Characterization of Osmotolerant Yeasts and Yeast-Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential Huxuan Wang, Zhongqiu Hu, Fangyu Long, Chen Niu, Yahong Yuan, and Tianli Yue

Yeasts and yeast-like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S-ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD-PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast-like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.

Abstract:

M: Food Microbiology & Safety

Keywords: apple orchards, apple juice processing plants, food spoilage, osmotolerant yeasts, yeast-like molds

The findings of this study will supplement existing literature and increase our understanding of the osmotolerant yeasts present in apple-related environments and their tolerance to high glucose concentrations.

Practical Application:

Introduction

Osmotolerant molds are generally capable of surviving in fruit juice concentrates. However, fruit juice concentrates that are stored in sealed plastic bags with minimal air pockets are less susceptible to spoilage by osmotolerant molds as these organisms are obligate aerobes (Combina and others 2008). Therefore, osmotolerant yeasts are primarily responsible for spoilage of fruit juice concentrates (Sancho and others 2000; Combina and others 2008). Osmotolerant yeasts are a group of microorganisms that are capable of growing in glucose concentrations of 50% (w/v) or higher (Tokuoka 1993). This group also includes a subset of yeasts termed as osmophilic yeasts to differentiate the 2 categories. Compared to osmotolerant yeasts, osmophilic yeasts can resist higher osmotic pressure and grow faster in high glucose (20% to 40%, w/v) than in basal glucose (2%, w/v; Tokuoka 1993). The most common osmotolerant yeasts found in fruit juice and concentrates are Candida tropicalis, C. stellata, C. glucosophila, Debaryomyces hansenii, Lodderomyces elongisporus Meyerozyma guilliermondii, Saccharomyces cerevisiae, S. pastorianus, Wickerhamomyces anomalus, Torulaspora delbrueckii, Zygosaccharomyces rouxii, Z. bailii, and Z. bisporus. (Deak and Beuchat 1993; Sancho and others 2000; Senses-Ergul and Ozbas 2006; MS 20150424 Submitted 3/12/2015, Accepted 5/26/2015. Authors Wang, Hu, Combina and others 2008; Rojo and others 2013). Recently, Long, Niu, Yuan, and Yue are with College of Food Science and Engineering, a batch of apple juice concentrate made by a plant in Shaanxi Northwest A&F Univ., Yangling, Shaanxi province, 712100, China. Direct inquiries exhibited signs of spoilage after transportation to the United to author Yue (Email: [email protected]). States. It is likely that the growth of spoilage microorganisms was

Apple juice concentrate is an important ingredient in consumer juice products and other beverages in the United States and other countries and therefore contributes substantially to the revenue of the food industry. China is by far the largest apple juice concentrate producer and supplier in the world, accounting for 60% of the global trade volume (Guo and others 2013). Shaanxi is the most important apple juice concentrate-producing province in China, responsible for more than 60% of the country’s production (Guo and others 2013). In addition to low pH (3.0 to 3.5), fruit juice concentrates also have low water activity (aw , 0.70 to 0.85) due to the high concentration of sugars. Consequently, they are rarely contaminated by the microorganisms that typically grow at aw values of more than 0.89 (Combina and others 2008). However, microorganisms such as osmotolerant yeasts and osmotolerant molds, which can grow at aw values of less than 0.80, might pose a threat to the stability of fruit juice concentrates (Combina and others 2008; Akdeniz and others 2013; Guo and others 2013; Rojo and others 2013).

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R  C 2015 Institute of Food Technologists

doi: 10.1111/1750-3841.12946 Further reproduction without permission is prohibited

promoted during transportation due to warm conditions in the shipping container. The contaminants were identified as strains of W. anomalus by our laboratory (unpublished work). The rapid identification of spoilage yeasts is of great importance to the food industry. Molecular-based techniques, faster, and more reliable than traditional tests, have been proposed to identify yeasts. Sequence analysis of the ITS-5.8S region or the D1/D2 domains in the large 26S subunit of rDNA has proven to be a suitable methodology for rapid and accurate yeast identification (EsteveZarzoso and others 1999; Couto and others 2005). Differentiation of yeasts at the subspecies level is also an important requirement for the food industry. A reliable polymorphism identification can be achieved by the classical taxonomy tests, which includes the fermentation tests, assimilation tests, and some other tests (Barnett and others 1983). On the other hand, RAPD-PCR technique has also been used for this purpose (Tofalo and others 2009). Both methods are useful for quality assurance typing of spoilage microorganisms and revealing the ecological complexity of the yeast flora associated with food and beverages (Fleet 2007). Stress factors such as low pH and high sugar content are effective in largely suppressing microorganisms. Yeasts in contrast, show a remarkable tolerance to low pH, and thus, are particularly associated to spoilage of fruit juice products (Martorell and others 2007). Therefore, sugar content of foods is a very important factor affecting yeast growth and it is essential to evaluate yeast spoilage potential by investigating their tolerance to high sugar concentrations. Another feature that may contribute to the spoilage capacity of yeasts is their ability to vigorously ferment hexose sugars, such as glucose and fructose (Martorell and others 2007). High fermentation can contribute to spoilage by causing swelling or explosions in packaging. In particular, juice products packaged in glass bottles are a public safety issue due to high fermentation of yeasts. Considerable effort has been put into the isolation and identification of osmotolerant yeasts from various high-sugar products (Ok and Hashinaga 1997; Senses-Ergul and Ozbas 2006; Combina and others 2008; Marvig and others 2014). Although identifying sources of osmotolerant yeast contaminants can help in the development of effective strategies to prevent spoilage, it has not been the focus of previous studies. In order to provide more information about the sources of osmotolerant yeast contaminants in apple juice concentrate, it is essential to understand the distribution of osmotolerant yeasts in apple-related environments (apple orchards and apple juice processing plants) and how they react towards the stress factor present in apple juice products such as high sugar concentrations. The main objectives of this study were to obtain a deep understanding of which osmotolerant yeasts are present in apple orchards and apple juice processing plants located in the Shaanxi province of China and to evaluate their spoilage potential by investigating their tolerance to high glucose concentrations (50% to 70%, w/v), similar to sugar concentrations found in apple juice products. Gas generation test in 50% (w/v) glucose was performed to estimate their capability of causing swelling or explosions in packaging. In addition, in order to obtain the information about polymorphism of yeast flora, the isolates belonging to the same species were typified in terms of their physiological and genomic properties.

