ldentification and characterization of Pichia membranifaciens Hmp-1 isolated from spoilage blackberry wine

WANG Ying, ZHAO Yan-cun, FAN Lin-lin, XlA Xiu-dong, Ll Ya-hui, ZHOU Jian-zhong

1 Institute of Agro-product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China

2 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China

Abstract The pellicle-forming yeast could cause the quality deterioration of wine. In this study, a pellicle-forming strain Hmp-1 was isolated from the spoilage blackberry wine, and identified as Pichia membranifaciens based on the morphology and partial nucleotide sequence of 26S rDNA. The effects offermentation conditions (ethanol, sulfur dioxide, sugar, and temperature)on the growth of P. membranifaciens strain Hmp-1 and Saccharomyces cerevisiae strain FM-S-115 (a strain used for the blackberry wine fermentation) were investigated, respectively. The results indicated that Hmp-1 had lower resistance to these factors compared to FM-S-115, and the growth of Hmp-1 was completely inhibited by 10% (v/v) or 50 mg L-1 SO2 during the fermentation of blackberry wine. These results suggested that Hmp-1 could effectively be controlled by increasing ethanolor SO2 concentration during the fermentation and storage of blackberry wine. Furthermore, the analysis based on gas chromatography-mass spectrometry (GC-MS) showed that Hmp-1 remarkably decreased kinds of volatile compounds in blackberry wine, especially aldehydes and esters. In addition, some poisonous compounds were detected in the blackberry wine fermented by FM-S-115 and Hmp-1. These results suggested that Hmp-1 was a major cause leading to the quality deterioration of blackberry wine.

Keywords: Picihia membranifaciens Hmp-1, blackberry wine, pellicle-forming spoilage, population dynamics, volatile compounds

1. lntroduction

Microbiological spoilage is a serious problem for the food industry, which renders the product unacceptable for consumers because of some abject factors, such as toxin,irritating odor, pellicle formation and anomal viscosity,thereby leading to huge economic losses (Loureiro and Malfeito-Ferreira 2003; Oelofse et al. 2008; Sturm et al.2014). Various yeasts, lactic acid bacteria (LAB) and acetic acid bacteria are widespread in the environment of wine production, some of which are detrimental to the wine quality (Bartowsky and Henschke 2008; Petri et al. 2013;Tristezza et al. 2016).

At present, various yeasts, such as Hanseniaspora,Pichia, Saccharomycodes, Schizosaccharomyces, and Zygosaccharomyces, could cause the quality deterioration of wine (Loureiro and Malfeito-Ferreira 2003; Suárez et al.2007; Enrique et al. 2008; Saez et al. 2011; Sturm et al.2014). Many of those yeasts could form a pellicle on the surface of bulk wines during storage, which caused cloudiness, haziness, and sediments (Sponholz 1992;Nakagawa et al. 2011). Wines with pellicle would lose commercial value.

Pichia membranifaciens is a kind of pellicle-forming yeast, and could form ascospores, which might cause a serious problem when temperature treatment is carried out in the industrial production offruit juice and wine (Veiga and Madeira-Lopes 2000). In addition, P. membranifaciens could produce volatile phenols, such as 4-ethylphenoland 4-ethylguaiacol. When these compounds are above certain levels, wine would produce abject odours (Saez et al. 2011).To the best of our knowledge, though some researches had found that P. membranifaciens was a kind offrequent spoilage yeast in the wine industry (Loureiro and Malfeito-Ferreira 2003; Saez et al. 2011), the growth dynamics of P. membranifaciens during fermentation period and its control measures had not been elucidated, especially in the blackberry wine.

Blackberry (Rubus fruticosus sp.) is known to contain high levels of phenolic compounds including anthocyanins,flavonols, chlorogenic acid and procyanidins, which have strong antioxidant activity (Elisia et al. 2007; de Souza et al.2014). Blackberry wine was produced by blackberry juice and yeast. This popular fruit wine is of particular interest because of its high phenolic contents and antioxidant properties (Johnson and Gonzalez de Mejia 2012; Ortiz et al.2013). In recent years, we studied the brewing technique of blackberry wine, and obtained a Saccharomyces cerevisiae FM-S-115 strain (patent no. ZL 201310098108.4, China).The strain was isolated from the spontaneous fermentation of blackberry wine, and had been used as an alcohol fermentation strain in the small-scale commercial production of blackberry wine.

