Journal of Applied Microbiology, 2024, 135(10), lxae245

https://doi.org/10.1093/jambio/lxae245
Advance access publication date: 26 September 2024
Research Article

Microbiological quality of vegan alternatives to dairy and

meat products in England during 2022–3
Caroline Willis1 ,* , Catherine Startin1 , Frieda Jorgensen1 , Lorraine Sadler-Reeves1 , Heather Aird2 ,
Sandra Lai3 , Corinne Amar4
1
UK Health Security Agency, Food Water and Environmental Microbiology Laboratory Porton, Porton Down, Salisbury SP4 0JG, United

Downloaded from https://academic.oup.com/jambio/article/135/10/lxae245/7778270 by guest on 09 January 2025

Kingdom
2
UK Health Security Agency, Food Water and Environmental Microbiology Laboratory York, York Biotech Campus, York YO41 1LZ, United
Kingdom
3
UK Health Security Agency, Food Water and Environmental Microbiology Services, Colindale, London NW9 5EQ, United Kingdom
4
UK Health Security Agency, Gastrointestinal Bacteria Reference Unit, National Infection Service, 61 Colindale Avenue, London NW9 5EQ,
United Kingdom
∗
Corresponding author. UK Health Security Agency, Food Water and Environmental Microbiology Laboratory Porton, Porton Down, Salisbury SP4 0JG, United
Kingdom. E-mail: caroline.willis@ukhsa.gov.uk

Abstract
Aims: Plant-based alternatives to meat and dairy products have become increasingly popular in the UK. Despite a public perception that they
have a relatively low microbiological risk, outbreaks of illness have been linked with these foods. This study aimed to assess the microbiological
safety and quality of vegan alternatives to dairy and meat products available in England.
Methods and results: Samples were collected between September 2022 and March 2023 from retail, production, and catering premises, and
tested for a range of bacterial pathogens and hygiene indicators using standard procedures. A total of 937 samples were tested, of which
92% were of a satisfactory microbiological quality, 3% were borderline, and 5% were unsatisfactory. Those interpreted as unsatisfactory were
due to elevated counts of Enterobacteriaceae and Escherichia coli (indicators of poor hygiene) rather than pathogenic microorganisms. Listeria
monocytogenes was present in five samples of tofu, all from the same producer (all at counts of <100 CFU g–1 ), while other Listeria species
were detected at counts of <20 CFU g–1 in two burgers and two ‘vegan chicken’ products. The majority of samples did not have pH and water
activity values that would significantly contribute to preventing microbial growth: 62.4% had pH > 5.0 and 82.4% had Aw > 0.94.
Conclusions: The majority of vegan products examined were of a satisfactory quality, but results demonstrate that microbiological control must
be maintained using appropriate processing and storage temperatures, and application of a safe length of shelf life.

Impact Statement
This study is one of the first to assess the hygiene and microbiological safety of vegan alternatives to meat and dairy products. While results
were largely satisfactory, it is important that producers and retailers understand the appropriate control measures to maintain safety throughout
shelf life.
Keywords: vegan; plant-based; microbiological quality; Bacillus cereus; coagulase-positive staphylococci; E. coli; Enterobacteriaceae; Listeria; Salmonella

Introduction (2015) produced a risk ranking model for pathogens in ready

Plant-based foods consumed as alternatives to meat and dairy to eat, non-processed foods of non-animal origin, which iden-
products have become increasingly popular in the UK in re- tified a strong risk for Shigella in fresh pods, legumes, and
cent years. Consumers are choosing plant-based alternatives grains, and moderate risks for Salmonella in nuts and nut
for environmental, lifestyle, and health reasons. A third of products as well as Shiga-toxin-producing Escherichia coli
British meat eaters reported reducing their meat consumption (STEC) and Staphylococcus aureus in fresh pods, legumes, and
in July 2018, and sales of meat-free foods grew by 40% be- grains.
tween 2014 and 2019 (Mintel 2020). In 2019, it was reported Vegan alternatives to milk of animal origin are commonly
that the UK had overtaken Germany as the nation with the produced by soaking nuts, grains, or pulses in water, while
highest number of new vegan food products launched (Mintel cheese alternatives may be made by soaking nuts, with or
2019). without a subsequent fermentation stage. In 2021, a Brie-style
While products of animal origin tend to be considered cashew nut cheese caused an outbreak of Salmonella Duis-
higher risk than plant-based products in terms of microbi- burg, affecting 20 people in the USA (Lewis et al. 2023). An
ological safety (Dewey-Mattia et al. 2018, Piglowski 2019), outbreak of listeriosis in France in 2022 was linked to nut-
there have been reports of foodborne outbreaks associated based cheese alternatives, and affected five people, includ-
with nuts (ECDC-EFSA 2020), seeds (EFSA 2011, Meinen et ing four pregnant women who delivered prematurely (Out-
al. 2019), flour (Vasser et al. 2021), and other plant-based break News Today 2023). An oat-based drink was linked
products (Farakos and Frank 2014). Da Silva Felicio et al. with two reports of illness in 2022 (Food Safety News 2022)