Samples A total of 120 samples including 30 air samples, 30 orchard soil samples, 30 apple samples, and 30 apple leaf samples were randomly collected from 10 apple orchards located in the Luochuan (LC) and Baishui (BS) counties of Shaanxi province. Air, orchard soil, apples, and apple leaves were collected in the front, middle and back of each orchard. Yeast Extract Peptone Dextrose (YPD) plates supplemented with 50 mg/L of chloramphenicol and 30 mg/L of streptomycin were exposed to air for 30 min to collect air samples in triplicates. Two kilograms of soil were collected from under and around apple trees and stored in aseptic bags. Thirty apples and 200 g of apple leaf were collected from different parts of apple trees and stored in aseptic bags. All samples were immediately transported to the laboratory. Samples were also collected from 2 apple juice-processing plants located in the LC and Qianxian (QX) counties of Shaanxi province. Both plants produce apple juice concentrate using apples from LC and BS counties. The flow chart of the manufacture of apple juice concentrate at both plants is shown in Figure 1. Aliquots of 2 L of juice were directly collected from the corresponding equipment at every stage, from juice extraction to aseptic packaging. Aliquots of 2 L of water were taken from the reservoir that stores the water used for washing apples and equipment or conveying apples. Two L of water were also collected from the evaporator inlet where condensate water is produced during juice concentration. Aliquots of 2 L of apple juice concentrate were sampled from the concentrate placed in sterile bag-in-boxes with a Vitop valve stored at 4 °C for several months. In addition, YPD plates supplemented with chloramphenicol (50 mg/L) and streptomycin (30 mg/L) were exposed to air for 30 min to collect air samples in the front, middle, and back of the production workshop in triplicates. All samples were immediately transported to the laboratory. Yeast isolation Approximately 10 g of apple leaf, apple skin, or soil were aseptically placed in a sterile conical flask containing 50 mL of sterile 0.85% (w/v) NaCl solution in duplicates. The conical flasks were placed on a shaking bed at 150 rpm for 15 min to wash the microorganisms from the samples into the NaCl solution. Then

Materials and Methods Reagents All reagents used were purchased from Beijing Solarbio Science & Technology Co., Ltd except where indicated in parentheses. Figure 1–Production procedure of apple juice concentrate in both plants. Vol. 80, Nr. 8, 2015 r Journal of Food Science M1851

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Osmotolerant yeasts and spoilage . . .

Osmotolerant yeasts and spoilage . . . aliquots of 5 mL of the NaCl solution were added to 95 mL of YPD broth containing 50 mg/L of chloramphenicol and 30 mg/L of streptomycin in triplicates. As for samples from the processing plants, 10 mL of water or apple juice was aseptically added to 40 mL of YPD broth with 50 mg/L of chloramphenicol and 30 mg/L of streptomycin in duplicates. Ten mL of on-line or stored apple juice concentrate was aseptically placed in 40 mL of YPD broth containing 30% (w/v) glucose in duplicates. We used 30% (w/v) glucose as it can prevent osmotic shock and allow sublethally injured cells to recover (Combina and others 2008; Rojo and others 2013). All YPD broths were statically incubated at 28 °C for 48 h as a yeast enrichment step. Then liquid cultures of all samples except the concentrate were serially decimal diluted using sterile 0.85% (w/v) NaCl solution whereas the liquid cultures of on-line or stored apple juice concentrate were serially decimal diluted using 30% (w/v) glucose solution (Combina and others 2008; Rojo and others 2013). Appropriate dilutions were spread onto YPD plates in triplicates and all plates were incubated at 28 °C for 3 d. Representative isolates of every colony type were purified by repetitive streak plating and maintained in YPD broth supplemented with glycerol (25% final concentration) at – 40 °C.

M: Food Microbiology & Safety

Selection of osmotolerant yeasts According to the definition of osmotolerant yeasts proposed by Tokuoka (1993), all isolates were inoculated on medium containing 50% (w/v) glucose to screen for osmotolerant yeasts. Malt Yeast Glucose (MYG50) agar reported by Beuchat (1993) was used as the selective medium after a modification that is, 50% (w/v) instead of 50% (w/w). All isolates were inoculated on MYG50 medium in triplicates and incubated at 28 °C for 28 d. The isolates capable of growth on MYG50 agar were termed as osmotolerant isolates. Molecular identification Osmotolerant isolates were grown aerobically in 5 mL of YPD broth at 28 °C for 48 h. The cultures were then transferred to an Eppendorf tube and centrifuged for 10 min at 22000 × g. The pellets of cells were collected and the supernatants were discarded. Genomic DNA extraction was carried out using E.Z.N.A.TM Yeast DNA Kit (Omega Bio-Tec, Norcross, Ga., U.S.A.) according to the manufacturer’s manual. The primers (Sunbiotech, Beijing, China) used for the amplification of the ITS1-5.8SITS2 regions were ITS1 (5´-TCCGTAGGTGAACCTGCGG3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´; EsteveZarzoso and others 1999). PCR reactions were performed in a total volume of 50 µL containing 1U Taq DNA polymerase (Takara, Dalian, Liaoning, China), 0.1 µmol of each primer, 0.1 mmol of each dNTP, 1× PCR reaction buffer, 2 mmol of MgCl2 , and 2 µL of extracted DNA. The amplification reaction was performed in a Peltier Thermal Cycler ALD1244 (Bio-Rad Laboratories, Hercules, Calif., U.S.A.) according to the method reported by Kurtzman (2006). Double distilled water was used instead of extracted DNA as negative control. All PCR products were sequenced in Beijing Sunbiotech Co., Ltd. Sequence similarity searches were performed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). When analyzing the 5.8S-ITS rDNA region, sequences with 99% nucleotide identity or higher were considered to be of the same species (Baffi and others 2011). Isolates whose sequences presented an identity score below 99% with the 5.8S-ITS sequences described in GenBank were additionally sequenced in the D1/D2 domains of 26S rDNA according to the protocol reported by

