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Canadian Journal of Microbiology, 2001, vol. 47, pp. 18-24

The effect of the addition  of proteases and glucanases  during yeast autolysis  on the production  and properties of yeast extracts

John Conway, Helene Gaudreau and Claude P. Champagne
 

ABSTRACT

Yeast extracts (YE) were produced with the addition of proteases or glucanases during bakers’ yeast (Saccharomyces cerevisiae) autolysis. Chemical composition, physical properties, and biological value of the YE were examined. Proteases had the highest impact on the turbidity and filterability of YE. All 11 proteases and two glucanases increased YE yields (% yeast solids solubilized) obtained from heated (80°C/15 min) bakers’ yeast creams (BYC). However, when proteases were added to native (unheated) BYC during autolysis, few increased YE yields, with papain being the most effective. The increased yields were generally related to increased levels of total nitrogen (TN) and a-amino nitrogen (a-AN) in the YE. Media were supplemented with the various yeast extracts, and the highest growth rates (µ_max) and biomass values (OD_max) of Lactobacillus acidophilus were noted. The best growth was obtained with YE produced with native BYC treated with a fungal protease, and results of this study show that some enzymes could be used to produce improved YE for microbiological media.

Key words: turbidity, filtration, amino acids, Lactobacillus.

INTRODUCTION

Yeast extracts (YE) are the soluble portion of yeast cell suspensions which have been submitted to controlled autolysis. These extracts are commercially used both as savoury food ingredients and in microbiological media (Kelly 1983; Difco 1984; Nagodawithana 1992). Yeast autolysis is generally slow and can extend to 72 h if the process is not accelerated in some way (Hernawan and Fleet 1995). Industrially, a 3 day incubation period is unattractive, and strategies have been devised to shorten the time taken for autolysis. Commonly, temperature is adjusted to 45–55°C and solvents or salts are added to the cell suspension which can reduce the required incubation to under 24 h (Hough and Maddox 1970; Kelly 1983; Chao et al. 1980; Akin and Murphy 1981; Breddam and Beenfeldt 1991). Papain and glucanases are used commercially in some processes (Kelly 1983), particularly nucleotide-rich YE (Reed and Nagodawithana 1991), but other enzymes have also been proposed for this purpose, and assays with b-glucanases, microbial proteases, pancreatic extracts, and lysozyme are reported (Belousova et al. 1995; Chao et al. 1980; Hobson and Anderson 1991; Kelly 1983; Knorr et al. 1979a, 1979b; Kollar et al. 1991; Ryan and Ward 1985, 1988). These previous studies focussed on the autolysis yield and chemical composition of the extracts, but none have examined the physical properties of the extracts (i.e., turbidity, filterability) or their ability to promote the growth of lactic cultures in microbiological media.

The beneficial effect of YE on the growth of lactic acid bacteria (LAB) is well documented (Smith et al. 1975; Bibal et al. 1988; Aeschlimann and Von Stockar 1990; Benthin and Villadsen 1996), which is unsurprising considering YE contain B-complex vitamins, peptides, and amino acids (Peppler 1982; Difco 1984; Hugenholtz et al. 1987; Jensen and Hammer 1993; Juillard et al. 1995). However, it should be noted that there are variations between strains of LAB with respect to their preference for amino acids and peptides (St-Gelais et al. 1993; Juillard et al. 1995).

It has been found that enzymatic treatment of the yeast cell suspension influences the degree of protein hydrolysis in the resulting YE (Belousova et al. 1995). Therefore, the growth-promoting abilities of YE on LAB could be affected by the addition of enzymes during autolysis.

The aim of this study was to determine the effect of proteases and glucanases on autolysis yields (% yeast solids solubilized), nitrogen content, turbidity, filterability of the YE, as well as their growth-promoting properties toward Lactobacillus acidophilus.

MATERIALS AND METHODS

Autolysis

Fresh bakers’ yeast cell suspensions were obtained from Lallemand Inc. (Montréal, Que.) and were recovered following centrifugation, containing approximately 18% solids at pH 5.2. These concentrated cell suspensions are typically called bakers’ yeast creams (BYC). In one series of experiments, BYC were heated at 80°C for 15 min prior to lysis, in order to denature yeast native enzymes. The heated or native BYC (250 mL) were placed in 500 mL bottom-baffled Erlenmeyer flasks (Bellco Glass; Vineland, N.J.), 1.5% ethyl acetate was added, and the autolysis was conducted at 48°C with agitation at 150 rpm (Lab Line Enviro-Shaker; Melrose Park, Ill.). Unless otherwise stated, the BYC were incubated for 24 h under these conditions.

