International Journal of Biological Macromolecules

Porous zein/cellulose composite scaffolds for Lactobacillus reuteri biofilms formation:

Synbiotics to enhance gastrointestinal tolerances of probiotics and regulate intestinal
microbiota
--Manuscript Draft--

Manuscript Number: IJBIOMAC-D-23-14468R1

Article Type: Research Paper

Section/Category: Carbohydrates, Natural Polyacids and Lignins

Keywords: porous zein/cellulose composite scaffolds, Lactobacillus reuteri biofilm, synbiotics

Abstract: Supplementing probiotics or indigestible carbohydrates is a usual strategy to prevent or

revert 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 probiotics. However, the role of scaffolds
containing indigestible carbohydrates in 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
understanding of the beneficial effects of carbohydrates and proteins as the scaffolds
of probiotics biofilms and the synergistic effects of biofilm-phenotype probiotics and
indigestible carbohydrates in gut microbiota modulation.

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Response to Reviewers

Manuscript Number: IJBIOMAC-D-23-14468

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.

Line 7 recalcitrant? Let's explain this part better

Answer: The word “recalcitrant” describes the difficulty of the unhealthy states to
manage or operate.

Line 12 why? It is not understandable to review

Answer: Thanks for your suggestion. 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”.

Line 14, why with indigestible carbohydrates? to explain better

Answer: Indigestible carbohydrates have the potential to be used as prebiotics (a
substrate that is selectively utilized by host microorganisms conferring a health
benefit, Nature Reviews Gastroenterology & Hepatology 14, 491–502 (2017))

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.

It is not understood please rewrite, there is a lack of bibliography. Interestingly

enough, biofilm as the most common growth mode of microorganisms in nature
possesses a self-produced protective barrier, named extracellular polymeric
substances (EPS)
Answer: Thanks for your suggestion. Reference 16 has been cited here.

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.”

2. Rationale and Context: a. The abstract effectively highlights the importance of

probiotics and non-digestible carbohydrates in maintaining gut health. It would be
valuable to include a sentence emphasizing the current gaps in knowledge or existing
challenges addressed by this study.
Answer: Thanks for your kind suggestion. The sentence, “However, the role of
scaffolds containing indigestible carbohydrates in biofilm formation has not been
studied.”, has been added in the Abstract.

3. Methodology and Experimental Design: a. Further details on the specific procedure

for preparing the porous zein/cellulose composite scaffolds would enhance the
scientific rigor of the abstract. Including key parameters or techniques used in the
scaffold preparation could contribute to the clarity of the methods section.
Answer: Thanks for your suggestion. We have tried our best to present all the key
parameters and techniques used in the scaffold preparation in the manuscript. The
thickness of the strips of the hydrogels is added in Section 2.2. Please let us know if
there is anything that you want to know.

4. Results and Findings: a. The abstract effectively communicates the enhanced

tolerances of biofilm-phenotype Lactobacillus reuteri in gastrointestinal conditions.
Providing a glimpse into the quantitative results or statistical significance, if available,
would strengthen the impact of the findings. b. Clarify if the study explored different
concentrations or variations of zein/cellulose composite scaffolds to optimize biofilm
formation.
Answer: We greatly appreciate this suggestion. The sentence “The biofilms showed
2.1 ~ 17.4 times higher tolerances in different gastrointestinal conditions.” has been
added to the Abstract. In addition, the influences of the concentration of zein and the
scaffold amount used in the culture solution on the biofilm formation are shown in
Figure 2. The results demonstrate that 0.3 wt% scaffold amount, 10% zein content,
and 20 h culture time are the best conditions for biofilm formation.

5. Significance and Novelty: a. The abstract mentions the synergistic effects of

biofilm-phenotype probiotics and non-digestible carbohydrates in gut microbiota
modulation. To enhance the abstract's impact, consider briefly discussing the novelty
of these findings in comparison to existing literature.
Answer: Thanks for your kind suggestion. The last three sentences, “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 understanding of the beneficial effects of carbohydrates and
proteins as the scaffolds of probiotics biofilms and the synergistic effects of
biofilm-phenotype probiotics and non-digestible carbohydrates in gut microbiota
modulation.”, discuss the novelty of the synergistic effects of biofilm-phenotype
probiotics and non-digestible carbohydrates in gut microbiota modulation. In addition,
the Abstract has been revised to strengthen the impact of our findings.

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.

