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Ijbiomac D 23 14468 r1 Reviewer
Ijbiomac D 23 14468 r1 Reviewer
Ijbiomac D 23 14468 r1 Reviewer
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Response to Reviewers
Thanks for all the suggestions. The whole manuscript has been checked and revised
carefully. We sincerely hope this revised manuscript meets the requirements of
International Journal of Biological Macromolecules. If there still exist some
problems, please feel at ease to reach us.
Reviewer #1:
Reviewer #1: The title is too long, please shorten
Answer: Thanks. The title has been shortened to “Porous zein/cellulose composite
scaffolds for Lactobacillus reuteri biofilms formation: Synbiotics to enhance
gastrointestinal tolerances of probiotics and regulate intestinal microbiota”.
Page 3 line 2 did he mean he receives little attention? I understand that almost no
attention is paid to the intestinal microbiome
Answer: The sentence means the gut microbial ecosystem gains a lot of attention.
Writing error states of the gut from the gut ecosystem angle
What does SCFA mean? If it is the first time you name it, you have to say what these
acronyms mean.
Answer: SCFA means short-chain fatty acid. The first time appears on Page 2.
Commensal Microbiota? I don't understand what you are referring to, please rewrite.
Answer: “Commensal Microbiota” is a conventional term, meaning microbiota living
together with the host.
Rewrite the last part someone else already did and not what you did in this work
Answer: Thanks. Three references (Ref. 17-19) have been cited here.
planktonic counterparts. They come from the seedling, what does the seedling have to
do with gastro-intestinal digestion, please explain
Answer: “planktonic counterparts” means planktonic L. reuteri bacteria. To
characterize the tolerance ability of L. reuteri bacteria in biofilm, planktonic L. reuteri
bacteria were cultured without porous protein/carbohydrate composite scaffold and
used as the control.
Page 8 line 22-37 what is the reason for these characteristics of the Network formed
by the compito
Answer: Porous protein/carbohydrate composite materials were used as the scaffolds
for L. reuteri biofilm formation. The morphology structures, especially the pore
structure and the porosity, have important influences on the biofilm formation as
shown in Fig. 2. Therefore, characterization of the scaffolds is necessary.
Page 9 line 5-7 in the pure cellulose scaffold broaden and shift to a lower
wavenumber 3419 cm-1 in the zein/cellulose composite scaffolds, which is due to the
introduction of zein into the cellulose and the formation of new hydrogen bonds
between zein and cellulose
It seems like the idea was cut, please rewrite
It is necessary to properly relate the FTIR with the SEM since there is no good
relationship in the text
There is a lack of bibliography in the last part of section 3.1.
Aromatic amino acids do not just emit at 1450. There have also been reports of what
they emit around 1600. Furthermore, this entire area can be obscured by the different
configurations of the secondary structure of Zein, whether cellulose is attached or not,
since this area is where amide one amide two amide three is found. So here I attribute
more to the secondary structure of the molecule.
In figure one, you are not showing the Zein spectrum and the cellulose spectrum alone,
I think it would be a good idea to put them
Answer: Thanks for your suggestion. The characteristic peaks of
aromatic compounds are indeed in the range of 1600 ~ 1450 cm-1 due to the C=C
skeleton stretching vibration. In addition, the characteristic peaks of amide one, amide
two, and amide three are in the range of 1800 ~ 1200 cm-1. And this entire area can
also be obscured by the different configurations of the secondary structure of zein.
Therefore, some modifications have been made in Section 3.1. The zein spectrum,
named zein, is the blue curve in Figure 1(b), and the cellulose spectrum, named 0, is
the chartreuse curve in Figure 1(b).
The next part should be in introduction since there is no discussion. The potential
health benefits of probiotics may not be realized because of the substantial reduction
in their viability during gastrointestinal digestion [28].
Answer: Thanks. The role of this sentence here is to bring up the results and the
discussion.
The conclusion of your research should be more concise since the conclusion as
written implies that it is part of the discussion, which is incorrect since it is a
conclusion, you should write only what results were reached with the formation of the
bio-films
Answer: Thanks for your kind suggestion. The conclusion section has been revised.
Reviewer #2:
1. Clarity and Structure: a. The MS provides a clear overview of the study's objective
and findings. However, it would be beneficial to include a concise statement outlining
the specific aims of the research. b. Consider providing a brief mention of the
methods employed in preparing the porous zein/cellulose composite scaffolds to
enhance the overall completeness of the abstract.
Answer: Thanks for your suggestion. We checked the Abstract carefully and found
that only one sentence introduces the roles of the porous zein/cellulose composite
scaffolds without the preparation method of the porous zein/cellulose composite
scaffolds. We think that this sentence is important. To concisely state the scaffold, the
sentence has been revised as “In this study, porous zein/cellulose composite scaffolds
provided nitrogen sources and carbon sources simultaneously at the solid/liquid
interfaces, being beneficial to the biofilm formation of Lactobacillus reuteri.”
6. Concluding Remarks: a. The abstract concludes with valuable insights into the
positive regulation ability of the "synbiotics." Including a concise statement
summarizing the broader implications of these findings for gut health would
contribute to the completeness of the abstract.
Answer: Thanks. The Abstract has been revised to summarize the broader
implications of our findings.
9. Biofilm Formation and SEM Analysis: a. The influence of zein content and
scaffold dosage on L. reuteri biofilm formation is well-documented. However,
consider providing additional details on the quantitative aspects of biofilm formation,
such as thickness or surface coverage, to complement the SEM images. b. The
influence of culture time on biofilm formation is discussed, but it would be helpful to
elaborate on the potential reasons behind the observed trends, such as nutrient
limitations.
Answer: Thanks for the kind suggestion. The biofilm thickness is hard to characterize
since biofilms clung to the pore surfaces. Vertical pore walls in the scanning view can
provide a chance to observe the thickness of the biofilm, like the 10% sample in
Figure 2 (b). However, this chance is not always presented in all the SEM samples.
By contrast, the surface coverage of biofilm on the scaffolds can be easily observed in
the SEM pictures. The sentence in Section 3.2, “This phenomenon may be due to the
lack of nutrients and the low pH and other accumulated metabolites of the culture
medium inhibiting the growth of L. reuteri as the culture time increased.”, explains
the decrease in the number of viable cells.
14. Figures and Tables: a. Ensure that figures and tables are appropriately referenced
in the text, and consider providing concise figure captions for better understanding.
In summary, the manuscript provides valuable insights into the effects of
zein/cellulose composite scaffolds and L. reuteri biofilms on gut health.
Enhancements in clarity, additional quantitative details, and further discussions on the
biological implications of the findings would strengthen the manuscript. Overall, the
MS is well-structured and provides a solid foundation for understanding the study.
Addressing the above points would further enhance the clarity and impact of the
abstract also.
Answer: Thanks for your kind suggestion. The Abstract has been revised to provide
the quantitative results and strengthen the impact of our findings.
Reviewer #3:
The paper titled "Porous zein/cellulose composite scaffolds for Lactobacillus reuteri
biofilms formation" delves into the pivotal realm of enhancing the gastrointestinal
tolerances of probiotics using innovative composite scaffolds. The abstract concisely
introduces the motivation behind the study, emphasizing the significance of
probiotics' survival in the gut and the pivotal role of biofilms in achieving this
objective. However, certain areas within the paper would benefit from minor revisions
for improved clarity and precision.
Here are my specific comments:
Major comments:
The language used throughout the paper is generally clear and comprehensible.
However, certain sections could be refined to enhance readability and coherence.
Answer: We greatly appreciate this suggestion. The manuscript has been carefully
revised.
The expansion of figure legends to include details on sample sizes and statistical tests
conducted would provide readers with essential information regarding the
experimental setup and data analysis. This enhancement would contribute to the
transparency and reproducibility of the study.
Answer: Thanks. The experimental data shown in the figures and table have error
bars or error ranges, which result from three parallel samples. These results were
statistically analyzed by GraphPad Prism and SPSS Statistics. The methods used for
statistical analysis are shown in Section 2.10.
Moreover, the conclusion section could be rephrased to succinctly highlight the most
significant findings reported in the study. Focusing on the enhanced gastrointestinal
tolerances of probiotics through biofilm formation on zein/cellulose composite
scaffolds, and the superior regulatory effects of the biofilm-phenotype probiotics
combined with non-digestible carbohydrates, would effectively encapsulate the key
takeaways from the research.
Answer: Thanks very much. The conclusion section has been revised.
Minor comments:
Page 3, lines 9-10: Change …strategies have been developed… to … strategies that
have been developed…
Answer: Thanks very much. The sentence has been revised as “Several strategies
have been developed to manipulate the gut microbiome to prevent the unhealthy states
or revert to healthy states caused by perturbations, including fecal microbiota
transplantation”.
Page 5, line 41: Authors state "… sterilized scaffolds were added to a 10 mL MRS
liquid medium…" The range of sterile scaffolds added, expressed in terms of weight
by volume ratio, should be specified by the authors.
Answer: Thanks for your reminder. The sentence has been revised as “The sterilized
scaffolds (0.1% ~ 0.5 wt.%) were added to a 10 mL MRS liquid medium with 1% L.
reuteri inoculation.”
Page 6, line 4: Authors state "…After culture duration,…" Authors must also specify
the duration or range of time.
Answer: As shown in Figure 2, the culture duration is in the range of 4 ~ 72 h. The
specific culture duration time is described in the corresponding Figure caption.
Page 7, line 36: Change the 2.9 sub-heading to Influences of L. reuteri biofilms on
SCFAs production.