M1852 Journal of Food Science r Vol. 80, Nr. 8, 2015

Couto and others (2005). This was done using the primer pair NL-1 (5´-GCATATCAATAAGCGGAGGAAAAG-3´) and NL4 (5´-GGTCCGTGTTTCAAGACG G-3´; Sunbiotech, Beijing, China).

Polymorphism analysis Physiological tests were performed to reveal polymorphism of the isolates belonging to the same species (Barnett and others 1983). Yeast physiological characteristics were determined according to the ability to ferment certain sugars semianaerobically and to assimilate various carbon or nitrogen compounds as the major source of carbon or nitrogen under aerobic conditions. Additional physiological tests also determined properties such as growth at different temperatures, growth in different concentrations of cycloheximide and glucose, urease activity, acid production, starch formation, and diazonium blue B (DBB) staining. All tests were performed in triplicates. RAPD-PCR analysis was performed to confirm the polymorphism obtained by the physiological tests. The 10-mer primer P24 (5´-GCGTGACTTG-3´) used in this study was provided by Sangon Biotech Co., Ltd., Shanghai, China. Reactions were carried out in 25 µL of reaction mix containing 2.5 µL of 10× PCR buffer, 2 mm MgCl2 , 0.15 mm of each dNTP, 20 pmol of primer, 1U of Taq polymerase (Takara, Dalian, Liaoning, China), and 50 ng of extracted DNA. The thermal cycler was set to the following program: initial denaturation at 95 °C for 5 min; 40 cycles of denaturing at 95 °C for 1 min, annealing at 36 °C for 1 min, an extension at 72 °C for 2 min; and a final extension period of 10 min at 72 °C. The RAPD-PCR products were visualized on 1.5% agarose gel after staining with ethidium bromide. The size of PCR products was determined with a DNA size marker of 100 bp. Distinguishing osmophilic isolates from osmotolerant isolates This test was performed using Yeast Malt (YM) agar as the basal medium. Glucose at different concentrations, 0% (control), 2%, 20%, 40%, and 50% (w/v) was added to the basal medium according to Tokuoka (1993). Actively growing cultures in YPD broth were centrifuged (3500 × g) at 4 °C for 15 min and the cells were washed thrice using Phosphate Buffered Saline (PBS) solution at pH 7.5 (adjusted by 5 M hydrochloric acid). After resuspending the pellet in the same buffer by vortex, cell concentration was calculated using a hemocytometer and standardized to 107 CFU/mL. Before inoculation, 1 mL of 107 CFU/mL cells in PBS was transferred to an Eppendorf tube and centrifuged for 15 min at 3500 × g. The supernatants were discarded and replaced by YM broth with glucose concentrations corresponding to the glucose concentrations of the culture plates (Deschuyffeleer and others 2011). The procedure mentioned above was also used for preparing the inoculums for subsequent tests. Aliquots of 5 µL of 107 CFU/mL suspension were spot-inoculated on each plate (n = 3). All plates were incubated at 28 °C for 7 d and examined daily for growth (Ok and Hashinaga 1997). The colony growth rate (mm/d) was defined as the ratio of the final colony diameter and incubation time. Influence of high glucose concentrations on the growth of osmotolerant and osmophilic isolates The medium used for performing this test was YPD broth supplemented with 0% (control), 50%, 55%, 60%, 65%, and 70%

Osmotolerant yeasts and spoilage . . . Table 1–Distribution and blast results of osmotolerant isolates in apple orchards and apple juice processing plants. City

Blast result

Accession number

Similarity (%)

Reference strain

Total

LCa BSb LC LC LC BS LC BS LC LC LC BS BS

Rhodotorula mucilaginosa Aureobasidium pullulans Aureobasidium pullulans Meyerozyma guilliermondii Saccharomyces cerevisiae Candida tropicalis Aureobasidium pullulans Pichia kudriavzevii Meyerozyma guilliermondii Debaryomyces hansenii Wickerhamomyces anomalus Pichia kudriavzevii Torulaspora delbrueckii

KC544481.1 KC544506.1 KC544497.1 KC544479.1 KC544499.1 KC544510.1 KC544477.1 KC544508.1 KC544478.1 KC544492.1 KC544480.1 KC756946.1 KC544495.1

99.82 99.64 99.27 99.65 99.25 99.39 99.26 99.79 99.64 99.18 99.11 99.14 99.35

AF444541.1 AY139394.1 JX156360.1 AY939792.1 AM262830.1 AY939810.1 JX462673.1 AY939808.1 AY939792.1 GQ458041.1 DQ249196.1 EU409797.1 HE616749.1

2

QXc QX QX QX QX QX QX QX LC LC LC LC LC LC LC LC LC LC LC LC QX QX QX QX QX LC LC LC LC QX LC LC QX LC LC LC QX LC LC LC LC QX LC QX QX QX QX QX LC LC QX QX QX LC