Received 19 July 2024; revised 21 August 2024; accepted 25 September 2024

© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International. This is an Open Access article distributed
under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution,
and reproduction in any medium, provided the original work is properly cited.
2 Willis et al.

and Bacillus cereus was identified in the implicated product. Microbiological examination
Plant-based beverages (coconut, almond, or cashew) were ob- Samples were examined using internationally recognized stan-
served to support the growth of Salmonella, Listeria monocy- dard methods and using culture media supplied either by E &
togenes, and Paenibacillus strains at a higher growth rate than O Laboratories Ltd, Bonnybridge, UK, or by Thermo Fisher
in bovine milk (Bartula et al. 2023), although Bacillus subtilis Scientific, Basingstoke, UK. Examinations comprised: detec-
grew equally fast in bovine milk and almond drink. The au- tion of Salmonella spp. (ISO 6579:2017; all ISO standards
thors of this study concluded that plant-based beverages may available at https://iso.org) by pre-enrichment in Buffered
present a significant risk for listeriosis and salmonellosis, and Peptone Water followed by enrichment in Muller–Kauffman
that recommendations for handling the products post-opening tetrathionate novobiocin and Rappaport-Vassiliades broths
should be carefully considered. and subculture onto xylose lysine deoxycholate and brilliant
Meat alternatives are formulated with a similar protein, green agars; detection and enumeration of Listeria spp., in-
fat, and moisture content to meat, and neutral pH levels, cluding L. monocytogenes (ISO 11290-1:2017 and 11290-
which provide suitable growth conditions for pathogenic and 2:2017) by enrichment in half Fraser followed by Fraser broth

Downloaded from https://academic.oup.com/jambio/article/135/10/lxae245/7778270 by guest on 09 January 2025

spoilage organisms. A study by Tóth et al. (2021) concluded and subculture onto chromogenic Listeria and Oxford agars;
that the raw materials used in meat analogues were more enumeration of the B. cereus group (based on BS EN ISO
perishable than meat products. Microbes proliferated faster 7932:2004;) using a surface inoculation technique on Man-
in samples containing meat alternatives, especially when the nitol egg yolk agar; enumeration of β-glucuronidase produc-
meals were cooled slowly. ing E. coli (based on BS ISO 16649-2:2001) using either a
Despite evidence of microbial contamination associated surface spread or a pour plate technique on tryptone bile
with these food types, production of plant-based vegan prod- agar; enumeration of coagulase-positive staphylococci (based
ucts is frequently seen as a low-risk process. The purpose on BS ISO 6888-1:2021) using a surface spread plate on
of this study was to investigate the microbiological quality Baird–Parker agar; enumeration of Enterobacteriaceae (using
of vegan foods (with a focus on ready-to-eat products) col- either BS ISO 21528-2:2017 or the TEMPO® EB technique
lected from retail, production, and catering premises during [(Biomerieux, Basingstoke, UK) (Owen et al. 2010)]; and enu-
2022–3, and to assess whether physical characteristics such meration of an aerobic colony count (ACC) by surface in-
as pH and water activity may be sufficiently low to control oculation (based on BS EN ISO 4833-2:2013 + A1:2022).
the growth of bacterial pathogens during the shelf life of these Single samples were collected for each product tested
products. and all presence/absence tests were performed on 25 g
aliquots.
The confirmation of identity of bacterial isolates was per-
formed in each of the individual testing laboratories as out-
Materials and methods lined in the standard methods earlier. Microbiological results
were interpreted as unsatisfactory, borderline, or satisfactory
Sample collection according to the guidelines for assessing the microbiological
Samples of plant-based alternatives to meat, fish, or dairy safety of ready-to-eat foods placed on the market (Health Pro-
products were collected over the period from 1 September tection Agency 2009). Regulation (EC) No. 2073/2005 (as
2022 to 31 March 2023 by environmental health practition- amended) on microbiological criteria for foodstuffs (Euro-
ers in accordance with the Food Standards Agency’s Food Law pean Commission 2005) was also used to interpret L. mono-
Practice Guidance (Food Standards Agency 2023). Sampling cytogenes results (Table 1).
officers were requested to focus particularly on ready-to-eat For a sub-set of the samples, pH and water activity were
items that did not require further processing prior to con- also determined (for the first five months of the study, all sam-
sumption. Samples were transported in insulated containers ples were tested for pH and all non-liquid samples for wa-
with sufficient frozen ice packs to maintain a temperature be- ter activity). The samples were first allowed to equilibrate to
tween 2◦ C and 8◦ C. All samples were examined by one of the room temperature. For pH determination of solid food sam-
three UK Health Security Agency (UKHSA) Food, Water and ples, a representative portion was taken and a pH electrode
Environmental Microbiology (FW&E) Laboratories in Eng- was placed either against a cut surface of the sample or into a
land located in London, Porton, or York. Microbiological test- homogenate of the sample. For liquid products, the pH elec-
ing commenced within 36 h of sampling. trode was placed directly into an aliquot of sample. Water
Data were collected by local authority staff on each individ- activity was determined using the Novasina LabMaster wa-
ual sample using a standardized questionnaire that included ter activity meter (Novatron Scientific Ltd, Horsham, UK) ac-
the type, name, and address of the business premises; date, cording to the manufacturer’s instructions.
time, and temperature of the sample at collection; a sample
description and type of product; the use-by date for the con-
sumer; country of origin; packaging type; and whether re- Characterisation of L. monocytogenes by whole
sampling had occurred in the same premises due to a previ- genome sequencing
ous poor microbiological result or because of physical obser- Cultures of L. monocytogenes were sent to the UKHSA Gas-
vations of hygiene concerns in the businesses’ environments. trointestinal Bacteria Reference Unit (GBRU) for confirma-
The information from the questionnaire was recorded via the tion and further characterisation by whole genome sequenc-
FW&E laboratory information management system (LIMS) ing (WGS), as described previously (Chattaway et al. 2016,
and extracted into Excel spreadsheets. To reduce bias on McLauchlin et al. 2021). Briefly, DNA from purified cultures
the microbiological results from examination of these foods, of L. monocytogenes was obtained by automated extraction
products known to be associated with incidents of foodborne (QIAsymphony DSP DNA Kit, Qiagen, Manchester, UK) ac-
illness were not included in this survey. cording to manufacturer’s instructions. Genomic DNA was
Microbiology of vegan foods 3