Answer: Thanks very much. The sentence has been revised to “2.9. Influences of L.
reuteri biofilms on SCFA production
”.
Page 11, lines 24-29: To clarify, reword the following sentence: …where the numbers
of viable cells between the 0 h group and the 3 h group exist significant differences…
Answer: Thanks very much. The sentence has been revised to “As shown in Fig. 4(a),
Fig. 4(b), and Fig. 4(c), planktonic L. reuteri decreased 0.23 Log CFU/mL, 0.14 Log
CFU/mL, and 0.10 Log CFU/mL after 3 h digestion in SGF with pH 2.5 and pH 3.5
and in SIF with pH 6.8, respectively, where the numbers of viable cells between the 0
h group and the 3 h group exist significant differences in these digestion conditions
(P<0.001, P<0.01, and P<0.05, respectively).”.
Page 11, lines 36-39: To clarify, reword the following sentence: …The numbers of
viable cells between the 0 h group and the 3 h group exist significant differences in
SGF with pH 3.5…
Answer: Thanks very much. The sentence has been revised to “In contrast, L. reuteri
in biofilm phenotype increased the viable cells with 0.11 Log CFU/mL, 0.31 Log
CFU/mL, and 0.27 Log CFU/mL in SGF with pH 2.5 and pH 3.5 and in SIF with pH
6.8 after 3 h digestion, where the numbers of viable cells between the 0 h group and
the 3 h group exist significant differences in SGF with pH 3.5 (P<0.01) and SIF with
pH 6.8 (P<0.001).” to make the expression clearer.
Page 12, line 4-5: Change … denaturant, and damaging DNA… to …denaturing and
damaging the DNA…
Answer: Thanks. The sentence has been revised to “In the gastrointestinal tracts, bile
salts secreted by the small intestine reduce bacterial survival by disrupting cell
membranes, leaking out intracellular material, inducing protein misfolding and
denaturing and damaging the DNA”.
Page 13, lines 51-52: Change …where cellulose exists the role of prebiotics…
to …where cellulose exists as the sole of prebiotics…
Answer: Thanks very much. The sentence has been revised to “where cellulose acts
as the prebiotics”.
Page 15, lines 41-42: Delete the sentence's repeated redundancy. …" Bilophila,
Bilophila, and Bifidobacterium…"
Answer: Here, Acinetobacter, Bilophila, Bilophila, and Bifidobacterium were the
most influenced bacteria in the control group, the P group, M group, and B/M group,
respectively. In other words, both the P group and the M group have the biggest
effects on Bilophila. Therefore, there should be two Bilophila.
Reviewer #4: This field is optional. If you have any additional suggestions beyond
those relevant to the questions above, please number and list them here.
Novelty is poor
Answer: Thanks for your evaluation. We have done our best to present the
significance and the novelty of this work in the manuscript. We would be grateful if
you have any specific suggestions.
Reviewer #5: This field is optional. If you have any additional suggestions beyond
those relevant to the questions above, please number and list them here.
The paper contains some wrong spelling errors, check and correct them
More details should be added in legends
Answer: Thanks very much. We have checked the whole manuscript and revised the
manuscript.
Reviewer #6
This field is optional. If you have any additional suggestions beyond those relevant to
the questions above, please number and list them here.
Reviewer doesn't find attractive findings and results in the paper. Bioactivity results
lack deep insight. If more molecular mechanism can be done, the paper will have high
publication merits.
Answer: Thanks for the suggestion. Good gastrointestinal tolerances are generally
acknowledged pre-conditions to exert the health benefits of probiotics. A lot of
research is devoted to increasing the gastrointestinal tolerances of probiotics. The
bioactivities of L. reuteri biofilms in gastrointestinal fluids and fecal fermentation
solutions have been studied in detail. We don’t understand what is more molecular
mechanism. We would appreciate your detailed suggestions about the molecular
mechanism.
Reviewer #7:
This field is optional. If you have any additional suggestions beyond those relevant to
the questions above, please number and list them here.
Similar researchs have been published much. In addition, experiment results are
superficial. More molecular indexs and muchenism should be done
Answer: Thanks for the suggestion. There are a lot of probiotics research. However,
the studies of probiotics biofilms, especially the preparation of probiotics biofilms, are
still in the early stages. Excluding the study of gastrointestinal tolerance, the gut
microbiota regulation ability and SCFAs production ability of L. reuteri biofilms on
the scaffolds in the fecal fermentation systems were also studied in detail. We think
that these results could demonstrate the advantage and the bioactivity of the prepared
L. reuteri biofilms on the scaffolds. We would appreciate your detailed suggestions
about the molecular index and mechanism.
Reviewer #8: This field is optional. If you have any additional suggestions beyond
those relevant to the questions above, please number and list them here.
The paper have many errors in writting, analysis. I don't suggest its publication
Answer: Thanks for your warning. The manuscript has been revised. We hope you
can reconsider your decision.
Abstract
unhealthy states of the gut by reshaping gut microbiota. One criterion that probiotics are
efficacious is the capacity to survive in the gastrointestinal tract. Biofilm is the common growth
mode of microorganisms with high tolerances toward harsh environments. Suitable scaffolds
are crucial for successful biofilm culture and large-scale production of biofilm-phenotype
formation has not been studied. In this study, porous zein/cellulose composite scaffolds
provided nitrogen sources and carbon sources simultaneously at the solid/liquid interfaces,
being beneficial to the biofilm formation of Lactobacillus reuteri. The biofilms showed 2.1 ~
fermentation, the biofilms combined with the zein/cellulose composite scaffolds act as the
“synbiotics” positively modulating the gut microbiota and the short-chain fatty acids (SCFAs),
where biofilms provide probiotics and scaffolds provide prebiotics. The “synbiotics” show a
more positive regulation ability than planktonic L. reuteri. These results provide an
probiotics biofilms and the synergistic effects of biofilm-phenotype probiotics and indigestible
1
Revised manuscript (clean version) Click here to view linked References
microbiota
Fei He, Xue-Ke Ma, Cheng-Kai Tu, Hui Teng, Xin Shao, Jie Chen, Meng-Xin Hu*
Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology,
* Corresponding author.
Tel: + 86 571 28008976; Fax: + 86 571 28008900; E-mail address: mengxinhu@zjgsu.edu.cn (Meng-Xin Hu)
1
ABSTRACT
unhealthy states of the gut by reshaping gut microbiota. One criterion that probiotics are
efficacious is the capacity to survive in the gastrointestinal tract. Biofilm is the common growth
mode of microorganisms with high tolerances toward harsh environments. Suitable scaffolds
are crucial for successful biofilm culture and large-scale production of biofilm-phenotype
formation has not been studied. In this study, porous zein/cellulose composite scaffolds
provided nitrogen sources and carbon sources simultaneously at the solid/liquid interfaces,
being beneficial to the biofilm formation of Lactobacillus reuteri. The biofilms showed 2.1 ~
fermentation, the biofilms combined with the zein/cellulose composite scaffolds act as the
“synbiotics” positively modulating the gut microbiota and the short-chain fatty acids (SCFAs),
where biofilms provide probiotics and scaffolds provide prebiotics. The “synbiotics” show a
more positive regulation ability than planktonic L. reuteri. These results provide an
probiotics biofilms and the synergistic effects of biofilm-phenotype probiotics and indigestible
2
Introduction
The gut microbial ecosystem is closely related to host health and gains a lot of attention [1,
2]. Perturbations to the human gut microbiome can disrupt the stability of the ecosystem,
possibly resulting in recalcitrant unhealthy states associated with diseases [3]. Several strategies
have been developed to manipulate the gut microbiome to prevent the unhealthy states or revert
supplementation with probiotics or indigestible carbohydrates [5], and more extensive dietary
modifications [6].
Among these strategies, supplementing probiotics and dietary fiber can reshape gut
microbiota and intestinal mucosa barrier and prevent or revert unhealthy states of the gut from
the gut ecosystem angle [7]. Probiotics are defined as "living microorganisms that, when given
in sufficient quantities, provide a health benefit to the host" [8]. Generally, probiotics are found
to maintain intestinal homeostasis through direct interaction with the intestine [9] and indirect
interaction through the produced metabolites, including SCFAs, with the intestine [10].
However, the criterion that a probiotic must have to be considered efficacious is the capacity to
healthy adults, which can promote the secretion of vitamins, inhibit the growth and colonization
microbiota composition in the host [12]. As with all orally consumed probiotics, the Gram-
gastrointestinal tract of the host, including low pH, effectors of the host immune system, as well
as competition with commensal and pathogenic bacteria, all of which can greatly reduce the
availability of live bacteria for healthy or therapeutic purposes [13]. Hence, it is important to
Interestingly enough, biofilm as the most common growth mode of microorganisms in nature
[16]. Previous studies have verified that Lactobacillus rhamnosus, Lactobacillus fermentum,
Lactobacillus acidophilus, and Lactobacillus casei can be encapsulated and form biofilms in
capsulates [17-19]. These probiotic biofilms show enhanced resistance to heat, acid, and storage
paracasei, benefiting from the protection of EPS and the biofilm phenotype [20]. Electrospun
probiotics [20-22]. However, the output of electrospun nanofibrous scaffolds is limited by the
existing production method. Seeking green, inexpensive, and friendly scaffolds suitable for
biofilm formation of probiotics is the key point for successful biofilm culture and large-scale
The goal of this work is to prepare porous protein/carbohydrate composite scaffolds with
zein and cellulose for L. reuteri biofilm formation. The effect of the chemical composition on
the morphology of the scaffolds was studied in detail. Different porous zein/cellulose composite
scaffolds were utilized for biofilm formation and the culture conditions were optimized. The
tolerances of two phenotypes of L. reuteri to simulated gastrointestinal fluids and bile salts were
compared in vitro. In an in vitro human fecal fermentation model, the differences between L.
reuteri biofilm and their planktonic counterparts impacting the intestinal flora and SCFAs were
also focused.
