foods

Review
Safety of Alternative Proteins: Technological, Environmental
and Regulatory Aspects of Cultured Meat, Plant-Based Meat,
Insect Protein and Single-Cell Protein
Joshua Hadi 1 and Gale Brightwell 1,2, *

1 AgResearch Ltd., Hopkirk Research Institute, Cnr University Ave and Library Road, Massey University,
Palmerston North 4442, New Zealand; Joshua.Hadi@agresearch.co.nz
2 New Zealand Food Safety Science and Research Centre, Massey University Manawatu (Turitea), Tennent
Drive, Palmerston North 4474, New Zealand
* Correspondence: Gale.Brightwell@agresearch.co.nz

Abstract: Food security and environmental issues have become global crises that need transformative
solutions. As livestock production is becoming less sustainable, alternative sources of proteins are
urgently required. These include cultured meat, plant-based meat, insect protein and single-cell
protein. Here, we describe the food safety aspects of these novel protein sources, in terms of their
technological backgrounds, environmental impacts and the necessary regulatory framework for
future mass-scale production. Briefly, cultured meat grown in fetal bovine serum-based media can
be exposed to viruses or infectious prion, in addition to other safety risks associated with the use of
genetic engineering. Plant-based meat may contain allergens, anti-nutrients and thermally induced





 carcinogens. Microbiological risks and allergens are the primary concerns associated with insect
protein. Single-cell protein sources are divided into microalgae, fungi and bacteria, all of which have
Citation: Hadi, J.; Brightwell, G.
specific food safety risks that include toxins, allergens and high ribonucleic acid (RNA) contents. The
Safety of Alternative Proteins:
Technological, Environmental and
environmental impacts of these alternative proteins can mainly be attributed to the production of
Regulatory Aspects of Cultured Meat, growth substrates or during cultivation. Legislations related to novel food or genetic modification
Plant-Based Meat, Insect Protein and are the relevant regulatory framework to ensure the safety of alternative proteins. Lastly, additional
Single-Cell Protein. Foods 2021, 10, studies on the food safety aspects of alternative proteins are urgently needed for providing relevant
1226. https://doi.org/10.3390/ food governing authorities with sufficient data to oversee that the technological progress in this area
foods10061226 is balanced with robust safety standards.

Academic Editor: Keywords: alternative proteins; cultured meat; plant-based meat; edible insects; single-cell protein;
Trine Kastrup Dalsgaard novel food; environmental issues; food safety

Received: 21 April 2021

Accepted: 26 May 2021
Published: 28 May 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
Global population is projected to reach 9.8 billion in the year 2050 [1]. This population
with regard to jurisdictional claims in
growth entails a projected livestock production of 455 million tons in 2050 [2], which
published maps and institutional affil- is 40% higher than the reported number in 2019 [3]. Currently, livestock production
iations. contributes to 14.5% of anthropogenic greenhouse gas (GHG) emission [4]. In particular,
livestock production releases methane and nitrous oxide gases, which have higher global
warming potential than carbon dioxide [5]. To avoid potential catastrophic events, global
temperature increase should be maintained within 1.5 ◦ C of the pre-industrial levels [6].
Copyright: © 2021 by the authors.
Considerable requirements for water and land further contribute to the environmental
Licensee MDPI, Basel, Switzerland.
footprints of livestock production [7,8].
This article is an open access article
Mitigation efforts include improved feeding practices for better forage digestibility,
distributed under the terms and manure management and diversification of crop and animal varieties [9,10]. Nevertheless,
conditions of the Creative Commons climate issues are a matter of great urgency and more radical solutions may be necessary
Attribution (CC BY) license (https:// to ensure food availability in an environmentally sustainable manner. Thus, the concept of
creativecommons.org/licenses/by/ alternative protein arises as an attempt to substitute conventional meats with other protein
4.0/). sources that require less intensive production means.

Foods 2021, 10, 1226. https://doi.org/10.3390/foods10061226 https://www.mdpi.com/journal/foods

Foods 2021, 10, 1226 2 of 29

Several examples of novel protein sources are cultured meat, plant-based meat, insect
protein and single-cell protein, which have gained interests from researchers and the
food industry in the past few years. To our knowledge, current research has focused
on the technological and industrial application of these alternative proteins, but their
safety aspects remain poorly described. In this review, we attempt to summarize their
current food safety status to support future developments of safe alternative proteins,
including brief descriptions of relevant technological backgrounds, environmental impacts
and regulatory framework.

2. Cultured Meat
2.1. Cell Type and Culture Media
Cultured meat, also known as in vitro, lab-grown or cell-based meat, is derived
from animal stem cells that are cultivated in controlled settings. Currently, the two main
stem cells considered to be the most suitable for culturing meat are embryonic stems
cells or satellite cells [11]. The main steps involved in the production of cultured meat
include the isolation of stem cells from an animal biopsy, followed by the proliferation
and differentiation of these isolated stem cells into desired tissues (for example, skeletal
muscles) in a cell culture medium [11,12]. In the process, the growing cells can be attached
to scaffolding materials, such as collagen-like gel polymers, which serve as a support
network for the tissue development [11,12]—potential polymers to be used as scaffolds are
listed elsewhere [13].
Key research milestones in the production of cultured meat include the patent for
in vitro meat filed by van Ellen et al. in 1999 [14] and the report on in vitro cultivation of fish
skeletal muscles in 2002 [15]. In 2013, the first cultured beef was produced by researchers
at Maastricht University and was sampled in London [16]. However, as only satellite
cells were used, the product only contained skeletal muscle fibers, but not other meat
components, such as fat and connective tissues [16]. Thus, for cultured meat to emulate
the complex structure of animal-based meats, additional cell sources have been proposed,
including adipocyte tissue-derived stem cells and endothelial cells [12]. As reviewed by
Ben-Arye and Levenberg, recapitulation of complex meat structures via tissue engineer-
ing needs to include skeletal muscles (myogenesis), extracellular matrix (fibrogenesis),
microvascular networks (vascularization) and intramuscular fats (adipogenesis) [17].
Given the need for an array of cell types in the production of cultured meat, there is a
practical advantage in utilizing pluripotent embryonic stem cells over their multipotent
counterparts derived from adult animals (for example, satellite cells). In 2018, Bogliotti et al.
reported on a stable culture of bovine embryonic stem cells [18]. However, ethical issues
may arise from the use of embryonic stem cells, and thus there is still a need for alternative
sources of stem cells. One of these sources is the induced pluripotent stem cells (iPSC)
derived from adult cells, which were first discovered in 2006 by Takahashi and Yamada [19].
About a decade later, researchers generated in vitro skeletal muscles from porcine iPSC [20].
The main findings presented in these studies [18–20], along with those from other relevant
publications, are summarized in Table 1.
Foods 2021, 10, 1226 3 of 29

Table 1. Relevant technologies to future developments of cultured meat.

Technology Relevance Main Finding Reference

Embryonic stem cells that are derived from First report on the derivation of stable bovine embryonic stem
Bovine embryonic stem cells livestock animals can be transformed into any cell cells in a culture containing fibroblast growth factor 2 and an [18]
type. inhibitor of Wnt signaling pathways.

1. Induction of pluripotent stem cells from mouse embryonic

or adult fibroblasts by the introduction of four
Pluripotent 1 stem cells derived from adult Ethical issues on the use of embryonic stem cells transcription factors (OCT3/4, Sox2, c-Myc and Klf4). [19]
fibroblasts (iPSC) may be circumvented. 2. Another transcription factor commonly associated with
pluripotency, namely NANOG, was dispensable.

Contractile porcine myotubes were produced through a

coordinated application of CHIR
Skeletal muscles derived from porcine iPSC Generation of livestock tissues from iPSC. [20]
99021 (inhibits GSK3B enzyme), 5-aza-cytidine (DNA
methylation inhibitor) and ectotopically expressed MYOD1.

1. IMP and MSC were successfully co-cultured using a

transwell chamber.
Co-culture system is required to produce a 2. In proliferative stage, MSCs accelerated the differentiation
Co-culture of IMP and MSC derived from chicken of IMPs, which resulted in a higher fat content in [21]
complex tissue resembling conventional meat.
co-cultured IMPs than single-cultured ones. Opposite
effect was observed in non-proliferative stage.

Descriptions of protocols for isolating pre-adipocyte

Method for culturing adipocytes in vitro allows (multipotent stem cell) from primary bovine adipose tissue and
Isolation of bovine PA and its adipogenic
for future development of cultured meat that for their subsequent differentiation in 2D culture media or on 3D [22]
differentiation
contains fat components. alginate scaffolds. Plant- and animal-based free fatty acids used
in the differentiation process.
1Pluripotent stem cells refer to those that can be transformed into any type of cell, as opposed to multipotent stem cells that can only be differentiated into specific cell types. iPSC, induced pluripotent stem cells;
GSK3B, glycogen synthase kinase 3-β; MSC, muscle satellite cells; IMP, intramuscular pre-adipocyte; PA, pre-adipocyte.
Foods 2021, 10, 1226 4 of 29

Stem cells must be cultured in a suitable medium. Currently, animal serum-based

media, particularly those containing fetal bovine serum (FBS), are the most commonly
used [12]. In cell culture media, serum provides the growing cells with a range of essential
nutrients, including hormonal and differentiation factors for cell proliferation (for example,
growth hormone and insulin), transport proteins (for example, transferrin and transcortin)
and growth factors (for example, epidermal, endothelial, fibroblast and insulin-like growth
factors) [23]. However, for safety and ethical reasons, there have been attempts to de-
velop serum-free media. While there are over 100 serum-free media formulations, growth
requirements vary with cell type, and thus a universal serum-free medium may not be
attainable [24].
Several serum-free media have been found to sustain the in vitro growth of bovine
muscle stem cells. Kolkmann et al. found that three commercial serum-free media (FBMTM ,
TesRTM and Essential 8TM ) were able to promote the growth of primary bovine myoblasts,
albeit the cell numbers after six days did not reach the level found in a standard growth
medium (Dulbecco’s Modified Eagle’s Medium with 30% serum) [25]. Nevertheless, cost
optimization will be needed for industrial-scale production of cultured meat, particularly
regarding the high price of growth factors in serum-free media, which could account
for up to 99% of the total cell culture medium cost (as estimated using ingredients of
Essential 8TM for a hypothetical 20,000 L batch) [26]. To alleviate these cost issues, a group
of researchers has proposed substituting serum with yeast extracts or several hydrolysates
derived from food by-products—these by-products were chicken carcass, cod backbone,
pork plasma, eggshell membrane or egg white powder—all of which were found to sustain
the proliferation of bovine skeletal muscle cells grown in serum-free media to a degree
comparable to cells cultured in full-serum conditions [27]. Cyanobacterial hydrolysates
have also been proposed as a nutrient source for culturing meat, albeit there is still a lack
of experimental data, and thus this remains a subject of future studies [28].

2.2. Potential Food Safety Risks of Cultured Meat: Virus, Prion and Foreign Genes
Given that cultured meat is almost exclusively produced in a laboratory, it can be
considered to be less susceptible to zoonotic diseases than conventional meat products.
However, there are gaps in the current knowledge of food safety related to cultured meat,
especially due to the fact that the majority of research efforts are focused on the optimization
of production means. The most apparent safety issues may arise from the use of animal
serum in the culture medium. As outlined by the European Medicines Agency [29] and the
United States Department of Agriculture [30], all bovine-derived serum should be free of
the following viruses: bovine viral diarrhea virus, reovirus 3, rabies virus, bluetongue virus,
bovine adenovirus, bovine parvovirus and bovine respiratory syncytial virus, regardless of
their geographical origin [31].
Another potential hazard in animal serum is the pathogenic and infectious prion
(PrPSC ), particularly due to possible cross-species and blood-related transmissions. PrPSC
is a misfolded prion protein commonly associated with transmissible spongiform en-
cephalopathies, such as scrapie in sheep, chronic wasting disease in deer, bovine spongi-
form encephalopathy (BSE) in cattle and variant Creutzfeldt-Jakob disease (vCJD) in
humans [32]. As an acquired form of Creutzfeldt-Jakob disease—the other forms being
sporadic and genetic—vCJD has been associated with the consumption of food products
derived from infected cattle. The most prominent example is the disease onset of vCJD in
the United Kingdom that peaked in the year 2000, which was about 6–7 years after the peak
of the BSE epidemic in the same region [33]. Another study confirmed that cross-species
transmission of PrPSC from BSE-infected cattle to humans was possible, using transgenic
mice as a model [34]. Others have also demonstrated that blood components may act as
vectors for prion disease transmission [35,36].
In addition, the production of cultured cells for human consumption is unprecedented
and would require further assessments, particularly regarding the use of genetic engineer-
ing. For example, the generation of iPSCs involves the introduction of exogenous genes
Foods 2021, 10, 1226 5 of 29

(i.e., transcription factors) into the genome of somatic cells (Table 1). Genetic engineering
may also be used to improve the means of production—for example, by increasing cell
culture density through the inhibition of Hippo signaling pathway, as demonstrated in a
patent application submitted by researchers at Memphis Meat [37].
The issues mentioned in this section are still mostly theoretical, as to our knowledge,
the current literature lacks studies that directly assess the food safety risks related to
cultured meat. Future research should aim at identifying the safety issues of cultured meat,
particularly within the context of mass-scale production. These may include the retention
and infectivity of viruses or PrPSC within the final structure of cultured meat and also the
health implications, if any, of ingesting meat analogues derived from genetically modified
cultured cells.

3. Plant-Based Meat
3.1. Ingredients and Processing Technologies
The early use of plant-based ingredients, such as fermented soybean cake (i.e., tofu
and tempeh) or wheat (i.e., seitan), as meat analogues could be traced back to the Asian
communities in the 10th century [38,39]. However, these products are not able to emulate
the sensorial properties of animal-based meat. Instead, texturized vegetable proteins
(TVP) can be used as a potential replacement of conventional meat and are commonly
derived from soy proteins [40], or to a lesser extent, from wheat glutens [41,42] and legume
proteins (for example, pea and chickpea) [43,44]. In association with the Institutes of Food
Technologists, Egbert and Borders proposed a composition of plant-based meat (Table 2),
which is consistent with the recipes used by several existing animal-free meat companies,
such as Impossible Food, Beyond Meat and Gardein [45,46].

Table 2. Ingredients of plant-based meat as proposed by Egbert and Borders in 2006 [47].

Ingredient Function Usage Level (%)

Water Distribution of ingredients, emulsification, juiciness 50–80
TVP = textured soy flour, textured soy concentrate, Water binding, texture/mouthfeel, appearance,
textured wheat gluten or textured protein protein fortification/nutrition and source of 10–25
combinations (for example, soy and wheat) insoluble fiber
Non-texture proteins = ISP, functional soy
Water binding, emulsification, texture/mouthfeel
concentrate, wheat gluten, egg whites * or whey 4–20
and protein fortification/nutrition
proteins *
Flavor (savory, meaty, roasted, fatty and serumy),
Flavors/spices flavor enhancement (for example, salt) and mask 3–10
cereal notes
Flavor, texture/mouthfeel, succulence and Maillard
Fat/oil 0–15
reaction/browning
Texture, water binding, potential fiber content and
Binding agents = wheat gluten, egg whites *, gums
determine processing conditions (depending on how 1–5
and hydrocolloids, enzymes or starches
and where they are added)
Coloring agents = caramel colors, malt extracts, beet
Appearance and eye appeal 0–0.5
powder and other FDA-approved colors (FD & C)
TVP, texture vegetable proteins; ISP, isolated soy proteins; FDA, Food and Drug Administration. * These ingredients are not plant-based
and their use in plant-based meat products requires clear labelling.

