attached to the collagen gels were recovered to observe expression of beta -casein, beta -lactoglobulin, and lactoferrin by reverse transcriptase polymeric chain reaction method. Expression of beta -casein was observed in the presence of insulin, hydrocortisone, and prolactin whereas transcription of beta -lactoglobulin and lactoferrin occured irrespective of hydrocortisone and prolactin. Immunoblot analysis demonstrated synthesis and secretion of lactoferrin in the fraction of recovered cells and the culture medium.
  • Nakamura, I, A Watanabe, H Tsunemitsu, NY Lee, H Kumura, K Shimazaki, Y Yagi
    PROTEIN EXPRESSION AND PURIFICATION 21 (3) 424 - 431 1046-5928 2001/04 [Not refereed][Not invited]
     
    Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation. (C) 2001 Academic Press.
  • H Kumura, T Sone, K Shimazaki, E Kobayashi
    JOURNAL OF DAIRY RESEARCH 67 (4) 631 - 636 0022-0299 2000/11 [Not refereed][Not invited]
  • S Watanabe, S Murata, H Kumura, S Nakamura, A Bollen, N Moguilevsky, K Shimazaki
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 274 (3) 756 - 761 0006-291X 2000/08 [Refereed][Not invited]
     
    Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The a-helix, p-structure, and unordered structure contents were found to be 17.8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly, A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined. (C) 2000 Academic Press.
  • MS Nam, DY Yu, M Kimura, H Kumura, K Shimazaki
    ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 13 282 - 282 1011-2367 2000/07 [Refereed][Not invited]
  • M Kimura, MS Nam, Y Ohkouchi, H Kumura, K Shimazaki, DY Yu
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 268 (2) 333 - 336 0006-291X 2000/02 [Refereed][Not invited]
     
    The antimicrobial activity of lactoferrin isolated from Korean native goat (RN goat) milk was studied and its antimicrobial domain was identified using synthetic peptides. Antimicrobial activity was assayed by a micro-method using 96-well microplates and a microplate reader. The amino acid sequence of the antimicrobial domain was suggested to be YQWQRRM-RKLGAPSIT and this sequence corresponds to amino acid residues 20 to 35 of KN goat lactoferrin. Five peptides with certain amino acid residues deleted were synthesized in an effort to identify the residues essential for antimicrobial activity and it was found that the part with the sequence RRMRK (24-28) is the region most important for this activity. On the other hand, the conformation of the peptides did not influence the antimicrobial activity. a 2000 Academie Press.
  • K Shimazaki, K Uji, T Tazume, H Kumura, T Shimo-Oka
    LACTOFERRIN: STRUCTURE, FUNCTION AND APPLICATIONS 1195 37 - 46 0531-5131 2000 [Refereed][Not invited]
     
    The affinity of lactoferrin for heparin is one its well-known properties. Certain consensus sequences have been proposed for other heparin-binding proteins, such as BBXB, BBBXXB or BXXBBXB, where B denotes a positively charged amino acid residue. The purpose of the present study was to identify the essential amino acid side chain groups of the lactoferrin molecule contributing to the interaction with heparin. The heparin-interacting properties of transferrin family proteins were compared by examining the heparin-binding activity of various peptides prepared by chemical synthesis. Each peptide was composed of 10-15 amino acid residues and was synthesized from Fmoc amino acid active esters on a preactivated cellulose membrane using the SPOTs(TM) system. Each of the peptides was incubated with heparin. To detect heparin-interaction, human vitronectin and alkaline phosphatase-conjugated antivitronectin monoclonal antibody were used. Peptides corresponding to partial sequences of human, bovine, pig and goat lactoferrins, human transferrin, chicken ovotransferrin and human melanotransferrin were studied. The results obtained were as follows: of the two BXBB sequences in the bovine lactoferrin N-lobe, KCRR (18-21) and RMKK (25-28), the latter was found to be essential for interaction with heparin; the BXBB sequence in the C-lobe did not interact with heparin; BXBB and BBXB sequences in human transferrin showed no interaction with heparin. These results were consistent with findings obtained in affinity chromatography experiments using an immobilized heparin column.
  • H Kumura, S Murata, T Hoshino, K Mikawa, K Shimazaki
    JOURNAL OF DAIRY SCIENCE 82 (10) 2078 - 2083 0022-0302 1999/10 [Refereed][Not invited]
     
