International Journal of Food Microbiology 242 (2017) 13–18

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Microbial counts of mealworm larvae (Tenebrio molitor) and crickets

(Acheta domesticus and Gryllodes sigillatus) from different rearing
companies and different production batches
D. Vandeweyer a,c,⁎, S. Crauwels b,c, B. Lievens b,c, L. Van Campenhout a,c
a
KU Leuven, Department of Microbial and Molecular Systems (M2S), Lab4Food, Technology Campus Geel, B-2440 Geel, Belgium
b
KU Leuven, Department of Microbial and Molecular Systems (M2S), Laboratory for Process Microbial Ecology and Bioinspirational Management (PME & BIM), Technology Campus De Nayer, B-
2860 Sint-Katelijne-Waver, Belgium
c
KU Leuven, Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: The rising interest in insects for human consumption and the changing regulations in Europe require a profound
Received 13 June 2016 insight into the food safety of insects reared and sold in Western society. The microbial quality of edible insects
Received in revised form 20 October 2016 has only been studied occasionally. This study aimed at generating an overview of intrinsic parameters (pH,
Accepted 7 November 2016
water activity and moisture content) and microbial quality of fresh mealworm larvae and crickets for several
Available online 9 November 2016
rearing companies and for several batches per rearer. In total, 21 batches obtained from 7 rearing companies
Keywords:
were subjected to analysis of intrinsic parameters, a range of plate counts and presence-absence tests for Salmo-
Edible insects nella spp. and Listeria monocytogenes. The microbial counts of the fresh insects were generally high. Different
Mealworm larvae rearing batches from a single rearing company showed differences in microbial counts which could not be ex-
Crickets plained by variations in intrinsic properties. The largest variations were found in numbers of bacterial endo-
pH spores, psychrotrophs and fungi. Salmonella spp. and L. monocytogenes were not detected in any of the
Water activity (aw) samples. Altogether, our study shows that large variations were found between batches from individual rearers.
Microbial counts As a consequence, no overall differences between rearers could be observed.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction foods. Hence, more research data on the microbial quality of edible in-
sects reared in Europe are necessary to support risk assessments per-
Interest in human consumption of edible insects (entomophagy) in formed by the European Food Safety Authority (EFSA) (Belluco et al.,
Western countries is increasing (Caparros Megido et al., 2014; Mlcek 2013). Additionally, more quantitative data concerning the microbial
et al., 2014) and more and more insect-based food products are being quality will be needed in order to establish microbial criteria for edible
marketed. Compared with Asian, African, Oceanian and Latin American insects in the future, similar to existing criteria for other food products
regions, Western society has no history of insect consumption (Regulation (EC) N° 2073/2005 on microbiological criteria for
(Siemianowska et al., 2013; van Huis, 2013; van Huis et al., 2013; Yen, foodstuffs).
2015). However, insects are a promising and valuable alternative to Presently, the nutrient composition of several insect species has al-
conventional protein sources such as meat. They provide an opportunity ready been studied extensively (Finke, 2002; Nowak et al., 2016;
to meet the increased protein demand of the growing world population Rumpold and Schlüter, 2013a; Sánchez-Muros et al., 2014;
(Mlcek et al., 2014; Premalatha et al., 2011; van Huis et al., 2013). More- Siemianowska et al., 2013). Microbiological data, however, are only
over, rearing insects for food has a smaller ecological footprint com- scarcely available, as highlighted in a recent EFSA opinion (EFSA
pared to traditional animal husbandry (Oonincx and de Boer, 2012; Scientific Committee, 2015). Moreover, the few studies available con-
Oonincx et al., 2010; van Huis et al., 2013). taining microbiological data (Giaccone, 2005; Grabowski et al., 2014;
Insects will be considered as Novel Food in Europe starting from Jan- Klunder et al., 2012; Rumpold et al., 2014) do not include analyses of
uary 2018, as stated in the new Regulation (EU) 2015/2283 on novel different production batches or insects from different rearing compa-
nies. So far, there is only one study including different production
⁎ Corresponding author at: Lab4Food, KU Leuven, Technology Campus Geel,
batches (Stoops et al., 2016), but they originate from only one rearing
Kleinhoefstraat 4, B-2440 Geel, Belgium. company. None of the studies available contain data on intrinsic proper-
E-mail address: [email protected] (D. Vandeweyer). ties of edible insects, such as pH and water activity (aw), although those

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.11.007
0168-1605/© 2016 Elsevier B.V. All rights reserved.
14 D. Vandeweyer et al. / International Journal of Food Microbiology 242 (2017) 13–18