7. Language and Terminology: Clarity and Detail in Morphology Analysis: a. The

explanation of the morphology changes in zein/cellulose composite scaffolds due to
zein concentration lacks clarity. The description is intricate and might benefit from
simplification. Consider providing a summary or key points for better comprehension.
b. It would be beneficial to include quantitative data, such as pore sizes at different
zein concentrations, to support the observed changes in the scaffolds.
Answer: Thanks for the suggestion. Section 3.1 has been revised. As seen from
Figure 1(a), small pores coexist with large pores in the scaffolds. It’s hard to give the
pore sizes of the scaffolds at different zein concentrations. Qualitative analysis is a
convenient way to discuss the morphology changes in the scaffolds.

8. FT-IR Analysis: a. The FT-IR analysis is informative, but consider providing a

brief summary or key findings to help readers grasp the main changes in the
zein/cellulose composite scaffolds. b. Discuss the biological significance of the
observed changes in the FT-IR spectra, linking them to the subsequent biofilm
formation.
Answer: Thanks for your suggestion. Section 3.1 has been revised and the biological
significance of the observed changes in the SEM pictures and the FT-IR spectra has
been linked to the biofilm formation in the last sentence. “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.”

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.

10. Ultrasonic Treatment: a. While the ultrasonic treatment section is informative,

consider discussing the potential limitations or challenges associated with this method
for releasing cells from biofilms.
Answer: Thanks for this good suggestion. The sentence, “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 our future work.”,
has been added here. Although it is not easy, we hope we can design a suitable
experiment in the next work and answer this question soon.

11. Gastrointestinal Tolerance: a. The gastrointestinal tolerance results are presented

clearly. However, provide a brief discussion on the potential implications of the
observed differences in tolerances between planktonic and biofilm L. reuteri for their
effectiveness as probiotics.
Answer: Thanks for this good suggestion. The sentence, “These observed differences
in tolerances between planktonic and biofilm L. reuteri verify that L. reuteri in
biofilm phenotype is more effective as probiotics.”, is added at the last of Section 3.4.

12. Influence on Gut Microbiota: a. The influence of L. reuteri biofilms on gut

microbiota diversity is well-documented. However, discuss the potential biological
significance of these changes and how they relate to the study's overall objectives. b.
The interpretation of the microbial communities at the genus level could be enhanced
by discussing known functions or roles of these genera in gut health.
Answer: Thanks. The sentence, “In our study, L. reuteri biofilm and the scaffold
synergistically affect the diversity and richness of the gut microbiota in vitro.”, is
added in the first paragraph of Section 3.5. In addition, the latter half of the second
paragraph in Section 3.5 presents the interpretation of the microbial communities at
the genus level.

13. SCFAs Production: a. The impact of L. reuteri biofilms on SCFAs production is

well-discussed. However, provide insights into the potential health implications of
increased SCFAs production, linking it back to the study's context.
Answer: The high richness of genus Lactobacillus, Bifidobacterium, Cloacibacillus,
and Bilophila in the P group, M group, and B/M group, especially the B/M group,
results from the L. reuteri bacteria and the scaffold. Here, the sentence, “Hence, the
B/M groups have double driving forces to strengthen SCFAs production.”

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 1: Change …planktonic L. reuteri biofilms and L. reuteri… to … L.

reuteri biofilms and planktonic L. reuteri…
Answer: Thanks very much. The sentence has been revised as “After the sequential
gastrointestinal digestion of L. reuteri biofilms and planktonic L. reuteri (≈ 1010
CFU),”.

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

Supplementing probiotics or indigestible carbohydrates is a usual strategy to prevent or revert

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

probiotics. However, the role of scaffolds containing indigestible carbohydrates in 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

understanding of the beneficial effects of carbohydrates and proteins as the scaffolds of

probiotics biofilms and the synergistic effects of biofilm-phenotype probiotics and indigestible

carbohydrates in gut microbiota modulation.

1
Revised manuscript (clean version) Click here to view linked References

[Prepared as an article for publication in International Journal of Biological Macromolecules]

Porous zein/cellulose composite scaffolds for Lactobacillus reuteri biofilms formation:

Synbiotics to enhance gastrointestinal tolerances of probiotics and regulate intestinal

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,

Zhejiang Gongshang University, Hangzhou 310018, China.

* Corresponding author.
Tel: + 86 571 28008976; Fax: + 86 571 28008900; E-mail address: mengxinhu@zjgsu.edu.cn (Meng-Xin Hu)
1
ABSTRACT

Supplementing probiotics or indigestible carbohydrates is a usual strategy to prevent or revert

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

probiotics. However, the role of scaffolds containing indigestible carbohydrates in 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

understanding of the beneficial effects of carbohydrates and proteins as the scaffolds of

probiotics biofilms and the synergistic effects of biofilm-phenotype probiotics and indigestible

carbohydrates in gut microbiota modulation.

