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Food Hydrocolloids 151 (2024) 109832
Available online 2 February 2024
0268-005X/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Renewable methanol utilizing bacteria as future meat analogue: An 
explorative study on the physicochemical and texturing properties of 
Methylobacillus flagellatus biomass and fractions 
Wanqing Jia a,d, Laurice Pouvreau b, Atze Jan van der Goot a, Timotheus Y. Althuis c, 
David Virant e, Aleksander J. Kruis e, Gregor Kosec e, Nico J. Claassens c, Julia K. Keppler a,* 
a Laboratory of Food Process Engineering, Wageningen University, P.O. Box 17, 6700 AA, Wageningen, the Netherlands 
b Wageningen Food and Biobased Research, Wageningen University, P.O. Box 17, 6700 AA, Wageningen, the Netherlands 
c Laboratory of Microbiology, Wageningen University, 6708 WE, Wageningen, the Netherlands 
d Farmless, 1068KL, Amsterdam, the Netherlands 
e Acies Bio d.o.o., Tehnološki Park 21, SI-1000 Ljubljana, Slovenia 
A R T I C L E I N F O 
Keywords: 
Single cell protein 
Gram-negative bacteria 
Meat analogue 
Shear cell 
Functional properties 
Future food 
A B S T R A C T 
There is an ongoing search for sustainable and functional alternative protein sources to animal proteins. Meth-
ylobacillus flagellatus (grown on renewable methanol) is known to give high protein yields with low resource 
requirements. However, the techno-functionality for food applications has not yet been explored. In this study, 
the biomass was processed by microfluidizer, centrifugation and acid precipitation. Unprocessed biomass (UB: 
protein content of 73 %) and processed biomass fractions were investigated on their composition, physico-
chemical and rheological properties. All the biomass fractions had comparable composition, a pink, meat-like 
colour and umami smell. Differences were observed in the rheological and structuring properties using shear 
cell: UB yielded layered but crumbly structure, while the acid precipitated fraction resulted in a firm consistent 
product. From a functional point of view, M. flagellatus is a promising protein source that can be used for meat 
analogues without extensive prior fractionation. 
1. Introduction 
Due to the increasing global demand for high-quality protein and the 
simultaneous growing awareness among consumers that the dispro-
portionate consumption of meat proteins is not sustainable, there is 
growing interest in the search for alternative protein sources. Plant 
proteins are promising, but often have deficiencies in functionality and 
sensory characteristics that limit their application for imitating the meat 
and dairy products (Kyriakopoulou et al., 2021). Recently, so-called 
single cell protein or cellular protein have been described as potential 
alternatives for plant proteins. In this category, fungi (e.g. Quorn™), 
yeast and bacteria are investigated, and some cases already commer-
cialized for their protein yield, composition, and functional properties as 
food ingredient (Kyriakopoulou et al., 2021; Nyyssölä et al., 2022; Ritala 
et al., 2017). However, while these are usually grown on glucose (for 
example from maize or sugar beet), growing bacteria on renewable 
methanol could be an alternative approach avoiding the dependence on 
crops as substrate (Cotton et al., 2020; Leger et al., 2021; Sakarika et al., 
2022). Methanol is a promising feedstock that can be made renewable 
from CO2, water, and electricity. Energy-efficient processes for pro-
duction of e-methanol, using hydrogen from water electrolysis as in-
termediate, are already developed and currently being scaled-up 
industrially (IRENA and Methanol Institute, 2021). In addition, meth-
anol can be renewably made from residual waste biomass and municipal 
solid waste, as these waste streams can be gasified and the generated 
syngas can be converted into bio-methanol. 
M. flagellatus is a promising obligate methylotroph Gram-negative 
strain with high protein content. It was selected as a prospective in-
dustrial strain due to its high growth rates and yields (Kruis et al., 2022). 
However, its compositional and functional properties for food applica-
tions have not been explored yet. One of the challenges of using mi-
crobial biomass for food is their high nucleic acid content. This is a 
concern because the ingestion of purine compounds derived from RNA 
degradation increases plasma uric acid concentrations, which can cause 
gout and kidney stones (Anaizi, 2023). 
* Corresponding author. 
E-mail address: julia.keppler@wur.nl (J.K. Keppler). 
Contents lists available at ScienceDirect 
Food Hydrocolloids 
journal homepage: www.elsevier.com/locate/foodhyd 
https://doi.org/10.1016/j.foodhyd.2024.109832 
Received 19 November 2023; Received in revised form 20 January 2024; Accepted 29 January 2024 
mailto:julia.keppler@wur.nl
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Food Hydrocolloids 151 (2024) 109832
2
The aim of the present study is to screen the composition, physico-
chemical, rheological, and texturing properties of unprocessed 
M. flagellatus biomass and its derived fractions. We hypothesize that 
these properties of the unprocessed microbial biomass and its derived 
fractions are influenced by the different processing steps and thus have 
different potential for food applications. Besides the above-mentioned 
analysis, microbial safety assessment for human consumption is also 
one of the main challenges for the food application, which is not part of 
this study. M. flagellatus is not known to be pathogenic, but it has not 
been tested and approved for food application yet. In this study we 
focussed on the compositional and techno-functional characterization of 
this bacterium for food applications. 
The Gram-negative bacteria cell wall is mainly composed of a lipo-
polysaccharide membrane (Kim et al., 2005). The cell wall can be 
fractured by high-pressure homogenization (Gomes et al., 2020), 
yielding disrupted biomass (DB) displaying potentially different prop-
erties than the reference “unprocessed” (washed, frozen and thawed) 
biomass (UB). High speed centrifugation is overall capable of generate 
an insoluble pellet fraction (IF) which is rich in bacteria cells and a 
soluble supernatant fraction (SF), which have different properties 
potentially. Finally, isoelectric precipitation (using acid) is often used as 
a method to recover soluble protein from plant and microalgae (la Cour 
et al., 2019; Veide Vilg & Undeland, 2017), and it is of interest to test if 
this also holds true for microbial protein. Thus, the acid-insoluble 
fraction (AIF) was produced by acid precipitation of the soluble frac-
tion SF. 
All fractions will be characterized with respect to composition 
including nucleic acid content, amino acid profile, nitrogen conversion 
factor, physicochemical-, and rheological properties. The results of the 
latter will be discussed comparatively with results from similar analyses 
reported in literature for various plant protein fractions to give a rough 
estimation on similarities and difference. Then the UB and the most- 
intensively processed fraction AIF, as well as soy protein concentrate, 
being an often used bulk ingredient for meat analogues as reference, are 
treated in the shear cell to test their ability to form a fibrous texture to 
understand the potential of those biomasses for use in meat analogue- 
like products. 
2. Material and methods 
2.1. Materials 
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	Renewable methanol utilizing bacteria as future meat analogue: An explorative study on the physicochemical and texturing pr ...
