Materials and Methods

MSc Project > Materials & Methods

This post is part of a series detailing the findings of my MSc research project which looked at the effects of different fermentation parameters on wort souring with Lactobacillus. If you haven’t already, take a look at the MSc project page for a full overview.

Wort preparation and reagents

Wort was prepared at specific gravities (SG) of 1.030, 1.040, 1.050 and 1.060 in 1 L Duran® bottles using extra light spray dried malt extract (DME) from Ritchies (Burton, UK) and distilled water. Each mixture was agitated at 300 rpm on magnetic stir plates for 10 min prior to being autoclaved at 121°C for 15 min at 0.1 MPa. The specific gravity of all wort produced was measured using an DMA 4100 M density meter (Anton Paar, Graz, Austria) after autoclaving. This was done to document variations in wort SG between batches as fresh wort was made when required. All reagents used were of analytical grade from either Sigma-Aldrich (Saint Louis, USA) or Thermo Fisher Scientific (Waltham, USA) unless otherwise stated.

Bacterial strains, isolation and culture conditions

The Lactobacillus species used in this study was Lactobacillus brevis strain WLP672 purchased from White Labs (San Diego, USA). The selection was based upon its availability from a commercial supplier to the brewing industry. The culture was stored at 4°C in the packaging provided by the manufacturer (35 ml vial) until required.

In order to isolate the Lactobacillus strain, both Raka-Ray and MRS (de Man, Rogosa and Sharpe) agar plates (Oxoid, Basingstoke, UK) were prepared as directed by the manufacturer. Selective Raka-Ray and MRS plates were also made by the addition of 0.1% cycloheximide (10 mg/L) prior to autoclaving. All media was autoclaved in 1 L Duran® bottles at 121°C for 15 min at 1 Bar. Plates were streaked with a loop from the supplied commercial cultures and incubated under aerobic conditions for 72 h at 30°C.

To propagate the strain for use in laboratory scale fermentations a 1.040 SG wort was prepared as described. The hot break material present after autoclaving was immediately removed by centrifuging the hot wort in sterile 50 ml centrifuge tubes for 3 min at 40,000 rpm. Six sterile 15 ml centrifuge tubes were filled with 10 ml of the autoclaved and centrifuged 1.040 SG wort then cooled to 20°C. Each centrifuge tube was subsequently inoculated with a loop of multiple colony forming units taken from a single agar plate. The L. brevis WLP672 colonies originated from three Raka-Ray with cycloheximide plates. The tubes were sealed and incubated under static conditions at 30°C for 24 h. The final propagation step was performed by adding each 10 ml culture to separate sterile 150 ml laboratory bottles filled with 100 ml of the same 1.040 SG wort at 20°C. The bottles were incubated under static conditions at 30°C for 24 h and if not used immediately were stored at 4°C. One L. brevis WLP672 culture was used as a stock solution and sub cultured by taking 10 ml and adding it to 100 ml of autoclaved and centrifuged 1.040 SG wort before incubating under static conditions at 30°C for 24 h.

Viable cell enumeration

A concentration curve for L. brevis WLP672 was developed using 10 ml of L. brevis WLP672 stock culture inoculated in 100 ml of autoclaved and centrifuged 1.041 SG wort that was incubated under static conditions at 30°C for 24 h. The measurement and serial dilutions were performed according to Campbell.1 The diluted L. brevis WLP672 samples were spread on MRS agar plates and incubated under aerobic conditions for 72 h at 30°C before counting the number of colonies on each plate. The colony forming units per ml (CFU/ml) was plotted against the OD600 measurements for a range of dilutions, resulting in a linear trendline that allowed estimates of bacterial concentration and inoculation rates for WLP672 cultures. For all OD600 measurements distilled water was used to zero the UV-Vis Spectrophotometer.

Lactobacillus souring trials

A series of small scale fermentation trials using L. brevis WLP672 were performed to investigate the effect of different variables on lactic fermentation. The variables used in each fermentation are shown in Table 1. Wort was prepared to the required specific gravities as detailed previously. For LF3, adjustment of wort pH to 5, 4.5 and 4 was done by adding DL-lactic acid prior to autoclaving. A further drop in pH was observed after autoclaving, yielding pH values of 4.84, 4.25 and 3.76. Sterile 50 ml centrifuge tubes were filled with 45 ml wort and inoculated at 20°C with the required volume of 24 h stock culture. The OD600 of the 24 h L. brevis WLP672 stock culture in conjunction with the calibration curve was used to estimate the volume necessary to achieve the desired inoculation rate (approximately 1×107 CFU/ml). The centrifuge tubes were then sealed and allowed to ferment in dark, static conditions at the temperatures specified (Table 1). Samples were taken at 24, 48 and 72 h. Each tube was centrifuged at 40,000 rpm for 3 min prior to analysis of pH, total acidity (TA) and specific gravity.