    The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-termal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.
  • E Kawai, A Idei, H Kumura, K Shimazaki, H Akatsuka, K Omori
    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1446 (3) 377 - 382 0167-4781 1999/09 [Refereed][Not invited]
     
    In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently. (C) 1999 Elsevier Science B.V. All rights reserved.
  • MS Nam, K Shimazaki, H Kumura, KK Lee, DY Yu
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 123 (2) 201 - 208 0305-0491 1999/06 [Refereed][Not invited]
     
    We purified lactoferrin from the colostrum of the Korean native goat (Capra hircus) by ion-exchange chromatography using CM-Toyopearl 650M followed by affinity chromatography on AF-Heparin Toyopearl 650M. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis suggested the molecular mass of Korean native goat lactoferrin is 82 kDa with an iron saturation of 30%: as estimated by spectroscopic analysis. Circular dichroism analysis shows goat lactoferrin molecule contains 24.5%, alpha-helix; 36.0%, beta-structure; 13.5%, beta-turn and 26.0%, unordered structure. Heparin binding affinity is the same as that of bovine lactoferrin, but lower than that of human lactoferrin. An analysis using synthetic peptides shows that the peptide from residue 22 to 31 - WQRRMRKLGA - exerts a positive heparin-binding ability. (C) 1999 Elsevier Science Inc. All rights reserved.
  • KI Shimazaki, MS Nam, T Tazume, H Kumura, K Mikawa, KK Lee, DY Yu
    PEPTIDE SCIENCE - PRESENT AND FUTURE 759 - 760 1999 [Refereed][Not invited]
  • K Shimazaki, T Tazume, K Uji, M Tanaka, H Kumura, K Mikawa, T Shimo-Oka
    JOURNAL OF DAIRY SCIENCE 81 (11) 2841 - 2849 0022-0302 1998/11 [Refereed][Not invited]
     
    A heparin-binding peptide was isolated from a proteolytic hydrolysate of bovine lactoferrin by affinity chromatography using an immobilized heparin column. Analysis of amino acid sequences at the N-terminus showed that this heparin-binding peptide is derived from the region beginning at the 17th amino acid residue of the bovine lactoferrin sequence. The molecular mass of this peptide was 3195.5 as measured by matrix-assisted laser desorption-time of flight mass spectrometry. This peptide is the same as the bactericidal peptide lactoferricin(R) B. In an aqueous environment, this peptide displays mainly a P-sheet structure and an unordered structure as assessed by measurements of circular dichroism spectra. When this peptide was mixed with heparin, a distinct spectral change was induced because of conformational alteration of the peptide. This spectral change was reversible. Analysis of data from peptide synthesis indicated that binding by the sequence Arg(28)-Met(29)-Lys(30)-Lys(31) of bovine lactoferrin is significant and that there is a synergistic contribution from Lys(18)-Cys(19)-Arg(20)-Arg(21), and Arg(38)-Arg(39).
  • J Matos, M Nardi, H Kumura, Monnet, V
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 64 (11) 4591 - 4595 0099-2240 1998/11 [Refereed][Not invited]
     
    We sequenced the pepP gene of Lactococcus lactis, which encodes an aminopeptidase P (PepP), and demonstrated that the X-prolyl dipeptidyl aminopeptidase PepX plays a more important role than PepP in nitrogen nutrition. PepP shares homology with methionine aminopeptidases and could play a role in the maturation of nascent proteins.
  • H Kumura, S Hirose, H Sakurai, K Mikawa, F Tomita, K Shimazaki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 62 (11) 2233 - 2235 0916-8451 1998/11 [Refereed][Not invited]
     
