研究者情報

マスター

アカウント（マスター）

• 氏名

  玖村　朗人(クムラ　ハルト), クムラ　ハルト

所属（マスター）

• 北方生物圏フィールド科学センター

所属（マスター）

• 北方生物圏フィールド科学センター

独自項目

syllabus

• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 食品、消化管、食品機能、腸内細菌叢、生理活性
• 2021, 食品科学特論, Advanced Review of Food Science, 修士課程, 農学院, 食品、消化管、食品機能、腸内細菌叢、生理活性
• 2021, 食品科学特論演習, Advanced Seminar on Food Science, 修士課程, 農学院, 食品、消化管、食品機能、腸内細菌叢、生理活性
• 2021, 農学フロンティア総論, Advanced Review of Agriculture Frontiers, 修士課程, 農学院, 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
• 2021, 畜産食品衛生学, Milk and Meat Hygiene, 学士課程, 農学部, 畜産食品、乳、肉、食品衛生、食中毒
• 2021, 牧場実習, Livestock-Farm Practice, 学士課程, 農学部, 草地、飼養管理、肉牛、馬
• 2021, 卒業論文, Graduation Thesis, 学士課程, 農学部, 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
• 2021, 基礎畜産物利用学Ⅰ, Fundamental Science of Animal Products I, 学士課程, 農学部, 乳、卵、食品科学
• 2021, 畜産科学演習Ⅰ, Seminar in Animal Science Ⅰ, 学士課程, 農学部, 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
• 2021, 畜産科学演習Ⅱ, Seminar in Animal Science Ⅱ, 学士課程, 農学部, 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
• 2021, 畜産科学概論, Introduction of Animal Science, 学士課程, 農学部, 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
• 2021, 応用食品科学, Applied Food Science, 学士課程, 農学部, 畜産食品、食用微生物
• 2021, 畜産資源開発学, Animal Product Science, 学士課程, 農学部, 畜産物利用、機能性食品
• 2021, 家畜疾病学, Disease of Domestic Animals, 学士課程, 農学部, 家畜、疾病、畜産動物、予防、診断、生産性
• 2021, 畜産物品質管理学実験, Laboratory Exercise in Quality Control of Animal Products, 学士課程, 農学部, 品質管理、食品衛生、畜産食品、食品化学、食の安全

researchmap

プロフィール情報

学位

• 博士（農学）（北海道大学）

プロフィール情報

• 姓

  玖村, クムラ

• 名

  朗人, ハルト

• ID各種

  200901030461029285

業績リスト

研究キーワード

• 微生物   食品   

研究分野

• ライフサイエンス / 食品科学

論文

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

  Napaporn Chintagavongse, Haruto Kumura, Toru Hayakawa, Jun-Ichi Wakamatsu, Koichi Tamano

  Journal of bioscience and bioengineering 2024年03月01日 

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

  Haruka Abe, Yang Zhai, Yu Toba, Hiroki Masumo, Toru Hayakawa, Haruto Kumura, Jun-Ichi Wakamatsu

  Food chemistry 441 138317 - 138317 2024年01月02日 

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

  Yang Zhai, Haruka Abe, Hung-Cheng Wang, Toru Hayakawa, Haruto Kumura, Jun-Ichi Wakamatsu

  Food chemistry 427 136755 - 136755 2023年11月30日 

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

  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年06月 

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

  Toru Hayakawa, Yu Kubono, Shuji Fujii, Jun‐ichi Wakamatsu, Haruto Kumura

  Animal Science Journal 94 1 2023年01月
• Zinc protoporphyrin IX predominantly exists as a complex non-enzymatically bound to apo-hemoglobin in Parma ham.

  Yang Zhai, Hung-Cheng Wang, Toru Hayakawa, Haruto Kumura, Jun-Ichi Wakamatsu

  Food chemistry 395 133604 - 133604 2022年11月30日 

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

  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年01月23日 

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

  Md. Kauser-Ul-Alam, Toru Hayakawa, Haruto Kumura, Jun-ichi Wakamatsu

  Meat Science 176 108467 - 108467 2021年06月 [査読有り]
   

  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.
• Water-extractable zinc protoporphyrin IX in Parma ham predominantly exists as complexes with hemoglobin and myoglobin

  Hung-Cheng Wang, Toru Hayakawa, Haruto Kumura, Jun-ichi Wakamatsu

  Food Bioscience 40 100870 - 100870 2021年04月 [査読有り]
  
• 鉄，亜鉛およびポルフィリンの推移からみた食肉中の亜鉛プロトポルフィリンIX形成機構

  Nihon Chikusan Gakkaiho 91 4 389 - 394 2020年11月25日 [査読有り]
  
• Lactococcus lactis subsp. cremoris Produces Zinc Protoporphyrin IX Both Aerobically and Anaerobically and Improves the Bright Red Color of Fermented Meat Products

  Md. Kauser-Ul-Alam, Yu Toba, Shoji Hioki, Toru Hayakawa, Haruto Kumura, Jun-ichi Wakamatsu

  Foods 9 11 1583 - 1583 2020年10月31日 [査読有り]
   

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

  Md Asaduzzaman, Momo Ohya, Haruto Kumura, Toru Hayakawa, Jun-ichi Wakamatsu

  Meat Science 165 108109 - 108109 2020年07月 [査読有り]
   

  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.
• Attempt at the adjunctive use of solid-state culture products and its freeze-dried powder from Aspergillus sojae for semihard cheese.

