Abstract
Lacticaseibacillus casei Zhang (L. casei Zhang) was used as an
auxiliary starter culture to explore its application in camel milk
fermentation. This study evaluated the effects of L. casei Zhang
supplementation on viable cell count, acidity, texture, insulin-like
growth factor 1 (IGF-1) retention, and metabolite profiles over a
21-day storage period. L. casei Zhang enhanced the retention rate of
active IGF-1 from 52.95% to 59.13% and mitigated the progression of
acidity (from 125 °T to 97.5 °T) compared with the control group.
Additionally, L. casei Zhang significantly improved viscosity and
promoted the formation of gel structures. Furthermore, its addition
significantly influenced the production of key metabolites, including
adenosine diphosphate, oleuropein, and threonine–tryptophan (P < 0.05).
These findings highlight the potential of L. casei Zhang as an
effective auxiliary starter culture for camel milk fermentation,
enhancing its physicochemical properties and modulating its metabolomic
profile.
Keywords: Fermented camel milk, Physicochemical properties, Metabolic
profile, Lacticaseibacillus casei Zhang
Highlights
* •
Fermented camel milk was demonstrated to be an effective carrier of
IGF-1.
* •
Lacticaseibacillus casei Zhang increased ADP and dipeptides in
fermented camel milk.
* •
Lacticaseibacillus casei Zhang affected purine and pyrimidine
metabolism during storage.
1. Introduction
The production and consumption of camel milk has a rich history,
tracing back to ancient times. Due to its geographical constraints,
camel milk is predominantly produced in the desert regions of the
Middle East, Africa, and Central Asia, where it is referred to as
‘desert gold’ by indigenous communities. With the growing development
of the camel milk industry, the camel milk market has gradually
expanded to Europe and North America. Camel milk is rich in vitamins,
minerals, unsaturated fatty acids, and lactoglobulin compared with
bovine milk ([31]Alhaj et al., 2022). Additionally, its high
concentrations of lysozyme, lactoperoxidase, and
N-acetyl-D-glucosaminidase provide protection against spoilage and
pathogenic bacteria, contributing to an extended shelf life ([32]Hamed
et al., 2024). Recent advances in the production of camel dairy
products have highlighted their health-promoting properties.
Researchers have identified anti-diabetic, hypoallergenic, anti-cancer,
and immune-protective effects that drive increased consumer interest
([33]El-Kattawy et al., 2021). Fermented camel dairy products exhibit
extended shelf life, improved digestibility, enhanced flavor, and
elevated levels of vitamins and short-chain fatty acids compared with
fresh camel milk ([34]El Hatmi et al., 2018; [35]Ho et al., 2022).
However, camel milk presents challenges, such as weak and fragile curd
formation during coagulation and prolonged fermentation time. Notably,
Bulca et al. demonstrated that microbial transglutaminase can enhance
the gel structure and sensory properties of camel milk yogurt
([36]Bulca et al., 2022). Furthermore, studies have shown that
Lactobacillus helveticus MB2–1 serves as an effective starter culture,
and in situ exopolysaccharides may act as probiotic stabilizer
substitutes in fermented dairy products ([37]Ge et al., 2022). These
findings suggest that incorporating probiotics and microbial enzymes is
a promising strategy for improving gel stability and overall quality of
camel milk-based products.
Lacticaseibacillus casei Zhang (L. casei Zhang) is a probiotic strain
isolated from Inner Mongolian kumiss that exhibits exceptional
gastrointestinal fluid tolerance, which is a prerequisite for the
viability and effectiveness of probiotics. A second critical criterion
for probiotics is achieving a ‘sufficient quantity’. To exert
beneficial effects, the concentration of viable cells in fermented
dairy products must exceed 10^6 CFU/mL. The final essential attribute
of probiotics is ‘efficacy’. Previous research has demonstrated that L.
casei Zhang harbors a rich repertoire of potential probiotic genes,
including those involved in glutathione synthesis, riboflavin
production, acid resistance, and extracellular polysaccharide secretion
([38]Sun et al., 2023). These genetic characteristics provide robust
theoretical support for its application in functional foods, fostering
the development of diverse probiotic functionalities, including
antioxidant activities. In addition to its genetic advantages, L. casei
Zhang exhibits significant bioactivity in practical applications.
Studies indicate that oral administration of L. casei Zhang may offer
therapeutic potential by modulating short-chain fatty acid (SCFAs) and
nicotinamide metabolism, thereby mitigating kidney injury and slowing
the progression of renal decline ([39]Zhu et al., 2021). Furthermore,
L. casei Zhang possesses protein hydrolysis capabilities that can be
applied in the preparation of fermented soymilk, where it contributes
to the regulation of histidine and lysine biosynthesis ([40]Wang et
al., 2012b, [41]Wang et al., 2012a). In conclusion, L. casei Zhang
demonstrates outstanding biological properties and multiple health
benefits, highlighting its potential for applications in functional
food products.