    The gene encoding an extracellular lipase from Pseudomonas fluorescens No, 33 was cloned and sequenced. A single open reading frame consisting of 1,428 nucleotides that encoded a mature protein of 476 amino acids was recognized. Sequence analysis showed that the deduced molecular weight of 50,209 agreed with the molecular weight of the purified lipase as measured by SDS-PAGE and the lipase lacked a signal peptide. The presence of a repeating motif, GXXGXDXXX, suggested that the lipase might be exported and secreted via a system that involves the ATP-binding cassette protein.
  • H Kumura, Y Hiramatsu, Y Ukai, K Mikawa, K Shimazaki
    ADVANCES IN LACTOFERRIN RESEARCH 443 85 - 89 0065-2598 1998 [Refereed][Not invited]
  • Shimazaki K, Kamio M, Nam MS, Harakawa S, Tanaka T, Omata Y, Saito A, Kumura H, Mikawa K, Igarashi I, Suzuki N
    Advances in experimental medicine and biology 443 41 - 48 0065-2598 1998 [Refereed][Not invited]
  • K Shimazaki, MS Nam, S Harakawa, T Tanaka, Y Omata, A Saito, H Kumura, K Mikawa, Igarashi, I, N Suzuki
    JOURNAL OF VETERINARY MEDICAL SCIENCE 58 (12) 1227 - 1229 0916-7250 1996/12 [Refereed][Not invited]
     
    Bovine lactoferricin(R) (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs(TM) and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of ''QWR''.
  • H KUMURA, K MIKAWA, Z SAITO
    JOURNAL OF DAIRY SCIENCE 76 (8) 2164 - 2167 0022-0302 1993/08 [Refereed][Not invited]
     
    The effects of some milk proteins on the thermostability of the lipase from Pseudomonas fluorescens 33 were investigated. All purified milk protein fractions except kappa-casein that dissolved in phosphate buffer were effective for thermostabilization of the lipase. Thermal behavior of the lipase containing beta-lactoglobulin was so specific that, after heating at 80 to 90-degrees-C, activity remained high and was comparable with that of unheated treatment. The thermostability of the lipase containing whey proteins in synthetic salts solution was extensively lowered, but that containing casein micelles retained 50% of original activity after heat treatment at 80-degrees-C for 10 min. Low temperature inactivation of the lipase was influenced by concomitant milk proteins.
  • H KUMURA, K MIKAWA, Z SAITO
    JOURNAL OF DAIRY RESEARCH 60 (2) 229 - 237 0022-0299 1993/05 [Refereed][Not invited]
     
    The extracellular proteinase from Pseudomonas fluorescens No. 33 was purified to electrophoretic homogeneity by a procedure including precipitation with HCl and (NH4)2SO4, and column chromatography. The enzyme was purified 170-fold giving a yield of 7% of the original activity. The molecular mass of the purified enzyme was 48000 by SDS-PAGE. The optimum pH and temperature for the hydrolysis of casein were 8.0-9.8 and 30-35-degrees-C respectively. The enzyme was more thermostable in synthetic milk salts solution than in 0.1 M-sodium phosphate buffer, but was heat-labile at 50-degrees-C in both buffer systems. The activity was inhibited by o-phenanthroline, Hg2+, Cu2+, Fe2+ and, to a lesser extent, Ni2+. Caseins were susceptible to the proteinase, but degradation patterns were dependent on the form of the casein.
  • H KUMURA, K MIKAWA, Z SAITO
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 48 (8) 431 - 434 0026-3788 1993 [Refereed][Not invited]
     
    Extracellular lipase from Pseudomonas fluorescens No. 33 was purified to electrophoretic homogeneity by a procedure including acid precipitation, hydrophobic-interaction chromatography, ion-exchange chromatography and gel filtration. The enzyme was purified about 4200- fold giving a yield of 35 % of the original activity. The molecular weight of the enzyme was 52,000 by SDS-PAGE: The optimum pH and temperature for the hydrolysis of butter oil emulsion were 7.5-8.5 and 45-degrees-C, respectively. The enzyme was stable over the pH range 5.5-7.5. It was remarkably more heat-labile at 40-degrees-C than at 50-60-degrees-C. The activity was inhibited by Zn2+ and Hg2+.
  • Influence of concomitant protease on the thermostability of lipase of psychrotropic bacteria.
    Kumura H, Mikawa K, Saito Z
    Milchwissenschaft 46 144 - 149 1991 [Refereed][Not invited]
  • H KUMURA, K MIKAWA, Z SAITO
    MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 46 (4) 215 - 218 0026-3788 1991 [Refereed][Not invited]
     
