Influence of packing materials and temperature on yeast activity

Romanian Biotechnological Letters Vol. 19, No. 4, 214 Copyright 214 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Abstract Influence of packing materials and temperature on yeast activity Received for publication, February 2, 214 Accepted, June2, 214 SONIA AMARIEI*, SORINA ROPCIUC*, GHEORGHE GUTT, MIRCEA OROIAN Faculty of Food Engineering, Stefan cel Mare University of Suceava, Romania *Address correspondence to: Stefan cel Mare University of Suceava, Faculty of Food Engineering, 13 Universitatii Street, 72229, Suceava, Romania. Tel.: +423216147; Fax: +423523267; Email: gutt_sonia@yahoo.com, sorinaropciuc@yahoo.com A commercial form of baking yeast Saccharomyces cerevisiae, was analyzed in terms of the influence of temperature and packing materials during storage. Yeast activity at different temperatures, from +3 ± -3 ºC, 22-24 C and up to 3 C over a period of 5 days was determined. As packing materials there have been studied: polyethylene, cellophane, OPP (oriented polypropylene) and aluminum, materials that can influence the fermentation activity of the yeast during storage. Results obtained showed that yeast stored at low temperatures (-1ºC) mantains the best yeast fermentative activity. A strongly significant correlation between the variables of fermentative activity and packing material with the correlation coefficient r =.996 is observed at individually packed yeast (cellophane foil). Packing materials have been analyzed in terms of water absorption, thickness and specific mass. Statistical correlations calculated by the Pearson matrix between the variables of yeast activity and packing material indicate that the cellophane mantains the fermentative activity at its best value (r =.996). Keywords:Saccharomyces cerevisiae, packing materials, fermentative activity. 1. Introduction Physical and chemical transformations of packed foods, the processes that occur throughout the entire period from packing to consumption are: solidification by drying, crystallization, formation of hydrates, autooxidation, non-enzymatic browning reactions, enzymatic transformations. These transformations that generate changes in organoleptic and toxicological aspect are closely related to packing and materials characteristics used in their manufacture. In commerce yeast can be found in several different forms: compressed yeast (fresh), active dry yeast, protected active dry yeast and instant dry yeast. Choosing a specific commercial form of yeast in order to obtain a quality bread is done by taking into consideration the quality of the yeast. In order to obtain the biomass extremely useful in backing industry, the yeast Saccharomyces cerevisiae is grown on carefully selected environments with the help of a complex of physical-chemical, biochemical, microbiological and thermoenergetic processes (1). The nutritional requirements of baking yeast are: carbohydrates, nitrogen, phosphorous, minerals, bio-stimulating substances, temperature of 3...35 C, the ph in the weakly acidic range and the absence of contamination substances. The yeast Saccharomyces cerevisiae is capable of fermenting even under anaerobic conditions but with an excess of fermentable carbohydrates (1-3). All strains of Saccharomyces cerevisiae can metabolize glucose, maltose and trehalose. Romanian Biotechnological Letters, Vol. 19, No. 4, 214 9475