factors have an important impact on the growth and survival of micro- meter (LabMaster aw, Novasina, Lachen, Switzerland), until water activ-
organisms (Madigan et al., 2009) and need to be taken into account ity and temperature (20 °C) were stable for 15 and 5 min, respectively.
when considering insects as a food matrix. Moisture content was calculated from the weight loss of 2 to 3 g from
The objective of this study is to investigate the microbial load and in- each subsample after oven-drying overnight at 105 °C.
trinsic properties of fresh mealworm larvae (Tenebrio molitor) and
crickets (Acheta domesticus and Gryllodes sigillatus) as a food product. 2.4. Microbial plate counts
In order to obtain a generalized view, different production batches
and rearing companies were included in the study. pH, moisture con- Since it was not clear whether pulverization of the insects before ho-
tent and aW were determined, as well as a range of plate counts and mogenization would affect microbial counts, a preliminary experiment
presence-absence tests for pathogens typically determined for foods. was executed. Several counts (mesophilic aerobic count, aerobic endo-
spores and Enterobacteriaceae, see below) were determined using a
2. Material and methods procedure with and without pulverization (as described in Section
2.2). Both approaches were performed on five subsamples of a meal-
2.1. Study materials worm sample obtained from company 4 (Table 1). Because pulveriza-
tion was found to be necessary for optimal extraction of micro-
Three insect species commonly reared for human consumption were organisms from their matrix (see Section 3.1), the step was included
investigated: Acheta domesticus (house cricket), Gryllodes sigillatus in all further analyses.
(banded cricket) and larvae of Tenebrio molitor (mealworm). Samples To obtain a primary dilution, 5 g of each pulverized subsample and
were obtained from seven rearing companies in Belgium and the Neth- 45 g of peptone physiological salt solution (PPS, 0.85% NaCl, 0.1% pep-
erlands, including five companies specialized in rearing for human con- tone, Biokar Diagnostics, Beauvais, France) were mixed together in a
sumption and two companies for pet food. For each company, three stomacher bag. After homogenization for 60 s in a Bagmixer®
production batches (i.e. rearing cycles) were sampled between March (Interscience, Saint Nom, France), a tenfold dilution series was prepared
and December 2015, resulting in 21 batches studied, consisting of 12 and plated using the pour-plate technique, according to the ISO stan-
mealworm and 9 cricket batches (Table 1). dards assembled by Dijk et al. (2015). Bacterial endospores and yeasts
and moulds were determined according to Dijk et al. (2007). Total via-
2.2. Sampling and sample preparation ble mesophilic and psychrotrophic aerobic counts were assessed after
aerobic incubation on Plate Count Agar (PCA, Biokar diagnostics) for re-
Samples of fully grown and living insects ready for consumption spectively 72 h at 30 °C and 10 days at 6.5 °C. Lactic acid bacteria (LAB)
were transported to the laboratory at ambient temperature and imme- were incubated on de Man, Rogosa & Sharpe agar (MRS, Biokar diagnos-
diately processed upon arrival. Prior to analysis, insects were sedated by tics) for 72 h at 30 °C, Enterobacteriaceae on Violet Red Bile Glucose agar
cooling (±4 °C, 1 h). Subsequently, three subsamples of 30 g were taken (VRBG, Biokar diagnostics) for 24 h at 37 °C, and yeasts and moulds on
aseptically from each batch and pulverized (Bosch CNHR 25, max Oxytetracycline Glucose Agar (OGA, Biokar diagnostics) supplemented
speed) as described previously (Stoops et al., 2016). with oxytetracycline (50 mg/550 ml OGA, Biokar diagnostics) for
5 days at 25 °C. Aerobic bacterial endospores were determined on PCA
2.3. Intrinsic properties for 24 h at 37 °C after a pasteurisation treatment of the 10−1 dilution
at 80 °C for 10 min.
All subsamples were subjected to measurements of pH, water activ-
ity (aw) and moisture content. pH was measured in threefold using a 2.5. Pathogen detection
digital pH meter (Portamess 911, Knick, Berlin, Germany with SI analyt-
ics electrode, Mainz, Germany). A single aw measurement was per- Pulverized samples were also used for detection of Salmonella spp.
formed on a 7 g aliquot of each subsample using a water activity and Listeria monocytogenes. Detection of Salmonella spp. was performed

Table 1
Sample information.

Sample ID Rearing company Batch Sampling month (2015) Insect type Species Purpose (human/pet food)

MW 1.1 1 1 March Mealworm T. molitora Human food

MW 1.2 1 2 May Mealworm T. molitor Human food
MW 1.3 1 3 September Mealworm T. molitor Human food
MW 2.1 2 1 March Mealworm T. molitor Human food
MW 2.2 2 2 June Mealworm T. molitor Human food
MW 2.3 2 3 October Mealworm T. molitor Human food
MW 3.1 3 1 May Mealworm T. molitor Pet food
MW 3.2 3 2 July Mealworm T. molitor Pet food
MW 3.3 3 3 November Mealworm T. molitor Pet food
MW 4.1 4 1 July Mealworm T. molitor Pet food
MW 4.2 4 2 August Mealworm T. molitor Pet food
MW 4.3 4 3 September Mealworm T. molitor Pet food
CR 1.1 5 1 March Cricket A. domesticusb Human food
CR 1.2 5 2 June Cricket A. domesticus Human food
CR 1.3 5 3 September Cricket A. domesticus Human food
CR 2.1 6 1 April Cricket A. domesticus Human food
CR 2.2 6 2 July Cricket A. domesticus Human food
CR 2.3 6 3 October Cricket A. domesticus Human food
CR 3.1 7 1 August Cricket G. sigillatusc Human food
CR 3.2 7 2 October Cricket G. sigillatus Human food
CR 3.3 7 3 December Cricket G. sigillatus Human food
a
T.: Tenebrio.
b
A.: Acheta.
c
G.: Gryllodes.
 D. Vandeweyer et al. / International Journal of Food Microbiology 242 (2017) 13–18 15