  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年06月01日 [査読有り][通常論文]
   

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

  Wakamatsu JI, Kawazoe H, Ohya M, Hayakawa T, Kumura H

  Meat science 161 107989 - 107989 2019年10月 [査読有り][通常論文]
   

  Zinc protoporphyrin IX (ZnPP) mainly contributes to the red color of dry cured ham without nitrites/nitrates. Here, we examined the effects of acids used for pH adjustment, pH, and microorganisms on ZnPP formation. The results showed that ZnPP formation and optimal pH were dependent upon the acid type. In the presence of microorganisms, the optimal pH for ZnPP formation shifted to higher values, with the amount of formed ZnPP markedly increased at the shifted optimal pH. Additionally, two bacterial strains isolated from incubated pork homogenate exhibited an enhanced ability to form ZnPP. Although the two isolated bacteria are not edible, inoculation with one bacterium into minced meat resulted in formation of large amounts of ZnPP and color closer to that of nitrite-added meat. These results suggest that appropriate food-grade bacterial strains can improve the color of various fermented meat products in the absence of nitrites/nitrates.
• Investigation of contributors to zinc protoporphyrin IX formation at optimum pH 5.5 in pork

  Mofassara Akter, Akiko Shiraishi, Haruto Kumura, Toru Hayakawa, Jun‐ichi Wakamatsu

  Animal Science Journal 90 6 774 - 780 2019年06月 [査読有り]
   

  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.
• ‘Lipase and protease production of dairy Penicillium sp. on milk‐protein‐based solid substrates’

  Haruto Kumura, Megumi Satoh, Taiki Machiya, Makoto Hosono, Toru Hayakawa, Jun‐ichi Wakamatsu

  International Journal of Dairy Technology 72 3 403 - 408 2019年04月26日 [査読有り]
   

  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.
• Optimal pH of zinc protoporphyrin IX formation in porcine muscles: Effects of muscle fiber type and myoglobin content

  Jun-ichi Wakamatsu, Mofassara Akter, Fumika Honma, Toru Hayakawa, Haruto Kumura, Takanori Nishimura

  LWT 101 599 - 606 2019年03月 [査読有り]
  
• 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年 [査読有り][通常論文]
  
• Adjunctive application of solid-state culture products from Aspergillus oryzae for semi-hard cheese.

  Kumura, H, Saito, C, Taniguchi, Y, Machiya, T, Takahashi, Y, Kobayashi, K, Kimura, A

  Journal of Advances in Dairy Research 05 03 2017年 [査読有り][通常論文]
  
• Prolactin and glucocorticoid signaling induces lactation-specific tight junctions concurrent with β-casein expression in mammary epithelial cells.

  Kobayashi K, Tsugami Y, Matsunaga K, Oyama S, Kuki C, Kumura H

  Biochimica et Biophysica Acta 1863 8 2006 - 2016 2016年08月 [査読有り][通常論文]
   

  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.
• Pro-inflammatory cytokine TNF-α is a key inhibitory factor for lactose synthesis pathway in lactating mammary epithelial cells.

  Kobayashi K, Kuki C, Oyama S, Kumura H

  Experimental Cell Research 340 2 295 - 304 2016年01月 [査読有り][通常論文]
   

  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.
• Early down-regulation of milk production after weaning by pup removal and prior to involution in mouse mammary glands

  Takaaki Uejyo, Chinatsu Kuki, Shoko Oyama, Haruto Kumura, Ken Kobayashi

  CELL AND TISSUE RESEARCH 359 2 643 - 653 2015年02月 [査読無し][通常論文]
   

  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.
• Stimulatory Effect of Brazilian Propolis on Hair Growth through Proliferation of Keratinocytes in Mice

  Shota Miyata, Yozo Oda, Chika Matsuo, Haruto Kumura, Ken Kobayashi

  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 62 49 11854 - 11861 2014年12月 [査読無し][通常論文]
   

  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.
• Underlying mechanisms involved in the decrease of milk secretion during Escherichia coli endotoxin induced mastitis in lactating mice

  Ken Kobayashi, Shoko Oyama, Takaaki Uejyo, Chinatsu Kuki, Md Morshedur Rahman, Haruto Kumura

  VETERINARY RESEARCH 44 119  2013年12月 [査読無し][通常論文]
   

  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.
• Histological analysis of mammary gland remodeling caused by lipopolysaccharide in lactating mice

  Ken Kobayashi, Takaaki Uejyo, Shoko Oyama, Md. Morshedur Rahman, Haruto Kumura

  Cell and Tissue Research 354 2 495 - 506 2013年11月 [査読無し][通常論文]
   

