Original Paper Antihypertensive Potentials of Blue, Dutch and Swiss
Milk Science Vol. 66, No. 2 2017 Original Paper
Antihypertensive potentials of Blue, Dutch and Swiss cheeses containing ValProPro and IleProPro peptides
Keiichi Matsuura1, Takanobu Gotou2,KyosukeKawaguchi3, Seiichi Mizuno1, Naoyuki Yamamoto4 (1Products Research & Development Laboratory, Asahi Soft Drinks Co., Ltd., Moriya-shi, Ibaraki 3020106 2R&D Section, Asahi Group Holdings Ltd., Azumabashi, Sumida-ku, Tokyo 1308602 3Core Technology Laboratories, Asahi Group Holdings Ltd., Moriya-shi, Ibaraki 3020106 4R&D Center, Asahi Group Holdings Ltd., Sagamihara-shi, Kanagawa 2520206)
Abstract The antihypertensive peptides, ValProPro (VPP) and IleProPro (IPP), were ˆrst isolated from Lactobacillus helveticus fermented milk; however, there are few reports on the antihypertensive eŠects of cheese. The objective of this study was to quantify the amounts of VPP and IPP in various cheeses and to discuss the potential of these antihyperten- sive eŠects. Fifteen European-type cheeses were selected and analyzed for these two antihypertensive peptides using an LCMS analysis. The Dutch cheeses, Reypenaer and Old Amsterdam, contained high amounts of VPP/IPP, 19.9/0.9 and 10.5/0.9 mg in 100 g of cheese, respectively. The Swiss cheeses, Emmental and Gruyere, also contained VPP/IPP at 8.0/1.1 and 5.2/1.2 mg in 100 g of cheese, respectively. Notably, some French-type Blue cheeses prepared with Penicillium roqueforti contained the highest amounts of both peptides (13.3/31.0 mg in Bleu d'Auvergne and 9.9/19.2 mg in Bleu des Causses in 100 g of cheese). The ratio of VPP to IPP was higher in Dutch and Swiss cheeses than in Blue cheeses. To understand the release of these peptides in Blue cheese, proteolytic enzymes, neutral protease I (NPI) and leucine amino peptidase (LAP), previously identiˆed as crucial proteolytic enzymes for IPP processing in Aspergillus oryzae, were searched in Penicillium usingBLAST.AhomologwithsimilaritytoNPIofA. oryzae was detected in extracellular metalloproteinase4ofPenicillium subrubescens with 36 identity. Moreover, LAP of A. oryzae was identi- cal to an unknown protein of P. roqueforti. These observations suggest that proteases of Penicillium with homology to A. oryzae NPI and LAP might be involved in the processing of antihypertensive peptides during Blue cheese ripening.
Key words: antihypertensive peptide, ValProPro and IleProPro, Penicillium roqueforti, Aspergillus oryzae, Lactobacillus helveticus, Blue cheese
the production of amino acids and peptides and Introduction the subsequent amino acid conversion to aromatic compounds. Among the LAB, Lactobacillus helveti- Microorganisms have proteolytic systems to cus has the highest proteolytic activity and can decompose various proteins to utilize peptides and release the most peptides. In the cheese-making amino acids as nitrogen sources for their growth. process, L. helveticus is generally used in mixed Lactic acid bacteria (LAB) and mold have been starter cultures for rapid acidiˆcation and accelera- traditionally used in the manufacturing of cheese tion of cheese ripening. and yogurt to add speciˆc ‰avors and tastes via Cheese and yogurt fermented with LAB and/or mold also have nutraceutical beneˆts, such as Corresponding author: Naoyuki Yamamoto (Tel0427697811，Fax0427697842， constipation relief1) and a cholesterol-lowering E-mail: naoyuki.yamamoto＠asahigroup-holdings.com) eŠect2). Our previous study reported the release March 10, 2017 received April 24, 2017 accepted of antihypertensive peptides, ValProPro (VPP) [doi:10.11465/milk.66.97] and IleProPro (IPP),inL. helveticus fermented 第巻 milk35). These two peptides are thought to be amount of hot water, pre-heated at 60°C, was processed from casein by speciˆc proteolytic added to the precipitate and the suspension was enzymes of L. helveticus67). These peptides have