Crude Enzymes from a Hericium Edible Mushroom Isolated in Japan: Variability in Milk-clotting Activity and the Ability to Coagulate Ultra-high-temperature Pasteurized Milk

Abstract

Eleven mycelial strains of the genus Hericium were isolated to investigate the variability in milk-clotting activity and ability to coagulate ultra-high-temperature pasteurized milk (UHT-milk) of their crude enzymes. As well, the antifungal activity of cheese prepared using the crude enzymes was assessed. Based on sequence analysis of ITS-5.8S ribosomal DNA, 8 strains were identified as H. erinaceus, 2 as H. abietis, and 1 as Hericium sp. Notable differences were observed among the species and within strains in terms of milk-clotting activity of the crude enzyme preparation. The UHT-milk coagulation ability was detected in 5 strains of H. erinaceus and showed no correlation with the potency of milk-clotting activity. Furthermore, all cheese samples prepared from low-temperature pasteurized milk using the crude enzymes from the 8 strains of H. erinaceus and ripened for 30 days at 13°C showed growth inhibitory activity toward Aspergillus niger NBRC 105649. Thus, the crude enzyme preparation from H. erinaceus may be useful for cheese production.

Introduction

In the production of cheese, rennet (chymosin), which is synthesized in the abomasum (a part of the stomach) of young ruminant animals, has traditionally been used for milk coagulation. Nonetheless, an inadequate supply of the enzyme, due to increased cheese production and concerns regarding the slaughter of animals, has prompted the search for microbes that produce milk-clotting enzymes with properties close to those of rennet. Milk-clotting enzymes derived from mold have been developed as replacements for rennet. Mass culture techniques for Rhizomucor miehei, Rhizomucor pusillus, and Cryphonectria parasitica have been established (Jacob et al., 2011). Milk-clotting enzymes derived from mold are advantageous for replacement of the costly calf rennet. Nevertheless, because of concomitant proteolytic activity, the cheese thus produced often tastes bitter (Sousa et al., 2001). Basidiomycetes may also be a useful source of milk-clotting enzymes without this problem. Irpex lacteus (Kobayashi et al., 1985, Kikuchi et al., 1988, Kobayashi and Kasamo, 1992, Chemeris et al., 2016), Phellinus chrysoloma, Kuehneromyces mutabilis, Ganoderma applanatum (Nerud et al., 1989), Schizophyllum commune (Okamura-Matsui et al., 2001), Coprinus lagopides (Shamtsyan et al., 2014), Termitomyces clypeatus (Majumder et al., 2015), Piptoporus soloniensis (El-Baky et al., 2011), Laetiporus sulphureus (Kobayashi and Kim, 2003), and Pleurotus ostreatus (Lebedeva and Proskuryakov, 2009, Yin et al., 2014) have been found to produce milk-clotting enzymes. Except for P. ostreatus, these Basidiomycetes are not commonly used in foods in Japan; moreover, some are suspected to cause infection in humans (Amitani et al., 1996, Buzina et al., 2005). Thus, Nakamura et al. (2014) explored milk-clotting-enzyme–producing strains among mushrooms consumed routinely and found high milk-clotting activity and low protease activity in three strains of Hericium erinaceum MAFF 435060, MAFF 430234 and NBRC 100328, indicating its potential use in cheese production. Furthermore, Sato et al. (2016) found that the milk-clotting enzyme in H. erinaceum MAFF 435060 has the ability to coagulate milk pasteurized at a low temperature (66°C, 30 min) as well as milk pasteurized at ultra-high temperatures (130°C, 2 s). Additionally, during the course of research, we found that microbial spoilage during ripening did not readily occur in cheeses prepared using the crude enzymes from H. erinaceum MAFF 435060 (unpublished results).

Although the classification of the genus Hericium remains unclear, phylogenetic classification based on ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequences revealed Hericium abietis, H. alpestre, H. americanum, H. coralloides, and H. erinaceus, among others, as the most promising species (Hallenberg et al., 2013). These findings have generated interest in the variability of milk-clotting activity as well as the UHT-milk coagulation ability of the crude enzymes among species and strains of the genus Hericium.

The purpose of this study was to examine the variability in milk-clotting activity and UHT-milk coagulation ability among species and strains of the genus Hericium newly isolated in Japan. Moreover, we evaluated the antifungal activity of the cheese produced using a crude enzyme preparation.