Table 1. Parameters for fermentation in barley malt wort with L. brevis WLP672

IDTemp. (°C)Initial wort SGInoculation rate (CFU/ml)Adjusted wort pH
LF120, 30, 371.0431×107
LF2301.031, 1.049, 1.0611×107
LF3301.0421×1074.84, 4.25, 3.76
LF4301.0410.2, 2 and 4×107

Measurement of pH, TA and D-/L-lactic acid

All wort and fermented samples were analysed for pH using a HI 83141 pH Meter (Hanna Instruments, Woonsocket, USA) and TA according to the ASBC Beer-8 Potentiometric Titration Method.2 The TA was reported as a g/L of a specific acid, in this case lactic acid, calculated as follows:

(1)   \begin{equation*}  TA = \frac{ (V_1 \times c_1 \times M)} {V_2}$ \end{equation*}

where
TA = Total acidity as g/L of organic acid
V_1 = Volume (ml) of NaOH to reach pH 8.2 endpoint
V_2 = Volume (ml) of wort or beer sample
c_1 = Molarity (mol/L) of NaOH
M = Molar mass (g/mol) of organic acid

For all TA measurements 25 ml wort/beer samples were titrated using 0.1 M NaOH to an end point of pH 8.2. To express TA as g/L of lactic acid (LA) a molar mass of 90 g/mol was used for lactic acid. Inputting these values, equation (1) becomes:

(2)   \begin{equation*}  TA = \frac{ (V_1 \times 0.1 \times 90)} {25}$ \end{equation*}

where
TA = Total acidity as g/L of LA
V_1 = Volume (ml) of NaOH to reach pH 8.2 endpoint

To account for the differences in initial wort TA and give an indication about the organic acid yield during lactic fermentation, acid produced as g/L of LA was calculated as follows:

(3)   \begin{equation*}  AP = TA_2 - TA_1 \end{equation*}

where
AP = Acid produced (g/L of LA)
TA_1 = TA (g/L of LA) of sample before fermentation
TA_2 = TA (g/L of LA) of sample after fermentation

Equation (3) makes the assumption that any organic acids already present in a sample were not altered during lactic fermentation. It has however been shown that there are some acids present in malt wort, albeit in low concentrations, such as malic acid that are degraded by LAB, including L. brevis.3 As changes in individual acid concentration weren’t measured, it’s unkown if any degradation actually took place. For the purposes of this study it’s therefore presumed that as the same bacterial strain was used throughout, if any degradation did occur, it was uniform across all fermentations or that the differences were negligably small. With this limitation in mind, acid production values were only used for comparative purposes between fermentations with different starting acid concentrations and shouldn’t be confused with the experimentally measured TA values.

Statistical analysis

All fermentations and corresponding analyses were carried out in triplicate, unless otherwise noted. All values are expressed as mean ± standard deviation. Regression analysis was carried out using GraphPad Prism 7 (La Jolla, USA) with a statistical significance value set at p = 0.05.

Microscopy

Bacterial cultures and fermented samples were wet mounted on glass microscope slides with cover slips and examined at 400x total magnification using a Unilux-12 bright field microscope (Kyowa, Sagamihara, Japan).

References

  1. Campbell, J. (2011) High-throughput assessment of bacterial growth inhibition by optical density measurements, Curr. Protoc. Chem. Biol., 2, 195-208. https://doi.org/10.1002/9780470559277.ch100115
  2. American Society of Brewing Chemists. Methods of Analysis, online. Beer-8a. Total acidity by potentiometric titration. Approved 1958, rev. 2016. The Society, St. Paul, MN, USA. https://doi.org/10.1094/ASBCMOA-Beer-8
  3. Nsogning, S. D., Fischer, S., Becker, T. (2018) Investigating on the fermentation behavior of six lactic acid bacteria strains in barley malt wort reveals limitation in key amino acids and buffer capacity, Food Microbiol., 73, 245-253. https://doi.org/10.1016/j.fm.2018.01.010

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