Commercial enzyme preparations (Table 1) were added at 0.5% (v/v for liquid preparations, and w/v for powders). Because of the variety of sources and form, and because the technical information provided by the suppliers (Table 1) did not provide a common activity unit (on a given substrate for given pH and temperature levels), we did not have a method of comparing the activity of the enzymes, in order to add them to the medium accordingly. Furthermore, the enzymes targeted different components of the cell (glucans or proteins), which simply made an addition based on units of activity unpractical. Therefore, it was decided to add the enzymes on a quantity basis (0.5% as described above) and use a high level of addition. In the various studies on yeast lysis with proteases, concentrations between 0.01 and 1% are found (Chao et al. 1980; Ryan and Ward 1985; Ryan and Ward 1988). Indeed, papain is effective at concentrations as low as 0.01%, but some patents recommend up to 1% (Chao et al. 1980). Thus, the concentration used in this study is in the high range of enzyme levels reported as being effective. In order to nevertheless provide a picture of the enzyme activities, the experimental plan included one series of assays on heated yeast suspensions under the pH and temperature conditions of the autolysis.

The autolysate was harvested and centrifuged for 15 min at 5000 × g (Beckman, rotor JA-10). The supernatant was collected, and approximately 300 mL of deionized water was mixed with the cell pellet. A second centrifugation was carried out in the same conditions. The two supernatants were combined and heated (80°C, 30 min). The heated YEs were placed in freeze-drying pans in order to obtain 1-cm-thick layers, and frozen at –40°C. They were then freeze-dried in a Lyo-Tech (LYO-SAN Inc., Lachute, Canada) unit at 24°C for 48 h, under a vacuum of at least 13 Pa. The YE powders were stored in sealed bottles at –18°C to avoid the rehydration of hygroscopic YE.

Chemical analyses

In order to determine yields, 15 mL of autolysate was first centrifuged at 5000 × g in a SS-34 Sorvall rotor, the supernatant collected, and its volume measured. An equivalent volume of deionized water was mixed with the cell pellet, and a second centrifugation was carried out. The supernatants, constituting the YE, were combined and their volume measured. The total solids in the yeast suspensions and in YE were determined by dry weights following drying at 100°C for 24 h. The yield was expressed as a percentage of the solids recovered in the YE (taking into account dilution due to two centrifugations) with respect to the total solids present in the BYC.

The a-amino nitrogen (a-AN) was determined by titration following reaction with formaldehyde (USP 1985). A 5% YE solution was prepared, and the pH adjusted to 7.0 with 0.1 M NaOH or 0.1 M HCl. A USP formaldehyde solution (37%) was adjusted to pH 9.0, and 10 mL of the formaldehyde was added to 25 mL of the YE solution. The mixture was agitated, and then titrated to pH 9.0 with 0.1 M NaOH. Glycine solutions served to verify and standardize the method. Total nitrogen (TN) and non-protein nitrogen (NPN) contents were determined by the Kjeldahl method (AOAC 1984) on a Tecator Kjeltec Auto 1030 Analyser. NPN samples were obtained following precipitation of proteins with 12% trichloroacetic acid (TCA), followed by a subsequent filtration with Whatman #42 paper. Results are expressed as mg of TN or a-AN per g of YE powder.

The pH was determined with a Radiometer (Copenhagen) model PHM 84.

Biological activity

The biological activity of the extracts was determined by following the optical density on an automated spectrophotometry unit (LabSystem Bioscreen C, Finland) as described by Champagne et al. (1999a). Lactobacillus acidophilus EQ57 was used for the evaluation of the growth-promoting properties of the YE. Production of inocula of Lb. acidophilus EQ57 were carried out as described by Champagne et al. (1999a). The basal growth medium was supplemented with 2 or 5 g/L of each YE powder (freeze-dried) with 350 mL placed in the Bioscreen 100-well plates. Data for maximum growth rate (mmax) were obtained from the OD curves of cultures in the presence of 5 g/L YE powder, while those of maximum biomass by optical density (ODmax) were obtained in media containing 2 g/L YE powder.

Statistical analyses

Two independent assays were made. In each replicate, at least two wells were prepared for each of the conditions, which served in preparing averages for each assay. These average values served as data for a given assay, and were used for the statistical analyses. Analyses of variance (ANOVA) were carried out on GraphPad Instat software (San Diego, Calif.), using the Student–Newman– Keuls test on the two replicates.