according to ISO 6579 (absence in 25 g) and detection of Listeria should be noted that these values only apply for the insect as a crushed
monocytogenes according to AFNOR BRD 07/4–09/08 (absence in matrix and can differ from the intrinsic properties of individual parts.
25 g). A single analysis was performed for all but two samples (one Therefore, conclusions on the distribution and growth rates of micro-or-
mealworm sample, one cricket sample), which were analysed in five- ganisms within the different parts (e.g. surface versus intestinal tract) of
fold as described in Regulation (EC) N° 2073/2005 on microbiological the insect cannot be made based on the data presented here.
criteria for foodstuffs. The preliminary experiment investigating the importance of pulver-
ization demonstrated that microbial counts obtained from non-pulver-
2.6. Statistics and multivariate analysis ized mealworm larvae are an underestimation of the total amount of
countable micro-organisms. With mean values of 7.0 ± 0.2 (standard
To determine statistical differences between production batches of error of the mean) log cfu/g for total aerobic count, 2.5 ± 0.2 log cfu/g
individual rearing companies, between different rearing companies, for aerobic endospores and 5.9 ± 0.2 log cfu/g for Enterobacteriaceae,
and between the different insect types studied, data were analysed counts from non-pulverized mealworms were strongly significantly
with SPSS Statistics 20 (IBM, New York, USA). To determine significant lower (p = 0.000 for all counts) than those obtained from pulverized
differences in intrinsic factors and microbial counts between rearing larvae (8.6 ± 0.1, 4.7 ± 0.1 and 7.7 ± 0.1 log cfu/g respectively). Pulver-
companies and between batches, One-way Kruskal-Wallis analysis izing insects as a first step in microbial analysis thus resulted in counts
was used, followed by a multiple comparison using the Dunn-Bonferoni that are 1.6 to 2.2 log cycles higher.
Post-Hoc test. For the Kruskal-Wallis tests, a distinction between weakly Average counts for mealworm larvae and crickets were generally
significant (0.05 b p b 0.10) and significant (p b 0.05) was adopted. high (Table 4), with total counts of at least 8 log cfu/g for all samples.
Mann-Whitney U tests were performed to analyse differences between For mealworm larvae, Klunder et al. (2012) found 7.7 log cfu/g for
mealworm larvae and crickets, with a level of significance of 0.05. In the total viable count, 6.8 log cfu/g for Enterobacteriaceae and 2.1 cfu/g for
preliminary experiment comparing pulverized and non-pulverized sub- bacterial spores in a single sample analysis. Those values are lower
samples, an Independent-Samples t-test with significance level 0.05 than the averages obtained in this work (see Table 4: 8.4, 7.4 and
was used. Additionally, all data (means) from intrinsic and microbiolog- 3.1 log cfu/g). That can be explained by the fact that insects were not
ical parameter analyses were used to perform multivariate non-metric pulverized in the study of Klunder et al. (2012), likely resulting in an in-
multidimensional scaling (NMDS) to visualize differences between complete extraction of countable micro-organisms from the matrix. A
batches, rearing companies and insects. Batches lacking one or more pa- complete recovery during analysis is of utmost importance as insects
rameters were excluded from this analysis. The NMDS plot was created are consumed or processed into foods as a whole, i.e. without prior re-
using the vegan package in R (v12.2.1) (Oksanen et al., 2012; R moval of the intestines. The same remark can be made for the data ob-
Development Core Team, 2006). tained from the cricket sample by Klunder et al. (2012). Sample
preparation in Stoops et al. (2016), however, was done the same way
3. Results and discussion as in our study and their values for total viable count, LAB, Enterobacte-
riaceae, and yeasts and moulds of mealworm larvae are highly compa-
3.1. Intrinsic properties and microbial counts rable to the data obtained in this work.
Psychrotrophic counts were never investigated before on edible in-
With total mean pH values of 6.7 and 6.4 for mealworm larvae sects, although it is an important parameter regarding chilled conserva-
(Table 2) and crickets (Table 3) respectively, a near-neutral pH was ob- tion of edible insects. Crickets contained on average 4.5 log cfu/g
served for both insect types. Together with a high mean water activity of psychrotrophic organisms, but the average for mealworm larvae was
0.96 for both insect types, the new food matrices can be considered as 6.6 log cfu/g (Table 4), with some batches even up to 9.1 log cfu/g
well-suitable for the growth of a broad range of micro-organisms. It (Table 2). That might involve a risk of spoilage in a chilled environment

Table 2
Intrinsic properties and microbial counts of fresh mealworm larvae (T. molitor) from different rearing companies and batches.1

Rearing Sample Intrinsic properties Microbial counts (log cfu/g)

company ID
pH aw Moisture Total viable Lactic acid Enterobacteriaceae Aerobic Psychrotrophic Yeasts and
content (%) aerobic count bacteria bacterial aerobic count moulds
endospores

