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Optimization of air-blast drying process for manufacturing Saccharomyces cerevisiae and non-Saccharomyces yeast as industrial wine starters

Optimization of air-blast drying process for manufacturing Saccharomyces cerevisiae and... Wine yeast (Saccharomyces cerevisiae D8) and non‑ Saccharomyces wine yeasts (Hanseniaspora uvarum S6 and Issatch- enkia orientalis KMBL5774) were studied using air‑ blast drying instead of the conventional drying methods (such as freeze and spray drying). Skim milk—a widely used protective agent—was used and in all strains, the highest viabilities following air‑ blast drying were obtained using 10% skim milk. Four excipients (wheat flour, nuruk, artichoke powder, and lactomil) were evaluated as protective agents for yeast strains during air‑ blast drying. Our results showed that 7 g lactomil was the best excipient in terms of drying time, powder form, and the survival rate of the yeast in the final product. Finally, 7 types of sugars were investigated to improve the survival rate of air ‑ blast dried yeast cells: 10% trehalose, 10% sucrose, and 10% glucose had the highest survival rate of 97.54, 92.59, and 79.49% for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. After 3 months of storage, S. cerevisiae D8 and H. uvarum S6 demonstrated good survival rates (making them suitable for use as starters), whereas the survival rate of I. orientalis KMBL5774 decreased considerably compared to the other strains. Air‑ blast dried S. cerevisiae D8 and H. uvarum S6 showed metabolic activities similar to those of non‑ dried yeast cells, regardless of the storage period. Air‑ blast dried I. orientalis KMBL5774 showed a noticeable decrease in its ability to decompose malic acid after 3 months of storage at 4 °C. Keywords: Wine yeast, Air‑ blast drying, Survival rate, Excipient, Lactomil during the initial stages of fermentation, affect the taste Introduction and aroma of wine, suggesting that suitable co-fermenta- Wine is one of the oldest fermented foods in history and tion using Saccharomyces yeasts mixed with non-Saccha- is produced as a result of complicated interplay between romyces yeast is an important factor in making wine of the metabolic reactions of various microorganisms such high quality (Ciani and Maccarelli 1998; Rojas et al. 2001; as yeast and lactic acid bacteria (Zagorc et al. 2001). Wine Jolly et al. 2006; Esteve-Zarzoso et al. 1998). yeast, Saccharomyces cerevisiae, has been used to make Several Korean wine-makers have widely utilized wine with high stability because of its high ethanol toler- S. cerevisiae Fermivin from Netherlands, S. cerevisiae ance and ability to inhibit bacteria and other undesirable W-3 from Japan, and S. cerevisiae EC1118 from Can- microorganisms during the fermentation process (Casey ada because these strains can be handled conveniently and Ingledew 1986; Philliskirk and Young 1975). On and offer reliable starter quality. Although most Korean the other hand, non-Saccharomyces yeasts, which grow wine has been made using these imported yeast start- ers, several studies have reported that indigenous yeasts *Correspondence: hpark@knu.ac.kr can also contribute to making distinctive wines based on School of Food Science and Biotechnology, Kyungpook National the grape cultivar and the geographical region (Heard University, 80 Daehakro, Daegu 41566, South Korea Full list of author information is available at the end of the article © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lee et al. AMB Expr (2016) 6:105 Page 2 of 10 and Fleet 1985; Mercado et  al. 2007; Querol et  al. 1992; Champagne et  al. 2012). Suitable agents can protect the Schütz and Gafner 1993; Hong and Park 2013). proteins and membranes of the microorganisms (Leslie The Campbell Early grape, which is the most domi - et al. 1995; Champagne and Gardner 2001). nant cultivar in Korea, has a high malic acid content In this study, we aimed to optimize the development of due to early harvesting for enhancing grape color. High Saccharomyces and non-Saccharomyces yeast starters at malic acid content lowers the quality of Korean wine the industrial level using air-blast drying, instead of the due to high acidity, which has resulted in the poor com- conventionally used freeze-drying method, as well as petitive value of indigenously manufactured wine against using various types of excipients and sugars at different imported wine (Kim et al. 1999; Lee et al. 2016). For this concentrations to enhance the survival rate of air dried- reason, isolating and utilizing indigenous yeasts instead yeast cells. Furthermore, the long-term storage proper- of imported yeast starters are necessary to make Korean ties of each dried-yeast strain and the metabolic activity wine competitive. Developing optimal industrial starter of air-blast dried yeast cells during storage at 4  °C were cultures for winemaking is essential for increasing the also investigated. prevalence of indigenous Korean yeast starter products. Previously, S. cerevisiae D8, Hanseniaspora uvarum S6, Materials and methods and Issatchenkia orientalis KMBL5774 were isolated Strains, media, and culture conditions from Korean Campbell Early grape cultivar and their Saccharomyces cerevisiae D8 (KACC 93245P), H. uvarum biological and physiological characteristics were studied. S6 (KACC 93248P) and I. orientalis KMBL5774 (KACC Kim et al. (2013b) reported that wine fermented by S. cer- 93124P) isolated from the Korean grape cultivar were evisiae D8 had higher color and taste scores compared to used in this study (Hong and Park 2013; Kim et al. 2013a; the wine fermented by S. cerevisiae W-3. Hong and Park Seo et  al. 2007). Each strain was cultured at 30  °C with (2013) described that wine fermented by H. uvarum S6 shaking (150  rpm) in sterilized YPD media composed (previously SS6) showed slower fermentation rate but of 1% yeast extract, 2% bacto-peptone, and 2% glucose had higher organic acid content and sensory evaluation and the cells were harvested for making the starters. All scores compared to wine fermented by S. cerevisiae W-3. strains were stored at −70  °C in 20% glycerol until they Seo et al. (2007) and Kim et al. (2008) reported that I. ori- were used for the experiments. entalis KMBL5774 could degrade malic acid during alco- hol fermentation, and co-fermentation with I. orientalis Protective agent conditions KMBL5774 and S. cerevisiae W-3 resulted in better color, Skim milk (5 and 10%) and 7 sugars (5 and 10% of glu- flavor, and taste compared to the fermentation using only cose, fructose, lactose, maltose, raffinose, sucrose, and S. cerevisiae W-3. trehalose) were used to evaluate the survival rate of air- The most important factors for developing microbial blast dried yeast cells. All protective solutions, including starters include maintenance of cell viability, capac- skim milk and sugars were sterilized at 121 °C for 15 min ity for long-term storage and the drying method used. before experiments. Four kinds of excipients—wheat Several studies have utilized freeze-drying (Lodato et  al. flour (CJ Cheiljedang Corp., Seoul, Korea), nuruk (Song - 1999; Ale et al. 2015; Abadias et al. 2001a), fluidized bed hak Agri. Corp., Gwangju, Korea), artichoke powder and drying (Bayrock and Ingledew 1997), and spray dry- lactomil (composed of lactose 89% and maltodextrin 11%; ing (Luna-Solano et  al. 2005; Isono et  al. 1995) to make Seo Kang Dairy & Food Co., LTD, Sacheon, Korea)—were starter products. Freeze-drying is disadvantageous owing used to process the yeast starters into an appropriate to the high expenses incurred, and fluidized bed drying powdered form. Artichoke was obtained from Gim- and spray drying are not suitable due to low viability in cheon, Korea, and it was processed by lyophilization and the starter cultures induced by the high temperature dur- grinding to be converted into powdered form. All excipi- ing drying. In contrast, air-blast drying can lower the cost ents were added at quantities of 2  g (lactomil amounts fivefold, result in comparatively less cell damage, as well ranged from 2 to 8  g) to compare their protective abil- as provide easier control of moisture in the starter com- ity and availability as a starter product in powdered form pared with other drying methods (Santivarangkna et  al. for each strain of the dried yeast. All excipients were used 2007). Even though air-blast drying has many advan- with the yeast pellet directly. tages for making yeast starters, only a few studies related to the air-blast dried yeast have been attempted. Simi- Air‑blast drying process lar to freeze-drying, the selection of protective agents Each yeast strain was cultured in 100 mL YPD broth and is very important in air-blast