2020 年 26 巻 1 号 p. 93-99
The aims of this study were to identify fungal growth inhibitory substances in cheese prepared using crude enzymes from Hericium erinaceum MAFF 435060 exhibiting milk-clotting activity and elucidate the conditions under which these substances are produced. Fungal growth inhibition caused by fatty acids, such as butanoic, hexanoic, octanoic, decanoic, and 9-decenoic acids, released by lipase activity of crude enzymes during ripening was estimated. Notably, the amounts of butanoic, hexanoic, and octanoic acids in ripened cheese exceeded their minimum growth inhibitory concentrations for Aspergillus niger NBRC 105649. Growth inhibition and high fatty acid content were observed in cheese prepared using homogenized milk as raw material, although homogenized milk in which milk fat was not encapsulated within the globule membrane was required. These results suggested that the use of crude enzymes from H. erinaceum MAFF 435060 may allow the production of cheese with a prolonged shelf life.
In cheese making, milk-clotting enzymes such as rennet (chymosin), which is obtained from the abomasum (a part of the stomach) of young ruminant animals, have been traditionally used. Alternatively, in recent years, milk-clotting enzymes obtained from molds such as Rhizomucor miehei, R. pusillus, and Cryphonectria parasitica have also been produced (Jacob et al., 2011), with Basidiomycetes being shown to serve as an abundant source of milk-clotting enzymes as well (Chemeris et al., 2016; El-Baky et al., 2011; Kikuchi et al., 1988; Kobayashi et al., 1985; Lebedeva and Proskuryakov, 2009; Majumder et al., 2015; Nakamura et al., 2014; Nerud et al., 1989; Okamura-Matsui et al., 2001; Shamtsyan et al., 2014; Yin et al., 2014). In particular, Nakamura et al. (2014) found that crude enzymes from the edible mushroom Hericium erinaceum MAFF 435060, known as lion's mane mushroom (Yamabushitake in Japanese), exhibited high milk-clotting activity and low protease activity. Sato et al. (2016) further revealed that crude enzymes from H. erinaceum MAFF 435060 had the ability to coagulate milk pasteurized at ultra-high temperatures (130 °C, 2 s) as well as milk pasteurized at a low temperature (66 °C, 30 min). It was also shown that cheese prepared using crude enzymes from H. erinaceum MAFF 435060, incorporating commercially available low-temperature pasteurized milk as the raw material, inhibited the growth of Aspergillus niger (Kishimoto et al., 2018).
For cheese prepared using rennet, particularly fresh-type cheese, the storage period is short and microbial spoilage often occurs during storage (Ledenbach and Marshall, 2009). The growth of various molds and bacteria on the cheese surface changes its color and smell and reduces cheese quality. In general, propionic acid and sorbic acid are used as preservatives to prevent microbial spoilage. These preservatives are recognized as safe, and their use is permitted within legal limit concentrations. Nevertheless, increasing consumer awareness regarding food safety has resulted in the increased popularity of food items that do not use preservatives. Accordingly, the development of bio-preservation techniques to prevent spoilage by microbes through the use of safe antibiotic substances derived from microorganisms is expected to comprise the next advance in cheese production. Therefore, the growth inhibition ability of cheese prepared using crude enzymes from H. erinaceum MAFF 435060 against A. niger NBRC 105649, as reported by Kishimoto et al. (2018), may be useful toward this goal. However, details concerning substances that inhibit the growth of A. niger NBRC 105649 and their formation mechanisms remain unclear. Thus, in this study, we aimed to identify fungal growth inhibitory substances in cheese prepared using crude enzymes from H. erinaceum MAFF 435060 and elucidate their formation mechanisms.
Chemicals and materials Malt extract and yeast extract were purchased from Kyokuto Seiyaku, Tokyo, Japan. Butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid were obtained from Fujifilm Wako Pure Chemical Co., Osaka, Japan. Rennet (CHY-MAX Powder-Extra, Christian Hansen Japan Co., Ltd., Tokyo, Japan) was used as chymosin for comparison. The commercial low-temperature pasteurized milk (66 °C, 30 min) was purchased from Takanashi Milk Products Co., Ltd., Yokohama, Japan. Raw milk and homogenized milk (45 000 rpm, 50 °C, 20 min) were provided by Megmilk Snow Brand Co., Ltd., Tokyo, Japan. Olive oil (Fujifilm Wako Pure Chemical Co.) and butter oil (Miyoshi Oil & Fat Co., Ltd., Tokyo, Japan) were used as a substrate for enzymatic reactions. Commercial cheeses were purchased from local stores. All other chemicals were of analytical grade.