1 MW 1.1 6.73 ± 0.02a 0.97 ± 0.00a 61.4 ± 0.4a,b 8.3 ± 0.0a 7.4 ± 0.0a 6.8 ± 0.1a 4.2 ± 0.1a,b 5.8 ± 0.0a,b⁎ 4.8 ± 0.1a
MW 1.2 6.75 ± 0.01a 0.97 ± 0.00a 60.8 ± 0.1a 8.4 ± 0.0a 7.4 ± 0.0a 7.2 ± 0.1a,b 5.0 ± 0.6a 4.8 ± 0.6a 4.5 ± 0.1a,b⁎
MW 1.3 6.76 ± 0.01a 0.95 ± 0.00b 62.7 ± 0.1b 8.3 ± 0.0a 7.7 ± 0.0b⁎ 7.6 ± 0.1b 2.6 ± 0.1b 7.0 ± 0.0b⁎ 4.2 ± 0.1b⁎
Mean 6.75 ± 0.01A 0.96 ± 0.00A 61.6 ± 0.5A 8.3 ± 0.0A 7.5 ± 0.1A 7.2 ± 0.2A 3.9 ± 0.7A 5.9 ± 0.6A 4.5 ± 0.2A
2 MW 2.1 6.73 ± 0.02a 0.97 ± 0.00a 64.8 ± 0.0a 8.5 ± 0.22,a 8.2 ± 0.02,a 6.9 ± 0.22,a 2.3 ± 0.1a,b⁎ 6.5 ± 0.02,a 5.3 ± 0.02,a
MW 2.2 6.68 ± 0.01b 0.97 ± 0.00a,b 65.3 ± 0.1b 8.2 ± 0.0b 7.6 ± 0.0b⁎ 7.4 ± 0.1a 2.0 ± 0.1a 6.6 ± 0.0a 5.3 ± 0.1a
MW 2.3 6.75 ± 0.01a 0.95 ± 0.00b 70.7 ± 0.3b 8.2 ± 0.0a,b 8.1 ± 0.0a,b⁎ 7.5 ± 0.1a 2.5 ± 0.2b⁎ 6.7 ± 0.1a 5.3 ± 0.1a
Mean 6.72 ± 0.02A 0.96 ± 0.01A 66.9 ± 1.9B⁎ 8.3 ± 0.1A 8.0 ± 0.2A 7.3 ± 0.2A 2.3 ± 0.1B⁎ 6.6 ± 0.1A 5.3 ± 0.0A
3 MW 3.1 6.68 ± 0.01a 0.96 ± 0.00a 64.3 ± 0.1a,b 8.0 ± 0.0a 7.4 ± 0.0a,b⁎ 7.1 ± 0.0a 4.2 ± 0.1a 5.3 ± 0.4a 5.6 ± 0.1a,b
MW 3.2 6.61 ± 0.00b 0.97 ± 0.00a 64.8 ± 0.1a 8.2 ± 0.1a,b 7.3 ± 0.0a 7.5 ± 0.1a,b 3.5 ± 0.7a 7.0 ± 0.1a,b 4.6 ± 0.1a
MW 3.3 6.71 ± 0.01a 0.96 ± 0.00a 63.4 ± 0.1b 9.3 ± 0.1b 8.1 ± 0.1b⁎ 8.3 ± 0.0b 4.1 ± 0.1a 9.1 ± 0.0b 7.5 ± 0.0b
Mean 6.67 ± 0.03A 0.96 ± 0.00A 64.2 ± 0.4A,B⁎ 8.5 ± 0.4A 7.6 ± 0.3A 7.6 ± 0.4A 3.9 ± 0.2A 7.1 ± 1.1A 5.9 ± 1.5A
4 MW 4.1 N.D.3 N.D. N.D. 8.3 ± 0.1a N.D. 7.8 ± 0.1a 2.2 ± 0.2a,b N.D. N.D.
MW 4.2 N.D. N.D. N.D. 8.3 ± 0.0a N.D. 7.8 ± 0.1a 1.7 ± 0.1a N.D. N.D.
MW 4.3 6.76 ± 0.02 0.97 ± 0.00 65.9 ± 0.1 8.3 ± 0.0a 8.2 ± 0.0 6.9 ± 0.1b⁎ 3.4 ± 0.1b 7.6 ± 0.0 6.0 ± 0.1
Mean 6.76A 0.97A 65.9B 8.3 ± 0.0A 8.2A 7.5 ± 0.3A 2.4 ± 0.5B⁎ 7.6A 6.0A
1
Data are the mean values of three replicates ± standard error of the mean; a,bMeans per sample with the same superscript (small letter) within the same columns from the same
rearing company do not differ significantly (p N 0.10); A,BMeans per rearing company with the same superscript (capital) within the same columns do not differ significantly (p N 0.05).
*Superscripts with an asterisk indicate a weak significance (0.05 b p b 0.10).
2
Mean value of two replicates ± standard error of the mean.
3
N.D. not determined.
16 D. Vandeweyer et al. / International Journal of Food Microbiology 242 (2017) 13–18

Table 3
Intrinsic properties and microbial counts of fresh crickets (A. domesticus or G. sigillatus) from different rearing companies and batches.1

Rearing Sample Intrinsic properties Microbial counts (log cfu/g)

company ID
pH aw Moisture Total viable Lactic acid Enterobacteriaceae Aerobic Psychrotrophic Yeasts and
content (%) aerobic count bacteria bacterial aerobic count moulds
endospores

5 CR 1.1 6.582,a 0.962,a N.D.3 8.3 ± 0.14,a,b⁎ 7.6 ± 0.34,a,b⁎ 8.0 ± 0.04,a,b⁎ 2.9 ± 0.04,a 5.3 ± 0.34,a 6.1 ± 0.04,a,b
CR 1.2 6.03 ± 0.01b 0.97 ± 0.00a 65.3 ± 0.1a 8.1 ± 0.1a 7.4 ± 0.2a 7.5 ± 0.2a 2.8 ± 0.0a,b⁎ 6.4 ± 0.7a 5.9 ± 0.0a
CR 1.3 6.61 ± 0.01a 0.98 ± 0.00b 73.3 ± 0.1b 8.8 ± 0.1b⁎ 8.1 ± 0.0b⁎ 8.3 ± 0.2b⁎ 2.6 ± 0.0b⁎ 4.9 ± 0.2a 7.2 ± 0.4b
Mean 6.41 ± 0.19A 0.97 ± 0.00A 69.3 ± 4.0A 8.4 ± 0.2A 7.7 ± 0.2A 7.9 ± 0.2A 2.8 ± 0.1A 5.5 ± 0.5A 6.3 ± 0.4A
6 CR 2.1 6.44 ± 0.01a 0.97 ± 0.00a 73.0 ± 0.1a 8.3 ± 0.1a 7.8 ± 0.1a,b⁎ 7.7 ± 0.1a 4.2 ± 0.9a 5.2 ± 0.5a 6.1 ± 0.4a
CR 2.2 6.34 ± 0.01b 0.99 ± 0.00b 69.9 ± 0.1b 8.2 ± 0.1a 8.0 ± 0.1a 7.4 ± 0.1a 3.9 ± 0.1a b3.0 ± 0.0b 7.1 ± 0.2a
CR 2.3 6.69 ± 0.01c 0.97 ± 0.00a,b 70.8 ± 0.2b 8.2 ± 0.04,a 7.7 ± 0.04,b⁎ 7.6 ± 0.04,a 4.3 ± 0.0a 3.7 ± 0.24,a,b 6.4 ± 0.04,a
Mean 6.49 ± 0.10A 0.98 ± 0.00A 71.3 ± 0.9A 8.2 ± 0.0A 7.8 ± 0.1A 7.6 ± 0.1A 4.1 ± 0.1B⁎ b4.0 ± 0.6A 6.5 ± 0.3A
7 CR 3.1 6.37 ± 0.01a 0.97 ± 0.00a 69.0 ± 0.6a,b 8.7 ± 0.2a 8.3 ± 0.0a 7.9 ± 0.2a 3.8 ± 0.1a,b⁎ 4.5 ± 0.34,a 6.2 ± 0.2a,b
CR 3.2 6.23 ± 0.01b 0.96 ± 0.00a,b 67.5 ± 0.1a 8.4 ± 0.1a 8.2 ± 0.1a 7.2 ± 0.1b⁎ 3.4 ± 0.3a b3.2 ± 0.1b⁎ 5.6 ± 0.1a
CR 3.3 6.37 ± 0.02a 0.92 ± 0.00b 70.0 ± 0.1b 8.6 ± 0.1a 7.9 ± 0.1b⁎ 7.6 ± 0.1a,b⁎ 4.9 ± 0.5b⁎ 4.1 ± 0.5a 7.2 ± 0.1b
Mean 6.32 ± 0.05A 0.95 ± 0.02A 68.9 ± 0.7A 8.6 ± 0.1A 8.1 ± 0.1A 7.6 ± 0.2A 4.0 ± 0.4B⁎ b3.9 ± 0.4A 6.3 ± 0.5A
1
Data are the mean values of three replicates ± standard error of the mean; a,b,cMeans per sample with the same superscript (small letter) within the same columns from the same
rearing company do not differ significantly (p N 0.05); A,BMeans per rearing company with the same superscript (capital) within the same columns do not differ significantly (p N 0.05).
*Superscripts with an asterisk indicate a weak significance (0.05 b p b 0.10).
2
Value of a single replicate.
3
N.D. not determined.
4
Mean value of two replicates ± standard error of the mean.