drying because intracellu- incubated at 30  °C for 16  h. After culturing, yeast cells lar accumulation of the appropriate solutes is related to were harvested by centrifugation (3578×g for 10  min) strain survival following air-blast drying (Kets et al. 1996; and rinsed twice in a 0.85% NaCl solution. The pellet was Lee et al. AMB Expr (2016) 6:105 Page 3 of 10 mixed with 2 g of various excipients such as wheat flour, glucose due to the ethanol fermentation (Jung and Park nuruk, and artichoke powder, and lactomil (2–8 g) as well 2005). Air-blast dried I. orientalis KMBL5774 was incu- as 1 mL protective agent solutions consisting of the skim bated in 10  mL YPD broth containing 2% malic acid at milk and sugars. The mixed yeast cell pellets were dried 30  °C with shaking (150  rpm) to measure the malic acid using Clear Air Oven (HB-509C, HanBaek, Bucheon, decomposition rate. Malic acid content was determined Korea) at 37 °C until the moisture content of dried yeast using the l-Malic Acid Assay Kit (K-LMALR; Mega - starter was <10%. After air-blast drying, the samples were zyme, Wicklow, Ireland) (Lee et al. 2016). immediately analyzed to determine their moisture con- tent and survival rate, then stored at 4  °C for 3  months, Statistical analysis after which their survival rate was determined. All experiments were carried out in at least triplicates and the results were analyzed using the Statistical Pack- Measurement of cell viability and moisture content age for the Social Sciences (SPSS, version 12.0 for Win- After air-blast drying, each sample was reconstituted to dows, Chicago, IL, USA) in order to obtain average and its original volume with distilled water for rehydration. standard deviations. Significance was determined to be Then, the serially diluted samples were spread on YPD p  <  0.05 using one-way analysis of variance (ANOVA), agar plates and incubated at 30  °C for 24  h. The white followed by Duncan’s multiple range test. colonies that formed on YPD agar were counted. The survival rate of each sample was calculated as (%) sur- Results vival  =  (N/N )  ×  100, where N represents the number Eec ff t of skim milk on the survival rate of air‑blast dried −1 of viable cell count after air-blast drying (cfu  mL ) and yeast cells N represents the number of viable cell count before air- Skim milk is typically used as a protective agent to pro- −1 blast drying (cfu  mL ). Moisture content of dried yeast tect cell membrane while drying microbial strains. It has starters was measured by determining the weight loss been suggested that milk proteins may cover the cells to after 10 h at 105 °C (AOAC 1990). prevent damage (Abadias et al. 2001b). In this study, the protective effect of skim milk on air-blast dried cells was Morphology of air‑blast dried yeast cells investigated. For this, 5–10% skim milk solutions were The morphologies of air-blast dried Saccharomyces and mixed with centrifuged yeast cells and the mixed cells non-Saccharomyces yeast cells were observed by scan- were air-blast dried at 37 °C for 2 h until dried cells were ning electron microscopy (SEM), as described by Hong- obtained in the appropriate powdered form. The sur - pattarakere et  al. (2013). The air-blast dried sample was vival rates of all air-blast dried yeast cell strains mixed affixed to “stubs” using double-sided metallic adhesive with skim milk increased in a dose-dependent man- tape and then coated with gold using sputter coater (WI- ner compared to that of the control (Fig.  1). When 10% RES-Coater-001). The morphology of the sample was skim milk was mixed with the dried yeast cells, the viable observed under a SU8220 scanning electron microscope count of S. cerevisiae D8, H. uvarum S6, and I. orienta- (Hitachi, Tokyo, Japan) that was operated at an accelerat- lis KMBL5774 increased to approximately 0.89, 0.71, and −1 ing voltage of 10 kV. Images were obtained under 2000× 1.03 log cfu mL , respectively. Thus, 10% skim milk was magnification. utilized in subsequent experiments. Metabolic activities of air‑blast dried yeast cells Eec ff t of various excipients on the survival rate Metabolic activities of yeast cells stored for 0–3  months and moisture content of air‑blast dried yeast cells after air-blast drying were analyzed and non-dried yeast The selection of the excipient is very important in gen - cells were used as the control. Air-blast dried S. cerevisiae erating a stable powdered form and shape for improving D8 and H. uvarum S6 were incubated in 100  mL YPD the stability and quality of the final product (Georgetti −1 broth containing 20% glucose (yeast extract 10  g  L , et  al. 2008). In this study, four excipients (wheat flour, −1 −1 peptone 20  g  L , and glucose 200  g  L ) at 30  °C with nuruk, artichoke powder, and lactomil) were utilized to shaking (150  rpm) to measure the glucose fermentation prevent cell membrane damage caused by the drying rate. A water trap apparatus containing conc. H SO was environment and the survival rates and moisture con- 2 4 attached to the top of each flask to trap water evaporated tents were determined. All samples were air-blast dried from the flask during the fermentation. The amount of until the moisture content reached  <10%. The air-blast CO produced was directly measured as the decrease dried yeast samples mixed with wheat flour showed the in the weight of the whole flask. The fermentation ratio longest drying time of 7 h and the lowest survival rate of was expressed as the percentage of the amount of CO 0.27–1.39%, whereas the samples mixed with lactomil produced per the theoretical CO production from the showed the shortest drying time of 2.5 h and the highest 2 Lee et al. AMB Expr (2016) 6:105 Page 4 of 10 Fig. 1 Viable yeast cell counts with 5 and 10% skim milk before (filled squares) and after (empty squares) air ‑blast drying for 2 h. All data are expressed as the mean ± SD (n = 3) survival rate of 1.01–3.40% (Table  1). On analysis of the added to 7  g lactomil was measured as 59.12%, which shape of the powdered form obtained by adding various was higher than the survival rate of the sample added excipients, we found that the samples mixed with wheat to 8 g lactomil. In case of H. uvarum S6 and I. orientalis flour and nuruk showed a lump form after air-blast dry - KMBL5774, the survival rates measured for 7–8 g of lac- ing, and the samples mixed with artichoke powder and tomil were not significantly different. Although the dry - lactomil could be easily collected due to their impalpable ing time of the samples added to 8  g lactomil was 0.3  h powdered form (Fig.  2). Based on the survival rate, dry- shorter than that of the samples added to 7  g lactomil, ing time, and the properties of the powdered form of air- after considering the excipient cost and the similar pro- blast dried yeast products, 2 g lactomil was considered as tective effect of 7–8  g of lactomil, 7  g was considered as the most suitable excipient for making yeast starters by the optimal amount of lactomil required for maintain- air-blast drying. Based on the results of the yeast samples ing the viability of air-blast dried yeast cells. Therefore, added to various excipients, the survival rates of air-blast subsequent experiments were carried out by adding 10% dried yeast cells in relation to the amount of lactomil skim milk and 7 g lactomil to the yeast cells, followed by added were investigated (Table  2). As the amount of lac- air-blast drying for 1.5 h. tomil added was increased, drying time for each sample with  <10% moisture content was reduced and the sur- Eec ff t of sugar additives as a protective agent on the vival rate of air-blast dried yeast cells increased in a dose- survival rate of air‑blast dried yeast cells dependent manner until the addition of 7  g lactomil. In To determine the protective effect of sugar on air-blast case of S. cerevisiae D8, the survival rate of the sample dried yeast cells, the survival rate of air-blast dried yeast Table 1 Eec ff ts of various excipients on the survival rate and moisture content of air-blast dried yeasts Excipient S. cerevisiae D8 H. uvarum S6 I. orientalis KMBL5774 Drying time (h) Survival rate Moisture content Survival rate Moisture content Survival rate Moisture content (%) (%) (%) (%) (%) (%) d d c Wheat flour 1.39 ± 0.04 9.53 ± 0.30 0.27 ± 0.04 9.21 ± 0.27 0.30 ± 0.02 9.31 ± 0.20 7 c c c Nuruk 2.23 ± 0.14 9.86 ± 0.23 0.36 ± 0.03 9.94 ± 0.19 0.33 ± 0.03 9.90 ± 0.35 4 b b b Artichoke powder 2.53 ± 0.08 9.89 ± 0.38 0.61 ± 0.04 9.50 ± 0.31 1.07 ± 0.08 9.73 ± 0.23 6 a a a Lactomil 3.40 ± 0.19 9.42 ± 0.12 1.01 ± 0.06 9.23 ± 0.16 1.65 ± 0.33 8.96 ± 0.14 2.5 Different letters within the same column indicate significant difference (p < 0.05) Lee et al. AMB Expr (2016) 6:105 Page 5 of 10 