Table 1. Interpretive criteria for microbiological test results.a

Microbiological quality (CFU g–1 )

Target Satisfactory Borderline Unsatisfactory Unacceptable/potentially hazardous

Salmonella Not detected in 25 g N/A N/A Detected in 25 g

L. monocytogenes <10 10–100 N/A >100
Listeria speciesb <10 10–100 >100 N/A
E. coli <20 20–100 >100 N/A
Enterobacteriaceae <100 100–104 >104 N/A
B. cereus group <103 103 to <105 >105 N/A

N/A: not applicable.

a
Health Protection Agency (2009) and European Commission (2005).
b
Not including L. monocytogenes.

Downloaded from https://academic.oup.com/jambio/article/135/10/lxae245/7778270 by guest on 09 January 2025

sequenced by the UKHSA Central Sequencing Laboratory: but packs were already opened at the point of sampling. A
sample was prepared using Nextera XT (Illumina Inc, San total of 90 samples (9.6%) were described as ‘loose’ (i.e. not
Diego, USA) and sequenced using the Illumina HiSeq 2500 pre-packed), and this information was not available for 76
platform with 2 × 100 bp reads (Illumina, Inc.). Short reads samples (8.1%).
were quality trimmed using Trimmomatic removing the se-
quence adaptor. Clonal complexes (CCs) were derived from
WGS analysis and were assigned using Metric Oriented Se- Results of microbiological testing
quence Typer (MOST) (Tewolde et al. 2016) in accordance Results of microbiological testing for each of the target bacte-
with the designation of the Institut Pasteur International Mul- rial groups/parameters are shown in Table 3. Combining the
tilocus sequence typing (MLST) database for L. monocy- results for all microbiological parameters, 866 (92%) were of
togenes (http://bigsdb.pasteur.fr/listeria/listeria.html). A core a satisfactory microbiological quality, 27 (3%) were border-
single-nucleotide polymorphism (SNP) alignment for each CC line, and 43 (5%) were unsatisfactory. The results interpreted
was generated using SnapperDB (Dallman et al. 2018). Pair- as unsatisfactory were due to elevated counts of Enterobac-
wise comparisons of SNP distances were performed between teriaceae and E. coli (indicators of poor hygiene) rather than
cultures within similar CCs (Dallman et al. 2018). Isolates the presence of pathogenic microorganisms.
linked within a 5 SNP single-linkage cluster were considered ACC were relatively low (<104 CFU g–1 ) in 88% of sam-
to be part of the same point source with each culture hav- ples, with a higher proportion of elevated counts seen in cheese
ing ≤5 SNPs difference with at least one other culture within (15% with ACC > 104 CFU g–1 ) and meat alternatives (16%)
that same cluster. Genomic data were stored in a customized compared to milk (2%) and other products (5%) (Table 4).
database (Gastro Data Warehouse), and pairwise comparisons This difference was significant (Fisher’s exact test: P < 0.002).
of SNP addresses were performed on isolates from vegan Counts were also more likely to be elevated in those sam-
products and cultures from clinical cases, which occurred in ples that were loose (unpackaged) or in open packages at the
the UK, as well as other isolates from food or the environ- time of sampling (24% with ACC > 104 CFU g–1 ) compared
ment. Sequence data are available through https://www.ncbi. to those that were in unopened packs (10.6%; Fisher’s exact
nlm.nih.gov/bioproject/?term=PRJNA248549. test: P = 0.0003; Table 4). A higher proportion of samples
that were unpackaged or in opened packages also had border-
Statistical methods line or unsatisfactory Enterobacteriaceae levels (17.4%) com-
pared to those in unopened packs (4.7%; Fisher’s exact test:
Descriptive analysis of the data was undertaken by using Mi-
P < 0.0001; Table 2).
crosoft Excel (Microsoft Corporation, Redmond, WA, USA).
Salmonella was not detected in any samples, B. cereus
Statistical analysis was carried out using Fisher’s exact test
was detected at borderline levels in two samples (Table 3),
(GraphPad Software). A probability value of <5% was de-
L. monocytogenes was present in five samples (all at counts
fined as significant.
of <100 CFU g–1 ), and other Listeria species were detected in
four samples (all at counts of <20 CFU g–1 ). Of the samples in
which L. monocytogenes was detected, two had Enterobacte-
Results
riaceae counts of >104 CFU g–1 and three had counts between
Sample types and origins 102 and 104 CFU g–1 .
Overall, 937 samples were collected by sampling officers from The five samples in which L. monocytogenes was detected
92 local authorities in England. Of these, 414 (44%) were were all samples of tofu from a single producer. One organic
meat substitutes, 246 (26%) were vegan cheeses, 138 (15%) natural tofu sample, collected from a retailer in January 2023,
were plant-based milks, 112 (12%) were other dairy alter- highlighted an initial problem (L. monocytogenes detected at
natives, 11 (1%) were fish alternatives, and 16 (2%) were a count of 20 CFU g–1 ), which led to follow-up sampling di-
other vegan items, including tofu, egg alternatives, and vegan rectly from the producer on a further three occasions: five
desserts. Of the 937 samples, 841 (89.8%) were from retail, follow-up samples of organic natural tofu were taken from the
88 (9.4%) were from producers , and 8 (0.9%) were from producer in early February 2023, with L. monocytogenes de-
catering. tected in three (of which one was at a count of 20 CFU g–1 and
The majority of samples (746; 79.6%) were pre-packed in two were at <20 CFU g–1 ); five samples of various tofu prod-
unopened packaging at the time of sampling (Table 2). A fur- ucts (natural, smoked, marinated tofu, and tofu burgers) were
ther 25 samples (2.7%) were described as being pre-packed, taken in late February, all of which were negative for Listeria;
4 Willis et al.