RESULTS AND DISCUSSION

Autolysis conditions

The autolysis conditions (pH/temperature/solvent) selected were those that are commonly used (Kelly 1983; Chao et al. 1980; Peppler 1982). A variety of enzymes were available (Table 1) and, in absence of standard activity units, it was our concern that the enzyme concentration would be a limiting factor. It was thus decided to use a high addition level. Yields after 24 or 48 h of incubation were not significantly different, which suggests that enzyme levels were not limiting.

Additionally, yields of YE obtained (Kelly 1983; Belousova et al. 1995), as well as their TN and a-AN contents, are similar to those reported in literature for cells suspensions at approximately 18% solids (Knorr et al. 1979a).

Table 1. Some characteristics of the enzymes used, as provided by the suppliers.

Table 2. Effect of the addition of proteases on solids and nitrogen yields of yeast autolysis after 24 h of incubation.

Table 3. Effect of the addition of glucanases on the degree of yeast autolysis (% total solids that are soluble) after 24 h of incubation.

Table 4. Effect of enzyme addition of filterability and turbidity of yeast extracts made from native bakers’ yeast creams.

Table 5. Effect of enzyme addition to native yeast cream on the bioactivity of the YE on Lactobacillus acidophilus EQ57.

Fig. 1. Relationship between the a-amino nitrogen content and

biomass levels of Lactobacillus acidophilus EQ57 reached in

growth media supplemented with YE obtained from native bakers’

yeast creams (BYC) treated with proteases.

Lysis yields of heated BYC

Initially, it was determined if the enzymes were active under these particular autolytic conditions and on this particular substrate (BYC). Since the pH and temperature conditions of yeast autolysis were set at 5.2 and 48°C respectively, many of the enzymes studied were not under optimal conditions (Table 1). In addition to BYC substrate, pH, and temperature conditions, ethyl acetate could potentially affect enzyme activity (Shetty and Kinsella 1978; Fenton 1982). Thus, a study was conducted on heated (80°C/15 min) BYC, in order to sharply reduce the effect of native yeast enzymes, and enable the determination of the activity of the adjunct enzymes. Heating at 80°C for 15 min effectively reduced autolysis yields by half, and all proteases increased YE yields from heated BYC (Table 2) although to varying degrees. Solvay fungal protease gave the greatest yield and TN increase, while Flavourzyme gave the greatest increase in a-AN. Thus, proteases evidently had very different activity patterns. All glucanases also appeared to increase yields on heated BYC, although only the increases with EDC b-1-3 glucanase and EDC fungal b-glucanase were judged to be significant (Table 3).

With respect to yields, the effects of the enzymes on the heated BYC were complete after only 8 h of incubation, which, again, suggests that the efficiency of the treatments was not related to enzyme concentration. The levels added were thus high, and commercial utilization would command reduced levels if autolysis is to be conducted over 24 h. Nevertheless, these results show that many enzymes are active on bakers’ yeast cells under conditions of YE manufacture, and assays on native yeast creams were consequently conducted.

Lysis yields with native BYC

Bakers’ yeast contains a variety of proteases (Hough and Maddox 1970; Aschstetter and Wolf 1985) that are very effective during autolysis. Indeed, YE yields obtained with enzymes added to heated BYC were all lower than the control treatment with native yeast creams (Table 2). As a consequence, most of the added enzymes did not significantly affect YE yields when added to native BYC. This was expected, as literature reports yield increases of only 5% to10% in the presence of added proteases (Kelly 1983; Peppler and Reed 1987) when autolysis promoters (e.g., ethyl acetate) are present. Solvay Papain had the most marked effect on autolysis on native yeast creams, although EDC Liquipanol and Solvay Fungal protease also show a tendency to increase yields (Table 2). Papain had previously been shown to be more effective than rennins, pepsin, trypsin, pancreatin, or Aspergillus protease in solubilizing yeast cells (Chao et al. 1980), and this study adds to the data on this aspect. This study emphasizes the effects of papain over currently available proteases, from a variety of sources and suppliers. We nevertheless wished to see if the proteolysis resulting from the addition of the proteases simply added to that occurring with the native yeast enzymes. If this were the case, yield and TN data with the native BYC would be increased proportionally to the activity of the enzymes on heated BYC, and correlations between yield and TN data of the YE obtained with native and heated BYC would then be high. The correlation between YE yields obtained with heated or native BYC was very low (R2 = 0.28), while the corresponding analysis with TN data also gave a low correlation level (R2 = 0.56). Therefore, the addition of proteases did not improve hydrolysis of the native BYC, suggesting that the native BYC enzymes are in sufficiently high concentration or that factors other than enzyme quantity affect the ultimate autolysis yields and TN content of the adjunct proteases in BYC during autolysis. One such factor could be product inhibition. The initial high solids of BYC (18%) enable the production of solutions having, in the case of EDC Equipanol, 62 g/L of peptides and free amino acids. It has indeed been shown that the accumulation of free amino acids and polypeptides formed during autolysis of bakers’ yeast can inhibit this process (Neklyudov et al. 1994). If this were the case, the selection of yeast strains destined for autolysis could be carried out on the basis of lack of end-product inhibition of their proteases. It could also be argued that autolysis conditions should be altered to enable the activity of the adjunct enzymes (Kelly 1983), for example papain at 65–85°C. This raises a concern, however, on the effectiveness of the native yeast enzymes at some conditions. Thus, studies on pH and temperature modulation during autolysis with native yeasts and adjunct proteases are warranted, particularly in order to initially assess native proteolysis with subsequent addition of adjunct proteases.

