Using RVA to measure pre-germination in barley and predict germination energy after storage

Pre-germination in barley

In the absence of dormancy, Canadian malting barley varieties have a propensity to pre-germinate in the field. Pre-germination is the premature sprouting of grain while still in the ear. Pre-germination can occur before full grain maturity is reached; this event is called ‘precocious germination’. More often, however, pre-germination is encountered as a consequence of prolonged spells of wet weather when mature grain remains uncut in the field; this event is called ‘pre-harvest sprouting’.

Although severely sprouted grain can be identified by visual inspection of barley, conducted during the selection processes, incipient germination may be more difficult to detect. The germination energy (GE) test does not indicate pre-germination in barley. Pre-germinated grain may show acceptable characteristics, including good germination percentage and may produce good quality malt. However, the capacity of barley to maintain a high level of germination energy during long-term storage is diminished by pre-germination. Barley initially accepted for malting may be rejected after several months in storage if it loses germination capacity and no longer has the high, uniform germination rates that produce good quality malt.

In 2005, the Canadian Grain Commission teamed up with industry partners to assess whether instrumental methods can be used to detect pre-germination in barley. It was determined that rapid visco analysis (RVA) was the best method to detect and measure the degree of pre-germination in barley.

The early stages of germination proceed without any visible changes to the kernel. However, there are numerous biochemical reactions that occur inside the kernel. One of the enzymes produced very early during germination is alpha-amylase, the enzyme responsible for catalyzing reactions leading to the conversion of starch into sugars used by the growing embryo. Since the level of alpha-amylase in sound grain is very low compared to its level in the germinating grain, the content of alpha-amylase in grain can be used as a marker of germination. RVA indirectly estimates the amount of alpha-amylase in barley by measuring the viscosity of ground barley in water.

Using RVA and interpreting RVA results

The test can be done using RVA instruments. The final viscosity of a mixture of ground barley and water is determined at 3 minutes after starting a specific heating and stirring protocol. The results are displayed as viscosity in centipoise (cP) or Rapid Visco Units (RVU).

Higher final viscosity values indicate lower amylase activity and no pre-germination in barley; in this case the risk of rapid germination loss during storage is very low. Low final viscosity values indicate higher amylase activity and moderate to high pre-germination; the risk of germination loss during storage is much greater.

Risk of raipd germination loss in storage RVA final viscosity
RVU (units) cP
Low ≥ 120 ≥ 1440
Intermediate ≥ 50 & < 120 ≥ 600 & < 1440
High < 50 < 600
1 RVU = 12cP

Samples with final viscosity values higher than 120 RVU (1440 cP) are normally considered sound and the probability that they will retain GE after storage is very high. However, the use of a single cut-off value to predict whether barley will retain its GE after storage may not be fully reliable because the retention of GE depends also on the storage conditions and moisture content of grain.

Samples with final viscosity values between 50 and 120 RVU (600-1440 cP) are moderately pre-germinated, and the risk that they will lose GE after storage is intermediate. The risk can be lowered if the samples are dry and/or they are stored in cool and dry conditions.

Samples with final viscosity values lower than 50 RVU (< 600 cP) units are pre-germinated, and the probability that they will lose GE after storage is high. They should be malted as soon as possible.

To predict safe storage time more accurately, not only the degree of pre-germination in barley, but also the storage conditions (temperature and relative humidity) and the initial moisture content of the grain have to be taken into account.

Testing for pre-germination using RVA – recommended practices

Apparatus

  1. Rapid Visco Analyser: StarchMaster viscometer (Newport Scientific, Pty. Ltd; Perten Instruments Inc.).
  2. Falling Number Laboratory Mill 311 with 0.8 mm screen or equivalent.
  3. NIR spectrometer or other suitable equipment for moisture content determination
  4. Laboratory balance accurate to ±0.01 g
  5. Bottle-top dispensette or other volumetric pipetting device capable of delivering 25.0 ml
  6. Sample canisters and paddles
  7. Robot Dispenser (optional)

Reagents

  1. Distilled water
  2. Calibration oil - to check the viscometer calibration. See Note 3.3.

Procedure

1. Instrument preparation

1.1 Switch on the RVA unit and allow a 30-min warm-up.

1.2 In order to ensure the accuracy of measurements, the calibration offset (zero viscosity) should be checked daily. Please follow the instructions in the Installation and Operation manual.

1.3 The calibration of the RVA unit should be checked with a standard oil of known viscosity once per month. See note 3.3. Heat the oil to between 60 and 70°C and weigh out 28.0 ± 0.1 grams. Measure the viscosity of the calibration oil using the CAL95 program (profile designed for a calibration check). The viscosity of the calibration oil should be within ± 5% of the recommended value provided with the standard.