and five more samples of different product types were taken

10 (11.1)

17 (19.3)
38 (4.1)
11 (4.5)

27 (6.5)

20 (2.7)
8 (10.5)

21 (2.5)
in March 2023, of which L. monocytogenes was detected in

>104

0
0
0

0
Enterobacteriaceae (CFU g–1 ) one marinated tofu sample (20 CFU g–1 ).
Samples in which other Listeria species were detected were
all meat substitutes: two burger samples contained L. welsh-
meri and L. innocua, respectively, while two ‘chicken’ prod-

10 (11.4)
102 –104

30 (3.2)

19 (4.6)

4 (16.0)
15 (2.0)

19 (2.3)

1 (12.5)
7 (2.8)

4 (3.6)

6 (6.5)

5 (6.6)
ucts both contained L. seeligeri.
0

0
Characterisation of L. monocytogenes
Isolates from the original tofu sample (collected in January
869 (92.7)
228 (92.7)

108 (96.4)

368 (88.9)

711 (95.3)

801 (95.2)
138 (100)

74 (82.2)
21 (84.0)

63 (82.9)

61 (69.3)
11 (100)

16 (100)

7 (87.5)
<102

2023), two of the follow-up samples (February) and the fi-

nal follow-up sample (March) were all of serotype 1/2a, ST37

Downloaded from https://academic.oup.com/jambio/article/135/10/lxae245/7778270 by guest on 09 January 2025

with whole genome sequences showing that they were identi-
cal strains with 0 SNP difference between them. In contrast,
Other spp.

the isolate from the third sample collected from the same pro-
Listeria species detected in 25 g

4 (0.4)∗

4 (1.0)

2 (8.0)
2 (0.3)

4 (0.5)
Number (%) of samples with specified result

duction premises in February was of serotype 4, ST145, which

0
0
0
0

0
0
was therefore unrelated to the 1/2a strain. Interrogation of the
UKHSA database did not identify any isolates from cases of
human illness that were closely related to these food isolates
L. monocytogenes

(at the <25 SNP level).

5 (0.5)

5 (1.2)

5 (0.7)

5 (5.7)
0
0
0
0

0
0

Determination of pH and water activity in vegan

products
A total of 772 samples were tested for pH. Of these, 41 (5.3%)
had a pH of <4.0; 108 (14.0%) had pH between 4.0 and 4.5;
141 (18.3%) had pH between 4.5 and 5.0; and the remainder
(62.4%) had pH > 5.0. Water activity was determined for
B. cereus > 10 CFU g–1

500 samples. Of these, 7 samples (1.4%) had Aw < 0.9; 81

Table 2. Microbiology results for vegan products categorized by type of product, packaging, and sampling setting.

