Material and experimental units

Ready-mix media, yeast extract, and other media components for microbial culture were purchased from HiMedia Laboratories, India. Chemicals for preparing washing solutions and for analysis experiments were provided by Xilong Scientific Co., Ltd, China with analytical grade. Otherwise, it will be stated.

Brewer's spent yeast slurry was kindly provided by HEINEKEN Vietnam Brewery Limited Company in Danang city, Vietnam.

Probiotic strains were preserved and provided by the Biotechnology Laboratory, The University of Danang, University of Science and Technology and Cell Laboratory, Institute of Biotechnology, Hue University of Science. They include 4C, sp., and BL21 (DE3).

Methods

Spent yeast slurry collected from the brewery was stored in a sterilized flask. To remove the beer liquor, brewer’s yeast slurry was refrigerated (4-10°C) overnight, and then the liquid at the top was dumped, retaining the sludge on the bottom. Before conducting the experiments, the brewer's spent yeast slurry was examined under a microscope and by culture on nutrient and MRS media to ensure the microbial purity of the yeast waste.

The cream yeast (harvested after giving an off-white liquid) was washed in buffer 1 (distilled water), buffer 2 (0.5% NaCl), and buffer 3 (0.1% Tween 80) at a weight ratio of 1:3 (cream yeast:buffer) before further processing. After gently stirring with a glass rod, the cream yeast was centrifuged at 3500 rpm for 15 mins at 4°C (Hettich Rotina 420 R, Germany). Washing buffers collected from each wash were subjected to tannin and iso-alpha acid determination.

Autolysis was carried out in sterilized glass jars in duplicate. The debittered yeast slurry was resuspended in autolysis buffer (1.5% (v/v) ethyl acetate and 0.086 M NaCl, pH 5.5) and adjusted to an OD value of 40 22. Autolysis was performed at 50°C in an incubator (Daihan Scientific IS-30, South Korea) with a shaking speed of 200 rpm. During autolysis, samples were taken, pasteurized at 80°C for 30 mins to terminate the enzyme activity, cooled to room temperature, and then centrifuged at 3500 rpm for 15 mins at 4°C for chemical analysis of the supernatant at the indicated time points.

Yeast cells at 0 h and 36 h of autolysis were observed under an Olympus optical microscope at 40X magnification of the objective lens.

Dry yeast weight and solid content were determined by drying the condensed solution in an oven (Memmert SF260plus, Germany) at 105°C to a constant weight. The total carbohydrate content was determined by Bertrand’s method 23. The Kjeldahl method was used to analyze the total nitrogen content in dried spent yeast cells. The protein content was estimated by multiplying the total nitrogen content by 6.2524. Samples are burned at high temperatures (500–600°C) in a muffle furnace to assess the amount of ash produced25. Then, the collected ash was dissolved in 3 M HCl solution, and the residual residue was filtered and burnt to determine the amount of acid insoluble ash. In addition, to ensure the results, the collected brewer’s spent yeasts were sent to the testing center TSL (TSL Science Co., Ltd) for analysis and double examination of some important parameters, such as protein, carbohydrate, lipid and calories.

The protein content in yeast extracts was determined by the Biuret method 26 with casein as the standard and a linear curve from 0-50 mg/mL (R is 0.9952). Alpha-amino nitrogen was measured by the ninhydrin method using glutamic acid as a standard and a linear curve from 0.2-0.6 mM (R is 0.9789) 6. The tannin content in the washings and in the yeast extracts was determined by UV‒Vis spectroscopy (Dlab SP-UV1100, China) as described previously27, 28, using tannic acid as a standard and a linear curve from 0.625-10 mg/L (R is 0.9992). The bitter substances in the samples were quantified by the European Brewery Convention (EBC) method9, 29.

The sonication treatments were carried out in continuous mode with the following parameters (Misonix XL-2000, New York, USA): approximately 1.3-1.5 g of dry yeast, 40 ml of D.I. water, ultrasound amplitude of 40%, temperature of 5°C, sonication time of 5 min, probe depth of 1 cm, and 60% of the maximum amplitudes30. The amount of tannin and bitterness units of this sonicated solution were determined as the total amount of these compounds per dry weight of yeast cells.

The yields of protein or α-amino nitrogen content extracted from dry yeast cells were calculated by the following formula:

C = (Amount of protein or α-amino nitrogen)/(weight of dry yeast slurry used) (mg/g or mmol/100 g) (1)

The percentage yield of bitterness removal was calculated by the following formula:

Y = (total EBU in washing solution per dry weight of yeast cells)/(EBU of yeast cells disrupted by sonication per dry weight of yeast cells)*100(%) (2)

The percentage yield of bitterness removal was calculated by the following formula:

Y = (total tannin amount in washing solution per dry weight of yeast cells *100 (%))/(tannin amount of yeast cells disrupted by sonication per dry weight of yeast cells) (3)

Extracellular protease activity and culture of microorganisms

The extracellular crude enzymes were collected by centrifugation of overnight cultures of microbial cells at 6000 rpm for 5 minutes at 4°C. Two hundred microliters of supernatant was placed into the hole in an agar plate containing 2% casein to investigate the microbial extracellular protease. Transparent circles due to casein degradation represented microbial protease activity.