cells (depending on the type and concentration of sugars as protective agents with 10% skim milk) were investi- gated (Table  3). In case of S. cerevisiae D8, the addition of 10% sugars (except for fructose) resulted in a survival rate of  >90%, and addition of 10% trehalose resulted in the highest survival rate of 97.54%. In case of H. uvarum S6, the high survival rates of all samples added to sugars resulted in considerably higher viability than that of sam- ples with no sugar addition. All samples added to 10% sugars had higher survival rate than those added to 5% sugars; in particular, 10% sucrose resulted in the highest survival rate of 92.59%. In case of I. orientalis KMBL5774, most sugar additions did not show a significant increase of the survival Malic acid content was determined rate compared to no sugar addition, but addition of 10% glu- cose and 10% fructose noticeably increased its survival rate to 79.49 and 65.17%, respectively. The morphology of air-blast dried yeast cells was observed using SEM (Fig. 3). The SEM images showed that each yeast cell was Fig. 2 Images of air‑blast dried yeast cells mixed with wheat flour, coated with skim milk, sugar, and lactomil and the cells nuruk, artichoke power, and lactomil. All excipients were added at were densely accumulated, which suggest that protective a concentration of 2 g and air‑blast dried until moisture content of agents and excipients protect yeast cells from the adverse samples was <10% drying environment. Table 2 Eec ff ts of the amount of lactomil added on the survival rate and moisture content of air blast dried yeasts Lactomil (g) S. cerevisiae D8 H. uvarum S6 I. orientalis KMBL5774 Drying time (h) Survival rate Moisture content Survival rate Moisture content Survival rate Moisture content (%) (%) (%) (%) (%) (%) e e c 2 3.40 ± 0.19 9.42 ± 0.12 1.01 ± 0.06 9.23 ± 0.21 1.65 ± 0.33 8.96 ± 0.15 2.5 e e c 3 5.35 ± 0.30 8.37 ± 0.19 2.46 ± 0.59 8.27 ± 0.13 3.11 ± 0.21 7.85 ± 0.11 2.5 d d b 4 32.70 ± 2.37 8.81 ± 0.24 6.33 ± 0.32 9.70 ± 0.17 11.79 ± 0.84 9.30 ± 0.21 2 c c b 5 39.31 ± 3.03 9.52 ± 0.21 9.52 ± 0.42 8.54 ± 0.15 13.66 ± 2.87 8.81 ± 0.13 2 c b b 6 43.40 ± 3.27 9.17 ± 0.18 17.18 ± 1.25 9.68 ± 0.29 16.33 ± 2.86 7.23 ± 0.09 2 a a a 7 59.12 ± 1.96 8.88 ± 0.24 29.65 ± 1.77 8.34 ± 0.14 23.71 ± 3.38 8.48 ± 0.16 1.5 b a a 8 53.45 ± 4.75 9.58 ± 0.31 29.60 ± 3.16 9.84 ± 0.32 23.68 ± 3.42 9.37 ± 0.22 1.2 Different letters within the same column indicate significant difference (p < 0.05) Table 3 Eec ff ts of the type and concentration of various sugars on the survival rate of air-blast dried yeasts Strains Conc. Survival rate (%) (%) Glucose Fructose Lactose Maltose Raffinose Sucrose Trehalose a b a a a a a S. cerevisiae D8 5 95.07 ± 8.45 64.31 ± 7.62 84.00 ± 12.17 88.80 ± 6.59 93.39 ± 15.32 94.38 ± 11.68 96.13 ± 5.94 a ab a a a a a 10 94.33 ± 6.66 74.28 ± 7.07 91.20 ± 9.53 94.03 ± 5.13 94.52 ± 10.46 93.73 ± 8.74 97.54 ± 6.77 b b ab b b ab ab H. uvarum S6 5 63.99 ± 5.81 67.67 ± 1.06 72.56 ± 8.75 67.20 ± 4.07 68.54 ± 2.35 69.66 ± 6.76 69.77 ± 1.55 ab ab ab ab ab a ab 10 71.71 ± 4.78 75.59 ± 11.05 85.81 ± 4.98 78.76 ± 17.82 84.68 ± 9.30 92.59 ± 11.17 73.38 ± 9.18 cd cd cd cd cd cd cd I. orientalis KMBL5774 5 41.89 ± 3.58 39.68 ± 5.99 43.04 ± 9.56 39.74 ± 4.62 41.03 ± 8.97 40.79 ± 2.63 44.30 ± 11.04 a b d bc d d cd 10 79.49 ± 9.25 65.17 ± 5.15 36.71 ± 4.56 54.17 ± 8.46 33.33 ± 4.62 37.78 ± 3.39 45.93 ± 6.06 Different letters within the same strains indicate significant difference (p < 0.05) Lee et al. AMB Expr (2016) 6:105 Page 6 of 10 Fig. 3 Images of air‑blast dried yeast cells observed by a scanning electron microscope (SEM) at ×2000 magnification. 10% skim milk and 7 g lacto ‑ mil were used in all samples. Ten percent trehalose, 10% sucrose, and 10% glucose were used as protective agents for S. cerevisiae D8 (a), H. uvarum S6 (b), and I. orientalis KMBL5774 (c), respectively Long‑term storability of air‑blast dried yeast starter samples showed similar fermentability on the second day products and completed the fermentation process on third day. Changes in the survival rate and viable count of air-blast Similarly, both air-blast dried H. uvarum S6 just after dried yeast cells were investigated in products that had drying and after 3 months of storage showed similar fer- been stored at 4  °C for 3  months (Fig.  4). All samples mentation rates compared to non-dried H. uvarum S6. were prepared based on the optimal conditions deter- Non-dried I. orientalis KMBL5774 initiated and com- mined in the present study. Air-blast dried S. cerevisiae pleted malic acid decomposition first, but the duration D8 and H. uvarum S6 continued to show a high survival of malic acid decomposition was not significantly dif - rate of 42.24 and 49.74% after 2  months, whereas the ferent from that observed for air-blast dried I. orientalis survival rate of air-blast dried I. orientalis KMBL5774 KMBL5774 immediately after drying. In case of air-blast rapidly decreased compared to the other yeast strains dried I. orientalis KMBL5774 after 3  months of storage and showed a survival rate of only 3.08% after 2 months at 4 °C, malic acid degradation was delayed by 12 h com- of storage. After 3  months, the viable count of S. cer- pared to that of the control. The results of the metabolic evisiae D8 and H. uvarum S6 decreased to 1.18 and 0.51 activities of air-blast dried yeast cells after 3  months of −1 log cfu  mL compared to the viable counts measured storage suggest that S. cerevisiae D8 and H. uvarum S6 immediately after air-blast drying. Therefore, air-blast retained their capacities and efficiencies as yeast starters, dried yeast cells of both strains, S. cerevisiae D8 and H. whereas reduction of the survival rate after long-term uvarum S6, have excellent potential as a starter product. storage possibly induced the decrease in the malic acid On the other hand, although I. orientalis KMBL5774 also degradation rate in I. orientalis KMBL5774. showed high survival rate immediately after air-blast dry- ing, further study on the long-term storage of I. orientalis Discussion KMBL5774 is necessary because it showed very low via- In this study, air-blast drying was established as a suit- ble count after 3 months of storage (2.38 log reduction). able substitute to conventional drying methods, such as freeze-drying or spray-drying for manufacturing yeast Changes in metabolic activities of air‑blast dried yeast cells starters for wine. Skim milk, generally used as a protec- The metabolic activities of each air-blast dried yeast tive agent in freeze-drying, was added at 5 and 10% to cell were investigated depending on the storage period air-blast dried yeast cells and the addition resulted in the (Fig.  5). Glucose fermentation ability of air-blast dried increase in the survival rate of air-blast dried yeast cells S. cerevisiae D8 and H. uvarum S6, and malic acid in a dose-dependent manner (Fig. 1). Ananta et al. (2005) decomposition ability of I. orientalis KMBL5774 were reported that when 20% reconstituted skim milk added to examined. All samples were prepared by the optimal Lactobacillus rhamnosus GG was spray dried at an outlet manufacturing process based on results obtained in the temperature of 80  °C, its survival rate was measured to present study and non-dried yeast cultures were used as be >60%. Abadias et al. (2001a) reported that freeze-dried control to compare the metabolic activity. Non-dried S. Candida sake showed the highest survival rate of 40% cerevisiae D8 decomposed glucose slightly faster com- when 10% skim milk and 10% lactose were added as pro- pared to both air-blast dried S. cerevisiae D8 immediately tective agents. Similarly, our study also showed that skim after drying and after 3  months of storage. However, all milk had a protective effect on air-blast dried yeast cells. Lee et al. AMB Expr (2016) 6:105 Page 7 of 10 Fig. 4 Changes in the survival rate (left panel) and viable count (right panel) of air‑blast dried yeast cells stored at 4 °C for 3 months. Empty circles, filled squares, and filled diamonds in the left panel represent the survival rates of S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respec‑ tively. Bars in the histogram (right panel) represent viable counts of yeast cells before (black) and after (gray) air‑blast drying and yeasts stored for 1 (white), 2 (diagonally patterned), and 3 (dotted) months. 10% skim milk and 7 g lactomil were used in all samples. 