vortexed. The supernatant was collected again inhibitory activities against angiotensin Iconvert- after a centrifugation at 4,000 rpm for 20 min. The ing enzyme (ACE)(kininase II; EC 3.4.15.1), supernatants were combined and ˆltered through a which plays a key role in the control of hyperten- Millipore ˆlter with a molecular sieve size of 0.45 sion8). The blood pressure lowering eŠects of mm. L. helveticus fermented milk have been reported Quantiˆcation of VPP and IPP: In cheese products, in many clinical trials on Japanese and European many kinds of peptides are released from milk subjects912). Mechanism of actions were casein by the proteolytic actions of LAB and mold. studied by pharmacokinetical13) and genetical14) Thus, the LCMS method was used to quantify approaches. Safety of these peptides were also VPP and IPP among the various peptides in cheese investigated by some studies15,16) Moreover, a products. The amounts of VPP and IPP extracted recent meta-analysis of many trials reported a by incubation in hot water (described above) were signiˆcant antihypertensive eŠect1719). analyzed according to a previously reported Many kinds of peptides are released in the method with some modiˆcations21). VPP and IPP ripening process of cheese with the involvement of were quantiˆed by the internal standard method 13 13 LAB and some molds. Speciˆcally, cheese with a VPP isotope ([ C5]Val[ C5]ProPro, m/ 13 manufactured by the ripening with L. helveticus is z 324.2) and IPP isotope (Ile[ C5]ProPro, m/z most likely to contain VPP and IPP. Moreover, 332.2) obtained from SCRUM Inc. (Tokyo, mold used in cheese ripening may have a potential Japan). The concentrations of the standards (VPP to release antihypertensive peptides. Aspergillus and IPP) were 0, 0.3, 0.6, 1.2 and 4.8 mg/mL, oryzae, a frequently used mold in the manufactur- and the internal standard (VPP isotope and IPP ing of traditional Japanese fermented foods, isotope) was 1.5 mg/mL. released a high amount of VPP and IPP from Homology search:BLAST(http:// blast.ncbi.nlm. casein20). Thus, in this study, we analyzed two nih.gov/Blast.cgi) searches were conducted using antihypertensive peptides in various cheeses sequences of NPI and LAP of A. oryzae, accession expecting to ˆnd an increase cheese ripening by numbers AAF046282222) and AF419160.123), proteolysis of LAB and mold by using the LCMS respectively. method21). Results Materials and Methods Quantiˆcation of VPP and IPP in various cheeses: Materials: Characteristics of various cheeses European cheeses, generally manufactured using including hard, semi-hard, Blue and cream-types, long ripening times to accelerate ripening, were purchased in a European market, are listed in obtained to analyze their peptide releases (Table Table 1. Information about the microorganisms 1).AsLactobacillus helveticus has the reported used in each product as starter cultures is also potential to release VPP and IPP during milk included in Table 1. fermentation, some cheeses contained high Preparation of peptides in cheese products:To amounts of VPP and IPP (Table 2). Dutch- and prepare the peptide fractions from cheese Swiss-type cheeses fermented with starter consist- products, each cheese was shredded and then ing of Lactobacillus helveticus contained relatively freeze-dried to produce cheese powder. An equal high amounts of VPP and IPP (Table 2). Amounts amount of hot water (pre-heated to 60°C) was of the two peptides in Dutch- and Swiss-type added to the cheese powder and homogenized. The cheeses were 100fold higher than that of the suspension was centrifuged at 4,000 rpm for 20 lowest one (Fromage d'a‹nois). For Dutch-type min after storing the suspension at 60°Cfor1hr. cheese, Reypenaer and Old Amsterdam, contained After collection of the supernatant, an equal VPP/IPP amounts of 19.9/0.9 and 10.5/0.9 mg in 第号
Table 1 Various kinds of cheeses for peptide analysis.
