Attributes of Lactobacillus acidophilus as Effected by Carao (Cassia grandis) Pulp Powder

Abstract

This study aimed to examine the prebiotic effect of Carao (Cassia grandis) pulp powder on the probiotic characteristics of Lactobacillus acidophilus regarding the viability, enzymatic activity, lysozyme resistance, bile and acid tolerances, and tolerance to gastric juices. Carao powder was used at 0% (control), 1%, 2%, and 3% (w/v). Acid and lysozyme tolerance were determined at 0, 30, 60, 90, and 120 min of incubation, whereas bile tolerance was analyzed at 0, 4, and 8 h. The gastric juice tolerance was determined at pH 2, 3, 4, 5, and 7 during 0 and 30 min of incubation. The protease was evaluated at 0, 12, and 24 h of incubation. The bacterial viability experiment was carried out for 10 h, taking readings every hour. Low-acidity conditions were used, and no significant differences were found between the control and the different Carao concentrations added to the L. acidophilus viability study. The Carao samples at 2% and 3% had significantly (p < 0.05) higher counts for bile and lysozyme resistance and higher protease activity when compared to control samples. On the other hand, Carao addition did not impact bacterial viability, acid tolerance, and gastric juice resistance. Thus, Carao pulp powder at different concentrations could act as a prebiotic source to enhance the development of L. acidophilus during gastrointestinal digestion.

Full text

Citation: Marcia, J.; Aleman, R.S.;
Montero-Fernández, I.; Martín-Vertedor,
D.; Manrique-Fernández, V.; Moncada,
M.; Kayanush, A. Attributes of
Lactobacillus acidophilus as Effected by
Carao (Cassia grandis) Pulp Powder.
Fermentation 2023,9, 408.
https://doi.org/10.3390/
fermentation9050408
Academic Editor: Alessandra Pino
Received: 1 April 2023
Revised: 20 April 2023
Accepted: 22 April 2023
Published: 24 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
fermentation
Article
Attributes of Lactobacillus acidophilus as Effected by Carao
(Cassia grandis) Pulp Powder
Jhunior Marcia 1, Ricardo Santos Aleman 2, Ismael Montero-Fernández 3, * , Daniel Martín-Vertedor 4,
Víctor Manrique-Fernández 5, Marvin Moncada 6and Aryana Kayanush 2
1Faculty of Technological Sciences, Universidad Nacional de Agricultura Road to Dulce Nombre de Culmí,
Km 215, Barrio El Espino, Catacamas 16201, Honduras
2School of Nutrition and Food Sciences, Louisiana State University Agricultural Center,
Baton Rouge, LA 70803, USA
3Department of Chemical Engineering and Physical Chemistry, Area of Chemical Engineering,
Faculty of Sciences, University of Extremadura, Avda. de Elvas, s/n, 06006 Badajoz, Spain
4Technological Institute of Food and Agriculture CICYTEX-INTAEX, Junta of Extremadura,
Avda. Adolfo Suárez s/n, 06007 Badajoz, Spain
5
Área de Nutrición y Bromatología, Departamento de Producción Animal y Ciencia de los Alimentos, Escuela
de Ingenierías Agrarias, Universidad de Extremadura, Avda. Adolfo Suárez s/n, 06007 Badajoz, Spain
6Department of Food, Bioprocessing & Nutrition Sciences and the Plants for Human Health Institute,
North Carolina Research Campus, North Carolina State University, Kannapolis, NC 27599, USA
*Correspondence: ismonterof@unex.es
Abstract:
This study aimed to examine the prebiotic effect of Carao (Cassia grandis) pulp powder
on the probiotic characteristics of Lactobacillus acidophilus regarding the viability, enzymatic activity,
lysozyme resistance, bile and acid tolerances, and tolerance to gastric juices. Carao powder was used
at 0% (control), 1%, 2%, and 3% (w/v). Acid and lysozyme tolerance were determined at 0, 30, 60,
90, and 120 min of incubation, whereas bile tolerance was analyzed at 0, 4, and 8 h. The gastric juice
tolerance was determined at pH 2, 3, 4, 5, and 7 during 0 and 30 min of incubation. The protease
was evaluated at 0, 12, and 24 h of incubation. The bacterial viability experiment was carried out for
10 h, taking readings every hour. Low-acidity conditions were used, and no significant differences
were found between the control and the different Carao concentrations added to the L. acidophilus
viability study. The Carao samples at 2% and 3% had significantly (p< 0.05) higher counts for bile and
lysozyme resistance and higher protease activity when compared to control samples. On the other
hand, Carao addition did not impact bacterial viability, acid tolerance, and gastric juice resistance.
Thus, Carao pulp powder at different concentrations could act as a prebiotic source to enhance the
development of L. acidophilus during gastrointestinal digestion.
Keywords: Lactobacillus acidophilus;Carao; enzymatic activity; lysozyme; gastric juices
1. Introduction
In recent decades, considerable scientific evidence has shown that the interaction of
microbiota with gastrointestinal digestion in humans is fundamental for health balance.
In this sense, studies that have been conducted that assess the use of microorganisms
such as Lactobacillus acidophilus have greatly benefited human health [
1
]. Clinical trials
have demonstrated that this microorganism could prevent and treat diarrhea (infantile,
acute, and associated with antibiotics) in children [
2
]. Lactobacillus acidophilus is also
effective in treating symptoms accompanying lactose intolerance, inflammatory bowel
diseases, modulation of the immune system, and colon cancer [
3
]. Furthermore, many
researchers have carried out studies for the inhibition of different digestive cancers when
these microorganisms are administered in adequate concentration. Isazadeh et al. (2021) [
4
]
indicated that L. acidophilus could inhibit the viability of colorectal cancer of Caco-2 cell line,
increasing the survival rate of the patients.
