A Simple and Rapid Protocol for Producing Yeast Extract from Saccharomyces Cerevisiae Suitable for Preparing Bacterial Culture Media

Original Article

A Simple and Rapid Protocol for Producing Yeast Extract from Saccharomyces cerevisiae Suitable for Preparing Bacterial Culture Media

Omid Zareia,b,c, Siavoush Dastmalchia,d and Maryam Hamzeh-Mivehrouda,d *

aBiotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. bDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. cStudent Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran. dDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.

Abstract

Yeasts, especially Saccharomyces cerevisiae, are one of the oldest organisms with broad spectrum of applications, owing to their unique genetics and physiology. Yeast extract, i.e. the product of yeast cells, is extensively used as nutritional resource in bacterial culture media. The aim of this study was to develop a simple, rapid and cost benefit process to produce the yeast extract. In this procedure mechanical methods such as high temperature and pressure were utilized to produce the yeast extract. The growth of the bacteria feed with the produced yeast extract was monitored in order to assess the quality of the product. The results showed that the quality of the produced yeast extract was very promising concluded from the growth pattern of bacterial cells in media prepared from this product and was comparable with that of the three commercial yeast extracts in terms of bacterial growth properties. One of the main advantages of the current method was that no chemicals and enzymes were used, leading to the reduced production cost. The method is very simple and cost effective, and can be performed in a reasonable time making it suitable for being adopted by research laboratories. Furthermore, it can be scaled up to produce large quantities for industrial applications.

Keywords: Baker’s yeast; Yeast extract; Bacterial growth; Saccharomyces cerevisiae.

Introduction content of yeasts known as “yeast extract” are also utilized for many important purposes. Yeast Yeasts are important eukaryotic extract is a processed yeast product, which microorganisms belong to the kingdom of comprises the soluble components of yeast cells fungi. From historical point of view, they have and is extensively used in food industries as food been used for wide variety of applications since flavoring, additives and vitamin supplementsas thousands of years ago such as fermentation, well as nutritional resource for bacterial culture baking and bioremediation (1-3) as well as media used in microbiology and biotechnology broad range of research activities in biological (9-10). Moreover, the importance of using sciences owing to their unique genetics and the yeast extract in industrial fermentation to physiology (4-8). In addition, nutrient cell produce the microbial biomass or products has been proven by numerous studies (11- * Corresponding author: E-mail: [email protected]; 14). Different yeast species especially [email protected] Saccharomyces cerevisiae (known as baker’s Zarei O et al. / IJPR (2016), 15 (4): 907-913 yeast) have been employed for the production of the same condition to make sure of removing the yeast extract (15-17). Yeast extract is produced insoluble cellcontents. Therecovered supernatant using variety of methods such as those based containing the soluble component was re- on autolysis and hydrolysis methods. The aim autoclaved and cooled again as described above. of different methods is to disrupt yeast cell wall The produced water soluble extract was stored at (for releasing the cell contents) and recover the –20 °C until preparation of the yeast extract in soluble fraction. Depending on the purpose of powder form. Subsequently, the soluble fraction production, the procedure for obtaining the yeast was subjected to mini spray dryer (B-290, Büchi extract varies. For instance, if the aim of yeast Labortechnik AG, Flawil, Switzerland) for extract production is to use it in food or cosmetic obtaining the powder form of the yeast extract. industries, several steps are required to obtain the food/cosmetic grade yeast extract. These Bacterial cell growth assessment steps include plenty of physical, chemical and The growth of the bacteria feed with the enzymatic methods for disrupting the yeast cell produced yeast extract was monitored in order wall, followed by several additional steps such to assess the quality of the product. To this as filtration, chromatography or other separation end, different bacterial culture media either in methods for removing insoluble materials in the form of solid or liquid were prepared. In order to obtain odorless, tasteless and colorless the first approach we used only yeast extract product (18-19). Although the quality of yeast as the sole source of nutrient in bacterial cell extract is improved by applying these additional culture media. For this purpose, solid and procedures but most of these steps are very liquid media were prepared using the following laborious, time consuming, expensive and non- protocol: For solid medium a mixture of 8g of essential where the aim of production is to use the produced yeast extract powder and 15 g agar the yeast extract for microbiological research (Merck, Darmstadt, Germany) was dissolved as one of the main components of bacterial cell in 1L deionized water and autoclaved (121 °C, culture media. 15 lb) for 15 min followed by dispensing in 8 Regardless of availability of numerous cm sterile plastic plates. As a negative control commercial yeast extracts for the preparation medium, a similar solid medium without yeast of microbial culture media, a simple method for extract powder was also prepared. For liquid preparing the high quality yeast extract will be of medium, 20 g of produced yeast extract powder great benefit. Taking into account the importance was dissolved in final volume of 1L deionized and wide range of application of yeast extract in water and was autoclaved as mentioned above. microbial fermentation media, our aim in the The cooled media was dispensed in sterile tubes. current study, was to develop a simple, rapid and A similar negative control medium (i.e. the cost benefit process to produce the yeast extract media without yeast extract) was also prepared. applicable in different laboratories in order to The bacteria used for culturing in the prepared meet the experimental needs as well to be used media were E. coli (DH5α) and Staphylococcus in industry as essential component of microbial aureus (PTCC 1112) as gram negative and gram fermentation media in large quantities. positive bacteria, respectively. For solid media, the bacteria were streaked out on the plate and Materials and Methods incubated overnight at 37 °C. In the case of liquid Preparation of the yeast extract media, a fresh overnight culture of bacteria was Aqueoussuspension of yeast cells was diluted 1:100 in medium and incubated at 37 prepared by adding 500 g of baker’s yeast °C while shaking at 180 rpm. At different time (Saccharomyces cerevisiae) to 2 L of deionized intervals (i.e. 2, 4, 8, 16, 24 and 48 h) samples water (milli-Q water). The prepared suspension were taken under sterile condition for monitoring was autoclaved at 115 °C for 10 min followed the growth of bacteria in the prepared media by by fast cooling on ice. Then, the cell debris was reading optical densities (OD) of the samples at separated by centrifugation at 400 gat 4 °C for 10 600 nm using UV spectrophotometer (Ultrospec min. The supernatant was re-centrifuged again at 2000, Amersham Pharmacia Biotech). Each