	1 Introduction
	2 Material and methods
	2.1 Materials
	2.2 Methods
	2.2.1 Biomass generation
	2.2.2 Fractionation and sample preparation
	2.2.3 Compositional analysis
	2.2.4 Biomass cell integrity
	2.2.5 Protein characterization
	2.2.5.1 Molecular weight distribution
	2.2.5.2 Protein secondary structure
	2.2.5.3 Thermograms
	2.2.5.4 Zeta potential and conductivity
	2.2.5.5 Water holding capacity and biomass solubility
	2.2.5.6 Colour
	2.3 Structuring properties
	2.3.1 Rheological properties
	2.3.2 Rheological properties with closed cavity rheometer
	2.3.3 Structure formation
	3 Result and discussion
	3.1 Appearance and colour
	3.2 Composition analysis
	3.2.1 Composition and amino acid profile
	3.2.2 Protein molecular weight profile
	3.2.3 Fraction composition and protein secondary structure
	3.3 Physico-chemical properties
	3.3.1 Thermograms by differential scanning calorimetry (DSC)
	3.3.2 Zeta potential and conductivity
	3.3.3 Water holding capacity
	3.4 Structuring properties
	3.4.1 Rheological properties with Discovery Hybrid Rheometer
	3.4.2 Rheological properties by closed cavity rheometer (CCR)
	4 The potential of using M. flagellatus for making meat analogue product
	5 Conclusion
	CRediT authorship contribution statement
	Declaration of competing interest
	Data availability
	Acknowledgement
	Appendix A Supplementary data
	ReferencesIQ 7000 Ultrapure Lab Water System, 
Merck KGaA, Darmstadt, Germany). 
2.2. Methods 
2.2.1. Biomass generation 
M. flagellatus OCB6 was modified to reduce the viscosity of the broth 
by deleting the genes responsible for exopolysaccharide formation 
(Kruis et al., 2022) using methods described by Hendrickson et al. 
(2010). 
The biomass of M. flagellatus was prepared by Acies Bio (Ljubljana, 
Slovenia) in 5 L lab-scale bioreactors (Sartorius) in fed-batch mode. A 
mineral medium containing KH2PO4, Na2HPO4, MgSO4, NH4SO4 and a 
trace element mixture was used. Methanol concentration was dynami-
cally maintained at 4–5 g/L throughout the cultivations via a feedback 
loop using an online methanol sensor. The pH was kept at 7 by automatic 
addition of ammonium hydroxide, which also served as source of ni-
trogen. The aeration rate was contolled at 2.0 VVM (volume air per 
volume of liquid per minute) with ambient air. The stirring speed was 
automatic regulated to maintain dissolved oxygen at 30 % during the 
first 10 h of growth, with a max speed of 1500 rpm. Once dry cell weight 
reached around 5 g/L, oxygen became limiting and max stirring was 
maintained till the end of the process. After the culture reached the 
stationary phase, the biomass was harvested by centrifugation, and 
washed twice with distilled water. The final wet pellet obtained after 
washing was stored at − 80 ◦C. 
2.2.2. Fractionation and sample preparation 
The fractionation process of the biomass was performed in two 
batches and is depicted in Fig. 1. The so called ‘unprocessed’ thawed 
biomass (UB) was taken as starting material, which was treated by ho-
mogenization, centrifugation, acid precipitation, and neutralization. For 
this, the 300 g UB was evenly distributed into four centrifugal bottles 
(1L) and Milli-Q water was added to reach a dilution factor of 10. To 
mitigate the viscous nature of the biomass, it was dispersed in water 
using a rotor-stator homogenizer (Ultra-Turrax IKA T18 basic, Germany) 
at a high speed of 10,000 rpm for approximately 30s. The method was 
adapted from a previous method reported by (Schröder et al., 2017). 
To stress the bacterial cell wall for a better release of protein, the 
dispersion was homogenized at 600 bar with a high-pressure homoge-
nizer to produce the so called ‘disrupted biomass’ (DB) (Microfluidizer® 
Processor MF 110Y with Y-shaped interaction chamber, F12Y; min. in-
ternal channel: 75 μm), (Microfluidics, Newton, Massachusetts, USA). 
DB was further centrifuged at 20,000 g for 30 min and the soluble su-
pernatant was collected and named as ‘soluble fraction’ (SF), while the 
pellet fraction was denoted as ‘insoluble fraction’ (IF). 
To test if the proteins can be further concentrated, acid precipitation 
was conducted with the SF. The highest protein recovery yield was 
achieved by acid precipitation at pH 4 (pH values between 2 and 6 were 
tested). Thus, acid precipitation was performed at pH 4 by adding 1M of 
HCl to the supernatant and the dispersion was mixed with a magnetic 
stirrer at 700 rpm. Afterward, the dispersion was centrifuged at 10,000 g 
for 10 min and the supernatant was freeze-dried for a mass balance 
calculation. The recovered pellet was redispersed into Milli-Q water to 
create a solution, which was stirred with a magnet at 700 rpm for 1 h and 
the pH was readjusted to 7 by adding 1 M NaOH. The solution named 
acid-insoluble fraction (AIF). All fractions (UB, DB, SF, IF and AIF) were 
collected and freeze-dried for a mass balance calculation and further 
analysis. 
Abbreviation 
AA Amino acid 
AIF Acid insoluble fraction 
ATR-FTIR Attenuated-total-reflection Fourier Transform Infrared 
C1 One carbon 
CCR Closed cavity rheometer 
CLSM Confocal laser scanning microscope 
DB Disrupted biomass 
DHR Discovery Hybrid Rheometer 
DSC Differential scanning calorimetry 
IF Insoluble fraction 
MW Molecular weight 
NCF Nitrogen conversion factor 
PPI Pea protein isolate 
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel 
electrophoresis 
SF Soluble fraction 
SPC Soy protein concentrate 
UB Unprocessed biomass 
WHC Water holding capacity 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
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2.2.3. Compositional analysis 
The fat content of freeze-dried UB, DB, SF, IF and AIF was deter-
mined with the AACC 30–25.01 method, and ash content was measured 
with the AACC 08-01 method. The soluble and the total nucleotides were 
analysed by NovoCIB (France). 
The full amino acid profile of the UB, IF and AIF including cysteine 
and tryptophan was analysed by Eurofins (The Netherlands): with the 
ISO 13903:2005; EU 152/2009 (F) method. The total amino acid content 
corresponds to the protein content of the sample. The nitrogen conver-
sion factor (NCF) for UB, IF and AIF was calculated as 5.35, 5.17 and 
5.06 based on the results of full amino acid profile. The same NCF of UB 
(5.35) was assumed for DB as there is no composition change expected 
during high-pressure homogenization. This NCF was also used for pro-
tein determination in SF, although it is expected to have a slightly higher 
NCF value than 5.35. 
Further, the total nitrogen content of the freeze-dried biomass frac-
tions was determined with the Dumas combustion method using a Ni-
trogen Analyzer (Flash EA 1112 Series, Thermo Scientific, The 
Netherlands), and protein concentrations were derived by the above 
mentioned NCF. The carbohydrate content of the biomass was calcu-
lated by the difference of ash, fat, protein, and nucleotides content. 
2.2.4. Biomass cell integrity 
To study the effect of freeze-drying on the biomass, a confocal laser 
scanning microscope (CLSM) (type 510; Zeiss, Oberkochen, Germany) 
was used to visualize the original as well as the freeze-dried UB. A 
mixture of 2 different staining dyes was used: 0.2 wt % Rhodamine-B 
(Rh-b) and 0.2 wt % of Bodipy for protein and fat staining respec-
tively. Excitation light in CLSM was provided by two lasers: a HeNe laser 
at 501 nm for Rhodamine B and 580 nm for Bodipy. Stained samples 
were mixed with a toothpick and left at 4 ◦C for about 2h prior to im-
aging. A 10 × EC Plan-Neofluar/0.5 objective lens was used to take the 
images. The ZEN software (Carl Zeiss Microscopy, Jena, Germany) was 
used to analyse the images. 