Currently, plant-based meats are mainly produced through thermoplastic extru-

sion [42,43,48–50]. This process can be categorized based upon the amount of water
added, i.e., low moisture (20–35%) or high moisture (50–70%) [51]. Both product types
are made in three main steps: (1) pre-conditioning of the raw materials outside of the
extruder; (2) heating and compression inside of the extruder; (3) cooling of the die and
processing of the final product (for example, cutting to desired pieces) [52]. In low-moisture
Foods 2021, 10, 1226 6 of 29

extrusion, unhydrated protein concentrates may be directly introduced into the extruder,
which would result in a final product that has a high water-binding capacity, and thus
can be used as a meat extender (for example, in sausages or beef patties). On the contrary,
high-moisture extrusion involves pre-extrusion hydration of the protein concentrates to a
moisture content of 60–70%, followed by heating in the extruder that induces the formation
of viscoelastic mass, and subsequently slow cooling to prevent the disruption of the newly
formed product due to excessive material expansion [45].
The inherent differences between plant and animal proteins present a challenge to
the production of plant-based meat products. As highlighted in one study, there were
differences in the physical and nutritional features between TVP and animal-based meats,
including their psychochemical properties, textural characteristics and amino acid retention
after thermal processing [53]. This concern may be partially addressed by the use of
potassium carbonate [50] and alginate [48] to achieve sensorial properties of plant-based
meat that are comparable to cooked ground beef. Essential nutrients that are often missing
from a vegetarian diet, such as vitamins B-12 and D, calcium, zinc, iron and long-chained
n-3 (omega-3) fatty acids, could also be added post-extrusion to increase the nutritional
value of plant-based meat [49]. However, future research is needed to further optimize the
processing conditions of plant-based meat, for example, through the use of computational
techniques to predict the sensorial properties of meat analogues as a function of operating
conditions. A group of researchers used a genetic algorithm to optimize the texture and
appearance of a meat analogue, i.e., extrusion process conditions that achieved the highest
water and oil binding capacity and that also achieved product brightness [54].
Shear cell technology can be used to structure proteins through the combination of sim-
ple shear and elevated temperature, either in a conical shear cell device or in a cylindrical
Couette cell [45]. The final structure depends on the process temperature, concentration of
dry matter content (soy protein concentrates) and presence of polysaccharide (for example,
soy fiber) [55]. Consistently, varying process conditions have been used for other types of
protein or protein–polysaccharide combinations, such as soy protein isolates (SPI) [55,56],
SPI and wheat gluten [57] or SPI and pectin [56,58]. Krintiras et al. have also demonstrated
that scale-up is possible from a small Couette cell (200 g of sample) [59] to a larger one
(approximately 7.5 kg of sample) [60], with the authors identifying no barriers to further
upscaling [60].
Other available technologies include electrospinning [61] and 3D printing [62], al-
though the feasibility of these techniques for the production of plant-based meat remains a
subject of future studies.

3.2. Food Safety Risks of Plant-Based Meat: Bacteria, Anti-Nutrients, Allergens, Thermally
Induced Carcinogens and Genetically Modified Soybean
Plant-based meat can carry pathogenic bacteria originating from the raw ingredients.
Although most of these bacteria could be inactivated by the heat produced during the ex-
trusion process, some endospore-forming bacteria, such as Clostridium spp. or Bacillus spp.,
may survive the heating regime [63]. In a research commissioned by the European Union
(i.e., LikeMeat project), Clostridium sporogenes spores (ATCC 19404) in protein ingredients
were rendered inactive (below detection level) by extrusion, albeit Bacillus amyloliquefaciens
(AB255669 and LMA008A; <1000 colony forming units/g) were detected in the final
protein extrudates, possibly due to re-contamination post-extrusion [63,64]. This notion
of re-contamination was further enhanced by the identification of Enterococcus durans,
Exigobacterium acetylicum, Acinetobacter spp. and Staphylococcus spp. in uninoculated sam-
ples [64]. Consistently, another study identified several bacterial contaminants in plant-
based meat, which predominantly consisted of Lactobacillus sakei, Enterococcus faecium and
Carnobacterium divergens [65].
Health risk of plant-based meat may also arise from the presence of anti-nutrients.
Legumes have been associated with anti-nutrients, such as protease inhibitors, α-amylase
inhibitors, lectins (phytohemagglutinin), polyphenols (particularly tannins) and phytic
acid [66]. While these anti-nutrients can have health benefits, they have also been asso-
Foods 2021, 10, 1226 7 of 29

ciated with negative physiological effects, including altered gut function (for example,
inactivation of digestive enzymes or reduced iron absorption) and endocrine disruption,
among others [66,67]. Nevertheless, as reviewed by Petroski and Minich, plant-based anti-
nutritional compounds (lectins, oxalate, phytate, goitrogens, phytoestrogens and tannins)
are mainly harmful when ingested in high quantities or in isolation, and they could be
inactivated through cooking [67]. Others also found that extrusion at 170–180 ◦ C reduced
the amount of trypsin inhibitor, phytic acid and tannins in a meal derived from maize,
soybean concentrate and cassava starch [68].
It is well-established that legumes contain allergens that cause mild to life-threatening
conditions. The majority of these allergens belong to three protein families—namely, stor-
age proteins (with two main superfamilies prolamins and cupins), pathogenesis-related
proteins (for example, Gly m 4 in soybean) or prolifins (for example, Gly m 3 in soy-
bean). Legume-related allergens elicit IgE-mediated immunological reactions, with clinical
manifestation primarily grouped into four categories: cutaneous (skin), gastrointestinal,
cardiovascular and respiratory. Detailed discussions on legume allergies are provided
elsewhere [69]. Given the uncertainty on whether thermal processing can reduce the
allerginicity of legume proteins, particularly those derived from soybeans and peas, ad-
ditional studies are warranted [70,71]. Several research reports suggest that treatments
with high pressure, ultrasound and pulsed light can reduce the activity of allergens in
soybean [72]. Nonetheless, clinical studies are also necessary to determine whether the
reduced activities of legume allergens post-processing lead to significant physiological and
immunological effects in live hosts, including humans.
While thermal processing is essential for reducing the activities of microorganisms,
anti-nutrients and allergens, it may also induce the formation of carcinogens—particularly
in processed meat products—for example, polycyclic aromatic hydrocarbons [73,74], ni-
trosamines [75,76] or heterocyclic aromatic amines [77,78]. In their review article, He et al.
mentioned unpublished data on the detection of N-nitrosodiethylamine (15 µg/kg) in one
sample of cooked plant-based meat [79]. Thus, more studies are required to determine the
potential safety risk of these chemicals in plant-based meat [79].
As of 2017, the global adoption of genetically modified soybeans (also known as
biotech soybean or GM soybean) reached 77%, which included glyphosate-tolerant vari-
ants and were primarily planted in Brazil, Argentina and the United States of America
(USA) [80]. Concerns arise from the potential accumulation of glyphosate residues in these
GM soybeans—or their associated food products—and the subsequent adverse effects upon
ingestion [81]. These health concerns have been highlighted in studies using non-human
animal models, such as Japanese quails [82] and crustacean Daphnia magna [83,84]. Current
literature contains no data on the accumulation of glyphosate in plant-based meat.
Another GM ingredient in plant-based meat is soy leghemoglobin, derived from
yeast (Pichia pastoris) expressing the leghemoglobin c2 gene from soybean (Glycine max).
Jin et al. reported no potential allergenicity and toxigenicity associated with the use of
recombinant soy leghemoglobin in food, which was a conclusion determined through a
literature search, bioinformatics analyses on the amino acid sequence of leghemoglobin
and 17 Pichia host proteins, and also in vitro pepsin digestibility of leghemoglobin [85].
Another study by Fraser et al. demonstrated no genotoxic effects of recombinant soy
leghemoglobin on Salmonella enterica subsp. enterica serovar Typhimurium and E. coli
(Ames test) as well as no adverse events in Sprague Dawley rats fed with the protein for
28 days at 750 mg/kg/day [86].

4. Insect Protein
4.1. Insect Species, Farming and Processing
Insects have been a part of the human diet for centuries, particularly in Asia and
Africa [87]. According to the Food and Agriculture Organization of the United Nations
(FAO), there are over 1900 insect species consumed around the world (Table 3) [88]. This
practice of eating insects, also known as entomophagy, is sustainable due to the high
Foods 2021, 10, 1226 8 of 29

amounts of protein and polyunsaturated fatty acid contained in edible insects, although
there are variations across species [89–92]. Insects are also more effective in converting
feed into edible body mass than farm animals [93]. These have made them an attractive
option for expanded production to improve global food security.

Table 3. Major groups of insects consumed around the world as reported by van Huis et al. in association with the Food
and Agriculture Organization of the United Nations (FAO) [88]. Several species from each class are mentioned, but this list
is not exhaustive.

Insect Class Insect Species Percentage (%) 2

Yellow mealworm (Tenebrio molitor), palm weevil
Coleoptera 1 (Rhynchophorus phoenicis, Rhynchophorus ferrugineus 31
and Rhynchophorus palmarum)
Caterpillars of butterflies or moths (Daphnis spp.,
Lepidoptera 1 Theretra, Imbrasia belina, Omphisa fuscidentalis, 18
Comadia redtenbacheri or Aegiale hesperiasis)
Weaver ants (Oecophylla spp.), yellow jacket wasps
Hymenoptera 1 (Vespula and Dolichovespula spp.) and honeybees 14
(Apis mellifera)
Crickets (Gryllus bimaculatus and Acheta domesticus)
Orthoptera 13
and locusts (Locusta migratoria)
Hemiptera (suborders Homoptera and Cicadas (Ioba, Playtypleura and Pycna), pentatomid
10
Heteroptera) bugs (Agonoscelis versicolor)
Termites (Macrotermes and Syntermes), dragonflies,
Isoptera, Odonata, Diptera and others 14
flies and others
1Edible insects listed are usually consumed during their larval stage. 2 Proportion of edible insects from each class, with the total reported
number of edible insects coming from 1900 species.

Most edible insects are harvested from the wild, but they can also be semi-domesticated
through habitat manipulation or reared in farms for a mass-scale production [88,94].
Wild harvesting and semi-domestication of insects are discussed in detail elsewhere [94],
whereas our review article focuses on farmed insects. For example, edible palm weevil
(Rhynchophorus ferrugineus) and crickets (Acheta domesticus, Gryllus bimaculatus,
Teleogryllus testaceus and Teleogryllus occipitalis) are produced by local farmers in Thai-
land [95]. These farmers use containers (plastic drawers, concrete block pens, plywood
boxes, etc.) to house the insects and nourish them with chicken feed (crickets) or plant-
based feeds (for example, sago palm trunks for palm weevil or cassava leaves for
crickets) [95].
Similar to other animals, insects require macronutrients (lipids, proteins and carbo-
hydrates) and micronutrients (essential sterols and vitamins), which can be derived from
animals, plants and yeast [96]. In particular, polyunsaturated acids, essential amino acids
and sterols must be supplied in the feeds, given that insects lack the ability to synthesize
these compounds in sufficient amounts [97]. As previously reported, the nutritional com-
position of feeds could influence the growth performance of mealworms (Tenebrio molitor,
Zophobas atratus or Alphitobius diaperinus), including their fatty acid profiles, lipid contents,
larval development time and progeny production [98–100]. As summarized by Morales-
Ramos et al., strategies to optimize artificial dietary supplements for farmed insects include
the determination of basic nutrient ratios (lipids, carbohydrates and proteins), feeding
adaptations (for example, encapsulation of liquid nutrients) and feeding refinement (for
example, feeding stimulants or nutritional adjustments for different growth stages) [97].
While organic wastes can be used as feeds for farmed insects, there are concerns about
the potential transmission of pathogens from these waste materials, the lack of adequate
nutrients present and the uncertain supply of organic wastes, particularly for a mass-scale
production [88]. A review article by Varelas provides extensive discussion on the mass
Foods 2021, 10, 1226 9 of 29

production of edible insects using food wastes as feeding substrates, including coverage of
topics on the composition of different substrates, fermentation processes and characteristics
of different insects fed with varying food wastes [101].
In addition to adequate nourishments, rearing conditions (for example, temperature,
humidity and population density) need to be optimized [102]. Currently, there is a lack
of data on the best method for processing and storing edible insects in a mass-scale
production, particularly given that nutritional needs and rearing conditions vary across
species [102]. Viral infections are another challenge for insect farmers, and this topic is
comprehensively assessed in a review article by Maciel-Vergara and Ros, which includes
discussion on the factors affecting the emergence of these pathogens in mass-scale rearing
systems and also the measures to prevent or manage infections that range from simple
sanitation interventions to advanced antiviral methods (for example, RNA interference
and transgenic technologies) [103].
Post-harvest processing of edible insects traditionally involves degutting and thermal
processes, such as boiling, frying, toasting, smoking, roasting and drying, which are
particularly important for eliminating microbial contaminants and increasing the shelf-
life of the final products [104]. More recently, other technologies have been used for the
extraction of substances from edible insects, including ultrasound, enzymatic hydrolysis,
supercritical carbon dioxide, sonication, soxhlet extraction and folch extraction [105].

4.2. Food Safety Risks of Edible Insects: Bacteria, Mycotoxins, Parasites, Allergens, Heavy Metals
and Anti-Nutrients
Microbial contents of edible insects are affected by growth conditions and species.
Metagenomic analyses revealed that microbial communities in yellow mealworm larvae
(T. molitor) [106,107] and grasshoppers (Locusta migratoria) [107] were dominated by bacte-
ria belonging to the phyla Proteobacteria and Firmicutes, although the two insect species
contained distinct bacterial genera. Further, the abundance of Actinobacteria and Bacteri-
oidetes differed considerably between the two studies [106,107]. Others have also reported
variations in the abundance and the type of bacteria present in edible insects supplied by
different companies [108,109]. These discrepancies may be attributed to differing farming
practices between companies, as highlighted by Li et al. that gut microbiota of T. molitor
larvae varied with rearing conditions (closed or open environment) [110].
Safety concerns can arise from the presence of pathogenic microorganisms in edible
insects. Several bacterial genera, including Cronobacter, Bacillus, Staphylococcus, Clostridium
and Haemophilus, have been identified in T. molitor or L. migratoria [106,107]. In another
study, Bacillus cereus and several potentially pathogenic bacterial genera (for example,
Salmonella, Vibrio and Acinetobacter) were detected in products derived from crickets
(A. domesticus), locusts (L. migratoria) and yellow mealworm larvae (T. molitor) [108]. Bac-
terial endospores of B. cereus were also isolated from T. molitor and A. domesticus, with
most strains (65%) containing cereulide plasmid, which may enable the production of a
heat-resistant toxin [111]. Others found that non-pathogenic Enterobacteriaceae dominated
in T. molitor [109,112,113]. Antimicrobial-resistance genes have also been reported in edible
insects, which included L. migratoria, A. domesticus and T. molitor [114–116].
The presence of fungi, such as those belonging to the genera Aspergillus, Penicillium
and Fusarium, in edible insects is a concern due to potential health risks associated with
mycotoxins [117,118]. As reported by Musundire et al., aflatoxin B1 was present in edible
stink bugs (Encosternum delegorguei), although the presence of the toxin depended on the
containers used to store the insects [119]. Interestingly, contamination of feeding substrates
with mycotoxins (for example, aflatoxin B1 , deoxynivalenol, ochratoxin or zearalenone) did
not seem to result in the accumulation of these toxins in several edible insect species, which
was potentially due to the ability of these insects to metabolize mycotoxins [120–123]. An-
other study showed that T. molitor could resist several mycotoxins introduced to their feeds,
albeit this depended on the fungal species from which the mycotoxins were derived [124].
These findings indicate that the digestion of mycotoxins may be specific to the insect species
Foods 2021, 10, 1226 10 of 29

and the type of mycotoxin, and thus future research is required, including studies on the
by-products of mycotoxin digestion by insects.
In addition to bacteria and fungi, parasitic pathogens may be transmitted via edible in-
sects. For example, potential human parasites have been identified in 91 insect farms across
six European countries, including Cryptosporidium spp., Isospora spp., Balantidium spp. and
Entamoeba spp. [125]. Diseases have been associated with the ingestion of infected insects, as
previously recorded for the transmission of Trypanosoma cruzi [126] and
Gongylonema pulchrum [127].
Safety risk of food-borne viral pathogens in edible insects is low, as one study reported
the absence of hepatitis A virus, hepatitis E virus and norovirus (genogroup II) in raw
yellow mealworms (T. molitor) and crickets (A. domesticus) [111]. A similar conclusion was
proposed in a report by the European Food Safety Authority (EFSA), in which a panel of
experts declared that viral infections in edible insects were specific for insects, and thus not
considered a hazard for humans [128].
These microbiological concerns can be mitigated by thermal interventions, such as
blanching and drying [113]. However, Klunder et al. found that heating was only effec-
tive against Enterobacteriaceae in T. molitor and A. domesticus, but not against bacterial
spores [129]. Instead, the authors proposed drying or acidification, including the use
of lactic acid fermentation to preserve composite meals of sorghum and T. molitor [129].
Interestingly, Rumpold et al. found that indirect cold plasma treatment was effective in
reducing the microbial load on the surface of T. molitor, albeit additional thermal (90 ◦ C)
and high pressure (600 MPa) treatments were required to achieve inactivation of the gut
microbiota [130].
Similar to plant-based meat, edible insects may be a source of allergens. These include
arginine kinase and tropomyosin, which are pan-allergens dominant in arthropods, as
evident from reports on IgE cross-reactivity between edible insects and house dust mite
or crustaceans [131]. However, the clinical significance of these allergens remains to be
established [131]. The effects of processing on the allergenicity of edible insects are also
unclear. In one study, enzymatic hydrolysis and thermal treatment eliminated cross-
reactivity and allerginicity of insect extracts (L. migratoria), as tested by immunoblots and
skin prick test [132]. Similarly, heat treatment reduced but did not eliminate the allergenicity
of mealworms (T. molitor, Zophobas atratus and Alphitobius diaperinus) in samples taken
from patients allergic to house dust mites and crustaceans [133]. Others demonstrated that
thermal processing of T. molitor did not change its IgE binding in a basophil activation
test nor in the skin reaction in a skin prick test (crustacean-allergic patients were used),
although the solubility of several proteins was altered [134]. Consistent with the data that
we collated in this review, conflicting results were also reported in a review article by Gier
and Verhoeckx [135].
Heavy metals can accumulate in edible insects, which is dependent on the growth
environments (wild or reared). Vijver et al. reported that cadmium, copper, lead and
zinc present in soils (naturally or artificially contaminated) were retained in the body of
T. molitor, with the extent of accumulation depending on the type of soil and metal [136].
Greenfield, Akala and van der Bank detected high concentrations of heavy metals in
Mopane worms (Imbrasia belina) taken from two sites at a South African national park: cad-
mium, copper and zinc concentrations were respectively 15–21, 2–2.5 and 0.4 times higher
than the recommended legal levels in the United Kingdom and European Union [137].
The concentration of manganese in I. belina was 20–67 times higher than the food safety
standard set by the United States Food and Drug Administration [137]. Others found
that contaminated feeds led to the accumulation of cadmium, lead or arsenic in soldier
fly larvae (Hermetia illucens) and yellow mealworms (T. molitor) [123,138], with one study
also suggesting that the accumulation rate was dependent on the insect species and type
of metal present [138]. On the contrary, another group of researchers demonstrated an
insignificant presence of arsenic, lead, chromium and mercury in reared grasshoppers
(Oxya chinensis subsp. formosana) [139].
Foods 2021, 10, 1226 11 of 29

Available data suggest that there is a minimal food risk associated with anti-nutritional
factors in edible insects. In India, a study found low levels of phenols and tannins (be-
low 0.52%) in aquatic edible insects—namely, Lethocerus indicus, Laccotrephes maculatus,
Hydrophilus olivaceous, Cybister tripunctatus and Crocothemes servillia [140]. Another study
conducted in Nigeria reported that the larvae of Cirina forda contained acceptable levels of
oxalate (4.11 mg/100 g) and phytic acid (1.02 mg/100 g) [141]. Similarly, EFSA declared
that the amounts of oxalic acid, phytic acid, hydrogen cyanide and polyphenols in T. molitor
were comparable to other foodstuffs [142]. However, researchers in Japan detected heat-
resistant thiaminase, which is a risk factor for vitamin B1 deficiency, in the pupae of
edible African silkworm (Anaphe spp.) [143]. Future investigations are required for other
insect species.