In the previous work of our laboratory, the pellicle-forming phenomenon was found on the surface of blackberry wine at the late stage offermentation and during storage. The pellicle formation led to quality deterioration of the wine.In order to control the pellicle-forming spoilage during fermentation and storage of wine, the objectives of this study were: (a) to identify the pellicle-forming yeasts in the blackberry wine; (b) to study the effects offermentation environments on their growth; and (c) to evaluate the negative effect of the spoilage yeast on the wine quality.

2. Materials and methods

2.1. lsolation of spoilage microbes in pellicles of blackberry wine

The blackberry wine was fermented by S. cerevisiae FM-S-115 strain, and stored in stainless steel vats in a traditional cellar (annualaverage temperature of 18.0°C) located in Jiangsu Academy of Agricultural Sciences, Nanjing, China.The pellicle samples were collected in sterile conditions, and transported to the laboratory at 4°C. The collected sample(5 g) was homogenized in a sterile porcelain mortar with 5 mlof sterile water. Diluted homogenates were plated on YPD agar plates (yeast extract 1% w/v, peptone 2% w/v,glucose 2% w/v, and agar 1.8% w/v; pH 5.8) and MRS agar plates (Oxoid Microbiology Co., Ltd., Beijing, China)plus 100 mg L-1natamycin, respectively. The number of colony forming units (CFU) on each plate was counted after incubating for 48-72 h at 28°C. Isolates with distinct colony morphology were recovered from YPD and MRS agar plates,respectively. The cellular morphology of isolates was viewed under the light microscope (YS100, Nikon Inc., Japan). In addition, the isolates were stored at -70°C in 20% sterile glycerol for further studies.

2.2. Confirmation of isolates associated with pellicle formation

This experiment was carried out to investigate the pellicleforming ability of the isolates. The blackberry wine was prepared (pH 3.35, 8.05% v/v alcohol, 0.85 g L-1reducing sugar) and sequentiallyfiltered through 0.8-μm and 0.45-μm sterile membranes (Merck Millipore Ltd., Billerica, MA, USA).

Two isolates (Hmp-1 and Hmp-2) and S. cerevisiae strain FM-S-115 were transferred to 10 mL YPD broth (Hmp-1 and FM-S-115) or MRS broth (Hmp-2), respectively, and incubated for 12 h at 28°C. Cells were harvested by centrifugation at 3 000×g for 10 min. The cells were inoculated in 100 mL flasks containing 50 mlof blackberry wine, and the cell concentration of each strain was adjusted to 106CFU mL-1.Then the flasks were incubated for 15 d at 28°C without shaking. All experiments were carried out in triplicate.

2.3. Molecular identification of the pellicle-formation isolate Hmp-1

Genomic DNA of the pellicle-forming yeast Hmp-1 was extracted with a DNA extraction Kit (Tiangen, China), and the D1/D2 domain of the large-subunit (26S) ribosomal DNA(rDNA) was amplified by polymerase chain reaction (PCR)as previously described (Van der Aa Kühle and Jespersen 2003). The following primers were used: NL-1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and NL-4 (5′-GGT CCG TGT TTC AAG ACG G-3′). Then the PCR products were directly sequenced (Invitrogen Inc., Shanghai, China).

Multiple sequence alignment was performed based on the partial nucleotide sequence of 26S rDNA D1/D2 domain using ClustalX 1.83 software (Thompson et al.1997). A phylogenetic tree was constructed using the neighbor-joining method (MEGA 5.05 software) (Tamura et al. 2011). Kluyveromyces lactis (HE660074.1) was used as an outgroup.

2.4. Population dynamics offM-S-115 and Hmp-1 during blackberry juice fermentation

S. cerevisiae strain FM-S-115 and the isolate Hmp-1 were cultured in YPD broth for 12 h at 28°C for the following experiment. Blackberry juice (pH 3.25, 8.51°Brix) was prepared from fresh blackberry, 10.01% (w/v) sugar and 75 mg L-1sulfur dioxide (SO2) were added, and sequentiallyfiltered through 0.8- and 0.45-μm sterile membranes (Merck Millipore Ltd., Billerica, MA, USA). The population dynamics offM-S-115 and Hmp-1 during blackberry wine fermentation was assessed using the dilution plate method as follows:100 mL flasks containing 50 mlof blackberry juice was inoculated with FM-S-115 (thefinal concentration(2.01±0.25)×106CFU mL-1), Hmp-1 ((2.01±0.31)×106CFU mL-1), or both FM-S-115 and Hmp-1 ((1.02±0.25)×106and(1.02±0.17)×106CFU mL-1), respectively, and the flasks were incubated under static conditions at 28°C. The population dynamics offM-S-115 and Hmp-1 were investigated after 0,2, 4, 6, 8, 10, 12, and 14 d, respectively. Because the colony morphologies offM-S-115 and Hmp-1 were remarkably different, two kinds of yeasts on each YPD agar plate could be counted according to their colony morphologies, respectively.All experiments were carried out in triplicate.