The field of fermenter strains in China is experiencing rapid
development, integrating both traditional and modern expertise to
unlock promising opportunities. Among these strains, L. casei Zhang,
which has been recognized for its probiotic properties in numerous
studies, can be incorporated as a primary or secondary starter in
various products to confer probiotic benefits. However, it is crucial
to acknowledge that the beneficial effects of L. casei Zhang are not
universally consistent across applications. For instance, the inclusion
of L. casei Zhang in Minas Frescal cheese resulted in changes in
multiple parameters and negatively impacted its sensory acceptance
([42]Dantas et al., 2016). Consequently, it is necessary to investigate
the specific effects of L. casei Zhang on different products. Bai et
al. utilized L. casei Zhang as an auxiliary starter to examine its
application in stirred yogurt and reported improvements in yogurt
viscosity, gel strength, and extracellular polysaccharide synthesis
([43]Bai et al., 2020). Similarly, researchers have observed that the
addition of L. casei Zhang enhanced the production of SCFAs in yogurt
during storage ([44]Peng et al., 2022). These findings suggest that L.
casei Zhang can significantly contribute to the production of
extracellular polysaccharides and SCFAs in fermented dairy products,
thereby improving yogurt texture and exerting prebiotic effects.
To further explore its potential, L. casei Zhang was incorporated as
the primary starter alongside YoFlex® Mild 1.0, in the preparation of
fermented camel milk. The resulting products were analyzed for pH, TA,
IGF-1 levels, water-holding capacity, viable cell counts, viscosity,
texture, and non-targeted metabolomic profiles to assess the impact of
L. casei Zhang during storage.
2. Materials and methods
2.1. Materials and instruments
The freeze-dried L. casei Zhang bacterial powder used in this study was
sourced from the Lactic Acid Bacteria Culture Collection at Inner
Mongolia Agricultural University, China. The strain has also been
deposited at the China General Microbiological Culture Collection
Center (CGMCC) under the strain number CGMCC No. 1697. The commercial
starter culture, YoFlex® Mild 1.0, was purchased from Kohansen
(Beijing, China). Camel milk was collected from the Inner Mongolia
Autonomous Region of the Alxa Left Banner Bayanhot City Pastoral Area.
MRS and M17 media were obtained from Haibo Company in Qingdao, and
vancomycin was purched from Luqiao (Beijing, China). Acetonitrile,
methanol, and formic acid were purchased from Thermo Fisher Scientific
(Shanghai, China).
2.2. Preparation of fermented camel milk
The fermented camel milk was prepared and divided into two groups. The
samples in both groups were inoculated with YoFlex® Mild 1.0 at a rate
of 0.03‰, and with L. casei Zhang at a concentration of
5.0 × 10^6 CFU/mL in the LcZ group. The production process of the
fermented camel milk samples was as follows. Initially, camel milk was
heated to 60 °C and homogenized at 20 MPa using an SRH 60–70
high-pressure homogenizer (China). Subsequently, the mixture was then
pasteurized at 95 °C for 5 min and immediately cooled to 37 °C in a
water bath. The samples were incubated at 37 °C for
constant-temperature fermentation. Fermentation was terminated when the
pH reached 4.50, and the samples were stored at 4 °C for 21 days. Three
parallel samples were prepared for each group. Samples were collected
on days 1, 7, 14, and 21 to measure various indicators.
2.3. Determination of viable cell counts
The method used was adapted from Sun et al. ([45]Sun et al., 2023). The
sample (1.0 g) was accurately weighed and placed in a sterilized glass
bottle, to which 9 mL of 0.9 % sterile NaCl saline was added. The
mixture was agitated for 15 min, after which 1 mL of the diluent was
transferred into 9 mL of saline for gradient dilution. Subsequently, an
appropriate dilution was then selected for pour-plating to determine
the viable cell count. Streptococcus salivarius subsp. thermophilus (S.
thermophilus) was cultured on M17 solid medium supplemented with 1.0%
lactose and incubated at 42 °C for 48 h. Lactobacillus delbrueckii
subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) was cultured on
MRS solid medium (pH = 5.2) at 37 °C for 48 h. L. casei Zhang was
cultured in an MRS-V solid medium (pH = 6.2) supplemented with 0.01‰
vancomycin at 37 °C for 48 h. All the aforementioned solid plates were
incubated under anaerobic conditions.
2.4. Determination of pH and acidity
2.4.1. pH
pH was measured using an SJ-3F pH meter (Shanghai Yi Electrical
Scientific Instruments Co., Ltd., China).
2.4.2. Ta
A precisely measured 10.00 g sample of fermented camel milk was
transferred into a 100 mL conical flask, followed by the addition of
20 mL of distilled water. The mixture was thoroughly agitated to ensure
complete dissolution of the solid components. Subsequently, 2 drops of
phenolphthalein indicator were added, and the solution was agitated
until the phenolphthalein indicator transitioned from colorless to red,
indicating a shift in pH from acidic to alkaline. Titration was
performed using the standard sodium hydroxide titration method
(0.1 mol/L) until the solution exhibited a slight red coloration that
persisted for 30 s, signaling that the endpoint was reached. Finally,
the volume of sodium hydroxide consumed was recorded and used to
calculate TA using the following formula:
[MATH: TA°T=VNaOH×CNaOH×1000/msample :MATH]
2.5. Determination of insulin-like growth factor 1 (IGF-1) content
The IGF-1 content of fermented camel milk was determined according to
the manufacturer's protocol provided with the kit (Shanghai
Enzyme-Linked Biology Co., China). After storage, the fermented camel
milk was centrifuged at 3000 × g for 10 min to remove suspended solids
and large particles. Subsequently, 200 μL of whey from the fermented
camel milk was combined with 800 μL of an acid/ethanol solution
(comprising 12.5 mL of 2 mol/L HCl and 87.5 mL of anhydrous ethanol).