  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.
• Lipopolysaccharide Disrupts the Milk-Blood Barrier by Modulating Claudins in Mammary Alveolar Tight Junctions

  Ken Kobayashi, Shoko Oyama, Atsushi Numata, Md. Morshedur Rahman, Haruto Kumura

  PLoS ONE 8 4 1 - 12 2013年04月23日 [査読無し][通常論文]
   

  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.
• Potential of polar lipids from bovine milk to regulate the rodent dorsal hair cycle

  H. Kumura, T. Sawada, Y. Oda, M. Konno, K. Kobayashi

  JOURNAL OF DAIRY SCIENCE 95 7 3629 - 3633 2012年07月 [査読有り][通常論文]
   

  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.
• Distinct behavior of claudin-3 and -4 around lactation period in mammary alveolus in mice

  Ken Kobayashi, Haruto Kumura

  HISTOCHEMISTRY AND CELL BIOLOGY 136 5 587 - 594 2011年11月 [査読無し][通常論文]
   

  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.
• Production and partial purification of proteases from Aspergillus oryzae grown in a medium based on whey protein as an exclusive nitrogen source

  H. Kumura, T. Ishido, K. Shimazaki

  JOURNAL OF DAIRY SCIENCE 94 2 657 - 667 2011年02月 [査読有り][通常論文]
   

  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.
• Continuous supply of OSCN- ions by lactoperoxidase system developed from lactose as the primary substrate and its antibacterial activities

  Takuya Ishido, Toki Nishiyama, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaiki, Hidemasa Motoshima, Kazuhiro Kawai

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 66 1 76 - 80 2011年 [査読有り][通常論文]
   

  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.
• Screening of Bifidobacterium spp. based on in vitro growth responses to bovine lactoferrin

  Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki

  INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY 45 3 453 - 458 2010年03月 [査読有り][通常論文]
   

  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.
• Growth performance of whey protein hydrolysates in the media on different strains of probiotic bacteria

  Seema Kafley, Woan-Sub Kim, Haruto Kumura, Kei-Ichi Shimazaki

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 65 3 245 - 248 2010年 [査読有り][通常論文]
   

  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.
• Growth promotion and cell binding ability of bovine lactoferrin to Bifidobacterium longum

  Md. Morshedur Rahman, Woan-Sub Kim, Toshiaki Ito, Haruto Kumura, Kei-ichi Shimazaki

  ANAEROBE 15 4 133 - 137 2009年08月 [査読有り][通常論文]
   

  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.
• Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins

  Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki

  BIOTECHNOLOGY LETTERS 31 6 863 - 868 2009年06月 [査読有り][通常論文]
   

  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.
• Potential of yeast from dairy origin to modulate immunoglobulin production

  Haruto Kumura, Shinya Takii, Ubune Iwase, Kei-ichi Shimazaki

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 64 1 10 - 13 2009年 [査読有り][通常論文]
   

  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.
• Growth and viability effects of cell-free extracts from dairy origin yeast on bifidobacteria

  Haruto Kumura, Yukie Ikezu, Erika Tajima, Kei-ichi Shimazaki

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 64 3 256 - 259 2009年 [査読有り][通常論文]
   

  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.
• UTILIZATION OF JAPANESE AROMATIC SUBSTANCES FOR MILK PRESERVATION AS ESTIMATED BY VAPOR CONTACT METHOD

  T. Khusniati, W. -S. Kim, S. Yanagisawa, H. Kumura, K. Shimazaki

  JOURNAL OF FOOD SAFETY 28 4 601 - 608 2008年11月 [査読有り][通常論文]
   

  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.
• Examination of bovine lactoferrin binding to bifidobacteria

  Md. M. Rahman, W. -S. Kim, T. Ito, H. Kumura, K. Shimazaki

  APPLIED BIOCHEMISTRY AND MICROBIOLOGY 44 5 478 - 481 2008年09月 [査読有り][通常論文]
   

  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.
• Autoaggregation and surface hydrophobicity of bifidobacteria

  Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki

  WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 24 8 1593 - 1598 2008年08月 [査読有り][通常論文]
   

  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.
• In vitro effects of bovine lactoferrin on autoaggregation ability and surface hydrophobicity of bifidobacteria

  Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kel-Lchi Shimazaki

  ANAEROBE 14 2 73 - 77 2008年04月 [査読有り][通常論文]
   

  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.
• Purification of bovine lactoferrin isoforms and their effects on growth of Lactobacillus acidophilus CH-2

  Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 62 1 6 - 8 2007年 [査読有り][通常論文]
   

  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.
• Profiles of bovine lactoferrin in the gastrointestinal tracts of rats as observed by ELISA, Western blotting and SELDI-affinity MS

  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 2007年 [査読有り][通常論文]
   

  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.
• Visualization of bovine lactoferrin binding to Bifidobacteria

  Morshedur R Md, Kim WS, Ito T, Kumura H, Shimazaki, K

  Bioscience Microflora 26 3 75 - 79 JAPAN BIFIDUS FOUNDATION 2007年 [査読有り][通常論文]
   

  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.
  