2.2.5. Protein characterization 
2.2.5.1. Molecular weight distribution. The protein molecular weight 
distribution of the freeze-dried UB, DB, SF, IF and AIF was determined 
using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- 
PAGE) as previously described (Jia et al., 2021). A protein solution of 
approximately 2 mg/mL was prepared in a falcon tube. The sample 
buffer (0.125M Tris/HCL buffer pH 6.8 containing 4 % w/v SDS, 40 % 
w/v glycerol, 0.02 % bromophenol blue) was diluted in a ratio of 1:1 
with 0.9 % NaCl-solution for the non-reduced samples and for the 
reduced samples β-mercaptoethanol was added in a concentration of 5 
%. The samples were suspended in sample buffer in a concentration of 
2.75 mg/ml sample buffer, vortexed and then heated for 30 min at 95 ◦C. 
In total 20 μL clear supernatant after centrifugation (15,000 g, 15 min) 
was loaded onto the gel. The experiment was performed at 200 V for 30 
min. Subsequently, the gels were stained with Coomassie Blue solution. 
Destaining was performed overnight using a water-methanol-acetic acid 
solution with an 80/10/10 % v/v ratio. The gel was scanned with a gel 
scanner (Biorad-GS900, Netherlands). 
2.2.5.2. Protein secondary structure. The protein secondary structure of 
the freeze-dried UB, DB, SF, IF and AIF was measured using a Fourier 
Transform Infrared (FTIR) spectrometer with a thermally controlled Bio 
ATR2 unit at 25 ◦C and a nitrogen cooled MCT detector (Confocheck™ 
system, BrukerOptics, Ettlingen, Germany) (Jia et al., 2022). A 2 % w/w 
protein solution was prepared by adding the freeze-dried powder to 5 
mL Milli-Q water, followed by vortexing to allow full hydration. In-
terferograms were accumulated over the spectral range 4000-400 cm− 1, 
with a resolution of 4 cm− 1. In total 60 scans at a resolution of 0.7 cm− 1 
were conducted. Independent duplicates with 20 μL of the sample were 
loaded into the cell for measurements. For evaluation of protein sec-
ondary structure, the measured spectra of the amide band І region 
(1580-1700 cm− 1) were vector-normalized using the Bruker OPUS 
software system (8.25, Ettlingen, Germany). The second derivative was 
calculated using 9 smoothing points. Besides, the whole spectra without 
normalization between 4000 and 900 cm− 1 were compared for each 
fraction. 
2.2.5.3. Thermograms. The thermal stability of freeze-dried UB, DB, SF, 
IF and AIF were analysed with differential scanning calorimetry (DSC) 
(TA instrument 250; TA Instruments, Newcastle, DE, USA). Approxi-
mately 8 mg of the sample was weighed in a high-volume pan and 32 μL 
of Milli-Q water was added. The pan was sealed and heated from 25 to 
130 ◦C using a heating rate of 5 ◦C/min. After 1 min, the pan was cooled 
down to 25 ◦C using a cooling rate of 20 ◦C/min. This heating and 
cooling process was repeated for a second time to make sure the peak 
indicated protein denaturation. Duplicates were measured for each 
sample. The onset denaturation temperature (onset Td), the peak tem-
perature of denaturation (Td), and the denaturation enthalpy (J/g 
Fig. 1. The scheme of biomass fractionation, including total and protein mass balance. UB, raw biomass; DB, disrupted biomass; SF, soluble fraction; IF, Pellet 
insoluble fraction; AIF, acid insoluble fraction. The mass balance was also expressed as total mass by dry matter (g DM/100g UB in DM), and total protein (g protein 
DM/100g UB in DM). The mass balance of each fraction was all originated from the initial raw material UB. 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
4
biomass) were collected by Trios data analysis software (TA 
Instruments). 
2.2.5.4. Zeta potential and conductivity. The zeta potential of the freeze- 
dried biomass UB, DB, SF, IF and AIF was measured as described by 
(Narong & James, 2006) using Zetasizer Nano ZS (Malvern Instruments 
Ltd., Malvern, Worcestershire, UK). The 2 % w/w solutions of the 
biomass were diluted approximately 100 times. The measurements were 
performed at 25 ◦C and the results were expressed as the average from 
measurements of two independent samples. The pH and conductivity 
were measured after overnight mixing using the Multi-Parameter Meter 
(HQ440D laboratory Dual Input, the Netherlands). 
2.2.5.5. Water holding capacity and biomass solubility. The amount of 
soluble dry matter and the water holding capacity (WHC) of the insol-
uble matter was measured based on an adapted method reported earlier 
(Möller et al., 2021). A 4 % w/w dispersion of the freeze-dried fractions 
UB, DB, SF, IF and AIF was made in a falcon tube by adding 0.4 g of the 
powder into 10 g of Milli-Q water. The samples were vortexed for 1 min 
and rotated overnight with a roller table. The pH value of all the frac-
tions was in between 6.2 and 6.3. Afterward, the samples were centri-
fuged at 4000 g for 30 min. The supernatant and the pellet were 
transferred into an aluminum tray and dried in an oven at 105 ◦C (Model 
E28, Binder, Germany) for at least 16 h. The weight of the pellet before 
(Mwet pellet) and after freeze drying (Mdry pellet) was measured. The dry 
masses of the original sample Moriginal and of the supernatant Msupernatant 
were measured. The WHC of the dry pellet and the solubility were 
calculated according to equations (1) and (2), respectively: 
WHC
(
g water
g dry pellet
)
=
Mwet pellet − Mdry pellet
Mdry pellet
(1) 
Solubility (%)=
Msupernatant
Moriginal
(2) 
2.2.5.6. Colour. The colour of UB, DB, SF, IF and AIF was measured 
with ColourFlexEZ (HunterLab) and the software of Easymatch QC for 
the data analysis. Approximately 5g of the sample were loaded onto the 
colour meter, and all measurements were performed in duplicates. 
2.3. Structuring properties 
2.3.1. Rheological properties 
Dispersions of 15 wt % concentration (dry matter) were prepared 
from the freeze-dried UB, DB, SF, IF and AIF. The viscosity was deter-
mined by a Discovery Hybrid Rheometer (DHR) (TA instrument, The 
Netherlands) combined with a Peltier Concentric Cylinders and a solvent 
trap (grooved bob with 14 mm diameter). A flow sweep was applied to 
the dispersions at 20 ◦C from 0.1 to 100 s− 1 to measure the viscosity. 
Afterward, a temperature sweep was performed by increasing the 
temperature from 20 to 95 ◦C at a rate of 5 ◦C/min, followed by a 
holding time of 5 min at 95 ◦C before cooling down to 20 ◦C at a rate of 
3 ◦C/min. The storage (G′) and loss modulus (G″) were recorded as a 
function of time. Subsequently, the heated samples were further exposed 
to an amplitude sweep from 0.1 to 100 % (at a frequency of 1 Hz). The 
storage (G′) and loss modulus (G″) dependency on strain were recorded. 