The mixture was then incubated at 25 °C for 30 min. Following
incubation, the mixture was centrifuged at 3500 g for 10 min. After
centrifugation, 500 μL of supernatant was collected. The solution was
subsequently neutralized with 200 μL of 0.855 mol/L Tris solution.
Following neutralization, the solution was diluted with IGF-1 buffer
solution in accordance with the ratio specified in the manufacturer's
instructions. Finally, the IGF-1 content was quantified by measuring
the optical density (OD) using a spectrophotometer and comparing the
results to a standard curve.
2.6. Viscosity analysis
The fermented camel milk sample was equilibrated at 25 °C to minimize
temperature fluctuations that could influence viscosity measurements.
Its viscosity was determined using a #2 rotor attached to a viscometer
(Brookfield DV-1, Brookfield Company, USA), operated at a fixed speed
of 100 rpm, with a torque range of 10–100%, over a measurement duration
of 30 s. The rotor was meticulously cleaned before and after each test
to ensure precision. Each sample was measured three times to ensure
repeatability and reliability of the results.
2.7. Water-holding capacity analysis
The water holding capacity of fermented camel milk was determined
according to a previously described method ([46]Sahan et al., 2008). A
20 g sample of fermented camel milk was weighed and placed in a funnel
lined with qualitative filter paper. The sample was maintained at 25 °C
for 120 min, after which the filtrate was collected and weighed
immediately.
[MATH: Water holding capacity%=1−Filtrate weightSample
weight×100 :MATH]
2.8. Texture analysis
The hardness, viscosity index, consistency, and cohesion of the
fermented camel milk samples were measured using a texture analyzer
(TA.XT.plus, Stable MicroSystems Company, UK). The appropriate test
probe was selected with the following parameters: pre-test speed of
1.5 mm/s, mid-test speed of 1.0 mm/s, post-test speed of 1.5 mm/s,
initial force of 2.0 g, compression degree of 20%, compression time of
5 s, and test distance of 20 mm.
2.9. Untargeted metabolomics analysis
2.9.1. Sample preparation
Fermented camel milk samples were stored at −80 °C and thawed at 4 °C.
Subsequently, 3 mL of the sample and 1 mL of acetonitrile (containing
400 ppm of 2-Chloro-L-phenylalanine) were added to a 5 mL centrifuge
tube and thoroughly homogenized. The mixture was centrifuged at 10000 ×
g for 10 min, and the supernatant was transferred to a 2 mL centrifuge
tube. The supernatant was concentrated to near dryness using a vacuum
concentrator, and 500 μL of 40% (v/v) acetonitrile solution was added
to resuspend the material. After filtration through a 0.22 μm organic
microporous membrane, metabolites were determined by LC-MS (SCIEX,
TripleTOF 6600, USA). Quality control (QC) samples were prepared by
mixing equal volumes of each sample. QC samples were injected into the
chromatographic system to ensure system stability and consistent
conditions. After every 10 samples were analyzed, a QC sample was used
to ensure accuracy. In addition, the effects of instrument drift and
other potential issues have been corrected.
2.9.2. Experimental conditions
Pretreated fermented camel milk samples were analyzed using a UPLC
system coupled with a SCIEX Q-TOF mass spectrometer (AB SCIEX 6600,
USA), and each sample was analyzed in triplicate. A 1 μL aliquot of
each sample was injected onto an ACQUITY HSS T3 column (Waters Corp,
2.1 mm × 100 mm × 1.8 μm; Waters, USA). The analysis was conducted at a
flow rate of 0.4 mL/min and column temperature of 40 °C. mobile phases
A (water: acetonitrile = 5:95 [v/v]) and B (acetonitrile) were prepared
and stored at 4 °C prior to use. The gradient elution system employed
was as follows: 1.0 min, 95% A; 6.0 min, 60% A; 18.0 min, 15% A;
18.5 min, 10% A; 22 min, 10% A; 22.5 min, 95% A; 25.0 min, 95% A, with
a total run time of 25.0 min. The MS analysis was performed according
to the method described by Shang et al. ([47]Shang et al., 2022) with
minor modifications. The declustering voltage was set at 40 V, gases 1
and 2 were maintained at 60 psi, and the curtain gas was set at 30 psi.
The source temperature was maintained at 600 °C, and the ion spray
voltage was set at 5000 V for the positive mode and −4500 V for the
negative mode.
2.10. Data analysis
The raw metabolome data were imported into Progenesis QI software
(Waters Corporation, USA) to generate a data matrix comprising
retention time, mass-to-charge ratio, and peak intensity. To enhance
the accuracy and reliability of the metabolomics data, the ‘tidyverse’
and ‘dplyr’ packages in R were used for data filtering. This process
involved the following steps: (1) removing features with identical
intensities across all samples, and (2) excluding features detected in
more than 30 % of samples within the same experimental group. The final
features were subsequently identified and classified by referencing
HMDB ([48]http://www.hmdb.ca/), Metlin
([49]https://metlin.scripps.edu/), and other internal databases. The
Human Metabolome Database (HMDB) was used for substance classification,
while the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was
used for pathway annotation. All experiments were performed in
triplicate, and one-way analysis of variance (ANOVA) was performed
using R (v 4.3.0) to determine statistical significance.