5. Single-Cell Protein: Microalgae, Fungi and Bacteria

Single-cell proteins, also known as microbial proteins, are commonly derived from mi-
croalgae, fungi or bacteria. In their review article, Ritala et al. summarized available studies
on potential fungal, microalgal and bacterial species for application in the production of
single-cell proteins, including patents from the years 2001 to 2016 [144]. However, the
safety aspects of this food category are still unknown, and thus this section was aimed at
identifying potential safety hazards associated with single-cell proteins, including allergens,
toxins and heavy metals.

5.1. Microalgal Protein: Species, Processing Technology and Food Safety Risks
Several of the most biotechnologically relevant microalgae include green algae
(Chlorella vulgaris), Haemotococcus pluvialis, Dunaliella salina and spirulina (Arthrospira maxima
or Arthrospira platensis), as shown in Figure 1 [145]—while spirulina is commonly categorized
as a product derived from microalgae (blue/green), it is biologically classified as belonging
to the phylum Cyanobacteria. As demonstrated in three studies, microalgae (A. platensis,
C. vulgaris, Chlorella pyrenodiosa, Isochrisis galbana or Tetraselmis spp.) contained high levels
of protein (wprotein /wdry mass ), albeit there were variations across species ranging from 27%
in I. galbana to 64% in A. platensis [146–148]. Similarly, the contents of polyunsaturated fatty
acids, carbohydrates and mineral elements (for example, calcium and potassium) vary with
different microalgal species [146,147]. These variations in the nutrient contents of microalgae
have also been reported in previous review articles [145,149,150].
Most commercial production of microalgae utilizes open-air systems, which can be di-
vided into four categories: big ponds, tanks, circular ponds and raceway ponds. However,
due to the disadvantages of these open systems, such as low productivity, contamination
risk, difficulty with biomass recovery and problems of temperature control, closed sys-
tems have been developed, including tubular, flat panels and others. Microalgal biomass
can be recovered by sedimentation, filtration, centrifugation or flotation. Subsequently,
the biomass can be used as a whole, for example, after being spray-dried [149]. Alterna-
tively, microalgal proteins can be extracted in two steps: (1) disrupting the cells through
mechanical (for example, bead mill, homogenizer or ultrasonication) or non-mechanical
(microwave, pulsed electric field, enzymatic, ionic liquid or chemicals) means; (2) sepa-
ration of protein phase from other cellular debris by means of centrifugation, filtration
or ultrafiltration, in combination with improving the dispersibility of protein in the aque-
ous solution [151]. A new technology of a three-phase partitioning system has also been
proposed to extract proteins from C. pyrenoidosa, in which the microalgal components are
divided into non-polar (upper), protein (middle) and polar (lower) phases [148].
 chanical (microwave, pulsed electric field, enzymatic, ionic liquid or chemicals) means;
(2) separation of protein phase from other cellular debris by means of centrifugation, fil-
tration or ultrafiltration, in combination with improving the dispersibility of protein in
the aqueous solution [151]. A new technology of a three-phase partitioning system has
also been proposed to extract proteins from C. pyrenoidosa, in which the microalgal com-
Foods 2021, 10, 1226 12 of 29
ponents are divided into non-polar (upper), protein (middle) and polar (lower) phases
[148].

Figure Microalgaethat
Figure 1. Microalgae thatcan
canbebe used
used as as sources
sources of protein
of protein for for human
human consumption:
consumption: (a) Haemotococcus
(a) Haemotococcus pluvialis
pluvialis with
with drop-
lets of astaxanthin
droplets within
of astaxanthin the the
within cells; (b) (b)
cells; Chlorella vulgaris;
Chlorella (c)(c)
vulgaris; Arthrospira maxima
Arthrospira maximaSAG
SAG21–99
21–99(spirulina).
(spirulina).Scale
Scalebar
bar == 15
15 µm.
µm.
Images were
Images were taken
taken from
from Wells
Wellset etal.
al. (2016)
(2016) published
published under
under the
the Creative
Creative Commons
Commons Attribution
Attribution 4.0
4.0 International
International License
License
(http://creativecommons.org/licenses/by/4.0/) [152].
(http://creativecommons.org/licenses/by/4.0/) [152].

harmful substances,
Despite their nutritional values, microalgae may also carry harmful substances, such
such
as heavy
heavy metals
metalsand
andtoxins,
toxins,particularly
particularlydue
duetoto
contaminations. Rzymski
contaminations. Rzymskiet al.
et found that
al. found
that certain food supplements derived from Spirulina spp. and Chlorella spp. contained
cadmium, mercury and lead, albeit at levels below the provisional tolerable weekly intake,
and this was likely due to the lack of quality control measures [153]. The contamination
risk is particularly high when wastewater is used as a growing substrate, given that mi-
croalgae can uptake these heavy metals from the environment, either passively via surface
sorption (living or non-living cells) or through metabolic-dependent activities (for example,
active transport) [154,155]. Further, the rate of heavy metal uptake varies across mi-
croalgal species; for example, cadmium accumulation ranged from 0.02 mgCd /gdry biomass
in I. galbana and 8–357 mgCd /gdry biomass in A. platensis to 1055.27 mgCd /gdry biomass in
Chaetoceros calcitrans [155].
Toxin can accumulate in microalgae, particularly when the environment is contam-
inated with toxin-producing cyanobacteria, such as Microcystis aeruginosa. Two studies
reported the contamination of microalgal dietary supplements (Aphanizomenon flos-aquae)
with cyanobacterial hepatoxins—namely, microcystins, as confirmed by toxin detection
(cPPIA, Adda-ELISA or LC/MS assays) and also the presence of microcystin-producing
genes mcyE or mcyB (PCR analysis) [156,157]. Indeed, A. flos-aquae has the capability of pro-
ducing cyanotoxins, such as anatoxin-a, cylindrospermopsin, microcystins and saxitoxins,
and thus the presence of these cyanotoxins in A. flos-aquae products may be the result of
direct toxin production by A. flos-aquae or cross-contamination with other toxin-producing
cyanobacteria [158]. Along with microcystins, anatoxin-a and β-methylamino-L-alanine
were detected in eight food products derived from Arthrospira or A. flos-aquae [159]. Interest-
ingly, it has been reported that while toxins (microcystins or polymethoxy-1-alkenes) were
absent in several dietary supplements derived from Arthrospira spp. and Chlorella spp.,
these products still exhibited cytotoxicity in human A549 cells [156] and adult zebrafish
(Danio rerio) [160], although the reason for this phenomenon was unknown [156,160].
Several authors have reported allergic reactions to microalgae, including anaphy-
laxis after the consumption of spirulina-derived products (A. platensis) [161,162] or acute
tubulointerstitial nephritis following the ingestion of Chlorella tablets [163]. Another
case report detailed severe allergic reactions in a girl after swimming in a lake with
blooming freshwater cynobacteria, and subsequent immunological tests showed IgE
cross-reactivity with extracts of several cyanobacterial species—namely, M. aeruginosa,
Synechocytis spp., Synechoccus spp., Pseudanabaena spp., Oscillatoria spp., Lyngbya spp. and
Arthrospira spp. [164]. The β-chain of the C-phycocyanin protein has been identified as
the main allergen in cyanobacteria [161,165], which may act alone or in a complex with
other phycobiliproteins [166]. One study also reported that other unidentified proteins
Foods 2021, 10, 1226 13 of 29

could potentially be allergens in several freshwater, marine and terrestrial cyanobacterial

species [165]. Allergens in non-cyanobacterial microalgae are still poorly understood.
Microbial contamination poses an additional safety issue associated with microalgal
food products. In one study, filamentous cyanobacterial contamination was detected in
commercial Chlorella tablets, along with several non-pathogenic bacteria [167]. Further,
genomic analyses have revealed the presence of several potentially pathogenic bacterial
genera in spirulina products (A. platensis)—namely, Pseudomonas, Flavobacterium, Vibrio,
Aeromonas, Clostridium, Bacillus, Fusobacterium and Enterococcus [168]. Another study re-
ported the detection of Clostridium endospores in commercial A. platensis products, includ-
ing toxin-producing and β-hemolytic isolates [169].
In addition to bacterial contamination, concerns about viral pathogens in microalgae
have been raised due to the isolation of Acanthocytis turfacea chlorella virus 1 (ATCV-1),
which is a chlorovirus commonly infecting green algae, in human oropharyngeal sam-
ples [170]. Subsequent analyses indicated that ATCV-1 resulted in decreased cognitive
functions in humans and mice [170], potentially due to fact that this virus persisted and
induced inflammatory factors in the macrophages [171]. The relevance of these findings to
the food industry needs to be ascertained in future studies.
As described above, the processing of microalgal biomass involves minimal heat
treatments. Thus, the removal of heavy metals, toxins, allergens and microorganisms from
microalgae can be challenging. Available methods include heavy metal-binding ligand
for removing heavy metals [172] or cold plasma for inactivating microorganisms [173].
Given that phycocyanin is a functional compound, it can be extracted from the cells of
cyanobacterial microalgae (for example, through a high-pressure extraction process) and
used as a separate product [174].
Pre-harvest preventative measures can also be taken with the use of microcystinase
A enzyme (MlrA) to reduce the prevalence of M. aeruginosa in the environment and its
concomitant production of microcystins. As reported by Liu et al., MlrA decreased the
cell viability of M. aeruginosa through the degradation of extracellular microcystin-LR,
inhibition of genes responsible for intracellular microcystin production (mycA, mycB, mycD
and mycG) and impairment of photosynthetic ability, including lowered expression of
photosynthetic genes psbB, psbD, rbcL and fbp [175]. The authors also found that the
activity of MlrA was selective, as it did not affect Synechocystis sp. PCC 6803, which is a
microcystin-nonproducing species [175].

5.2. Fungal Protein (Mycoprotein): QuornTM , Potential Allergens and Mycotoxins

In the late 1960s, an effort was conducted to search for alternative proteins from starch-
fermenting fungi, which led to the discovery of a filamentous fungus called
Fusarium graminearum A3/5 [176]. Thereafter, this fungal strain has been used for the
production of mycoprotein under the brand QuornTM . In 1998, the fungal species used in
QuornTM was re-classified as Fusarium venenatum based upon molecular phylogenetic, mor-
phological and mycotoxin analyses [177]. More recently, genomic analysis described the
difference between F. graminearum and F. venenatum, including genes that encode different
types of mycotoxin (type B trichothecene in F. graminearum and type A in F. venenatum) [178].
To date, F. venenatum A3/5 is not known to produce mycotoxins in the processing condi-
tions used, albeit regular monitoring is still used to ensure that the final product does not
contain these toxins [176].
Typical composition of QuornTM mycoprotein includes protein (45%), fiber (25%), fat
(13%) and carbohydrate (10%), as expressed per 100 g dry weight [179]. Manufacturing
processes of QuornTM mycoprotein begin with aerobic fermentation of F. venenatum in a
glucose-rich medium, along with other micronutrients, using an airlift bioreactor. After a
desired level of recirculating solids is achieved, the fermenter broth is heated to stop growth
and also to reduce the amount of RNA in the mycoprotein (approximately 1% w/w) through
the action of natural nuclease enzymes in the mycelium. Centrifugation is then used to
clarify the RNA-reduced fermenter broth, followed by vacuum chilling to achieve a final
Foods 2021, 10, 1226 14 of 29

mycoprotein at approximately 24% (w/w) total solid contents [176,179,180]. Subsequent

texturing can be done, for example, by the use of temperature (heat and chilled), pressure
and in combination with egg proteins [180].
The main food safety hazard associated with mycoprotein is allergens. While data
are limited, adverse reactions to mycoproteins have been reported in individuals with a
history of mold allergies. In one report, a 15-year-old male exhibited type I hypersensitivity
symptoms after consuming meatless chicken and subsequent skin prick test revealed that
the individual was also allergic to several mold species, with particularly strong reaction
to Fusarium vasinfectum [181]. Similarly, type I hypersensitivities were observed in a 27-
year-old female within a few minutes of ingesting QuornTM burger, with detected IgE
cross-reactivity with three mold species—namely, Alternaria alternate, Aspergillus fumigatus
and Cladosporium herbarm [182]. This IgE-mediated allergy to mycoprotein may be caused
by the acidic ribosomal protein P2, as previously determined in a 41-year-old male patient
showing allergic reactions to QuornTM product [183]. In 2018, a group of researchers
analyzed 2007 self-report adverse events related to QuornTM products from 1752 individual
people and subsequently found that the majority of these events involved allergic reactions
(hives and anaphylaxis) or gastrointestinal symptoms (vomiting and diarrhea) [184].
Future challenges in the mycoprotein research include finding sustainable carbon
sources for long-term production, particularly to replace the highly refined glucose syrup
currently in use. In addition to technological and industrial considerations, food safety must
also be taken into account [176]. As previously highlighted, the production of mycotoxins
varied with carbon sources [185,186], and thus these findings indicate a potential safety
risk that may arise from the use of an alternative carbon source in mycoprotein production.
Agri-food wastes can potentially be utilized as a nutrient source in the production
of mycoprotein. In one study, date wastes were used as a fermentation substrates for
F. venenatum, and the authors found that the resulting mycoproteins did not result in
allergic reactions in tested human subjects, that there was an absence of fumonisin gene
expression in the starter culture and that no mycotoxins (zearalenone and deoxynivalenol)
were detected in the fermentation medium, although low levels of lead (658 µg/kg), arsenic
(161 µg/kg) and cadmium (30.57 µg/kg) were reported [187]. Future research can aim at
assessing other waste materials for their potential use in mycoprotein production.
As summarized by Ritala et al., there are other fungal species considered for use as
mycoprotein [144]. Currently, primary concerns related to mycoprotein (QuornTM ) are
allergens, although mycotoxins may need to be considered when alternative carbon sources
(other than high-refined glucose syrup) are used for growing different fungal species or
heavy metals when the fermentation substrates are derived from agri-food wastes.

5.3. Bacterial Protein: Useful or Harmful Strains and RNA Contents

Bacterial protein contains about 50–80% protein on a dry weight basis [144]. Currently,
research efforts on bacterial proteins are mostly aimed at their utilization as feed in farms
or aquaculture, several of which are summarized in Table 4. Generally, bacteria can form
biomass through autotrophy (utilizing carbon dioxide as a carbon source as mediated
by light or chemical energy) or heterotrophy (non-carbon dioxide carbon source, for
example, acetic acid, methanol, methane or formic acid) [188]. Bacterial biomasses can be
grown in bioreactors using different growth substrates, including waste products from
varying industries—for example, rhizospheric diazotrophs on brewery wastewater [189],
Methylococcus capsulatus on methane gas [190] and Rhodopseudomonas palustris on latex
rubber sheet wastewater enriched with pineapple extracts [191]. Among the available
bacterial species, M. capsulatus has been found to promote health benefits in monogastric
animals, such as minks, pigs and chickens [190], and thus future research on its potential
as a protein source for humans is warranted. Feeding substrates supplemented with
M. capsulatus also alleviated the severity of colitis and improved gut function in mice [192].
Foods 2021, 10, 1226 15 of 29

Table 4. Several in vivo studies of bacterial protein fed to live animals.

