Isolation and identification of aroma-producing yeast strain from black glutinous rice wine.
MATERIALS AND METHODS
BGR yeast,10 strains, numbered [Q.sub.1] ~ [Q.sub.10], were collected in Guizhou, China; BGR samples were purchased from local market of Guiyang city, China.
Brewing process of BGR wine
The black glutinous rice was cleaned twice or three times r. Then it was soaked in 60 [degrees]C water for 3 hours. During cooking at atmospheric pressure, 85[degrees]C hot water was added in order to ensure that the rice yield rate was 160% to 200%.The steamed BGR was cooled to 28 to 30[degrees]C. About 1.0% (w/w) starter was added and mixed. The steamed BGR was fermented for 6 to 7 days at 28 to 30[degrees]C. After fermentation, the wine was pressed in time. The pressed wine was clarified for 2 to 4 days. The BGR wine was obtained after filtration and sterilization.
Primary screening of aroma-producing strains
The primary screening medium consisted of soluble starch 12 g, yeast extract 8 g, NaCl 5 g, agar 15 g and water 1L.A reagent 2.2 g [I.sub.2] and 4.4 g KI, dissolved in l00 ml of distilled water was prepared. Preparation of isolates singlel spores (ISS) was by mixing sterile water into activation agarslantculture-medium .The spores or bacterial cells were scraped and beaten. The spores were diluted to 1 x [10.sup.6] / ml after quantification using a hemocytometer. The isolates single spores were cultured at 30'C for 1 ~ 3 d after diluted and coated at the primary screening medium. Then the iodine reagent was pour on the primary screening medium. The transparent circle around the colonies were observed and measured by the colony diameter (D) and transparent circle diameter (d) . The higher the size was, the stronger the force of glucoamylase production. The experiment was carried out by five plates. Relationship between the activity and transparent circle expressed by the formula (Xu & Jiang, 2001):
log ([E]) / R) = k (d / D) ([DELTA] [C] / logt) (Formula: [E]--production enzyme concentration; R--cell volume; d--transparent circle diameter; D--diameter of the colony; [DELTA]--agar concentration; [C]--concentration of substrate; t--incubation time; k--constant.)
Secondary bran screening was performed as described by Chen (Chen, 1996)
A total of 20 g fresh bran was weighed, and mixed with water (60% w/v). After mixed for 30 min, the mixture sample was added into 500 ml flask with a 2 -3 cm thick, stoppered with cotton wool tampon. The sample was autoclaved at 0.10 MPa for 40 min, and exhausted to lower pressure. When the temperature dropped to 25 ~ 30 [degrees]C, the second sterilization was repeated for 40 min. After sterilization, the flask was removed and shook well. When the bran medium was cooled to 30 [degrees]C, 3ml ISS was inoculated. The sample was cultured at 32 [degrees]C for 72 h. Then the sample was put into the sterile bag, and dried at 40 [degrees]C for 12 h. The liquefaction power and saccharifying power were measured. BGR wine based on the traditional brewing process was fermented. The sugar, total acid content and aroma of fermentation starter were measured through sensory evaluation by five wine professional trained sensory tasters. Aroma components in the fermented starter with soft flavor were analyzed by GC/MS.
Determination of aroma compounds by using GC/ MS
The determination was performed as described by You et al.(You, Wang, Zhan & Huang, 2008). Fifty [micro]m thickness of DVB/CAR/ PDMS extraction head was selected. The sample was 120 ml/l alcoholicity with warm-up time 20min, extraction temperature 45 [degrees]C, extraction time 30 min. The concentration of electrolyte NaCl was 0.30 g/ml.
The analysis condition of GC was splitless mode, GC column HP-INNOWAX (30.0 m x 0.25 mm x 0.25 [micro]m), programmed temperature 40 [degrees]C HELD for 8 min, then with 5 [degrees]C /min rate to 70 [sup.i]C and held for 1 min, 5 [degrees]C / min rate to 185 [degrees]C, and then with 10 [degrees]C /min rate to 230 [degrees]C and held for 8 min, detector temperature 250 [degrees]C and inlet temperature was 280 [degrees]C. The MS condition was EI ionization source, with electron energy 70 eV, scan range 50 ~500 amu, ion source temperature 250 [degrees]C.