• Inhibitory activity of bovine milk osteopontin and its fragments on the formation of calcium phosphate precipitates

  Haruto Kumura, Natsuko Minato, Kei-Ichi Shimazaki

  JOURNAL OF DAIRY RESEARCH 73 4 449 - 453 2006年11月 [査読有り][通常論文]
  
• Parasiticidal activity of bovine lactoperoxidase against Toxoplasma gondii

  Tetsuya Tanaka, Shin Murakami, Haruto Kumura, Ikuo Igarashi, Kei-ichi Shimazaki

  BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 84 5 774 - 779 2006年10月 [査読無し][通常論文]
   

  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.
• Constitutive expression of human lactoferrin and its N-lobe in rice plants to confer disease resistance

  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 2005年04月 [査読有り][通常論文]
   

  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.
• Comparison of growth promoting effects on Bifidobacterium spp. by bovine lactoferrin hydrosates.

  Kim WS, Morshedur R Md, Kumura H, Shimazaki, K

  Bioscience Microflora 24 4 119 - 123 JAPAN BIFIDUS FOUNDATION 2005年 [査読有り][通常論文]
   

  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.
  
• Screening of dairy yeast strains for probiotic applications

  H Kumura, Y Tanoue, M Tsukahara, T Tanaka, K Shimazaki

  JOURNAL OF DAIRY SCIENCE 87 12 4050 - 4056 2004年12月 [査読有り][通常論文]
   

  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.
• Susceptibility of bovine osteopontin to chymosin

  H Kumura, A Miura, E Sato, T Tanaka, K Shimazaki

  JOURNAL OF DAIRY RESEARCH 71 4 500 - 504 2004年11月 [査読有り][通常論文]
  
• Susceptibilities against bovine lactoferrin with microorganisms isolated from mastitic milk

  NY Lee, K Kawai, Nakamura, I, T Tanaka, H Kumura, K Shimazaki

  JOURNAL OF VETERINARY MEDICAL SCIENCE 66 10 1267 - 1269 2004年10月 [査読無し][通常論文]
   

  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.
• Detection of bovine lactoferrin binding protein on Jurkat human lymphoblastic T cell line

  T Tanaka, H Morita, YC Yoo, WS Kim, H Kumura, KI Shimazaki

  JOURNAL OF VETERINARY MEDICAL SCIENCE 66 7 865 - 869 2004年07月 [査読無し][通常論文]
   

  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.
• Growth-promoting effects of lactoferrin on L-acidophilus and Bifidobacterium spp.

  WS Kim, M Ohashi, T Tanaka, H Kumura, GY Kim, IK Kwon, JS Goh, K Shimazaki

  BIOMETALS 17 3 279 - 283 2004年06月 [査読有り][通常論文]
   

  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.
• Partial characterization of dextran-degrading enzyme obtained from blue cheese

  Y Wang, A Suzuki, T Tanaka, H Kumura, K Shimazaki

  JOURNAL OF DAIRY SCIENCE 87 6 1627 - 1633 2004年06月 [査読有り][通常論文]
   

  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.
• The detection of bovine lactoferrin binding protein on Trypanosoma brucei

  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 2004年06月 [査読無し][通常論文]
   

  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).
• The detection of bovine lactoferrin binding protein on Toxoplasma gondii

  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 2003年12月 [査読有り][通常論文]
   

  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.
• Antiviral activity of lactoferrin against canine herpesvirus

  T Tanaka, S Nakatani, XN Xuan, H Kumura, Igarashi, I, K Shimazaki

  ANTIVIRAL RESEARCH 60 3 193 - 199 2003年11月 [査読無し][通常論文]
   

  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.
• Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii

  T Tanaka, Nakamura, I, NY Lee, H Kumura, K Shimazaki

  BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 81 5 349 - 354 2003年10月 [査読無し][通常論文]
   

  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.
• Expression and characterization of bovine lactoperoxidase by recombinant baculovirus

  T Tanaka, S Sato, H Kumura, K Shimazaki

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 10 2254 - 2261 2003年10月 [査読有り][通常論文]
   

  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.
• Preliminary study and characterization of dextran degradation by the water-soluble fraction extracted from blue cheese

  K Shimazaki, S Maki, Y Wang, M Sato, T Tanaka, H Kumura

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 58 7-8 379 - 382 2003年 [査読有り][通常論文]
   

  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.
• Liberation of free fatty acids from milk fat by lactic acid bacteria

  A Kimura, H Kumura, S Ishizuka, K Mikawa, K Shimazaki, Z Saito

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 58 11-12 609 - 611 2003年 [査読有り][通常論文]
   

  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.
• Fine structures of epitopic sites in human and bovine lactoferrin recognized by anti-bovine lactoferrin C-lobe monoclonal antibody

  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 2002年06月 [査読有り][通常論文]
   

  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.
• Casein digestion by Debaryomyces hansenii isolated from cheese