908 .A simple and rapid protocol for producing yeast extract experiment was performed in triplicate. autoclaving and centrifugation in order to obtain As a further evaluation approach, the soluble fraction. These steps (centrifugation bacterial growth was monitored in Luria Bertani and autoclaving) were repeated one more time broth media (a commonly used medium in for the complete removal of the cell debris. The microbiology) prepared using the yeast extract supernatant was applied to spray dryer to obtain produced in this study and those obtained from powdered yeast extract. commercial suppliers. The LB broth medium was prepared as follows: 5 g of yeast extract powder, Assessment of bacterial cell growth in solid 10 g tryptone (HiMedia, Cat. No: RM014) and liquid media and 5 g NaCl (Scharlau, Cat. No: SO0225) E. coli DH5α and Staphylococcus aureus, were dissolved in 1 L of deionized waterwith as the representatives for gram negative and subsequent autoclaving and dispensing in gram positive bacterial types, respectively, sterile tubes. In order to compare the bacterial were utilized to systematically determine the cell growth profile in medium prepared by our bacterial growth in both solid and liquid media produced yeast extract with similar commercial prepared by the produced yeast extract. The yeast extract products, three different yeast result showed that both bacteria can grow in extracts from well-known companies, namely, the solid medium containing the produced yeast Sigma (Cat. No.: 92144-F), Merck (Cat. extract as the sole source of nutritional support. No:1.03753) and Applichem (Cat. No: A3732) Figures 1a. and 1b. show the colonies of E. coli were used. A medium containing all ingredients DH5α and Staphylococcus aureus, respectively. except yeast extract was prepared and used as No colonies were observed for control media negative control medium. All the procedures for both bacteria (Figures 1c. and 1d.). Figures related to the bacterial culturing and monitoring 2a. and 2b. demonstrate the growth rate of E. of bacterial cell growth were conducted as coli DH5α and Staphylococcus aureus in liquid described above. broth media composed of the produced yeast extract as a sole source of energy, respectively. Product composition analysis The plots indicate that the quality of the yeast Quantitative compositional analysis of extract produced in this work is rich enough to produced yeast extract was performed by a be used as a sole source of nutrition for bacterial references laboratory (ASA laboratory, Tehran, cell growth. Iran). Total amount of protein, fat, ash, sodium The quality of the produced yeast extract chloride, total volatile nitrogen (TVN), moisture was also assessed as one of the LB broth media and pH were the chemical properties that were components and was compared with three analyzed. commercial yeast extracts. The results showed that both type of bacteria were capable of Statistical analysis growing in LB media prepared by the produced All the statistical analyses for comparing yeast extract comparable to that in LB media bacterial growth in LB broth media prepared prepared using three commercial yeast extracts using different yeast extracts were carried out (Figures 3a. and 4a.). The statistical analyses using t-test implemented in SPSS program indicated that there are no significant differences (version 21.0). A p-value of <0.05 was considered between growth rates of both type of bacteria in statistically significant. LB broth medium prepared either by our yeast extract product and three commercial ones. Results Comparing the bacterial growth rate in LB media prepared using the produced yeast extract Production of yeast extract with growth rate in control media devoid of In the current work, for the preparation any yeast extract revealed significant difference of the yeast extract, we used baker’s yeast for both type of bacteria. The difference (Saccharomyces cerevisiae). The aqueous was observed at 4–48 h after inoculation suspension of yeast was prepared followed by of E. coli and 8–48 h for Staphylococcus