(16.2%) had Aw between 0.9 and 0.94; and 412 (82.4%) had
3

2 (0.2)
2 (0.8)

2 (0.3)

2 (0.2)

Aw > 0.94.
0
0
0
0
0

0
0

0
0

The five tofu samples in which L. monocytogenes was de-

tected gave pH values between 5.3 and 6.3, and for two of
these samples where Aw was determined, the Aw was 0.97. A
sample of Camembert-style cheese with a borderline level of
B. cereus (2800 CFU g–1 ) had pH 5.2 and Aw 0.95–neither of
which would be sufficiently low to prevent the growth of B.
Other Listeria species comprised two L. seeligeri, one L. innocua, and one L. welshimeri.
–1

cereus. The second sample with a borderline B. cereus level

E. coli > 10 CFU g

(8800 CFU g–1 ) was a garlic and herb ‘soft cheese’ product,
and this had pH 4.4, which is sufficiently low to control B.
3 (0.3)
3 (1.2)

3 (0.4)

3 (0.4)
2

0
0
0
0
0

0
0

0
0

cereus growth during shelf life (Aw not determined for this
product).

Discussion
Number of samples (%)

This study is one of the first to assess the hygiene and microbi-
ological safety of vegan alternatives to meat and dairy prod-
246 (26.3)
138 (14.7)
112 (12.0)

414 (44.2)

746 (79.6)

841 (89.8)

ucts. Products of non-animal origin are recognized as being

11 (1.2)

16 (1.7)

90 (9.6)
25 (2.7)

76 (8.1)

88 (9.4)
8 (0.9)
937

associated with outbreaks of infection (EFSA 2013, Callejon

et al. 2015, Bennett et al. 2018). There are multiple oppor-
tunities for microbial contamination of plant-based ingredi-
ents before, during, and after harvest as well as during pro-
cessing and in retail, catering, and domestic environments. In
addition, these products may be subject to further processes
such as fermentation during production of the final product,
Other dairy alternatives

which may introduce additional opportunities for pathogen

Pre-packed—unopened

growth. However, results from this study have demonstrated

Cheese alternatives
Category of sample

Other vegan foods

Pre-packed—open
Meat alternatives

that the vast majority of plant-based alternatives to meat

Milk alternatives

Fish alternatives

Packaging type

and dairy products, sampled at retail, catering, or produc-

Total samples

tion in England, were of a satisfactory microbiological qual-

Not stated

Producer
Retailer

ity.
Caterer
Settings
Loose

The collection of appropriate samples for this study was de-

pendent on environmental health practitioners across England
∗
Microbiology of vegan foods 5

Table 3. Percentage of samples of vegan products with satisfactory, borderline, or unsatisfactory microbiological quality for different target bacteria.

Number (%) of samples

Target bacteria Number of samples tested Satisfactory Borderline Unsatisfactory

All targets 937 866 (92.4) 27 (2.9) 43 (4.6)

B. cereus 920 918 (99.9) 2 (0.2) 0
Enterobacteriaceae 937 869 (92.7) 30 (3.2) 38 (4.1)
E. coli 937 934 (99.7) 0 3 (0.3)
L. monocytogenes 937 934 (99.7) 3 (0.3) 0
Listeria species (other than L. monocytogenes) 937 937 (100) 0 0
Salmonella 927 927 (100) 0 0

Downloaded from https://academic.oup.com/jambio/article/135/10/lxae245/7778270 by guest on 09 January 2025

Table 4. Percentage of samples of vegan products with ACCs within the specified ranges.

Number (%) of samples with ACC (CFU g–1 ) in specified range

Category of sample Number of samples tested <104 104 to <105 105 to <106 106 to <107 ≥107

Cheese alternatives 245 207 (84.5) 9 (3.7) 10 (4.1) 5 (2.0) 14 (5.7)

Milk alternatives 136 133 (97.8) 2 (1.5) 0 1 (0.7) 0
Other dairy alternatives 112 103 (92.0) 4 (3.6) 0 0 5 (4.5)
Fish alternatives 11 10 (90.9) 0 1 (9.1) 0
Meat alternatives 412 344 (83.5) 23 (5.6) 18 (4.4) 11 (2.7) 16 (3.9)
Other vegan foods 16 16 (100) 0 0 0 0
Packaging type
Loose 90 67 (74.4) 11 (12.2) 5 (5.6) 3 (3.3) 4 (4.4)
Pre-packed—open 23 19 (82.6) 2 (8.7) 1 (4.3) 0 1 (4.3)
Pre-packed—unopened 744 665 (89.4) 22 (3.0) 20 (2.7) 9 (1.2) 28 (3.8)
Not stated 75 62 (82.7) 3 (4.0) 3 (4.0) 3 (4.0) 4 (5.3)