Bacterial Species Live Animal Main Finding Reference

Mice fed with the bacteria exhibited less
profound colitis symptoms and higher colonic
Methylococcus capsulatus (Bath) Female C57BL/6NTac mice epithelial layer (increased cell proliferation and [192]
mucin 2 transcription) than those in the control
groups.
Growth rate and feeding efficiency of fish fed
with bacterial protein was the same as those fed
Japanese yellow tail fish
M. capsulatus (Bath) with conventional fish meal but only up to a [193]
(Seriola quinqueradiata)
bacterial protein concentration of 20%, beyond
which both parameters were negatively affected.
Fish fed with bacterial protein (5% or 10%) had
Rainbow trout
similar feeding efficiency to the control groups,
Methylobacterium extorquens (Oncorchynchun mykiss [194]
with survival improved for fish in the 10%
Walbaum)
bacterial protein group.
Mice fed with the bacteria (experimental group)
had lower weight gain over 28 days of feeding
trial than those in the control groups, with PHB
detected in the excrements of the experimental
Cupriavidus necator H16 Sprague Dawley mice [195]
mice. Kidneys, ileums and stomachs of the
experimental mice were also heavier, potentially
due to the accumulation of PHB in the murine
organs.
Shrimps fed with either of two bacterial species
had higher feed conversion rates and individual
Rhodobacter capsulatus or White leg shrimp weights than those in the control group.
[196]
Rhodopseudomonas palustris (Penaeus vannamei) Tolerance of ammonia was also higher in
shrimps fed with R. palustris, relative to the
control group.
PHB, polyhydroxybutyrate.

To avoid metabolic overload during the production of high-value products, co-

cultures have been proposed, i.e., multi-species microbial consortia [197]. In one study,
Aspergillus niger H3 and Bacillus licheniformis were used to produce bacterial protein from
potato starch wastewater in a dual-step process: (1) A. niger H3 metabolized the fiber in the
potato wastewater through the action of cellulases; (2) B. licheniformis utilized the released
sugars from the fermentation of potato fibers to produce bacterial protein [198]. Others
used a hybrid system of purple non-sulfur bacteria and aerobic heterotrophic bacteria to
improve the production efficiency and nutritional quality of the bacterial protein produced,
including higher protein content and more favorable amino acid/fatty acid profiles, as
compared with when either of the bacteria was cultured alone [199].
Relative to other single-cell proteins (i.e., microalgal or mycoprotein), bacterial protein
has the highest nucleic acid content at 15–16% [200]. During the metabolism of nucleic
acid, purines (guanine and adenine) are degraded into uric acid. As humans lack the
uricase enzyme, which is involved in the metabolism of uric acid in mammals, uric acid
is usually excreted in the urine. However, impaired uric acid excretion leads to excessive
accumulation of uric acid in the human body, which is a major cause of gout or hyper-
uricemia [201,202]. Thus, it is imperative that the nucleic acid content in bacterial proteins
is reduced, for example, through heat treatments as used in the processing of mycoprotein.
Safety concerns can also arise from the use of toxin-producing bacterial species, such
as B. cereus [203]. However, given the availability of non-pathogenic alternatives, for
example, Bacillus subtilis [204], food manufacturers and researchers could circumvent the
issue of toxins in bacterial proteins with relative ease. Nevertheless, precautions should
still be taken, particularly when multispecies co-cultures are utilized. Another concern is
Foods 2021, 10, 1226 16 of 29

the presence of potentially harmful substances within the cells used to produce bacterial
proteins. For example, the bacterial strain Cupriavidus necator H16 contains non-nutritive
polyhydroxybutyrate in their cytoplasm, which could accumulate in the organs upon
ingestion, as previously reported for Sprague Dawley mice fed with the bacteria [195].
In addition, as wastewaters are commonly proposed as a growth substrate, surveil-
lance systems are required to monitor the presence of biological and chemical hazards. As
highlighted by Alloul et al., the cultivation of purple non-sulfur bacteria in wastewater was
flanked with a variety of other bacteria, such as those belonging to the genera Arcobacter,
Dysgonomonas and Acinetobacter, indicating potential quality control issues during the
bacterial protein production [196].

6. Environmental Impact of Alternative Proteins

The environmental impact of cultured meat may vary depending on the growth
medium used (Table 5). Regardless, current data suggest that the theoretical greenhouse
gas emission, water use, eutrophication and land use in culturing meat are lower than con-
ventional meat production, although cultured meat is still more energy intensive [28,205].
Similar to cultured meat, the environmental impact of insect-based food production de-
pends on the type of feed used [206–208], with nutritious waste-based feeds being the most
environmentally friendly [207].
Foods 2021, 10, 1226 17 of 29

Table 5. Life cycle analyses (LCA) of alternative proteins.

Energy Use GHG Emission Land Use

Protein Type Water Use or Eutrophication a Reference
(MJ/kg) (kg CO2 -eq/kg Product) (m2 a/kg) b
Cultured meat
Minced beef 1 26–33 1.90–2.24 0.36–0.52 m3 /kg meat (W) 0.19–0.23 [28]
CHO 2 106 7.5 7.9 g PO4 -eq/kg meat (E) 5.5 [205]
Plant-based meat
Beyond Burger® 54.15 3.35 28.84 m3 /kg meat (W) 3.97 [209]
Impossible Burger® NA 3.5 0.11 m3 /kgmeat (W); 1.3 g PO4 -eq/kg meat 2.5 [210]
Insect protein
Mealworm (T. molitor and Zophobas morio) 33.68 2.65 NA 3.56 [206]
Black soldier fly (H. illucens) 21.20–99.60 1.36–15.10 NA 0.032–7.03 [207]
Cricket (G. bimaculatans and A. domesticus) NA 2.29 0.43 m3 /kg cricket (W); 0.00047 kg P-eq and 0.020 kg N-eq/kg cricket (E) NA [208]
Single-cell protein
Spirulina tablets (A. platensis) 7.88–12.7 5.05–7.71 0.015–0.022 kg N-eq/kg tablet (E) NA [211]
Micoalgal protein (A. platensis) 1225.6–3338.3 78.1–196.3 3.2–3.3 m3 /kg protein meal (W); 49.2–85.3 kg N-eq/kg protein meal (E) 1.7–4.3 [212]
Microalgal protein (C. vulgaris) 217.1–4181.3 14.7–245.1 0.3–3.9 m3 /kg protein meal (W); 40.6–105.3 kg N-eq/kg protein meal (E) 1.9–5.4 [212]
Mycoprotein 60.07–76.8 5.55–6.15 NA 0.79–0.84 [213]
Bacterial protein (Cupriavidus necator) NA 0.81–1 0.0001–0.0038 m3 /kg protein (W); 0.000333 kg P-eq/kg protein (E) 0.029–0.085 [214]
2.5 m3 /kg protein (W);
Bacterial protein (hydrogen-oxidizing bacteria) 200 8 0.8 [215]
0.0025 kg P-eq/kg protein and 0.00035 N-eq/kg protein (E)
GHG, greenhouse gas; NA, not available. a Water use (W) is expressed in volumetric unit (m3 or L/weight of product), whereas eutrophication (E) is a measure of the amount of contaminants released into
freshwater or marine environments (g contaminant/weight of product). b Land use is expressed in annual area occupation (m2 a). 1 Cyanobacterial hydrolysate was assumed as the growth medium. 2 LCA was
conducted based on the proliferation of Chinese hamster ovary (CHO) in a growth medium mainly comprising basal medium and soy hydrolysate.
Foods 2021, 10, 1226 18 of 29

Available life cycle analyses on two commercial plant-based meats—namely, Beyond

Burger® and Impossible Burger® —suggest that these products are more environmentally
sustainable than conventional meats, as measured by their energy use, carbon emission,
land use, water use and eutrophication. The environmental impact of these plant-based
meats mainly occurs during the raw ingredient production [209,210].
Hydrogen-oxidizing autotrophic bacteria, such C. necator, can be turned into a sustain-
able protein source with a lower environmental impact than animal-based meat, including
beef, fish and poultry [215]. As electricity consumption is the main driver of bacterial pro-
tein production, energy sources should be optimized for a mass-scale production [214,215].
Similarly, the cultivation of microalgae is the most energy intensive stage, and thus it con-
tributes towards the majority of the environmental footprints associated with microalgal
protein production [211], particularly when an open raceway pond is used [212].
Smetana et al. conducted a comparative study of different alternative proteins and
found that cultured meat had the highest environmental impact (carbon emission and water
use), as compared with mycoprotein and insect-based protein, including when caloric and
protein contents were considered. However, the production of insect-based protein required
the largest amount of land occupation, relative to mycoprotein and cultured meat [213].
Consistent with our data (Table 5), another study by Smetana et al. reported that microalgal
proteins were more energy intensive, and thus had a higher carbon emission than other
protein sources, including cultured meat, insect, yeast and bacteria [216]. Interestingly, the
data collated in this review also indicate that cultured and plant-based meats have lower
eutrophication potential than insect and single cell proteins (Table 5). To our knowledge,
the two reports by Smetana et al. [213,216] are the only studies that directly compared the
environmental impacts of these alternative proteins, and thus more research is required to
establish a firm scientific framework for this issue. It is noteworthy that as functionalities
of different food types can vary (for example, protein content or nutrient availability),
direct comparison of the environmental impacts of different alternative proteins should be
conducted with prudence.

7. Regulatory Framework
The food legislative framework is yet another necessary platform to ensure the safety
of alternative proteins. Cultured meat, insect protein and single-cell protein are likely to
be regulated as novel foods. EFSA is the key administrative institution for food safety in
Europe and generally acts as expert consultants to the European Commission (EC). The
latest EC novel food legislation Regulation (EC) 2015/2283 came into effect in 2018 and
regulates the approval of foods derived from ingredients or production processes that
were not used within the European Union prior to 15 May 1997 [217]—a guide document
on the application process has also been published elsewhere [218]. Other relevant EC
regulations include food hygiene regulations, such as Regulation (EC) 852/2003 and (EC)
853/2004 [219]. Alternative proteins can also be regulated through the Food, Drug and
Cosmetic Act [220], including possible assessment for generally recognized as safe (GRAS)
status [221].
In 2019, the Food and Drug Administration (FDA) and the United States Department
of Agriculture’s Food Safety and Inspection Services (USDA-FSIS) announced a planned
collaboration to regulate cultured meat. Under the agreement, FDA will oversee cell
collection, cell banks, cell growth and cell differentiation, whereas USDA-FSIS will monitor
post-harvest processes, including the production and labelling of the final cell-based food
items [222]. Further, the Federal Meat Inspection Act and Poultry Product Inspection Act
have also been proposed as supporting legislations [13]. In Europe, cultured meat may
potentially be subjected to Regulations (EC) 1829/2003 and 1830/2003 [223,224], if GM
cells are used (for example, iPSC).
Similarly, insect and bacterial proteins are subjects to the novel food legislation Regu-
lation (EC) 2015/2283. In 2021, T. molitor was deemed safe for human consumption by the
EFSA Panel on Nutrition, Novel Foods and Food Allergens, in compliance with the Regula-
Foods 2021, 10, 1226 19 of 29

tions (EC) 2015/2283 [142]. Belluco, Halloran and Ricci summarized other supporting EC
regulations relevant to edible insects [225]. Microalgal products must obtain GRAS status
in the USA, for example, oil derived from Ulkenia sp. SAM2179, Haematococcus pluvialis
extract containing astaxanthin esters, dried biomass of A. platensis, DHA-rich single-cell oil
derived from Crypthecodinium cohni. In Europe, microalgal products that have historical
human consumptions prior to 15 May 1997, such as A. platensis, can be approved according
to the regular food safety standard Regulation (EC) 178/2002, but anything else is a subject
to the novel food regulation [226].
In contrast, plant-based meats are regulated in a similar manner as other non-animal
foods [227], particularly given that GM soybeans are deemed safe by the FDA [228] and
EFSA [229]. Certain plant-based meats that contain soy leghemoglobin may be subjected
to novel food regulation in Europe, although individual states can vary in their ways of
regulating soy leghemoglobin in plant-based meats. In the USA, soy leghemoglobin has
been declared as GRAS [86].

8. Research Gap and Future Outlook

Alternative proteins are a growing industry, and thus the global food sector should
initiate collaborative efforts to ensure the safety of foods in this category. The main focus
of these efforts should be to maintain food safety in a mass-scale production, including
aspects related to allergens, pathogens, chemical contaminants and the environmental
implications during production scale-ups. In this review, we have highlighted several
potential safety risks associated with cultured meat, plant-based meat, insect protein and
single-cell protein.
There is a lack of research on the safety of cultured meat, with most studies focusing on
technological improvements for better production means. Infectious prion and viruses are
potentially the main hazards related to cultured meat production using serum-based media.
Thus, future developments of methods for removing these contaminants are warranted,
such as the use of hollow fiber anion-exchange membrane chromatography to remove prion
from large volumes of cell culture media [230]. Concerns about the introduction of foreign
genes, such as during the conversion of somatic cells into iPSC, may be circumvented
by the use of small molecules as an alternative cell reprogramming system [231]. In the
current literature, it appears that antibiotics are not used in the production of cultured
meat, primarily based upon the notion that this alternative protein is produced in a highly
controlled and closely monitored environment [13,232]. However, to our knowledge,
there have not been any studies addressing this issue using verifiable data, and thus we
encourage the scientific community to investigate this issue further, including through the
provision of assessments of the safety measures used to control biological contaminants in
cultured meat without antibiotics.
Available data suggest that plant-based meat may contain allergens, anti-nutrients
or traces of glyphosate, although activities of these compounds may be reduced by heat
treatments. In the future, there is also a need for discussion of the health implications of
extensive processing (i.e., ultraprocessed) involved in the production of plant-based meat,
including potential development of carcinogens during the thermal treatments.
Allergens are one of the primary safety issues associated with the consumption of
insects, but the clinical significance of this is yet to be established. Future research can aim at
identifying the types of allergen present in different edible insect species, and subsequently
assessing their health effects across demographics, i.e., by age, allergy status, ethnicity,
etc. Microbiological content of insects also varies with species, and future mass-scale
production of edible insects would require careful selection of those species harboring
bacteria communities that are less pathogenic to humans.
Toxins pose a health risk related to single-cell protein. In microalgae, this is primarily
due to environmental cross-contamination, which indicates the importance of choosing
appropriate cultivation reservoirs. For mycoprotein, allergens are the main hazard, and
future research is necessary to identify the risk factors associated with mycoprotein allergies.
Foods 2021, 10, 1226 20 of 29

When bacteria are used as single-cell protein, careful selection of non-pathogenic bacterial
strains is paramount.
In the current literature, the regulatory framework for novel foods has been described
based upon food standards in Europe and the USA. As alternative proteins are a global
strategy to mitigate climate and environmental issues, the scientific community should
expand the scope of the discussion to include food standards in other parts of the world.
For example, Australia-New Zealand Food Standard Code Standard 1.5.1 describes the
pre-market assessment criteria for novel foods intended for sale in Australia and New
Zealand [233], or Schedule 25 lists the approved novel food products, including several
that are derived from microalgae [234]. Soy leghemoglobin has also been approved in these
two countries [235].

Author Contributions: G.B. conceived the idea of this review and provided general outlines. J.H.
conducted the literature investigation and wrote the original draft of this article. Both authors
contributed to the reviewing and editing of the original draft and subsequently agreed to the
published version of the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors would like to thank Tanushree Gupta and Delphine Rapp for their
comments on the original manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or
in the decision to publish the results.