Fermentation 2023,9, 408. https://doi.org/10.3390/fermentation9050408 https://www.mdpi.com/journal/fermentation

Fermentation 2023,9, 408 2 of 12
Certain foods, due to their chemical composition, are implemented as a prebiotic
source, because these are considered essential for people’s health. Therefore, prebiotic
foods play an important role in the microbiota, and are beneficial for the human gas-
trointestinal tract [
5
]. Prebiotics are defined as non-digestible compounds that serve to
modulate the composition and activity of the intestinal microbiota, which are metabo-
lized by microorganisms in the intestine, and confer a beneficial effect on the host [
6
].
Prebiotics are found in many fruits and vegetables, especially those that contain complex
carbohydrates, such as fiber and resistant starch [
6
], or non-digestible compounds, such as
inulin, that can stimulate the multiplication of microorganisms in the colon, modulating the
intestinal microbiota. Different investigations with prebiotic foods such as honey [
7
], meat
products [
8
], fermented soybean [
9
], or vegetable milk [
10
] have been shown to enhance
microbial development. As a result, new prebiotic sources are demanded by consumers to
be incorporated into their daily diet, and food industries are prioritizing the development
of these prebiotic products. Furthermore, prebiotics can stimulate the growth of healthy
bacteria in the intestine, reducing the risk of developing certain diseases [11].
Cassia grandis (Carao) fruits have been used in alternative medicine due to their charac-
teristic effect in humans and chemical composition [
12
]. These fruits have shown consider-
able amounts of alkaloids, flavonoids, and phenols, and excellent antioxidant capacity [
13
].
In the leaves, Carao has shown substantial amounts of phenolic compounds such as Gran-
disina, Kaempferol, Quercetin, and Flavonol [
14
]. Besides, the phytochemical characteristics
of this fruit, it has great antidiabetic potential due to its trypsin inhibitory effect [
15
]. In addi-
tion, Carao has been shown to improve acid and bile tolerance of Streptococcus
thermophilus
and Lactobacillus bulgaricus [
16
]. Due to all these characteristics, this fruit could be used as a
potential prebiotic against certain microorganisms. As a result, the current study aims to
study the prebiotic effect of Carao powder on enzymatic activity, lysozyme resistance, bile
and acid tolerances, and tolerance to gastric juices of Lactobacillus acidophilus, to determine
its potential to promote resistance in the digestive system from the mouth to the intestines.
2. Materials and Methods
2.1. Plant Material
The C. grandis (Carao) fruit was gathered from the Guapinol Biological Reserve, Mar-
covia Municipality, Choluteca Department (Honduras), between August and September
2021. The pulp was separated, and a solution of Carao pulp (10% w/w) was prepared
and then kept cryogenically (
−
80
◦
C). The obtained Carao aqueous solution was then
lyophilized (LIOTOP model L 101) for 48 h at a temperature of
−
75
◦
C and a chamber
pressure of 0.1 to 0.5 Pa. The freeze-dried Carao pulp powder was kept in plastic bags for
further use.
2.2. Experimental Design
The viability; acid, bile, lysozyme, and gastric juice tolerances; and protease activity
of Lactobacillus acidophilus LYO 50 (Danisco, Dairy Connection, Madison, WI, USA) as
affected by Carao powder were examined at 0% (control), 1%, 2%, and 3%. The bacterial
viability was studied in MRS broth. Acid tolerance was determined by adjusting the pH to
2, whereas bile tolerance was examined with Oxgall 0.3% (w/v) in MRS broth. Lysozyme
resistance was investigated in an electrolyte solution with lysozyme (100 mg/L), while
gastric juice tolerance was analyzed using pepsin and NaCl. Protease activity was deter-
mined spectrophotometrically at 340 nm in skim milk with o-phthaldialdehyde reagent.
The microbial growth was determined at 0, 2, 4, 6, 8, and 10 h of incubation. Acid tolerance
was determined at 0, 5, and 15 min, whereas bile tolerance was analyzed at 0, 4, and 8 h of
incubation. Lysozyme tolerance was determined at 0, 1, and 2 h of incubation, while gastric
juice tolerance was determined at pH 2, 3, 4, 5, and 7. The protease activity was evaluated
at 0, 12, and 24 h of incubation. L. acidophilus was incubated anaerobically (37
◦
C). The log
counts were measured in MRS agar with duplicate readings. All experiments were carried
out in triplicate.

Fermentation 2023,9, 408 3 of 12
2.3. Analytical Method
2.3.1. Bacterial Viability
The viability of L. acidophilus was examined by the procedure suggested by Lin and
Young (2000) [
17
], with some changes. The culture (inoculation of 10% (v/v)) was inoculated
in MRS broth (CriterionTM, Hardy Diagnostics, Santa Maria, CA, USA) containing 0.5%
lactose with 0.2% (w/v) sodium thioglycolate (Sigma-Aldrich, St. Louis, MO, USA) and the
pH was adjusted to 6.5. The cultured broths were incubated at 37
◦
C. An 11 mL sample was
collected at several periods (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h), 10-fold diluted in peptone
water, and plated in duplicate. Bacterial viability was evaluated in presence and absence of
Carao pulp at three different concentrations.