  H Kumura, K Takagaki, T Sone, M Tsukahara, T Tanaka, K Shimazaki

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 6 1370 - 1373 2002年06月 [査読無し][通常論文]
   

  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.
• Lactoferrin-binding proteins in Bifidobacterium bifidum

  WS Kim, T Tanaka, H Kumura, K Shimazaki

  BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 80 1 91 - 94 2002年02月 [査読有り][通常論文]
   

  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.
• Primary culture of porcine mammary epithelial cells as a model system for evaluation of milk protein expression

  H Kumura, A Tanaka, Y Abo, S Yui, K Shimazaki, E Kobayashi, K Sayama

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 9 2098 - 2101 2001年09月 [査読無し][通常論文]
   

  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.
• Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

  Nakamura, I, A Watanabe, H Tsunemitsu, NY Lee, H Kumura, K Shimazaki, Y Yagi

  PROTEIN EXPRESSION AND PURIFICATION 21 3 424 - 431 2001年04月 [査読無し][通常論文]
   

  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.
• Sequence analysis of porcine polymeric immunoglobulin receptor from mammary epithelial cells present in colostrum

  H Kumura, T Sone, K Shimazaki, E Kobayashi

  JOURNAL OF DAIRY RESEARCH 67 4 631 - 636 2000年11月 [査読無し][通常論文]
  
• Bovine lactoperoxidase and its recombinant: Comparison of structure and some biochemical properties

  S Watanabe, S Murata, H Kumura, S Nakamura, A Bollen, N Moguilevsky, K Shimazaki

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 274 3 756 - 761 2000年08月 [査読有り][通常論文]
   

  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.
• Antibacterial activities and structure of Korean native goat lactoferrin and its synthetic peptides

  MS Nam, DY Yu, M Kimura, H Kumura, K Shimazaki

  ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 13 282 - 282 2000年07月 [査読有り][通常論文]
  
• Antimicrobial peptide of Korean native goat lactoferrin and identification of the part essential for this activity

  M Kimura, MS Nam, Y Ohkouchi, H Kumura, K Shimazaki, DY Yu

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 268 2 333 - 336 2000年02月 [査読有り][通常論文]
   

  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.
• Approach to identification and comparison of the heparin-interacting sites of lactoferrin using synthetic peptides

  K Shimazaki, K Uji, T Tazume, H Kumura, T Shimo-Oka

  LACTOFERRIN: STRUCTURE, FUNCTION AND APPLICATIONS 1195 37 - 46 2000年 [査読有り][通常論文]
   

  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.
• Autolysis of the proteinase from Pseudomonas fluorescens

  H Kumura, S Murata, T Hoshino, K Mikawa, K Shimazaki

  JOURNAL OF DAIRY SCIENCE 82 10 2078 - 2083 1999年10月 [査読有り][通常論文]
   

  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.
• The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33

  E Kawai, A Idei, H Kumura, K Shimazaki, H Akatsuka, K Omori

  BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1446 3 377 - 382 1999年09月 [査読有り][通常論文]
   

  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.
• Characterization of Korean native goat lactoferrin

  MS Nam, K Shimazaki, H Kumura, KK Lee, DY Yu

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 123 2 201 - 208 1999年06月 [査読有り][通常論文]
   

  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.
• Comparison of secondary structure and antibacterial activity of bactericidal peptides derived from bovine and Korean native goat lactoferrin

  KI Shimazaki, MS Nam, T Tazume, H Kumura, K Mikawa, KK Lee, DY Yu

  PEPTIDE SCIENCE - PRESENT AND FUTURE 759 - 760 1999年 [査読有り][通常論文]
  
• Properties of a heparin-binding peptide derived from bovine lactoferrin

  K Shimazaki, T Tazume, K Uji, M Tanaka, H Kumura, K Mikawa, T Shimo-Oka

  JOURNAL OF DAIRY SCIENCE 81 11 2841 - 2849 1998年11月 [査読有り][通常論文]
   

  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).
• Genetic characterization of pepP, which encodes an aminopeptidase P whose deficiency does not affect Lactococcus lactis growth in milk, unlike deficiency of the X-prolyl dipeptidyl aminopeptidase

  J Matos, M Nardi, H Kumura, Monnet, V

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 64 11 4591 - 4595 1998年11月 [査読有り][通常論文]
   

  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.
• Molecular cloning and analysis of a lipase gene from Pseudomonas fluorescens No. 33

  H Kumura, S Hirose, H Sakurai, K Mikawa, F Tomita, K Shimazaki

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 62 11 2233 - 2235 1998年11月 [査読有り][通常論文]
   

  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.
• Expression of human lactoferrin in transfected rat mammary epithelial cells

  H Kumura, Y Hiramatsu, Y Ukai, K Mikawa, K Shimazaki

  ADVANCES IN LACTOFERRIN RESEARCH 443 85 - 89 1998年 [査読有り][通常論文]
  
• Structural and immunochemical studies on bovine lactoferrin fragments.

