As a member of ectomycorrhizal fungi, Tricholoma matsutake has a symbiotic relationship with its host, Pinus densiflora. To cultivate T. matsutake artificially, the co-cultivation of T. matsutake mycelia and bacteria from shiro was introduced. In this study, bacteria were isolated from soil samples in Bonghwa-gun, and seven bacterial isolates (B22_7_B05, B22_7_B06, B22_7_B07, B22_7_B08, B22_7_B10, B22_7_B13, and B22_7_B14) promoted the growth of T. matsutake mycelia (147.48, 232.11, 266.72, 211.43, 175.17, 154.62, and 177.92%, respectively). Sequencing of the 16S rRNA region of the isolated bacteria was performed. B22_7_B05 and B22_7_B10 were identified as Bacillus toyonensis, B22_7_B06 and B22_7_B08 as Paenibacillus taichungensis, B22_7_B07 and B22_7_B14 as P. gorilla, and B22_7_B13 as P. odorifer. These bacterial isolates were associated with the shiro community and are expected to contribute to the cultivation of T. matsutake.

Tricholoma matsutake is a traditional favorite food in East Asia, cultivated in fairy rings called “shiro,” which are found near Pinus densiflora. For effective artificial cultivation of Tri. matsutake, microorganisms from symbiotic fairy rings are co-cultivated. In this study, one bacterial isolate (Y22_B35) and two fungal isolates (Y22_F64 and Y22_F68) displayed growth-promoting effects on Tri. matsutake mycelium (158.47, 125.00, and 122.26% enhanced growth, respectively). For identification, 16S rRNA or ITS regions from the microorganisms¡¯ genomes were sequenced. Other sequences, including BenA, CaM, and RPB2 were sequenced in the fungal isolates. The bacterial isolate Y22_B35 was identified as Bacillus cereus. Y22_F64 and Y22_F68 were identified as Umbelopsis nana and Aspergillus parvulus, respectively. To identify the effects of the dominant microorganisms on Tri. Matsutake cultivation, metagenomic analyses were performed. Discovery of these Tri. matsutake mycelium growth-promoting microorganisms and metagenomics analyses are expected to contribute to our understanding of Tri. matsutake fruiting body growth and construction of biomimicry.

To cultivate pine mushroom (Tricholoma matsutake) artificially, co-cultivation with microorganisms has been introduced. Here, experiments were performed to assess the growth-promoting effect of bacteria on T. matsutake mycelia. Bacteria were isolated from soil samples collected in Yangyang County, Korea. Four of the bacterial isolates (Y22_B06, Y22_B11, Y22_B18, and Y22_B22) exhibited a growth-promoting effect on T. matsutake mycelia (154.67%, 125.91%, 134.06%, and 158.28%, respectively). To analyze the characteristics of the bacteria, especially the antifungal activity, -amylase and cellulase activity assays were performed. In comparison with the controls, the isolated bacteria exhibited low -amylase and cellulase activity. 16S rRNA gene sequencing was performed to identify the four bacterial isolates. The isolates belonged to the Terrabacteria group and were identified as Microbacterium paraoxydans, Paenibacillus castaneae, Peribacillus frigoritolerans, and P. butanolivorans. These bacterial isolates are expected to have contributed to the growth promotion of T. matsutake mycelia and the artificial cultivation of T. matsutake.