2.5. Effects offermentation conditions on growth dynamics of Hmp-1 and FM-S-115

In order to obtain a better adaptation to the test conditions,the Hmp-1 and FM-S-115 were cultured in 20 mlof YPD broth medium adjusted to pH 4.0 with citric acid (1 mol L-1) and supplemented with 50% of blackberry juice (pH 3.25, 8.51°Brix, sequentiallyfiltered through 0.8- and 0.45-μm sterile membranes). This medium was incubated for 3 d at 28°C without shaking. Cells were harvested by centrifugation at 3 000×g for 10 min and then inoculated in 100-mL flasks containing 50 mlof blackberry juice supplemented with 25 mlof sterile YPD broth. The cell concentration of each strain was adjusted to 106CFU mL-1.All flasks were incubated at 28°C without shaking. Then the fermentation conditions were modified with the variables described below.

To investigate the impact of ethanolon growth dynamics of Hmp-1 and FM-S-115, varying amounts of ethanol were added to flasks containing FM-S-115 or Hmp-1 culture to make up respectivefinal concentrations of 8, 10, 12, and 14% (v/v).

To investigate the impact of SO2on growth dynamics of Hmp-1 and FM-S-115, varying amounts of potassium metabisulphite were added to flasks containing FM-S-115 or Hmp-1 culture to make up respectivefinal concentrations of 25, 50, 75, 100 and 125 mg L-1SO2. The concentration of SO2was calculated based on the technical specifications of potassium metabisulphite.

To investigate the impact of sugar on growth dynamics of Hmp-1 and FM-S-115, varying amounts of sugar were added to flasks containing FM-S-115 or Hmp-1 culture to make up respectivefinal concentrations of 10, 15, 20, 25%and 30% (w/v).

To investigate the impact of temperature on growth dynamics of Hmp-1 and FM-S-115, the flasks containing FM-S-115 or Hmp-1 culture were respectively cultured at 15, 20, 25, 30 or 35°C.

The population densities offM-S-115 and Hmp-1 were respectively evaluated using the dilution plate method after 1, 5 and 10 d of incubation. All experiments were carried out in triplicate.

2.6. GC-MS analysis

This experiment included two treatments: (1) blackberry wine fermented by single S. cerevisiae FM-S-115; (2)blackberry wine fermented by both FM-S-115 and the isolate Hmp-1. Flavor compounds of each kind of blackberry wine were respectively extracted by headspace solid-phase microextraction (HS-SPME) technique (Jiang et al. 2013).In brief, 5 mlof wine sample was placed in a 15-mL SPME glass vial to gether with 3 g of NaCI. The vial was tightly capped, and incubated for 15 min at 50°C to equilibrate.Then the sample was extracted for 40 min at 45°C.

The gas chromatographic-mass spectrometry (GC-MS)analysis of the sample was carried out by using an Agilent 6890 GC equipped with an Agilent 5975 mass selective detector (MSD). Separations were performed using a DB-Wax column (30 m length, 0.25 mm i.d., 0.25 μmfilm thickness) (J&W Scientific, Folsom, CA, USA). Spectra were obtained on electron impact at 70 eV, scanning from 15 to 250 m/z at 2 scans s-1. The temperature program ranged from 40 to 250°C as following: 40°C for 10 min, from 40 to 200°C at 5°C min-1, 200°C for 1 min, from 200 to 250°C at 5°C min-1, andfinally at 250°C for 10 min. The carrier gas was helium at a flow rate of 1 mL min-1. A totalof 1 μlof each extract from wine was injected into splitless mode. The volatile compounds were identified using the software library of mass spectra database Willey 6.1 (NY, USA).

2.7. Statisticalanalysis

Results were means of three independent experiments±standard deviation (SD). Statisticalanalysis of data was performed by using SPSS 13.0 software (SPP Inc., Chicago,USA).