2.3.2. Bile Tolerance
The bile tolerance of L. acidophilus was evaluated using the Pereira & Gibson (2002) [
18
]
method, with slight modification. After the culture media broth was prepared, Carao pulp
powder, water, and 1.5 g bile salt (Oxgall salt 0.3%) were added and autoclaved. The culture
(inoculation of 10% (v/v)) was inoculated in MRS broth (CriterionTM, Hardy Diagnostics,
Santa Maria, CA, USA) containing 0.5% lactose with 0.2% (w/v) sodium thioglycolate
(Sigma-Aldrich) and bile salt Oxgall (bovine bile) (US Biological, Swampscott, MA, USA).
The cultured broths were incubated at 37
◦
C. An 11 mL sample was collected at several
periods (0, 4, and 8 h), 10-fold diluted in peptone water, and plated in duplicate. Tolerance
to specific acidity and resistance to gastric juices were evaluated in the presence and absence
of Carao pulp at three different concentrations.
2.3.3. Acid Tolerance
The acid tolerance of L. acidophilus was evaluated by inoculation of culture (10% (v/v))
into the acidified MRS broth containing 0.5% lactose, with 1 N HCl added to produce pH 2.0.
This acidified MRS broth containing the culture was incubated at a temperature of
37 ◦C
. A
1 mL sample was collected at several periods (0, 30, and 60 min). The protease activity was
evaluated in the presence and absence of Carao pulp at three different concentrations.
2.3.4. Protease Activity for Probiotics
L. acidophilus protease activity was evaluated by inoculation of culture (10% (v/v))
using the o-phthaldialdehyde (OPA) spectrophotometric test established by Oberg et al.
(1991) [
19
]. After incubation of L. acidophilus in sterile skim milk [
20
], L. acidophilus was
grown at 37
◦
C for 0, 12, and 24 h, then 2.5 mL of each sample was combined with 1 mL of
distilled water and 10 mL of 0.75 N trichloroacetic acid (TCA) to give a final concentration
of 7.7%. All samples were filtered using a Whatman Number 2 filter paper for 10 min
at ambient conditions. A double portion of each TCA filtrate was examined by the OPA
spectrophotometric test utilizing a spectrophotometer at 340 nm (Nicolet Evolution 100,
Thermo Scientific; Madison, WI, USA).
2.3.5. Tolerance to Simulated Gastric Juice
The tolerance of L. acidophilus to functional substances in synthetic gastric juice (SGJ)
was tested using the method described by García-Ruiz et al. (2014), Aleman et al. (2023), and
Liao et al. (2019) [
21
–
23
]. The SGJ was prepared using H
2
O, pepsin 0.32% (Sigma-Aldrich,
St. Louis, MO, USA), NaCl 0.2%, NaOH, and HCl for pH adjustment [
24
]. Lysozyme
resistance was evaluated in the presence and absence of Carao pulp at three different
concentrations. With 1 M HCl and 1 M NaOH, the simulated gastric juice was modified
to five concentration gradients (pH 7, 5, 4, 3, and 2). The culture was inoculated (10%
(w/v)) into SGJ, and incubated for 30 min under anaerobic conditions at 37
◦
C. Plates were
counted at 0 and 30 min of incubation to determine live bacteria. Bacterial viability was
measured by inoculating the bacteria in MRS broth, and numeration of L. acidophilus was
determined by plating the bacteria with MRS agar. Paz et al. (2022) [
16
] proposed these
methods as well.

Fermentation 2023,9, 408 4 of 12
2.3.6. Lysozyme Tolerance
The L. acidophilus resistance to lysozyme was evaluated according to Zago et al.
(2011) [
25
], with slight modification. The electrolyte solution was used to control the
lysozyme tolerance test and to imitate
in vivo
dispersion by saliva. Bacteria cultures were
inoculated (10% (w/v)) into sterile electrolyte solution (SES) of 0.22 g
·
L
−1
CaCl
2
, 6.2 g
·
L
−1
NaCl, 2.2 g
·
L
−1
KCl, and 1.2 g
·
L
−1
NaHCO
3
in the presence of lysozyme (100 mg
·
L
−1
)
(Sigma-Aldrich, CA USA). Tests comprised microbial cultures in SES without lysozyme.
Bacterial counting was performed on MRS agar after incubation (72 h at 37
◦
C). The survival
expectancy was determined by comparing the CFU·mL−1at 0, 30, 60, 90, and 120 min.
2.3.7. Enumeration of L. acidophilus
Preparation of the MRS broth of L. acidophilus included 1 L of distilled water being
added to 55 g of MRS broth powder (Difco, Becton, Dickinson and Co., Sparks, MD, USA).
Next, 1 N HCl was utilized to reduce the pH to 5.2. To thoroughly disperse the particles,
this medium was boiled under stirring, as well as sterilized at 121
◦
C for
15 min
[
26
,
27
].
Following plating into the inoculated medium, MRS broths were pipetted to various
formulations using 99 mL of sterilizing phosphate buffer 0.1% (w/v). Following 72 h,
these L. acidophilus plates were heated anaerobically at 37
◦
C. A Quebec Darkfield Colony
Counter was used to calculate (Leica Inc., Buffalo, NY, USA) [16].
2.4. Statistical Analysis
Data were analyzed using the General Linear Model (PROC GLM) of the Statistical
Analysis Systems (SAS). Differences of least square means were used to determine sig-
nificant differences at p< 0.05 for the main effect (Carao pulp concentration vs. control).