Media for the culture of each probiotic strain were prepared according to the corresponding recipe. 4C was grown in MRS medium supplemented with 1.5% NaCl (10 g/L meat extract; 5 g/L yeast extract; 10 g/L peptone; 20 g/L D-glucose; 8.3 g/L CHCOONa.3HO; 2.62 g/L KHPO.3HO; 0.12 g/L MgSO. HO; 0.06 g/L MnSO. HO; 1% Tween 80; 15 g/L NaCl)31. The growth medium of was YDP (20 g/L peptone; 10 g/L yeast extract; 20 g/L glucose) 32, and BL21 (DE3) was cultured in LB (10 g/L tryptone; 5 g/L yeast extract; 10 g/L NaCl)33. After autolysis, the solution was concentrated at 80-85°C with stirring, and in 6 hours, the concentration of soluble solids was 90.56 g/L. The concentration of produced yeast extract added to growth media was calculated based on the dry weight matter in the extracts. Commercial yeast extract was used as a control.

All strains of bacteria were grown in an incubator at a temperature of 30-37°C with shaking at 140-150 rpm. The change in cell density after 20-22 h of culture in different media is illustrated by optical density at 600 nm. The OD value shown in the graph is 10 times diluted to ensure an OD value within the measured range of the instrument.

The results obtained were evaluated for statistical significance by ANOVA, and Tukey's honest significant difference test or Duncan’s multiple range test was used for statistical analysis of differences in the mean of groups (p<0.05). All statistical analyses were carried out by using the Windows program SPSS version 10.0 (Chicago, IL) and Microsoft Excel 2019. The results are shown as the means and standard deviations obtained from at least three independent experiments.

Characteristics of brewer's spent yeast slurry

Brewer's spent yeast slurry was collected from a local Heineken brewery and stored in a sterilized container for transport to the laboratory. The characteristics of the brewer's yeast slurry were analyzed and are presented in Table 1.

The excessive moisture in the yeast slurry of approximately 90% has been a major obstacle in the reuse of yeast extracts due to the large volume required for storage. However, these yeast cells settle down at low temperatures. Therefore, the appropriate settling time at refrigerator temperature (4 - 10°C) to remove excessive liquor before performing further experiments was determined. The results showed that after 12 h of sedimentation, most of the spent yeasts settled and stuck quite firmly to the bottom of the container, and then the supernatant was taken out conveniently. The remaining yeast slurry had approximately 60% moisture.

The analysis of spent yeast composition showed that this was a rich source of protein (38.3-40.2% dry yeasts) and carbohydrate (49.2-50.7% dry yeasts), and thus, this has been a valuable source for many different applications (Table 1).

Tween 80 for decontaminating brewer’s spent yeast cells

Tannins and other bitter substances from hop resins are the main contaminants causing the bitter and astringent flavor 34 in the extract produced from brewers’ spent yeast slurry. Moreover, tannin and iso-alpha acid possess antibacterial activity35, 36, which is able to inhibit the growth of bacteria cultured in media containing produced yeast extract from them.

To remove impurities, three different types of wash buffers were employed in this study, including distilled water, 0.5% NaCl, and 0.1% Tween 80 solution. Compared with other wash buffers, the removal efficiency of tannin and bitter substances by Tween 80 was the most effective, except in the third wash to remove bitter substances. Especially for tannins, using Tween 80 gave significantly superior washing power with double as much amount in the 1 wash compared to the other wash solutions, and in the 2 wash, the tannin was removed by only Tween 80 (Figure 1A). After two washings with 0.1% Tween 80, the tannin removal effectiveness was 93.2% as opposed to the 25% of tannins removed by distilled water and 0.5% NaCl. The results also showed that the tannin content in the wash solution varied considerably in different batches (Figure 1A), probably due to the fluctuations of contaminated tannins. Tannins are not a strictly controlled indicator in brewing, and their amount depends on hop quality and barley bark. Meanwhile, bitterness is an important criterion of beer quality, so its content was relatively stable, and the variability was quite low (Figure 1B). The yield of bitterness removal after washing with distilled water, hypotonic 0.5% NaCl and 0.1% Tween 80 solution was 77.7%, 79.65%, and 83.6%, respectively. The total amount of bitterness removed after three washes was not statistically significant among the three washing solutions. However, in the 1 and 2 washes alone, washing with Tween 80 still prevailed, and thus, using a mild and safe detergent such as Tween 80 saves time in cleaning yeast cells for further applications. Together, these results showed that washing yeast slurry twice is adequate to remove unwanted contaminants from the spent yeast cells, where the tannin content was almost undetectable, and the bitterness content decreased significantly.