10% trehalose, 10% sucrose, and 10% glucose were used as protective agents for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. All data are expressed as the mean ± SD (n = 3) Fig. 5 Fermentation rate of air‑blast dried S. cerevisiae D8 (diamonds) and H. uvarum S6 (circles) (a) and malic acid content fermented by air ‑blast dried I. orientalis KMBL5774 (squares) (b). Empty figures represent non‑ dried yeast cells, filled figures represent air ‑blast dried yeast cells just after air ‑ blast drying, and gray figures represent air ‑blast dried yeast cells after 3 months of storage. All data are expressed as the mean ± SD (n = 3) Four excipients (wheat flour, nuruk, artichoke pow - yeast starter (Table  1). According to a study by Beker der, and lactomil) were evaluated to make an appropri- and Rapoport (1987), 12–13% moisture content is not ate powdered form of the final product of the air-dried suitable for storage of yeast, whereas at 8–10% moisture Lee et al. AMB Expr (2016) 6:105 Page 8 of 10 content, yeast retain a remarkable degree of cell viability SEM to confirm the accumulation of yeast cells (Fig.  3). during storage. Therefore, all samples were air-blast dried Pereira et al. (2003) reported that 10% trehalose was able until moisture content of each sample was  <10%. Dry- to reduce oxidative damage caused by dehydration in S. ing times for samples with the excipients wheat flour and cerevisiae and Garay-Arroyo et  al. (2004) reported that artichoke were relatively longer than those observed for S. cerevisiae could be easily adapted to various environ- the other excipients. This result could be attributed to the mental stresses, including oxidative stress, heat shock, water or moisture in these excipients, which could lead to freezing shock, osmotic, and ionic stress. A study by increased viscosity due to starch gelatinization and bind- Lemetais et  al. (2012) on S. cerevisiae showed that the ing to the gluten network in wheat flour (Fessas and Schi - plasma membrane is an essential structure for the sur- raldi 2001), and to the high dietary fiber content in the vival of cells during dehydration by air-drying. In a study artichoke (insoluble 18.11% and soluble 26.74%), which on the survival rate of H. uvarum lyophilized without could interact with the water held in the capillary struc- cryoprotectant and stored at −80  °C for 0–12  months, ture of the artichoke (Lintas and Capeloni 1988; López the viable counts showed a reduction of 2.47–2.82 log −1 et al. 1996). Nuruk, a Korean traditional starter prepared cfu  mL from the values recorded before freeze-dry- by the natural proliferation of fungi and other microor- ing (Pietrowski et al. 2015). A study by Kim et al. (2016) ganisms (Yoo et al. 2011), was also investigated as excipi- reported that S. cerevisiae D8, H. uvarum S6, and I. ori- ent for yeast starter. Although drying time for nuruk entalis KMBL5774 entrapped in 2% Ca-alginate beads by was shorter than wheat flour and artichoke, the physical air-blast drying showed 90.67, 90.81, and 87.04% survival properties of nuruk as well as that of wheat flour were not rate when 10% skim milk and 10% sugars (sucrose, treha- suitable for preparing the final starter product because lose, and glucose for S. cerevisiae D8, H. uvarum S6 and they led to the formation of lumps after air-blast drying I. orientalis KMBL5774, respectively) were used as pro- (Fig.  2). On the contrary, lactomil (consisting of lactose tectants. Miyamoto-Shinohara et al. (2010) reported that and maltodextrin) was considered as the most suitable I. orientalis had an 8.6 and 28.2% survival rate on freeze- excipient for yeast starter because it yielded a rapid dry- drying and liquid drying, respectively, without any pro- ing time of 2.5  h and the highest yeast cell survival rate tective agent. and formed a fine powder. Furthermore, the survival Long-term storage is the most important factor in rates of air-blast dried yeast cells, based on the amount of developing microbial starters for industrial use. In our lactomil added, were investigated and all samples mixed study, long-term storage effect of air-blast drying on each with 7  g lactomil statistically showed the highest and yeast strain stored at 4 °C for 3 months was investigated optimal survival rate (Table 2). (Fig. 4). Air-blast dried S. cerevisiae D8 and H. uvarum S6 Sugars have been widely used as protective agents due showed 1.18 and 0.51 log reductions, which means that to their low price, chemically innocuous nature, and gen- these strains retained a good viable count after 3 months eral utilization in the food industry (Peighambardoust of storage. However, since I. orientalis KMBL5774 et  al. 2011). The protective effects of various sugars on showed a 2.38 log reduction after 3  months of storage, the survival rate of microbial starters such as yeast and further study would be needed to improve its storability. bacteria have been determined in the last few decades Several studies have reported storability based on various (Jofré et al. 2014; Lodato et al. 1999; Niu et al. 2016). Our drying method and protectants. A study by Miyamoto- study showed that each strain showed its highest capac- Shinohara et  al. (2006) reported that freeze-dried S. cer- ity for survival with different optimal protective agents evisiae showed 0.010 log reduction per year for 20 years. (Table 3). S. cerevisiae D8 added to 10% trehalose showed Zayed and Roos (2004) demonstrated that 4% sucrose, the highest survival rate of 97.54% and most sugar- 4% trehalose, and 18% skim milk, used as protective based protectants (except for fructose) demonstrated solutions for freeze-dried Lactobacillus salivarius main- excellent protective effects with  >90% survival rate. H. tained the survival rate at 83–85% for 7 weeks of storage. uvarum S6 showed the highest survival rate (92.59%) In a study by Gardiner et  al. (2000), the survival rate of when 10% sucrose was added as a protectant and other spray-dried Lactobacillus paracasei NFBC 338 grown in sugars were also shown to demonstrate notable protec- 20% reconstituted skim milk was maintained at constant 9 −1 tive effects compared to no sugar addition. Contrary to at  ~1  ×  10 cfu  g during 2  months of storage at 4  °C, S. cerevisiae D8 and H. uvarum S6, only 10% glucose and while storage of L. salivarius UCC 118 under the same 10% fructose remarkably increased the survival rate of conditions showed 1 log reduction (from 7.2  ×  10 to 6 −1 I. orientalis KMBL5774, whereas other sugars demon- 9.5 × 10 cfu g ). strated protective effects that only slightly increased the Fermentation rates of air-blast dried S. cerevisiae D8 survival rate of I. orientalis KMBL5774. The morphol - and H. uvarum S6 and the malic acid decomposition ogy of air-blast dried yeast starters was also observed by ability of air-blast dried I. orientalis KMBL5774 after Lee et al. AMB Expr (2016) 6:105 Page 9 of 10 References 3  months of storage were analyzed to evaluate their Abadias M, Benabarre A, Teixidó N, Usall J, Viñas I (2001a) Eec ff t of freeze drying metabolic capacities (Fig.  5). 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Appl Environ Microbiol 66:2605–2612 Georgetti SR, Casagrande R, Souza CRF, Oliveira WP, Fonseca MJV (2008) Spray Author details drying of the soybean extract: effects on chemical properties and antioxi‑ School of Food Science and Biotechnology, Kyungpook National University, dant activity. LWT‑Food Sci Technol 41:1521–1527 80 Daehakro, Daegu 41566, South Korea. Department of Fermentation Heard GM, Fleet GH (1985) Growth of natural yeast flora during the fermenta‑ Biotechnology, Kyungpook National University, 80 Daehakro, Daegu 41566, tion of inoculated wines. Appl Environ Microbiol 50:727–728 South Korea. Fermented Food Science Division, Depertment of Agro‑food Hong YA, Park HD (2013) Role of non‑Saccharomyces yeasts in Korean wines Resources, NIAS, RDA, 166, Nongsaengmyeong‑ro, Iseo‑myeon, Wanju Gun, produced from Campbell Early grapes: potential use of Hanseniaspora Jeollabuk‑do 55365, South Korea. uvarum as a starter culture. Food Microbiol 34:207–214 Hongpattarakere T, Rattanaubon P, Buntin N (2013) Improvement of freeze‑ Acknowledgements dried Lactobacillus plantarum survival using water extracts and crude This study was supported by the Rural Development Administration, Republic fibers from food crops. Food Bioprocess Tech 6:1885–1896 of Korea (Research Grant PJ012425022016). Isono Y, Araya G, Hoshino A (1995) Immoblization of Saccharomyces cerevisiae for ethanol fermentation on γ‑alumina particles using a spray‑ dryer. Competing interests Process Biochem 30:743–746 The authors declare that they have no competing interests. Jofré A, Aymerich T, Garriga M (2014) Impact of different cryoprotectants on the survival rate of freeze‑ dried Lactobacillus rhamnosus and Lactobacillus Ethics approval casei/paracasei during long‑term storage. 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Optimization of air-blast drying process for manufacturing Saccharomyces cerevisiae and non-Saccharomyces yeast as industrial wine starters