  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 1998年 [査読有り][通常論文]
  
• Monoclonal antibody against bovine Lactoferricin(R) and its epitopic site

  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 1996年12月 [査読有り][通常論文]
   

  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''.
• INFLUENCE OF MILK-PROTEINS ON THE THERMOSTABILITY OF THE LIPASE FROM PSEUDOMONAS-FLUORESCENS-33

  H KUMURA, K MIKAWA, Z SAITO

  JOURNAL OF DAIRY SCIENCE 76 8 2164 - 2167 1993年08月 [査読有り][通常論文]
   

  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.
• PURIFICATION AND SOME PROPERTIES OF PROTEINASE FROM PSEUDOMONAS-FLUORESCENS NO-33

  H KUMURA, K MIKAWA, Z SAITO

  JOURNAL OF DAIRY RESEARCH 60 2 229 - 237 1993年05月 [査読有り][通常論文]
   

  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.
• PURIFICATION AND CHARACTERIZATION OF LIPASE FROM PSEUDOMONAS-FLUORESCENS NO 33

  H KUMURA, K MIKAWA, Z SAITO

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 48 8 431 - 434 1993年 [査読有り][通常論文]
   

  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年 [査読有り][通常論文]
  
• EFFECT OF PROTEASE ON CONCOMITANT LIPASE PRODUCED BY PSEUDOMONAS SP NO 33

  H KUMURA, K MIKAWA, Z SAITO

  MILCHWISSENSCHAFT-MILK SCIENCE INTERNATIONAL 46 4 215 - 218 1991年 [査読有り][通常論文]
   

  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.

MISC

• チーズの風味強化剤としての麹調製~酵素活性と香気特性を指標とした検討~

  川上倖奈, CHINTAGAVONGSE Napaporn, 原聡美, 玉野孝一, 早川徹, 若松純一, 玖村朗人, 三谷朋弘 ミルクサイエンス(Web) 72 (2) 2023年
• パルマハムで形成された水溶性ZnPPによる機構研究に対する新規な実験的モデルの確立

  YANG Zhai, 早川徹, 玖村朗人, 若松純一 日本食肉研究会総会提出議案及び大会講演要旨 62nd (CD-ROM) 2021年
• パルマハムから弱アルカリで抽出される亜鉛プロトポルフィリンIXの存在形態の解明

  阿部悠, 早川徹, 玖村朗人, 若松純一 日本食肉研究会総会提出議案及び大会講演要旨 62nd (CD-ROM) 2021年
• カルノシンは死後硬直条件下におけるアクトミオシンのATP分解を促進する

  岡田実那美, 若松純一, 玖村朗人, 早川徹 日本食肉研究会総会提出議案及び大会講演要旨 62nd (CD-ROM) 2021年
• ヒスチジンによるアクトミオシン加熱ゲル形成の抑制について

  早川徹, 窪野佑, 若松純一, 玖村朗人 日本バイオレオロジー学会誌(Web) 34 (2) 2020年
• L-ヒスチジンは低塩濃度条件下の天然アクトミオシンの加熱ゲル形成を抑制する

  窪野佑, 早川徹, 若松純一, 玖村朗人 食肉の科学 60 (1) 2019年
• パルマハムにおけるダークスポットの特性

  王鴻誠, 尾崎あかり, 早川徹, 玖村朗人, 若松純一 日本畜産学会大会講演要旨 125th 2019年
• ホエイ固形培地上における食用Penicillium属由来のリパーゼ産生

  佐藤恵実, 町谷泰紀, 早川徹, 曾根輝雄, 若松純一, 木村彰, 玖村朗人 日本農芸化学会大会講演要旨集(Web) 2017 ROMBUNNO.3A05p10 (WEB ONLY) 2017年03月05日 [査読無し][通常論文]
  
• Effects of prolactin, EGF and dexamethasone on mechanism for regulating beta-casein expression and secretion.

  S. Oyama, C. Kuki, H. Kumura, K. Kobayashi MOLECULAR BIOLOGY OF THE CELL 25 2014年12月 [査読無し][通常論文]
  
• Differential roles of prolactin and glucocorticoid in mammary alveolar tight junction formation during lactation in mice.

  K. Kobayashi, S. Oyama, C. Kuki, H. Kumura MOLECULAR BIOLOGY OF THE CELL 25 2014年12月 [査読無し][通常論文]
  
• Visualization of lactoferrin binding to bifidobacteria

  Rahman Md. Morshedur, Woan-Sub Kim, Toshiaki Ito, Haruto Kumura, Keiichi Shimazaki BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE 84 (3) 390 -390 2006年06月 [査読無し][通常論文]
  
• Comparison of Reactivity of Polyclonal and Monoclonal Antibodies against Bovine Lactoferrin and its Fragments (ラクトフェリン研究--基礎から応用への掛け橋)

  Tun Khin Mg., 田仲 哲也, 玖村 朗人 ミルクサイエンス 53 (4) 239 -241 2004年
• Growth Promotional Effects of Bovine Lactoferrin and its Hydrolysate on Bifidobacteria (ラクトフェリン研究--基礎から応用への掛け橋)

