INGREDIENTS POTENTIALLY TREATING LEAKY GUT: EFFECTS ON PHYSICO-

CHEMICAL, RHEOLOGICAL, MICROBIOLOGICAL, SENSORY, AND INTESTINAL

DYSFUNCTIONS CHARACTERISTICS OF YOGURT AND ITS INFLUENCE ON
ACID TOLERANCE, BILE TOLERANCE, PROTEASE ACTIVITY AND GROWTH
CHARACTERISTICS OF YOGURT’S STATER CULTURE

A Thesis Dissertation

Proposal

School of Nutrition and Food Sciences

by

Ricardo José Santos Alemán

Committee Members

Dr. Aryana Kayanush, Dr. Charles Boeneke & Dr. zhimin Xu

 Background

Prolonged inflammation initiates intestinal barrier dysfunctions that lead to the pathologies such
as obesity and leaky gut syndrome (LGS). Leaky gut syndrome, also known as intestinal
permeability, is a common disease that has impacted a considerable percentage of the population
in recent years (Mu et al., 2017). The incidence of inflammatory bowel and leaky gut diseases is
on the rise in countries that adopt a Western lifestyle. Its pathology is not well defined, but it is
associated with multifactorial causes (Mu et al., 2017). In summary, the intestinal barrier
functionality (Peterson & Artis, 2014) and gut microbial ecosystem (Wu et al. 2011) are critical
aspects influencing gut wellness. The significant consumption of fermented dairy products
providing probiotics has been related to a cure or prevention of inflammatory bowel diseases
(Aryana & Olson 2017). From a nutritional viewpoint, yogurt is widely and the health benefits
that they provide (Aryana & Olson, 2017) considered a healthy feed due to its protein, calcium,
vitamins, and probiotic content. Consequently, yogurt has been utilized as a carrier for
ingredients that are abundant in bioactive compounds. Yogurt originated from “yoğurmak”
(Turkish) has been a part of the human diet for Millennials. In 6000 BC, Indian Ayurvedic
scripts presented some recommendations towards the health benefits of yogurt. In the 20th
century, Stamen Grigorov reported scientific evidence concerning benefits to lactic acid bacteria.
Now, yogurt is additionally applied as a carrier for gut-friendly prebiotics and probiotics (Fisberg
and Machado, 2015). Due to its high nutritional value, yogurt is recommended to be consumed
daily. Dietary components such as probiotics, prebiotics, synbiotics, and polyphenols have been
shown to beneficially affect gut well-being (Wan, et al., 2018). Food fortification is one of the
most efficient mechanisms for promoting the quality of the nutrients in food. Lately, functional
foods have produced great awareness among an emergent market that is seeking healthy choices.
As a result, functional ingredients should be taken into consideration for incorporating into food
products. These ingredients could be medical herbs, amino acids or derivates, polyphenols,
minerals, and vitamins to name a few. L-glutamine, quercetin, slippery elm bark, marshmallow
root, N-acetyl-D-glucosamine (NAG), licorice root, maitake mushrooms, and zinc orotate have
shown potential benefits through gut microbiota and intestinal barrier functions (Aleman et al.,
2023). These ingredients alongside yogurt can in reducing intestinal barrier dysfunctions and
promote advantages to gut microbiota (Putt et al., 2017). These ingredients are commercially
available as powders, and it does not appear that there is any research performed on these
powders in yogurt systems. Yet, the addition of the previously-mentioned ingredients could
affect the yogurt's properties and potentially affect the physicochemical, rheological and
microbiological characteristics of yogurt. The nature and amount of such ingredients applied in
the yogurt matrix must not negatively influence sensory attributes or food safety characteristics
for consideration of industrial usage. As a result, the objective of this research was to study the
effect of these powders on the physico-chemical and microbiological properties of yogurt over
its shelf life.

Objectives
- To evaluate the Physico-chemical, microbiological, and sensory characteristics of yogurt as
affected by incorporated ingredients potentially treating leaky gut
 - To analyzed the acid tolerance, bile tolerance, protease activity and growth characteristics of
yogurt’s stater culture Streptococcus thermophilus & Lactobacillus bulgaricus as influenced by
ingredients potentially treating leaky gut.
- Study the effects of ingredients potentially treating leaky gut in intestinal barrier dysfunctions.

Experimental design

Functional plain yogurt will be produced using different ingredients (L-glutamine (Y1), quercetin (Y2),
slippery elm bark (Y3), marshmallow root (Y4), N-acetyl-D-glucosamine (Y5), licorice root (Y6), maitake
mushrooms (Y7), and zinc orotate (Y8) to evaluate the Physico-chemical, rheological, microbiological
and sensory characteristics. These ingredients will also be used in invitro studies of Streptococcus
thermophilus & Lactobacillus bulgaricus. All the ingredients will be applied in all studies as the
concentrations described: Y1=7 mg/L, Y2= 700 mg/L, Y3= 210 mg/L, Y4= 1340 mg/L, Y5= 210 mg/L, Y6=
210 mg/L, Y7= 42 mg/L, Y8= 70 mg/L.

Yogurt preparation

Yogurts were produced in the Louisiana State University creamery. The milk (11.3562 liters) was equally
divided into nine pails and the eight ingredients were randomly assigned to the eight pails. The control
had no ingredient. Ingredients were incorporated individually into the milk in the following amounts. L-
glutamine (7 mg/L of yogurt mix), quercetin (700 mg/L), slippery elm bark (210 mg/L), marshmallow root
(1340 mg/L), NAG (210 mg/L), licorice root (210 mg/L), maitake mushrooms (42mg/L), and zinc orotate
(70 mg/L). The ingredients were incorporated to the milk and the mix was stirred vigorously. Stirring
continued during the batch pasteurization process where the whole milk containing the ingredients was
heated to 85˚C for 30 min, followed by tempering to 41˚C. Freshly thawed pure cultures of S.
thermophilus STI- 06 and L. bulgaricus LB-12 were incorporated into the milk containing the ingredients
and mixed vigorously. After inoculation, the yogurt mix was poured into 355 mL labeled containers
(Reynolds RDC212-Del-Pak Combo-Pak, Alcoa Inc., Pittsburgh, PA) and incubated at 40˚C to pH 4.6
before cooling to 4˚C. Yogurts were manufactured in three separate trials.