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Copyright © 2016 by The Author(s)
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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10.1186/s13568-016-0278-9
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Abstract

Wine yeast (Saccharomyces cerevisiae D8) and non‑ Saccharomyces wine yeasts (Hanseniaspora uvarum S6 and Issatch- enkia orientalis KMBL5774) were studied using air‑ blast drying instead of the conventional drying methods (such as freeze and spray drying). Skim milk—a widely used protective agent—was used and in all strains, the highest viabilities following air‑ blast drying were obtained using 10% skim milk. Four excipients (wheat flour, nuruk, artichoke powder, and lactomil) were evaluated as protective agents for yeast strains during air‑ blast drying. Our results showed that 7 g lactomil was the best excipient in terms of drying time, powder form, and the survival rate of the yeast in the final product. Finally, 7 types of sugars were investigated to improve the survival rate of air ‑ blast dried yeast cells: 10% trehalose, 10% sucrose, and 10% glucose had the highest survival rate of 97.54, 92.59, and 79.49% for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. After 3 months of storage, S. cerevisiae D8 and H. uvarum S6 demonstrated good survival rates (making them suitable for use as starters), whereas the survival rate of I. orientalis KMBL5774 decreased considerably compared to the other strains. Air‑ blast dried S. cerevisiae D8 and H. uvarum S6 showed metabolic activities similar to those of non‑ dried yeast cells, regardless of the storage period. Air‑ blast dried I. orientalis KMBL5774 showed a noticeable decrease in its ability to decompose malic acid after 3 months of storage at 4 °C. Keywords: Wine yeast, Air‑ blast drying, Survival rate, Excipient, Lactomil during the initial stages of fermentation, affect the taste Introduction and aroma of wine, suggesting that suitable co-fermenta- Wine is one of the oldest fermented foods in history and tion using Saccharomyces yeasts mixed with non-Saccha- is produced as a result of complicated interplay between romyces yeast is an important factor in making wine of the metabolic reactions of various microorganisms such high quality (Ciani and Maccarelli 1998; Rojas et al. 2001; as yeast and lactic acid bacteria (Zagorc et al. 2001). Wine Jolly et al. 2006; Esteve-Zarzoso et al. 1998). yeast, Saccharomyces cerevisiae, has been used to make Several Korean wine-makers have widely utilized wine with high stability because of its high ethanol toler- S. cerevisiae Fermivin from Netherlands, S. cerevisiae ance and ability to inhibit bacteria and other undesirable W-3 from Japan, and S. cerevisiae EC1118 from Can- microorganisms during the fermentation process (Casey ada because these strains can be handled conveniently and Ingledew 1986; Philliskirk and Young 1975). On and offer reliable starter quality. Although most Korean the other hand, non-Saccharomyces yeasts, which grow wine has been made using these imported yeast start- ers, several studies have reported that indigenous yeasts *Correspondence: hpark@knu.ac.kr can also contribute to making distinctive wines based on School of Food Science and Biotechnology, Kyungpook National the grape cultivar and the geographical region (Heard University, 80 Daehakro, Daegu 41566, South Korea Full list of author information is available at the end of the article © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lee et al. AMB Expr (2016) 6:105 Page 2 of 10 and Fleet 1985; Mercado et  al. 2007; Querol et  al. 1992; Champagne et  al. 2012). Suitable agents can protect the Schütz and Gafner 1993; Hong and Park 2013). proteins and membranes of the microorganisms (Leslie The Campbell Early grape, which is the most domi - et al. 1995; Champagne and Gardner 2001). nant cultivar in Korea, has a high malic acid content In this study, we aimed to optimize the development of due to early harvesting for enhancing grape color. High Saccharomyces and non-Saccharomyces yeast starters at malic acid content lowers the quality of Korean wine the industrial level using air-blast drying, instead of the due to high acidity, which has resulted in the poor com- conventionally used freeze-drying method, as well as petitive value of indigenously manufactured wine against using various types of excipients and sugars at different imported wine (Kim et al. 1999; Lee et al. 2016). For this concentrations to enhance the survival rate of air dried- reason, isolating and utilizing indigenous yeasts instead yeast cells. Furthermore, the long-term storage proper- of imported yeast starters are necessary to make Korean ties of each dried-yeast strain and the metabolic activity wine competitive. Developing optimal industrial starter of air-blast dried yeast cells during storage at 4  °C were cultures for winemaking is essential for increasing the also investigated. prevalence of indigenous Korean yeast starter products. Previously, S. cerevisiae D8, Hanseniaspora uvarum S6, Materials and methods and Issatchenkia orientalis KMBL5774 were isolated Strains, media, and culture conditions from Korean Campbell Early grape cultivar and their Saccharomyces cerevisiae D8 (KACC 93245P), H. uvarum biological and physiological characteristics were studied. S6 (KACC 93248P) and I. orientalis KMBL5774 (KACC Kim et al. (2013b) reported that wine fermented by S. cer- 93124P) isolated from the Korean grape cultivar were evisiae D8 had higher color and taste scores compared to used in this study (Hong and Park 2013; Kim et al. 2013a; the wine fermented by S. cerevisiae W-3. Hong and Park Seo et  al. 2007). Each strain was cultured at 30  °C with (2013) described that wine fermented by H. uvarum S6 shaking (150  rpm) in sterilized YPD media composed (previously SS6) showed slower fermentation rate but of 1% yeast extract, 2% bacto-peptone, and 2% glucose had higher organic acid content and sensory evaluation and the cells were harvested for making the starters. All scores compared to wine fermented by S. cerevisiae W-3. strains were stored at −70  °C in 20% glycerol until they Seo et al. (2007) and Kim et al. (2008) reported that I. ori- were used for the experiments. entalis KMBL5774 could degrade malic acid during alco- hol fermentation, and co-fermentation with I. orientalis Protective agent conditions KMBL5774 and S. cerevisiae W-3 resulted in better color, Skim milk (5 and 10%) and 7 sugars (5 and 10% of glu- flavor, and taste compared to the fermentation using only cose, fructose, lactose, maltose, raffinose, sucrose, and S. cerevisiae W-3. trehalose) were used to evaluate the survival rate of air- The most important factors for developing microbial blast dried yeast cells. All protective solutions, including starters include maintenance of cell viability, capac- skim milk and sugars were sterilized at 121 °C for 15 min ity for long-term storage and the drying method used. before experiments. Four kinds of excipients—wheat Several studies have utilized freeze-drying (Lodato et  al. flour (CJ Cheiljedang Corp., Seoul, Korea), nuruk (Song - 1999; Ale et al. 2015; Abadias et al. 2001a), fluidized bed hak Agri. Corp., Gwangju, Korea), artichoke powder and drying (Bayrock and Ingledew 1997), and spray dry- lactomil (composed of lactose 89% and maltodextrin 11%; ing (Luna-Solano et  al. 2005; Isono et  al. 1995) to make Seo Kang Dairy & Food Co., LTD, Sacheon, Korea)—were starter products. Freeze-drying is disadvantageous owing used to process the yeast starters into an appropriate to the high expenses incurred, and fluidized bed drying powdered form. Artichoke was obtained from Gim- and spray drying are not suitable due to low viability in cheon, Korea, and it was processed by lyophilization and the starter cultures induced by the high temperature dur- grinding to be converted into powdered form. All excipi- ing drying. In contrast, air-blast drying can lower the cost ents were added at quantities of 2  g (lactomil amounts fivefold, result in comparatively less cell damage, as well ranged from 2 to 8  g) to compare their protective abil- as provide easier control of moisture in the starter com- ity and availability as a starter product in powdered form pared with other drying methods (Santivarangkna et  al. for each strain of the dried yeast. All excipients were used 2007). Even though air-blast drying has many advan- with the yeast pellet directly. tages for making yeast starters, only a few studies related to the air-blast dried yeast have been attempted. Simi- Air‑blast drying process lar to freeze-drying, the selection of protective agents Each yeast strain was cultured in 100 mL YPD broth and is very important