P-10 Effects of composition in longan must on the growth rates, cell biomass, and fermentation of wine yeasts Ni-orn Chomsri 1, Thirawan Chanrittisen 1, Pattharaporn Srisamatthakarn 1, Carola Schmitz 2 and Wasan Jaokondee 1 1 Food Science and Technology Department, Lampang Agricultural Research and Training Centre, 202 Pichai District, Amphur Muang, Lampang 52000, Thailand. 2 Food Science and Technology Department, Postbus 9101, 6700 HB Wageningen, Netherlands Abstract Two strains of wine yeasts species, Saccharomyces cerevisiae HK- 4 and S. bayanus EC 1118, were examined for growth rates and maximum cell biomass during fermentation of longan juice which prepared by different methods. Growth rate and maximum cell biomass of two strains increased in the early stage of fermentation. S. bayanus EC 1118 could grow faster than S. cerevisiae HK- 4, there by explaining it s early development in longan wine fermentations. The different longan must significantly affected the growth rate or cell biomass of the yeasts. S. bayanus EC 1118 gave rapid fermentation. At the end of fermentatation, the alcohol content, total soluble solid, and acidity as citric acid were 6.30-1060 %, 5.50-10.40 o Brix, and 3.9-10.3 g l -1, respectively.
Introduction Yeasts are responsible for the alcoholic fermentation of grape juice into wine. During fermentation they utilize sugars and other constituents of juice as substrates for their growth, converting these to ethanol, carbon dioxide and other metabolic end-products the contribute to the chemical composition and sensory quality of the wine. Obviously, some factors strongly affect alcoholic fermentation, and as a consequence, the quality of the wine [4,5,7]. The most important ones are the levels of the sulphur dioxide, the temperature of fermentation, the composition of fruit juice, inoculation with selected yeasts and the interaction with other microorganisms. One of these factors, the composition of must, directly affects the microbial reactions of the yeasts [4]. Research over the past 100 years has demonstrated that Saccharomyces cerevisiae is the principal species that dominates wine fermentation. Consequently, most fundamental research aimed at understanding the growth and metabolic activities of yeasts throughout fermentation have been concerned with S. cerevisiae. Although S. cerevisiae develops naturally in wine fermentation, many winemakers now inoculate selected strains of this species into the grape juice to encourage its rapid growth and predominance [5,9,10,16,17]. In Thailand, there are many alcoholic beverages which made by many kinds of Thai fruits. Considering Thai fruit wine industry presently began as SME, various fruits have been used for making fruit wine such as pineapple, longan, mulbery, mangosteen, and Ma-mao. Research related fruit wine over many years has mainly concerned in wine-making techniques with the introduction of processes. However, more developments of fruit wine production in Thailand is strongly needed to improve the quality of the fruit wines, these includes the preparation and treatment of the must, fermentation, aging, bottling, enzymic treatments, selected yeasts, and improvement of microbial starters. The aim of this paper is therefore to investigate the effect of different composition of longan wine must on the growth of Saccharomyces strains and how they interact during alcoholic fermentations.
Materials and methods Yeast strains The strain of Saccharomyces cerevisiae HK- 4 and S. bayanus EC 1118 came from Lallemand Inc. Fermentation experiments The medium was made from longan ; whole longan (WL), longan flesh (LF), longan with seed (LWS), longan without seed (LOS), that resulted four different compositions of longan must which was adjusted to obtain a final sugar concentration of 180 g l -1. Total acidity was adjusted to 5 g l -1 with citric acid. One hundred and fifty mg l -1 of potassium metabisulphite was used for killing wild yeast. Fermentations were conducted in 4 litre bottles filled with 3 litre of media and covered with a cotton cap. Each strain of yeast was inoculated in the bottle at the initial population of 1x10-5 cfu ml -1. Fermentations were incubated at 20+2 o C. Samples of the fermenting medium were taken out every 24 hours during the first eight days and every 72 hours thereafter for the investigation of yeast growth. The yeast growth was monitored by counting the viable yeast. The viable yeasts were determined by plating them on YM agar at an adequate dilution. Cell biomass was also determined as cell dry weight. For each strain, a calibration curve of cell dry weight against absorbance (610 nm) was constructed. Dry weights were determined by centrifuging at 10,000xg for 20 min. (Sorval Super TT21). Cell biomass retained by the centrifuge was washed three times with 10 ml of distilled water, then dried at 60 o C for 24 hours to constant weight. All fermentation were performed in duplicate without shaking. Maximal fermentation rate was the maximum slope obtained from the representation of alcohol production towards fermentation day and expressed as concentration of produced alcohol (in % v v -1 ) per day. Chemical analysis Total soulble solid and Ethanol was analysed by hand refractometer and Ebulliometer, respectively. Titratable acidity was analysed by titration with 0.1 N sodium hydroxide solution and phenolphthalein as the indicator [1].
