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Final report

Validation of a novel pasteurisation

approach for treating non-animal

by-product waste

This report describes a proposed validation approach

for the novel pasteurisation of non-Animal by

Product (ABP) materials.

Project code: OIN002-017

Research date: 2013/14 Date: September 2014

by-product waste 2

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Document reference : Report OIN002-017

WRAP, 2014, Validation of a novel pasteurisation approach for treating non-animal by-product waste, Prepared

by Richard Thwaites, The Food and Environment Research Agency

Document reference: [e.g. WRAP, 2006, Report Name (WRAP Project TYR009-19. Report prepared by…..Banbury, WRAP]

Front cover photography: Anaerobic Digestion tanks

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by-product waste 3

Contents

Contents ..................................................................................... 3

Executive Summary ................................................................... 4

2. Methods .................................................................... 5

2.1. Description of the pasteurisation system ........................... 5

2.2. Indicator organisms and conducting the pasteuriser test .... 6

2.3. Viability assays ............................................................... 7

3. Results ...................................................................... 7

3.1. The pasteuriser test ........................................................ 7

3.2. Viability of tomato seed ................................................... 8

3.3. Viability of P. brassiciae ................................................... 8

4. Discussion ................................................................. 9

Protocol for process validation of alternatives to pasteurisation for the

processing of non-animal by-product material ........................ 11

1. Preparation of indicator organisms ................................. 11

3. Viability assays ............................................................. 12

References ............................................................................... 14

by-product waste 4

Executive Summary

The pasteurisation requirements imposed by the PAS110:2010 specification for digestate

produced by anaerobic digestion are specified as 70°C for one hour. Previous reports

(WRAP OMK002-007 and OMK002-007S) concluded that alternatives to this may be

acceptable providing that they pass a suitable set of validation requirements and that

these should be based on an assessment of the viability of indicator organisms subjected

to the proposed alternative process. This previous work recommended that two indicator

organisms be used: The fungus Plasmodiophora brassicae and tomato seed.

The opportunity arose to test this validation approach on a digester which incorporates a

high temperature short time pasteurisation step after digestion. The pasteuriser comprises

a continuous flow system in which the separated liquor digestate is heated by a heat-

exchanger to 80°C for at least two minutes prior to cooling and storage. Capsules

containing tomato seed and propagules of P. brassicae, whose viability had previously

been confirmed, were introduced into the pasteurisation system and recovered through a

bypass valve. Indicator organisms were subsequently tested for viability. For tomato seed

this entailed germination in a glasshouse and for P. brassicae a bait-plant approach was

used in which brassica seedlings were incubated with the test material to allow

proliferation of P. brassicae cells which were then detected by real-time PCR.

Experimentation demonstrated that a six-week bait plant incubation was effective in

discriminating viable from non-viable P. brassicae material while a one week rapid

incubation was not. Tests indicated that the HTST pasteurisation system achieved 100%

kill of both tomato seed and P. brassicae.

A protocol for validating alternatives to pasteurisation based on detection of viable

indicator organisms is proposed, incorporating requirements for adequate controls and

selectivity for viable material.

by-product waste 5

1. Introduction

The PAS110:2010 specification for digestate produced in anaerobic digestion (AD) (BSI,

2010) imposes a near-universal requirement for pasteurisation to ensure adequate

reduction of microbial risk to humans, animals and crops. Although the specification

contains no requirement for monitoring of plant pathogens in digestate, it is generally

assumed that pasteurisation at 70°C for one hour is effective in reducing the viability of

the plant pathogens most likely to occur in feedstocks for AD. Research commissioned by

WRAP to investigate the effects of mesophilic AD both with and without pasteurisation on

the viability of a range of plant pathogens (project number OMK002-007; WRAP, 2013)

concluded that survival of all organisms tested was low providing the process

incorporated a pasteurisation step at 70°C for one hour. These data informed a set of

proposed recommendations for permitting alternatives for pasteurisation in a revised

PAS110 specification (WRAP report OMK002-007S; WRAP 2013b) which would require any

digestion process not incorporating a 70°C for one hour pasteurisation step to

demonstrate a defined level of kill for specified indicator organisms. These organisms

would be introduced into the system in sealed containers in a manner that they are

recoverable after the process in which pathogen kill is deemed to occur. The indicator

organisms specified in this proposed protocol were Plasmodiophora brassicae (the causal

agent of clubroot disease of brassicas) and tomato seed. In this respect the proposed

alternative validation method is similar to that specified by the German Waste Ordinance,

which uses P. brassicae and tomato seed as well as tobacco mosaic virus (TMV) as

indicator organisms (Anon, 1998).