3. Results
3.1. Determination of viable cell counts during storage time
The primary fermentation agent used in this experiment was YoFlex® Mild
1.0, which contained L. delbrueckii subsp. bulgaricus and S.
thermophilus. Consequently, changes in the viable cell counts of these
strains were monitored throughout the storage period. As storage time
increased, the viable cell counts of L. delbrueckii subsp. bulgaricus
and S. thermophilus gradually decreased, with significant differences
observed between the strains at different storage points (P < 0.05). On
D21, the viable cell counts of L. delbrueckii subsp. Bulgaricus in the
Control and LcZ groups were 6.39 ± 0.15 and 1.73 ± 0.01 × 10^6 CFU/mL,
respectively, whereas the corresponding counts for S. thermophilus were
1.14 ± 0.03 and 1.04 ± 0.10 × 10^8 CFU/mL, respectively. Notably, on
D1, the viable cell counts of L. casei Zhang in the LcZ group reached
6.05 ± 0.10 × 10^7 CFU/mL, significantly exceeding the threshold
required to confer expert-recognized probiotic benefits. Additionally,
notable observations were made regarding viable cell counts of L.
delbrueckii subsp. bulgaricus and S. thermophilus, which were lower in
the LcZ group than in the control group. This phenomenon may be
attributed to the presence of L. casei Zhang and the competitive
survival among these three strains, which utilized nutrients in the
fermented camel milk, resulting in a reduced number of L. delbrueckii
subsp. bulgaricus and S. thermophilus in the LcZ group compared with
the control group. Interestingly, the number of L. casei Zhang
exhibited a gradual upward trend as the storage time increased, with
significant differences observed among the various time points
(P < 0.05), warranting further investigation.
3.2. Changes of acidity and IGF-1 during storage time
Changes in acidity significantly influence the acceptability of
fermented camel milk products ([50]Ayyash et al., 2022). Throughout the
21-day storage period, the pH of both groups of fermented camel milk
exhibited a downward trend, with statistically significant differences
(P < 0.05). The pH changed substantially in the initial storage phase
(1–7 days), with the rate of decline decelerating after 14 days of
storage, as illustrated in [51]Fig. 2A. The pH of the LcZ group was
higher than that of the control group, although the difference was not
statistically significant (P > 0.05), indicating that the addition of
L. casei Zhang mitigated the downward trend in pH. The TA of both
groups of fermented camel milk exhibited an increasing trend throughout
the storage period, reaching peak values at D21 of 125 ± 2.13 and
97.5 ± 1.39 °T for the Control and LcZ groups, respectively. Notably,
significant differences in TA were observed between the two groups
throughout the storage period (P < 0.05). Donkor posited that 70–110 °T
was the optimal range for fermented milk ([52]Donkor et al., 2006), and
fermented milk exhibited superior organoleptic properties at this
point. This suggests that the fermented camel milk in the control group
may not meet consumer expectations. In contrast, the incorporation of
L. casei Zhang appeared to mitigate the formation of acidic compounds,
maintaining the acidity within an optimal range, thereby improving its
stability and sensory acceptability throughout the storage period.
Fig. 1.
[53]Fig. 1
[54]Open in a new tab
Changes in viable cell counts during storage of fermented camel milk.
[55]Fig. 1A and B represent the Control and LcZ groups, respectively.
Different capital letters represent differences between groups and
different lowercase letters represent differences within groups.
Statistical significance was set at P < 0.05.
Fig. 2.
[56]Fig. 2
[57]Open in a new tab
Changes in pH, TA, and IGF-1 during storage of fermented camel milk.
The broken line in [58]Fig. 2A represents pH, the bar graph represents
the IGF-1 activity retention rate (with camel milk set at 100 %), and
[59]Fig. 2B represents the trend of TA changes during storage.
Different capital letters indicate differences between groups, whereas
different lowercase letters indicate differences within groups.
Statistical significance was defined as P < 0.05.
IGF-1 content in each sample of the Control and LcZ groups during
storage was quantified. The IGF-1 content of camel milk was established
as the baseline (100%) and the activity retention rate of IGF-1 after
various storage periods was calculated. Changes in IGF-1 activity
retention rate during storage were also investigated. This primary
hypoglycemic effect of camel milk and fermented dairy products is
attributed to their IGF-1 content. The results indicated that, as
storage time increased, the IGF-1 activity retention rate in fermented
camel milk gradually decreased. After 21 days of storage, the IGF-1
activity retention rate in the control group decreased to
54.95 ± 1.68%, whereas that in the LcZ group was 59.13 ± 2.58%.
Throughout the storage period, IGF-1 activity retention rate in the LcZ
group consistently exceeded that in the control group. Although this
difference was not statistically significant (P > 0.05), the addition
of L. casei Zhang enhanced the IGF-1 activity retention rate.
3.3. Changes in viscosity and water-holding capacity during storage time
Viscosity is a crucial parameter for assessing the quality of fermented
camel milk, as high-quality fermented milk typically exhibits a
semi-solid consistency with a certain degree of firmness. However,
camel milk inherently resists coagulation, presenting a significant
challenge for its utilization in the production of fermented dairy
products. The changes in the viscosity of the two groups of fermented
camel milk samples during storage are shown in [60]Fig. 3A. As the
storage duration increased, the viscosity of the fermented camel milk
decreased to varying degrees, remaining below 80 mPa·s, whereas the
viscosity of the control group remained below 20 mPa·s throughout the
storage period. This study revealed that the addition of L. casei Zhang
facilitated the formation of a gel structure in fermented camel milk,
significantly enhancing its viscosity (P < 0.05). However, this
improvement has limited practical significance, owing to the
distinctive properties of camel milk. To address this limitation,
future studies should explore the incorporation of additives, such as
pectin to enhance the viscosity of fermented camel milk. Notably, the
gel structure of fermented milk plays a crucial role in its
water-holding capacity ([61]Zhang et al., 2024). Insufficient
water-holding capacity can lead to whey precipitation in fermented
camel milk, consequently affecting the texture and organoleptic
properties of the product. As depicted in [62]Fig. 3B, the
water-holding capacity of both groups of fermented camel milk samples
exhibited a declining trend from days 1 to 7, with no significant
changes observed thereafter (P > 0.05). Additionally, the incorporation
of L. casei Zhang reduced the water-holding capacity of fermented camel
milk; however, this alteration did not result in excessive syneresis of
the whey.
