Cryobiology 105 (2022) 1–9

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Cryobiology
journal homepage: www.elsevier.com/locate/cryo

Effects of freeze drying in complex lyoprotectants on the survival, and

membrane fatty acid composition of Lactobacillus plantarum L1 and
Lactobacillus fermentum L2
Ziyi Cheng, Xu Yan, Jingyi Wu, Peifang Weng, Zufang Wu *
College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315832, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of this study was to investigate the effects of freeze-drying protective agents on the viability, survival
Lactic acid bacteria and membrane fatty acid composition of Lactobacillus plantarum L1 and Lactobacillus fermentum L2. Cell survival
Freeze-drying rates of Lactobacillus plantarum L1 after freeze-drying without any additives was 6.57% (control group), 37.4%
Complex protective agents
with a single protective agent, as compared to 97.4% when L. plantarum L1 was freeze-dried in a solution of four
Unsaturated fatty acids
Cell enzyme activity
protectants (10% skim milk, 13% sucrose, 2% sorbitol, and 0.8% tyrosine (p < 0.05).) The L. fermentum L2 strain
had the highest survival rate 92.3% when was freeze-dried in a solution containing 10% skim milk, 7% trehalose,
2% sorbitol and 0.6% tyrosine (p < 0.05). Freeze drying in the presence of all four protective agents maintained
cell membrane integrity, as determined by reduced leakage of β-galactosidase and LDH, and increased ATPase
activity. LAB Incubation and freeze drying in the complex protective solution increased the content of unsatu­
rated fatty acids in the cell membrane such as oleic acid (C18:1) and C19cyc11 and it is speculated that this may
correlate with the improved outcomes.

1. Introduction
Probiotics are defined as ‘live microorganisms which confer a health
benefit on the host when administered in adequate amounts’ [1]. Lactic
acid bacteria (LAB) are the most common probiotics, and have been
widely used and are considered safe for human and animal consumption
[2,3]. LAB are also used as a starter because they improve the flavour of
foods and increase the nutritional value of foods. However, traditional
liquid starters are difficult to preserve and transport, which greatly af­
fects the production of fermented products, while freeze-dried LAB can
make up for these limitations [4]. Freeze drying gives LAB higher sur­
vival rates than spray-drying or fluidized bed drying [5,6]. However,
freeze-drying does damage microbial cell membranes and DNA, dena­
ture proteins (including enzymes) and can lead to cell death [7,8].
The current work was interested evaluating several protective agents
to improve the survival rate of LAB starters. A good protectant should be
non-toxic, easy to use and provide both cryo- and lyoprotection [9]. The
freeze-drying process can lyse membranes which lets enzymes escape
into the external environment and also have adverse effects on the ac­
tivity of LAB enzymes, such as β-galactosidase, lactate, dehydrogenase
and ATPase [10]. Enzymes are therefore important indicators of
bacterial vitality [11]. Freeze-drying also causes changes in the fatty
acid content of LAB cell membranes [12,13,14]. Wang et al. [15]
showed that the viability of L. plantarum after freeze-drying was posi­
tively correlated with the relative concentration of octadecenoic acid
(C18:1) and the ratio of unsaturated to saturated fatty acids (U/S).
Various substances including polyols, disaccharides, amino acids, pro­
teins, minerals and vitamins-complex media have been tested for pro­
tective effects [16,17]. Zayed et al. [18] showed that trehalose + sucrose
in addition to skim milk were the most efficient materials for Lactoba­
cillus salivarius, giving a survival rate of 83–85% after freeze-drying.
Chen et al. [19] found that skim milk powder, lactose, and sodium
ascorbate had a significant impact on variables and survival of cultures
after freeze-drying. On this basis, we investigated the synergistic effect
of the combination of various protective substances on Lactobacillus
plantarum L1 and Lactobacillus fermentum L2. For each laboratory strain,
the effect of the protectant on survival in the dry state may be unique.
The strains used in our study are Lactobacillus plantarum L1 and Lacto­
bacillus fermentum L2, which were isolated from pickled vegetables and
used in the fermentation of huyou juice and were supported to have
probiotic properties (unpublished observations). However, during
freeze-drying processes, the influences of protective agents on the

* Corresponding author.
E-mail address: wzfwpf@163.com (Z. Wu).