selecting samples according to specified instructions. While in- explain at least some of the elevated levels of this group
structions requested a focus on ready-to-eat products in par- of bacteria.
ticular, some samples did not appear to be ready to eat (e.g. The low ACCs in the majority of milk samples are likely
‘meat-free burgers’ might be expected to be heated prior to to reflect the frequent use of ultra-heat treatments or other
consumption). For these samples, interpretation according to high-temperature processes on these products to achieve mi-
the criteria in Table 1 may be excessively stringent, but even crobiological stability at ambient temperature during an ex-
taking this into account, the proportion of unsatisfactory sam- tended shelf life. In contrast, a higher proportion of the meat
ples was low. and cheese-style products had elevated ACCs, which is con-
Interpretation of results as borderline or unsatisfactory in- sistent with lower cooking temperatures (or no cooking) and
cluded consideration of Enterobacteriaceae levels. These are the use of fermentation processes during production of some
a group of Gram-negative bacteria that may be naturally of these products.
present in non-processed plant-based ingredients. While they It is recognized that ingredients commonly used in ve-
are readily killed by heat processes, it may be normal to find gan cheese and milk production, such as nuts, beans, and
these organisms in plant-based products that do not involve oats, may be frequently contaminated with spore-forming mi-
a heating stage during production. The majority of bacte- croorganisms, including B. cereus (Akbas and Ozdemir 2006,
ria in this group are non-pathogenic, but their presence in Kyrylenko et al. 2023). Nicholls et al. (2016) described an out-
cooked, ready-to-eat foods can be an indication of poor hy- break of B. cereus food poisoning amongst 182 children and
giene; moreover, these organisms can contribute to spoilage 18 staff attending nurseries in the UK, and reported that the
of foods, even at refrigeration temperatures. Therefore, for conditions used by the caterer to soak dried beans prior to
the purposes of this study, the criteria specified in Table 1 cooking in one of the products supplied to the nurseries was
were used for the interpretation of Enterobacteriaceae results. likely to have allowed growth of B. cereus to sufficient levels
On this basis, 3% of samples had a borderline level of En- to cause illness. Moreover, an outbreak of B. cereus food poi-
terobacteriaceae and 4% had an unsatisfactory level. This soning that affected 20 children in Norway was reported to
may be a slightly overcautious interpretation where prod- be linked to the consumption of porridge (Food Safety News
ucts have not undergone a heat process, but since informa- 2024). Production of plant-based dairy alternatives often in-
tion on production processes is not always available at the volves a heat-treatment step, which would be expected to re-
time of testing in the laboratory, a more cautious approach duce bacterial numbers in the final product. However, spore-
was considered to be appropriate. Moreover, a higher pro- forming bacteria such as Bacillus and Clostridium species are
portion of samples that were unpackaged or in opened pack- more likely to survive the heat process. Control of Bacillus
ages at the time of sampling had borderline or unsatisfac- growth in such products may include maintaining a low pH
tory Enterobacteriaceae levels compared to those in unopened and/or water activity, low storage temperature, and poten-
packs, indicating that poor hygiene/cross-contamination may tially modified atmosphere. Two samples of soft-style vegan
6 Willis et al.

cheese had borderline counts of B. cereus (>103 CFU g–1 ). One (Investigation, Methodology), Sandra Lai (Investigation), and
of these products had a pH level that would be low enough Corinne Amar (Investigation, Writing – review & editing)
to minimize the growth of Bacillus, but for the other prod-
Conflict of interest: None declared..
uct, neither the pH nor the water activity was sufficiently low
to control Bacillus growth. Therefore, storage at an appropri-
ate refrigeration temperature (with or without modified atmo- Funding
sphere) and maintenance of an appropriate length of shelf life
would be important to ensure the continued microbiological None declared.
safety of these products.
Listeria monocytogenes was detected at low counts in five
Data availability
tofu products from the same producer over a 2-month period.
According to microbiological criteria set out in EC 2073/2005 The data underlying this article are available in the article.
(as amended) (European Commission 2005), the detection of

Downloaded from https://academic.oup.com/jambio/article/135/10/lxae245/7778270 by guest on 09 January 2025