as well as the outgrowth of psychrotrophic pathogens such as L. Similar observations were made for crickets (Table 3; Fig. 1). For ex-
monocytogenes (which was not detected in this study though, see ample, for company 5, batch CR 1.3 diverged from the other batches,
Section 3.5) or Bacillus cereus (Hwang and Tamplin, 2005; Martínez et mainly for the yeast and mould count (p = 0.044). The insects from
al., 2007). batch CR 1.3 were younger (subadult) than those from the other two
batches, which may have influenced their microbiota (Yun et al.,
2014). Further, batch CR 2.2 from company 6 was different from the
3.2. Variation between batches of individual rearing companies other two batches, particularly for its low amount of psychrotrophs
(around or below detection limit) (p = 0.044) and the high number
Although insects are generally reared using established protocols, of yeasts and moulds (not significant). Company 7 provided samples
variation between batches from the same company was commonplace which were particularly different in the amount of psychrotrophs
(Fig. 1), especially for bacterial endospores, psychrotrophic counts, (weakly significant, p = 0.077) and yeasts and moulds (p = 0.027).
and yeasts and moulds (Tables 2 and 3). By contrast, variation in intrin- The highest amount of yeasts and moulds was observed in batch CR
sic properties was only marginal (Tables 2 and 3), suggesting that the 3.3, which may be related to the (slightly) lower water activity mea-
differences in microbiological parameters are caused by other variables sured (Madigan et al., 2009).
such as feed supply and/or rearing practices. For example, mealworm
larvae batches from company 1 showed a significantly lower endospore
count (p = 0.039) and a higher number of psychrotrophs (weakly sig- 3.3. Variation between rearing companies
nificant, p = 0.055) for one particular batch (MW 1.3) compared to
the other two batches. Furthermore, company 3 produced a mealworm Large differences between different insect batches from each indi-
batch (MW 3.3) which showed significantly higher counts than the vidual company resulted in large standard errors of the mean and
other two for almost all microbial parameters (p = 0.027 for total aero- hence few statistically significant differences between rearing compa-
bic count, Enterobacteriaceae, psychrotrophs, yeasts and moulds). That nies. For mealworm larvae (Table 2; Fig. 1), only (weakly) significant
was in line with deviating visual (moister) and olfactory (staler odour) differences between companies were found for the moisture content
observations for this sample. Additionally, batches MW 3.1 and MW 3.2 (ranging between 61.6 and 66.9%, p = 0.051) and the number of bacte-
differed from each other, numerically but not significantly, in counts for rial endospores (mean values between 2.3 and 3.9 log cfu/g, p = 0.059).
psychrotrophs and yeasts and moulds (Table 2). The findings on the Nevertheless, water activity has more impact on microbial growth in
overall variation are in agreement with Stoops et al. (2016), who partic- foods than moisture content and those values were highly comparable
ularly found variation in the number of bacterial endospores, ranging between companies (p = 0.861). The moisture content and the water
from not detected (b1.0 log cfu/g) to 3.5 log cfu/g. In our study, spore activity of insects are a result of the relative air humidity in the rearing
counts even ranged from 1.7 to 5.0 log cfu/g over all samples environment, the presence or absence of water supply and the moisture
investigated. content of the feed. Mealworm larvae are typically provided with water

Table 4
Total mean values for intrinsic properties and microbial counts of fresh crickets and mealworm larvae.1

Insect type Intrinsic properties Microbial counts (log cfu/g)

pH aw Moisture Total viable Lactic acid Enterobacteriaceae Aerobic bacterial Psychrotrophic Yeasts and
content (%) aerobic count bacteria endospores aerobic count moulds

Mealworm 6.70 ± 0.02A 0.96 ± 0.00A 64.4 ± 0.9A 8.4 ± 0.1A 7.7 ± 0.1A 7.4 ± 0.1A 3.1 ± 0.3A 6.6 ± 0.4A 5.3 ± 0.3A
larvae
Crickets 6.40 ± 0.07B 0.96 ± 0.01A 69.9 ± 0.9B 8.4 ± 0.1A 7.9 ± 0.1A 7.7 ± 0.1A 3.6 ± 0.3A 4.5 ± 0.4B 6.4 ± 0.2B
1
Data are the total mean values of all investigated batches per insect ± standard error of the mean. A,BMeans per insect with the same superscript do not differ significantly (p N 0.05).
 D. Vandeweyer et al. / International Journal of Food Microbiology 242 (2017) 13–18 17