3. Results

3.1. lsolation of pellicle-forming microbes in blackberry wine

On YPD agar plates, one kind of colonial morphology was found and named as Hmp-1. Its colonialand cellular morphology were shown in Fig. 1-A and A1. The colonialand cellular morphology of Hmp-1 were significantly different from those of S. cerevisiae FM-S-115 strain(Fig. 1-B and B1). On MRS agar plates, one kind of bacteria was found and named as Hmp-2. The colonialand cellular morphology of Hmp-2 were shown in Fig. 1-C and C1.Hmp-1 predominated in the pellicle formation of blackberry wine, and its population density was (8.21±0.23)×109CFU g-1. The population density of Hmp-2 was only(5.21±0.49)×104CFU g-1.

3.2. Confirmation of the isolate Hmp-1 associated with pellicle formation

The pellicle-forming ability of two isolates (Hmp-1 and Hmp-2)and S. cerevisiae strain FM-S-115 in blackberry wine was examined, respectively. After 15 d of incubation, Hmp-1 could form pellicle on the surface of blackberry wine (Fig. 2),whereas Hmp-2 and FM-S-115 could not form pellicle on the surface of blackberry wine.

3.3. Molecular identification of Hmp-1

The expected sequence of 26S rDNA D1/D2 domain (576 bp) was successfully amplified from Hmp-1. The sequence was deposited at GenBank database under an accession number KJ914549. Based on this sequence, a phylogenetic tree was constructed using neighbor-joining method, in which Hmp-1 was clustered with P. membranifaciens(Fig. 3). BLASTn analysis showed that the D1/D2 domain sequence of Hmp-1 shared 99% identity with those of P. membranifaciens (JX188208.1, DQ198957.1, EF564396.1 and DQ104727.1). Based on this result, Hmp-1 was classified as a member of P. membranifaciens.

Fig. 1 The colonial morphology and cellular morphology of the isolate Pichia membranifaciens Hmp-1 (A, A1), Saccharomyces cerevisiae FM-S-115 strain (B, B1), and the isolate Hmp-2 (C, C1).

Fig. 2 The pellicle-forming ability of Pichia membranifaciens Hmp-1 (A), Saccharomyces cerevisiae FM-S-115 (B), and Hmp-2(C) on the surface of blackberry wine. Hmp-1, S. cerevisiae FM-S-115, and Hmp-2 were cultivated for 15 d in 50 mlof blackberry wine at 28°C without shaking.

Fig. 3 The phylogenetic tree of Pichia membranifaciens Hmp-1 was constructed using neighbor-joining method based on the partial nucleotide sequence of 26S rDNA D1/D2 domain from yeasts related to Pichia species. Numbers at the branches denote the bootstrap percentages for 1 000 replicates. KJ914549 in parentheses indicates the accession numbers deposited in the GenBank database in this study, and the accession numbers for reference sequences are shown in parenthesis. The sequence of 26S rDNA D1/D2 domain from Kluyveromyces lactis (HE660074.1) was used as an outgroup. The scale indicates the evolutionary distance of the nucleotide substitutions per site.

3.4. Population dynamics offM-S-115 and Hmp-1 during blackberry juice fermentation

To analyze the growth dynamics and competitive position offM-S-115 and Hmp-1, their population densities were investigated when they were inoculated solely or jointly into blackberry juice (Fig. 4). The population density offM-S-115 increased about 100 times within 6 d after inoculation,and then decreased. The population density of Hmp-1 always decreased after alone inoculation, and then the living cells were not detected at 8-14 d. Compared to the growth dynamics of single inoculation, the growth dynamics offM-S-115 was unchanged at 1-6 d after inoculation in the joint inoculation blackberry juice, and then the cells density was lower than that of single inoculation blackberry juice.Moreover, we also found that the cells density of Hmp-1 decreased at 1-6 d after inoculation, and then sharply increased in joint inoculation blackberry juice, which was a very interesting phenomena.

Fig. 4 Population dynamics of Pichia membranifaciens Hmp-1 and Saccharomyces cerevisiae FM-S-115 strain in blackberry juice during fermentation. Blackberry juice was fermented at 28°C for 14 d., population dynamics of Hmp-1 in the blackberry juice inoculated solely; ■, population dynamics of Hmp-1 in the blackberry juice jointly inoculated with S. cerevisiae FM-S-115;, population dynamics of S. cerevisiae FM-S-115 in the blackberry juice jointly inoculated with Hmp-1;, population dynamics of S. cerevisiae FM-S-115 in the blackberry juice inoculated solely. Vertical bars represent the standard errors of the means.