L. monocytogenes in a 25 g portion of ready-to-eat food is
acceptable in any product that has left the control of the man- References
ufacturer, as long as the count does not exceed 100 CFU g–1 Akbas MY, Ozdemir M. Effectiveness of ozone for inactivation of Es-
during shelf life. However, at the end of the manufactur- cherichia coli and Bacillus cereus in pistachios. Int J of Food Sci
ing process, presence of L. monocytogenes, even at counts Tech 2006;41:513–9. https://doi.org/10.1111/j.1365-2621.2005.0
of <100 CFU g–1 , is unsatisfactory unless the shelf life is less 1099.x
Bartula K, Begley M, Latour N et al. Growth of food-borne
than 5 days or the pH and water activity are low enough to
pathogens Listeria and Salmonella and spore-forming Paenibacillus
control growth (i.e. pH ≤ 4.4 or Aw ≤ 0.92, or a combina- and Bacillus in commercial plant-based milk alternatives. Food Mi-
tion of pH ≤ 5.0 and Aw ≤ 0.94). While tofu is generally crobiol 2023;109:104143. https://doi.org/10.1016/j.fm.2022.1041
subjected to further cooking prior to consumption (so would 43
not normally be considered ready to eat), there are recipes Bennett SD, Sodha SV, Ayers TL et al. Produce-associated food-
available that use uncooked or lightly cooked tofu as ingre- borne disease outbreaks, USA, 1998–2013. Epidemiol Infect
dients. Therefore, while the risk of L. monocytogenes infec- 2018;146:1397–406. https://doi.org/10.1017/S095026881800162
tion from these tofu products is likely to be low, it is undesir- 0
able for L. monocytogenes to be present, particularly given the Callejón RM, M. Rodríguez-Naranjo I, Ubeda C et al. Reported food-
borne outbreaks due to fresh produce in the United States and Euro-
high (i.e. non-inhibitory) pH and Aw values in these product
pean Union: trends and causes. Foodborne Pathog Dis 2015;12:32–
types. Other Listeria species were detected in four different 38. https://doi.org/10.1089/fpd.2014.1821
meat-substitute products. These species are not pathogenic, Chattaway MA, Dallman TJ, Gentle A et al. Whole genome sequencing
but do indicate that the conditions during production and/or for public health surveillance of Shiga toxin-producing Escherichia
post-production storage and handling have allowed Listeria coli other than serogroup O157. Front Microbiol 2016;7:258. https:
to be introduced and to survive, and therefore highlight a po- //doi.org/10.3389/fmicb.2016.00258
tential risk of L. monocytogenes presence in these product Dallman T, Ashton P, Schafer U. et al. SnapperDB: a database solution
types. for routine sequencing analysis of bacterial isolates. Bioinformatics
Results from this study indicate that pH and water activity 2018;34:3028–9. https://doi.org/10.1093/bioinformatics/bty212
are rarely sufficiently low to control pathogen growth (only Da Silva Felício MT, Hald T, Liebana E et al. Risk ranking of pathogens
in ready-to-eat unprocessed foods of non-animal origin (FoNAO) in
37% of those tested had pH < 5.0 and 18% had Aw < 0.94).
the EU: initial evaluation using outbreak data (2007–2011). Int J
Therefore, these physical parameters are unlikely to have a Food Microbiol 2015;195:9–19. https://doi.org/10.1016/j.ijfoodmi
significant impact on the safety of products during shelf life, cro.2014.11.005
and microbiological control must depend on appropriate pro- Dewey-Mattia D, Manikonda K, Hall A et al. Surveillance for
cessing and storage temperatures, and determination of a safe foodborne disease outbreaks—United States 2009–2015. MMWR
length of shelf life for the majority of products of this type. Surveill Summ 2018;67:1–11. https://doi.org/10.15585/mmwr.ss67
While this study provides reassuring evidence that plant- 10a1
based alternatives to meat and dairy products consumed in ECDC-EFSA. Multi-Country Outbreak of Salmonella typhimurium
England are generally of low microbiological risk, it is im- and S. anatum Infections Linked to Brazil Nuts. 2020.
https://www.ecdc.europa.eu/sites/default/files/documents/salmo
portant that producers and retailers maintain an awareness
nella- typhimurium- rapid- outbreak- assessment- october- 2020.pdf.
of relevant risks and reliable controls.
(July 2023, date last accessed).
EFSA (European Food Safety Authority). EFSA panel on biological haz-
ards (BIOHAZ) panel; scientific opinion on the risk posed by Shiga
Acknowledgements
toxin-producing Escherichia coli (STEC) and other pathogenic bac-
The authors would like to thank colleagues in local authori- teria in seeds and sprouted seeds. EFSA J 2011;9:2424.
ties for sampling and investigations, and staff in the UKHSA EFSA (European Food Safety Authority). Scientific opinion on the
laboratories for testing and providing information involved in risk posed by pathogens in food of non-animal origin. Part 1 (out-
this survey. No ethical approval was required.. break data analysis and risk ranking of food/pathogen combina-
tions). EFSA J 2013;11:3025. https://doi.org/10.2903/j.efsa.2013.
3025
Author contributions European Commission. Commission regulation (EC) No.
2073/2005 on Microbiological Criteria for Foodstuffs. 2005.
Caroline Willis (Writing – original draft), Catherine Startin http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:
(Investigation, Writing – original draft), Frieda Jorgensen 32005R2073&from=en. (March 2022, date last accessed).
(Methodology, Writing – review & editing), Lorraine Sadler- Farakos S, Frank J. Challenges in the control of foodborne pathogens in
Reeves (Methodology, Project administration), Heather Aird low-water activity foods and spices. In: Gurtler J, Dyle M, Kornacki
Microbiology of vegan foods 7