Cheese Reported starter Milk Type Country, Area History
Bleu d'Auvergne Penicillium roquefortii Cow Blue Auvergne, France 1850s
Bleu des Causses Penicillium roquefortii Cow Blue Curuses, France 1925
Reypenaer VSOP Lactococcus lactic subsp. lactis Cow Semi-hard The Netherlands 1906 Lactococcus lactis subsp. cremoris Lactococcus lactis subsp. lactis biovar diacetylactis Leuconostoc mesenteroides subsp. cremoris Lactobacillus helveticus Lactobacillus lactis
Old Amsterdam Lactococcus lactic subsp. lactis Cow Semi-hard, The Netherlands 19th Century Lactococcus lactis subsp. cremoris Gouda type Lactococcus lactis subsp. lactis biovar diacetylactis Leuconostoc mesenteroides subsp. cremoris Lactobacillus helveticus Lactobacillus lactis
Emmental Streptococcus thermophilusis Cow Hard Switerland 14th Century Lactobacillus helveticus Lactobacillus bulgaricus Propionibacter freudenreichii Propionibacter shermani
Gruyere Lactobacillus helveticus Cow Semi-hard Switherland 19th Century Lactobacillus casei Lactobacillus rhamnosus
Fourme d'Ambert Penicillium roqueforti Cow Blue Auvergne, France 1972
Castello Blue Penicillium roqueforti Cow Blue Denmark 1893
Gouda Oud Lactococcus lactis subsp. lactis Cow Semi-hard The Netherlands Lactococcus lactis subsp. cremoris Lactococcus lactis subsp. lactis biovar diacetylactis Leuconostoc mesenteroides subsp. cremoris NSLAB
Cheddar Lactococcus lactis subsp. lactis Cow Semi-hard UK 1170 Lactococcus lactis subsp. cremoris NSLAB
Gouda Lactococcus lactis subsp. lactis Cow Semi-hard The Netherlands 12th Century Lactococcus lactis subsp. cremoris Lactococcus lactis subsp. lactis biovar diacetylactis Leuconostoc mesenteroides subsp. cremoris NSLAB
Blue de Bresse Penicillium roqueforti (Penicillium camanberti) Cow Blue France, Bresse 1951
Stilton Penicillium glaucum Cow Blue UK 1727
Gorgonzola dolce Lactobacillus bulgaricus Cow Blue Italy 12th Century Streptococcus thermophilus Penicillium glaucum Penicillium roqueforti
Fromage d'a‹nois Penicillium candidum Cow Brie-type France 19th Century Penicillium camemberti cream cheese Geotrichum candidum
NSLAB: Non-starter lactic acid bacteria 第巻
Table 2 Amounts of VPP and IPP in various cheeses.
Cheese Type VPP IPP VPP/IPP VPPeq EŠective dosage (mg/100 g) (mg/100 g) (mg/100 g) (g) Bleu d'Auvergne Blue 13.3 31.0 0.4 69.2 5.1
Blue des Causses Blue 9.9 19.2 0.5 44.4 7.9 Reypenaer VSOP Dutch 19.9 0.9 22.6 21.4 16.3
Old Amsterdam Dutch 10.5 0.9 11.3 12.2 28.7
Emmental Swiss 8.0 1.1 7.2 10.1 34.8 Gruyere Swiss 5.2 1.2 4.3 7.3 47.9
Fourme d'Ambert Blue 1.6 2.7 0.6 6.5 54.0
Castello Blue Blue 1.3 1.8 0.7 4.5 78.4 Gouda Oud Dutch 3.7 0.3 11.2 4.3 81.5
Cheddar English 2.1 0.8 2.7 3.6 98.3 Gouda Dutch 1.6 1.1 1.5 3.5 99.2
Bleu de Bresse Blue 0.6 1.1 0.5 2.5 138.3
Stilton Blue 0.4 0.8 0.4 1.8 191.7 Gorgonzola dolce Blue 0.1 0.3 0.4 0.6 616.2
Fromage d'a‹nois White 0.2 0.1 2.0 0.3 1023.4
VPPeq: (amount of VPP)＋(amount of IPP)×1.7 EŠective dosage: Minimum amount of cheese containing 3.4 mg of VPPeq to show antihypertensive eŠect calculated from a clini- cal study on Japanese subjects (7).