yeasts and moulds were significantly different. The difference in pH

was statistically significant (p = 0.000) but numerically rather small.
CR1_3 In line with Finke (2002), crickets contained significantly (p = 0.001)
CR3_2 more moisture (total mean value of 69.9%) than mealworm larvae
(64.4%). The significant difference (p = 0.001) found for psychrotrophic
counts might be due to a difference in starvation temperature. Further-
MW2_2
CR3_1 MW2_3 more, crickets show significantly higher results for the overall fungal
CR2_3 MW2_1
counts than mealworm larvae (p = 0.004). Interestingly, mealworm
CR1_2 larvae were dominated by moulds, whereas for crickets yeasts dominat-
CR3_3 CR2_1
MW1_3 ed the fungal biota. Correspondingly, mealworm larvae are generally
fed with cereal products, while crickets are reared on very diverse sub-
MW3_1
MW3_2 strates such as chicken or pig feed, organic vegetables and brewer's
MW4_3
spent grain.
MW1_1
MW1_2 Altogether, our results show that crickets and mealworms should be
considered as different food matrices in terms of intrinsic properties and
microbial quality. Therefore, when reporting on the microbial quality of
edible insects, it is important to distinguish between distinct insect
MW3_3 types and not to report average values over several types or to pool sam-
ples of different types, as was done in the past (e.g. Giaccone, 2005;
−0.04 −0.02 0.00 0.02 0.04 Grabowski et al., 2014).
MDS1
3.5. Compliance with microbial criteria for foodstuffs
Fig. 1. Non-metric multidimensional scaling (NMDS) of the samples analysed in this study.
Batches MW 4.1, MW 4.2, CR 1.1 and CR 2.2 were excluded from this analysis because of Many authors mention the possibility for insects to contain patho-
lacking parameters or data lower than the detection limit. All intrinsic and microbial genic micro-organisms such as Bacillus cereus, Campylobacter spp., Clos-
parameters (mean values) were included in the analysis for the other batches. Batches
from the same rearing company are represented by the same colour. The distance
tridium spp., Salmonella spp. and Staphylococcus aureus (Crippen et al.,
between samples on the plot reflects their similarity level: the more similar two 2012; EFSA Scientific Committee, 2015; Giaccone, 2005; Mpuchane et
samples are, the smaller their distance is. Sample IDs correspond with those in Table 1. al., 2006; Rumpold and Schlüter, 2013b; Rumpold et al., 2014; Stoops
et al., 2016; Templeton et al., 2006; Zheng et al., 2012). Furthermore, ed-
ible insects harbour a large amount of Enterobacteriaceae, a bacterial
family containing foodborne pathogens. The Belgian Superior Health
by offering a moist food product, such as carrots or apples. The varying Council and the Federal Agency for the Safety of the Food Chain (SHC
bacterial endospore load may be reflected by differing endospore num- and FASFC, 2014) and the Dutch Food and Consumer Product Safety Au-
bers in the feed, but this remains to be investigated. Notably, microbial thority (NVWA, 2014) both composed an opinion on food safety aspects
parameters for the two companies rearing mealworm larvae for pet of edible insects. The microbiological criteria referred to in those opin-
food (company 3 and 4; Table 2) did not differ significantly from ions are based on culture-dependent microbial counting, as is also the
those for the companies rearing for human consumption (company 1 case for other food products described in Regulation (EC) N° 2073/
and 2). However, psychrotrophs and yeasts and moulds show slightly 2005 on microbiological criteria for foodstuffs. Both opinions refer to
higher counts for the former. food safety criteria for Salmonella, Listeria monocytogenes and
For crickets (Table 3; Fig. 1), similar trends were observed. More spe- Escherichia coli for ready-to-eat meals, minced meat and live bivalve
cifically, our data indicate a weakly significant difference (p = 0.061) molluscs. The opinions suggest to use those criteria for edible insects,
between different companies in bacterial endospore counts (ranging since no specific criteria for insects exist yet. That led to the screening
from 2.8 to 4.1 log cfu/g). Soil, used as a substrate for crickets to lay of each batch on Salmonella spp. and L. monocytogenes in this work.
their eggs, can be a major source of contamination for bacterial endo- Both pathogens were not detected (in 25 g) in any of the samples,
spores (Klunder et al., 2012; Madigan et al., 2009). In contrast to the which corresponds to the findings from Giaccone (2005) and
moisture content of mealworm larvae, the moisture content of crickets Grabowski et al. (2014). Nevertheless, the recently updated circular
was similar among the different companies. Crickets are supplied with from the Belgian FASFC states that products available on the marked
pure water instead of a moist food product and that may result in should still be periodically tested on the presence of pathogens Salmo-
lower variations in moisture content. The cricket moisture content ob- nella and Listeria monocytogenes (FASFC, 2016).
tained in our study is comparable with findings in literature (Finke, In addition to food safety criteria, the opinions proposed process hy-
2002). Altogether, no significant differences in mean intrinsic properties giene criteria based on comparable food matrices, e.g. minced meat. All
or microbial counts were observed between the two cricket species total viable counts obtained in this study (8.2–9.3 log cfu/g) strongly ex-
Acheta domesticus (company 5 and 6) and Gryllodes sigillatus (company ceed the value for the low criterion (m = 5.7 log cfu/g) for minced meat
7). and even that for the high criterion (M = 6.7 log cfu/g). The relevance of
Athough the exact rearing history of the insect samples studied is criteria for other foodstuffs for edible insects can be questioned, howev-
unknown, some explanations for the observed differences can be for- er, and the development of criteria specific for insects is desirable.
mulated. Influencing factors are likely to be situated in the feed, the
rearing environment and an (optional) starvation procedure (Jones et 4. Conclusions
al., 2013; Yun et al., 2014). It is also plausible that rearing practices
and hygiene measures may result in a company-specific “house flora”, Our study provides a general view on the intrinsic properties and
which can also affect the insect microbiota (Li et al., 2016). microbial counts of three edible insect species. Large variations in mi-
crobial counts between batches originating from a single rearer resulted
3.4. Variation between different insect types in the variations between companies being smaller. Especially for aero-
bic bacterial endospores, aerobic psychrotrophic organisms, and yeasts
When comparing data from mealworm larvae and crickets (Table 4; and moulds, counts varied remarkably. Intrinsic properties of the in-
Fig. 1), pH, moisture content, psychrotrophic count and the amount of sects, on the other hand, varied to a smaller extent and cannot explain
18 D. Vandeweyer et al. / International Journal of Food Microbiology 242 (2017) 13–18