https://doi.org/10.1016/j.cryobiol.2022.01.003
Received 25 March 2021; Received in revised form 16 January 2022; Accepted 17 January 2022
Available online 20 January 2022
0011-2240/© 2022 Elsevier Inc. All rights reserved.
Z. Cheng et al.
Lactobacillus plantarum L1 and Lactobacillus fermentum L2 of our labo­
ratory have not been elucidated so far. In this study, we investigate the
influence of some protective agents on the freeze-drying outcomes for
the two strains of LAB including the influence of single versus combined
protective agent(s) on enzyme activities and of combined protectants on
cell membrane fatty acid composition.
2. Materials and methods
2.1. Strains
The study was performed using Lactobacillus plantarum L1 (Genbank:
MZ674414) and Lactobacillus fermentum L2 (Genbank: MZ674413),
which were isolated from pickled vegetables and maintained in the Food
Biotechnology Laboratory at Ningbo University, China.
2.2. Culture media and growth conditions
Based on previous studies (unpublished observations), L. plantarum
L1 was initially cultured in corn syrup broth medium composed of 4%
maltose, 0.4% tryptone, 10% corn syrup, 0.2% dipotassium hydrogen
phosphate, 0.1% Tween-80 and 0.5% sodium acetate. L. fermentum L2
was initially cultured in optimized deMan, Rogosa, Sharpe broth
(Sinopharm Chemical Reagent Co.,Ltd., Shanghai, China) composed of
5% maltose, 0.1% soy peptone, 15% corn syrup, 0.2% diammonium
hydrogen citrate, 0.1% Tween-80, 0.5% sodium acetate, 0.2% dipotas­
sium hydrogen phosphate, 0.058% magnesium sulfate, and 0.025%
manganese sulfate. All broth was sterilized by autoclaving at 121 ◦ C for
20 min before use. Both strains were statically cultured in a 37 ◦ C
constant temperature incubator. Growth was stopped after 18 h at the
end of the exponential phase and cells were harvested.
2.3. Protectants used in assays
The additives tested as protective agents were prepared in distilled
water and divided into four groups: (i) Skim milk as a basic protective
agent at a concentrations of 6%，8%，10%，12% and 14%; (ii) Sugars:
trehalose, lactose and sucrose at a concentration of 10%; (iii) Polyols:
glycerin, sorbitol and Tween-80 at a concentration of 3%; (iv) amino
acids (0.8%): tyrosine and L-glutamic acid at a concentration of 0.8%; (v)
Sterile distilled water served as the control. Skim milk was sterilized by
autoclaving at 115 ◦ C for 10 min before use. Sugars and amino acids
were filtered sterilized with 0.22 μm filter. Polyols and sterile distilled
water were sterilized by autoclaving at 121 ◦ C for 20 min.
2.4. Cell preparation and freeze-drying
Cells were concentrated by centrifugation (20 ◦ C, 7000×g for 20
min) and resuspended in 50 mM phosphate buffer (pH 6.8) for washing.
The bacterial suspension was then made by adding the protectants at a
ratio of 1:1 (w/v) between the bacterial precipitate and the protectant to
achieve the final concentration of the selected protectant solution. After
an equilibration time of 20 min at 20 ◦ C, each solution was added to 90-
mm glass petri dishes which were then placed in a freezer at − 80 ◦ C for
4 h, then freeze dried in a SCIENTZ-18 N, freeze-dryer (Ningbo scientz
biotechnology Co., Ltd, China) at a condenser temperature − 55 ◦ C and
at a chamber pressure of <0.06 mbar for 24 h.
2.5. Rehydration
After freeze-drying, the cells were immediately resuspended to their
original volume of bacterial culture (40 mL) with normal saline (0.85%).
The saline was added rapidly and with stirring gently. Then, the mixture
of bacterial cells and normal saline was shaken for 15 min to mix evenly
at room temperature (about 25 ◦ C).
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Cryobiology 105 (2022) 1–9
2.6. Determination of cell counts
Before centrifugation (mentioned in Section 2.4), a cell sample was
taken from each suspension and the number of Colony Forming Units
(cfu) was determined by the plate dilution method [20] using MRS agar
medium at 37 ◦ C for 48 h. The same method for cell counts was used
after rehydration. All experiments were carried out in triplicate.
cfu ​ after ​ freeze − drying
Survival ​ rate ​ (%) ​ = ​ × 100%
cfu ​ before ​ freeze − drying
2.7. Cell free extract and extracellular supernatant preparation
After rehydration and the removal of a sample for cell colony
counting, the remaining cells were harvested by centrifugation
(10,000×g, 4 ◦ C, 10 min), and the supernatant was used for the deter­
mination of extracellular enzymes. The effect of the protectants in the
supernatant on the extracellular enzyme activity was present but small,
so it may be possible to ignore this interference. The cells were washed
twice with 50 mM phosphate buffer (pH 6.8) and then resuspended in 4
mL of the same buffer. To prepare the cell free extract, lysozyme (BBI) at
a concentration of 10 mg mL− 1 was added and incubated at 37 ◦ C for 15