Influence of packing materials and temperature on yeast activity Among extrinsic factors, temperature is one of the important physical parameters in the optimization of the process. Changes in temperature affect the yield of conversion of the substrate into the desired product, on the nutritional requirements of yeast, on obtained biomass composition and on growth rate. The yeast Saccharomyces cerevisiae belongs to the mesophyll group, the optimum temperature is between 26ºC and 36ºC. At a temperature higher than 4 C, baking yeast does not multiply, and therefore the movement with a few degrees around the optimum of growth temperature not only affects the yield in resulted biomass and growth rate, but also the biochemical composition of yeast cell. Yeast cells can withstand very low temperatures to near absolute zero, surviving cold easily in a dry than in a wet environment. From experiments, it was observed that by lowering the temperature below C there is a reduction in the rate of metabolism. A decrease with 1ºC under the minimum temperature (-3 C) leads to a 5% decreasing in the rate of metabolism of nutrients, due to the folding of protein chains and masking active centers of enzymes so that they do not connect with the substrate and do not function as biocatalysts. At low temperatures, yeast losses intracellular water, and go into dormant life when metabolism is very slow, remaining viable for a long time (4-7). Water is important for yeast cell not only because it is the main constituent in quantitative terms, but it also represents about 8% of the living cell. It performs the functions of solvent for intracellular metabolites, maintaining cell shape and size. Yeast requires for normal growth an amount of free water to ensure a good transfer of nutrients into the cell. Aeration of the environment is important to provide continuously the yeast cell with oxygen, to remove the formed carbon dioxide that has an inhibitory effect on the process of reproduction, fast transport to the cells of added nutrients and maintainance of cells in a state of suspension. 2. Materials and methods The purpose of this study is to determine through physico-chemical analyses the yeast behavior in various types of packing materials throughout the validity term and the temperature influence during storage. For the determination it was taken a sample of compressed yeast resulted from the same batch with different grammage and packed in different materials. Thus, the following yeast samples were taken for analysis: -.5 kg yeast package, individually wrapped in OPP foil and then paraffin paper, stored in 1 kg carton boxes; -.5 kg yeast package, individually wrapped in sufite paper and then wrapped 5 pieces each in cellophane foil, forming 2.5 kg packets; -.5 kg yeast package, individually wrapped in sulfit paper and polyethylene foil, stored in 1 kg carton boxes; -.25 kg yeast package, wrapped in aluminum foil, waxed on the inside, then wrapped 8 packets in cellophane foil, with a weight of 2.4 kg. Physicochemical analyses were performed on the individually and in boxes wrapped yeast packages, with the amount of 1 kg, at different time intervals starting with the packing moment and up to 5 days from packing, thus determining the yeast activity and yeast cell vitality. 9476 Romanian Biotechnological Letters, Vol. 19, No. 4, 214

SONIA AMARIEI, SORINA ROPCIUC, GHEORGHE GUTT, MIRCEA OROIAN Yeast samples were analyzed also in terms of behavior at different temperatures: +3 ± -3ºC, 22-24ºC and 3ºC at the age of, 7, 14, 28, 36 and 5 days. The method of analysis used to determine the capacity of the yeast leavening fermentation after an hour, respectivelly two hours after the packaging was removed is done with a fermentograph. By reading fermentograms, fermentation capacity can be appreciated, known as yeast activity that is the total amount of released carbon dioxide, and also the duration of its fermentation, i.e. the time in which is obtained the optimum of the fermentation. The result was expressed in mg CO2/l. The influence of packing material on the capacity of yeast fermentation and the outer packaging of packaged yeast were studied. Also the influence of the packaging position, indoor, outdoor, in a row, centrum was the subject of determinations. The analyzed samples consisted of packets from the box, Cext and Cint, i.e. an external package and a package inside the string respectively, usually the third package in the range of five packages; in the packages in the outer cellophane, Text and Tint, with the same meanings as those from the box. Analyses were performed every four days in the case of the samples stored at temperatures above 22-24 C, respectively 3 C due to high temperature and rapid changes. In the case of the samples stored at +3 ± -3 C the analyses were performed once a week due to optimum storage conditions. 3. Results and discussions The thermal regime applied to compressed yeast during storage and transport period influences its conservability. Refrigeration leads to slowing down metabolic processes and the extension of shelf life. Packing materials were analyzed in terms of water permeability, water absorption, specific weight and thickness as in Table 1. Table 1. Characteristics of packing materials Material Water permeability Water absorption 24h % Specific weight g/m 2 Thickness m OPP very low.6.3 17 ± 2 2 ± 2 Cellophane high 19 33.5 23.3 Polyethylene impermeable <.15 21.6 - Paraffin paper impermeable - 53.2 ± 5 55 ± 5 Paraffined aluminum paper impermeable - 69 65 ± 5 The data on packing materials reveals different features presented by the four types of packing. Regarding the water absorption, cellophane has the highest value of 19% by comparison with the other materials that are impermeable or with a very low permeability. This property of cellophane can provide breathing of yeast, and facilitate the removal of water from the Romanian Biotechnological Letters, Vol. 19, No. 4, 214 9477