Hanseniaspora uvarum Pichia aff. fermentans Pichia kluyveri Candida zemplinina Torulaspora delbrueckii Pichia kudriavzevii Hanseniaspora uvarum Candida orthopsilosis Hanseniaspora opuntiae Candida tropicalis Pichia kluyveri Kluyveromyces marxianus Meyerozyma caribbica Candida glabrata Aureobasidium pullulans Candida tropicalis Kluyveromyces marxianus Rhodosporidium fluviale Candida glabrata Kluyveromyces marxianus Yarrowia lipolytica Yarrowia lipolytica Pichia kudriavzevii Pichia fermentans Yarrowia lipolytica Hanseniaspora opuntiae Saccharomyces cerevisiae Kluyveromyces marxianus Candida glabrata -d Saccharomyces cerevisiae Kluyveromyces marxianus Kluyveromyces marxianus Candida glabrata Saccharomyces cerevisiae – Saccharomyces cerevisiae Kluyveromyces marxianus Hanseniaspora opuntiae Hanseniaspora uvarum – – Candida tropicalis Zygosaccharomyces rouxii Zygosaccharomyces rouxii Zygosaccharomyces rouxii Zygosaccharomyces rouxii Zygosaccharomyces rouxii Zygosaccharomyces rouxii Kluyveromyces marxianus Pichia occidentalis Candida tropicalis Hanseniaspora uvarum

KC544511.1 KC544467.1 KC544470.1 KC544509.1 KC544496.1 KC544503.1 KJ739839.1 KC544482.1 KC544474.1 KC544487.1 KJ739840.1 KC544512.1 KC544483.1 KC544504.1 KC544497.1 KC544498.1 KC544505.1 KC544507.1 KC544516.1 KC544466.1 KC544493.1 KC544494.1 KC544469.1 KC544500.1 KC544463.1 KC544485.1 KC544484.1 KC544514.1 KC544489.1 – KC544486.1 KC544513.1 – KC544515.1 KC544491.1 KC544490.1 – KC544501.1 KC544462.1 KC544476.1 KC544471.1 – – KC544465.1 KC544461.1 KJ739842.1 KJ739843.1 KJ739844.1 KC544459.1 KC544460.1 KC544464.1 KJ739841.1 KC544468.1 KC544472.1

99.16 99.63 99.47 99.50 99.60 99.56 99.47 99.16 99.82 99.39 99.47 99.85 99.31 99.38 99.10 99.18 99.55 99.83 99.64 99.62 99.92 99.15 99.92 99.50 99.80 99.58 99.49 100 99.52 – 99.37 99.85 – 99.54 99.53 99.24 – 99.36 99.25 99.82 99.47 – – 99.75 99.48 99.13 99.13 99.48 99.82 99.31 99.81 99.29 99.47 99.48

KC254055.1 FN428873.1 JQ771710.1 EU183506.1 HE616749.1 KC601852.1 JQ771737.1 EU564208.1 AJ512453.1 AY939810.1 JQ771714.1 GU256755.1 HQ909093.1 JQ070075.1 AY139394.1 HQ398237.1 DQ249191.1 NR077089.1 AY939793.1 AJ508567.1 EU252546.1 EU252546.1 KC601854.1 FN376418.1 AF335977.1 FM199954.1 JX094776.1 GU256755.1 AY939793.1 – JX094776.1 GU256755.1 – DQ249191.1 AY939793.1 JX094776.1 – JX094776.1 AJ508567.1 AJ512453.1 EU004081.1 – – EU589206.1 KC146373.1 KC146373.1 KC146373.1 KC146373.1 KC146373.1 KC146373.1 AJ508567.1 EF550236.1 EU589206.1 EU004081.1

4

2 5

20

M: Food Microbiology & Safety

Isolates Source Apple orchard B-WHX-12-01 Air B-WHX-12-02 Air B-WHX-12-03 Soil B-WHX-12-04 Soil B-WHX-12-05 Soil B-WHX-12-06 Soil B-WHX-12-07 Leaf B-WHX-12-08 Leaf B-WHX-12-09 Apple surface B-WHX-12-10 Apple surface B-WHX-12-11 Apple surface B-WHX-12-12 Apple surface B-WHX-12-13 Apple surface Apple juice processing plant B-WHX-12-14 Air B-WHX-12-15 Air B-WHX-12-16 Air B-WHX-12-17 Air B-WHX-12-18 Air B-WHX-12-19 Air B-WHX-12-20 Air B-WHX-12-21 Air B-WHX-12-22 Air B-WHX-12-23 Air B-WHX-12-24 Air B-WHX-12-25 Air B-WHX-12-26 Air B-WHX-12-27 Air B-WHX-12-28 Air B-WHX-12-29 Air B-WHX-12-30 Air B-WHX-12-31 Air B-WHX-12-32 Air B-WHX-12-33 Air B-WHX-12-34 Apple juice B-WHX-12-35 Apple juice B-WHX-12-36 Apple juice B-WHX-12-37 Apple juice B-WHX-12-38 Apple juice B-WHX-12-39 Apple juice B-WHX-12-40 Apple juice B-WHX-12-41 Apple juice B-WHX-12-42 Apple juice Noned Enzymatic hydrolysis B-WHX-12-43 Enzymatic hydrolysis B-WHX-12-44 Enzymatic hydrolysis None Ultrafiltration B-WHX-12-45 Ultrafiltration B-WHX-12-46 Ultrafiltration B-WHX-12-47 Ultrafiltration None Preconcentration B-WHX-12-48 Preconcentration B-WHX-12-49 Preconcentration B-WHX-12-50 Preconcentration B-WHX-12-51 Preconcentration None On-line concentrate None Online concentrate B-WHX-12-52 Stored concentrate B-WHX-12-53 Stored concentrate B-WHX-12-64 Stored concentrate B-WHX-12-65 Stored concentrate B-WHX-12-66 Stored concentrate B-WHX-12-54 Stored concentrate B-WHX-12-55 Stored concentrate B-WHX-12-56 Washing water B-WHX-12-57 Washing water B-WHX-12-58 Washing water B-WHX-12-63 Washing water