Aroma-producing strain ITS Sequence analysis method
Yeast ITS sequence analysis was performed using the methods of Lehtonen (Lehtonen & Patrikainen, 2012). Universal primers ITS5 (5 ' GGAAGTAAAAGTCGTAACAAGG-3 ') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used to amplify genomic DNA and ITS-5. 8S rDNA sequences were obtained. Five ul amplification product was analyzed by agarose gel electrophoresis.
Primary screening of starch transparent circle
A total of 28 Saccharomycopsis and 212 Pichia strains identified were inoculated on starch plates and cultured at 28 [degrees]C for 24 h in the previous studies. After coloration, the diameters of saccharification transparent circle and colonies were measured to screen 12 yeasts with strong saccharification. Strain [Q.sub.10]-[M.sub.62] was cultured at 28[degrees]C for 24 h. Saccharification transparent circles were shown in figure1. Traits of higher capacity of glucoamylase production strains were shown in Table 1.
According to the formula log ([E])/R) = k (d/D) ([DELTA][C]/logt), the ratio of glycated transparent circle diameter and colony diameter (d/D) and enzyme concentrations were positively correlated. Therefore, the larger d/D, the stronger the production of glucoamylase(Xu et al., 2001). On the basis of saccharification transparent circle of screening test, 12 strains with high glucoamylase production capacity such as [Q.sub.8]-[M.sub.27], [Q.sub.9]-[M.sub.45], [Q.sub.3]-[M.sub.24] and so on were selected for further screening.
Bran medium secondary screening and BGR test
Twelve strains selected by primary screening were screened by secondary screening through bran medium, including bran solid starter extraction enzyme solution. The enzyme solution liquefaction and saccharification power were determined to compare the strain enzyme production power, saccharification power, acid production capacity, and aroma-producing capacity.
As seen from Table 2, [Q.sub.10]-[M.sub.62] strain showed higher saccharification enzyme production capacity than others, and contained the highest sugar level in the final starter. It suggested that the strain had significant influences on BGR saccharification, but relatively lower acid production capacity. Its aroma score was the highest. As a result, [Q.sub.10]-[M.sub.62] is an excellent aroma-producing strain.
GC / MS analysis
Yeast is the key functional fungi of wine-producing and aroma-producing. It directly affects the brewing process, thereby affects the wine production and aroma components (Hou, Wang, Li, Hu, Li & Gao, 2013). GC / MS has the abilities of efficient chromatographic separation and specific mass spectrometry identification and is commonly applied to the analysis of wine aroma components (Aznar, L O Pez, Cacho & Ferreira, 2001; D I AzMaroto, S A Nchez-Palomo & P E Rez-Coello, 2004). From the GC / MS analysis results in Table 3, 27 and 32 aroma components were detected in traditional and aroma-producing strains fermentation broths. The variety was less than aroma detected in light aroma type liquors by means of gas chromatography" olfactometry coupled with mass spectrometry, which was a total of 66 aroma compounds (Gao, Fan & Xu, 2014). Besides, a total of 39 aroma compounds were characterized by GC-O in Chinese rice wine Qu (Mo et al, 2010). Because different microorganisms and enzymes can produce different aroma components.
As we can see, there was a big difference between the two content of aroma components, like composition and ratio. There were a lot of microorganisms in traditional fermentation broth .Because of antagonistic, these microorganisms may inhibit the growth of some other microbes, such as aroma-producing strains. As a result, aroma composition and ratio were different. The kinds of aroma components in traditional fermentation broth were less than [Q.sub.10]-[M.sub.62] fermentation broth. Maybe there were more miscellaneous microorganisms in traditional fermentation broth and they may produce more methylalcohol. Methyl linolelaidate generated from methyl- esterification reaction of trans- linoleic acid with methylalcohol. The more methylalcohol benefited methyl- esterification reaction. Therefore, the content of methyl linolelaidate accounted for about 37% of the total aroma in traditional fermentation broth, but it was not seen in [Q.sub.10]-[M.sub.62] broth.