Results Effect of composition in longan musts on yeast growth The effect of longan composition in longan must with different preparations on the yeast growth is shown in Figure 1. During fermentation, the viable population of yeasts in all treatments increased from initial values of 10 5 cfu ml -1 to 10 9 cfu ml -1 and the growth rate of two yeast strains increased in early stage of fermentation. Yeast cell proliferated during ten days of longan must fermentation in all treatments as well. Exponential phase of fermentation is relatively short comparing with the stationary-decline phases significantly. S. cerevisiae HK- 4 exhibited faster growth than S. bayanus EC 1118 as shown in growth curves. Yeast count (log 10 cfu ml -1 ) 10 8 6 4 2 0 0 10 20 EC 1118 HK-4 Yeast count (log 10 cfu ml -1 ) 10 8 6 4 2 0 0 10 20 EC 1118 HK-4 Fermentation time (days) a Fermentation time (days) b Yeast count (log 10 cfu ml -1 ) 10 8 6 4 2 0 0 10 20 Fermentation time (days) c EC 1118 HK-4 Yeast count (log 10 cfu ml -1 ) 10 8 6 4 2 0 0 10 20 Fermentation time (days) d EC 1118 HK-4
Figure 1 Growth of yeast during fermentation of different longan must preparations. a) whole longan b) longan flesh c) longan with seed and d) longan without seed Effect of composition in longan must on specific growth rate(µ) and maximum cell biomass. To facilitate comparison, specific growth rate (µ) and maximum cell biomass data are expressed in Table 2. The highest specific growth rate of both S. cerevisiae HK- 4 and S. bayanus EC 1118 was occurred during 48 hours of fermentation for all treatments except the one which was the whole longan must. Maximum cell population was formed within an early kinetic stage. The maximum cell biomass varied with the fermentation since it was influenced by the composition of longan juice and yeast strains. This also affected to kinetics of sugar utilization and ethanol production by two species of wine yeast during fermentation of longan must (data not shown). The highest specific growth rate and the highest maximum cell biomass were formed in the must of longan without seed which was inoculated by S. bayanus EC 1118. Table 1 Specific growth rates (µ) and maximum cell biomass of yeasts during fermentation at 20 O C of different longan must preparations Longan preparation Specific growth rate (h 1 ) Maximum cell biomass (g l -1 ) EC 1118 HK-4 EC 1118 HK-4 Whole longan 0.51 0.18 22.67 24.12 Longan flesh 0.21 0.21 0.67 1.60 Longan with seed 0.67 0.23 9.10 0.28 Longan without seed 1.32 0.25 32.38 0.48
Effect of composition in longan must and yeast varieties on wine quality The effect of the composition in longan must and yeast varieties is presented in Table 2. Table 2 Chemical analysis of wine quality Longan preparation Alcohol (%) Total soluble solid ( O brix) Total acidity (%) EC 1118 HK-4 EC 1118 HK-4 EC 1118 HK-4 Whole longan 10.60 10.40 5.50 5.70 0.64 0.67 Longan flesh 8.20 6.80 9.20 10.40 0.96 1.03 Longan with seed 8.20 6.30 7.60 9.60 0.76 0.96 Longan without seed 10.60 10.50 6.00 6.00 0.39 0.46 Discussion The concept of inoculating juice with selective starter cultures of S. cerevisiae to encourage rapid and consistent fermentations has become widely accepted in the wine industry[5,6,16,17]. This technology comes the general assumption that the inoculated strain of S. cerevisiae would dominate the fermentation and overwhelm any contribution from indigenous yeasts which was accepted by Thai fruit wine industry. The different composition in longan juice may affect the yeast growth of wine fermentation was investigated as the assumption. The experiments were examined the growth kinetics using S. cerevisiae HK- 4 and S. bayanus EC 1118. They were monitored by measurement of viable yeast counts, cell dry weight, microscopic (total) yeast count, ethanol production and fermentable sugar utilization. S. cerevisiae EC 1118 was the dominant species and it also grew well in all longan juice. The growth curve indicated that the S. cerevisiae HK- 4 strain used in this study was slower growing than S. bayanus EC 1118. This would suggested that S. cerevisiae EC 1118
had better ability of sugar utilization for alcohol producing than S. cerevisiae HK- 4. However, chemical analysis during fermentation showed that the large amount of ethanol was produced at the stationary phase cell. Ethanol production was a major reason for the lowering of the yeast cells [3,5,11]. The higher the alcohol production, the lower the amount of yeast cells. It may indicate that S. cerevisiae HK- 4 delayed the onset of fermentation and increased the time to complete fermentation. This is because of the yeast strain could have utilized sugar or produced ethanol at a slow rate and caused a delay of fermentation. As the expected, the yeast growth varied according to the composition of longan must. Various authors have mentioned that the main variables which affect yeast growth might be supplying of nitrogenous substrates, presence of adequate vitamins, concentration of dissolved oxygen, and concentration of insoluble solids. Other factors that required consideration was fungicide residues from longans and yeast inhibitory or stimulatory substances produced by the growth of fungi or bacteria on the longan prior to harvest. Such variables could delay the onset of yeast growth, decrease the rate of growth, decrease the yield of growth and accelerate the death of yeast cell after growing ceased [4,5,6,13,14]. For different maximum cell biomass of two yeast strains, it was not able to conclude that it has a significant growth advantage over the other strain. On the other hand, S. bayanus EC 1118 exhibited higher specific growth rate than S. cerevisiae HK- 4 which represented that S. bayanus EC 1118 had better dynamic behavior and gave faster growth rate of yeast during fermentation than S. cerevisiae HK- 4 [2,8,15].. Different method of longan juice preparations appeared to be a major factor of alcohol production, sugar metabolism and acidity content in longan wine. In addition, it seemed that the juice prepared by longan flesh and longan with seed were not appropriate for wine fermentation because they had high acidity in end products. It was a possibility that both yeast species could produce less alcohol in the juice (6.30-8.20%) than the general wine fermentation. This also caused high sugar left in longan wine shown as TSS in Table 2. Moreover, the longan wine which made by both of the preparations had high acidity which may implied the contamination of other microorganism producing acid [12]. It also revealed that S. bayanus EC 1118 could not completely metabolize sugar and convert to ethanol in both juice.
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