min, followed by addition of 0.5 mL of 4 M NaCl solution and incubation
37 ◦ C for another 50 min. The cell suspension was then centrifuged at
10,000×g for 10 min. Resultant supernatant was used for enzyme assay
and protein determination.
2.8. Measurement of β-galactosidase
The β-galactosidase activity assay was carried out as described by
Splechtna et al. [21] and Sangwan et al. [22] with some modifications.
The chromogenic substrate o-nitrophenol-β-D-galactopyranoside
(ONPG, BBI) (3.125 mg mL− 1) was dissolved in 50 mM sodium phos­
phate buffer (pH 6.8). The cell free extract was equilibrated in a constant
temperature water bath at 37 ◦ C for 15 min before use. Then 1 mL of
sample was mixed with 5 mL of ONPG solution and incubated for 10 min
at 37 ◦ C ± 0.5 ◦ C. After 10 min the reaction was terminated by the
addition of 2 mL of 1 M Na2CO3 to the reaction mixture. In the control
group, Na2CO3 was first added then we added ONPG substrate. The
other steps were the same as before. The amount of o-nitrophenol
released was measured at 420 nm. One unit of enzyme activity (U) was
defined as the quantity of enzyme that would liberate 1 μmol of
o-nitrophenol (ONP) from ONPG per min under the assay conditions.
The enzyme assay was performed in triplicate.
2.9. Measurement of ATPase
The ATPase activity was measured with an ATPase assay kit (Nanjing
Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China). Reagents
and five centrifuge tubes A, B, C, D and E (control tubes, Na+k+, Mg2+,
Ca2+ and Ca2+Mg2+ tubes respectively) were prepared according to the
instructions. Samples and reagents were added to each tube according to
the operation steps. Enzymatic reaction and phosphorus determination
were carried out sequentially. Finally, the absorbance of the sample was
measured at 660 nm. The assays were performed in triplicate.
2.10. Measurement of lactate dehydrogenase
Lactate dehydrogenase (LDH) activity was determined using LDH
assay kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing,
China). Reagents and four centrifuge tubes A, B, C and D (blank tube,
standard tube, measuring tube and control tube respectively) were
prepared according to the instructions. The measurement method was
carried out according to the instructions. One unit of enzyme activity is
defined as 1 μmol pyruvate generated in the reaction system when each
gram of tissue protein interacts with the substrate at 37 ◦ C for 15 min.
Z. Cheng et al.
Fig. 1. Survival rates and number of viable bacteria after freeze-drying in solutions containing skim milk made up in sterile water. (a) Data for L. plantarum L1 and
(b) data for L. fermentum L2. Cells after freeze-drying were used with adding sterile water. Data are the means and standard deviations of 3 independent experiments.
Error bars indicate standard deviations (n = 3).
Finally, the absorbance of the sample was measured at 440 nm. The
experiments were performed in triplicate.
2.11. Analyses of fatty acid composition
After freeze-drying, the relative fatty acid composition of bacterial
membranes was determined by gas chromatography using the method
described by Bligh & Dyer [23]. Cells were washed three times in sterile
deionized water. After discarding the supernatant, 1.9 mL of
chloroform-methanol solution (Chloroform: Methanol 1:2 v/v, Sino­
pharm Chemical Reagent Co., Ltd) was added and shaken vigorously for
15 min. Then we added 0.625 mL chloroform and 0.625 mL sterile
deionized water, shaking thoroughly in a short time, followed by
centrifugation for 10min (4 ◦ C, 7000×g). Once the solution was strati­
fied, the lower liquid phase was absorbed and transferred to sterile
centrifuge tubes. Methylation and extraction were performed simulta­
neously at 4 ◦ C by adding 1 mL of sodium methoxide (1 M) in methanol,
(Sinopharm Chemical Reagent Co., Ltd) followed by shaking for 5 min
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Cryobiology 105 (2022) 1–9
and then adding 0.625 mL hexane (Sinopharm Chemical Reagent Co.,
Ltd). The solution was then centrifuged for 5 min (4 ◦ C, 7000×g), and
the upper phase removed, filtered by filter and stored at − 80 ◦ C in an
airtight glass bottle until analysis.
Analyses were performed on a gas chromatograph (7890 B–7000C,
Agilent, American) equipped with an automatic solid phase micro­
extraction sampling system (Gerstel, Germany) and connected to a solid
phase microextraction (DVB/CAR/PDMS, Supelco, USA). A capillary
column packed with polyethylene glycol (24217-U, 60 m * 0.32 mm *
1.8 μm, CNW, Germany) was employed. Helium was used as the carrier
gas (1.6 mL min− 1), and the injection volume was 2 μL. Injection was
done splitless for 2 min. Oven initial temperature was 35 ◦ C, raised to
240 ◦ C at 5 ◦ C min− 1, held 12 min at 240 ◦ C. Injection and detection