From the types of aroma components, 16 existed in both of the broths, and were accounted for more than half of the total detected components in traditional starter. From the relative content, 16 kinds of aroma components in both accounted for 33.34% of the total aroma components in traditional fermentation broth, and 75.62% of the total strain [Q.sub.10]-[M.sub.62] fermentation broth. The reason was that the traditional starter contains a variety of microorganisms and enzymes, which contributed to the wine flavor profile. Aroma components produced by [Q.sub.10]-[M.sub.62] accounted for 33.34% of the total aroma components in traditional fermentation broth. As a result, [Q.sub.10]-[M.sub.62] was the major aroma-producing strain in bGr wine.
ITS sequence analysis of aroma-producing strains [Q.sub.10]-[M.sub.62]
The result of ITS sequence is shown in Figure 2. The amplification stripe of [Q.sub.10]-[M.sub.62] ITS-5.8S rDNA was clear and had good specificity .It indicated that [Q.sub.10]-[M.sub.62] ITS sequences of PCR amplification was successful. Compared with the mark band, the [Q.sub.10]-[M.sub.62] sequence length was about 750 bp.
Characteristics of colony and thallus
As shown in Figure 3, the colony was round and flat with a white and raised middle. The surrounding area was white and radial. The colony surface was dry and rough with short hypha. The colony edge was irregular and odontoid. As can be seen in Figure 4, most lemon-shaped thallus was big and round at one end, and small pointed at the other end. Of course, some were round. They formed pseudohyphae with some spores with about 4.2 x 2.8[micro]m. The characteristics of colony and cell were very similar to Cyberlindnera jadinii (previously Pichia jadinii) (Fernandez, Cabral, Delgado, Farina & Figueroa, 2013).
Phylogenetic tree construction of [Q.sub.10]-[M.sub.62]
[Q.sub.10]-[M.sub.62] with fragment length 626 bp was amplified by ITS1-5.8S-ITS2 rDNA primers. ITS sequences were compared by Blast on NCBI. The results showed that [Q.sub.10]-[M.sub.62] had high homology with Cyberlindnera jadinii (Pichia jadinii) which was positive sequence similarity. MEGA5.1 maximum parsimony (MP) was used, with Candida as outgroup, to build phylogenetic tree (Numbers on the branch were obtained by 1000 Bootstrap Replications; CI = 0.757475 RI = 0.886115). As shown in figure 5, [Q.sub.10]-[M.sub.62] and Cyberlindnera jadinii were clustered into a group. Combined with characteristics of colony and thallus, [Q.sub.10]-[M.sub.62] was identified as Cyberlindnera jadinii, which was different from geotrichum candidum in white wine by (Zhou, Wang, Li, Hu, Hu & Wang, 2013).
The main aroma-producing yeast strain in BGR wine in Guizhou was identified as Cyberlindnera jadinii (previously Pichia jadinii). However, due to the limitation of time and other conditions, there are still many aspects that deserve our further study. For instance, the dynamic changes of microorganisms and substances content. How is the safety of pure fermentation? How to apply pure fermentation technology to the traditional process improvement of BGR wine made in Guizhou? Do functional ingredients such as pigment, polysaccharide and so on have influences on fermentation? All the problems urgently need to be studied.
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Wei Su [1,2,3,4],Zexiu Li , Chunzhi Xie , Bengang Xu , Yingchun Mu [1,3] and Shuyi Qiu 
 College of Liquor and Food Engineering, Guizhou University, Guiyang 550025, China.
 Southern Analysis and Test Center of Guizhou University, Guiyang 550025, China.
 Key Laboratory of Agricultural and Animal Products Store & Processing of Guizhou Province, Guiyang 550025, China.
 Qiannan Soil and Fertilizer Station, Duyun 558000, China.