Mycelia, cultivation, and preparation of crude enzymes H. erinaceum MAFF 435060 was used in this study owing to its high milk-clotting activity and the fungal growth inhibition ability of cheese prepared using crude enzymes obtained from this strain. MYS agar medium (pH 5.6) containing 1.0% malt extract, 0.4% yeast extract, 1.0% sucrose, and 1.5% agar was used for cultivation of the mycelia and measurement of fungal growth inhibition ability. Preparation of crude enzymes was conducted according to previously described methods (Kishimoto et al., 2018). Briefly, H. erinaceum MAFF 435060 was cultivated in 5 g sterilized wheat bran medium (water content of approximately 60%, w/w) at 25 °C for 14 d; then, 50 mL of 0.05 M McIlvaine buffer (pH 6.0) was added to extract crude enzymes. The prepared crude enzymes were used for the experiment after confirming that the milk-clotting activity was approximately 400 U/mL.
Preparation of cheese curd Cheese curd was prepared according to the method reported by Sato et al. (2016), using crude enzymes from H. erinaceum MAFF 435060. As a control, instead of crude enzymes, we used commercially available rennet (CHY-MAX Powder-Extra) adjusted to a concentration of 2 mg/mL. The prepared cheese curd was ripened for 7 to 45 d at either 13 °C, 25 °C, or 30 °C prior to being used in the study.
Preparation of cheese extract and gas chromatography–mass spectrometry (GC-MS) analysis The preparation of the cheese extract fraction was conducted as follows. First, 100 mL of n-hexane, diethyl ether, and distilled water were added to 6 g of ripened cheese, which was then homogenized using an Ace AM-8 homogenizer (Nihonseiki Ltd., Tokyo, Japan) for 2 min at 5 000 rpm. Then, 100 g of NaCl and 100 mL of 2.4 N HCl solution were added to the mixture and this was shaken vigorously prior to centrifugation (25 °C, 3 000 g for 10 min). The upper n-hexane-diethyl ether layer was then recovered. Extraction was conducted twice, and the upper n-hexane-diethyl ether layer was combined. Subsequently, 200 mL of a 1.2 N NaOH aqueous solution were added to the combined n-hexane-diethyl ether layer and mixed by shaking vigorously, whereupon the upper n-hexane-diethyl ether layer was recovered. To the lower layer, another 100 mL of n-hexane and diethyl ether were added for re-extraction. The upper n-hexane-diethyl ether layer was combined and concentrated after dehydration with anhydrous sodium sulfate and used as fraction 1. In the second step, 100 mL of n-hexane and diethyl ether were added to the remaining lower NaOH aqueous layer, and the emulsion layer from the first step shaken vigorously, and centrifuged; the lower aqueous layer was recovered. Subsequently, 200 mL of 1.2 N NaOH aqueous solution were added to the upper n-hexane-diethyl ether layer and emulsion layer, shaken vigorously and centrifuged, and the lower aqueous layer was collected and combined. Then, 100 mL of n-hexane and diethyl ether and 200 mL of 6 N HCl were added to the combined aqueous layer, shaken vigorously, and centrifuged. The recovered upper n-hexane-diethyl ether layer was then concentrated after dehydration and used as fraction 2. Finally, 200 mL of 1.2 N HCl were added to the upper n-hexane-diethyl ether layer, and the emulsion layer remaining after the recovery of the lower aqueous layer in the second step was shaken vigorously, and centrifuged. The recovered upper n-hexane-diethyl ether layer was then concentrated after dehydration and used as fraction 3.