Influence of packing materials and temperature on yeast activity product, helping the same time to the maintaining the vital functions for a longer period of time. Critical fermentative activity is recorded according to graphic expression in individual packages wrapped in OPP. The activity at every two hours at a temperature of 3 C and the yeast age for 12 days (V12 ) is of 48 mg CO2, the yeast vitality indicates the lowest value. The best fermentative activity stands in packages inside the box (125 mg CO2). Table 2.Yeast fermentative activity in OPP packing V V7( 3) V14 V21 V28 V36 V5 V( Results Act1_OP PCext 78 8 75 76 76 73 7 78 73 53 24 78 65 13-1 Act1_OP PCint 78 77 76 73 73 72 69 78 71 68 19 78 51 1-12 Act1_OP P.pac 78 78 75 74 72 75 66 12 76 6 3 78 73 27 49 25 Act2_OP Pext 12 11 113 114 11 12 111 89 44 12 18 - - 23 Act2_OP Pint 12 125 122 16 11 14 12 116 16-12 89 - - 48 Act2_OP Ppac 12 12 117 119 112 15 12 12 1 52 12 12 62 - - Cext = package outside the box; Cint = package inside the box (the third in a row of five packs); ext = package outside the foil; int = package inside the foil (the third in a row of five packs); pac. = individual yeast package, stored at the studied temperature. Table 3. Yeast fermentative activity in cellophane V5( V9( V13( V( 3) V4( 3) V8( 3) V12( 3) V12( 3) Results V( 3) V7( 3) V14 V21 V28 V36 V5 V( V5( V9( V13( V17( V( 3) V4( 3) V8( 3) V23( Act1_CE. Cext 78 77 77 77 77 74 7 78 77 73 44 42 78 75 39 11 Act1_CE. Cint 78 76 76 74 73 7 72 78 77 46 44 42 78 77 43 11 Act1_CE. Text 78 77 77 78 76 74 8 78 81 8 7 38 78 8 76 - Act1_CE. Tint 78 75 8 77 75 76 76 78 81 79 7 37 78 8 74 - Act1_CE. P.pac 78 8 8 78 8 8 77 78 88 75 44 7 78 85 59 - Act2_CE. Cext 12 125 12 123 12 11 11 12 121 12 74 66 12 123 72 - Act2_CE. Cint 12 116 117 113 15 114 12 12 82 77 69 12 125 74 - Act2_CE. Text 12 12 12 111 12 12 121 123 113 67 12 127 121 - Act2_CE. Tint 12 12 124 19 12 124 125 114 7 12 124 119-9478 Romanian Biotechnological Letters, Vol. 19, No. 4, 214

SONIA AMARIEI, SORINA ROPCIUC, GHEORGHE GUTT, MIRCEA OROIAN Act2_CE pppac 12 119 121 12 12 116 12 13 12 7-12 125 97 - The yeast activity by using cellophane as packing material indicates the highest fermentative activity in yeast aged 5 days at 22 C (121 mg CO2). The lowest fermentative activity was recorded at 23 days after production and stored at 22 C in individual packages (V23(). Table 4. Yeast fermentative activity in polyethylene V V7 V14 V21 V28 V36 V5 V ( Results Act1_PE Cext 78 77 76 72 73 72 69 78 73 35 1-78 66 2 - Act1_PE. Cint 78 76 75 72 74 72 75 78 65 28 1-78 5 19 - Act1_PE Ppac 78 75 75 73 76 73 68 78 76 6 33 11 78 71 3 1 Act2_PE. Pext Act2_PE. P tipla Act2_PE. Ppac 12 12 12 12 119 118 119 118 111 113 113 112 15 15 16 14 11 12 12 12 V5 ( V9 ( V13 ( V17 ( 118 63 - - 16 - - - 121 1 6 - V V4 V8 V12 12 11-14 12 9 - - 12 114 56 - The polyethylene foil as a packaging material for yeast indicates a high fermentative activity at 22 C and aged 5 days in individual packages. The lowest fermentative activity (1mg CO2) is observed in the packs wrapped 5 each in blisters with the age of 12 days and stored at a temperature of 3 C (V12 ). Table 5. Yeast fermentative activity in aluminum foil packaging V V7 V14 V21 V28 V36 V5 Results Act1_Al. Cext 76 75 75 72 7 72 69 76 72 47 25 76 48 5 Act1_Al.ti pla 76 76 76 7 71 7 66 76 74 25 14 76 65 43 Act1_Al.p ac 76 76 74 71 7 69 65 76 72 65 38 76 73 53 Act2_Al. Cext 12 118 118 111 18 1 12 113 8-12 83 - Act2_Al.ti pla 12 12 113 95 12 116 - - 12 11 8 Act2_Al.p ac 12 11 118 113 113 16 1 78 98-12 116 11 Packing yeast in paraffin aluminum foil on the inside has as maximum fermentative activity the value of 12 mg CO2, in freshly packed yeast (V) and storage temperature of 22 C. The lowest fermentative activity is noted at the same temperature of 22 C, but at 9, 13 and 17 days. The yeast reduces its for fermentative capacity at 3 C at 8 and 12 days of age. Statistically analyzing the conditioning between the variables of fermentative activity, reaction to the type of packaging and the age of yeast cells by Pearson correlation matrix (table 6), it is concluded that the best activity in yeast fermentation is mantained in the case of cellophane wrapped yeast, stored in cardboard boxes. The calculated Person correlation coefficient has the value r=.998 in the yeast packages inside the box (CEint). A very high V ( V5 ( V9 ( V13 ( V V4 V8 Romanian Biotechnological Letters, Vol. 19, No. 4, 214 9479