Table 1. Yogurt mix formulation

Addition Treatments
Ingredient
PY YI1 YI2 YI3 YI4 YI5 YI6 YI7 YI8
Milk (Kg) 3 3 3 3 3 3 3 3 3
**Ingredient N/A 7 700 210 1340 210 210 42 70
(mg/L)
Culture (ml) 6 6 6 6 6 6 6 6 6
Ratio used: 1:1
PY = plain yogurt (Control), YI1 = yogurt with L-glutamine, YI2 = yogurt with
Quercetin, YI3 = yogurt with Slippery Elm Bark, YI4 = yogurt with Marshmallow root,
YI5 = yogurt with N-acetyl-D-glucosamine (NAG), YI6 = yogurt with Licorice root, YI7
= yogurt with Maitake Mushrooms, YI8 = yogurt with Zinc Orotate.** Dosing based on
3 cups of yogurt daily.

Methods description for objective 1

Titratable acidity
It will be determined using 9 g of yogurt with 9 ml of distilled water and 0.5 ml of
phenolphthalein. Samples will be titrated with 0.1 N NaOH until to get a slight pink for
30 seconds and, the volume of 0.1 N NaOH used will be recorded.
pH
The pH will be measured by using an Thermo Orion 3 Star pH Benchtop Meter (Fisher
Scientific, Instruments, Pittsburgh, PA), it will be calibrated using pH 4.00 and 7.00
buffer solutions (Thermo Fisher Scientific, Beverly, MA) before reading the samples.
Syneresis
10 g of each sample will be weighted and then centrifuged in an AccuSpin™ 400 (Fisher
Scientific, Instruments, Pittsburgh, PA) at 5,000 rpm for 20 min. The clear supernatant
will be weighted. The ratio will be expressed following this formula:
(Weight of the clear supernatant / Initial weight of the sample) × 100 = Syneresis (%)
Color
The L*a*b* values will be determined using a Minolta CM 508d hand-held
spectrophotometer (Minolta Co., Ltd, Japan). It will be standardized with white and
black tiles. On average, five values will be taken per replication. Total color differences
(ΔΕ*) were calculated as follows:
ΔΕ* = (ΔL*)2 + (Δa*)2 + (Δb*)2
Where ΔL* = L*treatment – L*references, Δa* * = a*treatment – a*references, and Δb*
* = b*treatment – b*references. (references = day 0 values)
Viscosity
Viscosity will be measured by using a Brookfield DV-II viscometer (Brookfield
Engineering Lab Inc., Stoughton, MA) Samples will be measured at 5 ºC, with a helipath
stand mounted with a T-C spindle will be used at 20-30 rpm. The data will be acquired
using Wingather® software (Brookfield Engineering Lab Inc., Stoughton, MA). One
hundred points will be averaged per sample per replication.
Enumeration of Streptococcus thermophilus
Streptococcus thermophilus agar will be prepared using: to 1 L of distilled water the
following ingredients were weighted: 10 g of sucrose (Amresco, Solon, OH), 2 g of
K2HPO4 (Fisher Scientific, Fair Lawn, NJ), 5 g of Bacto yeast extract and 10 g of Bacto
Tryptone (Becton, Dickinson and Co., Sparks, MD) using individual plastic weighing
boats. Distilled water will be transferred from the graduated cylinder to a 2L Erlenmeyer
flask. The mix will be stirred to dissolve the ingredients. To reduce the pH to 6.8, 1 N
HCl will be added. Then, 12 g of agar (Fisher Scientific, Fair Lawn, NJ) will be added to
the medium and 6 ml of 0.5% bromocresol purple will be added (Fisher Scientific, Fair
Lawn, NJ). The media will be heated to boiling and it will be autoclaved at 121 ºC for 15
minutes (Dave and Shah 1996). Growth of Streptococcus thermophilus will be measured
by weighting out 11 g of yogurt sample will be diluted with 99 ml of sterilized MgCl2
KOH, getting different dilution. Then, 1 ml of each diluted sample will be pipetted into
petri dishes and then the media will be aseptically poured into the petri dish. Petri dishes
will be aerobically incubated at 37 ºC for 24 h. To enumerate the colonies a Quebec
Darkfield Colony Counter (Leica Inc., Buffalo, NY) will be used.
Enumeration of Lactobacillus bulgaricus
The Lactobacillus bulgaricus agar will be prepared using: for 1 liter of distilled water
use 15 g of agar (Fisher Scientific, Fair Lawn, NJ) and 55 g of Lactobacilli MRS broth
powder (Becton, Dickinson and Co., Sparks, MD). The pH will be adjusted to 5.2 using
1 N HCl. The media will be heated to boiling with agitation. Then, the media will be
autoclaved at 121 ºC for 15 minutes (Tharmaraj and Shah 2003). Growth of
Lactobacillus bulgaricus, will be measured and 11 g of yogurt sample will be diluted to
serial dilutions with 99 ml of sterilized MgCl2 KOH. 1 ml of each diluted sample will be
pipetted into petri dishes and then the media will be aseptically poured into the petri dish.
Petri dishes will be placed in a BBL GasPaks (BBL, Becton, Dickinson and Co.,
Cockeysville, MD) and then they will be incubated anaerobically at 43 ºC for 72 h. to
enumerate the colonies a Quebec Darkfield Colony Counter (Leica Inc., Buffalo, NY)
will be used.
Enumeration of Yeasts and Molds
The yogurt will be tested for yeast and molds before conducting the sensory evaluation
using the method by 3M Company, with slight modifications. These analyses will be
determined by making serial dilutions and duplicated in peptone water (0.1% wt/v) and
plated in 3M™ petrifilms for yeasts and molds (3M Microbiology, St. Paul MN). The
petrifilms will be placed on a flat surface and 1 ml of the cheese crackers dilution will be
placed on the center of the bottom film. The inoculums will be covered by the top film
and spread to an area of 20 cm2 using the plastic spreader supplied. The 3M™ Petrifilms
plates will be incubated for 72 hours at 22°C. After the incubation period, the colonies
will be counted.
Rheological Evalaution
The rheological properties of the yogurt will be carried out in triplicate by a rheometer
(AR 2000ex, TA Instruments-Waters LLC, New Castle, DE) and will be conducted by
using a parallel plate geometry (40-mm diameter) and the gap will set to 3mm. 1 g of
yogurt at 5 C will used for rheological evaluation. Three types of analysis will be carried
out: a) steady shear flow measurements b) frequency sweep c) hysteresis experiments for
thixotropic.
Small amplitude oscillatory shear (SAOS)
The frequency sweep will be carried out at 5oC, 0.1 to 100 rad/s and the following
viscoelastic parameters will be obtained: Storage Module (G', Pa), Loss Module (G', Pa)
and Phase Angle Tangent (Tan'). Also, the complex viscosity (n*) will be measured as a
function of frequency (u).
The Herschel - Bulkley model will be used to describe whether if the paste was shear
thickening or shear thinning (If τ < τ 0 the Herschel-Bulkley fluid behaves as a solid,
otherwise it behaves as a fluid. For n < 1 the fluid is shear-thinning, whereas for n > 1
the fluid is shear-thickening. If n = 1 and τ 0 = 0, this model reduces to the Newtonian
fluid (equation 3.13). In this model the parameters “𝑛” and “𝑘” are defined as in the
Power Law. As special cases, the model becomes Bingham Plastic when 𝑛 = 1, the Law
of Power is recovered when 𝜏𝑦 = 0 and when τy = 0 and n = 1, the Newtonian model is
obtained.