The mineral content of Tricholoma matsutake was evaluated for comparison of mineral contents according to the area of cultivation. Ten domestic and thirty Chinese (10 Yanji, 10 Yunnan and 10 Tibet) T. matsutake specimens were assessed using an atomic absorption spectrophotometer (AAS) and inductively coupled plasma mass spectrometer (ICP-MS). The Na, Mg, K, and Ca contents of domestic T. matsutake were 128.12±85.25 mg/kg, 218.52±105.35 mg/kg, 7,534.58±2,691.52 mg/kg, and 17.69±7.14 mg/kg, respectively, while those of Yanji T. matsutake were 124.89±57.24 mg/kg, 64.07±27.52 mg/kg, 1,439.18±311.04 mg/kg, and 10.88±4.52 mg/kg, respectively. The Na, Mg, K, and Ca contents of Yunnan T. matsutake were 90.78±23.23 mg/kg, 77.40±28.36 mg/kg, 1,446.29 ±126.33 mg/kg, and 28.42±5.18 mg/kg respectively, while those of Tibet T. matsutake were 143.50±41.54 mg/kg, 124.64±50.18 mg/kg, 3,530.95±2,714.99 mg/kg, and 21.05±8.71 mg/kg, respectively. The Cu contents of domestic, Yanji, Yunnan, and Tibet T. matsutake were 105.43±32.97 mg/kg, 19.92±8.95 mg/kg, 54.51±16.91 mg/kg, and 64.80±23.01 mg/kg, respectively. Both domestic and Chinese T. matsutake samples showed significantly different K, Mg, and Cu levels in this study. Therefore, a comparative evaluation of the K, Mg, and Cu contents of multiple domestic and Chinese T. matsutake varieties is needed to determine the appropriate area of cultivation in the future.

본 연구에서는 국산과 중국산 능이버섯과 송이버섯의 일반성분과 아미노산 조성을 분석하여 채취지역에 따른 능이버섯과 송이버섯의 식품학적 품질을 비교하고자 하였다. 송이버섯의 조회분 함량은 중국 티벳산이 5.52%, 운남 5.40%, 연길 6.95%, 한국 양양 6.40%로 시료 간에 차이를 나타내었다. 조지방 함량은 한국 양양산이 1.19%로 가장 낮았고, 조단백질 함량은 중국 티벳산이 16.83%로 가장 낮아 시료 간에 차이를 나타내었다. 송이버섯의 총아미노산 함량은 중국 연길 11,490.14±892.07 mg%, 중국 운남 8,000.03±2072.65 mg%, 티벳 6,815.48± 771.82 mg%, 한국 양양 6,074.74±814.86 mg% 순으로 나타났다. 능이버섯의 조회분, 조지방, 조단백질, 총아미노산 함량은 한국산과 중국산 시료 간에 차이를 나타내지 않았다. 본 실험결과 조회분 함량을 중국 남방지역(티벳, 운남) 송이버섯과 한국 양양 및 중국 북방(연길) 송이버섯의 구분지표로 활용할 수 있을 것으로 사료되며 무기질 성분에 대한 추가 실험을 통해 지표 무기질 도출이 필요한 것으로 판단된다.

There are about 15,000 kinds of mushrooms found worldwide and 2,000 kinds of edible mushrooms. There are about 1,500 kinds of mushrooms native to Korea, among which about 300 kinds of edible mushrooms are counted. In Korea, mainly Lentinus edodes (shiitake mushroom), Tricholoma matsutake (pine mushroom), and Sarcodon aspratus (Neungi mushroom) are edible, and they are imported from China due to insufficient production. The purpose of this study was to compare the nutritional quality of Chinese and Korean frozen Tricholoma matsutake and Sarcodon aspratus. The crude fat content of frozen Tricholoma matsutake was 1.1 ~ 1.5% in Chinese and 0.5% in Korea. Korea products were lower than Chinese ones. The content of crude protein in Chinese was 16.4 ~ 20.9% and that of Korea was 21.2%. In crude ash content, there are some differences between regions. The crude protein content of frozen Sarcodon aspratus was 18.9% in Chinese and 19.4% in Korea. The crude fat content was 1.2% in Chinese and 0.9% in Korea. There was no significant difference in the contents of crude ash.