Materials and Methods

Isolation and identification of mycelial strains    Fruiting bodies presumed to originate from the genus Hericium and growing on dead wood were harvested in 2015 in 4 prefectures: Yamanashi, Nagano, Wakayama, and Kochi. At the time of harvest, the spine length and spine arrangement typical of the genus Hericium were observed. To isolate the mycelium, cells from the central part of the fruiting body were cultured on MYS medium (pH 5.6) consisting of 1.0% malt extract (Kyokutoseiyaku, Tokyo, Japan), 0.4% yeast extract (Kyokutoseiyaku), 1.0% sucrose, and 1.5% agar or on potato dextrose agar (Nissui, Tokyo, Japan) at 25°C. The generated mycelia were purified and stored at −80°C. After culturing on MYS medium, the purified mycelia were subjected to ITS-5.8S rDNA base sequence analysis, BLAST analysis, and identification at Techno Suruga Lab Co., Ltd. (Shizuoka, Japan).

Cultivation of mycelia and preparation of crude enzymes    As described by Nakamura et al. (2014), the isolated strains were precultured on MYS plates at 25°C until the entire plate was covered with mycelia. Five discs were collected from each precultured plate using a sterilized cork-borer (7 mm in diameter). To 5 g of wheat bran in a 100-mL Erlenmeyer flask, 5.9 mL of water was added, followed by sterilization at 110°C for 20 min. The discs were inoculated into the sterilized wheat bran medium (water content of approximately 60%, w/w). After 14 days of cultivation at 25°C, 50 mL of 0.05 M McIlvaine buffer (pH 6.0) was added to the medium, and the suspension was kept overnight at 4°C. The suspension was filtered to remove the wheat bran and the mushroom mycelia using a metal mesh cloth (32-mesh). The filtrate was centrifuged (10,000 × g, 15 min, 7°C) and filtered to remove solid particles using No. 2 filter paper (Advantec, Tokyo, Japan). The filtrate served as the crude enzyme preparation. Similarly, H. erinaceum MAFF 435060, selected as a suitable strain for cheese making by Nakamura et al. (2014), was cultured, and the extracted crude enzyme was then used to prepare cheese.

Measurement of milk-clotting activity and UHT-milk coagulation ability    Milk-clotting activity of the crude enzymes extracted from the wheat bran culture of the isolated strain was determined as described by Arima et al. (1970). As a substrate solution, 5 mL of 10% (w/v) skim milk (Morinaga Co., Ltd., Tokyo, Japan) containing 0.01 M calcium chloride was incubated at 35°C. To this solution, we added 0.5 mL of a crude enzyme preparation, after which the time required for coagulated particles to form the curd was measured. Milk-clotting activity was expressed in Soxhlet units. Moreover, to determine the coagulation ability toward UHT-milk (130°C, 2 s), 5 mL of commercial UHT-milk (Meiji Co., Ltd., Tokyo, Japan) was incubated at 35°C, then 0.5 mL of a crude enzyme preparation was added, and the status of coagulation was checked after 40 min.

Preparation of cheese curd and measurement of antifungal activity    Cheese curd was made from 100 mL of low-temperature pasteurized milk (Takanashi Milk Products Co., Ltd., Kanagawa, Japan) as described by Sato et al. (2016). The milk was incubated at 35°C for 10 min before addition of 10 mL of a crude enzyme preparation from the isolated mycelial strains of H. erinaceus—or 1 mL of 2 mg/mL commercial rennet (CHY-MAX Powder-Extra, Christian Hansen Japan Co., Ltd., Tokyo, Japan) in place of the crude enzymes as the control—and mixed gently. The milk was then continuously incubated at 35°C until all milk was clotted completely. The clotted substance was diced into small cubes and then warmed to 50°C to accelerate the discharge of whey. The curd was poured into the mold and kept at 4°C for 3 h with a weight. After the whey was discharged, the hardened curd was immersed in 25% salted water for 1 h and placed in a tightly closed container to ripen at 13°C. The ripened cheese was stored at −20°C if necessary.

To evaluate the antifungal activity, an MYS plate was mixed with the test mold—Aspergillus niger NBRC 105649 spores—to a concentration of approximately 10⁵ spores per plate. A sample hole was made in the plate using a 7-mm ∅ dry-heat–sterilized cork-borer, then the hole was packed with 0.1 g of ripened cheese, and the plate was incubated at 25°C for 3 days. Next, the diameter of the circle of inhibition of growth was measured. The diameter of the cork-borer was subtracted from this value to determine the growth-inhibitory ability.