n =(𝜏 − 𝜏0)/ 𝑘 (𝛾)

Where:
- τ: Shear stress (Pa)
- k: Coefficient or flow consistency index (Pa.s)
- τ0: Creep threshold (Pa)
- γ: Deformation rate (1 / s)

Steady-shear
The steady shear flow measurements by applying an increasing shear rate of 0.01 to 100
(deformation range), which will be used for determining the linear viscoelastic region.
The Shear stress (t) and steady shear viscosity (n) will be measured as a function of the
increasing shear rate and the test will be done in automated mode. The power law model
will be used to characterize the flow behavior of flour suspensions. This model will be
used to describe experimental data as pseudoplastic or dilating fluids:

t = k Xn

Where X is the shear rate (s-1), k is the consistency coefficient (Pa) and n the flow
behavior index (dimensionless). The consistency index is related to the degree of
structuring of the system, and the flow index indicates whether it’s a Newtonian behavior
fluid or not (if n = 1, the fluid behaves Newtonian; if it is less than 1, it does as a
pseudoplastic fluid and if it is greater than 1 it behaves like a dilating fluid).
Thixotropic properties
The thixotropic properties of yogurt will be evaluated by a hysteresis experiments
(upward curve, plateau curve and downward curve) and the thixotropic tests will be used
to characterize and model the behavior of different the different flour suspensions. For
the test, a steel plate geometry (40 mm diameter) was used and a 0.5 mm gap will be
operated. During the test, the shear rate ramp will be increased from 0 to 100 s-1 in 1
min (ramp 1), and then the shear rate will be maintained (50 s-1) for 2 min (ramp 2).
Consecutively, the shear rate will be decreased from 100 to 0 s-1 for 1 min until it
stopped (ramp 3). The system response will be monitored throughout the cycle, and the
area will be measured and obtained of shear stress vs shear speed graph.
Consumer’s study
The sensory characteristics of the 9 treatments yogurt will be evaluated through a
consumer acceptance test. Two hundred sixty (225) untrained consumers will be
randomly chosen from Louisiana State University (LSU), Baton Rouge Campus. All the
participants will have to met with the following criteria: 18 years of age or older, not
allergic to dairy products, L-glutamine, quercetin, slippery elm bark, marshmallow root,
NAG, licorice root, maitake mushrooms, and zinc orotate; and willingness to participate
for approximately 7-10 minutes to complete the survey. Consumers will be required to
read and sign a consent form approved by the Louisiana State University Institutional
Review Board. The survey will be completed electronically using the Compusense
(Compusense® five, Release 5.6 with Compusense Inc., Guelph, Ontario) at the Sensory
laboratory located in the Animal and Food Science laboratories building of LSU.
Demographic information will be requested such as gender, race and age. Screening
questions, using a binomial scale (yes/no), regarding usual consumption of yogurt and
consideration of functional ingredients in the diet will be asked. Further, consumers will
rate color, aroma, flavor, consistency, and overall liking of the product based on the 9-
point hedonic scale (1 = dislike extremely, 5 = neither like nor dislike, 9 = like
extremely). Overall liking, a shortened emotions and purchase intent will be evaluated
before and after additional information about functional ingredients potentially treating
leaky gut in yogurt.

Methods description for objective 2

Bile Tolerance Test

Bile tolerance of Streptococcus thermophilus and Lactobaciulls bulgaricus will be
analyzed according to Pereira and Gibson (2002) with slight modifications. Yogurt’s
stater cultures will be evaluated for its ability to grow in MRS broth (Criterion™, Hardy
Diagnostics, Santa Maria, CA) and M17 broth (Criterion™, Hardy Diagnostics, Santa
Maria, CA) respectively supplemented with 0.2% (wt/v) of sodium thioglycolate (Acros
Organics, Fair Lawn, NJ)] with bile salt oxagall (0.2 wt/vol). Sodium thioglycolate will
only used in MRS broth as an oxygen scavenger to achieve microaerophilic conditions.
Stater cultures will be inoculated (10% [v/v]) into MRS broth or M17 broth with 0.3%
(wt/v) oxgall (bovine bile) (USBiological, Swampscott, MA) and incubated under
anaerobic conditions at 37 ºC and anaerobically at 43 ºC for 8 hours.
Acid Tolerance Test
The acid tolerance of Streptococcus thermophilus and Lactobaciulls bulgaricus will be
evaluated according Pereira and Gibson (2002) with slight modifications. Stater cultures
will be inoculated (10% [v/v]) into acidified MRS broth (Criterion™, Hardy
Diagnostics, Santa Maria, CA) previously adjusted to pH 2.0 with 1N HCl. The acidified
MRS broth mixtures will be incubated in a water bath at 37 ºC for 15 minutes. One
milliliter sample will be taken at various times (0, 30, 60, and 120 min), serially 10-fold
diluted in peptone water, and plated in duplicate onto MRS agar (Difco, Detroit, MI) and
M17 Agar (Difco, Detroit, MI).
Protease Activity
The extracellular protease activity of Streptococcus thermophilus and Lactobaciulls
bulgaricus will be determined by the o-phthaldialdehyde (OPA) spectrophotometric
assay according to the method described by Oberg et al., (1991). Streptococcus
thermophilus and Lactobaciulls bulgaricus will be inoculated (10% [v/v]) into sterile
skim milk (autoclaved at 12 ºC for 15 min), and incubated at 40 ºC for 0, 12 and 24
hours. After incubation, 2.5 m from each sample will be mixed with 1 ml distilled water
and transferred into test tubes containing 5 ml of 0.75N trichloroacetic acid (TCA)
(Fisher Scientific) and the test tu will be vortexed at the same time. After setting at room
temperature for 10 minutes the acidified samples will be filtered through a Whatman
Number 2 filter paper (Clifton, NJ). Non inoculated sterile skim milk will be prepared
similarly to use as a reference in a Duplicate aliquots from each TCA filtrate will be
analyzed by the o-phthaldialdehyde (OPA) spectrophotometric assay using an UV-Vis
spectrophotometer (Nicolet Evolution 100, Thermo Scientific; Madison, WI, USA). The
o-phthaldialdehyde final solution will be prepared by combining the following reagents
and diluting to a final volume of 50 ml with distilled water: 25 ml of 100 mM sodium
borate (Fisher Scientific); 2.5 ml 20% (wt/wt) SDS (Fisher Scientific); 40 mg of o-
phthaldialdehyde reagent (Alfa Aesar, Ward Hill, MA) dissolved in 1 ml methanol
(Sigma); and 100 µl of β-mercaptoe One hundred and fifty µl of each TCA filtrate will
be mixed with 3 ml of ophthaldialdehyde final solution in a 3 ml cuvette, and the
absorbance at 340 nm was read. Absorbance of the o-phthaldialdehyde final solution
with the non-inoculated sterile skim milk (reference) will be subtracted from each
sample reading. The o thanol (Sigma). -phthaldialdehyde final solution will be used as a
blank to calibrate the spectrophotometer.
Growth Test
Growth of Streptococcus thermophilus and Lactobaciulls bulgaricus will be analyzed
according to Aleman al., (2023). The growth will be monitored by measuring the optical
density at 600 nm (OD600) through an UV- Vis Spectrophotometer (Nicolet Evolution
100, Thermo Scientific; Madison, WI, USA) at 600 nm. Starter cultutres of
Streptococcus thermophilus and Lactobaciulls bulgaricus will be inoculated (10% [v/v])
into MRS broth (Criterion™, Hardy Diagnostics, Santa Maria, CA) and M17 broth
(Criterion™, Hardy Diagnostics, Santa Maria, CA) respectively, which will be
previously autoclaved at 121 ºC for 15 min with pH 6.5 and 6.8 respectively. The
inoculated MRS or M17 broth initial OD600 will be recorded, and there will be
incubated under anaerobically at 43 ºC for 72 h and anaerobic conditions at 37 ºC
respectively for 16 hours. The OD values will be collected hourly. The
spectrophotometer will be calibrated by using the broth as blank. An average of tw
values per treatment will be taken, that is two cuvettes per treatment. An estimate of
bacterial counts (CFU/mL) will be calculated from OD600 readings using a standard
curve.