The world mushroom market is continuously expanding due to the improvement of production technology and the continuous increase of demand. Production and consumption of mushrooms are increasing in Korea and imports are increasing year by year. Most of the imported mushrooms are from China. The Lentinus edodes (shiitake mushroom) are in dry form, but Tricholoma matsutake (pine mushroom) and Sarcodon aspratus (Neungi mushroom) are imported in frozen form. It is suspected that frozen Tricholoma matsutake and Sarcodon aspratus are coated with water to increase the weight excessively. In this study, we compared the moisture content and drip% of Chinese and Korean frozen Tricholoma matsutake and Sarcodon aspratus to determine the presence or absence of water coating. The moisture content of frozen Tricholoma matsutake was highest in Yunnan (China) at 94.7%, followed by Tibetan (China) at 92.5%, Yanji (China) at 90.6% and Korea at 88.5%. The moisture content of frozen Sarcodon aspratus was 92.4% in China and 91.6% in Korea. The drip% of frozen Tricholoma matsutake was 23.1% in Tibetan (China), 22.1% in Yunnan (China), 14.5% in Yanji (China) and 11.7% in Korea. The amount of drip% of Korea and Chinese frozen Sarcodon aspratus was 20.8% and 22.7%, respectively. Moisture content and drip% of frozen Sarcodon aspratus were similar to those of Chinese and Korea. Frozen Tricholoma matsutake imported from China showed high moisture content and drip% than Korea ones. These results indicated that water coating is possible in frozen Tricholoma matsutake imported from China.

Tricholoma matsutake is a representative mushroom species with a characteristic pleasant aroma. The characteristic aroma component is methyl cinnamate, which is also produced in many plants. In basil, cinnamic acid is produced from l-phenylalanine (l-Phe) by phenylalanine ammonia-lyase (PAL) and converted to methyl cinnamate by a cinnamate/p-coumarate carboxyl methyltransferase. Two PAL genes, Tmpal1 and Tmpal2, have been isolated from T. matsutake. In this study, we aimed to clarify the relationships between l-Phe, methyl cinnamate production, and PAL expression in the mycelium of T. matsutake strain NBRC 30605. For this purpose, methyl cinnamate content, PAL activity, and transcript levels of Tmpal1 and Tmpal2 were examined in the mycelia of T. matsutake supplemented with l-Phe. The mycelia were cultured in 20 mL of a liquid medium (2% glucose, 0.15% yeast extract, and 0.15% Bacto Soytone) at 20 °C for 45 d, supplemented with 0.5-6 mM l-Phe, and then grown for a further 15 d. Mycelia cultured without l-Phe supplementation for 60 d in the medium were used as a control. Crude extracts were prepared from the mycelia harvested for enzymatic, protein, and methyl cinnamate assays. Methyl cinnamate was measured using gas chromatography. PAL activity was assayed by measuring the rate of trans-cinnamic acid formation as the absorbance at 290 nm (ɛ290 = 10,000 M−1 cm−1). The transcript levels of Tmpal1 and Tmpal2 were examined by performing real-time reverse transcriptase-quantitative PCR on the total RNA. Methyl cinnamate was detected in very low levels in cultures without l-Phe supplementation, but its content per mg of protein increased markedly with increasing concentrations of l-Phe, especially at 4-6 mM. When 6 mM l-Phe was added to the culture medium, the methyl cinnamate content was approximately 55-fold higher than that of the control sample. The specific activity of PAL also increased in cultures supplemented with l-Phe, especially at 4-6 mM. When l-Phe was added to the culture medium, the methyl cinnamate content in the mycelia was relatively well correlated with PAL activity. These results indicated that supplementation with l-Phe, a precursor of methyl cinnamate, increases the specific activity of PAL, leading to an increase in methyl cinnamate production in the mycelia of T. matsutake. The transcript level of Tmpal1 did not change markedly with l-Phe supplementation. In contrast, the transcript level of Tmpal2 increased greatly in cultures supplemented with 4-6 mM l-Phe. These results suggested that the expression of Tmpal1 and Tmpal2 was controlled by different regulatory mechanisms and that they may have different biological functions in T. matsutake. In addition, the pattern of PAL activity in the presence of l-Phe was similar to that of the transcript level of Tmpal2, but not Tmpal1, suggesting that the increase in PAL activity was dependent on the increased transcription of Tmpal2.