9

2 3

4

0 7

4

Continued

Vol. 80, Nr. 8, 2015 r Journal of Food Science M1853

Osmotolerant yeasts and spoilage . . . Table 1–Continued. Isolates

Source

Apple juice processing plant B-WHX-12-59 Condensate water B-WHX-12-60 Condensate water B-WHX-12-61 Condensate water B-WHX-12-62 Condensate water

City

Blast result

Accession number

Similarity (%)

Reference strain

Total

QX QX LC LC

Pichia kudriavzevii Hanseniaspora opuntiae Hanseniaspora uvarum Candida tropicalis

KC544502.1 KC544475.1 KC544473.1 KC544488.1

99.34 99.82 99.82 99.18

KC601852.1 AJ512453.1 AF257273.1 AY939810.1

4

a

Luochuan county of Shaanxi province in China. county of Shaanxi province in China. Qianxian county of Shaanxi province in China. d No osmotolerant isolates were obtained. b Baishui c

Statistical analysis In the polymorphism analysis, similarities among banding patterns were established with the NTsys program, using the unweighted pair-group method with arithmetic averages (UPGMA) clustering based on the Dice correlation coefficient. The other data were analyzed using analysis of variance (ANOVA) in the SPSS software package (version 18.0). Statistical significance was determined at P < 0.05. Average and standard errors were calculated and reported for each experiment. A comparison was Gas generation YPD broth supplemented with 50% (w/v) glucose was used for performed using Duncan’s test (P < 0.05). performing the gas generation test (Martorell and others 2007). Durham’s fermentation tube was used to monitor the amount of Results and Discussion gas generated (Barnett and others 1983). Exactly 4.9 mL of media Although a total of 127 isolates were recovered from the apwas inoculated with 100 µL of cell suspension and incubated ple orchard environment, only thirteen strains were identified as statically at 28 °C for 7 d. The amount of gas inside the Durham’s osmotolerant isolates after screening. A total of 75 strains were fermentation tube was recorded daily. obtained from the processing plant environment, of which 53 (w/v) glucose (Martorell and others 2007). A total 99 mL of media in a conical flask was inoculated with 1 mL of cell suspension and incubated statically at 28 °C for 240 h. The increase in cell number was determined by measuring the optical density of cell cultures at 600 nm using a UV-1700 PharmaSpec spectrophotometer (Shimadzu, Kyoto, Japan) every 48 h (Tofalo and others 2009).

M: Food Microbiology & Safety

Figure 2–Cluster analysis of the polymorphism of different isolates belonging to the same species based on the physiological characteristics using UPGMA.

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strains were determined to be osmotolerant isolates. The ratio of osmotolerant isolates reached up to 70.67% (53/75) for the processing plant environment, whereas a smaller value of 10.24% (13/127) was obtained for the orchard environment, indicating that the processing plant environment might be a more suitable habitat for osmotolerant isolates. This result might be explained by the fact that the orchard environment lack sources of concentrated sugars necessary to select osmotolerant organisms whereas apple juice splatter on equipment, floors, and walls makes the plant environment relatively abundant in sugars. Several previous studies have also reported that osmotolerant yeasts were mainly recovered from high-sugar environments (Schneider and others 2003; Senses-Ergul and Ozbas 2006; Combina and others 2008; Saksinchai and others 2012; Marvig and others 2014). As shown in Table 1, osmotolerant isolates were obtained from sources like apple surfaces, soil, apple leaves, and air in the orchard environment. In case of the processing plant environment, osmotolerant isolates were present in all samples except in the online apple juice concentrate. This result is consistent with previous reports suggesting that the distribution of yeasts in orchard and processing plant environments was widespread (Sabate and others 2002; Vadkertiov´a and others 2012; Salom˜ao and others 2014). The highest numbers of osmotolerant isolates (twenty isolates) were recovered from the air in both plants (Table 1). This may be due to the transfer of isolates present on processing materials, walls, floors, and clothing of workers to the air through airflow. Asefa and others (2009) reported that some yeasts recovered from the air samples and some yeasts obtained from the installations and production materials belonged to the same species during the

production processes of dry-cured meat products, suggesting yeast transfer through airflow. Five osmotolerant isolates were found in only the apple juice extraction stage at the QX plant whereas osmotolerant isolates were discovered in all stages except online concentrate at the LC plant (Table 1). A possible explanation for this result might be that osmotolerant isolates in the extracted apple juice were not totally inactivated by the first pasteurization (96 °C, 30 s) and these residual isolates survived in the production line until the second concentration stage (80 to 85 °C, 3 min) in the LC plant. Similar to our findings, Salom˜ao and others (2014) also reported that yeasts were recovered from the stages before concentration during apple juice processing. All online concentrate tested negative for osmotolerant isolates whereas the concentrate that had been stored for a longer time without clear indications of spoilage contained osmotolerant isolates (Table 1). The contamination of apple juice concentrate by osmotolerant isolates in air might have happened at the time of encapsulation. A comparable result was report by Combina and others (2008), who found that concentrated grape juice prior to container filling contained fewer yeast counts (0.12 log CFU g-1 ) than the concentrate (4.40 to 7.06 log CFU g-1 ) stored in containers for an extended period of time. Thus, it is necessary to sterilize the concentrate again after encapsulation to prevent yeast contamination. Osmotolerant isolates were present in water samples collected from both plants (Table 1). Similarly, microorganisms such as Alicyclobacillus spp. have been recovered from the wash water and condensate water in concentrated apple juice-processing environments in previous studies (Chen and others 2006; Groenewald and others 2009). These findings indicate that the water source might be a potential pathway by which

Figure 3–Cluster analysis of the polymorphism of different isolates belonging to the same species based on the RAPD-PCR fingerprints using UPGMA. Vol. 80, Nr. 8, 2015 r Journal of Food Science M1855

M: Food Microbiology & Safety

Osmotolerant yeasts and spoilage . . .