the differences in microbial parameters completely. While culture-de- Mlcek, J., Rop, O., Borkovcova, M., Bednarova, M., 2014. A comprehensive look at the pos-
sibilities of edible insects as food in europe – a review. Polish J. Food Nutr. Sci. 64:
pendent analyses are very useful to quantify microbial loads and to 147–157. http://dx.doi.org/10.2478/v10222-012-0099-8.
compare counts with other food matrices or with microbiological Mpuchane, S., Allotey, J., Matsheka, I., Simpanya, M., Coetzee, S., Jordaan, A., Mrema, N.,
criteria, further research using culture-independent methods is needed Gashe, B.A., 2006. Carriage of micro-organisms by domestic cockroaches and implica-
tions on food safety. Int. J. Trop. Insect Sci. 26:166–175. http://dx.doi.org/10.1079/
to reveal the composition of the edible insect microbiota and variations IJT20063141.
in the composition between batches and companies. In addition, further Nowak, V., Persijn, D., Rittenschober, D., Charrondiere, U.R., 2016. Review of food compo-
research is needed to explore the relation between rearing conditions, sition data for edible insects. Food Chem. 193:39–46. http://dx.doi.org/10.1016/j.
foodchem.2014.10.114.
including hygiene, and the insect microbiota. NVWA, 2014. Advisory report on the risks associated with the consumption of mass-
reared insects.
Acknowledgements Oksanen, J., Blanchet, F.G., Kindt, R., Legendre, P., Minchin, P.R., O'Hara, R.B., Simpson, G.L.,
Solymos, P., Stevens, M.H.H., Wagner, H., 2012. Vegan: community ecology. R Package
Ver. 2.0-5. R Foundation for Statistical Computing, Vienna, Austria .
The authors thank all rearing companies who kindly provided insect Oonincx, D.G.A.B., de Boer, I.J.M., 2012. Environmental impact of the production of meal-
samples and A. Borremans for her assistance in laboratory work. worms as a protein source for humans - a life cycle assessment. PLoS One 7:e51145.
Funding: this PhD research was supported by Flanders Innovation & En- http://dx.doi.org/10.1371/journal.pone.0051145.
Oonincx, D.G.A.B., van Itterbeeck, J., Heetkamp, M.J.W., van den Brand, H., van Loon, J.J.A.,
trepreneurship (VLAIO) [Project 141129]. van Huis, A., 2010. An exploration on greenhouse gas and ammonia production by in-
sect species suitable for animal or human consumption. PLoS One 5:e14445. http://
References dx.doi.org/10.1371/journal.pone.0014445.
Premalatha, M., Abbasi, T., Abbasi, T., Abbasi, S.A., 2011. Energy-efficient food production
Belluco, S., Losasso, C., Maggioletti, M., Alonzi, C.C., Paoletti, M.G., Ricci, A., 2013. Edible in- to reduce global warming and ecodegradation: the use of edible insects. Renew. Sus-
sects in a food safety and nutritional perspective: a critical review. Compr. Rev. Food tain. Energy Rev. 15:4357–4360. http://dx.doi.org/10.1016/j.rser.2011.07.115.
Sci. Food Saf. 12:296–313. http://dx.doi.org/10.1111/1541-4337.12014. R Development Core Team, 2006. R: A Language and Environment for Statistical Comput-
Caparros Megido, R., Sablon, L., Geuens, M., Brostaux, Y., Alabi, T., Blecker, C., Drugmand, ing. R Foundation for Statistical Computing, Vienna, Austria .
D., Haubruge, É., Francis, F., 2014. Edible insects acceptance by Belgian consumers: Rumpold, B.A., Schlüter, O.K., 2013a. Nutritional composition and safety aspects of edible
promising attitude for entomophagy development. J. Sens. Stud. 29:14–20. http:// insects. Mol Nutr Food Res 57:802–823. http://dx.doi.org/10.1002/mnfr.201200735.
dx.doi.org/10.1111/joss.12077. Rumpold, B.A., Schlüter, O.K., 2013b. Potential and challenges of insects as an innovative
Crippen, T.L., Zheng, L., Sheffield, C.L., Tomberlin, J.K., Beier, R.C., Yu, Z., 2012. Transient gut source for food and feed production. Innov. Food Sci. Emerg. Technol. 17:1–11. http://
retention and persistence of Salmonella through metamorphosis in the lesser meal- dx.doi.org/10.1016/j.ifset.2012.11.005.
worm, Alphitobius diaperinus (Coleoptera: Tenebrionidae). J. Appl. Microbiol. 112: Rumpold, B.A., Fröhling, A., Reineke, K., Knorr, D., Boguslawski, S., Ehlbeck, J., Schlüter,
920–926. http://dx.doi.org/10.1111/j.1365-2672.2012.05265.x. O.K., 2014. Comparison of volumetric and surface decontamination techniques for in-
Dijk, R., Beumer, R., de Boer, E., Brinkman, E., van Dijk, J., Dijkstra, A., Uyttendaele, M., novative processing of mealworm larvae (Tenebrio molitor). Innov. Food Sci. Emerg.
Stegeman, H., Veenendaal, H., van Woerden, M., 2007. Microbiologie van Technol. 26:232–241. http://dx.doi.org/10.1016/j.ifset.2014.09.002.
voedingsmiddelen: Methoden, principes en criteria. fourth ed. Noordervliet Media, Sánchez-Muros, M.-J., Barroso, F.G., Manzano-Agugliaro, F., 2014. Insect meal as renew-
Houten . able source of food for animal feeding: a review. J. Clean. Prod. 65:16–27. http://dx.