in air-blast drying because intracellu- incubated at 30  °C for 16  h. After culturing, yeast cells lar accumulation of the appropriate solutes is related to were harvested by centrifugation (3578×g for 10  min) strain survival following air-blast drying (Kets et al. 1996; and rinsed twice in a 0.85% NaCl solution. The pellet was Lee et al. AMB Expr (2016) 6:105 Page 3 of 10 mixed with 2 g of various excipients such as wheat flour, glucose due to the ethanol fermentation (Jung and Park nuruk, and artichoke powder, and lactomil (2–8 g) as well 2005). Air-blast dried I. orientalis KMBL5774 was incu- as 1 mL protective agent solutions consisting of the skim bated in 10  mL YPD broth containing 2% malic acid at milk and sugars. The mixed yeast cell pellets were dried 30  °C with shaking (150  rpm) to measure the malic acid using Clear Air Oven (HB-509C, HanBaek, Bucheon, decomposition rate. Malic acid content was determined Korea) at 37 °C until the moisture content of dried yeast using the l-Malic Acid Assay Kit (K-LMALR; Mega - starter was <10%. After air-blast drying, the samples were zyme, Wicklow, Ireland) (Lee et al. 2016). immediately analyzed to determine their moisture con- tent and survival rate, then stored at 4  °C for 3  months, Statistical analysis after which their survival rate was determined. All experiments were carried out in at least triplicates and the results were analyzed using the Statistical Pack- Measurement of cell viability and moisture content age for the Social Sciences (SPSS, version 12.0 for Win- After air-blast drying, each sample was reconstituted to dows, Chicago, IL, USA) in order to obtain average and its original volume with distilled water for rehydration. standard deviations. Significance was determined to be Then, the serially diluted samples were spread on YPD p  <  0.05 using one-way analysis of variance (ANOVA), agar plates and incubated at 30  °C for 24  h. The white followed by Duncan’s multiple range test. colonies that formed on YPD agar were counted. The survival rate of each sample was calculated as (%) sur- Results vival  =  (N/N )  ×  100, where N represents the number Eec ff t of skim milk on the survival rate of air‑blast dried −1 of viable cell count after air-blast drying (cfu  mL ) and yeast cells N represents the number of viable cell count before air- Skim milk is typically used as a protective agent to pro- −1 blast drying (cfu  mL ). Moisture content of dried yeast tect cell membrane while drying microbial strains. It has starters was measured by determining the weight loss been suggested that milk proteins may cover the cells to after 10 h at 105 °C (AOAC 1990). prevent damage (Abadias et al. 2001b). In this study, the protective effect of skim milk on air-blast dried cells was Morphology of air‑blast dried yeast cells investigated. For this, 5–10% skim milk solutions were The morphologies of air-blast dried Saccharomyces and mixed with centrifuged yeast cells and the mixed cells non-Saccharomyces yeast cells were observed by scan- were air-blast dried at 37 °C for 2 h until dried cells were ning electron microscopy (SEM), as described by Hong- obtained in the appropriate powdered form. The sur - pattarakere et  al. (2013). The air-blast dried sample was vival rates of all air-blast dried yeast cell strains mixed affixed to “stubs” using double-sided metallic adhesive with skim milk increased in a dose-dependent man- tape and then coated with gold using sputter coater (WI- ner compared to that of the control (Fig.  1). When 10% RES-Coater-001). The morphology of the sample was skim milk was mixed with the dried yeast cells, the viable observed under a SU8220 scanning electron microscope count of S. cerevisiae D8, H. uvarum S6, and I. orienta- (Hitachi, Tokyo, Japan) that was operated at an accelerat- lis KMBL5774 increased to approximately 0.89, 0.71, and −1 ing voltage of 10 kV. Images were obtained under 2000× 1.03 log cfu mL , respectively. Thus, 10% skim milk was magnification. utilized in subsequent experiments. Metabolic activities of air‑blast dried yeast cells Eec ff t of various excipients on the survival rate Metabolic activities of yeast cells stored for 0–3  months and moisture content of air‑blast dried yeast cells after air-blast drying were analyzed and non-dried yeast The selection of the excipient is very important in gen - cells were used as the control. Air-blast dried S. cerevisiae erating a stable powdered form and shape for improving D8 and H. uvarum S6 were incubated in 100  mL YPD the stability and quality of the final product (Georgetti −1 broth containing 20% glucose (yeast extract 10  g  L , et  al. 2008). In this study, four excipients (wheat flour, −1 −1 peptone 20  g  L , and glucose 200  g  L ) at 30  °C with nuruk, artichoke powder, and lactomil) were utilized to shaking (150  rpm) to measure the glucose fermentation prevent cell membrane damage caused by the drying rate. A water trap apparatus containing conc. H SO was environment and the survival rates and moisture con- 2 4 attached to the top of each flask to trap water evaporated tents were determined. All samples were air-blast dried from the flask during the fermentation. The amount of until the moisture content reached  <10%. The air-blast CO produced was directly measured as the decrease dried yeast samples mixed with wheat flour showed the in the weight of the whole flask. The fermentation ratio longest drying time of 7 h and the lowest survival rate of was expressed as the percentage of the amount of CO 0.27–1.39%, whereas the samples mixed with lactomil produced per the theoretical CO production from the showed the shortest drying time of 2.5 h and the highest 2 Lee et al. AMB Expr (2016) 6:105 Page 4 of 10 Fig. 1 Viable yeast cell counts with 5 and 10% skim milk before (filled squares) and after (empty squares) air ‑blast drying for 2 h. All data are expressed as the mean ± SD (n = 3) survival rate of 1.01–3.40% (Table  1). On analysis of the added to 7  g lactomil was measured as 59.12%, which shape of the powdered form obtained by adding various was higher than the survival rate of the sample added excipients, we found that the samples mixed with wheat to 8 g lactomil. In case of H. uvarum S6 and I. orientalis flour and nuruk showed a lump form after air-blast dry - KMBL5774, the survival rates measured for 7–8 g of lac- ing, and the samples mixed with artichoke powder and tomil were not significantly different. Although the dry - lactomil could be easily collected due to their impalpable ing time of the samples added to 8  g lactomil was 0.3  h powdered form (Fig.  2). Based on the survival rate, dry- shorter than that of the samples added to 7  g lactomil, ing time, and the properties of the powdered form of air- after considering the excipient cost and the similar pro- blast dried yeast products, 2 g lactomil was considered as tective effect of 7–8  g of lactomil, 7  g was considered as the most suitable excipient for making yeast starters by the optimal amount of lactomil required for maintain- air-blast drying. Based on the results of the yeast samples ing the viability of air-blast dried yeast cells. Therefore, added to various excipients, the survival rates of air-blast subsequent experiments were carried out by adding 10% dried yeast cells in relation to the amount of lactomil skim milk and 7 g lactomil to the yeast cells, followed by added were investigated (Table  2). As the amount of lac- air-blast drying for 1.5 h. tomil added was increased, drying time for each sample with  <10% moisture content was reduced and the sur- Eec ff t of sugar additives as a protective agent on the vival rate of air-blast dried yeast cells increased in a dose- survival rate of air‑blast dried yeast cells dependent manner until the addition of 7  g lactomil. In To determine the protective effect of sugar on air-blast case of S. cerevisiae D8, the survival rate of the sample dried yeast cells, the survival rate of air-blast dried yeast Table 1 Eec ff ts of various excipients on the survival rate and moisture content of air-blast dried yeasts Excipient S. cerevisiae D8 H. uvarum S6 I. orientalis KMBL5774 Drying time (h) Survival rate Moisture content Survival rate Moisture content Survival rate Moisture content (%) (%) (%) (%) (%) (%) d d c Wheat flour 1.39 ± 0.04 9.53 ± 0.30 0.27 ± 0.04 9.21 ± 0.27 0.30 ± 0.02 9.31 ± 0.20 7 c c c Nuruk 2.23 ± 0.14 9.86 ± 0.23 0.36 ± 0.03 9.94 ± 0.19 0.33 ± 0.03 9.90 ± 0.35 4 b b b Artichoke powder 2.53 ± 0.08 9.89 ± 0.38 0.61 ± 0.04 9.50 ± 0.31 1.07 ± 0.08 9.73 ± 0.23 6 a a a Lactomil 3.40 ± 0.19 9.42 ± 0.12 1.01 ± 0.06 9.23 ± 0.16 1.65 ± 0.33 8.96 ± 0.14 2.5 Different letters within the same column indicate significant difference (p < 0.05) Lee et al. AMB Expr (2016) 6:105 