Osmotolerant yeasts and spoilage . . .

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osmotolerant isolates contaminate the apple juice production line. Percentage similarities of all osmotolerant isolates to the sequence of corresponding reference strains are listed in Table 1. These values range from 99% to 100%. Sixty-six osmotolerant isolates including Aureobasidium pullulans (a yeast-like mold species) were identified and represented 23 species in 14 genera (Table 1). Species found in the Chinese apple-related environments match well with species previously described in the apple-related environments of other countries. A. pullulans, D. hansenii, M. guilliermondii, Rhodotorula mucilaginosa, and W. anomalus were also recovered from apple orchards located in Northern Italy and southern Brazil (Camatti-Sartori and others 2005; Pelliccia and others 2011). Kluyveromyces marxianus, R. mucilaginosa, and Z. rouxii were also obtained from an apple juice processing plant located in Argentina (Brugnoni and others 2011). The species identified in this study that were not found in the apple-related environments of other countries might be part of an original microbiota because apple-related environments in different countries may have different microbiota (Baffi and others 2011). The most common osmotolerant isolates recovered were the species of K. marxianus, Hanseniaspora uvarum, S. cerevisiae, Z. rouxii, C. tropicalis, and Pichia kudriavzevii. The same species have been reported to be common spoilage microorganisms in fruit juice products (Tokuoka 1993; Sancho and others 2000; Combina and others 2008). Species such as C. glabrata, C. orthopsilosis, C. zemplinina, and H.opuntiae were recovered from the plant environment whereas they were not obtained from the orchard environment. This might be because these osmotolerant isolates originated from apple pickers, apple package cartons, and processing plant employees. In addition, strains of Z. rouxii were frequently recovered from stored apple juice concentrate (Table 1), posing a threat to the quality of this product. This phenomenon is in line with many previous studies, which reported that Z. rouxii was usually associated with the spoilage of high sugar products such as honey, syrup, and concentrated fruit juices (Combina and others 2008; Marvig and others 2014). It has been regarded as the most important spoilage organism due to its extreme osmotolerance and ability to adapt to high glucose concentrations (Vermeulen and others 2014). It is evident from Table 1 that more than one isolate was recovered from each of the 13 species including A. pullulans, C. glabrata, C. tropicalis, H. opuntiae, H. uvarum, K. marxianus, M. guilliermondii, P. kluyveri, P. kudriavzevii, S. cerevisiae, T. delbrueckii, Yarrowia lipolytica, and Z. rouxii. In order to reveal the polymorphism of osmotolerant isolates belonging to the same species, physiological tests and RAPD-PCR analysis were performed. As shown in Table 2, the fermentation or assimilation tests gave variable results for each of the 5 species (C. tropicalis, K. marxianus, P. kudriavzevii, S. cerevisiae, and Z. rouxii). Moreover, different patterns were observed for each of the 5 species in the dendrogram based on physiological characteristics or the generated digitized fingerprinters (Figure 2 and 3). Overall, limited polymorphism (1 to 3 biotypes) and close relatedness (greater than 95% in similarity) were revealed for the isolates belonging to each of the 13 species. This was possibly because apples from the same production area were used for producing the concentrate each year and selective pressures such as high sugar concentration and low pH were exerted by the plant environment. Comparable results have been reported by Tofalo and others (2009), who pointed out that pressures exerted by environmental conditions such as high osmotic pressure and high concentration of some important compounds accounted for reduced biodiversity of yeast species and strains during Vino cotto M1856 Journal of Food Science r Vol. 80, Nr. 8, 2015

production. A potential explanation for results showing that the isolates of A. pullulans, C. tropicalis, P. kudriavzevii, S. cerevisiae, and T. Delbrueckii obtained from the orchard environment displayed a biotype identical to the corresponding isolates recovered from the plant environment might be that some yeasts on the surface of apples were capable of resisting industrial washing (Pelliccia and others 2011). Thus, these isolates might enter the plant environments and be recovered again. The C. tropicalis strain obtained from the stored concentrate exhibited genomic patterns different from the C. tropicalis strains isolated from the air and water, as some osmotolerant yeasts may be able to adapt to environmental stress through gene mutation (Watanabe and others 2013; Wei and others 2013). Figure 4 shows the growth rate of B-WHX-12-10 (D. hansenii) and B-WHX-12-64 (Z. rouxii) in different glucose concentrations as examples of results observed for all species. All osmotolerant isolates grew in the medium supplemented with 2%, 20%, 40%, and 50% (w/v) glucose, and all of them except the isolates of Z.

Figure 4–Growth rate of isolates B-WHX-12-10 (A, D. hansenii) and BWHX-12-64 (B, Z. rouxii) on YM agar supplemented with 2%, 20%, 40%, and 50% (w/v) glucose at 28 °C. Results are shown as the mean of three independent experiments. Standard error bars of the means are included. Values below different letters are statistically different by Duncan’s test (P < 0.05).

