Characteristics of Morphological and Physiological Changes during the Autolysis Process of Saccharomyces cerevisiae FX-2

Abstract

In this paper, the autolysis process of Saccharomyces cerevisiae FX-2 (S. cerevisiae FX-2) via, a variety of endogenous enzyme, was investigated systematically by analyzing changes in physicochemical parameters in autolysate, surface morphology and the internal structure of the yeast cells. As an explicit conclusion, the arisen autolysis depended on the pH and the optimal pH was found to be 5.5. Based on the experimental data and the characteristics of mycelia morphology, a hypothesis is put forward that simple proteins in yeast vacuolar are firstly degraded for utilization, and then more membrane-bound proteins are hydrolyzed to release hydrolytic enzymes, which arouse an enzymatic reaction to induce the collapse of the cell wall into the cytoplasm.

Introduction

Yeast extract (YE) contains plenty of amino acids, peptides, nucleotides, B-complex vitamins, biotin and other nutrients, which is an important mixed nutrient source widely used in microbial fermentation, food industry, pharmaceutical industry and feed industry, etc [2, 5, 8]. Yeast autolysis is an effective technology for producing yeast extract.

Nowadays, international researchers mainly focus on adding exogenous enzymes [such as papain [24], β-glucanase [21], and lysozyme [13] to help increasing the biodegradation of macromolecular nutrient substances in yeast cells or nonpolar organic solvents (such as toluol, chloroform, ethyl acetate or amyl acetate) or inorganic salts (such as sodium chloride) [14, 18, 20] to accelerate autolysis. Though most of them have obtained better results by carrying out one of above methods, the negative impact due to adding these foreign objects cannot be ignored. On one hand, adding exogenous enzymes not only could add the cost but also increase risk of deterioration due to microbial contamination during processing [13]. On the other hand, adding solvents and sodium chloride are undesirable when yeast autolysate is intended for food products, even though adding these can induce rapid autolysis, it is unavoidable to prevent contamination and high concentrations which are harmful to health especially for hypertensive patients [4].

It should be noted that the dual requirements for a high solids yield and an acceptable composition in a yeast extract are somewhat contradictory: yeast extracts usually require a higher nitrogen (free amino acids and short peptides) content both in food-grade applications for the taste [16] and in microbial media for stimulation of microorganisms growth [24]. The best method which satisfies the requirements to produce yeast extract is by adjusting the environmental conditions (such as pH, temperature) to be suitable for yeast autolysis to make full use of hydrolytic enzymes system in living cells. Consequently, it is worthy to focus on studying the principle of autolysis process and figuring out key regulatory points to shorten the time without using undesirable additives, especially when autolysates are intended for human consumption.

A few studies have been found the cell wall retains its basic shape and integrity during autolysis of different kinds of yeasts [6], there is only a general understanding of the biological changes that occur at the end of yeast autolysis. However, the autolytic theories and dynamic changes about morphologic parameters of S. cerevisiae during autolysis has not been described detailedly.

In this study, active yeast suspensions were autolyzed in 10 L autolysis tank equipped with online pH and temperature controller, and timing sampling method was adopted to determine the relevant bio-chemical parameters during the process of dynamic autolysis. Thus contents of amino nitrogen (AN) and total nitrogen (TN) in autolysate which are important indicators of degrees of hydrolysis of proteins and all nitrogen-containing compounds, wet weight (WW) which discloses loss of all cellular contents, were detected in real time. In the meantime, the scanning electron microscope (SEM) and transmission electron microscope (TEM) were utilized to observe the appearance shapes and intracellular structures of yeast cell. The characteristic feature of autolysis process without additives and key points of regularization were revealed by dynamic analysis of changes in surface morphology, internal structural and physical and chemical parameters. This work is particularly an important guiding significance for the industrialization of YE, particularly in food industry.

 

Materials and Methods

Yeast cells

Fresh baker’s yeast cells (S. cerevisiae FX-2) were obtained from Angel Yeast Co. Ltd. The yeast was stored at 4℃ and was taken out just before running the experiment to avoid contamination and the reduction of its enzyme activities.