The commissioning of a digester facility by Barfoots Energy Ltd, which incorporated a

short-duration high-temperature pasteurisation stage, afforded an opportunity to test the

proposed protocol for alternatives to the 70°C for one hour heat treatment. P. brassiciae

and tomato seed were introduced into the pasteuriser in sealed tubes and recovered

when the allotted pasteurisation time had elapsed. Material was then tested for viability to

compare the level of kill achieved compared to untreated controls.

2. Methods

2.1. Description of the pasteurisation system

Barfoots Energy Ltd commissioned the design and build of a high temperature short time

(HTST) pasteuriser for anaerobic digestate based around a plate heat exchanger. Using

food and beverage industry standards for determining the number of pasteurisation units

equivalent to 70°C for one hour, it was calculated that the same level of pasteurisation

can be achieved by heating to 80°C for 2 min. This was proved to be the case in a

laboratory study and a field test following EU Regulation 142 (2011), in which Salmonella

and canine parvovirus were used as indicator organisms.

The unit is built from stainless steel, with the control systems and fail-safe mechanism

following standards required by the food treatment industry. Digestate from the

secondary digester with a solids content of 5% is passed through a screw auger to

remove fibre material. The separated liquor fraction is passed through a 4 mm filter then

enters a balance tank where it is agitated at low speeds to ensure uniform mixing of

material prior to pasteurisation. The inlet temperature is 40°C, the pasteurisation

temperature is 80°C and the outlet temperature is 50°C. The heat is provided in the form

of hot water generated from the Combined Heat and Power waste heat manifold at 85°C.

Before use, the pasteurisation unit will have been flushed out or cleaned, and will

therefore be full of water. The hot water pump is started and hot water recirculated

around the heat exchanger. Once pasteurisation temperature is reached the variable

speed pumps and flow meters ensure that the material passes through insulated holding

tubes for at least 2 min. At this point the pasteurised material is allowed to pass back

through the heat exchanger in order to heat up the incoming material. The pasteurised

material is under positive pressure compared to the untreated material in order to prevent

any contamination with untreated material in the unlikely event of leakage across the heat

exchanger. The pasteurised product which is at 50°C is then directed into a storage

lagoon.

In the event that either (a) the temperature sensors indicate that the correct temperature

has not been reached in the holding tubes, or (b) the flow meter indicates a residence

time of less than 2 min in the holding tubes then the material is automatically diverted

back to the holding tank for retreatment.

All temperature, flow rate and divert data are recorded on a chart recorder as well as on

loggers. For test purposes sample vials are introduced into the insulated holding tubes at

the first test point immediately after the heat exchanger once the water is up to

temperature and are recovered 2 min later at the second test point before the material

re-enters the heat exchanger.

2.2. Preparation of indicator organisms and conducting the pasteuriser test

Indicator organisms were packaged in 1.5 ml screw-capped polycarbonate tubes. For

tomato seed, each of the eight replicate samples contained 15 seed (cv Ailsa Craig; Mr

Fothergill’s Seeds) in a single tube. Viability of indicator organisms used was ascertained

using the methods described below before the test was administered. For P. brassicae,

symptomatic root material was packed into the sample tubes such that four tubes

contained a minimum of 3 g tissue in total. Thus – for P. brassicae – four tubes

constituted a single sample and all eight replicate samples were packaged in a total of 32

tubes. This was necessary as the aperture of the pasteuriser’s sample port was not large

enough to allow introduction of 3g samples in a single aliquot. After passage through the

pasteurisation unit the four subsamples were pooled for each of the eight replicate

samples. Control samples were treated in the same way.

Four control samples were included for each organism, two of which (labelled C1 and C2)

were stored at 4°C at Fera during the course of the test and two (C3 and C4) were

transported to the pasteuriser packed on ice but were not passed through the pasteuriser.

Samples C3 and C4 were then returned to Fera packed on ice along with the remaining

test samples. Eight test samples (labelled P1-P8) for each indicator organism were

transported to the facility on ice and passed through the pasteuriser as described above

before being transported back to Fera on ice. Vials were passed through the pasteuriser in

batches of four and the time taken for the first vial of each batch to pass from entry to

exit point was recorded.