    Lipase and protease produced by Pseudomonas sp. No. 33 originally isolated from raw milk were fractionated by ammonium sulphate and column chromatography on DEAE-Q Sepharose. The protease was further purified by column chromatography on Sephadex G-200. The purified protease preparation showed a purity index of 107, and was electrophoretically homogeneous with a molecular weight of 46,000 dalton. In the system free from protease, lipase retained higher lipolytic activity than that containing protease after heat treatment. Pseudo-LTI (low temperature inactivation) of lipase was observed irrespective of the presence of protease, which demonstrated that this phenomenon was not due to protease. However, the activity and thermostability of the lipase were affected by concomitant protease.

Books etc

  • 乳肉卵の機能と利用（新版）
    玖村 朗人 
    アイ・ケイコーポレーション 2018/09
  • 畜産物利用学
    文永堂 2011
  • 最新畜産物利用学
    朝倉書店 2006
  • 乳肉卵の機能と利用
    玖村 朗人 
    アイ・ケイコーポレーション 2005

MISC

Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013/04 -2017/03 
    Author : KUMURA HARUTO
     
    Monascus species, known as red-mold has the ability to produce diverse functional secondary metabolites such as lovastatin, monascin and ankaflavin which are responsible for health benefits including risk reduction of arteriosclerosis. It would be applicable for development of functional foods, however, some strains of Monascus sp. produce nephrotoxin, citrinin. Therefore, strain and culture condition should be carefully selected. In addition to the selection of test strains, we focused on the materials for culture substrate, which should be solid with convenience for preparation, if necessary, capability to add nutritional supplements, applicability to wide pH range and “ready to eat” property of the culture products. Following the screening of the suitale strain and culture condition, the resulting culture products were fed to experimental animals whether it could exert predicted biological effects.
  • 食用微生物代謝産物の機能性
    Date (from‐to) : 2009
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2006 
    Author : KUMURA Haruto
     
    In recent years, attention is being paid to the effect of fermented food products on the human health, especially probiotic microorganisms including lactic acid bacteria and Bifidobacterium spp. In this study, immunomodulatory effects and ability of promoting growth and improving viability of Bifidobacterium spp. of some yeast isolated from dairy origin were investigated. Despite statistical insignificance, oral administration of some yeasts resulted higher level of immunoglobulin A in the large intestine content of mice. Furthermore, spleen cells prepared from ovalbumin (OVA) immunized mice were incubated in the presence or absence of either OVA, lactic acid bacteria or the 12 yeast strains of 6 species. The results showed that the addition of OVA in the culture of spleen cells stimulated production of immunoglobulin E (IgE); conversely the addition of lactic acid bacteria or yeast cells debris to the culture suppressed IgE production. Although the suppression of IgE production and no induction of IFN-gamma were observed by all yeast strains tested; the level of IL-12 in the culture was found to be strain dependent. In addition, oral administration of a yeast and/or lactic acid bacteria to OVA immunized mice was conducted to monitor the serum IgE level. Reduction of serum IgE level in the experimental group was found to be undetectable as compared with control group. Ability of yeast cell to promote growth and improve viability during refrigeration storage of Bifidobacterium spp. was observed by the addition of yeast extracts to skim culture. Factor(s) associated with growth promotion of Bifidobacterium spp. was found to be thermostable because the effect was being existed even after boiling treatment with skim milk for 30 min. Yeast isolated from traditional food is considered as safe and an attractive source to design novel functional foods. Further investigation should be conducted f or beneficial application of yeast.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2002 -2004 
    Author : KIMURA Atsuo, MORI Haruhide, OKUYAMA Masayuki
     