송이버섯 균사체(Tricholoma matsutake)를 이용하여 발효시킨 발효대두와 Bacillus sp. Ja-8 균주 로 발효한 청국장 및 증자대두의 이화학적 특성을 비교 분석하였다. 유리아미노산의 총량은 송이버섯 균사체로 발효한 대두에서 3,379.01mg/100g으로 가장 높았으며, 증자대두에 비해 약 10배, 청국장에 비해서는 약 1.7배 더 높은 함량이었다. 총 isoflavone 함량은 증자대두가 1409.21μg/g, 송이버섯 균 사체로 발효한 대두가 1386.64μg/g, 청국장이 1256.64μg/g이었다. 총 isoflavone 가운데 isoflavone 비당체 구성 비율은 송이버섯 균사체로 발효한 대두에서 60%로 가장 높았으며, 청국장에서 14%, 증자 대두에서 2.4% 순이었다. 송이버섯 균사체 발효 대두의 총 유리 phenolics 함량은 14.2mg/g로 가장 높았고, 다음으로 청국장(13.5mg/g), 증자대두(9.8mg/g) 순이었다. 동결 건조한 대두 추출물을 에탄 올에 녹여 DPPH와 ABTs 라디칼 소거활성 및 FRAP를 측정한 결과, 100mg/mL 농도에서 시료들의 DPPH 라디칼 소거활성은 61.59-77.38%로 동일농도의 비타민 C와 유의차 없는 활성을 나타내었다. ABTs 라디칼 소거활성은 30mg/mL의 낮은 농도에서 송이버섯균사체 발효 대두 추출물의 활성이 63.22%로 가장 높았고, FRAP는 송이버섯균사체 발효물(341.02μmol)과 청국장(369.55μmol)에서 증자 대두(180.50μmol)에 비해 활성이 2배 정도 더 높았다. 결과적으로 송이버섯 균사체를 이용하여 대두 를 발효시킴으로서 isoflavone 비당체와 유리 phenolics 함량의 증가와 함께 항산화활성이 증가된 발 효대두 제조가 가능하였다.

본 연구는 도로건설이 송이생산지에 미치는 영향을 최소화하기 위해 강원도 양양군내 신설 예정인 고속도로 건설지 주변의 송이생산 소나무군락의 식생구조를 분석하여 보전 및 복원의 기초자료로 활용하고자 하였다. 고속도로 건설예정지의 영향권과 송이생산량을 고려하여 도로 주변에 총 20개 조사구를 설정하여 Classification기법중의 하나인 TWINSPAN을 이용하여 군락을 분리한 결과, 소나무군락(군락 I, II), 소나무-굴참나무군락(군락 III), 소나무-낙엽활엽수군락(군락 IV)의 4개 군락으로 최종 분리되었다. 군락별 종다양도는 1.7353±0.0341~1.9079±0.2471의 범위이었으며 종수는 평균 9.2±2.8, 교목층 출현 평균개체수는 9.6±5.0개체이었다. 식생밀도는 100m2당 4~29주(평균 9.55주), 평균상대공간지수는 35%이하이었으며 평균수령은 38±8.34년생이었으며 토양은 A0층의 깊이가 4~6cm, 토양산도는 4.70~5.63(평균 5.29) 송이 생육에 적정한 수준이었다. 송이는 소나무와 공생의 관계로 소나무군락의 식생구조와 매우 밀접한 관계를 가지고 있으므로 생태적 관리방안으로 적정밀도 조절, 아교목층과 관목층의 밀도조절, 교목층 낙엽활엽수의 제거 등을 제안하였다. 향후 도로건설시 송이생산지역내 관통도로를 최소화하고 송이생산지내 및 인근지역을 관통할 경우 숲내부 천이 및 식생구조 변화가 발생하지 않도록 생태적 관리 및 복원조치가 필요할 것이다.

In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3’- and 5’-RACE PCR and RT- PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