Methods description for objective 3

Simulated gastric and intestinal digestion of yogurt

The control yogurts and yogurts fortified with ingredients will be subjected to an in vitro gastric
and intestinal digestion as Minekus et al., 2014 with small modifications. For gastric digestion,
freeze-dried yogurt (0.2 g) will be dissolved in 4 mL of HCl (0.15 N) and then added with
simulated gastric fluid (Chemazone, Edmonton, Alberta, Canada) at a ratio of 1:1 (v/v). The
obtained solution will be then mixed with porcine pepsin enzyme (Sigma-Aldrich, St. Louis,
MO, USA) (2,000 U/mL). The obtained solution will be then adjusted to a pH of 3 using HCl
(0.1 N) and will be incubated at 37 °C for 3 h with continuous shaking. For the intestinal phase,
the solution obtained from gastric digestion will be mixed with simulated gastric fluid
(Chemazone, Edmonton, Alberta, Canada) at a ratio of 1:1 (v/v). The obtained solution will be
then mixed with pancreatin enzyme (Sigma-Aldrich, St. Louis, MO, USA) (100 U/mL) and
oxgall bite salt (bovine bile) (US Biological, Swampscott, MA, USA) (10 mM). The obtained
solution will be then adjusted to a pH of 3 using NaOH (0.1 N) and will be incubated at 37 °C
for 7 h with continuous shaking. All samples will be immediately collected after the invitro
digestion process with snap freezing using liquid nitrogen.
Antioxidant capacity
1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay
The DPPH radical scavenging test will be performed similarly to the technique illustrated by
Najgebauer-Lejko et al. (2011) with minor changes. The yogurt was freeze-dried in a freeze
dryer (Labconco Free zone, Kansas City, MO) for ~72 h, where the yogurt will be frozen to -20
℃ before being freeze-dried. The lyophilized yogurt will be grounded with a grinder (Retsch
GmbH, Germany) (501-700 mm). Later, 600 mg of grounded dried freeze-dried sample will be
placed in a 60 mL centrifuge tube with 40 mL of 80% methanol solution. The solution will be
mixed and sonicated for 15 min. The mixture will be then centrifuged for 8 min at 1700 g x
force. The supernatant will be used for antioxidant analysis. 100 ml of yogurt extracts will be
added with 3.0 ml of 0.1 mM DPPH reagent, and the obtained solution will be incubated in the
dark at 25 C for 2 h. Absorbance (Abs) will be recorded using a spectrophotometer (Genesys
10UV) at 515 nm. Distilled water will be used as the blank, and the methanol- DPPH reagent
solution (100:3.9) will be used as the control (3.9 ml). The radical scavenging activity will be
estimated as described in equation (1):
Antioxidant activity (%) = ((Absorbance control-Absorbance sample)/(Absorbance
control))×100
Ferric reducing antioxidant potential (FRAP) assay
The FRAP radical scavenging test will be conducted similarly to the method illustrated by
Benzie and Strain (1996) with minor modifications. 200 ml of yogurt extracts will be mixed with
1.8 ml of FRAP reagent, and the obtained solution will be incubated in the water bath at 37 C for
10 min. The FRAP reagent consisted of 8 mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) reagent, 300
mM acetate buffer, and 20 mM FeCl3 at a proportion of 1:10:1. Absorbance (Abs) will be
estimated using a spectrophotometer (Genesys 10UV) at 593 nm. FeSO47H2O (0.3–1.0 mM)
will be used for the calibration curve, and the results will be reported as mmol Fe2+ equivalent/l
(mmol Fe2+E/L).
Ferrous ion chelating (FIC) assay
The FIC radical scavenging test will be performed as the method described by Chan, Lim, and
Chew (2007). Yogurt extracts will be mixed with 0.1 mM iron(II) sulphate hydrate
(FeSO4xH2O) solution and 0.25 mM ferrozine solution at a ratio of 1:10:1. The obtained
solution will be incubated at 25 C for 10 min. Absorbance (Abs) will be estimated using a
spectrophotometer (Genesys 10UV) at 562 nm. Distilled water will be used as the blank, and
FeSO4xH2O (1 ml), ferrozine (1 ml), and distilled water (1 ml) solution will be used as the
control. The FIC ability will be calculated as illustrated in equation (1):
Caco-2 cell culture maintenance
The cells will be differentiated during 21 days and supplemented with Dulbecco's Modified
Eagle Medium with high glucose levels (Life Technologies, NY, USA), 10% (v/v) fetal bovine
serum (FBS) (Life Technologies, NY, USA), 1% (v/v) NEM non-essential amino acids solution
(PAA Laboratories GmbH, Pashing, Austria), and 1% (v/v) penicillin/streptomycin (InvivoGen,
San Diego, CA, USA) at 37 ºC and 5% CO2. The cells will be passaged (30–40 for all
experiments (Manassas, VA)) once a week at a confluence between 80-90%. Caco2 cells will be
subcultured with trypsin (2 × 105 cells per mL) onto 0.4 μm polycarbonate membrane Transwell
inserts (Corning, Inc; Lowell, MA). The medium will be changed every 2-3 days (Chen et al.,
2019).
Caco-2 Cell viability test
Caco-2 cells will be subcultured at 103 per well on 96-well plates with culture media integrated
with powdered yogurt (Control yogurt). Yogurt powder will be resuspended in growth media at
1:25, 1:50, 1:75, and 1:100 dilution ratios before its application to cell monolayers. The cells
were incubated for 0, 24, and 48 h at 37°C with 5% CO2. The highest concentration of nontoxic
yogurt-water solution will be used for subsequent analysis in the Caco2 cells. Caco-2 cells
treated with phosphate-buffered saline (PBS) will be considered as the negative control. Cells
will be washed as CellTiter 96 Aqueous One solution (Promega, Madison, WI) protocol with
tetrazolium compound and phenazine methosulfate solution. Cell density will be measured using
a BioRad Model 680 microplate reader at a 490 nm wavelength. Formazan will be used as an
indicator to estimate the number of living cells. TEER will be also measured after 48 h examine
the effect of powdered yogurt dilutions (1:25, 1:50, 1:75, and 1:100) on differentiated Caco-2
integrity. The cell viability essay and TEER measurements will be performed in triplicates within
cell batches and 6 measurements within 1 cell batch.
Induction of barrier dysfunction in Caco2 cells
The Caco-2 cells will be treated with lipopolysaccharide (LPS, 1 μg mL−1) and cytokines such