2.1. Materials
4
Cellulose (cotton pulp, Mη 10.0×104) was purchased from Hubei Chemical Fiber Group Co.
Ltd. Other chemicals were purchased from Aladdin. L. reuteri DSM 17938 was commercially
The preparation of porous zein/cellulose composite scaffolds is based on the reported work
dissolved in NaOH/urea aqueous solution and cooled to -12 ℃. Cellulose powder (3 wt%) was
added and stirred at -12 ℃ for 15 min to dissolve cellulose. Epichlorohydrin (ECH) with a
concentration of 9 % was dropped into the solution and stirred at room temperature for 1 h. The
gained solution was frozen at -20 ℃ for 20 h and thawed at room temperature to gain hydrogels,
which were then cut into thin strips with a thickness of about 1~2 mm and washed with distilled
water until the solution was neutral. After that, the hydrogels were soaked in distilled water for
3 days to reach swelling equilibrium. Finally, the hydrogels were frozen with liquid nitrogen
The morphology of zein/cellulose composite scaffolds after coating a layer of gold was
characterized by a scanning electron microscope (SEM, Phenom Pro). The chemical structures
The freeze-dried zein/cellulose composite scaffolds were rehydrated and autoclaved for 15
min at 121℃. The sterilized scaffolds (0.1% ~ 0.5 wt.%) were added to a 10 mL MRS liquid
medium with 1% L. reuteri inoculation. The anaerobic static culture was carried out at 37℃ to
5
Biofilms formed on zein/cellulose composite scaffolds were immersed in 2.5 %
glutaraldehyde solution for 6-8 h and then dehydrated with 30%, 50%, 70%, 80%, 90%, 95%,
100% ethanol solution in turn, where each dehydration time is 15 min. The dehydrated samples
were sprayed with gold for 90 s and the morphology of biofilms was observed with SEM.
Different from the quantitation of planktonic L. reuteri, the quantitation of live cells in
biofilms relies on cell detachment followed by conventional plate counting. First, cells were
detached from biofilms formed on scaffolds by ultrasonic treatment. After culture duration, the
culture solution was centrifuged at 5000 rpm for 5 min at 4℃. The supernatant was poured out
and sterile normal saline was added with equal volume. Then cells were detached through
ultrasonic treatment (100 W). The detached L. reuteri supernatant was serially diluted 10-fold
and 50 μL aliquots were plated onto agar plates. Colony-forming units (CFU) were counted
In vitro gastrointestinal tolerance assay for L. reuteri was performed according to the protocol
previously described, with some modifications [24]. Simulated gastric fluid (SGF), simulated
intestinal fluid (SIF), and bile salt solution were freshly prepared before each experiment. For
SGF, HCl was diluted with deionized water to gain solutions with pH 2.5 and 3.5, separately,
where pepsin concentration was 1 g/100 mL. For SIF, 6.8 g KH2PO4 was dissolved in 500 mL
deionized water, pH was adjusted to 6.8 with 0.4 g/100 mL NaOH solution, trypsin was then
added at a concentration of 10 g/100 mL, and finally the solution was diluted to 1 L with water.
For bile salt solutions, 0.03 g, 0.1 g, and 0.3 g pig bile salts were dissolved in 100 mL of
deionized water to obtain 0.03 %, 0.1 %, and 0.3 % artificial bile salt solutions. Before the
experiment, SGF, SIF, and bile salt solutions were filtered by microporous membranes (0.22
μm). Planktonic L. reuteri and L. reuteri biofilms were added into SGF, SIF, and bile salt
6
solutions, separately. After digestion at 37 ℃ on a shaker (100 r/min), viable cells were
determined by plate counting method with MRS agar plates. All experiments were done in
triplicate. Survival of L. reuteri in gastrointestinal fluids was expressed in terms of Log (N/N0),
where N0 and N represent the viable CFUs of samples before and after digestion.
After the sequential gastrointestinal digestion of L. reuteri biofilms and planktonic L. reuteri
(≈ 1010 CFU), the obtained digested products were added into human fecal solutions for in vitro
fermentation. Fecal sample collection and pretreatment followed previously reported work [22].
1 L of nutrient medium was composed of peptone 2 g, yeast extract 2 g, NaCl 0.1 g, K2HPO4
0.04 g, KH2PO4 0.04 g, MgSO4·7H2O 0.01 g, CaCl2·6H2O 0.01 g, NaHCO3 2 g, and 80-Tween
2 mL [25]. After autoclave sterilization at 121 °C for 30 min, heme 0.02 g, vitamin K1 10 mL,
bile salt 0.5 g, and cysteine hydrochloride 0.5 g were added. The human feces solution was
composed of 27 mL nutrient medium and 3 mL fecal bacteria extract, which was flushed by N2
for 5 min. The fermentation temperature was 37 °C and the rotation speed was 75 r/min. After
was added to the control group. The culture sustains 3 days, during which no nutritional medium
was added and 5 mL of fermentation broth was collected every day and stored in the -20 °C
refrigerator. After sampling, the solution was flushed by N2 again for 5 min and then the culture
continued. All the collected samples were analyzed via 16S rDNA by Majorbio Bio-Pharm
SCFAs in the collected samples were determined following the method described in
previously reported work with some modifications [22]. The sample (1 mL) was centrifuged at
12000 rpm for 10 min at 4 °C and the supernatant was collected and mixed with 100 μL of
concentrated hydrochloric acid and 5 mL of ethyl ether for 20 min. Then the mixture was
7
centrifuged at 3500 rpm for 10 min at 4 °C. The top layer was collected and mixed with 500 μL
of 1 M NaOH. After the second extraction and centrifugation, the water phase in the bottom
layer was collected. The bottom layer was filtered with a 0.22 μm filter after adding 100 μL
concentrated hydrochloric acid. The SCFAs in the aliquot were determined by HPLC. Column:
YMC-PackProC-18 (250×4.6 mm); mobile phase 0.025% phosphoric acid solution (pH 2.8):
acetonitrile = 95:5, flow rate 1.0 mL/min; injection volume 20 μL; detection wavelength 210
Statistical analysis was carried out and graphs were generated using GraphPad Prism
software (Version 9.0.0). Statistical differences between the two groups were analyzed with
unpaired two-tailed Student t-tests. Symbols ***, **, and * represent highly significant values
(P<0.001), moderately significant values (P<0.01), and weakly significant values (P<0.05),
respectively. In addition, the alpha diversity of gut microbiota was analyzed by SPSS Statistics.
The morphology of porous zein/cellulose composite scaffolds is shown in Fig. 1 (a). With
the increase in zein concentration, the number of large pores on the surface of the material
increased and the number of small pores decreased. When zein is not added, the strong
hydrogen bond interactions between cellulose molecules result in a powerful gel skeleton
network, which can withstand the growth of ice crystals during freeze-drying and give rise to a
lot of small pores [26]. Zein is rich in glycine, proline, glutamic acid, alanine, and arginine [27].
When zein was mixed into the cellulose solution, the -NH2, the -COOH, and the -CONH- in
zein protein interacted with the -OH of cellulose and formed intermolecular hydrogen bonds,
which destroy the strong hydrogen bond interactions between cellulose molecules. However,
among biopolymers, zein is a hydrophobic protein with a significant amount of non-polar amino
acids (about 50% of total amino acids) [28]. The intermolecular hydrogen bonds between
8
cellulose molecules and zein molecules were partly influenced by the non-polar amino acids of
zein molecules. The strength of the zein/cellulose composite gel skeleton network was
weakened compared to the pure cellulose gel skeleton network. Accordingly, big ice crystals
FT-IR spectra of the scaffolds are shown in Fig. 1 (b). The OH stretching vibration bands
around 3424 cm-1 in the pure cellulose scaffold broaden and shift to a lower wavenumber 3419
cm-1 in the zein/cellulose composite scaffolds, which is due to the introduction of zein into the
cellulose and the formation of new hydrogen bonds between zein and cellulose [27]. The alkyl
stretching of zein is in the range of 2834 cm-1 ~ 3004 cm-1, which can be found in the FT-IR
spectra of the zein/cellulose composite scaffolds. The peak at 1450 cm-1 is attributed to the
aromatic ring skeleton stretching vibration of aromatic amino acids (phenylalanine and tyrosine)
in zein molecules and the peak also can be found in the FT-IR spectra of the zein/cellulose
composite scaffolds but shift to 1459 cm-1. The results of FT-IR demonstrate that the composite
scaffolds are composed of both zein and cellulose. The interactions between zein and cellulose
at the molecular level closely affect the morphology of the zein/cellulose composite scaffolds.
