J Wood Sci (2010) 56:483–487 © The Japan Wood Research Society 2010 DOI 10.1007/s10086-009-1129-y ORIGINAL ARTICLE Masakazu Hiraide · Tadakazu Nakashima Takeshi Fujiwara The smell and odorous components of dried shiitake mushroom, Lentinula edodes VI: increase in odorous compounds of dried shiitake mushroom cultivated on bed logs Received: January 9, 2009 / Accepted: March 24, 2009 / Published online: July 29, 2010 Abstract Odor is one of the most important characteristics ing sales of ¥73 billion.1 The total accounted for 17% of the affecting consumer preference for dried shiitake mush- revenue from all forestry and forest products (¥432 billion) rooms [Lentinula edodes (Berk.) Pegler]. In our previous and 35% of the total mushroom output including matsutake studies, we found that the odor content of commercial dried mushrooms (¥209 billion). This confi rms that the shiitake products was too weak for most people, and that the odorous mushroom is one of the most important products in the compound content could be increased by adding amino industry. However, consumption of fresh shiitake mush- acids to sawdust media. Currently, however, bed-log cultiva- room has been decreasing since 2000, and that of dried tion is used to produce fruiting bodies for dried products. shiitake has been decreasing since 1993. The purpose of this study was to fi nd a method to increase As both fresh and dried shiitake mushroom are evalu- the content of odorous compounds in dried products culti- ated mainly by shape and size in Japanese markets, research vated on bed logs. Pressure injection of amino acids from efforts have concentrated on improving shape and increas- the side of the bed log was the most effi cient method, but ing production amounts. To raise consumer interest and put it had some problems. Hence, a simpler and less trouble- the brakes on decreasing consumption, attractive products some method was developed, i.e., injecting amino acid solu- are required. Odor is an important factor in evaluating tion from small bottles set in deep holes bored in the sides foods,2–5 and dried shiitake mushrooms in particular are of the bed logs. In fruiting bodies cultivated on bed logs known for their characteristic smell upon rehydration. We injected with amino acid solution by the improved method, previously reported that the smell of commercial products the mean contents of lentinic acid, a precursor of the was evaluated as weak by most people.6 In the case of odorous compound lenthionine, approximately doubled sawdust-medium cultivation, the odorous compound compared to that in the untreated logs, although the infi ltra- content of dried shiitake mushrooms could be increased by tion area of the solution injected by the improved method adding cysteine (Cys, or methionine) and glutamic acid was smaller than that by the former method. (Glu) to the medium.7 In bed-log cultivation also, the odorous compound content in fruiting bodies should be Key words Dried shiitake mushroom · Lentinic acid · increased as they are used to produce dried products in Bed-log cultivation Japan. Increasing this content in shiitake mushrooms culti- vated on bed logs requires a method such as the infi ltration of amino acids. Thus, this study investigated appropriate Introduction methods for adding amino acids to the bed logs. The amount of fresh shiitake mushroom [Lentinula edodes (Berk.) Pegler] produced in Japan in 2006 was 66 349 ton Materials and methods and that of dried shiitake mushroom was 3861 ton, generat- Methods for adding amino acids to sawdust medium M. Hiraide (*) · T. Nakashima · T. Fujiwara We used one strain of L. edodes, Forestry Mycology Code Forestry and Forest Products Research Institute, 1 Matsunosato, 140, derived from stock cultures of the Mushroom Science Tsukuba 305-8687, Japan Tel. +81-29-829-8341; Fax +81-29-874-3720 Lab., Forestry and Forest Products Research Institute e-mail: [email protected] (FFPRI). As a control, 1-kg samples of sawdust medium with a ratio of rice bran to beech sawdust set at 5:95 (w/w) Part of this article was presented at the 58th Annual Meeting of the and a moisture content of 65% (w/w) were used. Amino Japan Wood Research Society, Tsukuba, March 2008 acids, dissolved in 160 ml water, were added to media as 484 follows: Conc11 (Cys 0.4 g/kg (w/w) and Glu 3.0 g/kg), Methods for adding amino acid solution to bed logs Conc12 (Cys 0.52 and Glu 3.9), Conc13 (Cys 0.68, Glu 5.1), and Conc14 (Cys 0.8, Glu 6.0).7 Two addition methods were Three deep holes were bored at 10, 40 and 70 cm from the used: a portion of the