3.5. Effects offermentation conditions on growth dynamics of Hmp-1 and FM-S-115

As shown in Table 1, the growth of Hmp-1 was seriously inhibited with the increase of ethanol concentrations.When the ethanol concentration was 10% (v/v), the growth of Hmp-1 was completely inhibited. By contrast,S. cerevisiae FM-S-115 was stillable to proliferate when the ethanol concentration was 14% (v/v). Those results could suggest that Hmp-1 was more sensitive to high concentration ethanol, and its growth would be controlled through increasing ethanol concentration during wine storage.

The effects of SO2in different concentrations on the growth dynamics of Hmp-1 and FM-S-115 were presented in Table 2. We found that Hmp-1 was more sensitive to SO2than FM-S-115 based on their growth. When SO2concentration was 50 mg L-1, the growth of Hmp-1 was completely inhibited. By contrast, the counts offMS-115 were not obviously affected under different SO2concentration conditions (25-125 mg L-1) at the same fermentation time at P＞0.05.

The effects of sugar in different concentrations on growth dynamics of Hmp-1 and FM-S-115 were presented in Table 3. We observed that the growth offM-S-115 were not significantly affected under different sugar concentration conditions (10-25%, w/v) at the same fermentation time at P＞0.05. However, the growth of Hmp-1 was obviously inhibited when the sugar concentration was 25% (w/v). The results showed that Hmp-1 had lower resistance to high concentration of sugar compared to FM-S-115.

As shown in Table 4, the growth of Hmp-1 and FM-S-115 was all very slowly at 15°C. The viable count of Hmp-1 was 6.55 (Log10CFU mL-1), and that offM-S-115 reached to 6.79 (Log10CFU mL-1) on the 10th d, respectively. But in general, the cell densities of Hmp-1 and FM-S-115 were increased when the fermentation temperature increased from 15 to 30°C. The growth of Hmp-1 was obviously inhibited when the fermentation temperature was 35°C (P＜0.05).

3.6. Effect of Hmp-1 on volatile compounds of blackberry wine

The volatile compounds are important components of the flavor substances in blackberry wine. In this study,34 kinds of volatile compounds had been detected in the blackberry wine fermented by FM-S-115, and 28 kinds of volatile compounds had been detected in theblackberry wine fermented by both FM-S-115 and Hmp-1(Table 5). But only 16 kinds of volatile compounds had been simultaneously found in the two samples (Table 5).Compared to the blackberry wine fermented by FM-S-115,the kinds and contents of aldehydes and esters decreased in the blackberry wine fermented by both FM-S-115 and Hmp-1, but those of alcohols increased. In addition, 18 kinds of volatile compounds disappeared in this sample,such as hexanal, 2-furancarb oxaldehyde, hexanoic acid ethyl ester, octanal, 2-octenal, 2-decenaland 2-undecenal,and 14 kinds of peculiar volatile compounds were detected,such as benzothiazole, 2-heptanol, N-acetyl-cytidine,cyclotetrasiloxane octamethyl, and so on.

Table 1 Effects of ethanol in different concentrations on the growth dynamics of Pichia membranifaciens Hmp-1 and Saccharomyces cerevisiae FM-S-115 (Log10CFU mL-1) during fermentation

Table 2 Effects of SO2 in different concentrations on the growth dynamics of Pichia membranifaciens Hmp-1 and Saccharomyces cerevisiae FM-S-115 (Log10CFU mL-1) during fermentation

Table 3 Effects of sugar in different concentrations on the growth dynamics of Pichia membranifaciens Hmp-1 and Saccharomyces cerevisiae FM-S-115 (Log10CFU mL-1) during fermentation

Table 4 Effects of temperature on the growth dynamics of Pichia membranifaciens Hmp-1 and Saccharomyces cerevisiae FM-S-115 (Log10CFU mL-1) during fermentation

4. Discussion

Microbiological spoilage is a serious problem for the food industry, which degrades food products by producing toxins,irritating odor, and forming pellicle (Farkas et al. 2013;Parlapani et al. 2014, 2015). P. membranifaciens is a kind of pellicle-forming yeast, which can produce ascospores and might cause a serious problem when temperature treatment is carried out in the industrial production offruit juice and wine (Veiga and Madeira-Lopes 2000).