(Received: 08 February 2016; accepted: 19 April 2016)
* To whom all correspondence should be addressed. E-mail: firstname.lastname@example.org
Caption: Fig. 1. Saccharification transparent circle of [Q.sub.10]-[M.sub.62]
Caption: Fig. 2. PCR electrophoretogram of [Q.sub.10]-[M.sub.62] (A: Maker; J:ITS sequences of aroma-producing [Q.sub.10]-[M.sub.62])
Caption: Fig. 3. The colony photo of [Q.sub.10]-[M.sub.62]
Caption: Fig. 4. The thallus photo of [Q.sub.10]-[M.sub.62]
Caption: Fig. 5. Phylogenetic tree of [Q.sub.10]-[M.sub.62]
Table 1. Saccharificating properties of 12 strains selected by starch transparent circle method Number Strains Diameter of number saccharification transparent circles d (cm) 1 [Q.sub.8]-[M.sub.27] 1.70 [+ or -] 0.14 2 [Q.sub.9]-[M.sub.45] 1.60 [+ or -] 0.07 3 [Q.sub.6]-[M.sub.14] 1.48 [+ or -] 0.04 4 [Q.sub.9]-[M.sub.43] 1.80 [+ or -] 0.07 5 [Q.sub.3]-[M.sub.24] 1.72 [+ or -] 0.08 6 [Q.sub.6]-[M.sub.5] 1.62 [+ or -] 0.08 7 [Q.sub.10]-[M.sub.53] 1.66 [+ or -] 0.09 8 [Q.sub.9]-[M.sub.27] 1.68 [+ or -] 0.00 9 [Q.sub.10]-[M.sub.62] 1.86 [+ or -] 0.05 10 [Q.sub.10]-[M.sub.45] 1.72 [+ or -] 0.08 11 [Q.sub.10]-[M.sub.50] 1.94 [+ or -] 0.05 12 [Q.sub.10]-[M.sub.25] 1.88 [+ or -] 0.08 Number Diameter of d/D colonies D (cm) 1 0.50 [+ or -] 0.00 3.40 [+ or -] 0.28 2 0.44 [+ or -] 0.05 3.68 [+ or -] 0.47 3 0.40 [+ or -] 0.00 3.70 [+ or -] 0.11 4 0.52 [+ or -] 0.04 3.47 [+ or -] 0.19 5 0.50 [+ or -] 0.00 3.44 [+ or -] 0.17 6 0.46 [+ or -] 0.05 3.55 [+ or -] 0.32 7 0.50 [+ or -] 0.00 3.32 [+ or -] 0.18 8 0.50 [+ or -] 0.00 3.36 [+ or -] 0.09 9 0.56 [+ or -] 0.05 3.34 [+ or -] 0.24 10 0.54 [+ or -] 0.05 3.20 [+ or -] 0.20 11 0.56 [+ or -] 0.05 3.49 [+ or -] 0.29 12 0.52 [+ or -] 0.04 3.72 [+ or -] 0.11 Table 2. Saccharificating property comparison of different strains No. Strains Liquifying enzyme activity (U/g) 1 [Q.sub.8]-[M.sub.27] 68.58 [+ or -] 0.66 2 [Q.sub.9]-[M.sub.45] 69.40 [+ or -] 0.71 3 [Q.sub.6]-[M.sub.14] 68.51 [+ or -] 0.55 4 [Q.sub.9]-[M.sub.43] 68.57 [+ or -] 0.58 5 [Q.sub.3]-[M.sub.24] 69.51 [+ or -] 0.53 6 [Q.sub.6]-[M.sub.5] 69.45 [+ or -] 0.59 7 [Q.sub.10]-[M.sub.53] 68.80 [+ or -] 0.20 8 [Q.sub.9]-[M.sub.27] 69.43 [+ or -] 0.50 9 [Q.sub.10]-[M.sub.62] 83.77 [+ or -] 0.38 10 [Q.sub.10]-[M.sub.45] 68.51 [+ or -] 0.55 11 [Q.sub.10]-[M.sub.50] 71.27 [+ or -] 0.61 12 [Q.sub.10]-[M.sub.25] 70.16 [+ or -] 0.18 No. Saccharifying enzyme activity (U/g) Reducing sugar (g/100g) 1 454.88 [+ or -] 3.87 13.17 [+ or -] 0.31 2 419.61 [+ or -] 5.24 11.75 [+ or -] 0.21 3 291.44 [+ or -] 4.14 9.21 [+ or -] 0.19 4 407.66 [+ or -] 2.59 14.21 [+ or -] 0.20 5 