2.3. Viability assays

Tomato seed were tested for viability by sowing all 15 seeds from each vial in a single pot

in a standard germination compost. Pots were maintained in a glasshouse at 20-25°C for

25 days and were watered daily after which time germination rates were assessed.

P. brassicae viability was assessed by a combined bait plant and real-time PCR process.

One week bait plant tests were conducted by culturing seedlings of cabbage with roots

suspended in an aqueous suspension of the sample material. The six week bait plant test

was conducted using Chinese cabbage plants using the method described in WRAP

(2013). This process allows viable propagules of P. brassicae to infect bait plant roots

thus amplifying the quantity of pathogen material and reducing the likelihood of detecting

DNA from non-viable (ie heat-killed) cells in the subsequent PCR detection process. Two

bait plant protocols were undertaken to determine whether a short (one week) or longer

(six week) incubation with bait plants would yield the most robust results.

For PCR testing, roots of bait plants were removed after their allotted incubation time and

DNA extracted using the “Wizard” Food DNA extraction kit (Promega) incorporating an

automated washing process implemented on a Kingfisher ML magnetic DNA extraction

robot. DNA was subjected to real-time PCR using proprietary PCR assay and protocol at

Fera based on Taqman™ chemistry which specifically detects DNA of P. brassicae. A

second PCR was conducted on all samples in which primers amplifying the plant

cytochrome oxidase gene sequence (COX) were used as a positive control to determine

whether any P. brassicae negative results were due to inhibition of the PCR assay. All PCR

reactions were conducted in duplicate.

3. Results and discussion

3.1. The pasteuriser test

It was not possible to introduce remote dataloggers into the pasteuriser to record

temperatures reached inside the vials because of the restricted size of vials that could be

introduced via the sampling port. However, the pasteuriser’s own control system recorded

a water temperature of 84.5°C for the duration of the test. Transit times for vials through

the pasteuriser ranged from 1 minute 44s to 2 minutes 40s.

3.2. Viability of tomato seed

In each of the four control samples (not passed through the pasteuriser), 14 out of the

total 15 seeds sown germinated within one week of sowing. All seed were allowed a total

of 25 days to germinate, during which time none of the seed in the eight test samples

germinated.

3.3. Viability of P. brassiciae

When samples were incubated for one week, real-time PCR assays conducted on bait

plant roots yielded positive results from all samples, with no apparent reduction in

viability, although no amplification was observed from negative control plants which were

not inoculated with P. brassicae material. Different results were yielded from the six week

bait plant tests: all but one of the positive control samples were positive by PCR for the

presence of viable P. brassicae while none of the plants inoculated with test samples P1-

P8 had detectable levels of P. brassicae DNA. Table 1 shows the results of the PCR assays

conducted on six-week bait plant tests. Numbers quoted are Critical Threshold (Ct)

values, which give the number of PCR cycles required to yield a particular quantity of PCR

product. Lower Ct values therefore indicate the presence of higher amounts of target DNA

in the sample compared with samples yielding higher Ct values.

Table 1. Critical threshold (Ct) values obtained from amplification of DNA extracted from

bait plants incubated for six weeks with sample material. Two PCR assays were

performed; COX, which amplifies the plant cytochrome oxidase gene and the Clubroot

assay which is specific for P. brassicae DNA. NC denotes negative control bait tests (ie

bait plants not inoculated with P. brassicae). PC denotes positive control bait tests (bait

plants inoculated with viable P. brassicae material).

Sample name COX assay Clubroot assay

C1 24.603 No amplification

24.595 No amplification

C2 24.592 34.743

24.463 33.318

C3 24.359 31.252

24.132 30.549

C4 25.378 30.793

25.046 31.121

P1 23.920 No amplification








Sample name COX assay Clubroot assay









NC - A 23.652 No amplification


NC - B 24.886 No amplification


NC - C 23.105 No amplification


PC - A 20.672 16.408

20.601 16.404

PC - B 20.668 16.806

20.527 16.805

PC - C 20.613 16.803

20.787 16.792

Extraction control No amplification No amplification

No amplification No amplification

Water control No amplification No amplification

No amplification No amplification

Positive control (P. brassicae DNA) 21.688 21.081

22.315 21.044

4. Discussion

Results from tomato and P. brassicae viability tests indicate that the HTST pasteurisation