909 Zarei O et al. / IJPR (2016), 15 (4): 907-913

Figure 1. Growing bacteria on solid media prepared by the produced yeast extractas the sole source of energy for bacteria. (A) Colonies of Staphylococcus aureus as gram positive bacteria on the solid media prepared by the produced yeast extract. (B) Colonies of E. coli DH5α as gram negative bacteria on the solid media prepared by the produced yeast extract. (C and D) Yeast extract free solid media as control for culturing of Staphylococcus aureus and E. coli DH5α, respectively. As seen in the control plates, there are no colonies in the media. The experiments were conducted in triplicates.

aureus (p-value <0.05) (Figures 3b. and 4b.). Discussion

Analyses of yeast extract composition Yeast extract is the product of yeast cells, Quantitative analyses of the produced yeast containing amino acids, lipid, vitamins, minerals extract powder showed that the extract contains: and other soluble components withplenty protein (30%), fat (0.42%), sodium chloride of applications in food industry as well as (0.67%), ash (12.18%), total volatile nitrogen nutritional source for microbial culture in (9.2%) with moisture of 4.72% and pH of 6.29. molecular biology research area and fermentation

Figure 2. (A) Growth pattern of E. coli DH5αas gram negative bacteria (B) and Staphylococcus aureusas gram positive bacteria on liquid media prepared by the produced yeast extract (as sole source of energy) at different time intervals. As it is seen the number of bacteria increases during a 48 h period for both types of bacteria. The average ODs for three individual experiments at different times and the corresponding standard deviation are shown.

910 .A simple and rapid protocol for producing yeast extract

Figure 3. (A) Bacterial cell growth profile forE. coli DH5α as gram negative bacteria on Luria Bertani broth media prepared by the yeast extract produced in this study and commercial products (Applichem, Merck and Sigma). Yeast extract free medium was used as control. Triplicate determinationswere performed for each data point.For the purpose ofclarity the standard deviations are shown only for the medium prepared by our produced yeast extract as well as control media. (B) Histogram of E. coli DH5α growth in different LB medium. The results indicate that there is no significant difference between growth rate of bacteria in LB broth medium supplemented by the produced yeast extract and LB broth media containing three commercialyeast extracts (p-value >0.05). The analysis shows a significant difference between the growth rate of E. coli DH5αon the LB medium prepared by our produced yeast extract and control after 4, 8, 16, 24 and 48 h of inoculation (p-value <0.05). The significant differences are marked by asterisks (*).

industry (20-22).The two common approaches of the cellular constituents of a yeast cell by for yeast extract production are autolysis and the released endogenous digestive enzymes. hydrolysis, however, this is not a comprehensive Several strategies have been reported to improve categorization and many methods fall outside the autolysis process such as changing the of this classification. Autolysis, a natural event temperature, pH, and osmotic pressure, X-rays occurring after cell death, is self-degradation irradiation and mechanical comminuting or

Figure 4. (A) Bacterial cell growth profile forStaphylococcus aureus as gram positive bacteria on Luria Bertani broth media prepared by the yeast extract produced in this study and commercial products (Applichem, Merck and Sigma). Yeast extract free medium was used as control. Triplicate determinationswere performed for each data point.For the purpose ofclarity the standard deviations are shown only for the medium prepared by our produced yeast extract as well as control media. (B) Histogram of Staphylococcus aureus growth in different LB medium. The results indicate that there is no significant difference between growth rate of bacteria in LB broth medium supplemented by the produced yeast extract and LB broth media containing three commercialyeast extracts (p-value >0.05). The analysis shows a significant difference between the growth rate of Staphylococcus aureus on the LB medium prepared by our produced yeast extract and control after 8, 16, 24 and 48 h of inoculation (p-value <0.05). The significant differences are marked by asterisks (*).