The increased zein concentration not only results in large pores which are more beneficial for
L. reuteri spread into the inner of the scaffolds, but also provide more nitrogen sources for L.
Fig. 2 (a) shows the influences of the chemical composition and dosage of scaffolds on L.
reuteri biofilm formation. L. reuteri biofilms formed on the scaffolds increased with the zein
content in scaffolds and the scaffold dosage in the culture solution. As the ratio of zein/cellulose
was 10%, viable cell densities in biofilms on the composite scaffolds were significantly higher
than that on the pure cellulose scaffolds (P<0.01, P<0.001, and P<0.01 for 0.1%, 0.3 %, and
0.5%, respectively). The introduction of zein into the scaffolds provides extra nitrogen sources
9
and protein nutrients for L. reuteri growth, making L. reuteri biofilm grow better. The results
demonstrate that the composite of protein and carbohydrate in the scaffolds provides nitrogen
sources and carbon sources simultaneously at the solid/liquid interface and is more beneficial
to L. reuteri biofilm formation. SEM pictures in Fig. 2 (b) show dense L. reuteri biofilms were
formed on zein/cellulose composite scaffolds with 0.3% scaffold dosage in the culture medium.
In addition, the increasing scaffold dosage in the culture medium provides more surface and
space for L. reuteri adhesion and biofilm formation, resulting in more L. reuteri existing as the
biofilm phenotype (Fig. 2 (c)). The results are similar to L. paracasei biofilm formation on
electrospun cellulose acetate nanofibrous scaffolds [20]. When the scaffold dosage increased
from 0.1% to 0.3%, the number of viable cells of biofilms on scaffolds significantly increased.
Then, a further increase in the scaffold dosage cannot promote more biofilm formation, which
could result from the limited nutrition provided by the culture medium.
Furthermore, the culture time shows an influence on the biofilm formation similar to the
growth curve of planktonic L. reuteri (Fig. 2 (d) and Fig. S1). The number of viable cells on
the 10% zein/ cellulose composite scaffold increased from 7.94 Log CFU /mL to 9.15 Log CFU
/mL as the culture duration increased from 4 h to 20 h. Then the number of viable cells slightly
decreased and gradually stabilized. This phenomenon may be due to the lack of nutrients and
the low pH and other accumulated metabolites of the culture medium inhibiting the growth of
L. reuteri as the culture time increased. SEM pictures in Fig. 2 (e) demonstrate that only a small
amount of L. reuteri adhered on the surface of the scaffold after a 4 h culture duration. Then
obvious L. reuteri biofilm formed on the surface of the scaffold after an 8 h culture duration.
After that time, L. reuteri biofilm became more and more dense with the further increasing
culture duration.
10
The foregoing results indicate that L. reuteri biofilms formed on the porous zein/cellulose
composite scaffolds very well. The EPS matrix acts like a biological ‘glue’ [29] enabling L.
reuteri to adhere to the scaffold surface. Simply rinsing makes it hard to release cells from
biofilms. To determine the number of viable cells in biofilms, samples were handled by
ultrasonic treatment to release cells from biofilms. As shown in Fig. 3 (a), the number of viable
cells was the highest when the ultrasound time was 10 min, and there was a significant
difference compared with the ultrasound time of 5 min (P<0.01). Sufficient ultrasound time
caused more cells released from biofilms. However, the number of viable cells decreased as
ultrasound time was more than 10 min. As shown in SEM pictures in Fig. 3 (b), the residual L.
reuteri biofilms lessen when the ultrasonic time is more than 25 min, but the quantified number
of viable cells still is low. It’s because ultrasound is a relatively drastic method that could cause
cell rupture and death over a long time [30]. Therefore, the ultrasonic treatment combined with
the plate counting method to quantify the number of viable cells in biofilms results in low values
less than the true values. These results demonstrate that L. reuteri bacteria are not easy to release
from biofilms under simple solution conditions in vitro. Although the influence of L. reuteri
biofilms on the gut microbiota during in vitro fecal fermentation has been studied and shown
in this work, the release behaviors of probiotics from biofilms in vivo need further studied in
The potential health benefits of probiotics may not be realized because of the substantial
reduction in their viability during gastrointestinal digestion [31]. High tolerances of probiotics
toward low pH of gastric fluid, diverse digestive enzymes, and bile salts in the gastrointestinal
tracts are important guarantees to perform the probiotic functions. As shown in Fig. 4(a), Fig.
4(b), and Fig. 4(c), planktonic L. reuteri decreased 0.23 Log CFU/mL, 0.14 Log CFU/mL, and
0.10 Log CFU/mL after 3 h digestion in SGF with pH 2.5 and pH 3.5 and in SIF with pH 6.8,
11
respectively, where the numbers of viable cells between the 0 h group and the 3 h group exist
respectively). In contrast, L. reuteri in biofilm phenotype increased the viable cells with 0.11
Log CFU/mL, 0.31 Log CFU/mL, and 0.27 Log CFU/mL in SGF with pH 2.5 and pH 3.5 and
in SIF with pH 6.8 after 3 h digestion, where the numbers of viable cells between the 0 h group
and the 3 h group exist significant differences in SGF with pH 3.5 (P<0.01) and SIF with pH
6.8 (P<0.001). These results demonstrate that L. reuteri in biofilm phenotype has higher
tolerances than planktonic L. reuteri in SGF and SIF, showing stronger survival ability toward
low pH and diverse digestive enzymes. The longer the digestion time, the greater the impact of
SGF and SIF on the planktonic cells resulting in the death of L. reuteri. Viable cells of the
biofilm groups in SGF and SIF increased with the digestion time due to the EPS acting as
“protective clothing” for the embedded L. reuteri [32]. On the other hand, differentially
In the gastrointestinal tracts, bile salts secreted by the small intestine reduce bacterial survival
by disrupting cell membranes, leaking out intracellular material, inducing protein misfolding
and denaturing and damaging the DNA [33, 34]. Bile salts at a concentration of 0.3% are the
maximum that can be found in an average healthy person [35]. In our work, the negative effects
of bile salts on L. reuteri are shown in Fig. 4 (d)-(f’). The survival rates of L. reuteri both in the
planktonic state and in the biofilm state decrease with the concentration of bile salts and the
digestion time. Planktonic L. reuteri decreased 0.74 Log CFU/mL, 1.31 Log CFU/mL, and 2.62
Log CFU/mL after 3 h digestion in bile salts solution with 0.03%, 0.1%, and 0.3% concentration,
respectively, where the numbers of viable cells between the 0 h group and the 3 h group exist
decreases the viable cells with 0.20 Log CFU/mL, 0.47 Log CFU/mL, and 1.38 Log CFU/mL
after 3 h digestion in bile salts solution with 0.03%, 0.1%, and 0.3% concentration, respectively.
12
The survival ability of L. reuteri in biofilm phenotype is far beyond that of planktonic L. reuteri.
Many researchers verified that microbes including probiotics respond to bile salts exposure by
biofilm formation to avoid bactericidal effects of high bile concentration [36, 37]. On the other
hand, the EPS acting as “protective clothing” for the embedded L. reuteri limits the diffusion
of bile salts into the inner layer of EPS, which follows to prevent direct interactions with L.
reuteri cells.
In our work, L. reuteri biofilms benefit greatly from the biofilm phenotype and the
“protective clothing” of EPS. There are about 2.1 ~ 2.8 times more L. reuteri survival abilities
after exposure to SGF with pH 2.5 and pH 3.5 and in SIF with pH 6.8 after 3 h digestion in
biofilms compared with the planktonic counterparts. Moreover, the tolerance abilities of L.
reuteri biofilms are 3.5, 6.9, and 17.4 times higher than the tolerance abilities of planktonic L.
reuteri in bile salts solutions with different concentrations (0.03%, 0.1%, and 0.3%) after 3 h
biofilm L. reuteri verify that L. reuteri in biofilm phenotype is more effective than probiotics.
3.5. Influence of L. reuteri biofilms on gut microbiota during in vitro fecal fermentation
Table 1 shows the influence of L. reuteri in different phenotypes on the alpha diversity of
gut microbiota during in vitro fecal fermentation. Planktonic L. reuteri (P group), zein/cellulose
composite scaffold (M group), and L. reuteri biofilm (B/M group) decreased gut microbiota
diversity (decreased Shannon indices and increased Simpson indices) and richness (decreased
Sobs, Chao, and Ace indices) after 1-day fermentation compared to the Control group. The
short-term dietary intervention results in a slight but significant decrease in microbial diversity,
which could be called “transitory microbial stress” [38]. After 3 days of fermentation, the P
group, M group, and B/M group show increased gut microbiota diversity and richness compared
to the Control group, where the B/M group has the highest gut microbiota diversity and richness
and shows significant differences with the P group in Shannon indices and Sobs indices. The
13
results demonstrate that planktonic L. reuteri, zein/cellulose composite scaffold, and L. reuteri
biofilm on scaffold improve the community diversity, especially L. reuteri biofilm on scaffold.