process was effective in killing the indicator organisms and therefore, by implication,

effectively reduced risks from other agricultural pests and diseases examined in previous

studies (WRAP, 2013). It should be noted that the tests described here were administered

in capsules so that the seeds and inoculum were not in contact with the digestate liquor,

whereas in WRAP (2013) the seeds or inoculum were in mesh bags. The effects on

viability are therefore due mainly to the temperature treatment in the capsule. We tested

both one week and a six week bait incubation regimes for P. brassicae viability. PCR

assays conducted on material from one week bait incubations were unable to distinguish

pasteurised from control material. We considered it unlikely that viability had been

retained in all pasteurised samples and we attribute these positive PCR data to a carry-

over of DNA from heat-treated P. brassicae cells through the bait tests into the PCR

assays. There were no symptoms visible on bait plants after a one week incubation. A

longer, six week, bait inoculation was then used to allow adequate time for DNA from

non-viable cells to degrade as well as to permit development of symptoms and therefore

greater sensitivity of detection in post-bait PCR.

Real-time PCR assays conducted on six-week baited material detected viable P. brassicae

in all but one of the controls and none of the pasteurised material. The apparent non-

viability of one of the control samples is likely to be due to degradation of the sample

material before the six-week bait plant test was established. It should be noted that we

encountered some variation between bait plant tests, and our first six-week test failed to

show symptoms, possibly because of low levels of viable P. brassicae in the sample

material, although symptoms were observed on all PCR-positive plants in the second six-

week bait test. However, we do not think this indicates that the test is unsuitable as an

assay for viability of indicator organisms as it is highly specific for viable cells and, with

the inclusion of a real-time PCR test, highly sensitive. The advantage of analytical

methods which determine viability is that the data is highly robust and can accommodate

variation in approaches between laboratories providing adequate controls are

incorporated into the analytical design.

The German Waste Ordinance, which incorporates a facility for validation of alternatives

to pasteurisation, has a requirement for P. brassicae as an indicator species. This

regulation states a cut-off for reduction in P. brassicae inoculum based on a symptom-

scoring approach, ie plants are inoculated and proportion of those showing symptoms is

assessed. The method described here incorporates a DNA-based assay to determine

presence of P. brassicae in infected test plants, which is more sensitive and less open to

inter-lab variation than scoring symptoms alone. This bait plant-PCR approach provides a

robust assessment of whether the pathogen has survived at all during the pasteurisation,

and thus we propose a zero cut-off (ie no amplification of P. brassicae DNA must be

evident for the process to be deemed effective in killing the indicator organism). This this

gives greater certainty over the effectiveness of a discrete sanitation step and is also in

line with the EPPO guidelines for management of plant health risks in plant-derived

biowastes (EPPO, 2008). The latter was originally intended for composting systems, but

the hazards in AD are the same and similar indicator organisms (P. brassicae and tomato

seed) can be used.

Protocol for process validation of

alternatives to pasteurisation for the

processing of non-animal by-product

material

Process validation is required to substantiate methods, alternative to pasteurisation, that

claim to achieve inactivation of plant pests and pathogens equivalent to pasteurisation

requirements set out in the current PAS110 specification. Validation of these alternative

methods entails introduction of indicator organisms at the start of the process in which

inactivation is claimed to occur and recovery of these organisms such that they can be

transferred to a suitable analytical facility at which assessment of viability is then

undertaken. Indicator organisms shall be contained in discreet packages whose integrity

can be verified subsequent to recovery from the system and test administrators can be

certain that all test material has been recovered from the facility.

For the process undergoing validation to be deemed equivalent to pasteurisation at 70°C

for one hour it must be shown to inactivate both indicator organisms using the test

conditions described below.

1. Preparation of indicator organisms

Two indicator organisms must be used: tomato seed (cv. Ailsa Craig) and Plasmodiophora

brassicae. Viability of indicator organisms must be verified before the test proceeds, and

suitable methods for testing viability are described below.

Indicator organisms should be contained in capsules of a size and thermal conductance

that allows sufficient transfer of heat to the centre of the capsule during the test. The

dimensions and construction of the sample capsules may need to be determined

experimentally for each validation. Each sample replicate should contain:

For tomato seed: a minimum of 15 seeds

For P. brassicae: a minimum of 3 g gall tissue

Ideally each sample replicate is contained within a single capsule although it is permissible

to divide each replicate into subsamples contained in multiple capsules if necessitated by

the dimensions of the system undergoing validation. Subsamples can be pooled after

recovery from the system and before viability assays are undertaken.