The identification of each fatty acid contained in the second fraction extracted from cheese was performed by GC-MS using the GCMS-QP2010 Ultra system (Shimazu Co., Kyoto, Japan). The column used was Inert Cap Pure Wax (0.25 mm i.d. × 60 m, 0.25 µ m df; GL Science Inc., Tokyo, Japan). The flow rate of helium was 1 mL/min. The oven temperature cycle was programmed as follows: 40 °C held for 5 min, increased to 240 °C at a rate of 10 °C/min, and held at 240 °C for 20 min. Injection temperature, interface temperature, and ion source temperature were held at 250 °C, 200 °C, and 260 °C, respectively. Mass spectrometry, with an electron ionization voltage at 70 eV, scanned in the range of m/z 30 to 300 with a 0.2-s interval. Samples were methylated using a fatty acid methylation kit (Nacalai Tesque, Inc., Kyoto, Japan) prior to GC-MS analysis and identified by comparison with a mass spectral library (NIST 2008) and the results of analysis of methylated authentic compounds. Quantification of fatty acids in the cheese was carried out via the internal standard quantification method using 2-methyl pentanoic acid (3 mg/mL) as an internal standard. After methylation of 1 g of a cheese sample using the fatty acid methylation kit, fatty acids were subjected to quantification analysis.
Measurement of fungal growth inhibition ability MYS plates containing conidia or vegetative cells of A. niger NBRC 105649, A. oryzae NBRC 6215, Penicillium caseicolum NBRC 5849, P. roqueforti NBRC 5459, Saccharomyces cerevisiae NBRC 10217, or Candida albicans NBRC 1385 at a final concentration of approximately 1 × 105 cells/plate were used for measurements of fungal growth inhibition by cheese prepared using crude enzymes from H. erinaceum MAFF 435060. Sample holes were made in the plate using a dry heat-sterilized 7 mm in diameter cork borer and incubated over three days at 25 °C after placing 0.1 g of ripened cheese or commercially available cheese in the holes. The growth inhibition ability was determined by subtracting the diameter of the cork borer from the diameter of the inhibition circle confirmed after cultivation.
To examine the A. niger NBRC 105649 growth inhibition mediated by each fraction extracted from ripened cheese, 200 mg of each fraction were dissolved in 1 mL of dimethyl sulfoxide, and then 50 µL of the solution were dropped on dry heat-sterilized 8-mm paper disks (Advantec, Tokyo, Japan). The disk was placed on an MYS agar plate containing approximately 1 × 105 cells of conidia of A. niger NBRC 105649, and then the plate was incubated for three days at 25 °C. To determine the minimum inhibitory concentrations of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid authentic reagents for A. niger NBRC 105649, MYS agar medium containing approximately 1 × 105 cells of conidia of A. niger NBRC 105649 and a concentration of each fatty acid ranging from 296 to 1 795 mg/kg was dispensed in each well on sterilized microplates (AS ONE Corporation, Tokyo, Japan), and then the microplates were incubated for three days at 25 °C. The concentration at which the growth of the test organism was inhibited in all six wells of each concentration was taken as the minimum growth inhibitory concentration.
Measurement of lipase activity The substrate was prepared by adding 75 mL of 1% (w/v) gum arabic solution to 25 g of butter oil or olive oil and sonicating it at 28 kHz for 10 min for emulsification. Lipase activity of the crude enzymes from H. erinaceum MAFF 435060 was measured as follows. An aliquot of the substrate (5 mL), 4 mL of 0.05 M McIlvaine buffer (pH 6.0), and 1 mL of the crude enzymes were mixed and reacted at 35 °C for 24 h. Crude enzymes that had been thermally denatured at 100 °C for 10 min were used for the blank reaction. The mixture was heated for 30 min at 72 °C to stop the reaction. The reaction mixture was then titrated for neutralization with 0.05 N KOH solution, with lipase activity being determined from the titration volume. One unit was defined as the rate of producing 1 µ mol of oleic acid over 24 h.