Determination of AN, TN and WW

Samples (10 ml) of autolyzed suspension were centrifuged at 5,000 rpm for 10 min. The precipitation of cells was weighted by electronic scale. The wet weight of the cells was determined by subtracting the weight of the centrifuge tube. The supernatant liquor from this operation (termed the autolysate) were removed for analyses of AN, TN. AN concentration in the autolysis medium was estimated by the method of Malfatti formol titration method [7], and TN concentration by the method of micro Kjeldahl method [3].

Sensory analysis

Sensory evaluation was performed with a ranking test and a taste panel consisting of 10 professionals and 100 ordinary volunteers. The professionals were staff of Angel Yeast Co. Ltd. and ranged in age from 20 to 45 years. And ordinary volunteers who were selected randomly were shoppers of a large mall. Samples were numbered and served in mixed order, and were ranked from the most preferred to the least preferred by each volunteer.

Scanning electron microscopy (SEM)

10 ml suspension of the autolyzed yeast was centrifuged at 5,000 rpm for 10 minutes (min) to obtain autolyzing cells. Cell samples were fixed by 3% glutaraldehyde solution at 4℃ for 2 hours (h), rinsed in buffer for 10 min (3 times). The fixed samples were dehydrated firstly in ethanol gradients (30%, 50%, 70%, 90%, 100%) [25], then in anhydrous ethanol and acetone (v/v, 1:1), 100% acetone for 15 min, respectively, and the treated samples were transferred to dry in critical point drying apparatus (HCP-2, Hitachi, Japan). Coverslips were mounted on aluminum sample stubs, coated with gold and observed under SEM (JSM-750LV, JEOL, Japan).

Transmission electron microscopy (TEM)

Methods that cell samples being fixed by glutaraldehyde and washed by buffer were identical with that of SEM at the beginning. After that, samples were further fixed in 1% osmium tetroxide at 4℃ for 1.5 h, then rinsed in buffer 10 min (3 times). After being dehydrated with gradient ethanol and then treated in anhydrous ethanol and acetone (v/v, 1:1), 100% acetone for 15 min, respectively, the samples were embedded in Epon812 resin and acetone (v/v, 1:1) at room temperature for 3 h, then in 100% Epon812 resin for 1 h. The treated sample was solidified at 37℃ for 24 h and at 60℃ for 48 h. Ultrathin sections were prepared with an ultramicrotome (UltrotomeNOVA; LKB, Broma, Sweden), and stained with uranyl acetate and lead citrate for examination by TEM (Hitachi-7500, Hitachi, Japan).

 

Experimental Design

Optimization of pH for yeast autolyzing

Activity of hydrolytic enzymes in yeast cells, especially proteases, was greatly influenced by pH of autolysis environment [21]. There are several kinds of proteases in yeast’s vacuole, some studies showed slightly acidic or near neutral conditions were beneficial to yeast endogenous proteases’ activity. In addition, our previous experiments results have shown the optimal concentration of yeast suspensions and temperature of autolysis environment were 14% (wet weight for per volume) and 55℃, respectively [27]. So, experiments that fresh baker’s yeast cells were suspended in 0.05 mol/l sodium phosphate buffer, different pH (3.0–7.0), with the concentration being 14%, then autolyzed at 55℃ in 10 L autolysis tank equipped with pH online supervision instruments and temperature controller for 72 h with shaking at 100 rpm were designed to study the effects of pH on baker’s yeast autolysis and obtain the optimal pH. During autolysis, samples of the suspension were removed for analyses of AN, TN and WW. In addition, after 72 h, before autolysates were put into spray tower (LPG-25, Changzhou Erle Driers Co., Ltd., China) for YE powder, cytological changes of yeast cells were observed by SEM and TEM.

Study on autolysis process under the condition of optimal pH

14% fresh baker’s yeast cells suspensions were autolyzed at 55℃ and optimal pH in 10 L autolysis tank for 7 days (d) with shaking at 100 rpm in order to study the basic procedures of yeast autolysis. During the process of autolysis, besides detecting AN, TN, WW, samples of the suspension were also removed for cytological observation under SEM and TEM.