  Rahman Md. Morshedur, 玖村 朗人, 島崎 敬一 ミルクサイエンス 53 (4) 325 -327 2004年
• Comparison of activity and stability between native and recombinant bovine lactoperoxidase

  S. Sato, T. Tanaka, K. Nakao, H. Kumura, K. Shimazaki Australian Journal of Dairy Technology 58 209 2003年08月01日 [査読無し][通常論文]
   

  The comparison of activity and stability between native and recombinant bovine lactoperoxidase (LPO) is performed. LPO is known to suppress acid production of certain lactic acid bacteria during fermentation at low temperatures. It is found that both disulfide linkages and heme-binding are essential to the activity and stability of the LPO molecule.
• Structural and Immunochemical Studies of Bovine Anitmicrobial Peptide 'lactoferricin'

  SHIMAZAKI Kei-ichi, NAM Myoung Soo, HARAKAWA Shinji, TANAKA Tetsuya, OMATA Yoshitaka, SAITO Atsushi, KUMURA Haruto, MIKAWA Katsuhiko, IGARASHI Ikuo, SUZUKI Naoyoshi Peptide chemistry : proceedings of the ... Symposium on Peptide Chemistry 1996 197 -200 1996年10月01日
• 抗ラクトフェリンモノクローナル抗体の結合する部位の推定

  島崎 敬一, 神尾 真, NAM Myoung Soo, 玖村 朗人, 三河 勝彦, 原川 信二, 田仲 哲也, 小俣 吉孝, 齋藤 篤志, 五十嵐 郁夫, 鈴木 直義 日本分子生物学会年会プログラム・講演要旨集 19 399 -399 1996年08月01日
• ラクトペルオキシダーゼのフラグメント活性とその抗体反応性 : 食品

  渡邊 市紀子, 島崎 敬一, 劉 永春, 畑 克介, 玖村 朗人, 三河 勝彦, 中村 信吾, 東 市郎 日本農藝化學會誌 70 2 -2 1996年03月05日
• Pseudomonas fluorescens No.33が生産するリパーゼについて : 酵素

  廣瀬 修治, 玖村 朗人, 三河 勝彦, 島崎 敬一 日本農藝化學會誌 69 24 -24 1995年07月05日

書籍等出版物

• 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年09月
• 畜産物利用学

  文永堂 2011年
• 最新畜産物利用学

  朝倉書店 2006年
• 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年

所属学協会

• 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   

共同研究・競争的資金等の研究課題

• 真菌類を利用した新規生活習慣病予防食品素材の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2013年04月 -2017年03月 

  代表者 : 玖村 朗人, 石塚 敏

   

  紅麹菌は動脈硬化のリスク低減を目的とした機能性食品素材の開発に有効な二次代謝産物を産生する食用微生物である。しかし一方で肝腎障害に関わるシトリニンを同時に産生する場合があり、食品素材への紅麹菌の応用に際しては適切な菌株選択と厳密な培養制御が必要である。そこで申請者は広汎な条件設定にも対応可能な上に調製が容易で、得られる培養産物をready to eatで利用可能な固体培養基剤を用いて菌株や培養条件を精査し、得られる紅麹菌培養物を実験動物に給餌して機能性を評価する研究プログラムを立案した。
• 食用微生物代謝産物の機能性

  研究期間 : 2009年
• 発酵性乳製品由来の酵母を用いた新規プロバイオティクス製品開発を目指した基礎的研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 2004年 -2006年 

  代表者 : 玖村 朗人

   

  近年、発酵食品が健康に及ぼす効果について高い関心が寄せられ、乳酸菌やビフィズス菌を始めとする数々の微生物の機能に関する研究が行われている。本研究では新たなプロバイオティック微生物を探索するために発酵乳製品由来の酵母に着目し、その免疫調節作用と代表的な腸内細菌であるビフィズス菌の増殖や生存性に及ぼす効果を観察した。 まず酵母の投与が消化管や乳腺の免疫グロブリンA産生に及ぼす影響とオブアルブミン(OMへ)で免疫したマウスから調製した培養脾臓細胞の免疫グロブリンE(IgE)やサイトカイン産生に及ぼす影響について観察した。その結果、統計的な有意差はなかったものの、酵母添加飼料を投与した場合、大腸内容物中のIgA濃度が高値となる傾向が認められた。さらに本実験で供試した全ての酵母菌体(6菌種、46株)において培養脾臓細胞のIgE産生抑制効果が観察された。また、酵母菌体によって同細胞のIFN-γの産生は誘導されなかったが、IL-12産生については菌株によって異なる結果が得られた。マウスを用いたin vivo実験においては、酵母または乳酸菌の単独あるいは混合経口投与によってOVAで感作したマウスの血中IgE濃度を測定したが、この実験系においてはいずれの微生物においてもIgE産生抑制効果を観察することは出来なかった。 さらに脱脂乳でビフィズス菌を培養する際に酵母菌体抽出物を添加することによって、ビフィズス菌の増殖性やその後、冷蔵保存時の生存性を高めることが確認された。酵母菌体破砕物中のビフィズス菌増殖促進因子は沸騰条件で30分間処理した後にもその効果を一部維持した。 真核細胞である酵母は培養環境によって多様な遺伝子発現の誘導または抑制をみせる。それゆえ、酵母の培養条件によって未知の機能性を現わす可能性がある。本実験で用いた食品由来の安全な酵母を有効活用するためのさらなる研究が期待される。
• 触媒アミノ酸の変異により新しい機能を獲得したグリコシラーゼの研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 2002年 -2004年 