Influence of packing materials and temperature on yeast activity significant correlation between the variables of fermentative activity and packing material with correlation coefficient r =.996 is observed in individually wrapped yeast (CEpac). In polypropylene packing the Pearson correlation coefficient indicates a strongly significant correlation between the fermentative activity and packing. r =.995. Polyethylene and aluminum foil ensures the maintenance of the fermentative activity at average values, but one cannot notice a very high significant correlation for these types of packing material Table 6. Pearson correlation matrix between the variables fermentative activity in different packings and yeast age. The graphic representation of the Pearson correlation matrix (Figure 1) groups together the yeast activity in terms of variable ties intensity. Thus, in dial 1, counterclockwise, Act 1 of yeast in cellophane pack together with groups from Act1 of yeast packed in aluminum. At the intersection of dial 1 with dial 2 is noticeable the yeast activity in polyethylene packing, fact that indicates an proximity of the values of these two types of packing. Dials 2 and 3 contain yeast activity groups in polyethylene, polypropylene and aluminum packing. Dial 4 groups the activity at 2 hours in packages wrapped in cellophane. The analysis of the main components, the storage temperature and the age of the yeast (Figure 2 and Table 7) indicate very high significant correlations r=1. for fresh yeast obtained and stored at +3 C. Significant strong correlations are revealed at the age of 14, 21 and 28 days yeast stored at +3 C. Significant negative correlations are noticed when storing the yeast at 22 C 948 Romanian Biotechnological Letters, Vol. 19, No. 4, 214

SONIA AMARIEI, SORINA ROPCIUC, GHEORGHE GUTT, MIRCEA OROIAN and 3 C at the age of 12 and 23 days. The calculated Person correlation coefficient indicates strongly negative correlations, r=-.564 at the age of 12 days stored at 3 C. Figure 1. Graphic representation of how fermentative Figure 2. Analysis of the main components; age and activity is grouped in packing storage temperature of packed yeast Graphic representation of fermentative activity grouped in packing As a result of the analysis, it was found that yeast stored in conditions recommended by the manufacturer, i.e. -1 C, it maintains the best its organoleptic and physico-chemical properties. Table 7. Hierarchical cluster analysis of fermentative activity compared to Euclidean distance Observation 1-17 Class Act1_CE.Cext 1 Act1_Cepac 1 Act1_Al.Cext 1 Act1_OPPCext 1 Act1_Al.pac 2 Act2_PE.ext 2 Act2_PE.tipla 3 Act 1_OPPCint 3 Act2_PE.pac 3 Act2_Al.Cext 2 Act2_Al.tipla 4 Act2_Al.pac 5 Romanian Biotechnological Letters, Vol. 19, No. 4, 214 9481