solution was poured into a plastic bag top face on the bed-log side and one of two different 600-ml for infi ltration from the surface of the medium, and another amino acid solutions was injected into each bed log: Conc21 portion of the solution was directly injected into the medium (Cys 2 mg/ml and Glu 15 mg/ml) or Conc22 (Cys 3 and at four positions using syringes. The media were settled in Glu 22.5). a developing room after 4 months of cultivation; therefore, the test using the infi ltration method for a 4-month period Analysis of 1,2,4-trithiolane and lentinic acid was not carried out. The fruiting bodies obtained were dried at 60°C for 24 h.8 The 1,2,4-trithiolane content was analyzed using the same method as described in previous reports.8 The lentinic 10 Development of fruiting bodies on bed logs acid content was analyzed as follows. Lentinic acid was extracted from crushed samples of fruiting bodies with 0.1 Bed logs (Quercus serrata, approximately 12 cm in diameter N HCl. After protein was denatured using 75% acetonitrile and 90 cm long) cultivating L. edodes hyphae for approxi- (ACN), lentinic acid derivatized with 9-fl uorenylmethyloxy- mately 15 months outdoors were purchased from a pro- carbonyl chloride was analyzed consecutively by an HPLC ducer in Tsukuba, Japan. The bed logs were submerged system equipped with an ODS column (Kanto Chemical, φ × overnight, transferred indoors, and suspended from a rod Mightysil RP-18 GP 4.6 mm 15 cm) and a fl uorescent inserted through a hole in each log, which was bored in the detector. Analytical conditions were as follows: fl ow rate radial direction below 30 cm from the top face. 1 ml/min; eluant, 0.1 M CH3COOH/Na buffer (pH 3.8)/ ACN = 78/22(0 min) → 69/31(16) → 58/42(23) → 55/45(37) → 20/80(45) → 10/90(55); column temperature 38°C; excita- Methods for adding acid fuchsin solution to sound logs tion wavelength 263 nm, emission wavelength 313 nm. and bed logs To visualize the solution-infi ltration area, 0.1% acid fuchsin Results and discussion solution was used.9 On the top face of sound logs and bed logs, holes 1.2 cmφ were bored vertically to a depth of 3 cm. Timing of addition Additionally, two deep holes were drilled diagonally through the heartwood to just inside the bark on the opposite side The timing of addition was tested using sawdust media, at 10 and 50 cm from the top face. Two injection methods because the addition methods to bed log were still uncer- were used. In one, 1-l plastic bottles hanging 20 cm above tain. The timing of addition to media was assumed as follows: the top face were connected to the holes by tubes. In the at inoculation, during cultivation, and just before develop- other, the solution was poured into a 200-ml plastic bottle. ment of fruiting bodies. In the case of bed-log cultivation, The bottle was equipped with a conical cap, of which the amino acid addition at inoculation and early-stage cultiva- top was cut off, then the bottle was inverted and the cap tion would cause contamination, because it is performed part was inserted into the hole. After injection, the bed logs outdoors; therefore, the timing of addition to sawdust media were transversally sliced approximately 2 cm from the top was set at the end stage, namely, the shiitake mycelia spread or base face, photos of the disks were taken, and the infi ltra- through the whole media. On the other hand, the addition tion areas were measured using the GNU Image Manipula- methods to the media were assumed as follows: infi ltration tion Program (GIMP, http://www.gimp.org/index.html) and from the surface of the media or injected into the media. ImageJ (an image processing and analysis program, http:// Then, the timing of addition was assayed by both addition rsb.info.nih.gov/ij/). methods. Fig. 1a,b. Relation between a b 1,2,4-trithiolane content in dried 3 3 shiitake mushrooms and amino acid addition time. a Amino acid mol/g) solutions infi ltrated from the mol/g) μ μ surface of sawdust medium, and 2 2 b directly injected into the sawdust medium. The average contents were calculated by three repetitions. Solid circles, 1 1 Conc11; open circles, Conc12; solid triangles, Conc13; open 1,2,4-trithiolane content ( triangles, Conc14 1,2,4-trithiolane content ( 0 0 2 3 4 2 3 4 Addition time from inoculation (month) Addition time from inoculation (month) 485 The content of the odor indicator for dried fruiting 25 bodies (1,2,4-trithiolane) was measured in dried fruiting bodies from sawdust medium and was found to maintain 20 ) the same level regardless of the infi ltration time (Fig.