References
1. United Nations Department of Economic and Social Affairs. World Population Projected to Reach 9.8 Billion in 2050, and 11.2
Billion in 2100. Available online: https://www.un.org/development/desa/en/news/population/world-population-prospects-
2017.html (accessed on 30 March 2021).
2. Alexandratos, N.; Bruinsma, J. World Agriculture Towards 2030/2050: The 2012 Revision; FAO: Rome, Italy, 2012.
3. Organization for Economic Co-operation and Development/Food and Agricultural Organization. OECD-FAO Agricultural
Outlook 2020–2029; OECD Publishing, Paris/Food and Agriculture Organization of the United Nations: Rome, Italy, 2020.
4. Gerber, P.J.; Steinfeld, H.; Henderson, B.; Mottet, A.; Opio, C.; Dijkman, J.; Falcucci, A.; Tempio, G. Tackling Climate Change through
Livestock—A Global Assessment of Emissions and Mitigation Opportunities; FAO: Rome, Italy, 2013.
5. Gerber, P.J.; Hristov, A.N.; Henderson, B.; Makkar, H.; Oh, J.; Lee, C.; Meinen, R.; Montes, F.; Ott, T.; Firkins, J.; et al. Technical
Options for the Mitigation of Direct Methane and Nitrous Oxide Emissions from Livestock: A Review. Animal 2013, 7, 220–234.
[CrossRef]
6. Intergovernmental Panel on Climate Change. Special Report on Global Warming 1.5 ◦ C. Available online: https://www.ipcc.ch/
sr15/ (accessed on 30 March 2021).
7. Steinfeld, H.; Gerber, P.; Wassenaar, T.; Castel, V.; Rosales, M.; de Haan, C. Livestock’s Long Shadow: Environmental Issues and
Options; FAO: Rome, Italy, 2006.
8. Phelps, L.N.; Kaplan, J.O. Land Use for Animal Production in Global Change Studies: Defining and Characterizing a Framework.
Glob. Chang. Biol. 2017, 23, 4457–4471. [CrossRef]
9. Hristov, A.N.; Oh, J.; Lee, C.; Meinen, R.; Montes, F.; Ott, T.; Firkins, J.; Rotz, A.; Dell, C.; Adesogan, A.; et al. Mitigation of
Greenhouse Gas Emissions in Livestock Production: A Review of Technical Options for Non-CO2 Emissions; FAO: Rome, Italy, 2013.
10. Rojas-Downing, M.M.; Nejadhashemi, A.P.; Harrigan, T.; Woznicki, S.A. Climate Change and Livestock: Impacts, Adaptation,
and Mitigation. Clim. Risk Manag. 2017, 16, 145–163. [CrossRef]
11. Datar, I.; Betti, M. Possibilities for an in Vitro Meat Production System. Innov. Food Sci. Emerg. Technol. 2010, 11, 13–22. [CrossRef]
12. Post, M.J. Cultured Meat from Stem Cells: Challenges and Prospects. Meat Sci. 2012, 92, 297–301. [CrossRef]
13. Post, M.J.; Levenberg, S.; Kaplan, D.L.; Genovese, N.; Fu, J.; Bryant, C.J.; Negowetti, N.; Verzijden, K.; Moutsatsou, P. Scientific,
Sustainability and Regulatory Challenges of Cultured Meat. Nat. Food 2020, 1, 403–415. [CrossRef]
14. van Ellen, W.F.; van Kooten, W.J.; Westerhof, W. Industrial Scale Production of Meat from In Vitro Cell Cultures. WO1999031222A1,
18 December 1997.
15. Benjaminson, M.A.; Gilchriest, J.A.; Lorenz, M. In Vitro Edible Muscle Protein Production System (MPPS): Stage 1, Fish. Acta
Astronaut. 2002, 51, 879–889. [CrossRef]
16. Post, M.J. Cultured Beef: Medical Technology to Produce Food. J. Sci. Food Agric. 2014, 94, 1039–1041. [CrossRef] [PubMed]
17. Ben-Arye, T.; Levenberg, S. Tissue Engineering for Clean Meat Production. Front. Sustain. Food Syst. 2019, 3, 46. [CrossRef]
Foods 2021, 10, 1226 21 of 29

18. Bogliotti, Y.S.; Wu, J.; Vilarino, M.; Okamura, D.; Soto, D.A.; Zhong, C.; Sakurai, M.; Sampaio, R.V.; Suzuki, K.;
Izpisua Belmonte, J.C.; et al. Efficient Derivation of Stable Primed Pluripotent Embryonic Stem Cells from Bovine Blas-
tocysts. Proc. Natl. Acad. Sci. USA 2018, 115, 2090–2095. [CrossRef]
19. Takahashi, K.; Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by
Defined Factors. Cell 2006, 126, 663–676. [CrossRef] [PubMed]
20. Genovese, N.J.; Domeier, T.L.; Telugu, B.P.V.L.; Roberts, R.M. Enhanced Development of Skeletal Myotubes from Porcine Induced
Pluripotent Stem Cells. Sci. Rep. 2017, 7, 1–11.
21. Cui, H.X.; Guo, L.P.; Zhao, G.P.; Liu, R.R.; Li, Q.H.; Zheng, M.Q.; Wen, J. Method Using a Co-Culture System with High-Purity
Intramuscular Preadipocytes and Satellite Cells from Chicken Pectoralis Major Muscle. Poult. Sci. 2018, 97, 3691–3697. [CrossRef]
[PubMed]
22. Mehta, F.; Theunissen, R.; Post, M.J. Adipogenesis from Bovine Precursors. In Myogenesis: Methods in Molecular Biology;
Rønning, S.B., Ed.; Humana Press: New York, NY, USA, 2019; Volume 1889, pp. 111–125.
23. Brunner, D.; Frank, J.; Appl, H.; Schöffl, H.; Pfaller, W.; Gstraunthaler, G. Serum-Free Cell Culture: The Serum-Free Media
Interactive Online Database. ALTEX 2010, 27, 53–62. [CrossRef]
24. van der Valk, J.; Brunner, D.; De Smet, K.; Fex Svenningsen, Å.; Honegger, P.; Knudsen, L.E.; Lindl, T.; Noraberg, J.; Price, A.;
Scarino, M.L.; et al. Optimization of Chemically Defined Cell Culture Media—Replacing Fetal Bovine Serum in Mammalian in
Vitro Methods. Toxicol. In Vitro 2010, 24, 1053–1063. [CrossRef] [PubMed]
25. Kolkmann, A.M.; Post, M.J.; Rutjens, M.A.M.; van Essen, A.L.M.; Moutsatsou, P. Serum-Free Media for the Growth of Primary
Bovine Myoblasts. Cytotechnology 2020, 72, 111–120. [CrossRef]
26. Specht, L. An Analysis of Culture Medium Costs and Production Volumes for Cultivated Meat. Available online: https:
//www.gfi.org/files/sci-tech/clean-meat-production-volume-and-medium-cost.pdf (accessed on 2 February 2021).
27. Andreassen, R.C.; Pedersen, M.E.; Kristoffersen, K.A.; Beate Rønning, S. Screening of By-Products from the Food Industry as
Growth Promoting Agents in Serum-Free Media for Skeletal Muscle Cell Culture. Food Funct. 2020, 11, 2477–2488. [CrossRef]
[PubMed]
28. Tuomisto, H.L.; De Mattos, M.J.T. Environmental Impacts of Cultured Meat Production. Environ. Sci. Technol. 2011, 45, 6117–6123.
[CrossRef] [PubMed]
29. European Medicines Agency. Guideline on the Use of Bovine Serum in the Manufacture of Human Biological Medicinal Products.
Available online: https://www.gmp-compliance.org/gmp-news/ema-guideline-guideline-on-the-use-of-bovine-serum-in-
the-manufacture-of-human-biological-medicinal-products (accessed on 30 March 2021).
30. United States Department of Agriculture. 9 CFR 113.46—Detection of Cytopathogenic and/or Hemadsorbing Agents. Available
online: https://www.govinfo.gov/app/details/CFR-2012-title9-vol1/CFR-2012-title9-vol1-sec113-46/summary (accessed on
30 March 2021).
31. Hawkes, P.W. Fetal Bovine Serum: Geographic Origin and Regulatory Relevance of Viral Contamination. Bioresour. Bioprocess.
2015, 2, 1–5. [CrossRef]
32. Lee, J.; Kim, S.Y.; Hwang, K.J.; Ju, Y.R.; Woo, H.J. Prion Diseases as Transmissible Zoonotic Diseases. Osong Public Health Res.
Perspect. 2013, 4, 57–66. [CrossRef]
33. Geschwind, M.D. Prion Diseases. Contin. Lifelong Learn. Neurol. 2015, 21, 1612–1638. [CrossRef]
34. Scott, M.R.; Will, R.; Ironside, J.; Nguyen, H.O.B.; Tremblay, P.; DeArmond, S.J.; Prusiner, S.B. Compelling Transgenetic Evidence
for Transmission of Bovine Spongiform Encephalopathy Prions to Humans. Proc. Natl. Acad. Sci. USA 1999, 96, 15137–15142.
[CrossRef]
35. Houston, F.; McCutcheon, S.; Goldmann, W.; Chong, A.; Foster, J.; Sisó, S.; González, L.; Jeffrey, M.; Hunter, N. Prion Diseases Are
Efficiently Transmitted by Blood Transfusion in Sheep. Blood 2008, 112, 4739–4745. [CrossRef] [PubMed]
36. McCutcheon, S.; Blanco, A.R.A.; Houston, E.F.; de Wolf, C.; Tan, B.C.; Smith, A.; Groschup, M.H.; Hunter, N.; Hornsey, V.S.;
MacGregor, I.R.; et al. All Clinically-Relevant Blood Components Transmit Prion Disease Following a Single Blood Transfusion:
A Sheep Model of VCJD. PLoS ONE 2011, 6, e23169. [CrossRef]
37. Genovese, N.J.; Firpo, M.T.; Dambournet, D. Compositions and Methods for Increasing the Culture Density of a Cellular Biomass
within a Cultivation Infrastructure. Hippo Patent WO2018208628, 15 November 2018.
38. Joshi, V.; Kumar, S. Meat Analogues: Plant Based Alternatives to Meat Products—A Review. Int. J. Food Ferment. Technol. 2015,
5, 107. [CrossRef]
39. Ismail, I.; Hwang, Y.H.; Joo, S.T. Meat Analog as Future Food: A Review. J. Anim. Sci. Technol. 2020, 62, 111–120. [CrossRef]
[PubMed]
40. Malav, O.P.; Talukder, S.; Gokulakrishnan, P.; Chand, S. Meat Analog: A Review. Crit. Rev. Food Sci. Nutr. 2015, 55, 1241–1245.
[CrossRef] [PubMed]
41. Orcutt, M.W.; McMindes, M.K.; Chu, H.; Mueller, I.N.; Bater, B.; Orcutt, A.L. Textured Soy Protein Utilization in Meat and Meat
Analog Products. In Soy Applications in Food; Riaz, M.N., Ed.; CRC Press: New York, NY, USA, 2006; pp. 155–184.
42. Pietsch, V.L.; Emin, M.A.; Schuchmann, H.P. Process Conditions Influencing Wheat Gluten Polymerization during High Moisture
Extrusion of Meat Analog Products. J. Food Eng. 2017, 198, 28–35. [CrossRef]
43. Osen, R.; Toelstede, S.; Wild, F.; Eisner, P.; Schweiggert-Weisz, U. High Moisture Extrusion Cooking of Pea Protein Isolates: Raw
Material Characteristics, Extruder Responses, and Texture Properties. J. Food Eng. 2014, 127, 67–74. [CrossRef]
Foods 2021, 10, 1226 22 of 29

44. Sharima-Abdullah, N.; Hassan, C.Z.; Arifin, N.; Huda-Faujan, N. Physicochemical Properties and Consumer Preference of
Imitation Chicken Nuggets Produced from Chickpea Flour and Textured Vegetable Protein. Int. Food Res. J. 2018, 25, 1016–1025.
45. Kyriakopoulou, K.; Dekkers, B.; van der Goot, A.J. Plant-Based Meat Analogues. In Sustainable Meat Production and Processing;
Galanakis, C.M., Ed.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 103–126.
46. Bohrer, B.M. An Investigation of the Formulation and Nutritional Composition of Modern Meat Analogue Products. Food Sci.
Hum. Wellness 2019, 8, 320–329. [CrossRef]
47. Egbert, R.; Borders, C. Achieving Success with Meat Analogs. Food Technol. 2006, 60, 28–34.
48. Ajami, D.; Anderson, D.; Dill, J.; Geistlinger, T.; Mayoral, K.; Ngo, H.B.; Noriega, T.; Ryan, D.A.; Suarez-Trujillo, D.; Timmons, M.;
et al. Meat-Like Food Products. US 2017/0105438 A1, 20 April 2017.
49. Anderson, D.; Fuller, J.; Geistlinger, T. Nutrient Dense Meat Structured Protein Products. US 9526267 B2, 27 December 2016.
50. Geistlinger, T. Plant Based Meat Structured Protein Products. US 2015/0296834 A1, 22 October 2015.
51. Fellows, P.J. Extrusion Cooking. In Food Processing Technology; Fellows, P.J., Ed.; Woodhead Publishing Limited: Cambridge, UK,
2017; pp. 753–780.
52. Riaz, M.N. Texturized Soy Protein as an Ingredient. In Proteins in Food Processing; Yada, R.Y., Ed.; Woodhead Publishing Limited:
Cambridge, UK, 2004; pp. 517–558.
53. Samard, S.; Ryu, G.H. A Comparison of Physicochemical Characteristics, Texture, and Structure of Meat Analogue and Meats.
J. Sci. Food Agric. 2019, 99, 2708–2715. [CrossRef]
54. Tehrani, M.M.; Ehtiati, A.; Azghandi, S.S. Application of Genetic Algorithm to Optimize Extrusion Condition for Soy-Based Meat
Analogue Texturization. J. Food Sci. Technol. 2017, 54, 1119–1125. [CrossRef] [PubMed]
55. Grabowska, K.J.; Zhu, S.; Dekkers, B.L.; De Ruijter, N.C.A.; Gieteling, J.; Van Der Goot, A.J. Shear-Induced Structuring as a Tool to
Make Anisotropic Materials Using Soy Protein Concentrate. J. Food Eng. 2016, 188, 77–86. [CrossRef]
56. Dekkers, B.L.; Nikiforidis, C.V.; van der Goot, A.J. Shear-Induced Fibrous Structure Formation from a Pectin/SPI Blend. Innov.
Food Sci. Emerg. Technol. 2016, 36, 193–200. [CrossRef]
57. Grabowska, K.J.; Tekidou, S.; Boom, R.M.; van der Goot, A.J. Shear Structuring as a New Method to Make Anisotropic Structures
from Soy-Gluten Blends. Food Res. Int. 2014, 64, 743–751. [CrossRef] [PubMed]
58. Dekkers, B.L.; Hamoen, R.; Boom, R.M.; van der Goot, A.J. Understanding Fiber Formation in a Concentrated Soy Protein
Isolate—Pectin Blend. J. Food Eng. 2018, 222, 84–92. [CrossRef]
59. Krintiras, G.A.; Göbel, J.; Van Der Goot, A.J.; Stefanidis, G.D. Production of Structured Soy-Based Meat Analogues Using Simple
Shear and Heat in a Couette Cell. J. Food Eng. 2015, 160, 34–41. [CrossRef]
60. Krintiras, G.A.; Gadea Diaz, J.; Van Der Goot, A.J.; Stankiewicz, A.I.; Stefanidis, G.D. On the Use of the Couette Cell Technology
for Large Scale Production of Textured Soy-Based Meat Replacers. J. Food Eng. 2016, 169, 205–213.
61. Nieuwland, M.; Geerdink, P.; Brier, P.; Van Den Eijnden, P.; Henket, J.T.M.M.; Langelaan, M.L.P.; Stroeks, N.; Van Deventer, H.C.;
Martin, A.H. Food-Grade Electrospinning of Proteins. Innov. Food Sci. Emerg. Technol. 2013, 20, 269–275. [CrossRef]
62. Ramachandraiah, K. Potential Development of Sustainable 3D-Printed Meat Analogues: A Review. Sustainability 2021, 13, 938.
[CrossRef]
63. Wild, F.; Czerny, M.; Janssen, A.M.; Kole, A.P.W.; Zunabovic, M.; Domig, K.J. The Evolution of a Plant-Based Alternative to Meat:
From Niche Markets to Widely Accepted Meat Alternatives. Agro Food Ind. Hi Tech 2014, 25, 45–49.
64. European Commission. High Quality Meat-Like Products—From Niche Markets to Widely Accepted Meat Alternatives. Available
online: https://cordis.europa.eu/project/id/262560 (accessed on 30 March 2021).
65. Geeraerts, W.; De Vuyst, L.; Leroy, F. Ready-to-Eat Meat Alternatives, a Study of Their Associated Bacterial Communities. Food
Biosci. 2020, 37, 100681. [CrossRef]
66. Asgar, M.A.; Fazilah, A.; Huda, N.; Bhat, R.; Karim, A.A. Nonmeat Protein Alternatives as Meat Extenders and Meat Analogs.
Compr. Rev. Food Sci. Food Saf. 2010, 9, 513–529. [CrossRef] [PubMed]
67. Petroski, W.; Minich, D.M. Is There Such a Thing as “Anti-Nutrients”? A Narrative Review of Perceived Problematic Plant
Compounds. Nutrients 2020, 12, 2929. [CrossRef] [PubMed]
68. Omosebi, M.O.; Osundahunsi, O.F.; Fagbemi, T.N. Effect of Extrusion on Protein Quality, Antinutritional Factors, and Digestibility
of Complementary Diet from Quality Protein Maize and Soybean Protein Concentrate. J. Food Biochem. 2018, 42, e12508. [CrossRef]
69. Verma, A.K.; Kumar, S.; Das, M.; Dwivedi, P.D. A Comprehensive Review of Legume Allergy. Clin. Rev. Allergy Immunol. 2013,
45, 30–46. [CrossRef]
70. Cabanillas, B.; Jappe, U.; Novak, N. Allergy to Peanut, Soybean, and Other Legumes: Recent Advances in Allergen Characteriza-
tion, Stability to Processing and IgE Cross-Reactivity. Mol. Nutr. Food Res. 2018, 62, 1700446. [CrossRef] [PubMed]
71. Verma, A.K.; Kumar, S.; Das, M.; Dwivedi, P.D. Impact of Thermal Processing on Legume Allergens. Plant. Foods Hum. Nutr.
2012, 67, 430–441. [CrossRef]
72. Dong, X.; Wang, J.; Raghavan, V. Critical Reviews and Recent Advances of Novel Non-Thermal Processing Techniques on the
Modification of Food Allergens. Crit. Rev. Food Sci. Nutr. 2020, 61, 196–210. [CrossRef] [PubMed]
73. Singh, L.; Varshney, J.G.; Agarwal, T. Polycyclic Aromatic Hydrocarbons’ Formation and Occurrence in Processed Food. Food
Chem. 2016, 199, 768–781. [CrossRef] [PubMed]
Foods 2021, 10, 1226 23 of 29