Fig. 3.
[63]Fig. 3
[64]Open in a new tab
Changes in viscosity and water-holding capacity of fermented camel milk
during storage. [65]Fig. 3A and [66]Fig. 3B show the changes in the
viscosity and water-holding capacity of fermented camel milk during
storage. Different capital letters represent differences between
groups, and different lowercase letters represent differences within
groups. Statistical significance was defined as P < 0.05.
3.4. Textural changes in fermented camel milk during storage time
This study assessed the texture indices of fermented camel milk samples
in the Control and LcZ groups during storage and investigated the
effect of adding L. casei Zhang on the quality of fermented camel milk.
The measured parameters included the hardness, cohesion, consistency,
and viscosity index. These indicators reflect the textural
characteristics of fermented milk and influence its organoleptic
properties. The fat and protein content of camel milk, type and
concentration of fermentation agent, and fermentation time all
significantly impact the quality of fermented camel milk. However, in
this study, only the post-acidification of fermented camel milk and the
effect of L. casei Zhang on its texture during storage were considered.
The addition of probiotic L. casei Zhang enhances the hardness of
fermented camel milk, potentially due to the production of
extracellular polysaccharides, which improve texture ([67]Bai et al.,
2021). After 21 days of storage, the viscosity index of fermented camel
milk samples in the Control and LcZ groups reached peak values of
1.59 ± 0.01 and 1.54 ± 0.05 g·s, respectively. A higher viscosity index
indicates that liquid viscosity is less affected by fluctuations in
temperature, whereas a lower viscosity index indicates that viscosity
is more sensitive to temperature, resulting in significant changes.
This suggests that the addition of L. casei Zhang increased the
viscosity of fermented camel milk but reduced its viscosity index,
rendering it more sensitive to temperature.
3.5. Metabolomics analysis of fermented camel milk during storage
Based on previous results, the addition of L. casei Zhang improved the
IGF-1 retention rate, decelerated the decrease in pH during storage,
and enhanced the water-holding capacity and viscosity of fermented
camel milk. To elucidate the mechanisms underlying these changes,
metabolomic alterations in fermented camel milk in the control and LcZ
groups were investigated throughout the storage period. This study
primarily examined the changes induced by the addition of L. casei
Zhang to fermented camel milk, focusing on the differences between the
control and LcZ groups at various storage time points, and identifying
the metabolites responsible for these differences. As illustrated in
[68]Fig. 5E, 61 differential metabolites (VIP > 1.0, P < 0.05) appeared
only once during storage, with the highest number of unique metabolites
(45) observed on D7. As depicted in [69]Fig. 5A-D, after 1, 7, 14, and
21 days of storage, the addition of L. casei Zhang resulted in 69, 102,
23, and 59 metabolites, respectively, which differed significantly from
the control group (P < 0.05). According to the HMDB database
classification, these compounds primarily belong to amino acids,
peptides, and analogs, as well as carbohydrates and carbohydrate
conjugates. Previous research has demonstrated that when L. casei Zhang
reaches the late stage of growth in milk, the majority of the
significantly altered genes are related to sugar metabolism, amino acid
transport, and metabolism (Wang et al., 2012). Subsequently, KEGG
pathway enrichment analysis was performed for the differential
metabolites. As shown in [70]Table 1, the incorporation of L. casei
Zhang influenced 25 metabolic pathways, including carbohydrate, lipid,
amino acid, cofactor, vitamin, and nucleotide metabolism. These
findings align with those of previous studies, suggesting that the
addition of L. casei Zhang enhances amino acid and carbohydrate
metabolism. Furthermore, this study identified significant impacts on
vitamin and nucleotide metabolism, a result that distinguishes this
study from previous studies. Further analysis of these metabolic
pathways revealed that purine and pyrimidine metabolism consistently
appeared at each storage time point, suggesting that these two pathways
may play a central role in the continuous changes induced by the
addition of L. casei Zhang.
Fig. 5.
[71]Fig. 5
[72]Open in a new tab
Differential metabolites identified in fermented camel milk after 1, 7,
14, and 21 d of storage. [73]Figs. 5A-D display the heat maps of
differential metabolites between the Control and LcZ groups on days 1,
7, 14, and 21, whereas [74]Fig. 5E presents the upset plot of
differential metabolites between the Control and LcZ groups at each
time point.
Table 1.
Pathways enriched by differential metabolites at each storage time
point.