    Glycosylases are enzymes that hydrolyze the glycosidic linkage. These enzymes also catalyze the transglycosidation, in which the glycosyl residue is transferred to the acceptor substrate. The transglycosidation is an important reaction i)to produce oligosaccharides valuable for foods and ii)to synthesize bio-active sugar-chains. Transglycosidation and hydrolysis proceed in the same time, meaning that the substrate for transglycosidation as well as its product(s) is cleaved by hydrolysis even under conditions of transglycosidation. We have studied the reactions of glycosylase, and have found the phenomena that catalyzed the transglycosidation only. In this study, we analyze the mechanism of valuable phenomena and perform their application. The results are summarized as follows. 1)We have determined the catalytic residue of negatively charged by the method using suicide substrate. Mutant enzyme (synthase), of which catalytic residue was replaced, was produce and purified. The enzyme showed no hydrolytic reaction, only catalyzed the synthesis of oligosaccharide(s) from fluoride-substrate and acceptor. Acceptor of aryl glycoside is a good substrate, meaning that the hydrophobic interaction between aryl-group and subsite +2 is important. 2)Mutant enzyme, which recognized the plane-shaped substrate, a mimic compound of reaction intermediate, was constructed, and its ability of oligosaccharide-synthesis was studied. The low production was observed. We changed the substrate concentration, and succeeded in the improvement of yield. Addition of alcohol to reaction mixture was also effective, but the high concentration of alcohol decreased the production of oligosaccharide. We have found a glycosidase resistant for alcohol. Currently, the conversion of alcohol-stable enzyme to mutant enzyme of same type is trying.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1999 -2000 
    Author : SHIMAZAKI Kei-ichi, TANAKA Tetsuya, KUMURA Haruto, NAKAMURA Shingo
     
    The cDNA encoding bovine lactoperoxidase has been expressed in CHO cells. The recombinant lactoperoxidase was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. recombinant lactoperoxidase exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by CHO cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by CD measurements. The α-helix, β-structure and unordered structure content was found to be 17.8%, 54.2% and 28.0% for the natural lactoperoxidase and 18.6%, 50.1% and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined. Engineering of recombinant lactoperoxidase into a myeloperoxidase-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in myeloperoxidase. However, the resulting bovine lactoperoxidase mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of myeloperoxidase, underlining the complex nature of interactions in the heme vicinity.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1997 -1998 
    Author : 玖村 朗人
     
    本研究は平成8年度文部省科学研究費補助金を得て開始されたもので、前年度までに細胞の分離・培養条件をほぼ確立し、細胞の分化機能を観察するためのポリクローナル抗体も調製した。昨年度の時点では分離した乳腺細胞の培養はプラスチックシャーレ上で行なっており、RT-PCRによってカゼインの発現が窺われたもののウエスタンブロットではその発現が検出できなかった。 今年度はまず乳腺細胞の分化により好適といわれているコラーゲン・ゲルを用いる浮遊培養法に改変し、カゼインの発現を詳細に明らかにしようとした。無血清培地にインシュリン、コルチゾール、プロラクチン等の因子を様々に組み合わせて観察した結果、インシュリンの単独添加でもカゼインの発現が認められることがRT-PCRとウエスタンブロットによって明らかになった。他の動物種由来の乳腺細胞のカゼイン発現に必須であると考えられているプロラクチンを添加してもカゼインの発現は増強されなかった。このことから培養したブタ乳腺上皮細胞はin vitroにおいてなお、カゼインの合成能を有していること、そしてそれはこれまで明かにされてきたプロラクチンとは異なる系が関与していることが示唆された。さらに、本研究は遺伝子導入による分化機能を維持した乳腺細胞の株化を目的としていることから、pSVneoを6種類のリポフェクトアミンによって培養した乳腺細胞に導入し長期間G418存在下で細胞が生存しうるかどうかを検討した。その結果、どのリポフェクトアミンを用いた場合からも長期間安定にベクターが機能する細胞を得るまでには至らなかった。使用するブタの履歴を特定化することや遺伝子を導入する方法を再検討することなどが今後の課題である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1996 -1996 
    Author : 玖村 朗人
     