237.47 [+ or -] 4.14 7.52 [+ or -] 0.10 6 288.30 [+ or -] 3.27 8.85 [+ or -] 0.08 7 336.83 [+ or -] 3.23 8.85 [+ or -] 0.12 8 430.75 [+ or -] 5.14 12.22 [+ or -] 0.20 9 497.63 [+ or -] 5.29 12.75 [+ or -] 0.12 10 264.30 [+ or -] 2.07 7.43 [+ or -] 0.38 11 444.72 [+ or -] 3.53 11.58 [+ or -] 0.05 12 345.84 [+ or -] 2.57 8.35 [+ or -] 0.09 No. Black rice test Total acid Aroma (g/100g) 1 8.76 [+ or -] 0.16 76 [+ or -] 1 2 8.01 [+ or -] 0.05 76 [+ or -] 2 3 9.15 [+ or -] 0.34 71 [+ or -] 1 4 9.56 [+ or -] 0.05 74 [+ or -] 2 5 19.14 [+ or -] 0.18 75 [+ or -] 3 6 6.30 [+ or -] 0.20 74 [+ or -] 2 7 7.12 [+ or -] 0.17 73 [+ or -] 1 8 9.75 [+ or -] 0.05 75 [+ or -] 3 9 9.30 [+ or -] 0.19 80 [+ or -] 2 10 9.11 [+ or -] 0.14 75 [+ or -] 2 11 10.20 [+ or -] 0.19 76 [+ or -] 1 12 8.90 [+ or -] 0.09 72 [+ or -] 1 Table 3. GC/MS analysis result of aroma in traditional and aroma-producing fermentation starter Aroma composition Aroma content Aroma content in traditional in [Q.sub.10]- fermentation [M.sub.62] broth (%) fermentation broth (%) isopentyl alcohol * 10.679 3.551 acetylmethylcarbinol * 1.861 1.355 hydroxyacetone * 0.101 0.338 acetic acid * 10.745 6.739 methanoic * 0.319 1.709 2, 3 - butanediol * 1.835 22.675 isobutyric acid * 0.395 1.488 1, 3 - butanediol * 0.826 11.748 furfuryl alcohol * 0.29 1.017 isovaleric acid * 0.138 3.456 2 - hydroxy - 2 - ring 0.157 0.375 pentene 1 - ketone * phenethyl alcohol * 3.976 12.826 maltol * 0.101 0.368 4, 5 - dimethyl - 2 - 0.488 0.449 formyl furan * 1, 3 - dihydroxyacetone * 0.626 5.882 2,4-di-tert-butylphenol* 0.801 1.646 n-propanol 7.177 -- isobutanol 7.875 -- n-butyl alcohol 0.14 -- ethyl lactate 0.758 -- diethyl succinate 0.573 -- azulene 0.575 -- trans - 1 - methoxy - 4 - 0.216 -- (1 - allyl) benzene guaiacol 0.58 -- 5-hydroxymethylfurfural 8.405 -- mevalonic acid 2.221 -- methyl linolelaidate 37.025 -- sec-butyl alcohol -- 2.446 biose -- 2.743 methyl acrylate -- 0.345 furfuraldehyde -- 0.941 3 - methyl - 2 - (5 h) - -- 0.149 furan ketone butyrolactone -- 0.134 butyric acid -- 0.23 4 - hydroxy ethyl butyrate -- 0.563 4 - hydroxy - 5 - oxidation -- 0.772 caproic acid lactone 3-Heptalactone -- 0.461 ethyl tetradecanoate -- 1.106 succinaldehyde -- 0.486 pyranone -- 1.727 glycerol -- 0.762 ethyl palmitate -- 8.683 diethyl phthalate -- 3.458 Note :"--" means no date; "*" means common composition
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|Author:||Su, Wei; Li, Zexiu; Xie, Chunzhi; Xu, Bengang; Mu, Yingchun; Qiu, Shuyi|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Jun 1, 2016|
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