as interleukin-1β (IL-1β, 25 ng mL−1), tumor necrosis factor-α (TNF-α, 50 ng mL−1), and
interferon‐gamma (IFN-γ, 50 ng mL−1). The cytokines will be applied in the basolateral
compartment with supplemented growth media, whereas lipopolysaccharide will be applied to
both the apical and basolateral compartments. The isoflavone genistein will be used as a positive
control (Putt et al., 2017).
Transepithelial electrical resistance (TEER)
Transepithelial electrical resistance will be estimated to examine the Caco2 cells' integrity (Putt
et al., 2017). In both compartments, cells will be washed with Hank's Balanced Salt Solution (5
mM, HEPES) at 0.2 mL (apical side) and 1 mL (basolateral side). Consequently, the culture
media will be removed from plates and inserts. Later, the plates will be incubated for 25-30 min
at 37 °C with 5% CO2. After incubation, plates will be moved to a hot plate (37 °C). The
transepithelial electrical resistance will be recorded using an Epithelial Volt/Ohm Meter 3 with
an EVOM3 chopstick electrode (World precision instruments, Sarasota, FL) from 550–700 Ohm
cm2. TEER measurements without Caco2 cells will be considered blank. All experiments will be
performed in triplicates.
Paracellular permeability
The paracellular permeability will be evaluated similarly to Chelakkot et al. (2018) and Mohebali
et al. (2020) by estimating the flux of lucifer yellow (LY) and fluorescein isothiocyanate (FITC)-
dextran 4000 (FD) through the cell monolayers. FD (1 mg/mL) and LY (0.5 mg/mL) will be
added to HBSS/HEPES solution at 37 °C, and the HBSS/HEPES solution with FD and LY (0.2
mL) will be added into the apical compartment. In addition, 1 mL of HBSS will be added to the
basolateral compartment. The plates will be covered, incubated (37 °C), and shaken (150 rpm).
For every 4 h for 16 h, the basolateral solution (300 µL) will be moved into a clear-bottom and
black 96 well plate (Corning Costar, New York, NY, USA). Later, the basolateral compartment
will be rinsed with fresh HBSS (1 ml) at 37 °C. The fluorescence intensity will be recorded using
a PLX800 fluorescence spectrophotometer (BioTek, Winooski, VT, USA) at excitation/emission
wavelengths of 428/540 nm (LY) and 485/530 nm (FD). The apparent permeability coefficient
(Papp (cm/s)) will be calculated as equation (2):
Papp (cm s-1) = dQ/dt*1/(A*C) (2)
Where C is the initial amount of fluorescent marker on the apical side (mol/mL), dQ is the
concentration (fluorescent marker) on the basolateral side (mol/mL), A is the membrane surface
area (cm2), and dt is the flux per second (1/s).
Transmission electron microscopy (TEM)
The interaction between yogurt treatments (Q, MR, NAG, LG, ZN, MM, LR, SEB, and CY) and
caco2 cells will be examined by transmission electron microscopy, incubating for 48 h with
(Inflammatory Stimulus: Interleukin-1β (IL-1β), Tumor necrosis factor-α (TNF-α), Interferon‐
gamma (IFN-γ), lipopolysaccharide (LPS), and isoflavone genistein). Samples with
inflammatory stimuli were considered negative controls. Ultrathin areas (50 nm) will be cut by
DiATOME diamond knife (Reichert, Wien, Austria) with uranyl acetate solution and lead citrate.
TEM pictures (from 10 different clear spots) will be observed using a TEM microscope (Hitachi
H7000, 100 kV, Yokohama, Japan).
Immunofluorescence light microscopy
The immunofluorescence microscopy procedure will be performed according to Zeng et al.
(2016) with slight changes. The cells (4 × 105 cells/cm2) will be treated in the Lab-Tek II
chamber slide system (Nalge Nunc International) with control media, inflammatory stimulus
(IS), and yogurt samples (Q, MR, NAG, LG, ZN, MM LR, SEB, and CY) with (IS). Caco2 cells
will be washed with Hank's balanced salt solution with 3% paraformaldehyde and without Mg
and Ca. Cells will be overlaid (diluted at 1:50) with primary antibody ZO-1, occluding-1, and
claudin-1 (Zymed Laboratories, San Francisco, CA). The ZO-1, occludin-1, and claudin-1
staining of caco-2 cell monolayers will be conducted similarly to Putt et al. (2017) and Yokoo et
al. (2021). Cells will be washed and blocked for 35 min with 1.5% (w/v) bovine serum albumin
(BSA) in PBS for ZO-1, occludin-1, and claudin-1. Primary antibodies will be incubated
overnight at 4 °C for occludin, and 4 h at 37 °C for ZO-1 as described by Putt et al. (2017).
Claudin-1 will be incubated for 16 h at 4°C (Yokoo et al., 2021). After the incubation of ZO-1
and Occudin-1, goat anti-rabbit IgG antibody (Sigma) and (H+L) FTIC conjugate; (Sigma) will
be added at a ratio of 1:100 to Caco2 cells monolayer and incubated for 30 min (ZO-1 and
Occudin-1) and 1 h (Claudin-1). Fluorescence will be monitored using a fluorescent light
microscope with FITC-compatible media (Compound Microscope Leitz Optilux, Germany).
Immunofluorescence pictures will be obtained using ZEN 2010 software (Carl Zeiss AG,
Oberkochen, Germany). 10-15 fields will be viewed under balanced staining for all tight
junctions. The square cutout borders will be withdrawn from the pictures, and the image size
obtained was 80 um2.
Gene expression analysis of tight junction proteins
Cell RNA extraction will be conducted using Popović et al. (2020) with slight changes. Using
Ambion DNA-freeTM Kit (ThermoFisher Scientific, Waltham, MA), caco2 cells (5 × 106 cells)
will be collected with a centrifuge and DNaseІ. The reversed transcription verification will be
performed by the RevertAid RT kit (ThermoFisher Scientific, Waltham, MA). By fluorometric
quantitation, Qubit (ThermoFisher Scientific, Waltham, MA) will be used to determine RNA
levels. 1 µg of isolated RNA will be reverse transcribed to cDNA at a 15 uL reaction volume in
7500 real-time PCR using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).
Real-time quantitative polymerase chain reaction (RT-qPCR) will be conducted using iTaq™
Universal SYBR-Green Supermix on a Bio-Rad CFX96 system as described by the
manufacturer's method. The primers for ZO-1, claudin-1, occludin, ribosomal protein large P0,
and 18s RNA (RNA18S5) will be selected according to Putt et al. (2017). As Maubon et al.
(2007) and Vreeburg et al. (2011), gene expression will be normalized and standardized to a
mathematic mean using RNA18S5 and RPLP0.