Page 5 of 10 cells (depending on the type and concentration of sugars as protective agents with 10% skim milk) were investi- gated (Table  3). In case of S. cerevisiae D8, the addition of 10% sugars (except for fructose) resulted in a survival rate of  >90%, and addition of 10% trehalose resulted in the highest survival rate of 97.54%. In case of H. uvarum S6, the high survival rates of all samples added to sugars resulted in considerably higher viability than that of sam- ples with no sugar addition. All samples added to 10% sugars had higher survival rate than those added to 5% sugars; in particular, 10% sucrose resulted in the highest survival rate of 92.59%. In case of I. orientalis KMBL5774, most sugar additions did not show a significant increase of the survival Malic acid content was determined rate compared to no sugar addition, but addition of 10% glu- cose and 10% fructose noticeably increased its survival rate to 79.49 and 65.17%, respectively. The morphology of air-blast dried yeast cells was observed using SEM (Fig. 3). The SEM images showed that each yeast cell was Fig. 2 Images of air‑blast dried yeast cells mixed with wheat flour, coated with skim milk, sugar, and lactomil and the cells nuruk, artichoke power, and lactomil. All excipients were added at were densely accumulated, which suggest that protective a concentration of 2 g and air‑blast dried until moisture content of agents and excipients protect yeast cells from the adverse samples was <10% drying environment. Table 2 Eec ff ts of the amount of lactomil added on the survival rate and moisture content of air blast dried yeasts Lactomil (g) S. cerevisiae D8 H. uvarum S6 I. orientalis KMBL5774 Drying time (h) Survival rate Moisture content Survival rate Moisture content Survival rate Moisture content (%) (%) (%) (%) (%) (%) e e c 2 3.40 ± 0.19 9.42 ± 0.12 1.01 ± 0.06 9.23 ± 0.21 1.65 ± 0.33 8.96 ± 0.15 2.5 e e c 3 5.35 ± 0.30 8.37 ± 0.19 2.46 ± 0.59 8.27 ± 0.13 3.11 ± 0.21 7.85 ± 0.11 2.5 d d b 4 32.70 ± 2.37 8.81 ± 0.24 6.33 ± 0.32 9.70 ± 0.17 11.79 ± 0.84 9.30 ± 0.21 2 c c b 5 39.31 ± 3.03 9.52 ± 0.21 9.52 ± 0.42 8.54 ± 0.15 13.66 ± 2.87 8.81 ± 0.13 2 c b b 6 43.40 ± 3.27 9.17 ± 0.18 17.18 ± 1.25 9.68 ± 0.29 16.33 ± 2.86 7.23 ± 0.09 2 a a a 7 59.12 ± 1.96 8.88 ± 0.24 29.65 ± 1.77 8.34 ± 0.14 23.71 ± 3.38 8.48 ± 0.16 1.5 b a a 8 53.45 ± 4.75 9.58 ± 0.31 29.60 ± 3.16 9.84 ± 0.32 23.68 ± 3.42 9.37 ± 0.22 1.2 Different letters within the same column indicate significant difference (p < 0.05) Table 3 Eec ff ts of the type and concentration of various sugars on the survival rate of air-blast dried yeasts Strains Conc. Survival rate (%) (%) Glucose Fructose Lactose Maltose Raffinose Sucrose Trehalose a b a a a a a S. cerevisiae D8 5 95.07 ± 8.45 64.31 ± 7.62 84.00 ± 12.17 88.80 ± 6.59 93.39 ± 15.32 94.38 ± 11.68 96.13 ± 5.94 a ab a a a a a 10 94.33 ± 6.66 74.28 ± 7.07 91.20 ± 9.53 94.03 ± 5.13 94.52 ± 10.46 93.73 ± 8.74 97.54 ± 6.77 b b ab b b ab ab H. uvarum S6 5 63.99 ± 5.81 67.67 ± 1.06 72.56 ± 8.75 67.20 ± 4.07 68.54 ± 2.35 69.66 ± 6.76 69.77 ± 1.55 ab ab ab ab ab a ab 10 71.71 ± 4.78 75.59 ± 11.05 85.81 ± 4.98 78.76 ± 17.82 84.68 ± 9.30 92.59 ± 11.17 73.38 ± 9.18 cd cd cd cd cd cd cd I. orientalis KMBL5774 5 41.89 ± 3.58 39.68 ± 5.99 43.04 ± 9.56 39.74 ± 4.62 41.03 ± 8.97 40.79 ± 2.63 44.30 ± 11.04 a b d bc d d cd 10 79.49 ± 9.25 65.17 ± 5.15 36.71 ± 4.56 54.17 ± 8.46 33.33 ± 4.62 37.78 ± 3.39 45.93 ± 6.06 Different letters within the same strains indicate significant difference (p < 0.05) Lee et al. AMB Expr (2016) 6:105 Page 6 of 10 Fig. 3 Images of air‑blast dried yeast cells observed by a scanning electron microscope (SEM) at ×2000 magnification. 10% skim milk and 7 g lacto ‑ mil were used in all samples. Ten percent trehalose, 10% sucrose, and 10% glucose were used as protective agents for S. cerevisiae D8 (a), H. uvarum S6 (b), and I. orientalis KMBL5774 (c), respectively Long‑term storability of air‑blast dried yeast starter samples showed similar fermentability on the second day products and completed the fermentation process on third day. Changes in the survival rate and viable count of air-blast Similarly, both air-blast dried H. uvarum S6 just after dried yeast cells were investigated in products that had drying and after 3 months of storage showed similar fer- been stored at 4  °C for 3  months (Fig.  4). All samples mentation rates compared to non-dried H. uvarum S6. were prepared based on the optimal conditions deter- Non-dried I. orientalis KMBL5774 initiated and com- mined in the present study. Air-blast dried S. cerevisiae pleted malic acid decomposition first, but the duration D8 and H. uvarum S6 continued to show a high survival of malic acid decomposition was not significantly dif - rate of 42.24 and 49.74% after 2  months, whereas the ferent from that observed for air-blast dried I. orientalis survival rate of air-blast dried I. orientalis KMBL5774 KMBL5774 immediately after drying. In case of air-blast rapidly decreased compared to the other yeast strains dried I. orientalis KMBL5774 after 3  months of storage and showed a survival rate of only 3.08% after 2 months at 4 °C, malic acid degradation was delayed by 12 h com- of storage. After 3  months, the viable count of S. cer- pared to that of the control. The results of the metabolic evisiae D8 and H. uvarum S6 decreased to 1.18 and 0.51 activities of air-blast dried yeast cells after 3  months of −1 log cfu  mL compared to the viable counts measured storage suggest that S. cerevisiae D8 and H. uvarum S6 immediately after air-blast drying. Therefore, air-blast retained their capacities and efficiencies as yeast starters, dried yeast cells of both strains, S. cerevisiae D8 and H. whereas reduction of the survival rate after long-term uvarum S6, have excellent potential as a starter product. storage possibly induced the decrease in the malic acid On the other hand, although I. orientalis KMBL5774 also degradation rate in I. orientalis KMBL5774. showed high survival rate immediately after air-blast dry- ing, further study on the long-term storage of I. orientalis Discussion KMBL5774 is necessary because it showed very low via- In this study, air-blast drying was established as a suit- ble count after 3 months of storage (2.38 log reduction). able substitute to conventional drying methods, such as freeze-drying or spray-drying for manufacturing yeast Changes in metabolic activities of air‑blast dried yeast cells starters for wine. Skim milk, generally used as a protec- The metabolic activities of each air-blast dried yeast tive agent in freeze-drying, was added at 5 and 10% to cell were investigated depending on the storage period air-blast dried yeast cells and the addition resulted in the (Fig.  5). Glucose fermentation ability of air-blast dried increase in the survival rate of air-blast dried yeast cells S. cerevisiae D8 and H. uvarum S6, and malic acid in a dose-dependent manner (Fig. 1). Ananta et al. (2005) decomposition ability of I. orientalis KMBL5774 were reported that when 20% reconstituted skim milk added to examined. All samples were prepared by the optimal Lactobacillus rhamnosus GG was spray dried at an outlet manufacturing process based on results obtained in the temperature of 80  °C, its survival rate was measured to present study and non-dried yeast cultures were used as be >60%. Abadias et al. (2001a) reported that freeze-dried control to compare the metabolic activity. Non-dried S. Candida sake showed the highest survival rate of 40% cerevisiae D8 decomposed glucose slightly faster com- when 10% skim milk and 10% lactose were added as pro- pared to both air-blast dried S. cerevisiae D8 immediately tective agents. Similarly, our study also showed that skim after drying and after 3  months of storage. However, all milk had a protective effect on air-blast dried yeast cells. Lee et al. AMB Expr (2016) 6:105 Page 7 of 10 Fig. 4 Changes in the survival rate (left panel) and viable count (right panel) of air‑blast dried yeast cells stored at 4 °C for 3 months. Empty circles, filled squares, and filled diamonds in the left panel represent the survival rates of S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respec‑ tively. Bars in the histogram (right panel) represent viable counts of yeast cells before (black) and after (gray) air‑blast drying and yeasts stored for 1 (white), 2 (diagonally patterned), and 3 (dotted) months. 10% skim milk and 7 g lactomil were used in all samples. 