100 g of cheese, respectively. In contrast, Swiss- VPP and IPP, respectively, as a daily dose9). type cheeses, Emmental and Gruyere, contained Considering the ACE inhibitory activity, 1.1 mg of VPP/IPP amounts of 8.0/1.1 and 5.2/1.2 mg in IPP is equivalent to 1.9 mg of VPP (VPPeq).This 100 g of cheese, respectively. These results sug- means that the test sample contained 3.4 mg of gest that the high production of VPP and IPP in VPPeq (＝1.5 mg＋1.9 mg). So, the minimum European cheeses might be depend on starter amount of cheese to show antihypertensive eŠect culture containing L. helveticus that is used. on human was calculated (Table 2).Bleud'Auver- Interestingly, some of the French-type Blue gne and Bleu des Causses have the potential to cheeses prepared with Penicillium roqueforti con- have an antihypertensive eŠect on humans if 4.9 or tained the highest amounts of both peptides and 7.7 g, respectively, of each cheese is consumed were 13.3/31.0 in Bleu d'Auvergne and 9.9/19.2 daily. For Gruyere, the eŠective daily dosage was mg in Bleu des Causes of 100 g cheese (Table 2). calculated as 46.7 g. However, the amounts of VPP and IPP in other Key enzymes in Penicillium roqueforti:Proteolytic types of Blue cheeses prepared with Penicillium enzymes needed for the processing of VPP and roqueforti,suchasFourmed’Ambert, Castello IPP have been well-characterized and the catalytic Blue and Bleu de Bresse, were lower than those in pathway has been previously postulated7,24).To Bleu d’Auvergne and Bleu des Causses. The understand the release of both peptides in Blue amount of VPP was almost similar to that of IPP in cheese, proteolytic enzymes reported for the all Blue cheese, but was higher in Dutch and Swiss crucial role for IPP processing in Aspergillus oryzae cheeses (Table 2). (a frequently used mold in Japan25)) was searched The ˆfty percent inhibition of ACE activities by BLAST. In more detail, homologues of neutral were 9 mMforVPPand5mMforIPPinaprevious protease I (NPI) and leucine aminopeptidase study18). In the ˆrst clinical study on Japanese (LAP), previously identiˆed for IPP processing25), subjects, a test sample contained 1.5 and 1.1 mg of were searched in Penicillium. The results showed 第号
Fig. 1 Homology between neutral protease I (NPI) of Aspergillus oryzae and extracellular metalloproteinase-4 of Penicillium subrubescens (EMP). (A) Identical amino acids in both sequences are marked with a shadow. (B) Gluzincin superfami- ly (35) observed in NPI of A. oryzae is shown in a gray box and its homolog observed in EMP of P. subrubescens is also shown in a gray box.
a homolog with similarity to NPI of A. oryzae the key enzymes for VPP processing are not clear (most likely required for the processing of the C (Fig. 2). terminal end of IPP25)) was detected in the extracellular metalloproteinase4 sequence of Discussion Penicillium subrubescens (Fig. 1A) with 36 iden- tity.AsshowninFig.1B,thedomainofNPI, The potential of Swiss-type cheeses, manufac- known as the Gluzincin family, which is conserved tured by LAB including L. helveticus, to release in thermolysin-like proteinase, showed homology VPP and IPP has been previously reported2627). to the Cterminal region of the metalloproteinase Moreover, recent studies reported high amounts of 4inP. subrubescens with 41 identity. Moreover, VPP, IPP and other ACE inhibitory peptides in LAP of A. oryzae needed for amino terminal Parmigiano-Reggiano cheese (Italian cheese)28) processing of IPP25) was identical to an unknown and Wisconsin Cheddar cheese29). The present protein of P. roqueforti. Therefore, the proteases of studyshowedhighamountsofVPPandIPPin Penicillium with homology to A. oryzae NPI and Dutch and Blue cheeses, except for the above LAP might be involved in the processing of IPP reported cheeses. Cheeses manufactured with L. from casein during Blue cheese ripening, although helveticus may also have the potential to release 第巻
Fig. 2 Putative pathway of VPP and IPP processing in Dutch, Swiss and Blue cheeses. (A) C-terminal processing by endopep- tidases (pepO and pepO2) and N-terminal processing of VPP and IPP by aminopeptidase and X-prolyl dipeptidyl aminopeptidase (XPDAP) of Lactobacillus helveticus. (B) C-terminal processing by metalloproteinase-4 and N-terminal processing of IPP of Penicillium roqueforti.