Dijk, R., van den Berg, D., Beumer, R., de Boer, E., Dijkstra, A., Mout, L., Stegeman, H., doi.org/10.1016/j.jclepro.2013.11.068.
Uyttendaele, M., in't Veld, S., 2015. Microbiologie van voedingsmiddelen: Methoden, SHC, FASFC, 2014. Food safety aspects of insects intended for human consumption (Sci
principes en criteria. fifth ed. MYbusinessmedia, Capelle aan den Ijssel . Com dossier 2014/04; SHC dossier n° 9160).
EFSA Scientific Committee, 2015. Risk profile related to production and consumption of Siemianowska, E., Kosewska, A., Aljewicz, M., Skibniewska, K.A., Polak-Juszczak, L., Jarocki,
insects as food and feed. EFSA J 13. http://dx.doi.org/10.2903/j.efsa.2015.4257 60 pp. A., Jędras, M., 2013. Larvae of mealworm (Tenebrio molitor L.) as European novel food.
FASFC, 2016. Circular concerning the breeding and marketing of insects and insect-based Agric. Sci. 04:287–291. http://dx.doi.org/10.4236/as.2013.46041.
food for human consumption. Stoops, J., Crauwels, S., Waud, M., Claes, J., Lievens, B., Van Campenhout, L., 2016. Microbial
Finke, M.D., 2002. Complete nutrient composition of commercially raised invertebrates community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers
used as food for insectivores. Zoo Biol. 21:269–285. http://dx.doi.org/10.1002/zoo. (Locusta migratoria migratorioides) sold for human consumption. Food Microbiol. 53
10031. (Part B):122–127. http://dx.doi.org/10.1016/j.fm.2015.09.010.
Giaccone, V., 2005. Hygiene and health features of “minilivestock”. In: Paoletti, M.G. (Ed.), Templeton, J.M., De Jong, A.J., Blackall, P.J., Miflin, J.K., 2006. Survival of Campylobacter spp.
Ecological Implications of Minilivestock: Potential of Insects, Rodents, Frogs and in darkling beetles (Alphitobius diaperinus) and their larvae in Australia. Appl. Envi-
Snails. Science Publishers, Inc., Enfield (NH), USA, pp. 579–598. ron. Microbiol. 72:7909–7911. http://dx.doi.org/10.1128/AEM.01471-06.
Grabowski, N.T., Jansen, W., Klein, G., 2014. Microbiological status of edible insects sold as van Huis, A., 2013. Potential of insects as food and feed in assuring food security. Annu
pet feed in Germany. Conference “Insects to Feed the World”. Wageningen University Rev Entomol 58:563–583. http://dx.doi.org/10.1146/annurev-ento-120811-153704.
and Research Centre, Wageningen, The Netherlands . van Huis, A., Van Itterbeeck, J., Klunder, H.C., Mertens, E., Halloran, A., Muir, G., Vantomme,
Hwang, C.-A., Tamplin, M.L., 2005. The influence of mayonnaise pH and storage temper- P., 2013. Edible Insects: Future Prospects for Food and Feed Security. FAO (Food and
ature on the growth of Listeria monocytogenes in seafood salad. Int. J. Food Microbiol. Agricultural Organization of the United Nations), Rome .
102:277–285. http://dx.doi.org/10.1016/j.ijfoodmicro.2004.11.019. Yen, A.L., 2015. Insects as food and feed in the Asia Pacific region: current perspectives
Jones, R.T., Sanchez, L.G., Fierer, N., 2013. A cross-taxon analysis of insect-associated bac- and future directions. J. Insects as Food Feed 1:33–55. http://dx.doi.org/10.3920/
terial diversity. PLoS One 8:e61218. http://dx.doi.org/10.1371/journal.pone.0061218. JIFF2014.0017.
Klunder, H.C., Wolkers-Rooijackers, J., Korpela, J.M., Nout, M.J.R., 2012. Microbiological as- Yun, J.-H., Roh, S.W., Whon, T.W., Jung, M.-J., Kim, M.-S., Park, D.-S., Yoon, C., Nam, Y.-D.,
pects of processing and storage of edible insects. Food Control 26:628–631. http://dx. Kim, Y.-J., Choi, J.-H., Kim, J.-Y., Shin, N.-R., Kim, S.-H., Lee, W.-J., Bae, J.-W., 2014. Insect
doi.org/10.1016/j.foodcont.2012.02.013. gut bacterial diversity determined by environmental habitat, diet, developmental
Li, L., Xie, B., Dong, C., Wang, M., Liu, H., 2016. Can closed artificial ecosystem have an im- stage, and phylogeny of host. Appl. Environ. Microbiol. 80:5254–5264. http://dx.doi.
pact on insect microbial community? A case study of yellow mealworm (Tenebrio org/10.1128/AEM.01226-14.
molitor L.). Ecol. Eng. 86:183–189. http://dx.doi.org/10.1016/j.ecoleng.2015.09.015. Zheng, L., Crippen, T.L., Sheffield, C.L., Poole, T.L., Yu, Z., Tomberlin, J.K., 2012. Evaluation of
Madigan, M.T., Martinko, J.M., Dunlap, P.V., Clark, D.P., 2009. Brock Biology of Microorgan- Salmonella movement through the gut of the lesser mealworm, Alphitobius diaperinus
isms. 12th ed. Pearson/Benjamin Cummings, San Francisco, CA . (Coleoptera: Tenebrionidae). Vector-Borne Zoonotic Dis 12:287–292. http://dx.doi.
Martínez, S., Borrajo, R., Franco, I., Carballo, J., 2007. Effect of environmental parameters org/10.1089/vbz.2011.0613.
on growth kinetics of Bacillus cereus (ATCC 7004) after mild heat treatment. Int.
J. Food Microbiol. 117:223–227. http://dx.doi.org/10.1016/j.ijfoodmicro.2006.08.002.