Fungal growth inhibition by cheese prepared using crude enzymes from H. erinaceum MAFF 435060 Growth inhibition of several fungi by cheese prepared using crude enzymes from H. erinaceum MAFF 435060 or rennet was investigated after ripening at 13 °C for 30 d. The results are presented in Table 1. The growth of all tested strains (A. niger NBRC 105649, A. oryzae NBRC 6215, P. caseicolum NBRC 5849, P. roqueforti NBRC 5459, S. cerevisiae NBRC 10217, and C. albicans NBRC 1385) was inhibited by cheese prepared using crude enzymes. In contrast, no fungal growth inhibition was observed when cheese prepared using rennet was used.
| Strain | Growth inhibition (mm) | |
|---|---|---|
| Crude enzymesa | Rennet | |
| A. niger NBRC 105649 | 4.3 ± 0.9 | nd |
| A. oryzae NBRC 6215 | 5.7 ± 0.8 | nd |
| P. caseicolum NBRC 5849 | 3.9 ± 0.6 | nd |
| P. roqueforti NBRC 5459 | 4.1 ± 0.9 | nd |
| S. cerevisiae NBRC 10217 | 3.0 ± 0.6 | nd |
| C. albicans NBRC 1385 | 3.4 ± 0.9 | nd |
nd, Not detected.
Identification of fungal growth inhibitory substances in ripened cheese Fig. 1 shows the results of the growth inhibition of A. niger NBRC 105649 by each fraction extracted from 30-d ripened cheese that had been prepared using crude enzymes from H. erinaceum MAFF 435060. Growth inhibition of A. niger NBRC 105649 by fraction 2, but not by fractions 1 and 3, was observed. GC-MS analysis of fraction 2 identified the presence of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid, whereas long-chain fatty acids (≥ C12) were not detected. All of the authentic reagents of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid induced inhibition of A. niger NBRC 105649 growth; however, the minimum inhibitory concentrations differed between these compounds. The minimum inhibitory concentrations of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid for A. niger NBRC 105649 were 918, 908, 626, 1795, and 443 mg/kg, respectively.
Flowchart for the preparation of the cheese extract fractions, and inhibition of A. niger NBRC 105649 growth by the cheese extracts. (A), Fraction 1; (B), Fraction 2; (C), Fraction 3; (D), DMSO (solvent).
Changes in the growth inhibition of A. niger NBRC 105649 and fatty acid content of cheese prepared using either crude enzymes from H. erinaceum MAFF 435060 or rennet during the cheese ripening period are shown in Fig. 2. Growth inhibition of A. niger NBRC 105649 was confirmed at 15 days of ripening. Growth inhibition was strengthened with the ripening duration, with the amount of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid in the cheese also increasing simultaneously. Conversely, cheese prepared using rennet did not induce growth inhibition of A. niger NBRC 105649 even after 45 d of ripening and also showed little increase in the content of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid. Furthermore, the growth inhibition of A. niger NBRC 105649 by cheese prepared using crude enzymes from H. erinaceum MAFF 435060 became more potent over a short period when the cheese was ripened at 25 °C or 30 °C than when it was ripened at 13 °C (Fig. 3). The pH of cheese prepared using crude enzymes from H. erinaceum MAFF 435060 was 6.2 immediately after preparation, whereas it decreased to approximately 5.4 after ripening for 35 d.
Changes in A. niger NBRC 105649 growth inhibition and fatty acid content of cheese prepared using crude enzymes from H. erinaceum MAFF 435060 or rennet during ripening. (A) Changes in A. niger growth inhibition by the cheese. (○), Cheese prepared using the crude enzymes; (□), cheese prepared using rennet. (B) Changes in fatty acid content of cheese prepared using the crude enzymes. (●), Butanoic acid; (■), hexanoic acid; (◆) ,octanoic acid; (▴), decanoic acid; (×), decenoic acid. (C) Changes in fatty acid content of cheese prepared using rennet. Symbols are as in (B).Vertical bars represent standard deviation (n = 3). Ripening temperature was held at 13 °C.
Effect of ripening temperature of cheese prepared using crude enzymes from H. erinaceum MAFF 435060 on the growth inhibition of A. niger NBRC 105649. Cheese samples were ripened at 13 °C (●), 25 °C (■), or 30 °C(▴). Vertical bars represent standard deviation (n = 3).