2. Administering the process validation test Capsules should be introduced as near as practical to the start of the process claimed to

provide pasteurisation equivalence and must be in place within the process for the period

of time specified by the treatment under investigation. On removal, capsules must be

subjected to tests as described below, commencing within 24 h of removal from the

facility.

A minimum of 12 sample replicates of each indicator organism should be used as follows:

a minimum of eight should undergo the process being validated;

a minimum of two should be transported to the facility but not passed through the

facility and are thus positive controls to ensure viable material was provided to the

facility; and

a further minimum of two should be stored at 4°C (±2°C) at the analytical laboratory

as positive controls to account for any loss of viability during transit.

Samples should be placed on ice immediately after removal from the process undergoing

validation (or in the case of the two positive controls, kept on ice while the test is being

administered) and remain on ice for the duration of transit to the analytical laboratory.

3. Viability assays

Viability of tomato seed should be assessed by germination experiments in which all seed

are sown in growing suitable growing media under conditions conducive to germination. A

glasshouse or growth chamber maintained at 20-25°C is recommended. Replicates should

be sown in separate pots and seeds should be allowed to germinate for a minimum of 25

days. The number of germinated and non-germinated seed should be counted. The

process under validation is deemed to be sufficient to inactivate tomato seed as an

indicator organism if zero seed from the eight (or more) test replicates germinate AND if

>90% seed in each control replicate germinate within 20 days.

Viability of P. brassicae should be determined using a method that allows specific

detection of viable cells of P. brassicae. A combined bait-plant incubation and nucleic acid-

based detection should be used to ensure against false-positive results arising from

detection of non-viable cells. Chinese cabbage (Brassica oleracea pekinensis) is

recommended as the bait plant. Roots should be incubated with test material for a six

week period after which nucleic acid is extracted and subjected to analysis using a nucleic

acid amplification test specific for sequences of P. brassicae. Methods are described in

Noble et al. (2011).

Sufficient controls should be included throughout the analytical process to ensure all of

the following:

That negative results are not due to failure of the pathogen to colonise bait plant

roots. Inclusion of positive controls comprising gall material with known viability is

recommended.

That negative results in molecular assays are not due to inhibition of the detection

reaction by compounds co-purified during nucleic acid extraction. Additional DNA

amplification assays targeting plant genes, for example cytochrome oxidase (COX) are

recommended for all samples.

That negative results are not due to failure of the nucleic acid amplification reactions.

Positive controls comprising DNA extracted from P. brassicae gall material should be

included in all tests.

That positive nucleic acid amplification results are not the result of contamination of

amplification reagents with P. brassicae nucleic acid. Negative controls comprising

nucleic acid amplification reagents but without sample template should be included in

all tests.

The process under validation is deemed to be sufficient to inactivate P. brassicae as an

indicator organism if assays detect zero nucleic acid from P. brassicae in the eight (or

more) test replicates AND if the same assay method detects nucleic acid from P. brassicae

in all control samples AND if supporting data is obtained from control tests as described

above.

References

Anon, 1998. Ordinance on the utilisation of biowastes on land for agricultural, sivicultural

and horticultural purposes (Ordinance on Biowastes – BioAbfV). Federal Law Gazette

BGB1. I p. 2955. Germany.

BSI (2010) PAS110:2010 Specification for whole digestate, separated liquor and

separated fibre derived from the anaerobic digestion of source-segregated biodegradable

materials. British Standards Institution, London.

EPPO, 2008. Guidelines for the management of plant health risks of biowaste ofplant

origin. EPPO Bulletin 38(1)

WRAP, 2013. Investigation into the effects of anaerobic digestion processes on some

common agricultural pests and diseases in the UK. WRAP, UK. 58 pp.

Noble, R., Dobrovin-Pennington, A., Pietravalle, S., Weeks, R. and Henry, C. 2011.

Indicator organisms for assessing sanitization during composting of plant wastes. Waste

Management 31 1711-19.

WRAP, 2013b. A consideration of the PAS110:2010 pasteurisation requirements, and

possible alternatives. WRAP, UK. 35 pp.

www.wrap.org.uk/organics

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