In the present study, P. membranifaciens strain Hmp-1 wasfirst ever isolated from the pellicle of spoilage blackberry wine. The result was similar to that of Díaz et al. (2013) who found that P. membranifaciens remained active during the complete wine fermentation. However, a previous study had indicated that P. membranifaciens was not present during wine fermentation, and it was only found at the early stage of alcoholic fermentation (Combina et al. 2005). These contrary results reported in the literatures might be related to the differences in raw material, brewing environments,and the difference of P. membranifaciens strains.

The cell population of Hmp-1 was decreased throughout the fermentation period when Hmp-1 was alone inoculated in blackberry juice. However, when Hmp-1 was jointly inoculated with S. cerevisiae FM-S-115, the viable count of Hmp-1 was decreased within 6 d of incubation, and then a sharp increase was noted from 6th d of incubation. The possible reason for the difference of Hmp-1 growth dynamics was the decline of SO2concentration in blackberry juice,but the accurate reasons remained to be found in further research. The SO2concentration in wine is depended on carbonyland keto compounds, which are known to form covalent adducts (sulfonates) with SO2. There are many carbonyland keto compounds (acetaldehyde, pyruvic,α-ketoglutaric, galacturonic acids, and acetoin) in wine which were produced during the brewing offruit wine. Among them, acetaldehyde was identified as the most important SO2binder (Jackowetz and Mira de Ordu?a 2013). Bound forms of SO2are thought to have decreased preservative activity compared to free SO2(Rankine 1968).

Table 5 Chemical composition of volatile compounds from two kinds of blackberry wines fermented respectively with alone Saccharomyces cerevisiae FM-S-115 strain and both FM-S-115 and the isolate Hmp-1

In the present study, S. cerevisiae FM-S-115 showed strong adaptability and resistance to SO2compared to the spoilage yeast P. membranifaciens Hmp-1. The result was further confirmed that S. cerevisiae had strong competitive position compared to other microorganisms during spontaneous fermentation of wine (Maro et al. 2007;Wang and Liu 2013). Ethanol, a main product of wine fermentation, is well known as a kind of growth inhibitor of microorganisms. It had been reported that ethanol could damage mitochondrial DNA of yeast cells and lead to inactivation of some enzymes, such as hexokinase and dehydrogenase (Ibeas and Jimenez 1997). A previous study found that P. membranifaciens could not grow at a concentration of 6% (v/v) ethanol (Miguel et al. 2013). In this study, P. membranifaciens strain Hmp-1 could still grow and form pellicle in the blackberry wine containing 8.05%(v/v) ethanol. However, its growth could be completely inhibited when the concentration of ethanol was 10% (v/v)or more. Compared to Hmp-1, S. cerevisiae FM-S-115 showed stronger resistance to ethanol, and could still grow at a concentration of 14% (v/v) ethanol. This result was in agreement with previous reports (Fugelsang and Edwards 2007; Miguel et al. 2013; Contreras et al. 2014).

Flavor is one of the major factors which determine the nature and quality of wine (Selli et al. 2004). Some of volatile compounds come directly from fruits, others are produced by yeasts as a result of their metabolism during wine fermentation and aging (Fleet 2003). In this study, since the management practices and fermentation conditions were the same for all treatments, the difference of volatile compounds should be ascribed to the spoilage yeast Hmp-1. Some volatile compounds were only found in the blackberry wine fermented by both Hmp-1 and FM-S-115(Table 5), some of which might be harmful for consumers,such as benzothiazole. Benzothiazole and benthiazole derivatives were a group of xenobiotic heterocyclic chemicals, which had potential toxicity and mutagenic effects toward microorganisms and humans (Janna et al. 2011;Asimakopoulos et al. 2013; Pal et al. 2014). Aldehydes and esters are important flavor compounds, and their formation depends more on the yeast selected as starter rather than the raw materials used in fermentation (álvarez-Pérez et al. 2012; Cordente et al. 2012). In this study, 18 kinds of aldehydes and esters were detected in the blackberry wine fermented by alone FM-S-115, and their proportion was 53.35% in the total volatile compounds (Table 5).However, in the blackberry wine fermented by FM-S-115 with the spoilage yeast Hmp-1, only 9 kinds of aldehydes and esters were detected, and their proportion decreased to 34.06% (Table 5).

5. Conclusion

The present study demonstrated that P. membranifaciens strain Hmp-1 could form pellicle on the surface of blackberry wine, and lead to quality deterioration of blackberry wine.Its growth could effectively be controlled by increasing the concentrations of ethanolor SO2during blackberry wine fermentation and storage.

Acknowledgements

This work was supported by the Natural Science Foundation of Jiangsu Province, China (BK20170603).