 

Results

Effects of pH on yeast autolysis

In the process of autolysis, Fig. 1 revealed that the changing trends of concentrations of AN and TN under pH 3.0 condition as well as WW of autolyzed cells were quite different from those of other pH (4.0–7.0) condition, and AN and TN were kept at higher levels after autolysis for 68 h. Compared to other three pH conditions (5.0, 6.0 and 7.0), there were also two small differences in these trends: On one hand, the increasing trend of TN in pH 4.0 was similar to that of pH 5.0, 6.0 and 7.0 in first 36 h, after that, the former increased slowly, however, the other three had secondary quickly growth trends (Fig. 1A); one the other hand, WW of autolyzed yeast cells lost rapidly in the first 6 h, in pH 4.0, but a 6 h lag phase was displayed in Fig. 1C under 5.0, 6.0 and 7.0 conditions.

Fig. 1.The effect of pH on TN (A) and AN (B) contents of autolysate as well as WW (C) of autolyzed cell during autolysis.

Appearance of yeast cell changed more enormous after autolysis for 72 h in pH 3 than others ich became wizened and adhesive (Fig. 2). Yeast cell’s intracellular structure was observed by TEM. It was obviously to see that cytoplasm shrank away from the cytoderm in micrographs of pH 3 (Fig. 3A), but that was not found in the other autolysis processes in pH 4.0–7.0 (Fig. 3B–E). So it could infer that the cause of yeast intracellular changes in pH 3 might be different from other pH conditions.

Fig. 2.Changes of surface morphology in autolyzed yeast cells at different pH for 72 h. (A-E) images of cells autolyzed at pH 3.0, 4.0, 5.0, 6.0 and 7.0.

Fig. 3.Changes of TEM images in yeast autolysis at different pH for 24 h and 72 h. (A-E) images of cells autolyzed at pH 3.0, 4.0, 5.0, 6.0 and 7.0.

Sensory analysis were given in Table 1. Based on taste and aroma, the samples panelists liked from most to least was pH 6.0 > 5.0 > 7.0 > 4.0 > 3.0. And 68% volunteers supported and all professionals agreed that flavor of YE powder from pH 6 was the savouriest and mellowest which were just right suitable for the requirements of the food. Although yields of TN and AN were the highest in pH 3, flavor of YE powder was poor and taste bitter. So considering comprehensive variety of factors (flavor, yields of TN and AN), the optimal pH 5.5 was chosen to do further study.

Table 1.a Ranking test performed by 10 professionals. b Popularity rating came from 100 ordinary volunteers.

Study on autolysis process under pH 5.5

Bio-chemical changes during autolysis.

Fig. 4 displayed correlation analysis of biochemical factors including WW, TN and AN in autolyzed yeast cells. WW lost slowly in the first 6 h, and decreased rapidly during 6–12 h, then decreased slightly until reached the minimum. The initial WW of yeast cells is 5.649 g/10 ml, and the total weight loss at the end of the assay 168 h was 52%. On the contrary, the content of TN and AN increased rapidly in first 6 h, after that the acceleration gradually slowed. When autolysis time reached to 42–60 h, TN presented a high-speed growth again, but not found in AN. After 72 h, no appreciable changes were observed in contents of TN and AN, and reached their maximum, being 0.624 g/100 ml and 0.33 g/100 ml, respectively.

Fig. 4.Changes of contents of TN and AN in autolysate, and of WW during autolysis when pH 5.5 was employed.

The ratio of AN/TN in yeast autolysates increased quickly at first 12 h, shown as Fig. 5, and its stability was maintained from 12 to 36 h, then began to be a sharp drop until remain steady again after 60 h.

Fig. 5.Changes of value of AN/TN in yeast autolysates during autolysis when pH 5.5 was employed.

Morphology changes of yeast cell during autolysis

Morphology changes of yeast cells during autolysis were investigated. The external shape of yeast cells observed by SEM at high magnification (×10000) were exhibited in Fig. 6A –H, and it was clearly displayed that fresh cells were plump, smooth and like ellipsoids, with one or two buds on the surface (Fig. 6A). After one day for autolysis, there were not only a large number of slight wrinkles or folds but also plenty of small particles on the surface of cell walls (Fig. 6B). Furthermore, the size of wrinkles or folds increased but the number of small particles decreased with the autolysis proceeded (Fig. 6B–H) and it seen that the small particles were almost invisible on cell surface after seven days (Fig. 6F–H). Compared to the fresh cells, average diameter of the autolyzed cells reduced significantly (Table 2).