temperatures were 230 ◦ C. The fatty acid methyl esters were identified
by using a mass selective detector (Agilent 5973, Hewlett Packard) at a
scan rate of 273 scan s− 1. The electron impact energy was set at 70 eV,
and data were collected in the range of 50–500 atomic mass units. Re­
sults were expressed as relative percentages of each fatty acid that were
Fig. 2. Survival rates and number of viable
L. plantarum L1 following freeze-drying in
sterile water with 10% skim milk powder
and one additional protectant compound.
(a) Data for 10% milk powder and 10%
carbohydrate or 3% polyols or 0.8% amino
acids (b) data for 10% milk powder and
differing sucrose concentrations (c) data for
10% milk powder and differing sorbitol
concentrations (d) data for 10% milk pow­
der and differing sorbitol concentrations.
Vertical bars indicate standard errors; col­
umns with different letters indicate signifi­
cant differences (p < 0.05).
Z. Cheng et al.
calculated as the ratio of the surface area of the considered peak on the
total area of all peaks. Analyses were done in duplicate.
2.12. Statistical analyses
Statistical analyses were performed using SAS (SAS Institute Inc.,
Cary, NC, USA) using one-way ANOVA comparing all study groups.
Pearson correlation analysis was performed by SPSS (SPSS Inc., Chicago,
IL, USA, V 17.0.0). Values of p < 0.05 were considered as significant.
3. Results and discussion
3.1. Effect of skim milk alone on the survival rate of lyophilized
L. plantarum L1 and L. fermentum L2
Skim milk is a widely used protective agent for freeze-drying bac­
teria. It is generally thought to protect by reducing damage to the cell
membrane [24]. Tan et al. [25] showed that skim milk could stabilize
cell membrane components and provide a protective layer of proteins. It
cannot enter the cell membrane of intact bacteria but it can alter vis­
cosity and ice crystal formation [26]. In our study of L. plantarum L1 and
L. fermentum L2, 10% skim milk protected survival rate and viability
better than either higher or lower concentrations (Fig. 1a, Fig. 1b). The
survival rates of L. plantarum L1 and L. fermentum L2 in 10% skim milk
were 37.4% and 46.6%, respectively as compared to the control group
without skim milk which was only 6.57% and 8.35%, respectively. This
is in agreement with e.g. Archacka et al. [27] who obtained high survival
rates of freeze-dried (64.5 ± 0.5%) Lactococcus lactis cells with 10% skim
milk as the protective agent. Facco et al. [28] observed that skim milk
was the best protectant for the yeast W. anomalus IAL 4533. In our study,
the survival rates of LAB decreased at both lower and higher concen­
trations of skim milk. High osmotic pressure and rapid changes in os­
motic pressure can also damage the cell membrane of the bacteria,
thereby reducing survival rate [29,30]. As a single freeze-dried protec­
tive agent, skim milk was not sufficiently protective to be of use as an
LAB starter in the food industry. It is necessary to add other substances
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Cryobiology 105 (2022) 1–9
Fig. 3. Survival rates and number of viable
L. fermentum L2. Following freeze-drying in
sterile water with % skim milk powder and
one additional protectant compound. (a)
Data for 10% milk powder and 10% carbo­
hydrate or 3% polyols or 0.8% amino acids.
(b) Data for 10% milk powder and differing
sucrose concentrations (c) data for 10% milk
powder and differing sorbitol concentrations
(d) data for 10% milk powder and differing
tyrosine concentrations. Vertical bars indi­
cate standard errors; columns with different
letters indicate significant differences (p <
0.05).
Fig. 4. Survival rates and number of viable bacteria after freeze-drying in so­
lutions containing a combination of milk, sugar, polyol and amino acid A) data
for L. plantarum L1 in sterile water with 10% skim milk, 13% sucrose, 2%
sorbitol and 0.8% tyrosine and B) and L. fermentum L2 in a solution containing
10% skim milk, 7% trehalose, 2% sorbitol and 0.6% tyrosine in sterile water.
Vertical bars indicate standard errors; Data are the means and standard de­
viations of 3 independent experiments.
to form complex protective agents.
3.2. Effects of complex protective agent on L. plantarum L1 and
L. fermentum L2 survival rate during lyophilization
In this study, carbohydrates (trehalose, lactose, sucrose), polyols
(glycerin, Tween-80, sorbitol) and amino acids (L-glutamic acid, tyro­
sine) were evaluated in combination with skim milk as protective agents
for L. plantarum L1 and L. fermentum L2 as shown in Fig. 2 and Fig. 3. The
results showed that when bacteria were freeze dried in 10% skim milk
supplemented with a single additive (Figs. 2a and 3a) then the most
protective of the sugars was sucrose for L. plantarum and trehalose for
Z. Cheng et al. Cryobiology 105 (2022) 1–9