Influence of packing materials and temperature on yeast activity In case of hot storage, at temperatures above 3 C, the processes that occur take place fast. Thus, the lifetime of the yeast stored in OPP boxes is of only eight days, the rest having a maximum duration of twelve days. The cluster analysis of the fermentative activity (Table 8) divides the yeast activity in five classes, the best activity is preserved in cellophane packing and weakest activity in aluminum packing. Table 8. Pearson correlation matrix between storage temperature and yeast age The mathematical model of the statistical relationship between the average absorbance activity and cellophane water absorption. Fermentative activity= (883.96-82)*Water absorption in 24h -19 Mathematical equation logarithmic curve y = 76.98 ln(x) - 429.2 2 R =1.95 confidence interval Figure 3. Graphical representation of the mathematical model 9482 Romanian Biotechnological Letters, Vol. 19, No. 4, 214

SONIA AMARIEI, SORINA ROPCIUC, GHEORGHE GUTT, MIRCEA OROIAN The mathematical model of the relationship between the fermentative activity and the quality characteristic of packing material (figure 3), calculated at the confidence level of.95% has as ideal absorption the cellophane type packing. The point marked on the graphic with the value of 1215.412 is the best fermentative activity performed at the studied materials, the correlation ratio of logarithmic equation r 2 = 1. 4. Conclusion The used packing materials (aluminum, OPP and polyethylene) have a very low capacity to absorb water (for aluminum the value beeing practically zero), the process stopping migrations of the cell constituents during the storage of the yeast. This action affects yeast activity thus: most constitutive enzymes are activated, soluble minerals that provide enzymes activity are removed with the increase of the moisture. Yeast is unable to produce its own energy through sugars metabolism from the environment, and in the presence of oxygen sources, the yeast cell produces ATP (adenosine triphosphate). With the lack of oxygen and sugars sources, yeast enzymes that act as catalysts for chemical reactions are destroyed and no other reactions occur to produce energy (8-1). Yeast endogenous substances (protein, glycogen, lipids) provide metabolic substrate for a relative time, maximum 72 hours. Baking yeast consumes its endogenous substances and autolyze due to high temperature and reduced aeration conditions because of the packing materials and improper storage conditions (temperatures above 1 C). The packing material that ensures maintaining good values of fermentative characteristics is the cellophane wrap while the optimum temperature is +3 - +1 C. References 1. J. R. VAN DIJKEN, R.A. WEUSTHUIS, J. T. PRONK, Kinetics of growth and sugar consumption in yeasts. Antonie van Leeuwenhoek, 63, 343-352 (1993) 2. B. GROTE, T. ZENSE, B.HITZMANN, D-fluorescence and multivariate data analysis for monitoring of sourdough fermentation process. Food Control, 38,8-18 (214) 3. W. VISSER, W.A. SCHEFFERS, W.H. BATENBURG-VAN DER VEGTE, J.P. VAN DIJKEN, Oxygen Requirements of yeasts. Applied Environmental Microbiology. Energefics of the budding cycle of Saccharomyces cerevisiae during glucose-limited aerobic growth, Arches of Microbiology, 66,289-33 (199) 4. L.G.M. BOENDER, E.A.F. DE HULSTER, J.A.A. VAN MARIS, P.A.S. DARAN-LAPUJADE, J.T. PRONK, Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates. Applied and Environmental Microbiology, 75 (23),7 (29) 5. O.V. CARVALHO-NETTOA, M.F. CARAZZOLLEA, A. RODRIGUESA, W.O. BRAGANC G.G.L. COSTA, J.L. ARGUESOB, G.A.G. PEREIRA, A simple and effective set of PCR-based molecular markers for themonitoring of the Saccharomyces cerevisiae cell population during bioethanol fermentation. Journal of Biotechnology, 168, 71 79 (213) 6. H.A. SUK-JIN, S.R. KIM, H. KIM, J. DU, J.H.D. CATE, Y.S. JIN, Continuous co-fermentation of cellobiose and xylose by engineered Saccharomyces cerevisiae. Bioresource Technology, 149, 525 531 (213) 7. J. YIA, W. L. KERRB, Combined effects of freezing rate, storage temperature and time on bread dough and baking properties. Food Science and Technology, 42, (9), 1474 1483 (29) Romanian Biotechnological Letters, Vol. 19, No. 4, 214 9483

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