Previous study has shown that biofilms enhance the diversity and metabolic activity of
microbial communities [39]. In our study, L. reuteri biofilm and the scaffold synergistically
The microbial communities at the phylum and genus levels in the in vitro fecal fermentation
are shown in Fig. 5 (a) and Fig. 5 (b), separately. The Lactobacillus genus significantly
increased and the Acinetobacter genus decreased in the P groups and B/M groups compared to
the control groups. In addition, the Bifidobacterium genus decreased in the P groups, but
increased in the M groups and B/M groups, suggesting that the zein/cellulose composite
materials promoted the growth of Bifidobacterium where cellulose acts as the prebiotics. The
B/M group shows lower abundances of Klebsiella, Bilophila, and Bacteroides and a higher
abundance of Bifidobacterium than the P group after 1 day of fermentation. When the
fermentation time extends to 3 days, the B/M group shows lower abundances of Acinetobacter,
Klebsiella, and Bacteroides and higher abundances of Bifidobacterium and Bilophila than the
bacteremia, but can also cause soft tissue and urinary tract infections [40]. Klebsiella exists in
the intestinal tract and respiratory tract of normal people and is a kind of opportunistic pathogen,
which is prone to infection in people with low immunity and those undergoing surgical and
invasive iatrogenic procedures [41]. Bacteroides are symbiotic with humans and help break
down food to produce nutrients and energy that the body needs. However, when Bacteroidetes
enter parts of the body other than the gastrointestinal area, they can cause or exacerbate
infections such as abscesses [42]. On the other hand, human gut bacteria in the genus Bilophila
have been found to metabolize both trimethylamine and its precursors without the production
14
of trimethylamine-N-oxide and reduce the risk of cardiovascular disease induced by excessive
intake of meat [43]. Bifidobacterium is among the first microbes to colonize the human
intestine naturally, their abundance and diversity in the colon are closely related to host health
[44]. The results shown in Fig. 5 (a) and Fig. 5 (b) demonstrate that L. reuteri biofilms on
zein/cellulose composite scaffolds regulate gut microbiota more positively than planktonic L.
reuteri.
The Spearman correlation analysis in Fig. 5 (c) and Fig. 5 (d) shows the positive and negative
correlations between these gut microbes in the in vitro fecal fermentation. Principal coordinate
analysis of the gut microbes in Fig. 5 (e) demonstrates that the species composition of the
control group was different from that of the P groups, M groups, and B/M groups after the in
vitro fecal fermentation. The relative distances between the 1-day fermentation groups and the
3-day fermentation groups also were far away, indicating that there were differences in the
species composition between them and the fermentation time affects the species composition.
3.6. Influence of L. reuteri biofilms on SCFAs production during in vitro fecal fermentation
SCFAs are mainly composed of acetic acid, propionic acid, and butyric acid, which play an
important role in host physiology such as energy metabolism, glucose homeostasis, lipid
production regulation, and immune regulation [10, 45]. The influence of L. reuteri biofilms on
the concentration of SCFAs is shown in Fig. 6. After 1 day of fermentation, the total
concentration of SCFAs in the B/M group was 3.96 mg /mL, which was significantly higher
than that of the control group with 1.71mg /mL. After 3 days of fermentation, the total
However, the concentrations of acetic acid, propionic acid, and butyric acid in the P groups, M
groups, and B/M groups were significantly higher than the concentrations in the control groups
(P<0.0001). Each SCFA in the B/M groups had the highest concentration and was significantly
different from the P groups and the M groups (Fig. 6 (b)). Both the scaffolds and the supplied
15
L. reuteri and other SCFA production-related bacteria with increased abundances in the fecal
fermentation system enhanced the whole SCFA production. Hence, the B/M groups have
Redundancy analysis (RDA) in Fig. 6 (c) and Fig. 6 (d) demonstrate that microbial
correlated with SCFAs. After 1 day of fermentation, the biggest effects of the control group,
the P group, M group, and B/M group on the microbial communities were Acinetobacter,
Bilophila, Bilophila, and Bifidobacterium, respectively. On the other hand, the biggest effects
of the control group, the P group, M group, and B/M group on the microbial communities after
Bilophila was positively correlated with acetic acid and propionic acid. The cluster of
Lactobacillus, Cloacibacillus, and Bilophila was positively correlated with butyric acid.
4. Conclusion
In this study, zein and cellulose were dissolved in NaOH/urea aqueous solution at low
freeze-drying technique. The composite of protein (zein) and carbohydrate (cellulose) in the
scaffolds is more beneficial to L. reuteri biofilm formation than the pure carbohydrate scaffolds.
enhanced tolerances toward low pH of gastric fluid, diverse digestive enzymes, and bile salts
in the in vitro gastrointestinal conditions. The EPS layer acting as “protective clothing” for the
embedded L. reuteri limits the diffusion of low pH, enzymes, and bile salts into the inner layer
of EPS, which follows to prevent the direct interactions with L. reuteri cells and the killings of
L. reuteri cells. In human fecal fermentation, L. reuteri biofilms on the composite scaffolds
decrease the abundances of Acinetobacter, Klebsiella, and Bacteroides and increase the
16
abundances of Lactobacillus, Bifidobacterium, and Bilophila, showing a more positive
regulation ability of gut microbiota than planktonic L. reuteri. Also, L. reuteri biofilms on the
scaffolds result in the highest SCFA production in the human fecal fermentation solutions. The
scaffolds and the supplied L. reuteri and other SCFA production-related bacteria with increased
abundances in the fecal fermentation system synergistically enhanced the whole SCFA
scaffolds act as the “synbiotics” positively modulating the gut microbiota and the SCFAs, where
prebiotics, and play a more active role in regulating gut microbiota and producing SCFAs as
Fei He: Investigation, Methodology, Formal analysis, Data curation, Writing-original draft,
Teng: Investigation. Xin Shao: Investigation. Jie Chen: Methodology. Meng-Xin Hu:
Data availability
Acknowledgement
This project is financially supported by the Natural Science Foundation of Zhejiang Province
(Grant no. LTGN24C200005) and the Fundamental Research Funds for the Provincial
17
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Table 1. Alpha diversity after 1 day and 3 days of culture duration in vitro fecal fermentation of L. reuteri and L. reuteri biofilms.
22
0 5% 10% 20%
(a)
Fig. 1. (a) SEM images of zein/cellulose composite scaffolds with different zein contents
(zein/cellulose), top images ×200, bottom images ×500; (b) FT-IR spectra of zein, cellulose,
and zein/cellulose composite scaffolds with different zein concentrations.
23
0 5%
10% 20%
(b)
0.1% 0.2%
0.3% 0.5%
(c)
4h 8h 12 h
16 h 20 h 24 h
(e)
Fig. 2. (a) Effects of the zein/cellulose composite scaffolds amount added into culture solution
on the density of L. reuteri (culture time 20 h); (b) SEM images of L. reuteri biofilms formed
on zein/cellulose composite scaffolds with different zein content (scaffold amount amount 0.3%
24
and cultured time 20 h); (c) SEM images of L. reuteri biofilms formed on the zein/cellulose
composite scaffold (10% zein) with different scaffold amount added in the culture solution
(culture time 20 h); (d) the effect of culture time on the density of L. reuteri (scaffold amount
0.3% and zein content 10%); (e) SEM images of L. reuteri biofilms formed on the zein/cellulose
composite scaffold (10% zein) with different culture time.
25
5 min 10 min 15 min
(b)
Fig. 3. (a) The effect of ultrasonic time on the quantitative values of alive L. reuteri (scaffold
amount 0.3% and zein content 10%); (b) SEM images of L. reuteri biofilms on zein/cellulose
composite scaffolds (scaffold amount 0.3%, zein content 10%, and culture time 20 h) after
ultrasonic treatment (100 W) with different times.
26
27
Fig. 4. In vitro gastrointestinal tolerance of planktonic L.reuteri and L.reuteri biofilms on
zein/cellulose composite scaffolds (zein content 10%): (a) and (a’) SGF pH 2.5, (b) and (b’)
SGF pH 3.5, (c) and (c’) SIF pH 6.8, (d) and (d’) 0.03%, (e) and (e’) 0.1%, (f) and (f’) 0.3%.
28
(c) (d)
(e)
Fig. 5. Heatmaps exhibited the relative abundance of major bacteria profiling at (a) the phylum
level and (b) the genus level. Spearman correlation analysis diagram of the gut microbes in the
in vitro fecal fermentation after (c) 1 day and (d) 3 days. (e) Principal coordinate analysis of the
gut microbes in the in vitro fecal fermentation.
29
(c) (d)
Fig. 6. Effects of planktonic L. reuteri and L. reuteri biofilms on SCFAs production after (a) 1
day of fermentation and (b) 3 days of fermentation. Correlation between the SCFAs and
dominant microbes using the RDA ranking diagram of the genus horizontal species after (c) 1
day of fermentation and (d) 3 days of fermentation.
30
Declaration of Interest Statement
Macromolecules]
microbiota
Fei He, Xue-Ke Ma, Cheng-Kai Tu, Hui Teng, Xin Shao, Jie Chen, Meng-Xin Hu*
Food Safety Key Laboratory of Zhejiang Province, School of Food Science and
* Corresponding author.