Results and Discussion

Isolation and identification of mycelia from fruiting bodies    Fruiting bodies growing on a dead tree were harvested and a total of 11 mycelial strains were newly isolated. The characteristics of the harvested fruiting bodies and results of BLAST searches based on analysis of the ITS-5.8S rDNA sequence of the strains are presented in Table 1. All the harvested fruiting bodies were presumed to belong to the genus Hericium according to their morphological features. The BLAST results based on ITS-5.8S rDNA sequence analysis of the strains indicated that all the isolates belonged to the genus Hericium. The shared homology among 8 strains—WH01, WH02, WH03, WH04, WH06, WH07, WH08, and WH13—with H. erinaceus KUMC1008 was ≥99.8%; thus, these strains were identified as H. erinaceus. Two strains, WH20 and WH21, shared 99.5% homology with H. abietis CBS 125851, and thus the strains were identified as H. abietis. The homology between WH10 and H. coralloides ATCC52480 was 95.9%, which was a somewhat low value, and this strain was assumed to be closely related to H. coralloides. Therefore, the species could not be identified and was predicted to be a Hericium sp. The classification of the genus Hericium from Japan was investigated by Obatake et al. (2008) and reported as follows: Two species, H. erinaceus (Yamabushitake in Japanese) and H. abietis (Sangoharitakemodoki in Japanese), were identified, and Sangoharitake (in Japanese) had a fruiting body resembling that of Western-produced H. coralloides in morphological features and was closely related based on rDNA sequence analysis; however, crossing between single-nucleus mycelia was not confirmed. H. erinaceus is distributed in Japan and North America (Boddy et al., 2011), and has been used in folk medicine and in medicinal cuisine (Wang et al., 2014). This species is currently cultivated artificially in Japan.

Table 1. Characteristics of the harvested fruit bodies and results of BLAST searching based on analysis of the ITS-5.8S rDNA sequence of mycelial strains isolated in Japan.

Mycelial strain	Sampling place	Arrangements of spines	Length of spines	Presumed genusa)	BLAST match sequence
					Reference strain No. b)	Accession No.	Similarity(%)
WH01	Yamanashi	Spines in bundles	1–4 cm	Hericium	H. erinaceus KUMC1008	AY534593	100
WH02	Wakayama	Spines in bundles	1–4 cm	Hericium	H. erinaceus KUMC1008	AY534593	100
WH03	Yamanashi	Spines in bundles	1–4 cm	Hericium	H. erinaceus KUMC1008	AY534593	100
WH04	Yamanashi	Spines in bundles	1–4 cm	Hericium	H. erinaceus KUMC1008	AY534593	100
WH06	Yamanashi	Spines in bundles	1–4 cm	Hericium	H. erinaceus KUMC1008	AY534593	100
WH07	Yamanashi	Spines in bundles	1–4 cm	Hericium	H. erinaceus KUMC1008	AY534593	100
WH08	Yamanashi	Spines in bundles	1–2 cm	Hericium	H. erinaceus KUMC1008	AY534593	100
WH10	Yamanashi	Spines single, combilike	<1 cm	Hericium	H. coralloides ATCC52480	AY534584	95.9
WH13	Yamanashi	Spines in bundles	1–4 cm	Hericium	H. erinaceus KUMC1008	AY534593	99.8
WH20	Nagano	Spines in bundles	<1 cm	Hericium	Habietis CBS 125851	JN201334	99.5
WH21	Kochi	Spines in bundles	<1 cm	Hericium	H. abietis CBS 125851	JN201334	99.5

a)  Genus presumed from morphological features of the fruiting body.

b)  Highest-ranking strain in BLAST search with sequence of the strain.

Milk-clotting activity and UHT-milk coagulation ability of crude enzyme preparations    The milk-clotting activity of each crude enzyme preparation extracted from the wheat bran culture of isolated strains is shown in Table 2. The milk-clotting activities of crude enzyme preparations from WH01, WH02, WH03, WH04, WH06, WH07, WH08, and WH13, identified as H. erinaceus, were 85.8–333.8 U/mL. When the WH01 strain showing the highest activity and the WH13 strain showing the lowest activity were compared, an approximately 4-fold difference was observed. Milk-clotting activity was not detected in strains WH20 and WH21, which were identified as H. abietis. In addition, weak milk-clotting activity was detected in strain WH10 (Hericium sp.), equivalent to that of WH13, which showed the lowest activity among the H. erinaceus strains. These results revealed that the strength of milk-clotting activity varies considerably among species and strains. Chemeris et al. (2016) reported that milk-clotting enzymatic activity in a liquid culture of I. lacteus differs among strains.