VPP and IPP. However, no study on mold-fer- 36 homology (Fig. 1), but not in P. roqueforti. P. mented cheese has reported the production of VPP subrubescens was recently characterized as a new or IPP. species of Penicillium with e‹cient inulinase Given the high amount of VPP and IPP produc- activity30). One of the reasons for no homolog of tion in blue cheese, we attempted understand the NPI in P. roqueforti may be because of limited crucial enzymes in P. roqueforti using A. oryzae for genome sequences of P. roqueforti. Another reason comparison. In our previous study, A. oryzae pro- may be diŠerent genetic proˆles and gene expres- tease was selected for its potent proteolytic activity sions depending on the P. roqueforti strain used. for the e‹cient production of VPP and IPP from Generally, Blue cheese is manufactured by ripen- casein. So, the key proteolytic enzymes in A. oryzae ing with P. roqueforti; however, some Blue cheeses, were searched in P. roqueforti, and we expected to such as Bleu de Bresse, Stilton and Gorgonzola ˆnd similar enzymes in both molds by a BLAST dolce, had lower productions of VPP and IPP search. As a result, extracellular metalloproteinase (Table 2). There may be diŠerent proˆles of 4andLAPtype protease were selected as candi- proteolytic enzymes in diŠerent strains of P. dates for IPP processing. roqueforti. Moreover, current studies have revealed A non-annotated enzyme of P. roqueforti identi- adaptive horizontal gene transfer in P. roqueforti31) cal to LAP of A. oryzae by the BLAST search in the and the existence of highly diŠerentiated popula- present study must have LAP activity and the tions of P. roqueforti32), which may be one of the ability to process Nterminal processing of IPP25) reasons for the diŠerent amounts of VPP and IPP in Blue cheese. For the processing of the Ctermi- in diŠerent kinds of Blue cheeses. nal end of IPP, protease with homology to NPI In the present study, high amounts of VPP and in A. sojae was detected in P. subrubescens26) with IPP were detected in Dutch and Swiss-type 第号 cheeses. Our previous study indicated that L. 3.4 mg of VPP (＝ 1.5 mg ＋ 1.9 mg). Amount of helveticus has the highest proteolytic activity cheese which contains 3.4 mg of VPPeq and has towards milk casein among LAB and has the poten- potential to show antihypertensive eŠects was tial to release VPP and IPP in cheese. L. helveticus calculated (Table 2). The antihypertensive eŠect is traditionally used in the manufacturing of of Bleu d'Auvergne, Bleu des Causses, Reypenaer Swiss-type cheeses and long ripening-type Italian VSOP, or Old Amsterdam is expected only daily cheeses, such as Emmental, Gruyere and Par- consumption of 4.9, 7.7, 15.9 and 27.8 g of cheese migiano-Reggiano. In the tested all cheeses, VPP for 4 weeks, respectively. Moreover, antihyperten- and IPP were rich in the cheeses ripened with sive eŠect may be also expected for Swiss cheese starter culture containing L. helveticus.Bythe if 34.7 g of Emmental cheese or 46.7 g of Gruyere fermentation of milk with L. helveticus,almost cheese is consumed for 4 weeks. equal amounts of VPP and IPP were released Yearly cheese consumption in France, The mainly by the processing of bcasein4). However, Netherlands and Switzerland was reported as the amount of IPP in the Dutch and Swiss cheeses 26.8, 18.2 and 21.8 kg in 2015, respectively34). was lower compared to that of VPP (Table 2). This equates to a daily consumption of 73 g in Previous reports suggested no decompositions of France, 50 g in The Netherlands and 60 g in VPPandIPPbyanyL. helveticus proteases. These Switzerland. A recent meta-analysis of 18 clinical results strongly suggest that decomposition of IPP trials conducted in Japan and Europe revealed a might be because of the protease of other LAB signiˆcant reduction of systolic and diastolic blood containedinthestarterculture(see