References
1. Aleman, R.S., Paz, D., Cedillos, R., Tabora, M., Olson, D.W. and Aryana, K., 2023. Attributes of
Culture Bacteria as Influenced by Ingredients That Help Treat Leaky Gut. Microorganisms, 11:
893
2. Aryana, K. J., and D. W. Olson. 2017. A 100-year review: Yogurt and other cultured dairy
products. J. Dairy Sci. 100:9987–10013. https://doi.org/10.3168/jds.2017-12981
3. Fisberg, M., and R. Machado. 2015. History of yogurt and current patterns of consumption. Nutr.
Rev. 73:4-7. https://doi.org/10.1093/nutrit/nuv020
4. Mu Q., J. Kirby, C. Reilly, and X. Luo. 2017. Leaky Gut As a Danger Signal for Autoimmune
Diseases. Front Immunol. 8:598. https://doi.org/10.3389/fimmu.2017.00598
5. Peterson L., and D. Artis. 2014. Intestinal epithelial cells: regulators of barrier function and
immune homeostasis. Nat. Rev. Immunol. 14:141-153. https://doi.org/10.1038/nri3608
6. Pereira, D., & Gibson, G. (2002). Cholesterol Assimilation by Lactic Acid Bacteria and
Bifidobacteria Isolated from the Human Gut. Applied And Environmental Microbiology, 68(9),
4689-4693. doi: 10.1128/aem.68.9.4689-4693.2002
7. Putt K., R. Pei, H. White, and B. Bolling. 2017. Yogurt inhibits intestinal barrier dysfunction in
Caco-2 cells by increasing tight junctions. Food Funct. 8:406-414.
https://doi.org/10.1039/c6fo01592a
8. Oberg, C., Wang, A., Moyes, L., Brown, R., & Richardson, G. (1991). Effects of Proteolytic
Activity of Thermolactic Cultures on Physical Properties of Mozzarella Cheese. Journal Of
Dairy Science, 74(2), 389-397. doi: 10.3168/jds.s0022-0302(91)78180-0
9. Wan M., K. Ling, H. El-Nezami, and M. Wang. 2018. Influence of functional food components on
gut health. Crit Rev Food Sci Nutr. 59:1927-1936.
https://doi.org/10.1080/10408398.2018.1433629
10. Wu, G., J. Chen, C. Hoffmann, K. Bittinger, Y. Chen, S. Keilbaugh, M. Bewtra, D. Knights, W. A.
Walters, R. Knight, R. Sinha, E. Gilroy, K. Gupta, R. Baldassano, L. Nessel, H. Li, F. D.
Bushman, and J. D. Lewis. 2011. Linking Long-Term Dietary Patterns with Gut Microbial
Enterotypes. Science. 334:105-108. https://doi.org/10.1126/science.1208344Wyatt, D. A.
Leaky Gut Syndrome: A Modern Epidemic with an Ancient Solution?. Townsend Letter, 2014, 6,
68-72.
11. Thorning, T.K.; Bertram, H.C.; Bonjour, J.-P.; De Groot, L.; Dupont, D.; Feeney, E.; Ipsen, R.;
Lecerf, J.M.; Mackie, A.; McKinley, M.C. Whole dairy matrix or single nutrients in assessment of
health effects: Current evidence and knowledge gaps. Am. J. Clin. Nutr. 2017, 105, 1033–1045. 
12. Vegarud, G.E.; Langsrud, T.; Svenning, C. Mineral-binding milk proteins and peptides;
occurrence, biochemical and technological characteristics. Br. J. Nutr. 2000, 84, 91–98.
13. Boudraa, G.; Benbouabdellah, M.; Hachelaf, W.; Boisset, M.; Desjeux, J.F.; Touhami, M. Effect of
feeding yogurt versus milk in children with acute diarrhea and carbohydrate malabsorption. J.
Pediatr. Gastroenterol. Nutr. 2001, 33, 307–313.
14. Guarner, F.; Perdigon, G.; Corthier, G.; Salminen, S.; Koletzko, B.; Morelli, L. Should yoghurt
cultures be considered probiotic? Br. J. Nutr. 2005, 93, 783–786.
15. Adolfsson, O.; Meydani, S.N.; Russell, R.M. Yogurt and gut function. Am. J. Clin. Nutr. 2004, 80,
245–256.
16. Gijsbers, L.; Ding, E.L.; Malik, V.S.; de Goede, J.; Geleijnse, J.M.; Soedamah-Muthu, S.S.
Consumption of dairy foods and diabetes incidence: A dose-response meta-analysis of
observational studies. Am. J. Clin. Nutr. 2016, 103, 1111–1124.
17. Sayon-Orea, C.; Martínez-González, M.A.; Ruiz-Canela, M.; Bes-Rastrollo, M. Associations
between yogurt consumption and weight gain and risk of obesity and metabolic syndrome: A
systematic review. Adv. Nutr. 2017, 8, 146S–154S.
18. Dumas, A.-A.; Lapointe, A.; Dugrenier, M.; Provencher, V.; Lamarche, B.; Desroches, S. A
systematic review of the effect of yogurt consumption on chronic diseases risk markers in
adults. Eur. J. Clin. Nutr. 2017, 56, 1375–1392.
19. Fernandez, M.A.; Marette, A. Potential Health Benefits of Combining Yogurt and Fruits Based on
Their Probiotic and Prebiotic Properties. Adv. Nutr. 2017, 8, 155S–164S.
20. Bordoni, A.; Danesi, F.; Dardevet, D.; Dupont, D.; Fernandez, A.S.; Gille, D.; dos Santos, C.N.;
Pinto, P.; Re, R.; Rémond, D.; et al. Dairy products and inflammation: A review of the clinical
evidence. Crit. Rev. Food Sci. Nutr. 2015, 57, 2497–2525.
21. Meng, H.; Ba, Z.; Lee, Y.; Peng, J.; Lin, J.; Fleming, J.A.; Furumoto, E.J.; Roberts, R.F.; Kris-