Data are presented as mean
±
standa0072d error of means. Significant differences were
determined at α= 0.05.
3. Results and Discussion
3.1. Bacterial Viability
The bacterial viability of L. acidophilus over 10 h of incubation after the addition of
Carao pulp powder is shown in Figure 1. The Carao concentration effect and the interaction
effect (Carao concentration
×
hour) were not significant (p> 0.05), whereas the hour effect
was significant (p< 0.05) (Table 1). The interaction effect was not significant (p> 0.05),
meaning that the control and Carao samples followed the same trend. Control and Carao
samples increased in log counts over time. The log count increased from 8.83 to 9.38 from
0 to 10 h
for control samples. All Carao treatments followed a comparable viability tendency
to the control samples (Table 2). This is a good result, since the high antioxidant capacity of
this fruit [
12
] at this concentration does not cause a marked inhibition of the development
of L. acidophilus. Muramalla and Aryana (2011) [
28
] examined the viability of L. acidophilus
in MRS broth, and concluded that an increase in viability was reached in the first 3 h of
incubation. Paz et al. (2022) [
16
] also reported that Carao did not adversely impact the
viability of Streptococcus thermophilus and Lactobacillus bulgaricus.

Fermentation 2023,9, 408 5 of 12
Fermentation 2023, 9, x FOR PEER REVIEW 5 of 13
Figure 1. Viability of L. acidophilus as influenced by Carao concentration over 10 h.
Table 1. The p-value or F-value of Carao concentration, time, pH, and their interactions for bacterial
viability, bile tolerance, acid tolerance, resistance to gastric juices, protease activity, and lysozyme
resistance of Lactobacillus acidophilus LA-K.
Effect
L. acidophilus LA-K
Viability
Carao concentration
0.0770
Time (Hours)
<0.0001
Carao concentration × time
0.4756
Bile tolerance
Carao concentration
0.0057
Time (Hours)
<0.0001
Carao concentration × time
0.0045
Acid Tolerance
Carao concentration
0.095
Time (Minutes)
<0.0001
Carao concentration × time
0.5867
Resistance to gastric juices
Carao concentration
0.0786
pH
<0.0001
Carao concentration × pH
0.7845
Protease activity
Carao concentration
0.0155
Figure 1. Viability of L. acidophilus as influenced by Carao concentration over 10 h.
Table 1.
The p-value or F-value of Carao concentration, time, pH, and their interactions for bacterial
viability, bile tolerance, acid tolerance, resistance to gastric juices, protease activity, and lysozyme
resistance of Lactobacillus acidophilus LA-K.
Effect L. acidophilus LA-K
Viability
Carao concentration 0.0770
Time (Hours) <0.0001
Carao concentration ×time 0.4756
Bile tolerance
Carao concentration 0.0057
Time (Hours) <0.0001
Carao concentration ×time 0.0045
Acid Tolerance
Carao concentration 0.095
Time (Minutes) <0.0001
Carao concentration ×time 0.5867
Resistance to gastric juices
Carao concentration 0.0786
pH <0.0001
Carao concentration ×pH 0.7845
Protease activity
Carao concentration 0.0155
Time (Hours) <0.0001
Carao concentration ×time 0.4021
Lysozyme resistance
Carao concentration 0.0085
Time (Minutes) <0.0001
Carao concentration ×time 0.3945
The concentration and the type of plant influence microbial growth. For the most
part, medical plants’ phenolic content has reported inhibitory effects due to different
mechanisms of action, including the inhibition of acid production and the glucosyltrans-
ferase enzyme [29]. Plants such as Plantago major L,Erythroxylum novogranatense,Plowman
var truxillensey, and Camellia (extracted using 70% ethanol) have been shown to have an-
timicrobial activity towards L. acidophilus when the extracts are diluted to 25
µ
g
·
mL
−1
and 50
µ
g
·
mL
−1
[
29
]. Medical plant Tagetes eliptica (extracted using 70% ethanol) extract
has been shown to have an inhibition effect when extracts are diluted no lower than

Fermentation 2023,9, 408 6 of 12
62.5 mg·mL−1
[
30
]. Similarly, at concentrations greater than 1%, clove extract was reported
to have antimicrobial activity toward L. acidophilus in MRS broth [31].
Table 2.
Least squares means for bacterial viability, bile tolerance, acid tolerance, resistance to gastric
juices, protease activity, and lysozyme resistance of Lactobacillus acidophilus LA-K as influenced by
Carao concentration.
Test L. acidophilus LA-K
Bacterial Viabiliy
Carao 0% (Control) NS
Carao 1% NS
Carao 2% NS
Carao 3% NS
Bile tolerance
Carao 0% (Control) 10.20 A
Carao 1% 10.17 A
Carao 2% 10.24 B
Carao 3% 10.33 C
Acid Tolerance
Carao 0% (Control) NS
Carao 1% NS
Carao 2% NS
Carao 3% NS
Resistance to gastric juices
Carao 0% (Control) NS
Carao 1% NS
Carao 2% NS
Carao 3% NS
Protease activity
Carao 0% (Control) 0.300 A
Carao 1% 0.321 A
Carao 2% 0.339 B
Carao 3% 0.355 B
Lysozyme resistance
Carao 0% (Control) 6.06 A
Carao 1% 6.15 A
Carao 2% 6.36 B
Carao 3% 6.68 B
A, B: Means within the same column along with the same test with different letters differ statistically (p< 0.05).