Fig. 5. The intracellular and extracellular β-Galactosidase activity of L. plantarum L1 and L. fermentum L2 with different protective agents. Vertical bars indicate
standard errors; columns with different letters indicate significant differences (p < 0.05).

L. fermentum. As all experiments were conducted with 10% skim milk as
the basic protectant, the lactose content of the skim milk had no effect on
the final results of the screening experiments for the sugar protectant.
Both species gained most protection from the same polyol (sorbitol) and
amino acid (tyrosine). When solutions contained 10% skim milk but
only one additional component the protection was best with (13% su­
crose or 2% sorbitol or 0.8% tyrosine for L. plantarum L1) and (7%
trehalose or 2% sorbitol or 0.6% tyrosine for L. fermentum L2). Solutions
containing skim milk and three other protectants, the survival rates of L1
and L2 after freeze-drying were 97.4% and 92.3% with 3.08 × 1011 cfu/
mL and 5.23 × 1010 cfu/mL, viable cells respectively (Fig. 4).
The various components in the complex protective agent play their
own roles and have a synergistic effect in freeze-drying. Due to the
differences in the structure and size of microbial cells, the best protec­
tion effect can be achieved when the ratio and concentration of each
substance in the complex protective agents are coordinated. Organic
compound such as carbohydrates, polyols and amino acids are widely
used as high-efficiency protective agents in the production process of
LAB starters [31]. The hydroxyl groups of sugars or alcohols can
hydrogen bond with the phosphate groups of the lipids, thus effectively
replacing the hydrogen bonds that previously existed between the
phosphate groups and water molecules to protect the structure and
function completeness of cell membrane and protein [32]. The protec­
tive effect of disaccharides has been widely studied as due to stabiliza­
tion of cell membranes and prevention of intracellular ice-formation
[33,34]. The positive effect observed for skim milk and trehalose on the
viability of the two strains after freezing had also been reported in a
previous studies [35]. A study by Sae-Byuk Lee et al. [24] found that
freeze-dried L. plantarum JH287 cells with 10% sorbitol and 10% sucrose
survived better under harsh environments than the others. Sorbitol may
protect cells due to its free radical scavenging activity [36]. An increase
in the survival of freeze-dried species of Geotrichum candidum was re­
ported by Hamoudi et al. [37] using disaccharides such as trehalose and
sucrose at a concentration of 23%. Miao et al. [38] studied the
freeze-drying protective effect of different carbohydrates on Lactoba­
cillus rhamnosus and found that its survival rate was 99%~100% when
using trehalose. From the perspective of kinetics, trehalose is very
effective in promoting the formation of amorphous or glassy solids,
reducing the formation of ice crystals and the temperature at which
lipids bilayers transfer from the liquid crystal phase to the gel phase
which plays a protective role [39].
Most amino acids exist in the form of ions in the aqueous solution or
in the crystal state, and have strong hydrophilicity. The amino and
carboxyl groups of amino acids interact with water molecules to form
hydrogen bonds during the cooling process, which increases the vis­
cosity of the solution, so as to reduce cell damage [40]. Morichi [41]
believes that the reaction between the carboxyl group of the bacterial
protein and the amino group of the amino acid material stabilizes the
protein structure. Amino acids such as tyrosine and proline can enter the
cell and act as an intracellular buffer [42]. Research by Mattern et al.
[43] showed that phenylalanine and glycine can prevent protein
degradation during vacuum freeze-drying. Zhang et al. [44] showed a
significant survival improvement to 86.6% when glutathione (GSH) plus
vitamin C was added as cryo- and lyo-protectants. In the study by Facco
[28] on L. fermentum, adding 5% sodium glutamate allowed the recovery
of 100% of the bacterial cells after lyophilization. However, Jiang et al.
[45] reported that the addition of amino acids such as glutathione and
tyrosine did not significantly improve the freeze-dryingoutcomes of
Bifidobacterium breve. Taken together, these conclusions showed that
different microorganisms including various species have different
optimal conditions. Differences in genetic makeup, cell wall and mem­
brane composition may have contributed to the differences between