Fig. 6.Changes of surface morphology in autolyzed yeast cells during autolysis when pH 5.5 was employed. (A) images of fresh cells, (B-H) images of cells after 1, 2, 3, 4, 5, 6 and 7 days of autolysis.

Table 2.a LD referred to as “long diameter”. b SD referred to as “short diameter”.

TEM images of cells showed in Fig. 7 revealed that fresh cells were filled with structural elements being well pigmented and distributed. With the proceeding of autolysis, the structure got disordered and interspaces were larger. After 7 days of autolysis, Fig. 7D, a large number of voids appeared in intracellular structure and cells membranes have been completely cracked. However, during the whole process of the yeast autolysis, integrity of cells skeleton was nearly sustained.

Fig. 7.Changes of internal structure in autolyzed yeast cells during autolysis. (A) images of fresh cells, (B), (C) and (D) images of cells autolyzed at pH 5.5 after 1, 3, 7 days of autolysis.

 

Discussion

In this study, several typical characteristic changes have been found in the autolysis process of Saccharomyces cerevisiae FX-2:

First of all, compared to other pH conditions (pH 4.0–7.0), both appearance and internal structure were obviously different when pH 3 was employed. According to Cui Chun, et al. acid hydrolysis specificity was weaker than enzyme hydrolysis, that is to say the former’s hydrolysis rate was faster [1]. During the process of acid hydrolysis, a lot of sulfur-containing compounds were degraded by acid hydrolysis and furan derivatives, furfural, and pyrrole derivatives which made YE powder smell off-flavor [15]. So seen from experimental results, acid hydrolysis migh t occur under the condition of pH 3.

Moreover, the general data-changing trends between TN and AN were not entirely consistent, TN increased quickly at first 6 h, and the secondary quick growth appeared after a slow growth phase for 36 h, then it was to be stable at last (Fig. 4). By contrast, though changing trends of AN and TN were similar in the initial 36 h, the phenomenon of secondary quick growth (PSQG) did not come out correspondingly in the former (Fig. 4). At the same time, the value of AN/TN increased largely at the beginning 12 h, and then dropped sharply after keeping relatively stable level for 24 h (Fig. 5). All above observations indicated that the first quick growth of TN was caused by protein hydrolysis, whereas, PSQG was not come from protein hydrolysis, but mainly from hydrolysate of the other nitrogenous substances. Furthermore, besides protein hydrolyzing, hydrolysis of the other nitrogenous substances such as nucleic acids occurred gradually during the autolysis process from 12 to 36 h. It was worth mentioning, PSQG of TN occurred in pH 5.0, 6.0 or 7.0, but not found in pH 3.0 or 4.0.

According to the reported references, the vacuole can be considered to be equivalent to the lysosome of higher eukaryotes [23], and there are many kinds of hydrolytic enzymes in yeast vacuole such as proteases, lipases and nucleases being considered necessary for the autolytic process [11], moreover, vacuolar proteases are responsible for a large fraction of the total cellular proteolysis in yeast, particularly under conditions of nutrient deprivation [23]. In the early time of autolysis, most yeast cells are not lost their viability (data not shown), and proteases in these viable cells were activated and hydrolyzed intracellular protein to provide new sources of amino acids [11]. Remarkably, Hernawan et al. found that nucleic acid degradation was another characteristic reaction of yeast autolysis, and 80–90% RNA and 25–30% DNA were degraded after 5 days for autolysis, whereas just 25–29% RNA, and 7–15% DNA were lost after autolysis 1 days [6]. Similarly, Li Xiang also found stability of nucleic acid was better than protein’s [10].