74. Knize, M.G.; Salmon, C.P.; Pais, P.; Felton, J.S. Food Heating and the Formation of Heterocyclic Arohmatic Amine and Polycyclic
Aromatic Hydrocarbon Mutagens/Carcinogens. In Advances in Experimental Medicine and Biology; Jackson, L.S., Knize, M.G.,
Morgan, J.N., Eds.; Springer: Boston, MA, USA, 1999; Volume 459, pp. 179–193.
75. Herrmann, S.S.; Duedahl-Olesen, L.; Granby, K. Occurrence of Volatile and Non-Volatile N-Nitrosamines in Processed Meat
Products and the Role of Heat Treatment. Food Control 2015, 48, 163–169. [CrossRef]
76. Cantwell, M.; Elliott, C. Nitrates, Nitrites and Nitrosamines from Processed Meat Intake and Colorectal Cancer Risk. J. Clin. Nutr.
Diet. 2017, 3, 27. [CrossRef]
77. Abdulkarim, B.G.; Smith, J.S. Heterocyclic Amines in Fresh and Processed Meat Products. J. Agric. Food Chem. 1998, 46, 4680–4687.
[CrossRef]
78. Raza, A.; Shabbir, M.A.; Khan, M.I.; Suleria, H.A.R.; Sultan, S. Effect of Thermal Treatments on the Formation of Heterocyclic
Aromatic Amines in Various Meats. J. Food Process. Preserv. 2015, 39, 376–383. [CrossRef]
79. He, J.; Evans, N.M.; Liu, H.; Shao, S. A Review of Research on Plant-Based Meat Alternatives: Driving Forces, History,
Manufacturing, and Consumer Attitudes. Compr. Rev. Food Sci. Food Saf. 2020, 19, 2639–2656. [CrossRef]
80. Clive, J. Global Status of Commercialized Biotech./GM Crops in 2017: Biotech. Crop. Adoption Surges as Economic Benefits Accumulate in
22 Years; ISAAA Publications: Ithaca, NY, USA, 2017.
81. Bøhn, T.; Millstone, E. The Introduction of Thousands of Tonnes of Glyphosate in the Food Chain—An Evaluation of Glyphosate
Tolerant Soybeans. Foods 2019, 8, 669. [CrossRef]
82. Ruuskanen, S.; Rainio, M.J.; Uusitalo, M.; Saikkonen, K.; Helander, M. Effects of Parental Exposure to Glyphosate-Based
Herbicides on Embryonic Development and Oxidative Status: A Long-Term Experiment in a Bird Model. Sci. Rep. 2020, 10, 1–7.
[CrossRef]
83. Cuhra, M.; Traavik, T.; Dando, M.; Primicerio, R.; Holderbaum, D.F.; Bøhn, T. Glyphosate-Residues in Roundup-Ready Soybean
Impair Daphnia Magna Life-Cycle. J. Agric. Chem. Environ. 2015, 4, 24–36. [CrossRef]
84. Cuhra, M.; Traavik, T.; Bøhn, T. Life Cycle Fitness Differences in Daphnia Magna Fed Roundup-Ready Soybean or Conventional
Soybean or Organic Soybean. Aquac. Nutr. 2015, 21, 702–713. [CrossRef]
85. Jin, Y.; He, X.; Andoh-Kumi, K.; Fraser, R.Z.; Lu, M.; Goodman, R.E. Evaluating Potential Risks of Food Allergy and Toxicity of
Soy Leghemoglobin Expressed in Pichia pastoris. Mol. Nutr. Food Res. 2018, 62, 1700297. [CrossRef]
86. Fraser, R.Z.; Shitut, M.; Agrawal, P.; Mendes, O.; Klapholz, S. Safety Evaluation of Soy Leghemoglobin Protein Preparation
Derived From Pichia Pastoris, Intended for Use as a Flavor Catalyst in Plant-Based Meat. Int. J. Toxicol. 2018, 37, 241–262.
[CrossRef]
87. Ramos-Elorduy, J. Anthropo-Entomophagy: Cultures, Evolution and Sustainability. Entomol. Res. 2009, 39, 271–288. [CrossRef]
88. van Huis, A.; van Itterbeeck, J.; Klunder, H.; Mertens, E.; Halloran, A.; Muir, G.; Vantomme, P. Edible Insects: Future Prospects for
Food and Feed Security (No. 171); Food and Agriculture Organization of the United Nations: Rome, Italy, 2013.
89. Raksakantong, P.; Meeso, N.; Kubola, J.; Siriamornpun, S. Fatty Acids and Proximate Composition of Eight Thai Edible Terricolous
Insects. Food Res. Int. 2010, 43, 350–355. [CrossRef]
90. Ghosh, S.; Lee, S.M.; Jung, C.; Meyer-Rochow, V.B. Nutritional Composition of Five Commercial Edible Insects in South Korea.
J. Asia. Pac. Entomol. 2017, 20, 686–694. [CrossRef]
91. Kouřimská, L.; Adámková, A. Nutritional and Sensory Quality of Edible Insects. NFS J. 2016, 4, 22–26. [CrossRef]
92. Pieterse, E.; Pretorius, Q. Nutritional Evaluati on of Dried Larvae and Pupae Meal of the Housefly (Musca domestica) Using
Chemical-and Broiler-Based Biological Assays. Anim. Prod. Sci. 2014, 54, 347–355. [CrossRef]
93. Van Huis, A. Potential of Insects as Food and Feed in Assuring Food Security. Annu. Rev. Entomol. 2013, 58, 563–583. [CrossRef]
[PubMed]
94. Yen, A.L. Insects as Food and Feed in the Asia Pacific Region: Current Perspectives and Future Directions. J. Insects Food Feed
2015, 1, 33–55. [CrossRef]
95. Hanboonsong, Y.; Jamjanya, T.; Durst, P.B. Six-Legged Livestock: Edible Insect Farming, Collection and Marketing in Thailand; Food
and Agriculture Organization of the United Nations: Bangkok, Thailand, 2013.
96. Cortes Ortiz, J.A.; Ruiz, A.T.; Morales-Ramos, J.A.; Thomas, M.; Rojas, M.G.; Tomberlin, J.K.; Yi, L.; Han, R.; Giroud, L.; Jullien, R.L.
Insect Mass Production Technologies. In Insects as Sustainable Food Ingredients; Dossey, A.T., Rojas, M.G., Morales-Ramos, J.A.,
Eds.; Academic Press: Cambridge, UK, 2016; pp. 153–201.
97. Morales-Ramos, J.A.; Rojas, M.G.; Coudron, T.A. Artificial Diet Development for Entomophagous Arthropods. In Mass Production
of Beneficial Organisms: Invertebrates and Entomopathogens; Morales-Ramos, J.A., Rojas, M.G., Shapiro-Ilan, D.I., Eds.; Academic
Press: San Diego, CA, USA, 2013; pp. 203–240.
98. van Broekhoven, S.; Oonincx, D.G.A.B.; van Huis, A.; van Loon, J.J.A. Growth Performance and Feed Conversion Efficiency of
Three Edible Mealworm Species (Coleoptera: Tenebrionidae) on Diets Composed of Organic by-Products. J. Insect Physiol. 2015,
73, 1–10. [CrossRef] [PubMed]
99. Morales-Ramos, J.A.; Rojas, M.G.; Shapiro-Llan, D.I.; Tedders, W.L. Use of Nutrient Self-Selection as a Diet Refining Tool in
Tenebrio molitor (Coleoptera: Tenebrionidae). J. Entomol. Sci. 2013, 48, 206–221. [CrossRef]
100. Morales-Ramos, J.A.; Rojas, M.G.; Shapiro-Ilan, D.I.; Tedders, W.L. Self-Selection of Two Diet Components by Tenebrio molitor
(Coleoptera: Tenebrionidae) Larvae and Its Impact on Fitness. Environ. Entomol. 2011, 40, 1285–1294. [CrossRef] [PubMed]
Foods 2021, 10, 1226 24 of 29

101. Varelas, V. Food Wastes as a Potential New Source for Edible Insect Mass Production for Food and Feed: A Review. Fermentation
2019, 5, 81. [CrossRef]
102. Hawkey, K.J.; Lopez-Viso, C.; Brameld, J.M.; Parr, T.; Salter, A.M. Insects: A Potential Source of Protein and Other Nutrients for
Feed and Food. Annu. Rev. Anim. Biosci. 2021, 9, 333–354. [CrossRef] [PubMed]
103. Maciel-Vergara, G.; Ros, V.I.D. Viruses of Insects Reared for Food and Feed. J. Invertebr. Pathol. 2017, 147, 60–75. [CrossRef]
104. Mutungi, C.; Irungu, F.G.; Nduko, J.; Mutua, F.; Affognon, H.; Nakimbugwe, D.; Ekesi, S.; Fiaboe, K.K.M. Postharvest Processes
of Edible Insects in Africa: A Review of Processing Methods, and the Implications for Nutrition, Safety and New Products
Development. Crit. Rev. Food Sci. Nutr. 2019, 59, 276–298. [CrossRef] [PubMed]
105. Wade, M.; Hoelle, J. A Review of Edible Insect Industrialization: Scales of Production and Implications for Sustainability. Environ.
Res. Lett. 2019, 15, 123013. [CrossRef]
106. Wynants, E.; Crauwels, S.; Lievens, B.; Luca, S.; Claes, J.; Borremans, A.; Bruyninckx, L.; Van Campenhout, L. Effect of Post-
Harvest Starvation and Rinsing on the Microbial Numbers and the Bacterial Community Composition of Mealworm Larvae
(Tenebrio molitor). Innov. Food Sci. Emerg. Technol. 2017, 42, 8–15. [CrossRef]
107. Stoops, J.; Crauwels, S.; Waud, M.; Claes, J.; Lievens, B.; Van Campenhout, L. Microbial Community Assessment of Mealworm
Larvae (Tenebrio molitor) and Grasshoppers (Locusta migratoria migratorioides) Sold for Human Consumption. Food Microbiol. 2016,
53, 122–127. [CrossRef]
108. Osimani, A.; Garofalo, C.; Milanović, V.; Taccari, M.; Cardinali, F.; Aquilanti, L.; Pasquini, M.; Mozzon, M.; Raffaelli, N.;
Ruschioni, S.; et al. Insight into the Proximate Composition and Microbial Diversity of Edible Insects Marketed in the European
Union. Eur. Food Res. Technol. 2017, 243, 1157–1171. [CrossRef]
109. Costa, S.; Pedro, S.; Lourenço, H.; Batista, I.; Teixeira, B.; Bandarra, N.M.; Murta, D.; Nunes, R.; Pires, C. Evaluation of Tenebrio
molitor Larvae as an Alternative Food Source. NFS J. 2020, 21, 57–64. [CrossRef]
110. Li, L.; Xie, B.; Dong, C.; Wang, M.; Liu, H. Can Closed Artificial Ecosystem Have an Impact on Insect Microbial Community? A
Case Study of Yellow Mealworm (Tenebrio molitor L.). Ecol. Eng. 2016, 86, 183–189. [CrossRef]
111. Vandeweyer, D.; Lievens, B.; Van Campenhout, L. Identification of Bacterial Endospores and Targeted Detection of Foodborne
Viruses in Industrially Reared Insects for Food. Nat. Food 2020, 1, 511–516. [CrossRef]
112. Vandeweyer, D.; Crauwels, S.; Lievens, B.; Van Campenhout, L. Microbial Counts of Mealworm Larvae (Tenebrio molitor) and
Crickets (Acheta domesticus and Gryllodes sigillatus) from Different Rearing Companies and Different Production Batches. Int. J.
Food Microbiol. 2017, 242, 13–18. [CrossRef] [PubMed]
113. Vandeweyer, D.; Lenaerts, S.; Callens, A.; Van Campenhout, L. Effect of Blanching Followed by Refrigerated Storage or Industrial
Microwave Drying on the Microbial Load of Yellow Mealworm Larvae (Tenebrio molitor). Food Control 2017, 71, 311–314. [CrossRef]
114. Milanović, V.; Osimani, A.; Roncolini, A.; Garofalo, C.; Aquilanti, L.; Pasquini, M.; Tavoletti, S.; Vignaroli, C.; Canonico, L.;
Ciani, M.; et al. Investigation of the Dominant Microbiota in Ready-to-Eat Grasshoppers and Mealworms and Quantification of
Carbapenem Resistance Genes by QPCR. Front. Microbiol. 2018, 9, 3036. [CrossRef] [PubMed]
115. Osimani, A.; Garofalo, C.; Aquilanti, L.; Milanović, V.; Cardinali, F.; Taccari, M.; Pasquini, M.; Tavoletti, S.; Clementi, F.
Transferable Antibiotic Resistances in Marketed Edible Grasshoppers (Locusta migratoria migratorioides). J. Food Sci. 2017,
82, 1184–1192. [CrossRef] [PubMed]
116. Osimani, A.; Cardinali, F.; Aquilanti, L.; Garofalo, C.; Roncolini, A.; Milanović, V.; Pasquini, M.; Tavoletti, S.; Clementi, F.
Occurrence of Transferable Antibiotic Resistances in Commercialized Ready-to-Eat Mealworms (Tenebrio molitor L.). Int. J. Food
Microbiol. 2017, 263, 38–46. [CrossRef] [PubMed]
117. Wynants, E.; Crauwels, S.; Verreth, C.; Gianotten, N.; Lievens, B.; Claes, J.; Van Campenhout, L. Microbial Dynamics during
Production of Lesser Mealworms (Alphitobius diaperinus) for Human Consumption at Industrial Scale. Food Microbiol. 2018,
70, 181–191. [CrossRef]
118. Braide, W.; Oranusi, S.; Udegbunam, L.; Oguoma, O.; Akobondu, C.; Nwaoguikpe, R. Microbiological Quality of an Edible
Caterpillar of an Emperor Moth, Bunaea Alcinoe. J. Ecol. Nat. Environ. 2011, 3, 176–180.
119. Musundire, R.; Osuga, I.M.; Cheseto, X.; Irungu, J.; Torto, B. Aflatoxin Contamination Detected in Nutrient and Anti-Oxidant
Rich Edible Stink Bug Stored in Recycled Grain Containers. PLoS ONE 2016, 11, e0145914. [CrossRef] [PubMed]
120. Van Broekhoven, S.; Mota Gutierrez, J.; De Rijk, T.C.; De Nijs, W.C.M.; Van Loon, J.J.A. Degradation and Excretion of the Fusarium
Toxin Deoxynivalenol by an Edible Insect, the Yellow Mealworm (Tenebrio molitor L.). World Mycotoxin J. 2017, 10, 163–169.
[CrossRef]
121. Camenzuli, L.; van Dam, R.; de Rijk, T.; Andriessen, R.; van Schelt, J.; van der Fels-Klerx, H.J.I. Tolerance and Excretion of the
Mycotoxins Aflatoxin B1, Zearalenone, Deoxynivalenol, and Ochratoxin A by Alphitobius diaperinus and Hermetia illucens from
Contaminated Substrates. Toxins 2018, 10, 91. [CrossRef] [PubMed]
122. Bosch, G.; Van Der Fels-Klerx, H.J.; De Rijk, T.C.; Oonincx, D.G.A.B. Aflatoxin B1 Tolerance and Accumulation in Black Soldier
Fly Larvae (Hermetia illucens) and Yellow Mealworms (Tenebrio molitor). Toxins 2017, 9, 185. [CrossRef]
123. Purschke, B.; Scheibelberger, R.; Axmann, S.; Adler, A.; Jäger, H. Impact of Substrate Contamination with Mycotoxins, Heavy
Metals and Pesticides on the Growth Performance and Composition of Black Soldier Fly Larvae (Hermetia illucens) for Use in the
Feed and Food Value Chain. Food Addit. Contam. Part A 2017, 34, 1410–1420. [CrossRef] [PubMed]
Foods 2021, 10, 1226 25 of 29