3.2. Bile Tolerance
The effect of Carao pulp power on L. acidophilus’ tolerance to bile (to resist Oxgall
salt) in MRS broth is shown in Figure 2. The main effects (Carao concentration and time)
and interaction effect were significant (p> 0.05) (Table 1). The interaction effect was not
significant (p> 0.05), meaning that the control and Carao samples did not follow the same
trend (Table 2). Control and 1% and 2% Carao samples decreased in log counts over time,
whereas 3% Carao samples increased in log counts over time. Theegala et al. (2021) [
32
]
reported a similar growth for L. acidophilus evaluated in MRS broth. Bile salts are known to
alter eukaryotic gene expression, denature proteins, damage membranes, chelate calcium
and iron, and disrupt DNA in probiotics’ immunity activity [
33
]. At 5, 6, 7, and 8 h, 2% and
3% Carao broths had significantly (p> 0.05) higher counts than control samples. Adding
inulin to yogurt enhanced the capacity of L. acidophilus in yogurt to resist bile salts [
34
], and
incorporating flaxseed into MRS broth with 0.3% Oxgall salt also improved the survivability
of L. acidophilus [
32
]. The addition of 5.3 g
·
L
−1
of Carao improved resistance to Oxgall salt
(0.03%) in M17 and MRS broth for Streptococcus thermophilus and Lactobacillus delbrueckii
ssp. bulgaricus, respectively [
16
]. Alginate–milk microspheres can encapsulate L. bulgaricus
and increase the survivability for 1 and 2 h in 1% and 2% porcine bile salt solutions [
35
]. In
Cassia fistula, polysaccharides with encapsulating properties have been reported [
36
]. It is

Fermentation 2023,9, 408 7 of 12
possible that polysaccharides in Carao could have an encapsulating effect on L. acidophilus,
resulting in improved bile tolerance.
Fermentation 2023, 9, x FOR PEER REVIEW 8 of 13
Figure 2. Bile tolerance of L. acidophilus as influenced by Carao concentration over 8 h.
3.3. Acid Tolerance and Resistance to Gastric Juices
The acid tolerance and resistance of L. acidophilus to gastric juices are shown in Figure
3 and Figure 4, respectively. The results at different pH values are shown in Figure 4. Acid
tolerance was examined through a 15 min bacterial count to examine the effects of Carao
concentration on the survival of L. acidophilus under stomach acid condition. Under nor-
mal conditions, the transit time through the gastrointestinal system ranges from 2 to 4 h,
and varies depending on the individual [37]. The gastric juice resistance was analyzed at
different pH values (2, 3, 4, 5 and 7) for bacterial viability, to investigate the influence of
Carao concentration on the survival of L. acidophilus in the different parts of the digestive
system (esophagus, small intestine, and large intestine). For acid tolerance, the Carao con-
centration and interaction effect (Carao concentration × time) were not signific ant (p < 0.05),
whereas the time effect was significant (p > 0.05) (Table 1). The interaction effect was not
significant (p > 0.05), meaning that the control and Carao samples followed the same trend
(Table 2). Control and Carao samples decreased in log counts over time. The log counts
decreased from 0 to 10 min, and remained stable from 10 to 15 min for the Carao treatments
and control. For gastric juice resistance, the Carao concentration and interaction effect
(Carao concentration and pH) were not significant (p < 0.05), whereas the pH effect was
significant (p > 0.05) (Table 1). The log counts were lower from pH 2 to 4 and higher from
pH 5 to 7 for the Carao treatments and control. Not surprisingly, these results are con-
sistent with other studies showing that Lactobacillus strains showed lower counts when
exposed to pH values of 4.0, and higher viability at higher pH values [38]. It was observed
that adding Carao into MRS broth did not influence the acid tolerance and gastric juice
resistance of L. acidophilus. The high levels of H+ ions can disrupt hydrogen bonding, pro-
moting cell denaturation and destroying activity by producing complications for associ-
ated cell membrane energetics [39]. Probiotics must persist under the acidic gastric condi-
tions from the digestive system to colonize in the small intestine. Thereby, functional in-
gredients must, at least, not negatively affect the probiotics in order to survive the acidic
gastric environment. Paz et al. (2022) [16] reported that C. grandis improved the acid toler-
ance of S. thermophilus, and it did not impact the acid tolerance of L. bulgaricus.
Figure 2. Bile tolerance of L. acidophilus as influenced by Carao concentration over 8 h.