10% trehalose, 10% sucrose, and 10% glucose were used as protective agents for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. All data are expressed as the mean ± SD (n = 3) Fig. 5 Fermentation rate of air‑blast dried S. cerevisiae D8 (diamonds) and H. uvarum S6 (circles) (a) and malic acid content fermented by air ‑blast dried I. orientalis KMBL5774 (squares) (b). Empty figures represent non‑ dried yeast cells, filled figures represent air ‑blast dried yeast cells just after air ‑ blast drying, and gray figures represent air ‑blast dried yeast cells after 3 months of storage. All data are expressed as the mean ± SD (n = 3) Four excipients (wheat flour, nuruk, artichoke pow - yeast starter (Table  1). According to a study by Beker der, and lactomil) were evaluated to make an appropri- and Rapoport (1987), 12–13% moisture content is not ate powdered form of the final product of the air-dried suitable for storage of yeast, whereas at 8–10% moisture Lee et al. AMB Expr (2016) 6:105 Page 8 of 10 content, yeast retain a remarkable degree of cell viability SEM to confirm the accumulation of yeast cells (Fig.  3). during storage. Therefore, all samples were air-blast dried Pereira et al. (2003) reported that 10% trehalose was able until moisture content of each sample was  <10%. Dry- to reduce oxidative damage caused by dehydration in S. ing times for samples with the excipients wheat flour and cerevisiae and Garay-Arroyo et  al. (2004) reported that artichoke were relatively longer than those observed for S. cerevisiae could be easily adapted to various environ- the other excipients. This result could be attributed to the mental stresses, including oxidative stress, heat shock, water or moisture in these excipients, which could lead to freezing shock, osmotic, and ionic stress. A study by increased viscosity due to starch gelatinization and bind- Lemetais et  al. (2012) on S. cerevisiae showed that the ing to the gluten network in wheat flour (Fessas and Schi - plasma membrane is an essential structure for the sur- raldi 2001), and to the high dietary fiber content in the vival of cells during dehydration by air-drying. In a study artichoke (insoluble 18.11% and soluble 26.74%), which on the survival rate of H. uvarum lyophilized without could interact with the water held in the capillary struc- cryoprotectant and stored at −80  °C for 0–12  months, ture of the artichoke (Lintas and Capeloni 1988; López the viable counts showed a reduction of 2.47–2.82 log −1 et al. 1996). Nuruk, a Korean traditional starter prepared cfu  mL from the values recorded before freeze-dry- by the natural proliferation of fungi and other microor- ing (Pietrowski et al. 2015). A study by Kim et al. (2016) ganisms (Yoo et al. 2011), was also investigated as excipi- reported that S. cerevisiae D8, H. uvarum S6, and I. ori- ent for yeast starter. Although drying time for nuruk entalis KMBL5774 entrapped in 2% Ca-alginate beads by was shorter than wheat flour and artichoke, the physical air-blast drying showed 90.67, 90.81, and 87.04% survival properties of nuruk as well as that of wheat flour were not rate when 10% skim milk and 10% sugars (sucrose, treha- suitable for preparing the final starter product because lose, and glucose for S. cerevisiae D8, H. uvarum S6 and they led to the formation of lumps after air-blast drying I. orientalis KMBL5774, respectively) were used as pro- (Fig.  2). On the contrary, lactomil (consisting of lactose tectants. Miyamoto-Shinohara et al. (2010) reported that and maltodextrin) was considered as the most suitable I. orientalis had an 8.6 and 28.2% survival rate on freeze- excipient for yeast starter because it yielded a rapid dry- drying and liquid drying, respectively, without any pro- ing time of 2.5  h and the highest yeast cell survival rate tective agent. and formed a fine powder. Furthermore, the survival Long-term storage is the most important factor in rates of air-blast dried yeast cells, based on the amount of developing microbial starters for industrial use. In our lactomil added, were investigated and all samples mixed study, long-term storage effect of air-blast drying on each with 7  g lactomil statistically showed the highest and yeast strain stored at 4 °C for 3 months was investigated optimal survival rate (Table 2). (Fig. 4). Air-blast dried S. cerevisiae D8 and H. uvarum S6 Sugars have been widely used as protective agents due showed 1.18 and 0.51 log reductions, which means that to their low price, chemically innocuous nature, and gen- these strains retained a good viable count after 3 months eral utilization in the food industry (Peighambardoust of storage. However, since I. orientalis KMBL5774 et  al. 2011). The protective effects of various sugars on showed a 2.38 log reduction after 3  months of storage, the survival rate of microbial starters such as yeast and further study would be needed to improve its storability. bacteria have been determined in the last few decades Several studies have reported storability based on various (Jofré et al. 2014; Lodato et al. 1999; Niu et al. 2016). Our drying method and protectants. A study by Miyamoto- study showed that each strain showed its highest capac- Shinohara et  al. (2006) reported that freeze-dried S. cer- ity for survival with different optimal protective agents evisiae showed 0.010 log reduction per year for 20 years. (Table 3). S. cerevisiae D8 added to 10% trehalose showed Zayed and Roos (2004) demonstrated that 4% sucrose, the highest survival rate of 97.54% and most sugar- 4% trehalose, and 18% skim milk, used as protective based protectants (except for fructose) demonstrated solutions for freeze-dried Lactobacillus salivarius main- excellent protective effects with  >90% survival rate. H. tained the survival rate at 83–85% for 7 weeks of storage. uvarum S6 showed the highest survival rate (92.59%) In a study by Gardiner et  al. (2000), the survival rate of when 10% sucrose was added as a protectant and other spray-dried Lactobacillus paracasei NFBC 338 grown in sugars were also shown to demonstrate notable protec- 20% reconstituted skim milk was maintained at constant 9 −1 tive effects compared to no sugar addition. Contrary to at  ~1  ×  10 cfu  g during 2  months of storage at 4  °C, S. cerevisiae D8 and H. uvarum S6, only 10% glucose and while storage of L. salivarius UCC 118 under the same 10% fructose remarkably increased the survival rate of conditions showed 1 log reduction (from 7.2  ×  10 to 6 −1 I. orientalis KMBL5774, whereas other sugars demon- 9.5 × 10 cfu g ). strated protective effects that only slightly increased the Fermentation rates of air-blast dried S. cerevisiae D8 survival rate of I. orientalis KMBL5774. The morphol - and H. uvarum S6 and the malic acid decomposition ogy of air-blast dried yeast starters was also observed by ability of air-blast dried I. orientalis KMBL5774 after Lee et al. AMB Expr (2016) 6:105 Page 9 of 10 References 3  months of storage were analyzed to evaluate their Abadias M, Benabarre A, Teixidó N, Usall J, Viñas I (2001a) Eec ff t of freeze drying metabolic capacities (Fig.  5). 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Appl Environ Microbiol 66:2605–2612 Georgetti SR, Casagrande R, Souza CRF, Oliveira WP, Fonseca MJV (2008) Spray Author details drying of the soybean extract: effects on chemical properties and antioxi‑ School of Food Science and Biotechnology, Kyungpook National University, dant activity. LWT‑Food Sci Technol 41:1521–1527 80 Daehakro, Daegu 41566, South Korea. Department of Fermentation Heard GM, Fleet GH (1985) Growth of natural yeast flora during the fermenta‑ Biotechnology, Kyungpook National University, 80 Daehakro, Daegu 41566, tion of inoculated wines. Appl Environ Microbiol 50:727–728 South Korea. Fermented Food Science Division, Depertment of Agro‑food Hong YA, Park HD (2013) Role of non‑Saccharomyces yeasts in Korean wines Resources, NIAS, RDA, 166, Nongsaengmyeong‑ro, Iseo‑myeon, Wanju Gun, produced from Campbell Early grapes: potential use of Hanseniaspora Jeollabuk‑do 55365, South Korea. uvarum as a starter culture. Food Microbiol 34:207–214 Hongpattarakere T, Rattanaubon P, Buntin N (2013) Improvement of freeze‑ Acknowledgements dried Lactobacillus plantarum survival using water extracts and crude This study was supported by the Rural Development Administration, Republic fibers from food crops. Food Bioprocess Tech 6:1885–1896 of Korea (Research Grant PJ012425022016). Isono Y, Araya G, Hoshino A (1995) Immoblization of Saccharomyces cerevisiae for ethanol fermentation on γ‑alumina particles using a spray‑ dryer. Competing interests Process Biochem 30:743–746 The authors declare that they have no competing interests. Jofré A, Aymerich T, Garriga M (2014) Impact of different cryoprotectants on the survival rate of freeze‑ dried Lactobacillus rhamnosus and Lactobacillus Ethics approval casei/paracasei during long‑term storage. 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AMB Express – Springer Journals

Published: Nov 8, 2016

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