Enrichment pathway Class Time
Amino sugar and nucleotide sugar metabolism Carbohydrate Metabolism D1
Fructose and mannose metabolism Carbohydrate Metabolism D1
Biosynthesis of unsaturated fatty acids Lipid Metabolism D7
Butanoate metabolism Other D7
Citrate cycle (TCA cycle) Carbohydrate Metabolism D7
Propanoate metabolism Other D7
Tryptophan metabolism Amino Acid Metabolism D7
Glycerophospholipid metabolism Lipid Metabolism D21
beta-Alanine metabolism Amino Acid Metabolism D1,D7
Cysteine and methionine metabolism Amino Acid Metabolism D1,D7
Glycosaminoglycan biosynthesis - chondroitin sulfate / dermatan sulfate
Other D1,D7
Pantothenate and CoA biosynthesis Metabolism of Cofactors and Vitamins
D1,D7
Retinol metabolism Metabolism of Cofactors and Vitamins D1,D21
Riboflavin metabolism Metabolism of Cofactors and Vitamins D1,D21
Caffeine metabolism Other D7,D14
Alanine, aspartate and glutamate metabolism Amino Acid Metabolism
D1,D7,D14
Arginine and proline metabolism Amino Acid Metabolism D1,D7,D21
Galactose metabolism Carbohydrate Metabolism D1,D7,D21
Pentose phosphate pathway Carbohydrate Metabolism D1,D7,D21
Phenylalanine metabolism Amino Acid Metabolism D1,D7,D21
Phenylalanine, tyrosine and tryptophan biosynthesis Amino Acid
Metabolism D1,D7,D21
Tyrosine metabolism Amino Acid Metabolism D1,D7,D21
Ubiquinone and other terpenoid-quinone biosynthesis Lipid Metabolism
D1,D7,D21
Purine metabolism Nucleotide Metabolism D1,D7,D14,D21
Pyrimidine metabolism Nucleotide Metabolism D1,D7,D14,D21
[75]Open in a new tab
Enrichment pathway, pathways enriched through the KEGG database; Class,
classification of pathways; Time, D1, D7, D14, and D21 present the
difference pathways between Control and LcZ groups at day 1, day 7, day
14, and day21.
3.6. Core differential metabolites
The addition of L. casei Zhang induced alterations in the metabolome of
fermented camel milk. To elucidate the relationship between the changes
in numerous differential metabolites, this study focused on the
metabolites that differed at each storage time point following the
addition of L. casei Zhang, which were defined as core differential
metabolites. In total, 6 core differential metabolites were identified:
adenosine diphosphate (ADP), oleuropein, arginyl-arginine (Arg-Arg),
isoleucyl-proline (Iso-Pro), lysyl-lysine (Lys-Lys) and
threoninyl-tryptophan (Thr-Try). The latter four metabolites are
dipeptides, which are typically derived from protein digestion,
absorption, and degradation, as well as from a combination of free
amino acid enzymatic reactions ([76]Minen et al., 2023). As illustrated
in [77]Fig. 6A and F, the ADP and Thr-Try levels in the LcZ group were
significantly higher than those in the control group at each storage
time point (P < 0.001), indicating that the addition of L. casei Zhang
significantly increased ADP and Thr-Try levels in fermented camel milk.
The Thr-Try levels in the LcZ and control groups followed a similar
trend, reaching their peak at D7 and subsequently decreasing to
118.52 ± 3.59 and 11.71 ± 2.10, respectively. Except for oleuropein,
the other core differential metabolites exhibited an overall upward
trend following the addition of L. casei Zhang to the fermented camel
milk.
Fig. 6.
[78]Fig. 6
[79]Open in a new tab
Core differential metabolites identified after 1, 7, 14, and 21 days of
storage. [80]Fig. 6A-F show ADP, Arg-Arg, Iso-Pro, Lys-Lys, oleuropein,
and Thr-Try, respective. Significant differences were evaluated using
t-tests; *: P < 0.05, **: P < 0.01, and ***: P < 0.001.
4. Discussion
During the fermentation and storage periods, the pH of the fermented
milk decreased due to the continuous production of lactic acid by L.
delbrueckii subsp. bulgaricus and S. thermophilus. After one day of
storage, the pH of the control and LcZ groups fell below 4.50, which is
outside the optimal range for the growth of these strains.
Consequently, the viable cell counts of L. delbrueckii subsp.
bulgaricus and S. thermophilus gradually decreased throughout the
storage period. The combined fermentation of the two strains led to an
increased in the production of amino acids and oligopeptides, which are
precursors of flavor compounds. This, in turn, promoted the synthesis
of additional flavor compounds, enhancing the flavor and taste of
yogurt ([81]Kaneko et al., 2014). Previous research has demonstrated
that the addition of L. casei Zhang, either alone or in combination
with Bifidobacterium lactis V9, can effectively shorten the
fermentation time, reduce the loss of IGF-1 ([82]Wang et al., 2024),
and contribute to functional products targeting hypertension and
diabetes. Furthermore, these strains have been shown to significantly
influence purine metabolism, offering valuable insights into the role
of L. casei Zhang in driving metabolic changes in fermented camel milk
during storage. For fermented camel milk to confer the health benefits
of L. casei Zhang, it must contain a minimum of one million viable
cells per gram of product. This level must be maintained throughout the
shelf life, rather than at a specific point in time. Our study
demonstrated that the incorporation of L. casei Zhang decreased the
number of viable L. delbrueckii subsp. bulgaricus and S. thermophilus.