3.3. Acid Tolerance and Resistance to Gastric Juices
The acid tolerance and resistance of L. acidophilus to gastric juices are shown in Figure 3
and Figure 4, respectively. The results at different pH values are shown in Figure 4. Acid
tolerance was examined through a 15 min bacterial count to examine the effects of Carao
concentration on the survival of L. acidophilus under stomach acid condition. Under normal
conditions, the transit time through the gastrointestinal system ranges from 2 to
4 h
, and
varies depending on the individual [
37
]. The gastric juice resistance was analyzed at
different pH values (2, 3, 4, 5 and 7) for bacterial viability, to investigate the influence of
Carao concentration on the survival of L. acidophilus in the different parts of the digestive
system (esophagus, small intestine, and large intestine). For acid tolerance, the Carao
concentration and interaction effect (Carao concentration
×
time) were not significant
(
p< 0.05
), whereas the time effect was significant (p> 0.05) (Table 1). The interaction effect
was not significant (p> 0.05), meaning that the control and Carao samples followed the
same trend (Table 2). Control and Carao samples decreased in log counts over time. The
log counts decreased from 0 to
10 min
, and remained stable from 10 to 15 min for the
Carao treatments and control. For gastric juice resistance, the Carao concentration and
interaction effect (Carao concentration and pH) were not significant (p< 0.05), whereas the
pH effect was significant (p> 0.05) (Table 1). The log counts were lower from pH 2 to 4
and higher from pH 5 to 7 for the Carao treatments and control. Not surprisingly, these
results are consistent with other studies showing that Lactobacillus strains showed lower
counts when exposed to pH values of 4.0, and higher viability at higher pH values [
38
]. It
was observed that adding Carao into MRS broth did not influence the acid tolerance and
gastric juice resistance of L. acidophilus. The high levels of H
+
ions can disrupt hydrogen
bonding, promoting cell denaturation and destroying activity by producing complications
for associated cell membrane energetics [
39
]. Probiotics must persist under the acidic
gastric conditions from the digestive system to colonize in the small intestine. Thereby,
functional ingredients must, at least, not negatively affect the probiotics in order to survive
the acidic gastric environment. Paz et al. (2022) [
16
] reported that C. grandis improved the
acid tolerance of S. thermophilus, and it did not impact the acid tolerance of L. bulgaricus.

Fermentation 2023,9, 408 8 of 12
Fermentation 2023, 9, x FOR PEER REVIEW 9 of 13
Figure 3. Acid tolerance of L. acidophilus as influenced by Carao concentration over 15 min. Average
of three replicates. Error bars represent SE.
Figure 4. Gastric juices resistance of L. acidophilus as influenced by Carao concentration over different
pH levels (2,3,4,5, and 7). * Average mean log CFU/mL of 0 min and 30 min. Average of three repli-
cates. Error bars represent SE.
3.4. Protease Activity
Proteolysis in fermented milk is of great importance for several aspects: it can deter-
mine the survival of the probiotic cultures, it contributes to the formation of flavor and
odor compounds, it confers rheological properties, and it allows the formation of bioactive
peptides [40]. The protease activity of L. acidophilus is shown in Figure 5. Proteolysis is the
degradation of proteins by the action of the proteolytic system of lactic acid bacteria
(LAB), which produces small peptides and free amino acids that are essential for probiotic
growth and activity [41]. The Carao concentration and time effects were significant (p >
0.05), whereas the interaction effect (Carao concentration × time) was not significant (p <
0.05) (Tabl e 1). The interaction effect was not significant (p > 0.05), meaning that the control
and Carao samples followed the same trend. Control and Carao samples increased in log
counts over time. The protease activity of L. acidophilus showed an increase after 24 h for
control and Carao treatments (Table 2 ). The proteolytic activity of L. acidophilus is mainly
Figure 3.
Acid tolerance of L. acidophilus as influenced by Carao concentration over 15 min. Average
of three replicates. Error bars represent SE.
Fermentation 2023, 9, x FOR PEER REVIEW 9 of 13
Figure 3. Acid tolerance of L. acidophilus as influenced by Carao concentration over 15 min. Average
of three replicates. Error bars represent SE.
Figure 4. Gastric juices resistance of L. acidophilus as influenced by Carao concentration over different
pH levels (2,3,4,5, and 7). * Average mean log CFU/mL of 0 min and 30 min. Average of three repli-
cates. Error bars represent SE.
3.4. Protease Activity
Proteolysis in fermented milk is of great importance for several aspects: it can deter-
mine the survival of the probiotic cultures, it contributes to the formation of flavor and
odor compounds, it confers rheological properties, and it allows the formation of bioactive
peptides [40]. The protease activity of L. acidophilus is shown in Figure 5. Proteolysis is the
degradation of proteins by the action of the proteolytic system of lactic acid bacteria
(LAB), which produces small peptides and free amino acids that are essential for probiotic
growth and activity [41]. The Carao concentration and time effects were significant (p >
0.05), whereas the interaction effect (Carao concentration × time) was not significant (p <
0.05) (Tabl e 1). The interaction effect was not significant (p > 0.05), meaning that the control
and Carao samples followed the same trend. Control and Carao samples increased in log
counts over time. The protease activity of L. acidophilus showed an increase after 24 h for
control and Carao treatments (Table 2 ). The proteolytic activity of L. acidophilus is mainly
Figure 4.
Gastric juices resistance of L. acidophilus as influenced by Carao concentration over different
pH levels (2,3,4,5, and 7). * Average mean log CFU/mL of 0 min and 30 min. Average of three
replicates. Error bars represent SE.
3.4. Protease Activity
Proteolysis in fermented milk is of great importance for several aspects: it can deter-
mine the survival of the probiotic cultures, it contributes to the formation of flavor and
odor compounds, it confers rheological properties, and it allows the formation of bioactive
peptides [
40
]. The protease activity of L. acidophilus is shown in Figure 5. Proteolysis is
the degradation of proteins by the action of the proteolytic system of lactic acid bacteria
(LAB), which produces small peptides and free amino acids that are essential for probiotic
growth and activity [
41
]. The Carao concentration and time effects were significant (p> 0.05),
whereas the interaction effect (Carao concentration
×
time) was not significant (p< 0.05)
(Table 1). The interaction effect was not significant (p> 0.05), meaning that the control and
Carao samples followed the same trend. Control and Carao samples increased in log counts
over time. The protease activity of L. acidophilus showed an increase after 24 h for control
and Carao treatments (Table 2). The proteolytic activity of L. acidophilus is mainly because of
the synthesis of serine-like proteinase [
42
]. Carao treatments showed no significant differ-
ence (p> 0.05) from control samples at 0 h and 12 h, whereas 2% Carao and 3% Carao had

Fermentation 2023,9, 408 9 of 12
significantly higher protease activity at 24 h. Paz et al. (2022) [
16
] also reported that Carao
pulp increases the protease activity of Streptococcus thermophilus and Lactobacillus bulgaricus
after 24 h in skim milk. During milk fermentation, the proteolytic system of probiotic
cultures plays a key role [
40
], since the proteolysis carried out by each microorganism is
initiated by a single extracellular proteinase. Danisco, Dairy Connection, Madison, WI was
studied in absence (control) and in presence of three different concentrations of Carao pulp
powder (1%, 2%, and 3%). Acid and lysozyme tolerance were determined.