Keywords: porous zein/cellulose composite scaffolds, Lactobacillus reuteri biofilm, 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 states or revert

to healthy states caused by perturbations, including fecal microbiota transplantation [4],

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

survive in the gastrointestinal tract [11].

Lactobacillus reuteri is an important member of the intestinal microflora of newborns and

healthy adults, which can promote the secretion of vitamins, inhibit the growth and colonization

of pathogenic microbes by producing antimicrobial molecules, and remodel the commensal

microbiota composition in the host [12]. As with all orally consumed probiotics, the Gram-

positive bacterium L. reuteri encounters numerous challenges as it transits through 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 important to

enhance the tolerance of L. reuteri toward the gastrointestinal environment. As reported,

microencapsulated L. reuteri in polysaccharides and proteins show enhanced survival in the

3
gastrointestinal environment, where polysaccharides and proteins act as a protective barrier to

L. reuteri from harsh environments [14, 15].

Interestingly enough, biofilm as the most common growth mode of microorganisms in nature

possesses a self-produced protective barrier, named extracellular polymeric substances (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 formation of

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

production of biofilm-phenotype probiotics.

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. Materials and methods

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

obtained (Bio Gaia Probiotics, Sweden).

2.2. Preparation of porous zein/cellulose composite scaffolds

The preparation of porous zein/cellulose composite scaffolds is based on the reported work

with some modifications [23]. Zein (5 %, 10 %, and 20 % of cellulose concentration) was

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.

2.3. Structures of scaffolds

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

of zein/cellulose composite scaffolds were characterized by Fourier transform infrared (FT-IR,

Nicolet iS5, Thermo Scientific).

2.4. Biofilm culture

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

form biofilms on the scaffolds.

2.5. Morphology of biofilms

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.

2.6. Quantitation of live cells in biofilms

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

after 48 h of colony growth at 37°C.

2.7. In vitro gastrointestinal tolerance assay

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.

2.8. Influences of L. reuteri biofilms on in vitro human fecal microbiota

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

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 16S rDNA by Majorbio Bio-Pharm

Technology Co. Ltd. (Shanghai, China).

2.9. Influences of L. reuteri biofilms on SCFA production

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

nm; column temperature 30 °C.

2.10. Statistical Analysis.

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.

3. Results and discussion

3.1. Effect of zein concentration on the structure of zein/cellulose composite scaffolds

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

formed during freeze-drying giving rise to a lot of large pores.

--- Fig. 1 ---

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.

3.2. L. reuteri biofilms formed on porous zein/cellulose composite scaffolds

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.

--- Fig. 2 ---

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.

3.3. Effect of ultrasonic time on quantification of viable cells in L. reuteri biofilms

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

our future work.

--- Fig. 3 ---

3.4. Tolerances of L. reuteri biofilms toward gastrointestinal environments

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

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

expressed genes in tolerance by biofilms as compared to their planktonic counterparts make L.

reuteri in biofilm-phenotype possess enhanced tolerances toward harsh environments [20].

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

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.
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

digestion, respectively. These observed differences in tolerances between planktonic and

biofilm L. reuteri verify that L. reuteri in biofilm phenotype is more effective than probiotics.

--- Fig. 4 ---

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

affect the diversity and richness of the gut microbiota in vitro.

--- Table 1 ---

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

P group. Acinetobacter commonly causes nosocomial infections primarily in

immunocompromised patients, predominantly aspiration pneumonia 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 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.

--- Fig. 5 ---

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

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
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

double driving forces to strengthen SCFAs production.

Redundancy analysis (RDA) in Fig. 6 (c) and Fig. 6 (d) demonstrate that microbial

communities Lactobacillus, Bifidobacterium, Cloacibacillus, and Bilophila were positively

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

3 days of fermentation were Lachnoclostridium, Lactobacillus, Bifidobacterium, and

Lactobacillus, respectively. The cluster of Lactobacillus, Bifidobacterium, Cloacibacillus, and

Bilophila was positively correlated with acetic acid and propionic acid. The cluster of

Lactobacillus, Cloacibacillus, and Bilophila was positively correlated with butyric acid.

--- Fig. 6 ---

4. Conclusion

In this study, zein and cellulose were dissolved in NaOH/urea aqueous solution at low

temperatures to prepare porous protein/carbohydrate composite scaffolds through a vacuum

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.

Compared to planktonic L. reuteri, L. reuteri in biofilms on the composite scaffolds showed

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

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 microbiota and producing SCFAs as

compared with planktonic L. reuteri.