911 Zarei O et al. / IJPR (2016), 15 (4): 907-913 employing chitosan, chemical inducers, like ii) In extraction process, no enzyme was used organic solvents and common salts (23-26). and therefore the final cost of production would Autolysis process is associated with some be reduced. disadvantages such as low extraction yield, iii) The number of required steps in the difficulty in solid-liquid separationdue to risk extraction process was decreased which speeds of microbial contamination resulted from long up the yeast extraction production compared to term incubation. In hydrolysis method, different the other available tedious and time consuming exogenous enzymes, acids or alkalis are used to methods. disrupt the cell contents (27-29). By applying the exogenous enzymes in hydrolysis process Conclusion the cost of manufacturing will be increased, moreover, using acids or alkalis in this process In summary, we developed a simple, fast and will complicate the purification processes. cost benefit method for producing yeast extract Studies show that various mechanical from baker’s yeast (Saccharomyces cerevisiae). procedures have been employed in some This method can be used in research laboratories protocols to improve the auto-or hydrolysis to reduce the research costs, and scaled up processes, but they have not been used alone for to produce large quantities for industrial yeast extract production (30-31). In the current applications where microbial culturing is needed. work, we developed a fast mechanical method to produce the yeast extract from baker’s yeast Conflict of interest (Saccharomyces cerevisiae) in order to be used The authors have no conflict of interest to as nutrient agent in bacterial culture media. In declare. this method, high temperature and pressure were applied to the suspension of yeast cells References followed by rapid cooling. It triggers loss of cell membrane integrity by inducing a shock (1) Mensour N, Margaritis A, Briens C, Pilkington H and for yeast cells, which cannot be regarded as Russell I. Application of immobilized yeast cells in the either autolysis or hydrolysis based method. brewing industry. Prog. Biotechnol. (1996) 11: 661-71. (2) Fleet GH. Yeasts in foods and beverages. impact on Subsequently, the soluble fraction of the extract product quality and safety. Curr. Opin. Biotechnol. was subjected to spray dryer for obtaining yeast (2007) 18: 170-5. extract in the powder form, which is more (3) Makkar RS, DiNovo AA, Westwater C and suitable for storage. The quality of the produced Schofield DA. Enzyme-mediated bioremediation of yeast extract was very promising concluded from organophosphates using stable yeast biocatalysts. J. Bioremed. Biodeg. (2013) 4: 2. the growth pattern of bacterial cells in media (4) Dato L, Branduardi P, Passolunghi S, Cattaneo D, prepared from this product. It is important to Riboldi L, Frascotti G, Valli M and Porro D. Advances note thatthe produced yeast extract was capable in molecular tools for the use of Zygosaccharomyces of being used by bacteria as a sole source of bailii as host for biotechnological productions and energy in bacterial solid and liquid media construction of the first auxotrophic mutant. FEMS (Figures 1. and 2.). The produced yeast extract Yeast Res. (2010) 10: 894-908. (5) Delneri D. Barcode technology in yeast application is comparable with the three commercial yeast to pharmacogenomics. FEMS Yeast Res. (2010) 10: extracts in terms of bacterial growth properties 1083-9. in LB media (Figures 3 and 4). The advantages (6) Salari R, Rajabi O, Khashyarmanesh Z, Fathi Najafi M of our developed method for the production of and Fazly Bazzaz BS. Characterization of encapsulated yeast extract compared to the existing protocols berberine in yeast cells of Saccharomyces cerevisiae. Iran J. Pharm. Res. (2015) 14: 1247-56. are as followings: (7) Krivoruchko A, Siewers V and Nielsen J. Opportunities i) No chemicals like acids, alkalis or salts for yeast metabolic engineering: Lessons from were used in extraction process, which in turn synthetic biology. Biotechnol. J. (2011) 6: 262-76. eliminates complication in the downstream (8) de Jong B, Siewers V and Nielsen J. Systems biology processes due to removing the added reagents, of yeast. enabling technology for development of cell and decreases the cost of production. factories for production of advanced biofuels. Curr.

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