This phenomenon occurs because L. casei Zhang competes these strains
for nutrients in camel milk, such as lactose, amino acids, and
vitamins, leading to reduced growth due to nutrient limitation during
storage. Previous research has demonstrated that with increased storage
time, regardless of the matrix being milk, goat milk, or a mixture
thereof, the viable cell counts of L. delbrueckii subsp. bulgaricus and
S. thermophilus exhibit a downward trend ([83]Dimitrellou et al.,
2019), which is consistent with our findings. In the later stages of
storage, as the viable cell counts of L. delbrueckii subsp. bulgaricus
and S. thermophilus and the pH decreased, the growth of L. casei Zhang
was not significantly inhibited. As a result, the viable cell counts of
L. casei Zhang exhibited a consistent upward trend throughout the
storage period.
Our findings revealed that not all strains exhibited beneficial effects
on fermented camel milk. For instance, when certain strains were used
as starter cultures, the pH remained consistently low (around 3.3)
throughout the storage period ([84]Ayyash et al., 2018), rendering the
product unsuitable for consumer consumption. These observations
underscore the importance of thoroughly investigating the changes in
fermented camel milk during storage to ensure its quality and
acceptability. Throughout the storage period, the pH exhibited a
decreasing trend ([85]Fig. 2A). At the end of storage, the pH of the
LcZ group was 4.39 ± 0.03, which was significantly higher than that of
the control group (P < 0.05), indicating that L. casei Zhang can
mitigate post-acidification in fermented camel milk when co-fermented
with the commercial starter YoFlex® Mild 1.0. The researchers found
that after adding Lacticaseibacillus rhamnosus, Lacticaseibacillus
casei, or Lactiplantibacillus plantarum, the pH of fermented camel milk
dropped more rapidly, but its antioxidant capacity and flavor were
better ([86]Shori, 2024), indicating that the addition of L. casei
Zhang may also help improve the probiotic properties of fermented camel
milk. This may be due to the higher abundance of L. delbrueckii subsp.
bulgaricus and S. thermophilus in the control group, which is capable
of producing a larger number of acidic compounds, leading to a
continuous decrease in pH, lower than that of the LcZ group. The pH of
most fermented milk products ranges from 4.0 to 4.6. This pH range is
ideal for fermenting camel milk, as it not only imparts a distinctive
flavor to the milk, but also inhibits contamination by undesirable
strains owing to the lower pH. Therefore, the pH of the both groups
remained within the optimal range for shelf life throughout the storage
period. As shown in [87]Fig. 2B, titrated acidity (TA) in both groups
increased over time, with statistically significant differences
(P < 0.05), consistent with pH changes. Previous studies utilizing L.
casei Zhang in yogurt observed that as storage time increased, pH
decreased, whereas TA increased ([88]Wang et al., 2021), which aligns
with the findings of this study. The pH and TA results indicate that L.
casei Zhang exhibits weak acid-producing ability, which may contribute
to extending the shelf life of fermented camel milk and possesses
potential application value. IGF-1 is structurally like insulin,
promotes glucose utilization, and enhances insulin sensitivity, thereby
aiding in blood sugar regulation. They also play indispensable roles in
growth and development ([89]Bailes & Soloviev, 2021). Under acidic
conditions, IGF-1 undergoes protein denaturation, which results in
decreased activity ([90]Bhalla et al., 2022). Therefore, exploring the
activity of IGF-1 during the storage of fermented camel milk is
essential as it is a prerequisite for its efficacy. Throughout the
storage period, the IGF-1 activity retention rate in the LcZ group was
higher than that in the control group, although the difference was not
statistically significant (P > 0.05). This observation indicated that
when the pH was between 4 and 6, there was no significant effect on the
activity of IGF-1. The activity of IGF-1 in both groups remained above
50%, demonstrating that fermented camel milk can serve as an effective
carrier for IGF-1. The researchers found that Lactococcus lactis
[91]KX881782, when applied to fermented camel milk, can effectively
inhibit α-glucosidase and help reduce blood sugar ([92]Ayyash et al.,
2018). This finding is consistent with the results of this study,
indicating that the addition of L. casei Zhang can reduce the loss of
IGF-1 and contribute to its hypoglycemic effects.
Camel milk exhibits difficulty in curd formation during fermentation
because of its low casein and calcium ion content. This study
incorporated L. casei Zhang into YoFlex® Mild 1.0 to investigate the
effects of L. casei Zhang on the viscosity and water-holding capacity
of fermented camel milk. The viscosity of both groups decreased during
storage, with the viscosity of the LcZ group being significantly higher
than that of the control group (P < 0.05), indicating that the addition
of L. casei Zhang facilitated the formation of a stable gel structure
in the fermented camel milk. The Lactiplantibacillus plantarum MWLp-12
and Limosilactobacillus fermentum MWLf-4 strains were introduced into
milk, and it was observed that the addition of these two strains
promoted protein hydrolysis, influenced the water retention and network
structure of the protein, and enhanced the textural characteristics of
fermented milk ([93]Wang et al., 2022). In this study, the gradual
decrease in pH throughout storage suggests that L. casei Zhang
possesses a limited capacity to produce acid, resulting in a gradual
decrease in pH throughout the storage period. As the pH decreases, the
colloidal calcium phosphate in fermented camel milk continues to
dissolve, causing alterations in the cross-linked structure of the
protein, thereby leading to whey syneresis and ultimately manifesting
as a decrease in viscosity. However, the viscosity of the fermented
camel milk prepared in this study was lower than 80 mPa·s,
substantially below 900 mPa·s, which is considered to have the optimal
viscosity according to Li et al. ([94]Li et al., 2020). This
discrepancy can be attributed to the inherent characteristics of camel
milk. Future research should consider the addition of coagulants to
increase the viscosity of fermented camel milk.