2.3.2. Rheological properties with closed cavity rheometer 
Viscoelastic properties were measured with a Closed Cavity 
Rheometer device (CCR) (RPA elite, TA instruments, USA). Approxi-
mately 2 g of the sample powder was mixed with Milli-Q water and a 
total mass of 5 g sample (40 % DM) was made. Besides, a comparison 
was made for UB, SF and AIF by adding 1 % salt to the Milli-Q water 
before mixing with the sample to understand the effect of salt on the 
rheological properties. After 30 min hydration time, the samples were 
then placed in between two plastic foils in the closed cavity (disk 
geometry). A pressure of 4 bars was applied to prevent water evapora-
tion. The complex modulus (G*) was calculated by the software based on 
the measured storage modulus (G′) and loss modulus (G″). Due to the 
limitation of the sample availability, the measurement was performed 
only once. 
2.3.3. Structure formation 
Structure formation was only tested for freeze-dried UB and AIF 
fractions (60 g for each fraction) because they represent the lowest and 
the highest processed fractions. A high-temperature conical shear cell 
(HTSC) (Wageningen University, Wageningen, the Netherlands) was 
used for the structuring experiments (Grabowska et al., 2016). Heating 
and cooling were performed using an external oil bath. A biomass 
mixture of 40 wt % concentration was prepared by firstly mixing 0.9 g 
sodium chloride and 53.1 g demineralized water. Then the 36 g sample 
(dry matter) was mixed with water, and subsequently hydrated for 30 
min. Afterward, the hydrated sample was transferred to the preheated 
HTSC and then sheared at 30 rpm at 140 ◦C for 15 min. 
3. Result and discussion 
Unprocessed biomass of M. flagellatus and the various fractions 
derived from it were characterized with respect to their physicochemical 
and rheological properties, to evaluate their potential use for food 
applications. 
3.1. Appearance and colour 
To assess whether freezing and freeze-drying already affected the 
integrity of the cell wall, the wet unprocessed biomass (UB) was 
compared before and after freeze-drying: the protein (red colour) and fat 
(yellow colour) distribution for the untreated biomass and freeze-dried 
UB was investigated using CLSM (Fig. 2). 
Large numbers of rod-like particles can be identified for the un-
treated biomaterial at 10 μm scale (Fig. 2 A2, B2), which are most likely 
the bacteria cells, since the methylotrophic bacteria have a rod-shape 
(Kalyuzhnaya et al., 2012). However, it is hard to distinguish between 
protein and fat, because thecolour of the particles was in between red 
and yellow. This indicates that both protein and lipids are present in 
these particles and likely still located within the cell. After freeze-drying, 
the individual rod-like particles were less visible, and the CLSM image 
was dominated with red colour (proteins) and some yellow particles 
(fat). This suggests that some of the individual cells in UB are broken 
after freezing, freeze-drying or resuspension, which released the protein 
and fat from the rod-like bacteria. The fat coalesced after release and 
formed domains that were larger than the original microbe. It is known 
that the freezing and thawing rate without cryoprotectants can affect 
bacteria cells, because intracellular ice crystals being formed upon 
freezing can rupture cells either physically or through osmotic pressure 
changes (Lorv et al., 2014). Because all samples obtained during frac-
tionation were similarly frozen, freeze-dried, and resuspended, it can be 
assumed that this will have influenced the cell wall integrity of all 
samples similarly and that differences can be ascribed to their individual 
processing history. 
Next to the cell wall integrity, also the colour of the samples was 
analysed: The colour intensities of all fractions were similar except for 
the lowest lightness with AIF fraction (Supplementary Fig. S1). All 
fractions had a pinkish hue. The results confirmed that the difference in 
appearance between the fractions was small and not significantly 
affected by fractionation. This means that the colour was not caused by 
pigments with specific properties that might have accumulated specif-
ically in the soluble or the insoluble fraction. 
W. Jia et al. 
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Food Hydrocolloids 151 (2024) 109832
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3.2. Composition analysis 
3.2.1. Composition and amino acid profile 
Table 1 shows the true protein content based on amino acid analysis, 
ash, fat, total nucleotides, and rest (potentially carbohydrate) contents 
of UB, DB, SF, IF and AIF. The corresponding amino acid analysis of the 
protein fraction, which was also used to calculate the nitrogen conver-
sion factor for further analyses with Dumas, is shown in Table 2. 
UB contained 73 % protein, 2 % fat, 5 % ash, 5 % soluble nucleotides 
and 14 % others (potentially carbohydrates). A high protein content of 
approximately 74 % dry mass has been reported for hydrogen-oxidizing 
bacteria (Volova & Barashkov, 2010). However, in that study, a higher 
lipid concentration of 6–9 % was observed, a lower carbohydrate con-
centration of 5–6 % and a higher DNA/RNA content of 12–13 %. These 
differences could be caused by using different types of bacteria, but it is 
also possible that different analysis methods were used. The protein 
content in the biomass based on amino acid analysis (approx. 73 % 
protein with NCF of 5.06 due to the presence of nucleotides) is com-
parable to commercial plant protein fractions, such as soy protein 
concentrate (approx. 62 % with NCF of 5.7) and pea protein isolate 
(approx. 69 % with NCF of 5.45) (Moreno et al., 2020; Peng, 2021). 
Thus, further protein fractionation is likely not required for many food 
applications that require a high protein content. However, different 
processing methods were still applied to the UB to understand the 
Fig. 2. CLSM micrographs of the biomass before freeze-drying at a scale of 40 μm (A1) and 10 μm (A2) and the freeze-dried UB at a scale of 40 μm (B1) and 10 μm 
(B2). Red: proteins, Yellow: lipids. of freeze-dried powder from UB, DB, SF, IF and AIF fractions. 
Table 1 
The biomass composition with protein, carbohydrate, ash, and fat for UB, DB, SF, IF and AIF in dry matter (g/100g powder). The nitrogen to protein conversion factor 
(NCF) was derived by amino acid analysis (Table 2) for UB, IF and AIF. 
Composition Total Nitrogen g/ 
100g 
Proteina g/ 
100g 
Non-Protein Nitrogen g/ 
100g 
NCF Fat g/ 
100g 
Ash g/ 
100g 
Total nucleotides g/100g 
b 
Carbohydrates g/ 
100gc 
UB 13.7 ± 0.1 73.1 ± 0.3 1.6 5.35 2.0 ± 0.2 5.5 ± 0.4 5.0 14.4 
DB 13.2 ± 0.3 69.8 ± 1.2 N.A 5.35 1.8 ±
0.01 
5.4 ± 0.1 4.8 18.2 
SF 13.4 ± 0.03 71.7 ± 0.8 N.A 5.35 2.1 ±
0.04 
5.7 ± 0.3 5.3 15.4 
IF 13.3 ± 0.3 67.0 ± 2.4 2.2 5.17 1.2 ±
0.01 
4.7 ± 0.5 3.8 23.3 
AIF 14.0 ± 0.2 70.9 ± 0.9 2.3 5.06 0.7 ± 0.1 5.0 ± 1.3 5.0 18.5 
a Protein content of UB, IF and AIF was obtained from total amino acid analysis, thus it is true protein content. 
b The soluble nucleotides were also measured for the fractions UB, DB, SF, AIF and IF as follows: 0.08, 0.08, 0.99, 0.11, 0.05 g/100g powder (dry matter). Due to the 
sample limitation, only one measurement was obtained, thus no standard deviation was obtained. 
c The carbohydrate content was calculated by the difference (mean value of each component was used). 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
6
influence of the processing steps on the composition (e.g. carbohydrates 
and nucleotides) and on the functional properties of the biomass. 