Tel: + 86 571 28008976; Fax: + 86 571 28008900; E-mail address: mengxinhu@zjgsu.edu.cn (Meng-Xin Hu)
1
ABSTRACT
unhealthy states of the gut by reshaping gut microbiota. One criterion that probiotics are
efficacious is the capacity to survive in the gastrointestinal tract. Biofilm is the common
growth mode of microorganisms with high tolerances toward harsh environments. Suitable
scaffolds are crucial for successful biofilm culture and large-scale production of biofilm-
biofilm formation has not been studied. In this study, porous zein/cellulose composite
scaffolds provided nitrogen sources and carbon sources simultaneously at the solid/liquid
interfaces, being beneficial to the biofilm formation of Lactobacillus reuteri. The biofilms
showed 2.1 ~ 17.4 times higher tolerances in different gastrointestinal conditions. In human
fecal fermentation, the biofilms combined with the zein/cellulose composite scaffolds act as
the “synbiotics” positively modulating the gut microbiota and the short-chain fatty acids
(SCFAs), where biofilms provide probiotics and scaffolds provide prebiotics. The “synbiotics”
show a more positive regulation ability than planktonic L. reuteri. These results provide an
synbiotics
2
Introduction
The gut microbial ecosystem is closely related to host health and gains a lot of attention [1,
2]. Perturbations to the human gut microbiome can disrupt the stability of the ecosystem,
possibly resulting in recalcitrant unhealthy states associated with diseases [3]. Several
strategies have been developed to manipulate the gut microbiome to prevent the unhealthy
Among these strategies, supplementing probiotics and dietary fiber can reshape gut
microbiota and intestinal mucosa barrier and prevent or revert unhealthy states of the gut from
the gut ecosystem angle [7]. Probiotics are defined as "living microorganisms that, when
given in sufficient quantities, provide a health benefit to the host" [8]. Generally, probiotics
are found to maintain intestinal homeostasis through direct interaction with the intestine [9]
and indirect interaction through the produced metabolites, including SCFAs, with the intestine
[10]. However, the criterion that a probiotic must have to be considered efficacious is the
healthy adults, which can promote the secretion of vitamins, inhibit the growth and
commensal microbiota composition in the host [12]. As with all orally consumed probiotics,
the gastrointestinal tract of the host, including low pH, effectors of the host immune system,
as well as competition with commensal and pathogenic bacteria, all of which can greatly
reduce the availability of live bacteria for healthy or therapeutic purposes [13]. Hence, it is
(EPS) [16]. Previous studies have verified that Lactobacillus rhamnosus, Lactobacillus
fermentum, Lactobacillus acidophilus, and Lactobacillus casei can be encapsulated and form
biofilms in capsulates [17-19]. These probiotic biofilms show enhanced resistance to heat,
acid, and storage environments. Our previous research demonstrates that Lactobacillus
paracasei in biofilms shows excellent tolerance under different harsh environments compared
to planktonic L. paracasei, benefiting from the protection of EPS and the biofilm phenotype
[20]. Electrospun nanofibrous scaffolds show excellent properties in facilitating the biofilm
limited by the existing production method. Seeking green, inexpensive, and friendly scaffolds
suitable for biofilm formation of probiotics is the key point for successful biofilm culture and
The goal of this work is to prepare porous protein/carbohydrate composite scaffolds with
zein and cellulose for L. reuteri biofilm formation. The effect of the chemical composition on
the morphology of the scaffolds was studied in detail. Different porous zein/cellulose
composite scaffolds were utilized for biofilm formation and the culture conditions were
and bile salts were compared in vitro. In an in vitro human fecal fermentation model, the
differences between L. reuteri biofilm and their planktonic counterparts impacting the
2.1. Materials
4
Cellulose (cotton pulp, Mη 10.0×104) was purchased from Hubei Chemical Fiber Group Co.
Ltd. Other chemicals were purchased from Aladdin. L. reuteri DSM 17938 was commercially
The preparation of porous zein/cellulose composite scaffolds is based on the reported work
dissolved in NaOH/urea aqueous solution and cooled to -12 ℃. Cellulose powder (3 wt%) was
added and stirred at -12 ℃ for 15 min to dissolve cellulose. Epichlorohydrin (ECH) with a
concentration of 9 % was dropped into the solution and stirred at room temperature for 1 h.
The gained solution was frozen at -20 ℃ for 20 h and thawed at room temperature to gain
hydrogels, which were then cut into thin strips with a thickness of about 1~2 mm and washed
with distilled water until the solution was neutral. After that, the hydrogels were soaked in
distilled water for 3 days to reach swelling equilibrium. Finally, the hydrogels were frozen
with liquid nitrogen and freeze-dried to obtain porous zein/cellulose composite scaffolds.
The morphology of zein/cellulose composite scaffolds after coating a layer of gold was
The freeze-dried zein/cellulose composite scaffolds were rehydrated and autoclaved for 15
min at 121℃. The sterilized scaffolds (0.1% ~ 0.5 wt.%) were added to a 10 mL MRS liquid
medium with 1% L. reuteri inoculation. The anaerobic static culture was carried out at 37℃ to
5
Biofilms formed on zein/cellulose composite scaffolds were immersed in 2.5 %
glutaraldehyde solution for 6-8 h and then dehydrated with 30%, 50%, 70%, 80%, 90%, 95%,
100% ethanol solution in turn, where each dehydration time is 15 min. The dehydrated
samples were sprayed with gold for 90 s and the morphology of biofilms was observed with
SEM.
Different from the quantitation of planktonic L. reuteri, the quantitation of live cells in
biofilms relies on cell detachment followed by conventional plate counting. First, cells were
detached from biofilms formed on scaffolds by ultrasonic treatment. After culture duration,
the culture solution was centrifuged at 5000 rpm for 5 min at 4℃. The supernatant was poured
out and sterile normal saline was added with equal volume. Then cells were detached through
ultrasonic treatment (100 W). The detached L. reuteri supernatant was serially diluted 10-fold
and 50 μL aliquots were plated onto agar plates. Colony-forming units (CFU) were counted
In vitro gastrointestinal tolerance assay for L. reuteri was performed according to the
protocol previously described, with some modifications [24]. Simulated gastric fluid (SGF),
simulated intestinal fluid (SIF), and bile salt solution were freshly prepared before each
experiment. For SGF, HCl was diluted with deionized water to gain solutions with pH 2.5 and
3.5, separately, where pepsin concentration was 1 g/100 mL. For SIF, 6.8 g KH2PO4 was
dissolved in 500 mL deionized water, pH was adjusted to 6.8 with 0.4 g/100 mL NaOH
solution, trypsin was then added at a concentration of 10 g/100 mL, and finally the solution
was diluted to 1 L with water. For bile salt solutions, 0.03 g, 0.1 g, and 0.3 g pig bile salts
were dissolved in 100 mL of deionized water to obtain 0.03 %, 0.1 %, and 0.3 % artificial bile
salt solutions. Before the experiment, SGF, SIF, and bile salt solutions were filtered by
microporous membranes (0.22 μm). Planktonic L. reuteri and L. reuteri biofilms were added
6
into SGF, SIF, and bile salt solutions, separately. After digestion at 37℃ on a shaker (100
r/min), viable cells were determined by plate counting method with MRS agar plates. All
expressed in terms of Log (N/N0), where N0 and N represent the viable CFUs of samples
reuteri (≈ 1010 CFU), the obtained digested products were added into human fecal solutions
for in vitro fermentation. Fecal sample collection and pretreatment followed previously
reported work [22]. 1 L of nutrient medium was composed of peptone 2 g, yeast extract 2 g,
NaCl 0.1 g, K2HPO4 0.04 g, KH2PO4 0.04 g, MgSO4·7H2O 0.01 g, CaCl2·6H2O 0.01 g,
NaHCO3 2 g, and 80-Tween 2 mL [25]. After autoclave sterilization at 121 °C for 30 min,
heme 0.02 g, vitamin K1 10 mL, bile salt 0.5 g, and cysteine hydrochloride 0.5 g were added.
The human feces solution was composed of 27 mL nutrient medium and 3 mL fecal bacteria
extract, which was flushed by N2 for 5 min. The fermentation temperature was 37 °C and the
rotation speed was 75 r/min. After 24 h, 5 mL of gastrointestinal digested solution was added,
and 5 mL of sterile normal saline was added to the control group. The culture sustains 3 days,
during which no nutritional medium was added and 5 mL of fermentation broth was collected
every day and stored in the -20 °C refrigerator. After sampling, the solution was flushed by N2
again for 5 min and then the culture continued. All the collected samples were analyzed via
SCFAs in the collected samples were determined following the method described in
previously reported work with some modifications [22]. The sample (1 mL) was centrifuged
at 12000 rpm for 10 min at 4 °C and the supernatant was collected and mixed with 100 μL of
7
concentrated hydrochloric acid and 5 mL of ethyl ether for 20 min. Then the mixture was
centrifuged at 3500 rpm for 10 min at 4 °C. The top layer was collected and mixed with 500
μL of 1 M NaOH. After the second extraction and centrifugation, the water phase in the
bottom layer was collected. The bottom layer was filtered with a 0.22 μm filter after adding
100 μL concentrated hydrochloric acid. The SCFAs in the aliquot were determined by HPLC.
Column: YMC-PackProC-18 (250×4.6 mm); mobile phase 0.025% phosphoric acid solution
(pH 2.8): acetonitrile = 95:5, flow rate 1.0 mL/min; injection volume 20 μL; detection
Statistical analysis was carried out and graphs were generated using GraphPad Prism
software (Version 9.0.0). Statistical differences between the two groups were analyzed with
unpaired two-tailed Student t-tests. Symbols ***, **, and * represent highly significant values
(P<0.001), moderately significant values (P<0.01), and weakly significant values (P<0.05),
respectively. In addition, the alpha diversity of gut microbiota was analyzed by SPSS
Statistics.
The morphology of porous zein/cellulose composite scaffolds is shown in Fig. 1 (a). With
the increase in zein concentration, the number of large pores on the surface of the material
increased and the number of small pores decreased. When zein is not added, the strong
hydrogen bond interactions between cellulose molecules result in a powerful gel skeleton
network, which can withstand the growth of ice crystals during freeze-drying and give rise to
a lot of small pores [26]. Zein is rich in glycine, proline, glutamic acid, alanine, and arginine
[27]. When zein was mixed into the cellulose solution, the -NH2, the -COOH, and the -
CONH- in zein protein interacted with the -OH of cellulose and formed intermolecular
hydrogen bonds, which destroy the strong hydrogen bond interactions between cellulose
8
molecules. However, among biopolymers, zein is a hydrophobic protein with a significant
amount of non-polar amino acids (about 50% of total amino acids) [28]. The intermolecular
hydrogen bonds between cellulose molecules and zein molecules were partly influenced by
the non-polar amino acids of zein molecules. The strength of the zein/cellulose composite gel
skeleton network was weakened compared to the pure cellulose gel skeleton network.