1.4 The samples should be be analyzed using the Stirring Number (SN) profile or equivalent. The test temperature should be 95±1°C. Initial stirring (0-10 sec) 960 rpm; stirring for the remainder of the test (10 sec – 3 min) is 160 rpm. The duration of test is 3 min.

2. Sample preparation and analysis

2.1 Determine the moisture content (%) of the whole grain by NIR or another suitable method.

2.2 Grind a representative sample (~300g) using the Falling Number Laboratory Mill 3100 or a suitable mill to pass a 0.8-mm sieve. Clean the grinder thoroughly after each sample.

2.3 Thoroughly stir the ground sample before weighing out a sub-sample. Weigh the appropriate amount of ground sample into a canister. Use the moisture content determined in step 2.1 and the “moisture conversion table” provided below to determine the amount of sample in grams to be weighed into a canister.

2.4 Add exactly 25.0 ml of distilled water using a bottle-top dispensette or other suitable volumetric pipetting device.

2.5 Mix the contents of the canister with a spatula to disperse the sample. Ensure that no lumps remain at the bottom of the canister where the paddle cannot reach. Use the paddle to scrape off the sample from the spatula and place the paddle into the canister.

2.6 Insert the canister into the instrument and initiate the test by pressing the tower down.

2.7 Record the Final Viscosity as displayed in cP (centipoise) units. Divide by 12 to convert into RVU.

2.8 Remove the canister upon completion of test with an insulated glove and discard.

2.9 Run each sample in duplicate.

Alternative procedure using Robot Dispenser

Please refer to the manual for installation and operation of Robot Dispenser. The Robot should be filled with fresh distiller water every day and the calibration check should be performed every day.

Conduct steps 2.1 and 2.2 as previously.

2.3a Place an empty canister on the balance and add ~4.00±0.15g of ground barley. Follow the instructions displayed in the window.

2.4a Set the control dial to the appropriate moisture content of the sample and press the dial to continue. Wait until the Robot will dispense the appropriate amount of water.

Continue through steps 2.5-2.9 as outlined above.

Alternative procedure using Robot Dispenser
M.C. (%) Weight (g) M.C. (%) Weight (g)
8.0 3.74 12.5 3.93
8.1 3.74 12.6 3.94
8.2 3.75 12.7 3.94
8.3 3.75 12.8 3.94
8.4 3.76 12.9 3.95
8.5 3.76 13.0 3.95
8.6 3.76 13.1 3.96
8.7 3.77 13.2 3.96
8.8 3.77 13.3 3.97
8.9 3.78 13.4 3.97
9.0 3.78 13.5 3.98
9.1 3.78 13.6 3.98
9.2 3.79 13.7 3.99
9.3 3.79 13.8 3.99
9.4 3.80 13.9 4.00
9.5 3.80 14.0 4.00
9.6 3.81 14.1 4.00
9.7 3.81 14.2 4.01
9.8 3.81 14.3 4.01
9.9 3.82 14.4 4.02
10.0 3.82 14.5 4.02
10.1 3.83 14.6 4.03
10.2 3.83 14.7 4.03
10.3 3.84 14.8 4.04
10.4 3.84 14.9 4.04
10.5 3.84 15.0 4.05
10.6 3.85 15.1 4.05
10.7 3.85 15.2 4.06
10.8 3.86 15.3 4.06
10.9 3.86 15.4 4.07
11.0 3.87 15.5 4.07
11.1 3.87 15.6 4.08
11.2 3.87 15.7 4.08
11.3 3.88 15.8 4.09
11.4 3.88 15.9 4.09
11.5 3.89 16.0 4.10
11.6 3.89 16.1 4.10
11.7 3.90 16.2 4.11
11.8 3.90 16.3 4.11
11.9 3.90 16.4 4.11
12.0 3.91 16.5 4.12
12.1 3.91 16.6 4.12
12.2 3.92 16.7 4.13
12.3 3.92 16.8 4.13
12.4 3.93 16.9 4.14

3. Notes

3.1 Analyze each sample within a few minutes after adding water.

3.2 A new canister should be used for each analysis

3.3 Certified Viscosity Standard may be obtained from the Cannon Instrument Company. Please note that some RVA units are calibrated at 25°C. In this case, the calibration check should be done with CAL25 profile and appropriate oil.

Summary of the collaborative study conducted in 2005.

For more information and/or full report of the study please contact:

Marta Izydorczyk, Ph.D.
Grain Research Laboratory, Canadian Grain Commission
303 Main Street
Winnipeg, MB R3C 3G8

Tel: (204) 983-1300
Email: marta.izydorczyk@grainscanada.gc.ca

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