124. Guo, Z.; Doll, K.; Dastjerdi, R.; Karlovsky, P.; Dehne, H.W.; Altincicek, B. Effect of Fungal Colonization of Wheat Grains with
Fusarium Spp. on Food Choice, Weight Gain and Mortality of Meal Beetle Larvae (Tenebrio molitor). PLoS ONE 2014, 9, e100112.
[CrossRef]
125. Gał˛ecki, R.; Sokół, R. A Parasitological Evaluation of Edible Insects and Their Role in the Transmission of Parasitic Diseases to
Humans and Animals. PLoS ONE 2019, 14, e0219303. [CrossRef]
126. Pereira, K.S.; Schmidt, F.L.; Barbosa, R.L.; Guaraldo, A.M.A.; Franco, R.M.B.; Dias, V.L.; Passos, L.A.C. Transmission of Chagas
Disease (American Trypanosomiasis) by Food. In Advances in Food and Nutrition Research; Taylor, S.L., Ed.; Academic Press:
San Diego, CA, USA, 2010; Volume 59, pp. 63–85.
127. Molavi, G.H.; Massoud, J.; Gutierrez, Y. Human Gongylonema Infection in Iran. J. Helminthol. 2006, 80, 425–428. [CrossRef]
128. ESFA Scientific Committee. Risk Profile Related to Production and Consumption of Insects as Food and Feed. EFSA J. 2015,
13, 4257. [CrossRef]
129. Klunder, H.C.; Wolkers-Rooijackers, J.; Korpela, J.M.; Nout, M.J.R. Microbiological Aspects of Processing and Storage of Edible
Insects. Food Control 2012, 26, 628–631. [CrossRef]
130. Rumpold, B.A.; Fröhling, A.; Reineke, K.; Knorr, D.; Boguslawski, S.; Ehlbeck, J.; Schlüter, O. Comparison of Volumetric and
Surface Decontamination Techniques for Innovative Processing of Mealworm Larvae (Tenebrio molitor). Innov. Food Sci. Emerg.
Technol. 2014, 26, 232–241. [CrossRef]
131. Ribeiro, J.C.; Cunha, L.M.; Sousa-Pinto, B.; Fonseca, J. Allergic Risks of Consuming Edible Insects: A Systematic Review. Mol.
Nutr. Food Res. 2018, 62, 1700030. [CrossRef] [PubMed]
132. Pali-Schöll, I.; Meinlschmidt, P.; Larenas-Linnemann, D.; Purschke, B.; Hofstetter, G.; Rodríguez-Monroy, F.A.; Einhorn, L.;
Mothes-Luksch, N.; Jensen-Jarolim, E.; Jäger, H. Edible Insects: Cross-Recognition of IgE from Crustacean- and House Dust
Mite Allergic Patients, and Reduction of Allergenicity by Food Processing. World Allergy Organ. J. 2019, 12, 100006. [CrossRef]
[PubMed]
133. Van Broekhoven, S.; Bastiaan-Net, S.; De Jong, N.W.; Wichers, H.J. Influence of Processing and in Vitro Digestion on the Allergic
Cross-Reactivity of Three Mealworm Species. Food Chem. 2016, 196, 1075–1083. [CrossRef]
134. Broekman, H.; Knulst, A.; Den Hartog Jager, S.; Monteleone, F.; Gaspari, M.; De Jong, G.; Houben, G.; Verhoeckx, K. Effect of
Thermal Processing on Mealworm Allergenicity. Mol. Nutr. Food Res. 2015, 59, 1855–1864. [CrossRef] [PubMed]
135. de Gier, S.; Verhoeckx, K. Insect (Food) Allergy and Allergens. Mol. Immunol. 2018, 100, 82–106. [CrossRef]
136. Vijver, M.; Jager, T.; Posthuma, L.; Peijnenburg, W. Metal Uptake from Soils and Soil-Sediment Mixtures by Larvae of Tenebrio
molitor (L.) (Coleoptera). Ecotoxicol. Environ. Saf. 2003, 54, 277–289. [CrossRef]
137. Greenfield, R.; Akala, N.; Van Der Bank, F.H. Heavy Metal Concentrations in Two Populations of Mopane Worms (Imbrasia belina)
in the Kruger National Park Pose a Potential Human Health Risk. Bull. Environ. Contam. Toxicol. 2014, 93, 316–321. [CrossRef]
138. Van Der Fels-Klerx, H.J.; Camenzuli, L.; Van Der Lee, M.K.; Oonincx, D.G.A.B. Uptake of Cadmium, Lead and Arsenic by Tenebrio
molitor and Hermetia illucens from Contaminated Substrates. PLoS ONE 2016, 11, e0166186.
139. Hyun, S.H.; Kwon, K.H.; Park, K.H.; Jeong, H.C.; Kwon, O.; Tindwa, H.; Han, Y.S. Evaluation of Nutritional Status of an Edible
Grasshopper, Oxya chinensis formosana. Entomol. Res. 2012, 42, 284–290. [CrossRef]
140. Shantibala, T.; Lokeshwari, R.K.; Debaraj, H. Nutritional and Antinutritional Composition of the Five Species of Aquatic Edible
Insects Consumed in Manipur, India. J. Insect Sci. 2014, 14, 1536–2442. [CrossRef] [PubMed]
141. Omotoso, O.T. Nutritional Quality, Functional Properties and Anti-Nutrient Compositions of the Larva of Cirina forda (Westwood)
(Lepidoptera: Saturniidae). J. Zhejiang Univ. Sci. B 2006, 7, 51–55. [CrossRef] [PubMed]
142. Turck, D.; Castenmiller, J.; De Henauw, S.; Hirsch-Ernst, K.I.; Kearney, J.; Maciuk, A.; Mangelsdorf, I.; McArdle, H.J.; Naska,
A.; Pelaez, C.; et al. Safety of Dried Yellow Mealworm (Tenebrio molitor Larva) as a Novel Food Pursuant to Regulation (EU)
2015/2283. EFSA J. 2021, 19, e06343. [PubMed]
143. Nishimune, T.; Watanabe, Y.; Okazaki, H.; Akai, H. Thiamin Is Decomposed Due to Anaphe Spp. Entomophagy in Seasonal
Ataxia Patients in Nigeria. J. Nutr. 2000, 130, 1625–1628. [CrossRef]
144. Ritala, A.; Häkkinen, S.T.; Toivari, M.; Wiebe, M.G. Single Cell Protein-State-of-the-Art, Industrial Landscape and Patents
2001–2016. Front. Microbiol. 2017, 8, 2009. [CrossRef]
145. Gouveia, L.; Batista, A.P.; Sousa, I.; Raymundo, A.; Bandarra, N.M. Microalgae in Novel Food Products. In Food Chemistry Research
Developments; Papadoupoulus, K.N., Ed.; Nova Science Publishers: New York, NY, USA, 2008; pp. 75–112.
146. Tokuşoglu, Ö.; Ünal, M.K. Biomass Nutrient Profiles of Three Microalgae: Spirulina Platensis, Chlorella Vulgaris, and Isochrisis
Galbana. J. Food Sci. 2003, 68, 1144–1148. [CrossRef]
147. Schwenzfeier, A.; Wierenga, P.A.; Gruppen, H. Isolation and Characterization of Soluble Protein from the Green Microalgae
Tetraselmis sp. Bioresour. Technol. 2011, 102, 9121–9127. [CrossRef]
148. Waghmare, A.G.; Salve, M.K.; LeBlanc, J.G.; Arya, S.S. Concentration and Characterization of Microalgae Proteins from Chlorella
pyrenoidosa. Bioresour. Bioprocess. 2016, 3, 1–11. [CrossRef]
149. Chacón-Lee, T.L.; González-Mariño, G.E. Microalgae for “Healthy” Foods-Possibilities and Challenges. Compr. Rev. Food Sci. Food
Saf. 2010, 9, 655–675. [CrossRef]
150. Becker, E.W. Micro-Algae as a Source of Protein. Biotechnol. Adv. 2007, 25, 207–210. [CrossRef] [PubMed]
Foods 2021, 10, 1226 26 of 29

151. Amorim, M.L.; Soares, J.; dos Reis Coimbra, J.S.; de Oliveira Leite, M.; Albino, L.F.T.; Martins, M.A. Microalgae Proteins:
Production, Separation, Isolation, Quantification, and Application in Food and Feed. Crit. Rev. Food Sci. Nutr. 2020, 1–27.
[CrossRef] [PubMed]
152. Wells, M.L.; Potin, P.; Craigie, J.S.; Raven, J.A.; Merchant, S.S.; Helliwell, K.E.; Smith, A.G.; Camire, M.E.; Brawley, S.H. Algae as
Nutritional and Functional Food Sources: Revisiting Our Understanding. J. Appl. Phycol. 2017, 29, 949–982. [CrossRef] [PubMed]
153. Rzymski, P.; Niedzielski, P.; Kaczmarek, N.; Jurczak, T.; Klimaszyk, P. The Multidisciplinary Approach to Safety and Toxicity
Assessment of Microalgae-Based Food Supplements Following Clinical Cases of Poisoning. Harmful Algae 2015, 46, 34–42.
[CrossRef]
154. Markou, G.; Wang, L.; Ye, J.; Unc, A. Using Agro-Industrial Wastes for the Cultivation of Microalgae and Duckweeds: Contamina-
tion Risks and Biomass Safety Concerns. Biotechnol. Adv. 2018, 36, 1238–1254. [CrossRef] [PubMed]
155. Suresh Kumar, K.; Dahms, H.U.; Won, E.J.; Lee, J.S.; Shin, K.H. Microalgae—A Promising Tool for Heavy Metal Remediation.
Ecotoxicol. Environ. Saf. 2015, 113, 329–352. [CrossRef]
156. Heussner, A.H.; Mazija, L.; Fastner, J.; Dietrich, D.R. Toxin Content and Cytotoxicity of Algal Dietary Supplements. Toxicol. Appl.
Pharmacol. 2012, 265, 263–271. [CrossRef]
157. Vichi, S.; Lavorini, P.; Funari, E.; Scardala, S.; Testai, E. Contamination by Microcystis and Microcystins of Blue-Green Algae Food
Supplements (BGAS) on the Italian Market and Possible Risk for the Exposed Population. Food Chem. Toxicol. 2012, 50, 4493–4499.
[CrossRef]
158. Lyon-Colbert, A.; Su, S.; Cude, C. A Systematic Literature Review for Evidence of Aphanizomenon Flos-Aquae Toxigenicity in
Recreational Waters and Toxicity of Dietary Supplements: 2000–2017. Toxins 2018, 10, 254. [CrossRef]
159. Roy-Lachapelle, A.; Solliec, M.; Bouchard, M.F.; Sauvé, S. Detection of Cyanotoxins in Algae Dietary Supplements. Toxins 2017,
9, 76. [CrossRef] [PubMed]
160. Henao, E.; Murphy, P.J.; Falfushynska, H.; Horyn, O.; Evans, D.M.; Klimaszyk, P.; Rzymski, P. Polymethoxy-1-Alkenes Screening
of Chlorella and Spirulina Food Supplements Coupled with in Vivo Toxicity Studies. Toxins 2020, 12, 111. [CrossRef] [PubMed]
161. Petrus, M.; Culerrier, R.; Campistron, M.; Barre, A.; Rougé, P. First Case Report of Anaphylaxis to Spirulin: Identification of
Phycocyanin as Responsible Allergen. Allergy 2010, 65, 924–925. [CrossRef]
162. Le, T.M.; Knulst, A.C.; Röckmann, H. Anaphylaxis to Spirulina Confirmed by Skin Prick Test with Ingredients of Spirulina Tablets.
Food Chem. Toxicol. 2014, 74, 309–310. [CrossRef] [PubMed]
163. Yim, H.E.; Yoo, K.H.; Seo, W.H.; Won, N.H.; Hong, Y.S.; Lee, J.W. Acute Tubulointerstitial Nephritis Following Ingestion of
Chlorella Tablets. Pediatr. Nephrol. 2007, 22, 887–888. [CrossRef] [PubMed]
164. Geh, E.N.; De La Cruz, A.A.; Ghosh, D.; Stelma, G.; Bernstein, J.A. Sensitization of a Child to Cyanobacteria after Recreational
Swimming in a Lake. J. Allergy Clin. Immunol. 2016, 137, 1902–1904.e3. [CrossRef] [PubMed]
165. Lang-Yona, N.; Kunert, A.T.; Vogel, L.; Kampf, C.J.; Bellinghausen, I.; Saloga, J.; Schink, A.; Ziegler, K.; Lucas, K.; Schuppan, D.;
et al. Fresh Water, Marine and Terrestrial Cyanobacteria Display Distinct Allergen Characteristics. Sci. Total Environ. 2018,
612, 767–774. [CrossRef]
166. Geh, E.N.; Ghosh, D.; McKell, M.; de la Cruz, A.A.; Stelma, G.; Bernstein, J.A. Identification of Microcystis Aeruginosa Peptides
Responsible for Allergic Sensitization and Characterization of Functional Interactions between Cyanobacterial Toxins and
Immunogenic Peptides. Environ. Health Perspect. 2016, 123, 1159–1166. [CrossRef]
167. Görs, M.; Schumann, R.; Hepperle, D.; Karsten, U. Quality Analysis of Commercial Chlorella Products Used as Dietary
Supplement in Human Nutrition. J. Appl. Phycol. 2010, 22, 265–276. [CrossRef]
168. Vardaka, E.; Kormas, K.A.; Katsiapi, M.; Genitsaris, S.; Moustaka-Gouni, M. Molecular Diversity of Bacteria in Commercially
Available “Spirulina” Food Supplements. Peer J. 2016, 4, e1610. [CrossRef]
169. Hoekstra, D.T.; Volschenk, H.; Collins, M.; McMaster, L.D. An Investigation of Clostridium Species Present in Nutraceutical
Preparations of Arthrospira Platensis (Spirulina) for Human Consumption. J. Appl. Phycol. 2011, 23, 777–787. [CrossRef]
170. Yolken, R.H.; Jones-Brando, L.; Dunigan, D.D.; Kannan, G.; Dickerson, F.; Severance, E.; Sabunciyan, S.; Talbot, C.C.;
Prandovszky, E.; Gurnon, J.R.; et al. Chlorovirus ATCV-1 Is Part of the Human Oropharyngeal Virome and Is Associated with
Changes in Cognitive Functions in Humans and Mice. Proc. Natl. Acad. Sci. USA 2014, 111, 16106–16111. [CrossRef]
171. Petro, T.M.; Agarkova, I.V.; Zhou, Y.; Yolken, R.H.; Van Etten, J.L.; Dunigan, D.D. Response of Mammalian Macrophages to
Challenge with the Chlorovirus Acanthocystis Turfacea Chlorella Virus 1. J. Virol. 2015, 89, 12096–12107. [CrossRef]
172. Groves, B.M.; Whitbeck, E.J.; Henderson, F.M. Methods of Removing Heavy Metals from Food Products. WO2012009234A2,
8 July 2011.
173. Beyrer, M.; Pina-Perez, M.C.; Martinet, D.; Andlauer, W. Cold Plasma Processing of Powdered Spirulina Algae for Spore
Inactivation and Preservation of Bioactive Compounds. Food Control 2020, 118, 107378. [CrossRef]
174. Seo, Y.C.; Choi, W.S.; Park, J.H.; Park, J.O.; Jung, K.H.; Lee, H.Y. Stable Isolation of Phycocyanin from Spirulina Platensis
Associated with High-Pressure Extraction Process. Int. J. Mol. Sci. 2013, 14, 1778–1787. [CrossRef]
175. Liu, H.; Guo, X.; Liu, L.; Yan, M.; Li, J.; Hou, S.; Wan, J.; Feng, L. Simultaneous Microcystin Degradation and Microcystis
Aeruginosa Inhibition with the Single Enzyme Microcystinase A. Environ. Sci. Technol. 2020, 54, 8811–8820. [CrossRef]
176. Whittaker, J.A.; Johnson, R.I.; Finnigan, T.J.A.; Avery, S.V.; Dyer, P.S. The Biotechnology of Quorn Mycoprotein: Past, Present and
Future Challenges. In Grand Challenges in Fungal Biotechnology; Nevalainen, H., Ed.; Springer: Cham, Germany, 2020; pp. 59–79.
Foods 2021, 10, 1226 27 of 29