Autolysis is a process that occurs when yeast cells die at temperatures above 45°C. This process is caused by a series of autologous enzymes that breakdown the yeast cell wall and membrane, releasing the substances inside the cell into the solution, known as yeast extract. Figure 2 displays yeast cell wall cleavage after 36 hours of autolysis at 50°C. Under the microscope, it was observed that yeast cells no longer had clear borders but instead appeared rough with holes. (Figure 2B).

To reassess the removal efficiency of tannin and bitter substances by different wash buffers, the content of these two substances in the autolysates of yeast cells washed by different buffers was measured. The results shown in Figure 3 were compatible with the washing efficiency by Tween 80 observed in Figure 1. The tannins and bitterness units in the autolysates of yeast cells washed with Tween 80 were significantly lower than those in other extracts (Figure 3A&B). Next, the growth of 4C in a medium supplemented with autolysates from yeast cells that had been washed with various buffers was observed 4C is a gram-positive bacterium sensitive to inhibition by tannins and iso-alpha acids37, 38. The sensitivity was verified as the cell biomass of 4C reached its peak when the medium was supplemented with extracts from yeast cells washed with Tween 80 (as shown in Figure 3D), even though there was no difference in protein and α-amino nitrogen content among the extracts (Figure 3C). The 4C density increased by 16.5% and 10.2% with Tween 80-pretreated extract compared to that washed with distilled water or 0.5% NaCl, respectively.

The residual tannins in the autolysate of yeast cells washed with NaCl solution were significantly lower, only approximately 50% of those washed with distilled water (Figure 3A). This observation contradicted the findings in Figure 1A, in which tannins washed out by 0.5% NaCl solution were substantially lower than those washed by 0.1% Tween 80 but on par with those washed with distilled water. Yeast cells washed with 0.5% NaCl and distilled water were thus predicted to have the same tannin content in their autolysate. The reason for this unexpected result is probably due to Na+ strengthening the connections between tannins and proteins or carbohydrates 39, so it is challenging to remove tannins adhering to yeast cell walls or cell membranes during the subsequent washing and autolysis process. Next, the tannin complexes and cell debris are probably precipitated from the autolysis fluid through centrifugation and discarded, so the tannin in the extract was low.

The effect of time on autolysis efficiency

The economic efficiency of the production of extracts from spent yeast cells is affected by autolysis time. Next, the change in soluble solids concentration, protein, and α-amino nitrogen content during autolysis of brewer's spent yeasts washed with 0.1% Tween 80 was evaluated.

After 6 hours of autolysis, the concentration of soluble solids and protein content within the autolysate increased considerably. Concurrently, there was a significant reduction in the mass of yeast cells (Figure 4). Then, the yeast cell mass and soluble solids content barely changed (Figure 4A). Protein content markedly dropped after 12 hours of autolysis. This is because protease secreted from disintegrated yeast cells degrades protein and causes the concentration of α-amino nitrogen to be enriched (Figure 4B).

MRS (for ) and YPD (for ) media were prepared according to the reported recipes but replaced with yeast extracts at different collection times instead of commercial ones. The media were then used for the cultures of respective probiotics. Here, two strains of probiotics 4C and sp. possessing different abilities to secrete extracellular protease were utilized. In particular, the extracellular protease of was very low compared to as evaluated by casein degradation (Figure 5A&B). Casein breakdown by 4C resulted in a substantially larger translucent circle (27.5 mm) than that by (3.5 mm). As a result, the demand for amino acids or hydrolyzed proteins in the culture medium of the two strains may also be different. The density of showed almost no difference after 20 h of culture in different media, except for the control (Figure 5A). The cells that were grown in the medium containing the extract collected at 12 hours or earlier had a slower growth rate compared to those grown in the medium with yeast extract autolyzed for 24 hours or more, which showed a 10% higher yeast biomass ( < 0.01). It is likely that the lower activity of extracellular proteases in contributes to this phenomenon. To facilitate nutrient absorption and growth, it may be advantageous to add partially hydrolyzed protein or amino acids from extracts during longer autolysis times.

The combined findings demonstrated that the extract obtained following a 24-hour autolysis process was suitable for the culture of a variety of probiotic strains with a range of extracellular protease activities. Therefore, in subsequent investigations, the composition and efficacy of the microbial culture were compared to those of commercial yeast extracts by using brewer’s spent yeast extract obtained after 24 hours of autolysis.

Probiotic culture by extract supplementation.

Through the growth of (DE3) in suitable media with the addition of brewer's spent yeast extract, the ability to utilize the extracts for microbial culture was assessed. As a control, microorganisms were grown in media without or with commercial yeast extracts. The amount of yeast extract in the culture medium was adjusted to match the commercial product based on dry matter content.

The addition of yeast extract had a favorable effect on the proliferation of microbial cells (Figure 6). Particularly for BL21 (DE3), which is frequently employed in the manufacture of recombinant proteins, bacterial biomass increased by 300% compared to the medium without yeast extract. The biomass of and also increased by 34.5% and 67.7%, respectively, by the addition of yeast extract. For all the examined microorganism strains, the density of microbial cells that reproduced in the medium supplemented with an extract from brewers' spent yeast cells was equivalent to that in the medium supplied with commercial yeast extract.