Table 1). pressure with dosages over 3.4 mg of VPP eq17). Moreover, non-starter lactobacilli (NSLAB), The antihypertensive eŠect was higher in Asian deˆned as LAB originating from factory environ- studies than in the Caucasian one, but a reduction ment or milk may in‰uence the production of VPP of －3.73 mm Hg for systolic blood pressure was and IPP because of the cooperative eŠect of detected on the mixed subject analysis. In contrast, Lactococcus lactis and NSLAB has been suggested 0.10.3 g of Na is contained in 50 g of hard-type for the formation of cheese aroma33). cheese, such as Gruyeres, Swiss- and Dutch-type In this study, various types of cheeses were cheeses. But, a daily uptake of less than 0.3 g Na analyzed the amount of antihypertensive peptides has a small impact on systolic blood pressure. for the consideration of potentials of various These considerations indicate that daily consump- cheeses to help blood pressure control (Table 2). tion of the Bleu d'Auvergne, Bleu des Causses, Animal studies to evaluate the antihypertensive Reypenaer VSOP, Old Amsterdam, Emmental or eŠects of cheese are di‹cult because the oral Gruyere might have signiˆcant antihypertensive administration of cheese on SHR is not controlled eŠects due to the uptake of VPP and IPP. and the eŠect cannot be extrapolated to humans. The antihypertensive eŠects of Blue, Dutch The antihypertensive eŠect of VPP and IPP was and Swiss type cheeses, containing eŠective ˆrst reported in a clinical study conducted on dosages of VPP and IPP (Table 1) are in agree- Japanese subjects by consecutive uptakes of both ment with the meta-analysis17). Current studies peptides for 4 and 8 weeks9). In the ˆrst clinical have revealed a signiˆcant eŠect of VPP and study on Japanese subjects, the test sample con- IPP on improvements of central blood pressure and tained 1.5 and 1.1 mg of VPP and IPP, respective- arterial stiŠness in hypertensive subjects3536). ly, as a daily dosage9). ACE inhibitory activity, Moreover, small clinical studies have shown that which showed 50 inhibition against ACE, was casein hydrolysate containing VPP and IPP 5 mMforIPPandwas1.7fold higher than that improved vascular endothelial function37) and for VPP (9 mM)4). So, amount of IPP contained in arterial compliance in postmenopausal women34). the test sample of the ˆrst study was equivalent Arterial stiŠness is monitored by central blood to 1.9 mg of VPP (VPPeq＝1.1 mg×1.7).It pressure and arterial compliance, which are key reveals that antihypertensive eŠect on Japanese markers for the risk of cardiovascular events subjects might be expected by a daily dosage of including atherosclerosis, myocardial infarction, 第巻
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ブルー，オランダ，スイスチーズにおける血圧降下ペプチド， ValProPro と IleProPro，の遊離
松浦啓一1・後藤孝信2・川口恭輔3・水野征一1・山本直之4 (1アサヒ飲料株式会社，商品開発研究所，茨城県守谷市 3020106) (2アサヒグループホールディングス株式会社，研究開発部門，東京都墨田区 1308602) (3アサヒグループホールディングス株式会社，コアテクノロジー研究所，茨城県守谷市 3020106) (4アサヒグループホールディンクス株式会社，R&D センター，神奈川県相模原市 2520303）
Lactobacillus helveticus 発酵乳から最初に単離された血圧降下ペプチド，ValProPro (VPP）と IleProPro (IPP） に関して様々なチーズについて分析した。15種類のヨーロッパタイプのチーズを選び，これら 2 種類の血圧降下ペプ チドを LCMS を用いて分析した。オランダチーズのライペナーとオールドアムステルダムには高含量の VPP/IPP を， 100 g 当たりに19.9/0.9と10.5/0.9 mg づつ含んでいた。スイスチーズのエメンタールとグリュイエールには同様に VPP / IPP をそれぞれ100 g 当たりに8.0/ 1.1と 5.2/ 1.2 mg づつ含んでいた。注目すべき点として，Penicillium roqueforti で製造される幾つかのフランスチーズ（ブルー・ド・オーベルニュとブルー・ド・カース）にも，高濃度の 両ペプチドが，100 g 当たりにそれぞれ，13.1/31.0と9.9/19.2 mg づつ含んでいた。VPP に対する IPP の比はドイツ やスイスチーズにおいて，ブルーチーズのものに比べて低かった。ブルーチーズにおける両ペプチドの産生の理由を理 解するために，以前，Aspergillus oryzae 内の IPP の加工に重要な蛋白質分解酵素であるニュートラルプロテアーゼ I（NPI）とロイシンアミノペプチダーゼ（LAP）について Penicillium 属での存在を BLAST 検索した。その結果，A. oryzae の NPI に36の相同性をもつホモログが Penicillium subrubescens の菌体外金属プロテアーゼ4 に確認された。 さらに，A. oryzae の LAP は P. roqueforti の未同定の蛋白質と同一であった。これらのことから，A. oryzae の NPI と LAP に相同性を持つ Penicillium のプロテアーゼがブルーチーズの熟成中の血圧降下ペプチドの生成に関与することが 示唆された。