Fatty acid content and fungal growth inhibition by commercial cheese The results of growth inhibition of A. niger NBRC 105649 and fatty acid content in commercial cheese as well as cheese prepared using crude enzymes from H. erinaceum MAFF 435060 or rennet are presented in Table 2. Similar to cheese prepared using rennet, commercial cheese did not induce A. niger NBRC 105649 growth inhibition. Moreover, the contents of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid in both types of cheese were below the minimum inhibitory concentrations for A. niger NBRC 105649 and substantively lower than those in cheese prepared using crude enzymes from H. erinaceum MAFF 435060, in which the amount of butanoic acid, hexanoic acid, and octanoic acid exceeded the A. niger NBRC 105649 minimum inhibitory concentrations.
| Sample | Growth inhibitiona | Fatty acid (mg/kg)b | ||||
|---|---|---|---|---|---|---|
| Butanoic | Hexanoic | Octanoic | Decanoic | 9-Decenoic | ||
| Commercial cheese | ||||||
| Bleu d'Auvergne AOP | − | 514 ± 48 | 366 ± 47 | 221 ± 49 | 459 ± 71 | 49 ± 20 |
| Roquefort AOP Société | − | 208 ± 24 | 197 ± 52 | 202 ± 119 | 896 ± 318 | 29 ± 13 |
| Mozzarella | − | 120 ± 77 | 34 ± 20 | 23 ± 10 | 156 ± 71 | 14 ± 10 |
| Parmigiano Reggiano | − | 402 ± 76 | 248 ± 20 | 126 ± 5 | 351 ± 50 | 5 ± 5 |
| Le Rustique Petit Munster | − | 145 ± 27 | 38 ± 7 | 26 ± 5 | 156 ± 26 | 7 ± 5 |
| Cheese prepared using | ||||||
| Crude enzymes from H. erinaceum | + | 6 342 ± 491 | 3 966 ± 62 | 1 714 ± 118 | 1 424 ± 148 | 265 ± 52 |
| Rennet | − | 87 ± 10 | 39 ± 15 | 22 ± 8 | 23 ± 8 | 3 ± 3 |
Lipase activity of crude enzymes from H. erinaceum MAFF 435060 The results of lipase activity in enzymatic reactions using butter oil or olive oil as substrate, along with the fatty acid content in the reaction mixture, are presented in Table 3. Notably, lipase activity was observed in enzyme reactions using butter oil, but not olive oil, as substrate. Products of the former enzymatic reactions included butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid.
| Enzyme | Substrate | Lipase activity (U/mL) | Fatty acid (mg/L)a, b | ||||
|---|---|---|---|---|---|---|---|
| Butanoic | Hexanoic | Octanoic | Decanoic | 9-Decenoic | |||
| Blank | Butter oil | nd | 20 ± 2 | 5 ± 2 | 2 ± 0 | 6 ± 5 | <1 |
| Crude enzymes | Butter oil | 76 | 109 ± 18 | 60 ± 6 | 26 ± 1 | 68 ± 8 | 4 ± 0 |
| Crude enzymes | Olive oil | nd | not analyzed | ||||
nd, Not detected.
Influence of homogenization of raw milk on fatty acid content and fungal growth inhibition by cheese Growth inhibition of A. niger NBRC 105649 and fatty acid content of cheese prepared using homogenized milk or raw milk as raw material and ripened for 30 d at 13 °C were evaluated; the results are summarized in Table 4. Cheese prepared from homogenized milk induced A. niger NBRC 105649 growth inhibition and showed high content of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid, with the first three exceeding the minimum inhibitory concentrations. In contrast, cheese prepared from raw milk as raw material did not induce A. niger NBRC 105649 growth inhibition and showed butanoic acid, hexanoic acid, and octanoic acid contents lower than the minimum inhibitory concentrations.
| Material | Growth inhibitiona | Fatty acid (mg/kg)b | ||||
|---|---|---|---|---|---|---|
| Butanoic | Hexanoic | Octanoic | Decanoic | 9-Decenoic | ||
| Raw milk | − | 208 ± 67 | 101 ± 38 | 39 ± 18 | 198 ± 38 | 8 ± 2 |
| Homogenized milk | + | 4 889 ± 560 | 3 200 ± 237 | 788 ± 72 | 814 ± 86 | 105 ± 23 |
Preventing microbial spoilage and improving shelf life represents a challenge for cheese manufacturing (Ledenbach and Marshall, 2009). To address this issue, the present study focused on determining the growth inhibition ability against A. niger NBRC 105649 of cheese prepared using crude enzymes from H. erinaceum MAFF 435060. To examine its potential as a bio-preservative, we identified the fungal growth inhibitory substances contained in the resultant cheese and elucidated the conditions facilitating their production. Butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid were identified in fraction 2 extracted from cheese that exhibited growth inhibition of A. niger NBRC 105649. In comparison, Parmesan cheese has been reported to contain butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid as primarily volatile free fatty acids (Kim and Lindsay, 1990).