Two glucanases also tend to increase yields, when added to native yeast creams, but the effect was not judged to be significant (Table 3). This is in contrast with that of Ryan and Ward (1985, 1988), who found sharp increases in yeast solubilization using glucanases. Close examination of their data shows, however, that the discrepancy is not due to lower yields with glucanase, but rather to our having higher yields with the control. YE yields vary as a function of the strain (Belousova et al. 1995), and this could partially account the differences between our data and that of Ryan and Ward (1985, 1988).

Extending the incubation to 48 h did not significantly affect the autolysis yields (P = 0.49). Therefore, although all the enzymes seemed active on yeast, very few generated a significant improvement of yield under classic autolysis conditions. Again, proteolysis may have been limited by the accumulation of free amino acids and peptides (Neklyudov et al. 1994), and prevented improvement of yields during the 24–48 h incubation period.

pH

The initial pH of the native yeast cream was 5.2, and pH was determined after 24 and 48 h of autolysis. After 24 h of incubation the pH varied between 5.33 and 5.53 in the various treatments, with an average of 5.45, and pH values after 48 h were on the average 0.08 units lower than those at 24 h. Therefore, a rise in pH of approximately 0.25 unit occurred during the first 24 h, but a slight drop was then noted between 24 and 48 h. It may be supposed that such shifts in pH probably have a minor effect on autolysis yields (Champagne et al. 1999b).

Nitrogen content of the YE

In addition to yield, there were marked differences in nitrogen contents of YE obtained between heated or native BYC. The increased yields noted with proteases on heated BYC, when compared to the control, was related to increased levels of TN in the YE (Table 2). Glucanases had less effect on the TN or a-AN contents of YE obtained from either the heated or native BYC. As can be calculated from Table 2, the proteolytic activity on the average increased by 4.6 mg/g the TN content of the YE obtained from the heated BYC, while the average TN increase of glucanase-treated equivalents were of 3.7 mg/g, which is 20% less. This is logical considering the fact that YE treated with glucanases should have proportionally higher contents in soluble sugars resulting from the hydrolysis of the membrane polysaccharides (Ryan and Ward 1988).

The YE resulting from native BYC had, on average, 15% more TN than those obtained from heated BYC (Table 2). The level of protein hydrolysis was also lower when heated BYC were used. Thus, a-AN represented, on average, 55% of TN in YE obtained from native BYC, while it represented 44% with heated BYC. This could be a result of the destruction of peptidases synthesized by bakers’ yeast during the heat treatment of BYC (Aschstetter and Wolf 1985). The fact that Flavourzyme, which contains peptidases, had the highest a-AN content of the YE obtained from heated BYC (Table 2) lends support to this hypothesis. It is noteworthy that the proteases had different effects on TN and a-AN. Thus, on heated creams, papain increases TN but not a-AN, whereas the opposite is found with Flavourzyme (Table 2). This could be related to the presence of peptidases in Flavourzyme allowing completion of the protein hydrolysis initiated by the proteases. The adjunct proteases did not have significant effects on TN or a-AN when native BYC were used.