Fig. 6. The intracellular and extracellular Lactate dehydrogenase (LDH) activity of L. plantarum L1 and L. fermentum L2 with different protective agents. Vertical bars
indicate standard errors; columns with different letters indicate significant differences (p < 0.05).

5
Z. Cheng et al. Cryobiology 105 (2022) 1–9

Fig. 7. The ATPase activity of L. plantarum L1 and L. fermentum L2 with different protective agents. Vertical bars indicate standard errors; columns with different
letters indicate significant differences (p < 0.05).

strains [46].
Table 1
Cell membrane fatty acid composition of freeze-dried L. plantarum L1 with
3.3. Effect of different protective agent on cell enzyme activity of different freeze-dried protective agents.
L. plantarum L1 and L. fermentum L2 after freeze-drying
Fatty acids No protective Single protective Compound
species (%) agent agent protective agent
The activity of β-glucosidase, lactate dehydrogenase and ATPase of
C11:0 1.21 ± 0.12b 5.11 ± 0.07a 5.1 ± 0.08a
freeze-dried L. plantarum L1 and L. fermentum L2 were shown in Fig 5–7. C14:0 5.78 ± 0.13a 5.51 ± 0.03a 1.56 ± 0.11b
With the addition of protective ingredients, the activities of intra and C16:0 50.51 ± 0.78a 43.17 ± 0.34b 24.54 ± 0.29c
extra cellular β-galactosidase and LDH of L. plantarum L1 and C16:1 ND 1.01 ± 0.025b 1.96 ± 0.096a
L. fermentum L2 maintained better than those freeze-dried bacteria C18:0 15.19 ± 0.14a 7.64 ± 0.07b 5.85 ± 0.12c
C18:1 12.52 ± 0.13c 14.5 ± 0.18b 25.58 ± 0.1a
without protection. β-Galactosidase is a key enzyme in the process of
C18:2n6c 3.38 ± 0.06a 1.54 ± 0.027b 0.96 ± 0.06c
lactose metabolism to help LAB exert its probiotic function [47]. It can C18:3 0.91 ± 0.03c 3.11 ± 0.06b 5.95 ± 0.05a
decompose lactose into galactose and glucose, which means that its C19cyc11 ND 5.01 ± 0.08b 17.63 ± 0.12a
activity is one of the factors that reflect the activity of the starter. LDH is C21:0 1.43 ± 0.02b 5.21 ± 0.032a 5.61 ± 0.024a
a significant enzyme that catalyzes the reduction of pyruvate to lactic UFA/SFA 0.227 ± 0.01c 0.41 ± 0.022b 1.221 ± 0.013a
Survival rate (%) 6.57c 31.42b 97.34a
acid, and its activity reflects energy metabolism and the strain’s ability
to produce acid [48]. The cell membrane damage can be judged by Note: a,b and c represented significant differences in cell membrane fatty acids
measuring the activity of β-galactosidase and LDH exuded from the cell. in the three groups. (p < 0.05). ND = not detected. Single protective agent refers
The extracellular β-galactosidase and LDH levels indicated that the to 10% skim milk. No protective agent means that distilled water replaces the
protective agent.
addition of the protective agent reduced the permeability of the cell
membrane, reduce the leakage of intracellular enzymes, and improved
the viability of LAB. The milk proteins of skim milk may have protected
Table 2
by forming a protein film outside the bacteria, immobilizing freeze-dried Cell membrane fatty acid composition of freeze-dried L.fermentum L2 with
enzymes and effectively prevent the leakage of intracellular substances different freeze-dried protective agents.
caused by cell wall protein damage [49]. In addition, the disaccharides
Fatty acids No protective Single protective Compound
as substrates of glycosidase may improve the stability of the enzyme by species (%) agent agent protective agent
binding to the enzyme or inducing the activity of the enzyme [50]. The
C12:0 1.75 ± 0.05a ND ND
activity of ATPase is directly related to the life activities of biological C14:0 4.75 ± 0.08c 5.97 ± 0.08b 6.68 ± 0.11a
cells. It can be seen from Fig. 7 that the ATPase activity of each group C14:1 4.56 ± 0.11a 1.29 ± 0.02b ND
with the protective agent is higher than that of the control group, and C16:0 42.48 ± 0.14a 40.01 ± 0.25a 24.03 ± 0.18b
the group with the complex protective agent has the highest ATPase C16:1 14.33 ± 0.11a 12.52 ± 0.17c 13.34 ± 0.07b
C18:0 13.67 ± 0.08a 12.01 ± 0.11b 7.71 ± 0.12c
activity. Na+-K+-ATPase is a protein in the bilayer of the cell plasma
C18:1 3.37 ± 0.04c 6.94 ± 0.037b 15.57 ± 0.18a
membrane which acts as a carrier and an enzyme. It catalyzes ATP hy­ C18:2 ND 5.35 ± 0.065a 1.3 ± 0.022b
drolysis for energy supply, maintains membrane potential on both sides C18:3 ND ND 10.76 ± 0.12a
of the cell membrane, regulates cell osmotic pressure and provides C19cyc11 ND ND 8.35 ± 0.39a
power for nutrient absorption [51]. Castro et al. [52] found that the ratio C22:1n9 ND 3.09 ± 0.62b 4.02 ± 0.053a
UFA/SFA 0.37 ± 0.034c 0.49 ± 0.01b 1.39 ± 0.043a
of K+ and Na+ in the cells of Lactobacillus bulgaricus without added
Survival rate (%) 8.67c 30.17b 92.31a
protective agent changed after freeze-drying, indicating that the cell
membrane permeability of the strain was damaged after freeze-drying, Note: a,b and c represented significant differences in cell membrane fatty acids
in the three groups. (p < 0.05). ND = not detected. Single protective agent refers
and the transfer of ions inside and outside the cell occurred.
to 10% skim milk. No protective agent means that distilled water replaces the
Ca2+-Mg2+-ATPase is an important substance that drives the regulation
protective agent.
of ion transport inside and outside the membrane, maintains cell os­
motic pressure and transmits cell signals [53].
Compared with a single 10% skim milk protective agent, the complex
protective agent with three additional protectants (sugars, polyols,
amino acids) was more effective, presumably due to synergistic effects.
These results are in agreement with those obtained with Gluconaceto­
bacter xylinus [54], as similar changes were found in addition with

6
Z. Cheng et al. Cryobiology 105 (2022) 1–9

Table 3
Correlation analysis of main membrane fatty acids and freeze-drying survival rate of L. plantarum L1 under complex protective agents.
C11:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2n6c C18:3 C19cyc11 C21:0 UFA/SFA Survival rate (%)