    本研究課題であるブタ乳腺上皮細胞の分離・樹立を遂行するためには、1)乳腺組織からの上皮細胞の分離・培養条件の検討、2)in vitroにおける乳蛋白質合成能の確認、3)樹立株の構築のために適用する遺伝子導入法の検討ならびにその条件の最適化が不可欠である。ブタ乳腺上皮細胞の分離はこれまで報告例がなく、さらに遺伝子導入時の転換効率を考慮すれば、まずは他の細胞の混入が極力回避される上皮細胞の分離法の確立が望ましい。そこで、その条件を種々検討した結果、以下のようになった。 ブタ乳腺組織を細切した後、コラゲナーゼ、デイスパーゼ、ヒアルロニダーゼ、インスリン、DNaseを添加した199倍地を用いて37℃で5時間振とう処理した。一旦細胞を遠心分離によって回収し、DNase、プロナーゼを添加した199倍地を用いて37℃1時間処理した。回収した細胞をDNase、BSA、199培養液を加えて浮遊させ、パーコールを用いた密度勾配遠心分離法によって乳腺細胞の層を採取した。さらに、細胞をナイロンメッシュでろ過し、大きな細胞塊を取り除くと共に、その細胞浮遊液を静置しても沈まず分散した細胞も除去した。このようにして得た乳腺細胞の画分をインスリン、EGF、プロゲステロン、グルココルチコイドを単独あるいは併用で実験を行った結果、マウスやラットとは異なり、ブタ乳腺細胞の増殖にはグルココルチコイドが必要であることが明らかとなった。 細胞のin vitroにおける乳蛋白質の発現は未確認である。しかし、ブタミルクよりα-、β-カゼイン、β-ラクトグロプリンをカラムクロマトグラフィーによって精製し、マウスに免疫した結果、特に力価の高い抗体として抗β-カゼイン血清を得ることができたため、これを用いて分離した細胞の分化能に関する観察が可能であると考えられる。

Educational Activities

Teaching Experience

  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 食品、消化管、食品機能、腸内細菌叢、生理活性
  • Advanced Review of Food Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 農学院
    キーワード : 食品、消化管、食品機能、腸内細菌叢、生理活性
  • Advanced Seminar on Food Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 農学院
    キーワード : 食品、消化管、食品機能、腸内細菌叢、生理活性
  • Advanced Review of Agriculture Frontiers
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 農学院
    キーワード : 農学の最先端研究、食品、バイオマス、エネルギー、水土資源、農業経済、持続的生産、生物共生
  • Milk and Meat Hygiene
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 畜産食品、乳、肉、食品衛生、食中毒
  • Livestock-Farm Practice
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 草地、飼養管理、肉牛、馬
  • Graduation Thesis
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 家畜、家禽、遺伝学、繁殖、家畜栄養、家畜管理、家畜行動、食肉科学、酪農食品科学、副生物
  • Fundamental Science of Animal Products I
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 乳、卵、食品科学
  • Seminar in Animal Science Ⅰ
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 遺伝繁殖学，畜牧体系学，動物機能栄養学，細胞組織生物学，応用食品科学
  • Seminar in Animal Science Ⅱ
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 遺伝繁殖学，畜牧体系学，動物機能栄養学，細胞組織生物学，応用食品科学
  • Introduction of Animal Science
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 畜産科学、細胞組織生物学、応用食品科学、遺伝繁殖学、動物機能学、畜牧体系学
  • Applied Food Science
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 畜産食品、食用微生物
  • Animal Product Science
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 畜産物利用、機能性食品
  • Disease of Domestic Animals
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 家畜、疾病、畜産動物、予防、診断、生産性
  • Laboratory Exercise in Quality Control of Animal Products
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 品質管理、食品衛生、畜産食品、食品化学、食の安全

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