Turbidity and filtration rate

Some commercial applications, microbiological media for example, require that YE produce clear low-turbidity solutions. Since some media must be sterilized by 0.22 m filtration, YE must not contain compounds that could clog the filters. Thus, turbidity and filterability are functional parameters that warrant examination.

Turbidity and particularly filterability of the YE obtained were found to be very variable, and few treatments had effects that were judged to be statistically significant. No clear trend could be detected between proteases and glucanases. However, proteases gave the two lowest turbidity values, while a glucanase gave the highest (Table 4). The opposite was found with filtration rates. It is noteworthy that the five samples having the highest filtration rates were obtained from native BYC treated with proteases. Thus, the best relationship seems to be with proteolysis. In this sense, YE solutions could have similar properties as beers, in which hazing is partially protein related (Hough et al. 1982). However, very poor correlations (R2 < 0.3) were found between turbidity or filterability and the proteolysis indicators (TN or a- AN). Therefore, more work needs to be done on how enzymes influence the turbidity and filterability of YE.

Growth-promoting properties

Two features of the Lactobacillus acidophilus EQ57 growth curves were examined: (1) maximum growth rate (mmax) and (2) highest biomass level, as determined by maximum optical density (ODmax).

The adjunct enzymes did not highly affect the yields but this was not the case with the biological value of the YE. The mmax was generally reached in the early exponential growth phase. None of the glucanases had a significant effect on mmax (Table 5). The highest mmax values were obtained with YE obtained with native yeast creams treated with fungal proteases (EDC and Solvay) as well as with EDC protease 180 (Table 5). The best YE for biomass production were also obtained in this fashion. There was a poor correlation between mmax and ODmax data (R2 = 0.11), which suggests that the two growth characteristics are influenced differently by YE composition. This being noted, the two products giving the highest growth rates (EDC and Solvay Fungal proreases) also enabled the highest biomass levels to be reached. Therefore, it is possible to select adjunct enzymes that can generate YE having both biological properties. Data on yields and TN contents of YE obtained from native BYC had cast a doubt on the activity of the adjunct enzymes on the autolytic process when the yeast’s native enzymes were active. However, it was determined that the adjunct enzymes could act on yeast cells under the conditions of autolysis (heated BYC; Table 2) and that the products were different with respect to turbidity, filterability (Table 4), and biological activity (Table 5). Therefore, the adjunct enzymes significantly modified the properties of the YE, even though yields were little affected. The TN values in the YE obtained with native BYC were similar (Table 2), but results suggest that proteolysis was different (turbidity), as were the amino acid and peptide components (growthpromoting properties). Papain, for example, preferably hydrolyses peptide bonds that include lysine, arginine, and phenylalanine (Kelly 1983). The LAB require many amino acids for growth (Jensen and Hammer 1993; Desmazeaud and de Roissart 1994; Juillard et al. 1995; Benthin and Villadsen 1996), and the nitrogen content of YE is a major factor in their growth stimulation (Smith et al. 1975; Benthin and Villadsen 1996). Thus the LAB are good tools to evaluate YE for the effect of proteases on BYC.

Some LAB can use free amino acids, while others prefer peptides (Jensen and Hammer 1993; St-Gelais et al. 1993; Juillard et al. 1995; Benthin and Villadsen 1996; Mierau et al. 1996; Juillard et al. 1998). Thus, various regression analyses were carried out to determine if the chemical composition of the YE had an effect of their biological properties. The best correlation was obtained between a-AN and Biomass (Fig. 1). Thus, it can be hypothesized that enzymes generating the highest hydrolysis of the proteins give the products with the highest biological activity for Lactobacillus acidophilus EQ57. The advantage of adding peptidases to promote this hydrolysis is not clear. The YE obtained from Flavourzyme did give a good biomass level, and more data is necessary in this respect.

The Lactobacillus acidophilus EQ57 strain used in this study was previously found to detect differences in YE biological activities, but its requirements differ from those of the lactococci (Champagne et al. 1999a). Thus, results of this study show that some enzymes (particularly EDC fungal; Table 5) could be used to produce improved YE for microbiological media, but it must be determined if this conclusion can extend to many other organisms. Previous studies (Champagne et al. 1999a), suggest that it is unlikely that a single YE-production process could generate products universally efficient in microbiology. However, these results show that it is possible to use enzymes to improve YE for a designed strain, and YE suppliers could find it worthwhile to develop products customized for clients producing large volumes of a given culture.

ACKNOWLEDGEMENTS

The technical support of N. Gardner and N. Chagnon is gratefully acknowledged.

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