The various processing steps (Fig. 1) did not have a large effect on the 
overall composition of the biomass. The protein content was around 70 
g/100g for all fractions. The centrifugation of disrupted biomass (DB) 
yielded two fractions: the insoluble fraction (IF) and the supernatant 
fraction (SF). The lowest protein content of 67.0 g/100g was found for 
the IF, whereas this fraction also had the highest carbohydrate content 
(23.3 g/100g). Since the SF had a lower carbohydrate content of 15.4 g/ 
100g, it can be concluded that the centrifugation process removed 28 % 
of carbohydrates from the DB (18.2 g/100g) into the IF (23.3 g/100g). 
Since the supernatant fraction was slightly turbid, this large decrease 
might have been caused by phase separation phenomena between the 
different polymers present (Van De Velde et al., 2015), which could be 
interesting to explore in future studies. 
The slightly lower nucleotide content in the IF (3.8 g/100g) corre-
lates with the higher amount in the SF (5.7 g/100g). The fat content of 
DB and SF was similar with approx. 2 g/100g, and acid precipitation of 
SF resulted in a lower fat content of 0.7 g/100g in acid precipitated 
fraction (AIF), which indicates that fat could be removed to a certain 
extent by the acid precipitation process. The ash content was similar in 
all samples. 
Acid precipitation is a common method to purify protein from plant- 
based materials, such as soy protein or pea protein (Peng, 2021; Yang 
et al., 2021). Bacterial biomasses contain mainly ribosomal proteins and 
enzymes, while plant seeds contain storage proteins (Hendrickson et al., 
2010). The remaining carbohydrates (or others) are most likely 
composed of polysaccharides, even though this strain was modified to 
reduce exopolysaccharide formation. Hower some exopolysaccharides 
may still be attached to the cell material and may lead to higher viscosity 
and are difficult to separate from the proteins just by acid precipitation 
(Mahapatra et al., 2013). Identification of the type of carbohydrates 
present could help to develop strategies for effective removal of (exo) 
polysaccharide (in case their removal is necessary from a functional 
point of view). Polysaccharides were reported to be extracted by ultra-
sound in combination with formamide and NaOH or various combina-
tions of ultrasound and heat (Dai et al., 2016). 
The average amino acid(AA) composition for UB, IF and AIF is 
shown in Table 2 alongside the composition of soy protein concentrate 
(SPC), pea protein isolate (PPI) and casein for comparison. Overall, the 
analysis revealed that all fractions had a well-balanced amino acid 
profile, though they are somewhat low in cysteine, which was in line 
with previous findings for other hydrogen-oxidizing, Gram-negative 
bacteria (Nyyssölä et al., 2022; Volova & Barashkov, 2010). The nucleic 
acid content varied slightly between 3.8 and 5 g/100 g. In the case of 
mycoprotein, which is already used commercially for food, a reduction 
from 10 to 2 g/100 g dry matter is achieved through post-processing, to 
reach the 18 yrs 
Amino acid (AA) g/100g protein g/100g protein g/100g protein g/100g protein g/100g protein g/100 g protein g/100g 
protein 
Essential AA 
Threonine 5.1 5.4 5.0 3.9 3.7 4.2 2.3 
Methionine 2.1 1.9 1.9 1.3 1.1 2.5 2.2 
Phenylalanine 4.6 4.7 4.7 5.0 5.5 4.6 3.8 
Histidine 2.2 2.0 2.3 2.4 2.5 3.3 1.5 
Lysine 7.2 7.1 7.3 6.4 7.5 7.3 4.5 
Valine 6.3 6.2 6.6 4.8 5.3 5.7 3.9 
Isoleucine 5.0 5.0 5.4 4.5 4.9 2.2 3.0 
Leucine 8.6 8.4 8.9 7.9 8.4 9.4 5.9 
Total Essential AA 41.1 40.8 42.2 36.1 38.9 39.3 27.1 
Non-essential AA 
Alanine 7.9 8.0 8.0 4.5 4.4 3.0 N.A 
Arginine 6.2 5.8 6.2 7.4 8.7 3.2 N.A 
Aspartic Acid 11.3 11.6 11.2 11.4 11.7 7.1 N.A 
Glutamic Acid 12.5 11.7 12.1 19.9 17.4 22.2 N.A 
Glycine 6.3 6.7 6.1 4.2 4.1 1.9 N.A 
Proline 4.0 3.7 4.0 5.2 4.5 10.4 N.A 
Serine 3.9 4.2 3.9 5.2 5.1 5.7 N.A 
Tyrosine 3.8 4.6 4.0 3.6 4.1 4.8 N.A 
Cysteine + Cystine 0.8 0.5 0.4 1.48a 1.1 0.4 N.A 
Tryptophan 2.1 2.5 2.0 1.2 0.7 1.3 N.A 
Total non-essential AA 58.9 59.2 57.9 62.5 61.1 60.0 N.A 
a Only cysteine was analysed for SPC and PPI. 
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Food Hydrocolloids 151 (2024) 109832
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3.2.3. Fraction composition and protein secondary structure 
The complete ATR-FTIR spectra give an overview of the sample 
fraction composition (Fig. 4A). The interaction of infrared light with 
molecular bonds represents typically the following components: carbo-
hydrates (C–O bond at 900-1150 cm− 1), lipids (CH2 bond at 2800-3000 
cm− 1), proteins (C–O and NH at 1400-1700 cm− 1) and nucleotides 
(1650 cm− 1) (Mosharaf et al., 2018; Nicolaou et al., 2010), although 
multicomponent samples result in superpositions of different signals. 
The spectra confirmed the composition of the fractions described in 
Table 2, except for the acid precipitated sample (AIF). AIF has signifi-
cantly less signal intensity in the amide I (1600-1800 cm− 1) and amide II 
(1470-1570 cm− 1) region (primarily protein) than the other fractions, 
whereas the protein content of all samples (Table 2) is approximately the 
same. It can be assumed that the strong signal of the other fractions is 
caused by the substances with similar absorption to proteins. For 
example, nucleotides have an overlapping absorption at 1650 cm− 1 and 
cell wall fragments not only consist of carbohydrates but also have a 
distinct absorption at 1640 cm− 1 and 1540 cm− 1 due to the presence of 
amides (Jiang et al., 2004). A similar effect was observed for the CH2 
signal, where lipids but also cell wall insoluble lipopolysaccharides give 
a signal (Kim et al., 2005). 
The secondary derivative of the amide I band (Fig. 4B) can be used to 
understand if the protein conformation was altered by the fractionation 
process. Since the protein composition did not change (Fig. 3), these 
variations only reflect the mechanical or chemical treatment of the 
sample. The results show a strong α-helix signal and a low intensity for 
β-sheets for all samples. An absorption intensity shift is seen in the 
intramolecular β-sheets: UB shows a strong signal at 1630 cm− 1, 
whereas all the other signals of the samples were shifted towards a 
higher wavelength (1635 cm− 1). This indicates changes in the hydrogen 
bonds stabilizing the β-sheets, which could be caused by the homoge-
nization treatment applied to all the fractions except UB (i.e. confor-
mational changes caused by the high pressure). No significant protein- 
protein aggregates (intermolecular β-sheets at 1620 cm− 1) are seen for 
the fractions, except for IF (signal at 1625 and 1610 cm− 1), but we 
cannot exclude the occurrence of complexes between proteins and 
polysaccharides or other types of aggregates with FTIR. Probably any 
aggregates with a low solubility end up in the pellet (IF) after centri-
fugation, but we also observed some turbidity in the soluble fraction 
(SF). However, no further analysis of protein aggregates was conducted 
in the current study and their presence cannot be confirmed or excluded 
at this stage. Finally, minor changes were found in the α-helix region, 
with AIF having the lowest signal intensity at 1655 cm− 1. However, the 
α-helix is known to overlap with superimposed signals from, for 
example, nucleotides at 1650 cm− 1. Thus, there were only minor 
conformational differences between the different samples, suggesting a 
limited effect of the processing history on the protein structure. 