  代表者 : 木村 淳夫, 森 春英, 奥山 正幸, 玖村 朗人

   

  グリコシラーゼは糖質のグリコシド結合を加水分解する酵素の総称である。これらの酵素は加水分解の他に、基質のグリコシド残基をアクセプターに転移する反応(糖転移反応)を示す。転移反応は応用性が高く、i)清涼飲料水などの食品に含まれる機能性オリゴ糖の生産やii)多彩な生理活性をもつ複合糖鎖の合成に利用されている。しかし、加水分解と糖転移反応は切り離すことはできず、同時に進行する。従って、転移糖の生成条件であっても基質の加水分解が行われ、かつ、転移反応の生成物も分解されてしまう。我々は、グリコシラーゼの構造と機能を解析してきた。この研究過程で糖転移反応のみを抽出できる現象を見出した。本課題の目的は、本現象の機構を分子解析し応用に結びつけることであり、成果を以下に示す。1)対象としたグリコシラーゼについて自殺基質の手法で求核残基を推定後、本残基の点置換酵素(合成酵素)を発現・精製した。フルオライド基質について合成法を確立させ、大量調製を行った。得られた合成酵素は、いずれも分解活性を示さず、フルオライド基質とアクセプター基質を合成させる反応のみを行った。合成の至適pHはアルカリ側へシフトし、本現象は一般的に観察された。アクセプター特異性を調べたところ、アリールなどの疎水基を有する合成配糖体が最も高い収率を与えた。これは、サブサイト+2による疎水グループへの認識能が高いことを意味している。2)「歪み発生のアミノ酸変異酵素」を作製・精製・性質解析を行い、オリゴ糖合成試験を行った。収率が低いことが判明したので、反応条件を検討し、基質濃度の変化に効果を認めた。また、有機溶媒の添加も有効であったが、高濃度では活性低下が生じた。有機溶媒に耐性な糖質分解酵素が得られているので、これを歪み発生型酵素にするような発展研究を行いたい。
• ウシラクトペルオキシダーゼとその組み換え体を用いた構造と機能の解析

  日本学術振興会：科学研究費助成事業

  研究期間 : 1999年 -2000年 

  代表者 : 島崎 敬一, 田仲 哲也, 玖村 朗人, 中村 信吾

   

  牛乳に含まれる酸化還元酵素・ラクトペルオキシダーゼの示す抗菌・抗ウイルス活性を自由に操作出来るならば、医薬品や食品へ応用する際に非常な利点となると考えられる。そこで組換えラクトペルオキシダーゼを調製し、牛乳由来のラクトペルオキシダーゼと比較することで、活性に寄与する高次構造、およびヘム近傍の微細構造についての詳細な情報を得ることを目的としてこれまでに以下の実験を行った。 〇ラクトペルオキシダーゼの検出に用いる目的で、ラクトペルオキシダーゼに対するポリクローナルおよびモノクローナル抗体を作成した。モノクローナル抗体については、ラクトペルオキシダーゼ分子内のエピトープ部位も決定した。 〇ウシ乳腺細胞からラクトペルオキシダーゼ遺伝子をクローニングし、CHO細胞を用いて組換え体を発現させ、さらにイオン交換クロマトグラフィーにて精製した。発現した組換え体の確認は、抗ラクトペルオキシダーゼ抗体および活性測定にて行った。 〇円偏光二色性スペクトルの測定により、牛乳ラクトペルオキシダーゼと高次構造が同じであることを確かめた。一方、牛乳ラクトペルオキシダーゼのN-末端アミノ酸部分は組換え体に比べて15〜20残基短かいことを見いだし、ミルク中に分泌される際の機構に新たな知見を加えた。 〇牛乳から分離したラクトペルオキシダーゼおよび組換えラクトペルオキシダーゼをペプシン分解した後、ヘム結合フラグメントを逆相クロマトグラフィーにより分離することに成功した。現在、そのフラグメントのアミノ酸配列を決定中であり、ヘムとの結合様式を推定する手掛かりとなるものと期待される。
• ブタ乳腺上皮細胞の培養と樹立株の構築

  日本学術振興会：科学研究費助成事業

  研究期間 : 1997年 -1998年 

  代表者 : 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。
• ブタ乳腺上皮細胞の分離・樹立

  日本学術振興会：科学研究費助成事業

  研究期間 : 1996年 -1996年 

  代表者 : 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。