Accordingly, big ice crystals formed during freeze-drying giving rise to a lot of large pores.
FT-IR spectra of the scaffolds are shown in Fig. 1 (b). The OH stretching vibration bands
around 3424 cm-1 in the pure cellulose scaffold broaden and shift to a lower wavenumber
3419 cm-1 in the zein/cellulose composite scaffolds, which is due to the introduction of zein
into the cellulose and the formation of new hydrogen bonds between zein and cellulose [27].
The alkyl stretching of zein is in the range of 2834 cm-1 ~ 3004 cm-1, which can be found in
the FT-IR spectra of the zein/cellulose composite scaffolds. The peak at 1450 cm-1 is
attributed to the aromatic ring skeleton stretching vibration of aromatic amino acids
(phenylalanine and tyrosine) in zein molecules and the peak also can be found in the FT-IR
spectra of the zein/cellulose composite scaffolds but shift to 1459 cm-1. The results of FT-IR
demonstrate that the composite scaffolds are composed of both zein and cellulose. The
interactions between zein and cellulose at the molecular level closely affect the morphology
of the zein/cellulose composite scaffolds. The increased zein concentration not only results in
large pores which are more beneficial for L. reuteri spread into the inner of the scaffolds, but
also provide more nitrogen sources for L. reuteri growth and biofilm formation.
Fig. 2 (a) shows the influences of the chemical composition and dosage of scaffolds on L.
reuteri biofilm formation. L. reuteri biofilms formed on the scaffolds increased with the zein
content in scaffolds and the scaffold dosage in the culture solution. As the ratio of
zein/cellulose was 10%, viable cell densities in biofilms on the composite scaffolds were
9
significantly higher than that on the pure cellulose scaffolds (P<0.01, P<0.001, and P<0.01
for 0.1%, 0.3 %, and 0.5%, respectively). The introduction of zein into the scaffolds provides
extra nitrogen sources and protein nutrients for L. reuteri growth, making L. reuteri biofilm
grow better. The results demonstrate that the composite of protein and carbohydrate in the
scaffolds provides nitrogen sources and carbon sources simultaneously at the solid/liquid
interface and is more beneficial to L. reuteri biofilm formation. SEM pictures in Fig. 2 (b)
show dense L. reuteri biofilms were formed on zein/cellulose composite scaffolds with 0.3%
In addition, the increasing scaffold dosage in the culture medium provides more surface
and space for L. reuteri adhesion and biofilm formation, resulting in more L. reuteri existing
as the biofilm phenotype (Fig. 2 (c)). The results are similar to L. paracasei biofilm formation
on electrospun cellulose acetate nanofibrous scaffolds [20]. When the scaffold dosage
increased from 0.1% to 0.3%, the number of viable cells of biofilms on scaffolds significantly
increased. Then, a further increase in the scaffold dosage cannot promote more biofilm
formation, which could result from the limited nutrition provided by the culture medium.
Furthermore, the culture time shows an influence on the biofilm formation similar to the
growth curve of planktonic L. reuteri (Fig. 2 (d) and Fig. S1). The number of viable cells on
the 10% zein/ cellulose composite scaffold increased from 7.94 Log CFU /mL to 9.15 Log
CFU /mL as the culture duration increased from 4 h to 20 h. Then the number of viable cells
slightly decreased and gradually stabilized. This phenomenon may be due to the lack of
nutrients and the low pH and other accumulated metabolites of the culture medium inhibiting
the growth of L. reuteri as the culture time increased. SEM pictures in Fig. 2 (e) demonstrate
that only a small amount of L. reuteri adhered on the surface of the scaffold after a 4 h culture
duration. Then obvious L. reuteri biofilm formed on the surface of the scaffold after an 8 h
10
culture duration. After that time, L. reuteri biofilm became more and more dense with the
The foregoing results indicate that L. reuteri biofilms formed on the porous zein/cellulose
composite scaffolds very well. The EPS matrix acts like a biological ‘glue’ [29] enabling L.
reuteri to adhere to the scaffold surface. Simply rinsing makes it hard to release cells from
biofilms. To determine the number of viable cells in biofilms, samples were handled by
ultrasonic treatment to release cells from biofilms. As shown in Fig. 3 (a), the number of
viable cells was the highest when the ultrasound time was 10 min, and there was a significant
difference compared with the ultrasound time of 5 min (P<0.01). Sufficient ultrasound time
caused more cells released from biofilms. However, the number of viable cells decreased as
ultrasound time was more than 10 min. As shown in SEM pictures in Fig. 3 (b), the residual L.
reuteri biofilms lessen when the ultrasonic time is more than 25 min, but the quantified
number of viable cells still is low. It’s because ultrasound is a relatively drastic method that
could cause cell rupture and death over a long time [30]. Therefore, the ultrasonic treatment
combined with the plate counting method to quantify the number of viable cells in biofilms
results in low values less than the true values. These results demonstrate that L. reuteri
bacteria are not easy to release from biofilms under simple solution conditions in vitro.
Although the influence of L. reuteri biofilms on the gut microbiota during in vitro fecal
fermentation has been studied and shown in this work, the release behaviors of probiotics
The potential health benefits of probiotics may not be realized because of the substantial
reduction in their viability during gastrointestinal digestion [31]. High tolerances of probiotics
toward low pH of gastric fluid, diverse digestive enzymes, and bile salts in the gastrointestinal
11
tracts are important guarantees to perform the probiotic functions. As shown in Fig. 4(a), Fig.
4(b), and Fig. 4(c), planktonic L. reuteri decreased 0.23 Log CFU/mL, 0.14 Log CFU/mL,
and 0.10 Log CFU/mL after 3 h digestion in SGF with pH 2.5 and pH 3.5 and in SIF with pH
6.8, respectively, where the numbers of viable cells between the 0 h group and the 3 h group
exist significant differences in these digestion conditions (P<0.001, P<0.01, and P<0.05,
respectively). In contrast, L. reuteri in biofilm phenotype increased the viable cells with 0.11
Log CFU/mL, 0.31 Log CFU/mL, and 0.27 Log CFU/mL in SGF with pH 2.5 and pH 3.5 and
in SIF with pH 6.8 after 3 h digestion, where the numbers of viable cells between the 0 h
group and the 3 h group exist significant differences in SGF with pH 3.5 (P<0.01) and SIF
with pH 6.8 (P<0.001). These results demonstrate that L. reuteri in biofilm phenotype has
higher tolerances than planktonic L. reuteri in SGF and SIF, showing stronger survival ability
toward low pH and diverse digestive enzymes. The longer the digestion time, the greater the
impact of SGF and SIF on the planktonic cells resulting in the death of L. reuteri. Viable cells
of the biofilm groups in SGF and SIF increased with the digestion time due to the EPS acting
as “protective clothing” for the embedded L. reuteri [32]. On the other hand, differentially
In the gastrointestinal tracts, bile salts secreted by the small intestine reduce bacterial
survival by disrupting cell membranes, leaking out intracellular material, inducing protein
misfolding and denaturing and damaging the DNA [33, 34]. Bile salts at a concentration of
0.3% are the maximum that can be found in an average healthy person [35]. In our work, the
negative effects of bile salts on L. reuteri are shown in Fig. 4 (d)-(f’). The survival rates of L.
reuteri both in the planktonic state and in the biofilm state decrease with the concentration of
bile salts and the digestion time. Planktonic L. reuteri decreased 0.74 Log CFU/mL, 1.31 Log
CFU/mL, and 2.62 Log CFU/mL after 3 h digestion in bile salts solution with 0.03%, 0.1%,
and 0.3% concentration, respectively, where the numbers of viable cells between the 0 h
12
group and the 3 h group exist significant differences in these digestion conditions (P<0.0001).
L. reuteri in biofilm phenotype decreases the viable cells with 0.20 Log CFU/mL, 0.47 Log
CFU/mL, and 1.38 Log CFU/mL after 3 h digestion in bile salts solution with 0.03%, 0.1%,
and 0.3% concentration, respectively. The survival ability of L. reuteri in biofilm phenotype
is far beyond that of planktonic L. reuteri. Many researchers verified that microbes including
probiotics respond to bile salts exposure by biofilm formation to avoid bactericidal effects of
high bile concentration [36, 37]. On the other hand, the EPS acting as “protective clothing”
for the embedded L. reuteri limits the diffusion of bile salts into the inner layer of EPS, which
In our work, L. reuteri biofilms benefit greatly from the biofilm phenotype and the
“protective clothing” of EPS. There are about 2.1 ~ 2.8 times more L. reuteri survival abilities
after exposure to SGF with pH 2.5 and pH 3.5 and in SIF with pH 6.8 after 3 h digestion in
biofilms compared with the planktonic counterparts. Moreover, the tolerance abilities of L.
reuteri biofilms are 3.5, 6.9, and 17.4 times higher than the tolerance abilities of planktonic L.
reuteri in bile salts solutions with different concentrations (0.03%, 0.1%, and 0.3%) after 3 h
biofilm L. reuteri verify that L. reuteri in biofilm phenotype is more effective than probiotics.