Etherton, P.M.; Rogers, C.J. Consumption of Bifidobacterium animalis subsp. lactis BB-12 in
yogurt reduced expression of TLR-2 on peripheral blood-derived monocytes and pro-
inflammatory cytokine secretion in young adults. Eur. J. Nutr. 2017, 56, 649–661.
22. Meyer, A.L.; Elmadfa, I.; Herbacek, I.; Micksche, M. Probiotic, as well as conventional yogurt, can
enhance the stimulated production of proinflammatory cytokines. J. Hum. Nutr. Diet. 2007, 20,
590–598.
23. Pei, R.; DiMarco, D.M.; Putt, K.K.; Martin, D.A.; Gu, Q.; Chitchumroonchokchai, C.; White, H.M.;
Scarlett, C.O.; Bruno, R.S.; Bolling, B.W. Low-fat yogurt consumption reduces biomarkers of
chronic inflammation and inhibits markers of endotoxin exposure in healthy premenopausal
women: A randomised controlled trial. Br. J. Nutr. 2017, 118, 1043–1051.\
24. Xu, L.Q.; Pranantyo, D.; Neoh, K.-G.; Kang, E.-T.; Fu, G.D. Thiol reactive maleimido-containing
tannic acid for the bioinspired surface anchoring and post-functionalization of antifouling
coatings. ACS Sustain. Chem. Eng. 2016, 4, 4264–4272.
25. Yu, J.-Y.; Ha, J.Y.; Kim, K.-M.; Jung, Y.-S.; Jung, J.-C.; Oh, S. Anti-Inflammatory Activities of Licorice
Extract and Its Active Compounds, Glycyrrhizic Acid, Liquiritin and Liquiritigenin, in BV2 Cells and Mice
Liver. Molecules 2015, 20, 13041-13054. https://doi.org/10.3390/molecules200713041
26. Rao, R.; Samak, G. Role of Glutamine in Protection of Intestinal Epithelial Tight Junctions. J.
Epithel. Biol. Pharmacol. 2012, 5, 47–54. 
27. Suzuki, T.; Hara, H. Quercetin Enhances Intestinal Barrier Function through the Assembly of
Zonula [Corrected] Occludens-2, Occludin, and Claudin-1 and the Expression of Claudin-4 in
Caco-2 Cells. J. Nutr. 2009, 139, 965–974.
28. Ried, K.; Travica, N.; Dorairaj, R.; Sali, A. Herbal formula improves upper and lower
gastrointestinal symptoms and gut health in Australian adults with digestive disorders. Nutr.
Res. 2020, 76, 37–51.
29. Bonaterra, G.A.; Bronischewski, K.; Hunold, P.; Schwarzbach, H.; Heinrich, E.U.; Fink, C.; Aziz-
Kalbhenn, H.; Muller, J.; Kinscherf, R. Anti-inflammatory and Anti-oxidative Effects of
Phytohustil((R)) and Root Extract of Althaea officinalis L. on Macrophages in vitro. Front.
Pharm. 2020, 11, 290.
30. Ollig, J.; Kloubert, V.; Weßels, I.; Haase, H.; Rink, L. Parameters Influencing Zinc in Experimental
Systems in Vivo and in Vitro. Metals. 2016, 6, 71. https://doi.org/10.3390/met6030071
31. Jayachandran, M.; Xiao, J.; Xu, B. A Critical Review on Health Promoting Benefits of Edible
Mushrooms through Gut Microbiota. Int. J. Mol. Sci. 2017, 18, 1934
32. Zhai, Z.; Wang, J.; Huang, B.; Yin, S. Low-fat yogurt alleviates the pro-inflammatory cytokine IL-
1β-induced intestinal epithelial barrier dysfunction. J. Dairy Sci. 2019, 102, 976-984.
33. Minekus, M.; Alminger, M.; Alvito, P.; Ballance, S.; Bohn, T.; Bourlieu, C.; Carriere, F.; Boutrou,
R.; Corredig, M.; Dupont, D. A standardised static in vitro digestion method suitable for food—An
international consensus. Food Funct. 2014, 5, 1113–1124.
34. Najgebauer-Lejko, D.; Sady, M.; Grega, T.; Walczycka, M. The impact of tea supplementation on
microflora, pH and antioxidant capacity of yoghurt. Int. Dairy J. 2011, 21, 568–574.
35. Benzie, I.F.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of
“Antioxidant power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76.
36. Chan, E.W.C.; Lim, Y.Y.; Chew, Y.L. Antioxidant activity of Camellia sinensis leaves and tea from
a lowland plantation in Malaysia. Food Chem. 2007, 102, 1214–1222. 
37. Chen, Y.; Zhang, H.; Liu, R.; Mats, L.; Zhu, H.; Pauls, K.P.; Deng, Z.; Tsao, R. Antioxidant and
anti-inflammatory polyphenols and peptides of common bean (Phaseolus vulgaris L.) milk and
yogurt in Caco-2 and HT-29 cell models. J. Funct. Foods 2019, 53, 125–135.
38. Putt, K.K.; Pei, R.; White, H.M.; Bolling, B.W. Yogurt inhibits intestinal barrier dysfunction in Caco-
2 cells by increasing tight junctions. Food Funct. 2017, 8, 406–414.
39. Chelakkot, C.; Ghim, J.; Ryu, S.H. Mechanisms regulating intestinal barrier integrity and its
pathological implications. Exp. Mo. Med. 2018, 50, 103. 
40. Mohebali, N.; Ekat, K.; Kreikemeyer, B.; Breitrück, A. Barrier Protection and Recovery Effects of Gut
Commensal Bacteria on Differentiated Intestinal Epithelial Cells In Vitro. Nutrients 2020, 12, 2251.
https://doi.org/10.3390/nu12082251