Pohl et al. (2011) reported that many fatty acids, including butanoic acid, hexanoic acid, octanoic acid, and decanoic acid, demonstrate antifungal activity. In the present study, we found that cheese prepared using crude enzymes from H. erinaceum MAFF 435060 increased the growth inhibition of A. niger NBRC 105649 through ripening, while also increasing the butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid content. In particular, the contents of butanoic acid, hexanoic acid, and octanoic acid increased beyond their minimum inhibitory concentrations for A. niger NBRC 105649; thus, these three fatty acids were considered to primarily contribute to growth inhibition. Additionally, all five types of commercial cheeses analyzed contained concentrations of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic lower than the minimum inhibitory concentrations for A. niger NBRC 105649, with none of these cheeses inducing fungal growth inhibition.
Furthermore, when ripened at 25 °C or 30 °C, as opposed to 13 °C, cheese prepared using crude enzymes from H. erinaceum MAFF 435060 induced a more potent growth inhibition of A. niger NBRC 105649 over a short period and produced butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid, which suggested the involvement of lipase contained in the crude enzymes. Although lipase activity was observed in butter oil, this activity was not observed when olive oil was used as a substrate, which suggested that crude enzymes possess substrate specificity. Milk and butter fat contain a relatively high concentration of glycerides, which bind to butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid through the sn-3 position. Rennet paste and the sublingual lipase of ruminant animals specifically hydrolyze the ester bond of triglycerides with butanoic acid, hexanoic acid, and octanoic acid, thereby producing an sn-1,2 diacylglyceride and the fatty acid that was bound to the sn-3 position (Sun et al., 2007). Therefore, we speculated that the lipase contained in crude enzymes from H. erinaceum MAFF 435060 also acts weakly at the sn-1 and sn-2 positions and works specifically on the sn-3 position to promote the release of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic acid.
In turn, cheese prepared using raw milk as raw material with crude enzymes and subsequent ripening did not induce A. niger NBRC 105649 growth inhibition and had low contents of butanoic acid, hexanoic acid, octanoic acid, decanoic acid, and 9-decenoic. In contrast, cheese prepared using homogenized milk as raw material induced growth inhibition of A. niger NBRC 105649 and had high contents of the listed fatty acids. This suggested that the morphology of the fat globule membrane of milk also constitutes an important factor affecting the release of fatty acids by lipase action. Consistent with our findings, Haiya et al. (1989) reported that the homogenization treatment destroys the fat globule membrane of milk, exposing milk fat not covered with the membrane and rendering it susceptible to lipase action. Conversely, the commercially available rennet used in the present study was purified, resulting in its lipase activity being extremely low, which likely explained the resultant lower fatty acid content observed in this study.
Together, our findings indicate that through the use of homogenized milk as raw material and milk-clotting enzymes possessing lipase activity, such as crude enzymes from H. erinaceum MAFF 435060, it might be possible to develop cheese with high butanoic acid, hexanoic acid, and octanoic acid contents that exhibits an improved shelf life. In addition, lipase has been utilized to enhance cheese flavor (Aravindan et al., 2007; Hasan et al., 2006). We recognize that the sensory evaluation of cheese prepared using crude enzymes from H. erinaceum MAFF 435060 is extremely important. The cheese prepared on a small scale in this study had no serious faults in flavor and taste after ripening. Unfortunately, at this stage, the preparation of cheese using crude enzymes has not attained a scale adequate to perform sensory evaluation. The flavor characteristics of cheese prepared using crude enzymes from H. erinaceum MAFF 435060 would be clarified while conducting research on scaled-up cheese production.
Acknowledgments This work was supported by JSPS KAKENHI Grant Number 15K00780.