3.5. Influence of L. reuteri biofilms on gut microbiota during in vitro fecal fermentation
Table 1 shows the influence of L. reuteri in different phenotypes on the alpha diversity of
zein/cellulose composite scaffold (M group), and L. reuteri biofilm (B/M group) decreased
gut microbiota diversity (decreased Shannon indices and increased Simpson indices) and
richness (decreased Sobs, Chao, and Ace indices) after 1-day fermentation compared to the
Control group. The short-term dietary intervention results in a slight but significant decrease
in microbial diversity, which could be called “transitory microbial stress” [38]. After 3 days
13
of fermentation, the P group, M group, and B/M group show increased gut microbiota
diversity and richness compared to the Control group, where the B/M group has the highest
gut microbiota diversity and richness and shows significant differences with the P group in
Shannon indices and Sobs indices. The results demonstrate that planktonic L. reuteri,
zein/cellulose composite scaffold, and L. reuteri biofilm on scaffold improve the community
diversity, especially L. reuteri biofilm on scaffold. Previous study has shown that biofilms
enhance the diversity and metabolic activity of microbial communities [39]. In our study, L.
reuteri biofilm and the scaffold synergistically affect the diversity and richness of the gut
microbiota in vitro.
The microbial communities at the phylum and genus levels in the in vitro fecal
fermentation are shown in Fig. 5 (a) and Fig. 5 (b), separately. The Lactobacillus genus
significantly increased and the Acinetobacter genus decreased in the P groups and B/M
groups compared to the control groups. In addition, the Bifidobacterium genus decreased in
the P groups, but increased in the M groups and B/M groups, suggesting that the
acts as the prebiotics. The B/M group shows lower abundances of Klebsiella, Bilophila, and
Bacteroides and a higher abundance of Bifidobacterium than the P group after 1 day of
fermentation. When the fermentation time extends to 3 days, the B/M group shows lower
Bifidobacterium and Bilophila than the P group. Acinetobacter commonly causes nosocomial
and catheter-associated bacteremia, but can also cause soft tissue and urinary tract infections
[40]. Klebsiella exists in the intestinal tract and respiratory tract of normal people and is a
kind of opportunistic pathogen, which is prone to infection in people with low immunity and
those undergoing surgical and invasive iatrogenic procedures [41]. Bacteroides are symbiotic
14
with humans and help break down food to produce nutrients and energy that the body needs.
However, when Bacteroidetes enter parts of the body other than the gastrointestinal area, they
can cause or exacerbate infections such as abscesses [42]. On the other hand, human gut
bacteria in the genus Bilophila have been found to metabolize both trimethylamine and its
the first microbes to colonize the human intestine naturally, their abundance and diversity in
the colon are closely related to host health [44]. The results shown in Fig. 5 (a) and Fig. 5 (b)
The Spearman correlation analysis in Fig. 5 (c) and Fig. 5 (d) shows the positive and
negative correlations between these gut microbes in the in vitro fecal fermentation. Principal
coordinate analysis of the gut microbes in Fig. 5 (e) demonstrates that the species composition
of the control group was different from that of the P groups, M groups, and B/M groups after
the in vitro fecal fermentation. The relative distances between the 1-day fermentation groups
and the 3-day fermentation groups also were far away, indicating that there were differences
in the species composition between them and the fermentation time affects the species
composition.
3.6. Influence of L. reuteri biofilms on SCFAs production during in vitro fecal fermentation
SCFAs are mainly composed of acetic acid, propionic acid, and butyric acid, which play an
important role in host physiology such as energy metabolism, glucose homeostasis, lipid
production regulation, and immune regulation [10, 45]. The influence of L. reuteri biofilms
on the concentration of SCFAs is shown in Fig. 6. After 1 day of fermentation, the total
concentration of SCFAs in the B/M group was 3.96 mg /mL, which was significantly higher
than that of the control group with 1.71mg /mL. After 3 days of fermentation, the total
15
concentration of SCFAs decreased, which could be attributed to the volatilization of SCFAs.
However, the concentrations of acetic acid, propionic acid, and butyric acid in the P groups,
M groups, and B/M groups were significantly higher than the concentrations in the control
groups (P<0.0001). Each SCFA in the B/M groups had the highest concentration and was
significantly different from the P groups and the M groups (Fig. 6 (b)). Both the scaffolds and
the supplied L. reuteri and other SCFA production-related bacteria with increased abundances
in the fecal fermentation system enhanced the whole SCFA production. Hence, the B/M
Redundancy analysis (RDA) in Fig. 6 (c) and Fig. 6 (d) demonstrate that microbial
correlated with SCFAs. After 1 day of fermentation, the biggest effects of the control group,
the P group, M group, and B/M group on the microbial communities were Acinetobacter,
Bilophila, Bilophila, and Bifidobacterium, respectively. On the other hand, the biggest effects
of the control group, the P group, M group, and B/M group on the microbial communities
and Bilophila was positively correlated with acetic acid and propionic acid. The cluster of
Lactobacillus, Cloacibacillus, and Bilophila was positively correlated with butyric acid.
4. Conclusion
In this study, zein and cellulose were dissolved in NaOH/urea aqueous solution at low
freeze-drying technique. The composite of protein (zein) and carbohydrate (cellulose) in the
scaffolds is more beneficial to L. reuteri biofilm formation than the pure carbohydrate
showed enhanced tolerances toward low pH of gastric fluid, diverse digestive enzymes, and
16
bile salts in the in vitro gastrointestinal conditions. The EPS layer acting as “protective
clothing” for the embedded L. reuteri limits the diffusion of low pH, enzymes, and bile salts
into the inner layer of EPS, which follows to prevent the direct interactions with L. reuteri
cells and the killings of L. reuteri cells. In human fecal fermentation, L. reuteri biofilms on
showing a more positive regulation ability of gut microbiota than planktonic L. reuteri. Also,
L. reuteri biofilms on the scaffolds result in the highest SCFA production in the human fecal
fermentation solutions. The scaffolds and the supplied L. reuteri and other SCFA production-
related bacteria with increased abundances in the fecal fermentation system synergistically
enhanced the whole SCFA production accordingly. L. reuteri biofilms combined with the
zein/cellulose composite scaffolds act as the “synbiotics” positively modulating the gut
microbiota and the SCFAs, where biofilms provide probiotics and cellulose belonging to
indigestible carbohydrates provide prebiotics, and play a more active role in regulating gut
Fei He: Investigation, Methodology, Formal analysis, Data curation, Writing-original draft,
Teng: Investigation. Xin Shao: Investigation. Jie Chen: Methodology. Meng-Xin Hu:
Data availability
Acknowledgement
17
This project is financially supported by the Natural Science Foundation of Zhejiang
Province (Grant no. LTGN24C200005) and the Fundamental Research Funds for the
18
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22
Table 1. Alpha diversity after 1 day and 3 days of culture duration in vitro fecal fermentation of L. reuteri and L. reuteri biofilms.
23
0 5% 10% 20%
(a)
Fig. 1. (a) SEM images of zein/cellulose composite scaffolds with different zein contents
(zein/cellulose), top images ×200, bottom images ×500; (b) FT-IR spectra of zein, cellulose,
and zein/cellulose composite scaffolds with different zein concentrations.
24
0 5%
10% 20%
(b)
0.1% 0.2%
0.3% 0.5%
(c)
4h 8h 12 h
16 h 20 h 24 h
(e)
Fig. 2. (a) Effects of the zein/cellulose composite scaffolds amount added into culture
solution on the density of L. reuteri (culture time 20 h); (b) SEM images of L. reuteri biofilms
formed on zein/cellulose composite scaffolds with different zein content (scaffold amount
25
amount 0.3% and cultured time 20 h); (c) SEM images of L. reuteri biofilms formed on the
zein/cellulose composite scaffold (10% zein) with different scaffold amount added in the
culture solution (culture time 20 h); (d) the effect of culture time on the density of L. reuteri
(scaffold amount 0.3% and zein content 10%); (e) SEM images of L. reuteri biofilms formed
on the zein/cellulose composite scaffold (10% zein) with different culture time.
26
5 min 10 min 15 min
(b)
Fig. 3. (a) The effect of ultrasonic time on the quantitative values of alive L. reuteri (scaffold
amount 0.3% and zein content 10%); (b) SEM images of L. reuteri biofilms on zein/cellulose
composite scaffolds (scaffold amount 0.3%, zein content 10%, and culture time 20 h) after
ultrasonic treatment (100 W) with different times.
27
28
Fig. 4. In vitro gastrointestinal tolerance of planktonic L.reuteri and L.reuteri biofilms on
zein/cellulose composite scaffolds (zein content 10%): (a) and (a’) SGF pH 2.5, (b) and (b’)
SGF pH 3.5, (c) and (c’) SIF pH 6.8, (d) and (d’) 0.03%, (e) and (e’) 0.1%, (f) and (f’) 0.3%.
29
(c) (d)
(e)
Fig. 5. Heatmaps exhibited the relative abundance of major bacteria profiling at (a) the
phylum level and (b) the genus level. Spearman correlation analysis diagram of the gut
microbes in the in vitro fecal fermentation after (c) 1 day and (d) 3 days. (e) Principal
coordinate analysis of the gut microbes in the in vitro fecal fermentation.
30
(c) (d)
Fig. 6. Effects of planktonic L. reuteri and L. reuteri biofilms on SCFAs production after (a) 1
day of fermentation and (b) 3 days of fermentation. Correlation between the SCFAs and
dominant microbes using the RDA ranking diagram of the genus horizontal species after (c) 1
day of fermentation and (d) 3 days of fermentation.
31
Supplementary Material