CRediT authorship contribution statement

Fei He: Investigation, Methodology, Formal analysis, Data curation, Writing-original draft,

Visualization. Xue-Ke Ma: Investigation, Methodology. Cheng-Kai Tu: Investigation. Hui

Teng: Investigation. Xin Shao: Investigation. Jie Chen: Methodology. Meng-Xin Hu:

Conceptualization, Methodology, Project administration, Writing - Review & Editing,

Visualization, Funding acquisition.

Declaration of competing interest

The authors declare no conflicts of interest.

Data availability

Data will be made available on request.

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

Universities of Zhejiang (3090JYN9922001G-016).

17
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21
Table 1. Alpha diversity after 1 day and 3 days of culture duration in vitro fecal fermentation of L. reuteri and L. reuteri biofilms.

Culture Time Samples Shannon Sobs Simpson Ace Chao

Control 3.5538±0.0609b 568.5±21.92 0.1260±0.0288c
a
638.52±29.33a 629.55±31.26a
P 3.1107±0.0346cd 490.5±2.12d 0.1419±0.0238c 559.97±15.32c 553.67±21.54c
1 day
M 3.2317±0.0370c 519.5±3.53c 0.1370±0.0230c 598.69±0.42b 590.34±2.64abc
B/M 3.0920±0.0354cd 505.0±4.24cd 0.1442±0.0204c 582.89±0.02bc 584.54±0.06bc
Control 2.8885±0.0683d 510.5±6.36cd 0.2044±0.0100b 604.72±18.90ab 602.67±22.69ab
P 3.6105±0.1420b 525.0±2.82c 0.0740±0.0080d 604.25±6.23ab 602.65±5.97ab
3 days
M 3.7217±0.1413ab 519.5±2.12c 0.0674±0.0114d 604.64±5.79ab 623.24±4.93ab
B/M 3.8463±0.1427a 547.0±4.24b 0.0571±0.0074d 615.07±11.24ab 604.42±15.90ab

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

20 min 25 min 30 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

Declaration of Interest Statement

All the authors declare that they have no conflict of interest.

Revised manuscript (with changes marked)

[Prepared as an article for publication in International Journal of Biological

Macromolecules]

Porous zein/cellulose composite scaffolds for Lactobacillus reuteri biofilms formation:

Synbiotics to enhance gastrointestinal tolerances of probiotics and regulate intestinal

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, Zhejiang Gongshang University, Hangzhou 310018, China.

* Corresponding author.
Tel: + 86 571 28008976; Fax: + 86 571 28008900; E-mail address: mengxinhu@zjgsu.edu.cn (Meng-Xin Hu)
1
ABSTRACT

Supplementing probiotics or indigestible carbohydrates is a usual strategy to prevent or revert

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 probiotics. However, the role of scaffolds containing indigestible carbohydrates in

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

understanding of the beneficial effects of carbohydrates and proteins as the scaffolds of

probiotics biofilms and the synergistic effects of biofilm-phenotype probiotics and

indigestible carbohydrates in gut microbiota modulation.

Keywords: porous zein/cellulose composite scaffolds, Lactobacillus reuteri biofilm,

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

states or revert to healthy states caused by perturbations, including fecal microbiota

transplantation [4], 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 survive in the gastrointestinal tract [11].

Lactobacillus reuteri is an important member of the intestinal microflora of newborns and

healthy adults, which can promote the secretion of vitamins, inhibit the growth and

colonization of pathogenic microbes by producing antimicrobial molecules, and remodel the

commensal microbiota composition in the host [12]. As with all orally consumed probiotics,

the Gram-positive bacterium L. reuteri encounters numerous challenges as it transits through

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

important to enhance the tolerance of L. reuteri toward the gastrointestinal environment. As

reported, microencapsulated L. reuteri in polysaccharides and proteins show enhanced

3
survival in the gastrointestinal environment, where polysaccharides and proteins act as a

protective barrier to L. reuteri from harsh environments [14, 15].

Interestingly enough, biofilm as the most common growth mode of microorganisms in

nature possesses a self-produced protective barrier, named extracellular polymeric substances

(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

formation of 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 production of biofilm-phenotype probiotics.

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. Materials and methods

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

obtained (Bio Gaia Probiotics, Sweden).

2.2. Preparation of porous zein/cellulose composite scaffolds

The preparation of porous zein/cellulose composite scaffolds is based on the reported work

with some modifications [23]. Zein (5 %, 10 %, and 20 % of cellulose concentration) was

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.

2.3. Structures of scaffolds

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 of zein/cellulose composite scaffolds were characterized by Fourier transform

infrared (FT-IR, Nicolet iS5, Thermo Scientific).

2.4. Biofilm culture

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

form biofilms on the scaffolds.