177. O’Donnell, K.; Cigelnik, E.; Casper, H.H. Molecular Phylogenetic, Morphological, and Mycotoxin Data Support Reidentification
of the Quorn Mycoprotein Fungus as Fusarium Venenatum. Fungal Genet. Biol. 1998, 23, 57–67. [CrossRef] [PubMed]
178. King, R.; Brown, N.A.; Urban, M.; Hammond-Kosack, K.E. Inter-Genome Comparison of the Quorn Fungus Fusarium Venenatum
and the Closely Related Plant Infecting Pathogen Fusarium Graminearum. BMC Genomics 2018, 19, 1–19. [CrossRef]
179. Finnigan, T.; Needham, L.; Abbott, C. Mycoprotein: A Healthy New Protein With a Low Environmental Impact. In Sustainable
Protein Sources; Nadathur, S.R., Scanlin, L., Wanasundara, J.P.D., Eds.; Academic Press: London, UK, 2017; pp. 305–325.
180. Finnigan, T.J.A. Mycoprotein: Origins, Production and Properties. In Handbook of Food Proteins; Phillips, G.O., Williams, P.A., Eds.;
Woodhead Publishing Limited: Cambridge, UK, 2011; pp. 335–352.
181. Sandhu, M.S.; Hopp, R.J. Type I Hypersensitivity Reaction to Ingestion of Mycoprotein (Quorn) in a Patient with Mold Allergy.
Pediatr. Asthma Allergy Immunol. 2009, 22, 5–6. [CrossRef]
182. Katona, S.J.; Kaminski, E.R. Sensitivity to Quorn Mycoprotein (Fusarium venenatum) in a Mould Allergic Patient. J. Clin. Pathol.
2002, 55, 876–877. [CrossRef] [PubMed]
183. Hoff, M.; Trüeb, R.M.; Ballmer-Weber, B.K.; Vieths, S.; Wuethrich, B. Immediate-Type Hypersensitivity Reaction to Ingestion
of Mycoprotein (Quorn) in a Patient Allergic to Molds Caused by Acidic Ribosomal Protein P2. J. Allergy Clin. Immunol. 2003,
111, 1106–1110. [CrossRef] [PubMed]
184. Jacobson, M.F.; DePorter, J. Self-Reported Adverse Reactions Associated with Mycoprotein (Quorn-Brand) Containing Foods.
Ann. Allergy Asthma Immunol. 2018, 120, 626–630. [CrossRef]
185. Brzonkalik, K.; Herrling, T.; Syldatk, C.; Neumann, A. The Influence of Different Nitrogen and Carbon Sources on Mycotoxin
Production in Alternaria Alternata. Int. J. Food Microbiol. 2011, 147, 120–126. [CrossRef]
186. Bouras, N.; Strelkov, S.E. Influence of Carbon Source on Growth and Mycotoxin Production by Isolates of Pyrenophora Tritici-
Repentis from Wheat. Can. J. Microbiol. 2010, 56, 874–882. [CrossRef]
187. Hashempour-Baltork, F.; Hosseini, S.M.; Assarehzadegan, M.A.; Khosravi-Darani, K.; Hosseini, H. Safety Assays and Nutritional
Values of Mycoprotein Produced by Fusarium Venenatum IR372C from Date Waste as Substrate. J. Sci. Food Agric. 2020,
100, 4433–4441. [CrossRef]
188. Linder, T. Making the Case for Edible Microorganisms as an Integral Part of a More Sustainable and Resilient Food Production
System. Food Secur. 2019, 11, 265–278. [CrossRef]
189. Lee, J.Z.; Logan, A.; Terry, S.; Spear, J.R. Microbial Response to Single-Cell Protein Production and Brewery Wastewater Treatment.
Microb. Biotechnol. 2015, 8, 65–76. [CrossRef] [PubMed]
190. Øverland, M.; Tauson, A.H.; Shearer, K.; Skrede, A. Evaluation of Methane-Utilising Bacteria Products as Feed Ingredients for
Monogastric Animals. Arch. Anim. Nutr. 2010, 64, 171–189. [CrossRef] [PubMed]
191. Kornochalert, N.; Kantachote, D.; Chaiprapat, S.; Techkarnjanaruk, S. Use of Rhodopseudomonas Palustris P1 Stimulated Growth
by Fermented Pineapple Extract to Treat Latex Rubber Sheet Wastewater to Obtain Single Cell Protein. Ann. Microbiol. 2014,
64, 1021–1032. [CrossRef]
192. Kleiveland, C.R.; Hult, L.T.O.; Spetalen, S.; Kaldhusdal, M.; Christofferesen, T.E.; Bengtsson, O.; Romarheim, O.H.; Jacobsen, M.;
Leaa, T. The Noncommensal Bacterium Methylococcus Capsulatus (Bath) Ameliorates Dextran Sulfate (Sodium Salt)-Induced
Ulcerative Colitis by Influencing Mechanisms Essential for Maintenance of the Colonic Barrier Function. Appl. Environ. Microbiol.
2013, 79, 48–57. [CrossRef] [PubMed]
193. Biswas, A.; Takakuwa, F.; Yamada, S.; Matsuda, A.; Saville, R.M.; LeBlanc, A.; Silverman, J.A.; Sato, N.; Tanaka, H. Methanotroph
(Methylococcus capsulatus, Bath) Bacteria Meal as an Alternative Protein Source for Japanese Yellowtail, Seriola quinqueradiata.
Aquaculture 2020, 529, 735700. [CrossRef]
194. Hardy, R.W.; Patro, B.; Pujol-Baxley, C.; Marx, C.J.; Feinberg, L. Partial Replacement of Soybean Meal with Methylobacterium
Extorquens Single-Cell Protein in Feeds for Rainbow Trout (Oncorhynchus mykiss Walbaum). Aquac. Res. 2018, 49, 2218–2224.
[CrossRef]
195. Kunasundari, B.; Murugaiyah, V.; Kaur, G.; Maurer, F.H.J.; Sudesh, K. Revisiting the Single Cell Protein Application of Cupriavidus
Necator H16 and Recovering Bioplastic Granules Simultaneously. PLoS ONE 2013, 8, e78528. [CrossRef]
196. Alloul, A.; Wille, M.; Lucenti, P.; Bossier, P.; Van Stappen, G.; Vlaeminck, S.E. Purple Bacteria as Added-Value Protein Ingredient
in Shrimp Feed: Penaeus vannamei Growth Performance, and Tolerance against Vibrio and Ammonia Stress. Aquaculture 2021,
530, 735788. [CrossRef]
197. Jones, J.A.; Wang, X. Use of Bacterial Co-Cultures for the Efficient Production of Chemicals. Curr. Opin. Biotechnol. 2018, 53, 33–38.
[CrossRef]
198. Liu, B.; Li, Y.; Song, J.; Zhang, L.; Dong, J.; Yang, Q. Production of Single-Cell Protein with Two-Step Fermentation for Treatment
of Potato Starch Processing Waste. Cellulose 2014, 21, 3637–3645. [CrossRef]
199. Alloul, A.; Muys, M.; Hertoghs, N.; Kerckhof, F.M.; Vlaeminck, S.E. Cocultivating Aerobic Heterotrophs and Purple Bacteria for
Microbial Protein in Sequential Photo- and Chemotrophic Reactors. Bioresour. Technol. 2021, 319, 124192. [CrossRef]
200. Anupama; Ravindra, P. Value-Added Food: Single Cell Protein. Biotechnol. Adv. 2000, 18, 459–479. [CrossRef]
201. Johnson, R.J.; Lanaspa, M.A.; Gaucher, E.A. Uric Acid: A Danger Signal From the RNA World That May Have a Role in
the Epidemic of Obesity, Metabolic Syndrome, and Cardiorenal Disease: Evolutionary Considerations. Semin. Nephrol. 2011,
31, 394–399. [CrossRef] [PubMed]
Foods 2021, 10, 1226 28 of 29

202. Maiuolo, J.; Oppedisano, F.; Gratteri, S.; Muscoli, C.; Mollace, V. Regulation of Uric Acid Metabolism and Excretion. Int. J. Cardiol.
2016, 213, 8–14. [CrossRef] [PubMed]
203. Kurbanoglu, E.B.; Algur, O.F. Single-Cell Protein Production from Ram Horn Hydrolysate by Bacteria. Bioresour. Technol. 2002,
85, 125–129. [CrossRef]
204. Wongputtisin, P.; Khanongnuch, C.; Khongbantad, W.; Niamsup, P.; Lumyong, S. Screening and Selection of Bacillus spp. for
Fermented Corticate Soybean Meal Production. J. Appl. Microbiol. 2012, 113, 798–806. [CrossRef]
205. Mattick, C.S.; Landis, A.E.; Allenby, B.R.; Genovese, N.J. Anticipatory Life Cycle Analysis of In Vitro Biomass Cultivation for
Cultured Meat Production in the United States. Environ. Sci. Technol. 2015, 49, 11941–11949. [CrossRef] [PubMed]
206. Oonincx, D.G.A.B.; de Boer, I.J.M. Environmental Impact of the Production of Mealworms as a Protein Source for Humans—A
Life Cycle Assessment. PLoS ONE 2012, 7, e51145. [CrossRef] [PubMed]
207. Smetana, S.; Palanisamy, M.; Mathys, A.; Heinz, V. Sustainability of Insect Use for Feed and Food: Life Cycle Assessment
Perspective. J. Clean. Prod. 2016, 137, 741–751. [CrossRef]
208. Halloran, A.; Hanboonsong, Y.; Roos, N.; Bruun, S. Life Cycle Assessment of Cricket Farming in North-Eastern Thailand. J. Clean.
Prod. 2017, 156, 83–94. [CrossRef]
209. Heller, M.C.; Keoleian, G.A. Beyond Meat’s Beyond Burger Life Cycle Assessment: A Detailed Comparison between a Plant-Based and an
Animal-Based Protein Source; Univesity of Michigan: Ann Arbor, MI, USA, 2017.
210. Khan, S.; Dettling, J.; Loyola, C.; Hester, J.; Moses, R. Environmental Life Cycle Analysis: Impossible Burger 2.0; Quantis: Boston, MA,
USA, 2019.
211. Ye, C.; Mu, D.; Horowitz, N.; Xue, Z.; Chen, J.; Xue, M.; Zhou, Y.; Klutts, M.; Zhou, W. Life Cycle Assessment of Industrial Scale
Production of Spirulina Tablets. Algal Res. 2018, 34, 154–163. [CrossRef]
212. Smetana, S.; Sandmann, M.; Rohn, S.; Pleissner, D.; Heinz, V. Autotrophic and Heterotrophic Microalgae and Cyanobacteria
Cultivation for Food and Feed: Life Cycle Assessment. Bioresour. Technol. 2017, 245, 162–170. [CrossRef] [PubMed]
213. Smetana, S.; Mathys, A.; Knoch, A.; Heinz, V. Meat Alternatives: Life Cycle Assessment of Most Known Meat Substitutes. Int. J.
Life Cycle Assess. 2015, 20, 1254–1267. [CrossRef]
214. Sillman, J.; Uusitalo, V.; Ruuskanen, V.; Ojala, L.; Kahiluoto, H.; Soukka, R.; Ahola, J. A Life Cycle Environmental Sustainability
Analysis of Microbial Protein Production via Power-to-Food Approaches. Int. J. Life Cycle Assess. 2020, 25, 2190–2203. [CrossRef]
215. Järviö, N.; Maljanen, N.-L.; Kobayashi, Y.; Ryynänen, T.; Tuomisto, H.L. An Attributional Life Cycle Assessment of Microbial
Protein Production: A Case Study on Using Hydrogen-Oxidizing Bacteria. Sci. Total Environ. 2021, 776, 145764. [CrossRef]
216. Smetana, S.; Aganovic, K.; Irmscher, S.; Heinz, V. Agri-Food Waste Streams Utilization for Development of More Sustainable
Food Substitutes. In Designing Sustainable Technologies, Products and Policies; Springer: Cham, Germany, 2018; pp. 145–155.
217. European Commission. Regulation (EU) 2015/2283 of the European Parliament and of the Council of 25 November 2015.
Available online: https://eur-lex.europa.eu/legal-content/en/TXT/?uri=CELEX%3A32015R2283 (accessed on 29 March 2021).
218. Turck, D.; Bresson, J.L.; Burlingame, B.; Dean, T.; Fairweather-Tait, S.; Heinonen, M.; Hirsch-Ernst, K.I.; Mangelsdorf, I.;
McArdle, H.; Naska, A.; et al. Guidance on the Preparation and Presentation of an Application for Authorisation of a Novel Food
in the Context of Regulation (EU) 2015/2283. EFSA J. 2016, 14, e04594.
219. van der Spiegel, M.; Noordam, M.Y.; van der Fels-Klerx, H.J. Safety of Novel Protein Sources (Insects, Microalgae, Seaweed,
Duckweed, and Rapeseed) and Legislative Aspects for Their Application in Food and Feed Production. Compr. Rev. Food Sci. Food
Saf. 2013, 12, 662–678. [CrossRef]
220. US Food and Drug Administration. Federal Food, Drug, and Cosmetic Act (FD&C Act). Available online: https://www.fda.gov/
regulatory-information/laws-enforced-fda/federal-food-drug-and-cosmetic-act-fdc-act (accessed on 29 March 2021).
221. US Food and Drug Administration. Generally Recognized as Safe (GRAS). Available online: https://www.fda.gov/food/food-
ingredients-packaging/generally-recognized-safe-gras (accessed on 29 March 2021).
222. United States Department of Agriculture. USDA and FDA Announce a Formal Agreement to Regulate Cell-Cultured Food
Products from Cell Lines of Livestock and Poultry. Available online: https://www.usda.gov/media/press-releases/2019/03/07
/usda-and-fda-announce-formal-agreement-regulate-cell-cultured-food (accessed on 29 March 2021).
223. European Commission. Regulation (EC) No. 1829/2003 of the European Parliament and of the Council of 22 September 2003 on
Genetically Modified Food and Feed. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=uriserv%3AOJ.
L_.2003.268.01.0001.01.ENG&toc=OJ%3AL%3A2003%3A268%3ATOC (accessed on 29 March 2021).
224. European Commission. Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003
Concerning the Traceability and Labelling of Genetically Modified Organisms and the Traceability of Food and Feed Products
Produced from Genetically Modified Organisms. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=
uriserv%3AOJ.L_.2003.268.01.0024.01.ENG&toc=OJ%3AL%3A2003%3A268%3ATOC (accessed on 29 March 2021).
225. Belluco, S.; Halloran, A.; Ricci, A. New Protein Sources and Food Legislation: The Case of Edible Insects and EU Law. Food Secur.
2017, 9, 803–814. [CrossRef]
226. Enzing, C.; Ploeg, M.; Barbosa, M.; Sijtsma, L. Microalgae-Based Products for the Food and Feed Sector: An Outlook for Europe; European
Commission Joint Research Centre: Ispra, Italy, 2014.
227. Rubio, N.R.; Xiang, N.; Kaplan, D.L. Plant-Based and Cell-Based Approaches to Meat Production. Nat. Commun. 2020, 11, 1–11.
[CrossRef] [PubMed]
Foods 2021, 10, 1226 29 of 29

228. US Food and Drug Administration. GMO Crops, Animal Food, and Beyond. Available online: https://www.fda.gov/food/
agricultural-biotechnology/gmo-crops-animal-food-and-beyond (accessed on 29 March 2021).
229. European Commission. The Commission Authorises Eight Genetically Modified Crops for Use as Food and Feed. Available
online: https://ec.europa.eu/commission/presscorner/detail/en/mex_21_190 (accessed on 29 March 2021).
230. Chou, M.L.; Bailey, A.; Avory, T.; Tanimoto, J.; Burnouf, T. Removal of Transmissible Spongiform Encephalopathy Prion from
Large Volumes of Cell Culture Media Supplemented with Fetal Bovine Serum by Using Hollow Fiber Anion-Exchange Membrane
Chromatography. PLoS ONE 2015, 10, e0122300. [CrossRef]
231. Qin, H.; Zhao, A.; Fu, X. Small Molecules for Reprogramming and Transdifferentiation. Cell. Mol. Life Sci. 2017, 74, 3553–3575.
[CrossRef] [PubMed]
232. Chriki, S.; Hocquette, J.F. The Myth of Cultured Meat: A Review. Front. Nutr. 2020, 7, 7. [CrossRef] [PubMed]
233. Food Standards Austrilia New Zealand. Australia New Zealand Food Standards Code—Standard 1.5.1—Novel Foods. Available
online: https://www.legislation.gov.au/Details/F2017C00324 (accessed on 29 March 2021).
234. Food Standards Austrilia New Zealand. Australia New Zealand Food Standards Code—Schedule 25—Permitted Novel Foods.
Available online: https://www.legislation.gov.au/Details/F2017C00543 (accessed on 29 March 2021).
235. Food Standards Austrilia New Zealand. A1186—Soy Leghemoglobin in Meat Analogue Products. Available online: https:
//www.foodstandards.gov.au/code/applications/Pages/A1186.aspx (accessed on 29 March 2021).