Osmotolerant yeasts and spoilage . . . Table 2–Physiological characteristics of the 13 species with more than one isolate representing each species. Osmotolerant isolate species

Fermentation tests Glucose D-Galactos Maltose Sucrose Cellobiose Lactose Raffinose Inulin Melibiose Melezitose Soluble starch Assimilation tests D-Galactose L-sorbose D-glucosamine D-ribose D-xylose L-Arabinose D-Arabinose L-Rhamnose Sucrose Maltose Cellobiose Lactose Raffinose Inulin Soluble starch Melibiose Melezitose Glycerol Erythritol Xylitol D-Sorbitol D-Mannitol Galactitol Inositol DL-Lactic acid Succinic acid Citric acid Methonal Ethomal Nitrate Ethylamine L-Lysine Growth at/in 37°C 45°C Cycloheximide (0.01%) Cycloheximide (0. 1%) D-Glucose 50% (w/w) D-Glucose 60% (w/w) Urease activity Acid production Starch formation DBB a

A. C. C. H. H. K. M. P. P. S. T. Y. Z pullulans glabrata tropicalis opuntiae uvarum marxianus guilliermondii kluyveri kudriavzevii cerevisiae delbrueckii lipolytica rouxii +b − − − − − − − − − −

+ + + V − − − − − − −

+ − − − + − − − − − −

+ − − − + − − − − − −

+ + − + − V − + − − −

+ + − + − − + + − − −

+ − − − − − − − − − −

− − − − − − − − − −

+ + Vc + − − + − − − −

+ + − + − − + − − − −

− − − − − − − − − −

+ − V V − − − − − − −

+ − + + + − + + + − + + + + + + + + + + − − − − − − − + + + +

− − − − − − − − − − − − − − − − − + − − − − − − + − − − + − − −

+ + − + + − − − + + V − − − + − − V − + + + − − + + + − + − + +

− − − − − − − − − − + − − − − − − − − − − − − − − − − − − − + +

− − − − − − − − − − + − − − − − − − − − − − − − − − − − − − + +

+ + − + + + − − + + + + V + − − − + − + + V − − + + + − + − + +

+ + + + + + + + + + + − + + − + + + − + + + + − − + + − + − + +

− − − − − − − − − − − − − − − − − + − − − − − − − + + − + − + +

− − + V − − − − − − − − − − − − − V − − − − − − + + + − + − + +

+ − − − − − − − + V − − + − + − − + − − V V − − + + − − + − − −

+ + − − − − − − + − − − + + − − − + − − + + − − − − − − + − − +

+ + − + − − − − − − − − − − − − − + + − + + − − + + + − + − + +

+ + − − − − − − + + − − − − − − − V − − + + − − − − − − + − + +

+ − − − + − + − − −

+ + − − + − − − − −

+ − + + V − − − − −

+ − + + + − − − − −

− − + + + − − − − −

+ − + + − − − − − −

+ − + + + − − − − −

− − − − − − − − − −

+ − − − − − − − − −

+ − − − + − − − − −

+ − − − + − − − − −

+ − + + − − + − − −

+ − − − + + − − − −

-a − − − − − − − − − − +

No growth.

b Growth. c

Variable results for different isolates belonging to the same species.

rouxii gradually grew slower with increasing glucose concentration from 2% to 50% (w/v). The strains of Z. rouxii showed a faster growth rate in 20% to 40% (w/v) glucose than in 2% (w/v) glucose. Based on the definition proposed by Tokuoka (1993), the isolates of Z. rouxii in this study could be classified as osmophilic, whereas the other species had characteristics of osmotolerant yeasts. This

result is consistent with those of previous studies that reported only strains of Z. rouxii were osmophilic (Tokuoka and others 1985; Tokuoka 1993). Figure 5 shows the growth curves of B-WHX-12-64 (Z. rouxii, osmophilic) and B-WHX-12-63 (H. uvarum, osmotolerant) in different glucose concentrations as examples of results observed for all

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Substrate

Osmotolerant yeasts and spoilage . . . species. A general decline in optical density values at 600 nm and an extension of lag phase duration were observed for all osmotolerant isolates, with an increase in glucose concentration from 50% to 70% (w/v). Such a trend might be attributed to the inhibition of the glucose utilization due to the osmotic pressure generated by high glucose concentration (Tofalo and others 2009). However, osmophilic isolates identified as Z. rouxii had the same lag phase of 48 h in 50% to 70% (w/v) glucose, displaying lesser sensitivity to high glucose concentration than osmotolerant isolates. Similarly, Z. rouxii has previously been reported to be the most sugar-tolerant yeast, capable of growth in more than 5 M glucose whereas the growth of other yeast species was inhibited in 3.2 to 4.75 M glucose (Martorell and others 2007). It is noteworthy that the species A. pullulans, C. tropicalis (from apple juice concentrate), C. glabrata, C. orthopsilosis, C. zemplinina, D. hansenii,