All these experimental data and references demonstrated that proteases played a leading role in the early process of yeast autolysis (from 0 to 36 h), which mainly hydrolyzed simple proteins in vacuole at first, then worked on the combined proteins in membrane, and resulted in the vacuole membrane damage (Fig. 7B), and lots of hydrolytic enzymes were released into cytoplasm from vacuoles. Being hydrolyzed by these proteases, the mitochondrial membrane and nuclear membrane were destroyed, then RNA and DNA which contacted with nuclease released from the vacuoles were degradated rapidly, leading to the second sharp growth of TN. However, literatures have reported that optimal pH value of nuclease was 5.0–6.0, and the activity of nuclease would be affected greatly when pH 3.0 or 4.0 was employed. So PSQG of TN was not observed under pH 3.0 and 4.0 conditions, it proved that PSQG of TN was caused by hydrolysis of nucleic acids again. Moreover, when cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3'-, 5'- and 2'-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases, and 5'-ribonucleotide, in particular, can enchance the flavor of production [26]. Oliveira found when aiming at the maximum ribonucleic acid extraction and yeast extract production, the optimization pH value was 5.1 [17]. Hence sensory evaluation of YE powder from experiments of pH 5.0 and pH 6.0 were better than others.

What’s more, WW declined quickly after a short lag phase (approximate 6 h) when the employed pH value was from 5.0 to 7.0 (Fig. 1 and 4). It could be deduced that under the condition of high acidity, acid hydrolysis might occur in yeast cells, and the cell membrane and vacuole membrane permeability increased because of being destroyed, neutral and alkaline protease inactivation or degradation and macromolecular substances such as protein, nucleic acid hydrolyzed and released into autolysate, resulted in the osmotic pressure difference across the membrane, thereby causing lots of intracellular water lost and plasmolysis (Fig. 3B). As a consequence, when pH 3.0 was employed, WW declined quickly at first and the rate of WW decrease was faster than others. While under mild conditions (pH 5.0–7.0), yeast autolysis is mainly caused by endogenous enzymes. And from above analysis, protein only in vacuole was gradually degradated at the initial time, and hydrolysate released slowly, which brought out tardy decrease of WW at the beginning of autolysis. However, after the vacuole membrane rupture, a large number of macromolecular materials were hydrolyzed by their corresponding hydrolysis enzymes, and more hydrolysate released into autolysate which also caused the osmotic pressure difference. Furthermore, it was obvious seen that WW descending rate in pH 4 was higher than others (pH 5.0, 6.0, and 7.0) at first 6 h, which indicated that, in this condition, on the one hand, a slightly higher acidity led to cell contents slightly acid hydrolysis, and on the other hand, endogenous enzymes had hardly negative effect on activity and degraded protein, nucleic acid, and so on.

At last, some studies found that yeast cell wall had been degradated in some extent during autolysis [12]. However in our study, unconspicuous damage on the framework of yeast cells was observed by TEM (at a magnification of 10000×) regardless of serious loss in intracellular substances (Fig. 7C) or plasmolysis (Fig. 3B), and the destruction of the cell wall maybe local, small-scale, instead of a large area of rupture in the course of autolysis. This local change, which made the cell wall get substantially loosened, but still maintain the properties of a semipermeable membrane [19], is responsible for extensive efflux of intracellular small molecules hydrolyzate rather than other macromolecules.

pH had a remarkable effect on yeast autolysis. It was found that high acidity of autolysis environment (pH value lower than 4.0) led to yeast cells destroyed by acid hydrolysis, only when the pH value greater than 4.0, activities of most endogenous enzymes of yeast cells would not be affected and play a key role in enzymatic hydrolysis during autolysis. It was found that, in contrast to the enzyme hydrolysis, in acid hydrolysis, yields of AN and TN was higher and taste of YE powder worse, so based on experimental data, optimal pH 5.5 was employed in the yeast extract production for food.

All the informations from experiments of pH 5.0–7.0 condition indicated that there may be 4 steps during the process of yeast autolysis: (1) Simple proteins in yeast vacuolar were degraded at the beginning of yeast autolysis because of nutrient deficiencies. (2) More membrane-bound protein began to be degraded, at the same time, vacuolar membrane was destroyed, leading to a lot of hydrolytic enzymes being released into the cytoplasm. (3) These activated enzymes reacted with their corresponding substrates resulted in hydrolysis of the internal cell structure including part of compositions of cell wall. So, selective permeability of cell membrane and wall was weakened, leading to the nitrogen content of autolysate increasing again. (4) Hydrolytic enzymes were consumed by themselves and autolysis came to the end, only remaining cell ghosts.