본 연구는 효과적인 송이산 관리를 위한 기초자료 확보를 위하여 강원도 양양군을 사례로 송이 생육환경특성을 고려한 소나무비오톱지도 작성을 목적으로 수행하였다. 연구방법은 송이와 관련한 국내 외 관련연구 고찰을 통해 송이 생육환경 특성을 도출하고, 송이 생산지의 식물군집구조와 토양환경을 파악하여 소나무비오톱 지도화 기준을 도출하였다. 지도화 기준은 송이가 발생할 수 있는 중요 요인을 지형 조건과 토양 조건, 식생 조건으로 구분하여 도출하였다. 전체 비오톱유형은 송이생산 가능지, 송이생산 잠재지로 크게 구분하였으며, 송이생산 가능지는 송이생산이 가능한 지형구조 및 토양구조 내 분포하는 소나무 비오톱으로 관목층에 기타 관목성상의 수종이 우점하는 흉고직경 30cm 미만의 단층구조의 소나무 비오톱과 아교목층에 소나무가 우점하는 다층구조의 소나무비오톱이 해당되었다. 송이생산잠재지는 송이생산이 가능한 지형구조 내에 분포하고 있으나 흉고직경 30cm 이상의 노령화된 소나무가 우점하는 단층구조의 소나무비오톱, 아교목층에 참나무류가 우점하는 소나무 비오톱, 관목층에 참나무류가 우점하는 단층구조의 소나무 비오톱 등 식생구조가 부적절한 지역으로 향후 식생관리를 통해 송이 발생 유도가 가능한 지역이었다. 연구결과 소나무비오톱은 송이생산 가능지 6개 유형, 송이생산 잠재지 9개 유형, 송이생산 불가능지 등 총 16개 유형으로 구분되었다. 송이생산 가능지는 전체 소나무림 중 총 9.8km2, 15.48%이었고, 송이생산 잠재지는 20.52km2, 32.42%이었으며, 송이생산 불가능지는 32.97km2, 52.10%이었다.

[Introduction] Matsutake mushroom (Tricholoma matsutake), an ectomycorrhizal fungus has low activity for polysaccharide hydrolyzing enzymes such as amylase, cellulase and hemicellulase. Therefore, only glucose and a few other monoand di-saccharides can be used to grow this fungus. Then, we examined the possibility for the mass production of the mycelia using the saccharified rice bran as growth substrate. [Methods and Results] For the selection of Kouji-mold, 32 strains of Aspergillus spp. were tested. These fungi were cultivated in potato dextrose liquid (PDL) medium added rice bran and amylase activities were assayed. As a result, Kouji-mold from Isesou Mugi Kouji (Asp. oryzae) was selected to use for saccharification of rice bran. When the saccharified rice bran was used as the growth substrate, the mycelical growth was enhanced. In this mushroom, the dry weight mycelia increased at about 3.0 times (control : 50mg, saccharified rice bran : 160mg, 60days, 24℃) by using saccharified rice bran compared with that of the control without saccharified rice bran. From there results, we see that saccharified rice bran is a useful culture material for the mass production of matsutake mycelia. Chemical analysis in saccharified rice bran solution was done with TLC and HPLC. The large amounts of glucose and maltose were detected in the saccharified solution. Now, we are trying to hydrolyze of cellulose and hemicelluloses components in rice bran using Asp. kawachii or Asp. saitoi isolated from Shouchu kouji.

Effect of trehalose for the mycelial growth of Tricholoma matsutake were examined. When T. matsutake Z-1 strain was cultured in the partially modified matsutake liquid (PMML) medium and the Hamada matsutake liquid (HML) medium supplemented with trehalose at 24 ℃ for 80days, the vegetative mycelial dry weights showed higher value compared with those of PMML medium and HML medium supplemented with glucose (control). The range of the effect of 1.0-8.0% carbohydrate substrate on vegetative mycelial growth was investigated. The optimal concentration for mycelial growth was 2.0% for the glucose medium but 8.0% for the trehalose medium. To evaluate the potential of the production of trehalase from T. matsutake, the extracellular trehalase activity during the vegetative mycelial growth was measured. The activity of the extracellular trehalase increased during vegetative mycelial growth of T. matsutake and was maximal 70 days after inoculation. This extracellular enzyme was investigated for the purification and the characterization. The partially purified trehalase was obtained from about 1.53l static culture filtrate, with 19.1% recovery, and about 2,940 fold purification. The molecular mass was about 62.6kDa (SDS-PAGE) and 70kDa (Gel-filtration). The enzyme was most active around 40℃ and pH 5.0 and stable over a pH of 4.5~ 6.5 for 30min at 37℃. The enzyme readily hydrolyzed trehalose having α -1,1 glucosidic bond. However, it did not hydrolyze disaccharides such as maltose, iso-maltose, cellobiose, saccharose and lactose.