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M. caribbica, M. guilliermondii, W. anomalus, T. delbrueckii, and Z. rouxii were capable of growth in 70% (w/v) glucose within 192 h. This result is pertinent as previous studies report that fruit juice concentrates may contain about 70% (w/v) sugar (Steels and others 1999) and the aforementioned isolates may be spoilage agents in facilities with unsuitable hygiene. Previous studies have also implicated some of these isolates in the spoilage of fruit juice concentrates (Tokuoka 1993; Sancho and others 2000; Combina and others 2008). However, the pathway by which these isolates could contaminate concentrates is not clearly understood. Based on the results that fewer isolates were recovered in the production stages and no isolates were discovered in the online concentrate (before packaging), it might be deduced that osmotolerant yeasts are susceptible to heat and would not survive in the production line after 2 pasteurization steps (96 °C, 30 s) and a 2 stage evaporation process (90 to 95°C and 80 to 85 °C, 3 min). Our ongoing research showed that these isolates could be completely inactivated by heating at 80 °C for 15 s in apple juice concentrate (unpublished work). Previous studies also reported that D values of yeast rarely exceed 1 min at 55 °C in juice products (T¨or¨ok and King 1991; Splittstoesser 1996). Therefore, there is evidence to support our hypothesis that these isolates probably cannot contaminate the apple juice concentrate because of the processes of the production line. Based on the results that the highest amount of osmotolerant isolates were obtained from the air in both plants and almost all of them were capable of growth in 70% (w/v) glucose, we inferred that the osmotolerant isolates in air might contaminate apple juice concentrate at the time of encapsulation. Because of lack of air pressure controls for the filling facility and cooling of the online concentrate to 10 to 20 °C before encapsulation, the osmotolerant isolates in air might contaminate the filling valve through adhesion to the valve surface and then these isolates would enter the sterile bags with the flow of the concentrate (Brugnoni and others 2011). Thus, in order to avoid osmotolerant yeast contamination, it is essential to take measures such as air purification and air pressure control for the filling facility. These isolates generally grow slowly in apple juice concentrate at a suitable temperature due to the high glucose concentration and low pH values (Rojo and others 2013). However, rapid changes in atmospheric temperature might cause the condensation of moisture on the surface of concentrate when they are shipped abroad, thereby accelerating the growth of the microorganisms in the concentrate (Tokuoka 1993; Rojo and others 2013). Another feature that may contribute to the spoilage capacity of yeasts is their ability to vigorously ferment hexose sugars such as glucose and fructose (Martorell and others 2007). High fermentation can contribute to spoilage by causing swelling or explosions in packaging (Steels and others 1999; Martorell and others 2007). As shown in Table 3, the isolates belonging to the species C. glabrata, C. orthopsilosis, C. zemplinina, S. cerevisiae, W. anomalus, T. delbrueckii, and Z. rouxii had the ability to generate gas in 50% (w/v) glucose within 24 h, exhibiting faster sugar fermentation in comparison with the other isolates. In particular, the isolate belonging to Z. rouxii was able to generate gas more than two thirds of the whole volume of Durham’s tube within 24 h, displaying the fastest glycometabolism rate.

Figure 5–Growth curve of isolates B-WHX-12-64 (A, Z. rouxii) and B-WHX- Conclusion 12-63 (B, H. uvarum) in YPD broth supplemented with 50%, 55%, 60%, Study of the distribution of osmotolerant yeasts in apple-related 65%, and 70% (w/v) glucose at 28 °C for 10 d. Results are the mean of 2 environments (apple orchards and apple juice processing plants) independent experiments. Standard error bars of the means are included. Symbols: represent glucose concentrations: (), 50% (w/v); (), 55% indicated that osmotolerant yeasts are mainly present in the air of plant environment. The air of plant environments were also a (w/v); (), 60% (w/v); (), 65% (w/v); (•), 70% (w/v).

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Osmotolerant yeasts and spoilage . . . Table 3–Results of gas generation test for osmotolerant isolates in 50% (w/v) glucose. Isolates B-WHX-12-01 B-WHX-12-02 B-WHX-12-04 B-WHX-12-05 B-WHX-12-06 B-WHX-12-08 B-WHX-12-10 B-WHX-12-11 B-WHX-12-10 B-WHX-12-17 B-WHX-12-21 B-WHX-12-26 B-WHX-12-30 B-WHX-12-31 B-WHX-12-34 B-WHX-12-37 B-WHX-12-15 B-WHX-12-16 B-WHX-12-57 B-WHX-12-39 B-WHX-12-61 B-WHX-12-42 B-WHX-12-53

Identity R. mucilaginosa A. pullulans M. guilliermondii S. cerevisiae C. tropicalis P. kudriavzevii D. hansenii W. anomalus T. delbrueckii C. zemplinina C. orthopsilosis M. caribbica K. marxianus R. fluvial Y. lipolytica P. fermentans P.aff. fermentans P. kluyveri P. occidentalis H. opuntiae H. uvarum C. glabrata Z. rouxii

24 -a − − + − − − + ++ ++ ++ − − − − − − − − − − + +++

48 − − +b ++ ++ − − + ++ +++ +++ − + − − + − − − + + ++ +++

72 − − ++c ++ +++ + − ++ +++ +++ +++ + + − − + + − + +++ ++ ++ +++

Time (h) 96 − − ++ +++ +++ +++ − +++ +++ +++ +++ +++ ++ − − ++ ++ + ++ +++ +++ +++ +++

120 − − ++ +++ +++ +++ − +++ +++ +++ +++ +++ ++ − − +++ ++ ++ +++ +++ +++ +++ +++

144 − − +++d +++ +++ +++ − +++ +++ +++ +++ +++ +++ − − +++ +++ ++ +++ +++ +++ +++ +++

168 − − +++ +++ +++ +++ − +++ +++ +++ +++ +++ +++ − − +++ +++ +++ +++ +++ +++ +++ +++

No gas generation. generation less than a third of the whole volume of Durham’s fermentation tube. Gas genJGDSeration between a third and two thirds of the whole volume of Durham’s fermentation tube. d Gas generation more than two thirds of the whole volume of Durham’s fermentation tube. b Gas c

significant source of contamination for apple juice concentrate. Our future studies will focus on measures to prevent osmotolerant isolates in the air of plant environments from contaminating apple juice concentrate at the time of encapsulation and adjusting the key stress factors that play an important role in inhibiting the growth of osmotolerant isolates in apple juice concentrate.

Acknowledgments This work was supported by the China State “12th-Five-Year Plan” Scientific and Technological Scheme (2012BAD31B01).

Author Contributions Huxuan Wang designed the experiment and interpreted the results. Zhongqiu Hu, Fangyu Long, and Chen Niu helped to execute the work and collected the test data. Yahong Yuan and Tianli Yue helped to interpret the results and revise the manuscript.

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