C11:0 (1) 1
C14:0 (2) − 0.547 1
C16:0 (3) − 0.717 0.976 1
C16:1 (4) 0.874 − 0.885 − 0.965 1
C18:0 (5) − 0.983 0.691 0.832 − 0.948 1
C18:1 (6) 0.615 − 0.996 − 0.991 0.921 − 0.749 1
C18:2n6c (7) − 0.973 0.726 0.859 − 0.963 0.999* − 0.781 1
C18:3 (8) 0.826 − 0.924 − 0.985 0.996 − 0.915 0.953 − 0.934 1
C19cyc11 (9) 0.718 − 0.975 − 1.000** 0.966 − 0.833 0.99 − 0.86 0.985 1
C21:0 (10) 0.996 − 0.619 − 0.776 0.913 − 0.996 0.683 − 0.99 0.873 0.777 1
UFA/SFA (11) 0.625 − 0.995 − 0.992 0.926 − 0.757 1.000** − 0.789 0.956 0.992 0.692 1
Survival rate (%) (12) 0.633 − 0.994 − 0.994 0.93 − 0.764 1.000* − 0.795 0.959 0.998* 0.699 1.000** 1

Note：*p < 0.05 **p < 0.01.

0.575% glutamic acid + 0.075% trehalose + 6.4% skim milk as a pro­
tective agent.
3.4. Effect of different protective agents on membrane fatty acid
composition of L. plantarum L1 and L. fermentum L2 after freeze-drying
The fatty acid components of the cell membrane play a role in
maintaining the integrity of the cell membrane and regulating the ac­
tivity of membrane proteins [55]. The relative percentages of the
membrane fatty acids of L. plantarum L1 and L. fermentum L2 were
determined for cells placed with different protective agents during
freeze-drying (Table 1, Table 2). A total of ten and eleven different fatty
acids representing more than 85% of the total fatty acid contents, were
observed in the membranes of L. plantarum L1 and L. fermentum L2,
respectively. As seen in Table 1, significant differences in the contents of
some membrane fatty acids appeared, depending on types of protective
agents. For L. plantarum L1, the percentages of palmitoleic acid (C16:1),
oleic acid (C18:1), linolenic acid (C18:3), lactobacillic acids (C19cyc11,
here considered an unsaturated fatty acid) were obviously higher (p＜
0.05), whereas palmitic acid (C16:0), stearic acid (C18:0) relative con­
tents were significantly lower (p < 0.05) in cells treated with different
protective agents (single 10% skim milk protective agent and complex
protective agent and no protective agents control). For L. fermentum L2,
the percentages of oleic acid (C18:1), linolenic acid (C18:3), lactoba­
cillic acids (C19cyc11), erucic acid (C22:1n9) were obviously higher (p
＜0.05), whereas myristoleic acid (C14:1), palmitic acid (C16:0), stearic
acid (C18:0) relative contents were significantly lower (p < 0.05). These
results led to the calculation of the ratios between unsaturated and
saturated fatty acids (U/S) (Table 1, Table 2). For both strains, the ratio
of unsaturated fatty acid and saturated fatty acid (UFA/SFA) was highest
added with complex protective agents during freeze-drying compared
with the control group or single protective agents. The freeze-drying
survival rate of LAB is closely related to the composition of cell mem­
brane fatty acids [56]. It is found from the Tables that the fatty acid
unsaturation (U/S value) of the cell membrane after adding with the
complex protective agent is 1.22 and 1.39, which are 5.37 and 3.76
times that of no protective agent, and 2.98 and 2.84 times adding with a
single protective agent, respectively. The increase of unsaturated fatty
acid content in cell membrane may improve the cell membrane fluidity
and the freeze-drying survival rate [57]. Therefore, the higher
freeze-drying survival rate of the complex protective agents group may
be related to the increase of the unsaturated fatty acid content of the cell
membrane [58]. This showed that the complex protective agents could
greatly maintain the unsaturation of the cells and the fluidity to reduce
cell activity or even death due to external conditions during the
freeze-drying process. The unsaturation index of fatty acids determines
the viscosity and thickness of the cell membrane. High content of un­
saturated fatty acids can improve the resistance of cell membranes to
lyophilization [59]. The presence of cis-double bonds in unsaturated
fatty acids prevents the fatty acid molecules from being aligned neatly
and enhances the cell membrane fluidity. LAB uses LDH to adjust the
ratio of saturated fatty acids to unsaturated fatty acids, which is
consistent with the previous results of LDH activity [49]. In addition,
fatty acids carry proteins to promote the transfer between solute and
regulate the activity of membrane-bound enzyme NA+-K+-ATPase [60].
Oleic acid (C18:1) has a special cis structure that can cause changes in
the hydrophobic core of the lipid bilayer, leading to an increase of cell
membrane fluidity [61]. Lactobacillic acid (C19cyc11) is a type of
cyclopropane fatty acid whose formation is considered to be a late
synthetic modification of phospholipids [62]. It is recognized that
cyclopropane fatty acids are formed “in situ” at the expense of unsatu­
rated fatty acids by conversion of the unsaturated position into a