3.3. Physico-chemical properties 
3.3.1. Thermograms by differential scanning calorimetry (DSC) 
Thermograms of the different biomass fractions were recorded to 
understand their melting behaviour. The term melting refers to protein 
denaturation as well as to the melting of cell wall components. The heat 
flow changes of UB, DB, SF, IF and AIF fraction are shown in Fig. 5, and 
the melting temperatures (Tm) as well as enthalpies are listed in Table 3. 
Three different, albeit weak endothermic peaks were observed for all 
fractions during heating for the first run.The melting temperature (Tm) 
of the first peak was detected between 70 and 76 ◦C, and 89–98 ◦C for 
the second peak, and 124–126 ◦C for the third peak. Thermograms of 
Gram-negative bacteria report melting of ribosomal proteins between 47 
and 85 ◦C (Mackey et al., 1991). But next to proteins, also the individual 
melting of different cell components might cause some of these peaks: 
the Tm around 95 ◦C and higher could be linked to the melting of cellular 
DNA combined with dehydration and depolymerization of cell wall 
components. It is noteworthy that the observed enthalpies were mostly 
far below 0.8 J/g biomass, which would correspond to less than 1 J/g 
protein and is lower than values found for other processed proteins. 
Depending on the processing history, soy protein isolates with a similar 
protein content gave an enthalpy of 1.3–6.9 J/g protein (Peng et al., 
2020). For pea protein a total enthalpy of 2.2 J/g protein was found 
(Tanger et al., 2022). 
Fig. 3. Protein molecular weight (MW) distribution by SDS-PAGE without reducing agent (A) and with reducing agent (B) for the freeze-dried samples of UB, DB, SF, 
IF and AIF. 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
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Fig. 4. A) ATR-FTIR spectra of the sample fractions UB, DB, SF, IF and AIF and B) second derivative of the amide I band of the same samples. 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
9
Enthalpy changes could be caused by variances in the overall cell 
material composition, given that the enthalpy is strongly dependent on 
the concentration of the individual component. The decreasing of the Tm 
of almost all samples relative to the UB could be caused by either further 
damaging the cell wall components and proteins by the microfluidizer 
treatment (as hypothesized based on the FTIR analysis Fig. 4), or by 
liberating the proteins from the cell wall material. A second temperature 
run was performed with each sample and the first and the third peak 
both disappeared during this second run, indicating that these were 
irreversibly damaged by the heat treatment during the first run. 
3.3.2. Zeta potential and conductivity 
The absolute zeta potential (Fig. 6 A) of all samples is ranging be-
tween |50| and |40| mV. Based on the amino acid composition, the 
charge at neutral pH for free ionizable groups were roughly estimated to 
be around |10| mV and |20| mV (Pace et al., 2009), which is far lower 
than the measured values. Dependent on the carbohydrate composition, 
some bacterial polysaccharides or cells can be strongly negatively 
charged (e.g. |35| mV for Paenibacillus (Boukhelata et al., 2019), |44| 
mV for E.coli bacterial cells (Halder et al., 2015), or in a range of |30| 
and |35| mV for an K. pneumaniae strain (Zając et al., 2023). 
Gram-negative bacteria were found to be more highly charged than 
Gram-positive bacteria probably because of the highly charged lipo-
polysaccharides in the outer membrane layer (Zając et al., 2023). We 
thus assumed that the carbohydrates (e.g. lipopolysaccharides from the 
cell wall) of all samples contributed towards the high negative zeta 
potential. 
The lowest absolute zeta potential (|40| mV) was found for the su-
pernatant (SF) among all the fractions. An explanation could be the 
minor compositional changes of the fractions, as SF had the lowest 
carbohydrate content, and at the same time the highest ash content (6 g/ 
100g) (including salts as counter ions) and the highest fat content (2 g/ 
100g) (i.e., uncharged molecules). Also, the conductivity of SF (8 μS/ 
cm) is significantly higher than for all the other fractions (Fig. 6 B), 
likewise reflecting a higher salt concentration. 
Besides, all the fractions have a higher zeta potential compared to 
various plant proteins, although such results are highly dependent on 
the composition and processing history of the fractions. For plant pro-
tein, soy protein concentrate (SPC) was reported to have a zeta potential 
between |20| and |30| at pH 7 and pea protein isolate (PPI) had a zeta 
potential value between |17| and |25| at pH 7. Besides, it was also found 
that the amount and type of salt added during SPC and PPI fractionation 
directly influenced the zeta potential of the fraction (Gravel et al., 2023; 
Helmick et al., 2021; Liu et al., 2011; Peng, 2021). 
3.3.3. Water holding capacity 
The water holding capacity (WHC) of the different fractions is shown 
in Fig. 6 C. WHC is an important parameter for meat analogue appli-
cation, which links to the juiciness of the structure (Cornet et al., 2021). 
Again, the unprocessed UB fraction has the highest WHC of approxi-
mately 20 g/g dry pellet. Interestingly, a lower WHC was observed for all 
the processed biomass of DB, SF and IF. The WHC of all the biomass 
fractions is in general much higher compared to plant protein sources 
such as rapeseed, soy and lupine protein (less than 10 g/g dry pellet) (Jia 
et al., 2021; Peng, 2021; Peters, 2016a,b) or microalgae (3.1 g/g sample) 
(Waghmare et al., 2016). Specifically, commercial SPC and PPI were 
Fig. 5. A) Heat flow of the sample fractions UB, DB, SF, AIF and IF by heating 
from 20 to 130 ◦C for the first run, and B) heat flow of the sample fractions for 
the second run. 
Table 3 
The onset of melting temperature (onset Tm), melting peak temperature (Tm) and 
melting enthalpy (J/g) with respect to the different fractions of UB, DB, SF, AIF, 
and IF. Values are listed as mean and standard deviation of two independent 
measurements. 