J (eds). The Microbiological Safety of Low Water Activity Foods 2017. Euro Surveill 2019;24:1800543. https://doi.org/10.2807/15
and Spices. New York: Springer, 2014, 15–34. 60-7917.ES.2019.24.36.1800543
Food Safety News. Oatly Recalls Drink Because of Bacillus cereus; 2 Mintel. Veganuary: UK Overtakes Germany as World’s Leader for
Sick with 27 Other Complaints Filed. 2022. https://www.foodsafe Vegan Food Launches. 2019. https://www.mintel.com/press-centr
tynews.com/2022/04/oatly- recalls- drink- because- of- bacillus- cereu e/veganuary- uk- overtakes- germany- as- worlds- leader- for- vegan- fo
s- 2- sick- with- 27- other- complaints- filed/#more- 213286. (August od-launches/. (March 2024, date last accessed).
2023, date last accessed). Mintel. Plant-Power: UK Sales of Meat-free Foods Shoot Up 40%
Food Safety News. Norway Solves Outbreak Involving Young Chil- Between 2014–19. 2020. https://www.mintel.com/press-centre/plan
dren. 2024. https://www.foodsafetynews.com/2024/02/norway-solv t- based- push- uk- sales- of- meat- free- foods- shoot- up- 40- between- 2
es- outbreak- involving- young- children/. (March 2024, date last ac- 014-19/. (March 2024, date last accessed).
cessed). Nicholls M, Purcell B, Willis C et al. Investigation of an outbreak of
Food Standards Agency. Food and Feed Law Codes of Practice. vomiting in nurseries in south-east England, May 2012. Epidemiol
2023. https://www.food.gov.uk/about- us/food- and- feed- codes- of- Infect 2016;144:582–90. https://doi.org/10.1017/S0950268815001
practice. (February 2024, date last accessed). 491

Downloaded from https://academic.oup.com/jambio/article/135/10/lxae245/7778270 by guest on 09 January 2025

Health Protection Agency. Guidelines for Assessing the Microbiolog- Outbreak News Today. France: Listeria Infections Reported
ical Safety of Ready-to-Eat Foods Placed on the Market. 2009. Linked to Jay and Joy Vegetable Alternatives to Cheese. 2023.
https://www.gov.uk/government/uploads/system/uploads/attachm https://outbreaknewstoday.com/france- listeria- infections- reported-
ent_data/file/363146/Guidelines_for_assessing_the_microbiolog linked- to- jay- joy- vegetable- alternatives- to- cheese- 53832/. (March
ical_safety_of_ready- to- eat_foods_on_the_market.pdf. (March 2024, date last accessed).
2022, date last accessed). Owen M, Willis C, Lamph D. Evaluation of the TEMPO most proba-
Kyrylenko A, Eijlander RT, Alliney G et al. Levels and types of mi- ble number technique for the enumeration of Enterobacteriaceae in
crobial contaminants in different plant-based ingredients used in food and dairy products. J Appl Microbiol 2010;109:1810–6.
dairy alternatives. Int J Food Microbiol 2023;407:110392. https: Piglowski M. Pathogenic and non-pathogenic microorganisms in the
//doi.org/10.1016/j.ijfoodmicro.2023.110392 rapid alert system for food and feed. Int J Environ Res Public Health
Lewis K, Vasser M, Garman K et al. Notes from the field: multistate, 2019;16:477. https://doi.org/10.3390/ijerph16030477
multiserotype outbreak of Salmonella infections linked to cashew Tewolde R, Dallman T, Schaefer U et al. MOST: a modified MLST
brie—United States, 2021. Morb Mortal Wkly Rep 2023;72:589– typing tool based on short read sequencing. PeerJ 2016;4:e2308.
90. https://doi.org/10.15585/mmwr.mm7221a4 https://doi.org/10.7717/peerj.2308
McLauchlin J, Aird H, Amar C et al. Listeriosis associated with pre- Tóth AJ, Dunay A, Battay M et al. Microbial spoilage of plant-based
prepared sandwich consumption in hospital in England, 2017. Epi- meat analogues. Appl Sci 2021;11:8309. https://doi.org/10.3390/ap
demiol Infect 2021;149:e220. https://doi.org/10.1017/S095026882 p11188309
1001916 Vasser M, Barkley J, Miller A et al. Notes from the field: multistate
Meinen A, Simon S, Banerji S et al. Salmonellosis outbreak with novel outbreak of Escherichia coli O26 infections linked to raw flour—
Salmonella enterica subspecies enterica serotype (11:z41:e,n,z15) at- United States, 2019. Morb Mortal Wkly Rep 2021;70:600–1. https:
tributable to sesame products in five European countries, 2016 to //doi.org/10.15585/mmwr.mm7016a4

Received 19 July 2024; revised 21 August 2024; accepted 25 September 2024

© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International. This is an Open Access article distributed under the
terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in
any medium, provided the original work is properly cited.