Fermentation 2023, 9, x FOR PEER REVIEW 10 of 13
because of the synthesis of serine-like proteinase [42]. Carao treatments showed no signif-
icant difference (p > 0.05) from control samples at 0 h and 12 h, whereas 2% Carao and 3%
Carao had significantly higher protease activity at 24 h. Paz et al. (2022) [16] also reported
that Carao pulp increases the protease activity of Streptococcus thermophilus and Lactobacil-
lus bulgaricus after 24 h in skim milk. During milk fermentation, the proteolytic system of
probiotic cultures plays a key role [40], since the proteolysis carried out by each microor-
ganism is initiated by a single extracellular proteinase. Danisco, Dairy Connection, Madi-
son, WI was studied in absence (control) and in presence of three different concentrations
of Carao pulp powder (1%, 2%, and 3%). Acid and lysozyme tolerance were determined.
Figure 5. Protease activity of L. acidophilus as influenced by Carao concentration over 24 h. * Average
of three replicates. A–C Values with different leers are significantly different between control and
Carao treatments (p < 0.05). Error bars represent SE. * No significant differences between control and
Carao treatments (p < 0.05). ** No significant differences between 0 h and 12 h (p < 0.05).
3.5. Lysozyme Resistance
Resistance to lysozyme is shown in Figure 6. Lysozyme is a crucial element of anti-
microbial activity in saliva, making it an essential component of mouth immune activity.
The mechanism of action of this enzyme on, especially, Gram-positive bacteria is by hy-
drolyzing 1,4-beta-linkages between N-acetylglucosamine and N-acetylmuramic acid in
the bacterial membrane [43]. The Carao concentration and time effects were significant (p
> 0.05), whereas the interaction effect (Carao concentration × time) was not significant (p <
0.05) (Table 1). The interaction effect was not significant (p > 0.05), meaning that the control
and Carao samples followed the same trend. Control and Carao samples decreased in log
counts over time. For control and Carao treatments, the log counts decreased from 0 to 60
min, and remained stable from 60 to 120 min. The 2% and 3% Carao electrolyte dispersions
reported significantly (p > 0.05) higher viability than control samples (Table 2). Carao has
substantial portions of sucrose [44], and this substrate could be used for the survivability
of this probiotic. Furthermore, Carao can also inhibit the digestive enzyme activity of pan-
creatic lipase [15]. As a hypothesis, Carao could act as a barrier between the cell membrane
and the lysozyme to protect L. acidophilus, leading to higher viability in electrolyte solu-
tions with Carao treatments, and possibly inhibiting the hydrolysis of 1,4-beta-linkages
between N-acetylglucosamine and N-acetylmuramic acid. Nevertheless, this mechanism
of action still needs to be confirmed, and more research on this topic is encouraged.
Figure 5.
Protease activity of L. acidophilus as influenced by Carao concentration over 24 h. * Average
of three replicates.
A–C
Values with different letters are significantly different between control and
Carao treatments (p< 0.05). Error bars represent SE. * No significant differences between control and
Carao treatments (p< 0.05). ** No significant differences between 0 h and 12 h (p< 0.05).
3.5. Lysozyme Resistance
Resistance to lysozyme is shown in Figure 6. Lysozyme is a crucial element of an-
timicrobial activity in saliva, making it an essential component of mouth immune activity.
The mechanism of action of this enzyme on, especially, Gram-positive bacteria is by hy-
drolyzing 1,4-beta-linkages between N-acetylglucosamine and N-acetylmuramic acid in
the bacterial membrane [
43
]. The Carao concentration and time effects were significant
(
p> 0.05
), whereas the interaction effect (Carao concentration
×
time) was not significant
(p< 0.05) (Table 1). The interaction effect was not significant (p> 0.05), meaning that the
control and Carao samples followed the same trend. Control and Carao samples decreased
in log counts over time. For control and Carao treatments, the log counts decreased from
0 to 60 min, and remained stable from 60 to 120 min. The 2% and 3% Carao electrolyte
dispersions reported significantly (p> 0.05) higher viability than control samples (Table 2).
Carao has substantial portions of sucrose [
44
], and this substrate could be used for the
survivability of this probiotic. Furthermore, Carao can also inhibit the digestive enzyme
activity of pancreatic lipase [
15
]. As a hypothesis, Carao could act as a barrier between
the cell membrane and the lysozyme to protect L. acidophilus, leading to higher viability
in electrolyte solutions with Carao treatments, and possibly inhibiting the hydrolysis of
1,4-beta-linkages between N-acetylglucosamine and N-acetylmuramic acid. Nevertheless,
this mechanism of action still needs to be confirmed, and more research on this topic
is encouraged.