Peak Fractions UB DB SF IFb AIFa 
1st run 
Peak 1 
Onset Tm,1 
(◦C) 
66.9 ±
0.8 
65.1 ±
0.8 
64.8 ±
0.7 
62.3 ±
1.3 
72.6 ±
1.1 
Tm,1 (◦C) 75.8 ±
0.5 
70.4 ±
0.1 
70.8 ±
0.2 
70.0 ±
1.7 
75.9 ±
1.1 
Enthalpy 
(J/g) 
0.5 ±
0.2 
0.2 ±
0.03 
0.2 ±
0.01 
0.8 ±
0.02 
0.06 ±
0.001 
1st run 
Peak 2 
Onset Tm,2 
(◦C) 
90.8 ±
0.6 
89.7 ±
0.01 
89.9 ±
0.1 
88.6 ±
0.4 
83.1 ±
2.4 
Tm,1 (◦C) 97.6 ±
0.3 
94.5 ±
0.4 
94.3 ±
0.1 
93.3 ±
0.1 
89.7 ±
0.9 
Enthalpy 
(J/g) 
0.3 ±
0.01 
0.3 ±
0.02 
0.4 ±
0.01 
0.2 ±
0.01 
0.4 ±
0.05 
1st run 
Peak 3 
Onset Tm,3 
(◦C) 
123.7 
± 1.9 
121.0 
± 0.7 
118.6 
± 0.1 
120.0 
± 0.2 
119.5 
± 0.4 
Tm,1 (◦C) 125.7 
± 0.4 
124.9 
± 0.01 
123.6 
± 0.03 
124.8 
± 0.1 
124.0 
± 0.4 
Enthalpy 
(J/g) 
0.1 ±
0.02 
0.2 ±
0.02 
0.2 ±
0.01 
0.3 ±
0.01 
0.3 ±
0.01 
2nd run 
Peak 4 
Onset Tm,1 
(◦C) 
79.9 ±
0.1 
78.7 ±
0.9 
79.9 ±
0.4 
78.2 78.0 ±
0.1 
Tm,1 (◦C) 83.4 ±
0.1 
83.6 ±
0.2 
83.9 ±
0.04 
82.5 83.6 ±
0.01 
Enthalpy 
(J/g) 
0.04 ±
0.01 
0.1 ±
0.06 
0.04 ±
0.004 
0.05 0.1 ±
0.05 
2nd run 
Peak 5 
Onset Tm,1 
(◦C) 
89.6 ±
0.5 
89.0 ±
0.7 
88.4 ±
0.1 
90.0 N.D 
Tm,1 (◦C) 94.4 ±
0.2 
92.9 ±
1.2 
91.9 ±
0.2 
90.3 N.D 
Enthalpy 
(J/g) 
0.1 ±
0.01 
0.2 ±
0.1 
0.03 ±
0.01 
0.1 N.D 
a For AIF fraction in the first run, an additional peak was found with the onset Tm 
(61.0 ± 1.0 ◦C), Tm (65.5 ± 1.3 ◦C) and enthalpy (0.1 ± 0.01 J/g). 
b During the second run of IF fraction, only a single measurement was per-
formed, thus no standard deviation is known. 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
10
reported to have a WHC of approximately 4 g/g sample. Besides, the 
extraction methods were reported to have a big impact on the WHC, e.g. 
PPI extracted by alkali-isoelectric precipitationis reported to result in a 
lower WHC of 2.4–2.6 g/g sample compared to the commercial PPI. 
The complexity of the biomass composition makes it hard to explain 
the high WHC and the differences between the fractions. The WHC is 
affected by the material composition as well as the mechanic processing 
history. Polysaccharides from cyanobacteria were reported to have a 
comparable high WHC of 20–25 g/g (Gongi et al., 2022, p. 227). Thus, it 
is likely that the high WHC is mainly derived from both the protein and 
polysaccharides. However, other factors play into the decrease of the 
WHC between the UB and all the other fractions as well, for example 
mechanical processing also affects the integrity of the bacterial cell wall 
and further research is recommended to elucidate this. Furthermore, the 
structure of the resulting pellet also influences the amount of water that 
the pellet can hold (Jia et al., 2021; Peters, 2016a,b). Here, a gel like 
pellet was observed after centrifugation, and water was trapped inside 
the gel-like structure. In addition, a high solubility of the biomass was 
observed for all fractions (above 80 %) (in supplementary data Fig. S2). 
3.4. Structuring properties 
3.4.1. Rheological properties with Discovery Hybrid Rheometer 
The viscosity of the different fractions (UB, DB, SF, IF and AIF) are 
shown in Fig. 7A. All fractions appeared as fluid under 15 % DM. The 
viscosity decreased with an increased shear rate, which indicates a 
shear-thinning behaviour under room temperature. Besides, the viscos-
ity of the different fractions are all higher compared to commercial spray 
dried soy protein concentrate (was not highest compared to the other 
fractions, the longest LVE regime indicates that it has the resistant gel 
against deformation upon oscillation. Commercial SPC had a γc of 
slightly higher than 1 % (Peng, 2021). Pea protein fractions prepared by 
two aqueous purification processes were had a γc of 1–10 % (Kornet 
et al., 2021). 
3.4.2. Rheological properties by closed cavity rheometer (CCR) 
CCR is suggested as a tool to understand the rheology behaviour of 
plant protein during thermo-mechanical processing, such as inside an 
extruder (Cornet et al., 2022; Pietsch, 2019). Here the oscillation at 1 % 
strain and 1 Hz frequency was measured for all the freeze-dried biomass 
fractions in CCR. In addition, soy protein concentrate (SPC) with a 
similar protein content of 67 % was used as a reference (denominated as 
“reference”). The complex modulus G* was measured by heating 
(40–150 ◦C) was measured (Fig. 7D) by cooling (150–50 ◦C) (Fig. 7E). 
In general, the heating of all the fractions resulted in a constant re-
gion of a G*-value from 40 to 70 ◦C, followed by an increase from 70 to 
100 ◦C, followed by a decrease from 100 to 135 ◦C and stabilized from 
135 to 150 ◦C. An increase of the G*-value from ~10,000 Pa (70 ◦C) to 
20,000 Pa (100 ◦C) further confirmed heat-induced gelation as indicated 
in Fig. 7B and the denaturation as suggested by the DSC peak 1 and 2 at 
approximately 70 and 90 ◦C (Fig. 5). The G*-value decreased after 
reaching the maximum value at 100 ◦C though the DSC peak was 
observed at 120 ◦C. The decrease of the G*-value at high temperature 
was known to be associated with the degradation of crosslinked struc-
tures (Emin et al., 2017), for example cell wall material. Thus, the 
depolymerization effect was most likely most important in determining 
the rheological behavior. The AIF fraction showed a similar trend, but 
this dispersion had a much higher G*-value compared to the other 
fractions. The result suggest that the acid precipitation enhanced the 
rheological properties, although the composition of this fraction 
remained roughly similar to the other samples. The commercial SPC 
showed a different trend, which started with a steady decrease from 40 
to 120 ◦C, and then largely decreased from 120 to 150 ◦C. Besides, SPC 
showed highest G*-value throughout the whole heating ramp, indicating 
the highest mechanical strength. This value is similar to the reported 
data (Jia et al., 2021). 
Though the SPC showed the highest G*-value during the whole 
heating ramp, the G*-value of SPC during cooling enhanced only to 100 
kPa at the end of cooling (50 ◦C), which was much less compared to AIF 
(250 kPa) as well as DB, SF, and IF (165 kPa). Again, the lowest G*-value 
was found for UB (57 kPa). As previously discussed, this could be due to 
the lower mechanical processing of this fraction, and thus lower initial 
accessibility of the proteins. 