2.5. Morphology of biofilms

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.

2.6. Quantitation of live cells in biofilms

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

after 48 h of colony growth at 37°C.

2.7. In vitro gastrointestinal tolerance assay

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

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.

2.8. Influences of L. reuteri biofilms on in vitro human fecal microbiota

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 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

16S rDNA by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

2.9. Influences of L. reuteri biofilms on SCFA production

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

wavelength 210 nm; column temperature 30 °C.

2.10. Statistical Analysis.

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.

3. Results and discussion

3.1. Effect of zein concentration on the structure of zein/cellulose composite scaffolds

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.

--- Fig. 1 ---

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.

3.2. L. reuteri biofilms formed on porous zein/cellulose composite scaffolds

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%

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.

--- Fig. 2 ---

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

further increasing culture duration.

3.3. Effect of ultrasonic time on quantification of viable cells in L. reuteri biofilms

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 our future work.

--- Fig. 3 ---

3.4. Tolerances of L. reuteri biofilms toward gastrointestinal environments

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

expressed genes in tolerance by biofilms as compared to their planktonic counterparts make L.

reuteri in biofilm-phenotype possess enhanced tolerances toward harsh environments [20].

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

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

digestion, respectively. These observed differences in tolerances between planktonic and

biofilm L. reuteri verify that L. reuteri in biofilm phenotype is more effective than probiotics.

--- Fig. 4 ---

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
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.

--- Table 1 ---

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 P group. Acinetobacter commonly causes nosocomial

infections primarily in immunocompromised patients, predominantly aspiration pneumonia

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

precursors without the production 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.

--- Fig. 5 ---

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

groups have double driving forces to strengthen SCFAs production.

Redundancy analysis (RDA) in Fig. 6 (c) and Fig. 6 (d) demonstrate that microbial

communities Lactobacillus, Bifidobacterium, Cloacibacillus, and Bilophila were positively

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 3 days of fermentation were Lachnoclostridium, Lactobacillus, Bifidobacterium, and

Lactobacillus, respectively. The cluster of Lactobacillus, Bifidobacterium, Cloacibacillus,

and Bilophila was positively correlated with acetic acid and propionic acid. The cluster of

Lactobacillus, Cloacibacillus, and Bilophila was positively correlated with butyric acid.

--- Fig. 6 ---

4. Conclusion

In this study, zein and cellulose were dissolved in NaOH/urea aqueous solution at low

temperatures to prepare porous protein/carbohydrate composite scaffolds through a vacuum

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. Compared to planktonic L. reuteri, L. reuteri in biofilms on the composite scaffolds

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

the composite scaffolds decrease the abundances of Acinetobacter, Klebsiella, and

Bacteroides and increase the 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 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

microbiota and producing SCFAs as compared with planktonic L. reuteri.

CRediT authorship contribution statement

Fei He: Investigation, Methodology, Formal analysis, Data curation, Writing-original draft,

Visualization. Xue-Ke Ma: Investigation, Methodology. Cheng-Kai Tu: Investigation. Hui

Teng: Investigation. Xin Shao: Investigation. Jie Chen: Methodology. Meng-Xin Hu:

Conceptualization, Methodology, Project administration, Writing - Review & Editing,

Visualization, Funding acquisition.

Declaration of competing interest

The authors declare no conflicts of interest.

Data availability

Data will be made available on request.

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

Provincial Universities of Zhejiang (3090JYN9922001G-016).

18
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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.

Culture Time Samples Shannon Sobs Simpson Ace Chao

Control 3.5538±0.0609b 568.5±21.92 0.1260±0.0288c
a
638.52±29.33a 629.55±31.26a
P 3.1107±0.0346cd 490.5±2.12d 0.1419±0.0238c 559.97±15.32c 553.67±21.54c
1 day
M 3.2317±0.0370c 519.5±3.53c 0.1370±0.0230c 598.69±0.42b 590.34±2.64abc
B/M 3.0920±0.0354cd 505.0±4.24cd 0.1442±0.0204c 582.89±0.02bc 584.54±0.06bc
Control 2.8885±0.0683d 510.5±6.36cd 0.2044±0.0100b 604.72±18.90ab 602.67±22.69ab
P 3.6105±0.1420b 525.0±2.82c 0.0740±0.0080d 604.25±6.23ab 602.65±5.97ab
3 days
M 3.7217±0.1413ab 519.5±2.12c 0.0674±0.0114d 604.64±5.79ab 623.24±4.93ab
B/M 3.8463±0.1427a 547.0±4.24b 0.0571±0.0074d 615.07±11.24ab 604.42±15.90ab

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

20 min 25 min 30 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
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