Fermentation 2023,9, 408 10 of 12
Fermentation 2023, 9, x FOR PEER REVIEW 11 of 13
Figure 6. Lysozyme resistance of L. acidophilus as influenced by Carao concentration over 120 min.
4. Conclusions
The results showed that the Carao fruit could be used as a potential prebiotic for Lac-
tobacillus acidophilus because it does not affect bacterial viability, acid tolerance, and re-
sistance to gastric juices, since, due to its high antioxidant capacity, there is a viability
trend comparable to that of the fruit. In addition, the addition of 2% and 3% Carao im-
proved bile tolerance. For the tolerance to gastric juices, there was no influence of Carao
on the tolerance to acid or on the resistance to gastric juices of Lactobacillus. Regarding its
protease activity, Carao in concentrations of 2 and 3% presented significant activity at 24
h, as well as resistance to L. acidophilus lysozyme in MRS broth and electrolyte solution.
Due to this, Carao could act as a barrier between the cell membrane and the protective
lysozyme of L. acidophilus, so Carao could have prebiotic properties against L. acidophilus.
For future research, it is suggested to examine the probiotic characteristics of Carao in vivo,
to enable its precise application in prebiotic or symbiotic scenarios.
Author Contributions: Conceptualization, R.S.A., J.M., and I.M.-F.; methodology, R.S.A., J.M.,
M.M., V.M.-F., A.K.; and I.M.-F.; software, R.S.A. and I.M.-F.; formal analysis, J.M. (most of the re-
search), R.S.A., and I.M.-F.; resources, R.S.A. and I.M.-F.; data curation, I.M.-F., R.S.A., and J.M.;
writing—original draft preparation, R.S.A., J.M., V.M.-F., and I.M.-F.; writing—review and editing,
R.S.A., J.M., M.M., A.K., V.M.-F., D.M.-V., and I.M.-F.; project administration, R.S.A., I.M.-F., and
D.M.-V.; funding acquisition, R.S.A., A.K., D.M.-V., and I.M.-F. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the the European Regional Development Fund (FEDER) and
Universidad Nacional de Agricultura (Honduras).
Data Availability Statement:.The authors confirm that the data supporting the findings of this study
are available within the article and the raw data that support the findings are available from the
corresponding author, upon reasonable request.
Acknowledgments: We wish to thank the School of Food Sciences, Louisiana State University Ag-
ricultural Center; the Faculty of Technological Sciences, Universidad Nacional de Agricultura Road
to Dulce, Catacamas, Olancho, Honduras; the Research Groups of the Junta de Extremadura (ref.
GR21121); and the European Regional Development Fund (FEDER) for their help in the develop-
ment of this work. This research was also funded by USDA Hatch funds (LAB94511).
Conflicts of Interest: The authors state that they have no conflict of interest.
Figure 6. Lysozyme resistance of L. acidophilus as influenced by Carao concentration over 120 min.
4. Conclusions
The results showed that the Carao fruit could be used as a potential prebiotic for
Lactobacillus acidophilus because it does not affect bacterial viability, acid tolerance, and
resistance to gastric juices, since, due to its high antioxidant capacity, there is a viability
trend comparable to that of the fruit. In addition, the addition of 2% and 3% Carao improved
bile tolerance. For the tolerance to gastric juices, there was no influence of Carao on the
tolerance to acid or on the resistance to gastric juices of Lactobacillus. Regarding its protease
activity, Carao in concentrations of 2 and 3% presented significant activity at 24 h, as well
as resistance to L. acidophilus lysozyme in MRS broth and electrolyte solution. Due to this,
Carao could act as a barrier between the cell membrane and the protective lysozyme of
L. acidophilus, so Carao could have prebiotic properties against L. acidophilus. For future
research, it is suggested to examine the probiotic characteristics of Carao
in vivo
, to enable
its precise application in prebiotic or symbiotic scenarios.
Author Contributions:
Conceptualization, R.S.A., J.M. and I.M.-F.; methodology, R.S.A., J.M.,
M.M., V.M.-F., A.K. and I.M.-F.; software, R.S.A. and I.M.-F.; formal analysis, J.M. (most of the
research), R.S.A. and I.M.-F.; resources, R.S.A. and I.M.-F.; data curation, I.M.-F., R.S.A. and J.M.;
writing—original
draft preparation, R.S.A., J.M., V.M.-F. and I.M.-F.; writing—review and editing,
R.S.A., J.M., M.M., A.K., V.M.-F., D.M.-V. and I.M.-F.; project administration, R.S.A., I.M.-F. and
D.M.-V.; funding acquisition, R.S.A., A.K., D.M.-V. and I.M.-F. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was funded by the the European Regional Development Fund (FEDER) and
Universidad Nacional de Agricultura (Honduras).
Data Availability Statement:
The authors confirm that the data supporting the findings of this study
are available within the article and the raw data that support the findings are available from the
corresponding author, upon reasonable request.
Acknowledgments:
We wish to thank the School of Food Sciences, Louisiana State University
Agricultural Center; the Faculty of Technological Sciences, Universidad Nacional de Agricultura
Road to Dulce, Catacamas, Olancho, Honduras; the Research Groups of the Junta de Extremadura
(
ref. GR21121
); and the European Regional Development Fund (FEDER) for their help in the develop-
ment of this work. This research was also funded by USDA Hatch funds (LAB94511).
Conflicts of Interest: The authors state that they have no conflict of interest.

Fermentation 2023,9, 408 11 of 12
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