4. The potential of using M. flagellatus for making meat 
analogue product 
The macrostructure of the sheared and heated UB, AIF, and SPC 
samples at 40 % DM using shear cell are shown in Fig. 8. Both UB and 
AIF formed intact pancakes with an umami smell, which had some 
similarities with meat. Some water was found on the surface of the AIF 
sample, whereas hardly any water was found on the UB sample’s sur-
face. The UB sample formed a layered structure broken into pieces when 
taken from the shear cell. For the AIF sample, a pancake like structure 
was formed, which was porous with many holes. Hardly any fine fibrous 
structure was present with the UB and AIF sample, which might be 
associated with the low cysteine content (Table 2). Cysteine is a sulfur- 
containing amino acid, which is known to be associated with the 
network structure formation due to the S–S group crosslinking between 
proteins (Dinani, van der Harst, Boom, & van der Goot, 2023). The SPC 
showed more fibrous structure after the same treatment in shear cell. 
This result is not surprising since SPC has a much higher cysteine content 
(approximately two times more than UB and nearly four times more than 
AIF). Furthermore, SPC is known to contain two immiscible components 
of protein and polysaccharides, which is important for the fibrous 
structure formation (Grabowska et al., 2016). The differences in the 
structure formed for UB and AIF fractions might be explained by the 
differences between their composition, techno-functional properties, 
and processing history. It is noticed that the AIF had a higher carbo-
hydrate and lower protein content compared to UB (Table 1), as well as 
higher viscosity and a higher viscoelastic property (Fig. 7). The microbe 
cells might still be intact for UB due to less strong mechanical treatment 
Table 4 
The G’-, G″-value and Tan δ at the onset of heating, holding regions and end of cooling region by the temperature ramp is recorded for the different fractions (the last 
data point of each region). The critical strain γc during amplitude sweep is determined at the end of the LVE region. 
Fractions Onset heating 20 ◦C (T = 0 min) Holding (95 ◦C) 
(T = 20 min) 
End of cooling 20 ◦C (T = 50 min) Critical strain γc 
G’ (Pa) G’’ (Pa) Tan δ G’ (Pa) G’’ (Pa) Tan δ G’ (Pa) G’’ (Pa) Tan δ % 
UB 315.2 201.8 0.6 825.8 214.2 0.3 9951.9 3563.3 0.4 8.4 
DB 76.3 49.5 0.6 1501.6 313.7 0.2 17816.3 5525.5 0.3 38.7 
SF 7.2 7.2 1.0 1492.0 142.9 0.1 13752.7 3190.9 0.2 44.5 
IF 460.7 273.1 0.6 2066.2 531.9 0.3 16411.0 5662.8 0.3 8.4 
AIF 546.1 201.3 0.4 2974.8 417.5 0.1 19923.0 5179.0 0.3 10.7 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
13
compared to intensively processed AIF fraction, hindering a continuous 
homogenous protein network. 
While the solid appearance of AIF and the layered and crumbly 
texture of UB certainly needs further process optimization steps to 
improve the final texture towards a more stable and fibrous texture, it is 
still promising that some structure can already be formed based on the 
unprocessed biomass. Furthermore, although similar in composition, UB 
and AIF differ in their appearance, which gives them a different appli-
cation range (e.g. UB potentially for meat analogue production and AIF 
as more homogenous gelling agent). It remains to be tested if plant 
protein blends (e.g. from soy or wheat) with the bacterial biomass can 
further improve the texture towards forming meat analogues, by for 
example introducing another phase and/or a higher cysteine 
concentration. 
From a food safety point of view, a meat analogue with the presented 
bacterial biomass and 40 % dry matter would contain 2 g nucleotides/ 
100 g meat analogue (based on Table 1), which is within the 2 g/day (w/ 
w) upper limit recommended by WHO for novel foods from single cell 
protein. But further reduction steps should be evaluated in the future 
(Coelho et al., 2020, p. 1115). Besides, from the consumer acceptance 
point of view, the pinkish colour of the biomass (that turned brown on 
the surface after heating in the shear cell), as well as the umami flavour 
(sausage smell), suits the target product, lessening the need for colour-
ants or artificial flavourings. Nevertheless, a more in-depth analysis of 
the involved pigments needs to be conducted to better understand their 
nature. 
5. Conclusion 
We screened the physicochemical- and rheological properties of 
M. flagellatus unprocessed biomass and different fractions. Overall, a 
high protein content of 73 %, very high zeta-potential and high-water 
holding capacity was evident. Thus, we observed promising structural 
and rheological properties for food applications, further supported by 
the pleasant smell and colour of the biomass fractions. In particular, the 
unprocessed biomass of M. flagellatus showed properties suitablefor 
meat analogues and should be further investigated. 
Interestingly, the various processing steps applied for protein frac-
tionation and removal of nucleotides had a significant effect on the 
rheological properties and structure formation of the unprocessed 
biomass versus acid precipitated fraction. However, there were only 
minor changes in the overall composition (such as protein and nucleo-
tides), primarily with respect to carbohydrate content. Since we could 
not find a clear correlation between the rheological properties and the 
carbohydrate content, we thus assume that either the individual car-
bohydrate composition and/or the fractionation and mechanical/ 
chemical treatment modified the rheological properties of the fractions. 
Next to that, a negative effect of salt on the rheological properties 
seemed evident. The presented results were an explorative study to 
assess the functional potential of microbial biomass M. flagellatus for 
future applications. Further research is still required to better explain 
the underlying phenomena and modulate the material and composition. 
Future research should also address food safety aspects, including 
confirmation that no traces of methanol feedstock is left in the biomass. 
We note that we do not expect any methanol in the biomass because it is 
probably all consumed during cultivation, and possible traces can be 
easily removed due to its volatility. While it is not known as a pathogen, 
studies are needed to confirm the safety of this promising microbial 
protein source for food application as well as its taste. In addition, 
regulatory approval is needed to apply M. flagellatus as a food ingredient. 
CRediT authorship contribution statement 
Wanqing Jia: Writing – original draft, Methodology, Formal anal-
ysis, Data curation, Conceptualization. Laurice Pouvreau: Writing – 
review & editing, Supervision, Funding acquisition, Conceptualization. 
Fig. 8. Structuring properties of freeze-dried UB (A1, A2 and A3), AIF (B1, B2 and B3) and SPC (C1 and C2) (dry matter of 40 %), corresponding to a protein 
concentration of approximately 28 % in the shear cell. 
W. Jia et al. 
Food Hydrocolloids 151 (2024) 109832
14
Atze Jan van der Goot: Writing – review & editing. Timotheus Y. 
Althuis: Writing – review & editing, Investigation. David Virant: 
Writing – review & editing. Aleksander J. Kruis: Writing – review & 
editing. Gregor Kosec: Writing – review & editing. Nico J. Claassens: 
Writing – review & editing, Supervision, Funding acquisition, Concep-
tualization. Julia K. Keppler: Writing – original draft, Supervision, 
Funding acquisition, Formal analysis, Conceptualization. 
Declaration of competing interest 
The authors declare that they have no known competing financial 
interests or personal relationships that could have appeared to influence 
the work reported in this paper. 
Data availability 
Data will be made available on request. 
Acknowledgement 
This project was funded by the Protein Transition Investment Theme 
of Wageningen University. Bacterial biomass was kindly provided by 
Acies Bio (Ljubljana, Slovenia). The authors would like to thank Pan-
agiotis Voudouris (WFBR) for the support for the CLSM images. The 
authors also would like to thank Frederique Catsman and Aleksandra 
Pawlik (WFBR) for the support of the rheology measurements. The 
graphical abstract was made in BioRender.com. 
Appendix A. Supplementary data 
Supplementary data to this article can be found online at https://doi. 
org/10.1016/j.foodhyd.2024.109832. 
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