Table 2. Milk-clotting activity and UHT-milk coagulation ability of crude enzymes from the isolated mycelial strains.

Mycelial strain	Milk clotting activity (U/mL)	UHT-milk coagulation ability
WH01	333.8±103.0	Absent
WH02	310.6±48.3	Present
WH03	260.2±49.8	Present
WH04	251.6±49.7	Present
WH06	218.2±20.2	Absent
WH07	174.9±9.2	Present
WH08	169.0±36.5	Present
WH10	70.6±39.4	Absent
WH13	85.8±25.2	Absent
WH20	ND	NT
WH21	ND	NT

Values are the mean ±standard deviation (n=4). Mean of the enzyme activity in four independent cultures extracts. Absent, milk without coagulation; Present, milk with strong coagulation.

ND, not detected; NT, not tested.

An example of coagulation of UHT-milk by a crude enzyme preparation is shown in Fig. 1. As shown in Fig. 1(B), clearly separate curd and whey were produced by crude enzymes from 5 strains—WH02, WH03, WH04, WH07, and WH08—and their UHT-milk coagulation ability was confirmed. In contrast, the coagulation ability toward UHT-milk was not detected in strains WH01, WH06, WH10, and WH13 (Table 2). The status of UHT-milk coagulation ability differed among the strains of H. erinaceus, and showed no correlation with the potency of milk-clotting activity. According to these results, we estimated that the nature of the milk-clotting enzymes in H. erinaceus is strain-dependent. Sato et al. (2016) found that the milk-clotting enzyme in H. erinaceum MAFF 435060 has coagulation ability not only toward milk pasteurized at a low temperature but also toward UHT-milk, and demonstrated that the milk-clotting effect is not due to degradation of κ-casein. It is known that rennet does not have coagulation ability toward UHT-milk (McMahon et al., 1993). The UHT-milk coagulation ability identified in crude enzyme preparations from WH02, WH03, WH04, WH07, and WH08 is expected to bring about technological innovation to the currently challenging process of cheese production from UHT-milk as a source.

Fig. 1.

An example of UHT-milk coagulation by a crude enzyme preparation. (A) Milk without coagulation; (B) Milk with effective coagulation.

Antifungal activity of cheese prepared using crude enzymes from H. erinaceus    We found that microbial spoilage during ripening did not readily occur in cheeses prepared with crude enzymes from H. erinaceus MAFF 435060. The changes in growth inhibition of A. niger NBRC 105649 during the ripening of cheese prepared using the crude enzymes from H. erinaceum MAFF 435060 were compared with those of cheese prepared using rennet (Fig. 2). When rennet was used, A. niger growth was not inhibited during the ripening period. In contrast, for cheeses prepared using the crude enzymes from H. erinaceus, the potency of growth inhibition gradually increased with the number of ripening days. We also compared the growth inhibition ability toward A. niger of the cheese prepared using the crude enzymes (from the 8 strains of H. erinaceus, which all created successful curd, and ripened for 30 days at 13°C) with the inhibition exerted by the cheese prepared using rennet (Fig. 3). Potent growth inhibition was observed for all 8 strains, indicating that cheese prepared using crude enzymes from H. erinaceus has antifungal activity. For cheese prepared using rennet, particularly fresh-type cheese, the storage period is short, and microbial spoilage often occurs during storage (Ledenbach et al., 2009). The use of crude enzymes from H. erinaceus for cheese-making may improve the shelf-life of cheese; thus, further studies on the mechanism of antifungal activity are needed.

Fig. 2.

Inhibition of A. niger growth by cheese prepared using rennet (A) or crude enzymes from H. erinaceum MAFF435060 (B). Cheese samples were tested before ripening (a) and after ripening at 13°C for 15 days (b), 30 days (c), and 45 days (d).

Fig. 3.

Growth inhibition abilities toward A. niger of the cheese prepared using the crude enzymes from 8 strains of H. erinaceus and rennet.

Error bars indicate standard deviation (n = 3). Means reflect the growth inhibition shown by 3 independent cheese samples.

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