Table 4
Correlation analysis of main membrane fatty acids and freeze-drying survival rate of L.fermentum L2 under complex protective agents.
C12:0 C14:0 C14:1 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C19cyc C22:1 UFA/S Survival rate
11 n9 FA (%)

C12:0 (1) 1
C14:0 (2) − 0.238 1
C14:1 (3) 0.962 − 0.495 1
C16:0 (4) 0.603 − 0.918 0.799 1
C16:1 (5) 0.892 0.227 0.733 0.177 1
C18:0 (6) 0.457 − 0.973 0.684 0.985 0.006 1
C18:1 (7) − 0.726 0.841 − 0.887 − 0.986 − 0.336 − 0.944 1
C18:2 (8) − 0.688 − 0.541 − 0.462 0.164 − 0.942 0.331 0 1
C18:3 (9) − 0.5 0.96 − 0.718 − 0.992 − 0.054 − 0.999* 0.959 − 0.285 1
C19cyc11 (10) − 0.5 0.96 − 0.718 − 0.992 − 0.054 − 0.999* 0.959 − 0.285 1.000** 1
C22:1n9 (11) − 0.6 − 0.634 − 0.357 0.276 − 0.897 0.437 − 0.115 0.993 − 0.393 − 0.393 1
UFA/SFA (12) − 0.588 0.926 − 0.787 − 1.000* − 0.158 − 0.988 0.983 − 0.183 0.995 0.995 − 0.295 1
Survival rate (%) − 0.606 0.917 − 0.801 − 1.000** − 0.18 − 0.985 0.987 − 0.161 0.992 0.997* − 0.273 1.000* 1
(13)

Note：*p < 0.05 **p < 0.01.

7
Z. Cheng et al.
cyclopropane ring due to the transfer of a methyl group from S-adeno­
syl-L-methionine (SAM) to the double bond catalyzed by the cyclopro­
pane fatty acids synthase [63,64]. Some authors have suggested that
cyclopropane fatty acids increase membrane fluidity by preventing close
packing of lipids in the cell membrane like unsaturated fatty acids to
enhance the resistance and permeability of the membrane and promote
the exchange of intracellular and extracellular media [60,65]. As a
result, complex protective agents had different carbon sources, polyols
and amino acids which showed important modifications in their mem­
brane fatty acid composition.
3.5. Relationship between fatty acid composition and cell viability after
freeze-drying
In order to seek a possible relationship between fatty acid composi­
tion and cell viability after freeze-drying, all these results of L. plantarum
L1 and L. fermentum L2 were subjected to Pearson correlation analysis
calculated by SPSS software, respectively (Table 3, Table 4). We
analyzed the correlation between different kinds of fatty acids, UFA/SFA
and survival rate under complex protective agents. For L. plantarum L1
and L. fermentum L2, significant positive correlations were observed
between the UFA/SFA ratio and the survival rates after freeze-drying
and also between C19cyc11 and the survival rates. It is consistent
with the results of Li et al. [66] and Zhang et al. [67] that high content of
unsaturated fatty acid and cyclopropane fatty acid improved survival
rates. In addition, C19cyc11 was negatively correlated with palmitic
acid (C16:0) under the action of complex protectants. This result was in
agreement with the previous reports [67,68]. However, in some studies
[69], a significant proportion of oleic acid (C18:1) was converted to
C19cyc11 which is contrary to our results. Our study showed the content
of stearic acid (C18:0) sharply decreased and the content of oleic acid
(C18:1) increased significantly. We speculated that it may be because
that stearic acid (C18:0) is first converted to oleic acid (C18:1) and then
cyclopropanated to form C19cyc11 even though there is no significant
negative correlation under the action of complex protectants. This may
be determined by the differences between strains, and it also showed
that there are differences in the biosynthesis of cyclopropane fatty acids
between different strains.
4. Conclusion
In this study, we investigated which freeze-drying protective agents
had a significant impact on the survival rates and number of viable
bacteria for L. plantarum L1 and L. fermentum L2. The results showed that
10% skim milk, 13% sucrose, 2% sorbitol, 0.8% tyrosine significantly
improved the survival of L. plantarum L1 while 10% skim milk, 7%
trehalose, 2% sorbitol and 0.6% tyrosine was protective for L. fermentum
L2. We explored the effects of freeze drying on enzyme activity and
revealed that complex protective agents could maintain the integrity of
cell membranes as determined by reduced leakage of β-galactosidase
and LDH and increased the ATPase activity and viability of LAB strains.
The increase of unsaturated fatty acids content in cell membrane such as
oleic acid (C18:1) and acid C19cyc11 may have enhanced the cell
membrane fluidity and improved the resistance to freeze-drying for both
L. plantarum L1 and L. fermentum L2. These results provide the basis for
understanding the influence of complex protective agent on cell
viability.
Declaration of competing interest
The authors have declared no conflict of interest.
Acknowledgements
The authors gratefully acknowledge the support of the Key R & D
project of Zhejiang province (Grant No. 2018C02047)
8
Cryobiology 105 (2022) 1–9
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