41. Zeng, J.; Jiang, J.; Zhu, W.; Chu, Y. Heat-killed yogurt-containing lactic acid bacteria prevent
cytokine-induced barrier disruption in human intestinal Caco-2 cells. Ann. Microbiol. 2016, 66,
171–178.
42. Beguin, P.; Errachid, A.; Larondelle, Y.; Schneider, Y.J. Effect of polyunsaturated fatty acids on
tight junctions in a model of the human intestinal epithelium under normal and inflammatory
conditions. Food Funct. 2013, 4, 923–931.
43. Popović, N.; Brdarić, E.; Đokić, J.; Dinić, M.; Veljović, K.; Golić, N.; Terzić-Vidojević, A. Yogurt
produced by novel natural starter cultures improves gut epithelial barrier in
vitro. Microorganisms 2020, 8, 1586.
44. Maubon, N.; Le Vee, M.; Fossati, L.; Audry, M.; Le Ferrec, E.; Bolze, S.; Fardel, O. Analysis of
drug transporter expression in human intestinal Caco-2 cells by real-time PCR. Fund. Clin.
Pharmacol. 2007, 21, 659–663.
45. Vreeburg, R.A.M.; Bastiaan-Net, S.; Mes, J.J. Normalization genes for quantitative RT-PCR in
differentiated Caco-2 cells used for food exposure studies. Food Funct. 2011, 2, 124–129.
46. Karimi, S.; Ghanbarzadeh, B.; Roufegarinejad, L.; Falcone, P.M. Polysaccharide extracted
from Althaea officinalis L. root: New studies of structural, rheological and antioxidant
properties. Carbohydr. Res. 2021, 510, 108438.
47. Xu, D.; Hu, M.-J.; Wang, Y.-Q.; Cui, Y.-L. Antioxidant Activities of Quercetin and Its Complexes for Medicinal
Application. Molecules 2019, 24, 1123. https://doi.org/10.3390/molecules24061123
48. Manian, R; Anusuya, N; Siddhuraju, P; Manian, S. The antioxidant activity and free radical
scavenging potential of two different solvent extracts of Camellia sinensis (L.) O. Kuntz, Ficus
bengalensis L. and Ficus racemosa L. Food Chem 2008, 107, 1000–1007.
49. Srinivasan, B.; Kolli, A.R.; Esch, M.B.; Abaci, H.E.; Shuler, M.L.; Hickman, J.J. TEER
Measurement Techniques for In Vitro Barrier Model Systems. J. Lab. Autom. 2015, 20, 107–126.
50. Zucco, F.; Batto, A.; Bises, G.; Chambaz, J.; Chiusolo, A.; Consalvo, R.; Cross, H.; Dal Negro,
G.D.; de Angelis, I.; Fabre, G.; et al. An inter-laboratory study to evaluate the effects of medium
composition on the differentiation and barrier function of Caco-2 cell lines. Altern. Lab.
Anim. 2005, 33, 603–618.
51. Valdez, J.C.; Cho, J.; Bolling, B.W. Aronia berry inhibits disruption of Caco-2 intestinal barrier
function. Arch. Biochem. Biophys. 2020, 688, 108409. 
52. Aleman, R.S.; Moncada, M.; Aryana, K.J. Leaky Gut and the Ingredients That Help Treat It: A
Review. Molecules 2023, 28, 619. https://doi.org/10.3390/molecules28020619
53. Zhang, J.; Li, Q.; Wu, L.; Xu, S.; Lu, R. Protective effect of surface‐layer proteins from four
Lactobacillus strains on tumor necrosis factor‐α‐induced intestinal barrier dysfunction. J. Sci.
Food Agric. 2022, 102, 4446-4453.
54. Li, N.; Lewis, P.; Samuelson, D.; Liboni, K.; Neu, J. Glutamine regulates Caco-2 cell tight junction
proteins. Am. J. Physiol. Gastrointest. Liver Physiol. 2004, 287, G726–G733.
55. Amasheh, M.; Schlichter, S.; Amasheh, S.; Mankertz, J.; Zeitz, M.; Fromm, M.; Schulzke, J.D.
Quercetin Enhances Epithelial Barrier Function and Increases Claudin-4 Expression in Caco-2
Cells. J. Nutr. 2008, 138, 1067–1073.
56. Shi, L.-E.; Li, Z.-H.; Zhang, Z.-L.; Zhang, T.-T.; Yu, W.-M.; Zhou, M.-L.; Tang, Z.-X. Encapsulation
of Lactobacillus bulgaricus in carrageenan-locust bean gum coated milk microspheres with
double layer structure. LWT Food Sci. Technol. 2013, 54, 147–151.
57. Chen, Y.; Liu, D.; Wang, D.; Lai, S.; Zhong, R.; Liu, Y.; Yang, C.; Liu, B.; Sarker, M.R.; Zhao, C.
Hypoglycemic activity and gut microbiota regulation of a novel polysaccharide from Grifola
frondosa in type 2 diabetic mice. Food Chem. Toxicol. 2019, 126, 295–302.
58. Ain, N.U.; Wu, S.; Li, X.; Li, D.; Zhang, Z. Isolation, Characterization, Pharmacology and Biopolymer
Applications of Licorice Polysaccharides: Review. Materials 2022, 15, 3654.
https://doi.org/10.3390/ma15103654
59. Hashemifesharaki, R.; Xanthakis, E.; Altintas, Z.; Guo, Y.; Gharibzahedi, S.M.T. Microwave-
assisted extraction of polysaccharides from the marshmallow roots: Optimization, purification,
structure, and bioactivity. Carbohydr. Polym. 2020, 240, 116301.
60. Cheng, Y.; Liu, Y.; Chen, D.; Zhou, Y.; Yu, S.; Lin, H.; Liao, C.K.; Lin, H.; Xu, P.; Huang, M. Dual
effects of quercetin on protein digestion and absorption in the digestive tract. Food Chem. 2021,
358, 129891.