The three-dimensional network structure of casein undergoes continuous
modifications in its gel structure during fermentation and storage of
fermented camel milk, consequently altering its water-holding capacity.
Whey precipitation occurs when the water holding capacity is
insufficient, thereby affecting the texture and organoleptic properties
of the product. Previous research has demonstrated that yogurt
subjected to high-amplitude ultrasound treatment exhibits a
significantly enhanced water-holding capacity and viscosity, as well as
reduced syneresis compared with non-sonicated samples ([95]Wu et al.,
2000). Consequently, we incorporated high-pressure homogenization into
the experimental protocol to ensure that the fermented camel milk
maintained desirable organoleptic properties during the storage period.
As shown in [96]Fig. 3B, the water holding capacity of the control
group ranges between 65 and 85 %, which was significantly different
from that of the LcZ group (P < 0.05). The water-holding capacity of
the LcZ group was lower than that of the control group, likely due to
the stable network structure formed by the interaction of casein and
microbial metabolites in the gel. As the storage duration increased,
the bacterial content in fermented camel milk decreased. The bacterial
content (including viable and non-viable bacteria) was higher in the
LcZ group than in the control group. The accumulation of bacteria
increases the viscosity of the sample, which can be readily disrupted
in an acidic environment, thereby reducing the water-holding capacity
of the fermented camel milk. Additionally, the degradation of
hydrophobia subunits by lactic acid bacteria enhances the electrostatic
repulsion within the gel, resulting in uneven distribution of gel
particles and a coarse microstructure, which further diminishes the
water-holding capacity of the gel.
Fig. 4.
[97]Fig. 4
[98]Open in a new tab
Texture changes in fermented camel milk during storage. [99]Fig. 4A-D
display the consistency, cohesion, viscosity, and hardness. No
significant differences were observed between the two groups at any
time point (P > 0.05).
These results demonstrated that the incorporation of L. casei Zhang
enhanced the viscosity of fermented camel milk, increased the retention
of IGF-1 activity, and reduced the rate of acidity change. To explore
the mechanisms underlying these effects, we investigated the
metabolites present in fermented camel milk. The study revealed that
the addition of L. casei Zhang primarily altered the production of
amino acids, peptides, and analogs, as well as carbohydrates and
carbohydrate conjugates. During camel milk fermentation, S.
thermophilus metabolizes lactose to produce lactic acid, thereby
lowering the pH and facilitating the growth and protein utilization of
L. delbrueckii subsp. bulgaricus. Concurrently, L. delbrueckii subsp.
bulgaricus promotes the growth of S. thermophilus through the secretion
of nutrients, such as amino acids. In this process, the protease
produced by the strain hydrolyzes the proteins into amino acids and
peptides. The proteases secreted by different strains exhibit varying
specificities, resulting in the production of distinct final products.
This phenomenon may account for the observed differences in amino
acids, peptides, and analogs as differential metabolites in the present
study. These strains exhibit varying utilization efficiencies and
metabolic pathways for carbohydrates, leading to a diversity of
metabolites that subsequently influence the pH and texture of fermented
camel milk ([100]Li et al., 2020). L. casei Zhang, an extensively
studied probiotic strain, has been shown to produce various beneficial
substances, including capric acid, caprylic acid, caproic acid, and
valeric acid when incorporated into yogurt ([101]Peng et al., 2022).
The present study found that the addition of L. casei Zhang resulted in
increased concentrations of Thr-Try, ADP, Thr-Thr, Iso-Pro, Cys-Try,
and Arg-Arg in fermented camel milk.
KEGG enrichment analysis was performed on the differential metabolites
at each time point ([102]Table 1). The results indicated that two
metabolic pathways, purine, and pyrimidine, were consistently present
at each storage time point, suggesting that the addition of L. casei
Zhang significantly influenced these pathways in fermented camel milk.
Purine metabolism is a critical biochemical metabolic pathway involved
in the synthesis, degradation, and recycling of purine, which provides
energy for cells and participates in intracellular signal transduction
([103]Yin et al., 2018). A detailed analysis of all metabolites within
this pathway, it was determined that the difference in purine
metabolism was attributable to changes in ADP production ([104]Fig.
6A), with the addition of L. casei Zhang significantly increased the
ADP content of the fermented camel milk (P < 0.05). This phenomenon may
be attributed to the larger total number of viable cells in the LcZ
group, which consumed more nutrients, thereby promoting the conversion
of ATP to ADP and leading to ADP accumulation. ADP is a central
molecule in cellular energy metabolism, in which ATP is converted to
ADP during the energy consumption processes, subsequently releasing
energy. An increase in ADP content leads to a reduction in cellular ATP
levels, consequently inhibiting purine synthesis, which may have
beneficial implications for patients with uric acid-related disorders
and other conditions ([105]James et al., 2023). Pyrimidine metabolism
plays a crucial role in nucleic acid synthesis and cellular metabolic
regulation. Disruptions in this metabolic pathway can result in various
pathological conditions including cancer and immunodeficiency. Hao et
al. ([106]Hao et al., 2023) observed that purine metabolism, pyrimidine
metabolism, and ABC transporters were involved in the storage of
fermented milk beverages. Purine metabolism is essential for LAB
growth, as well as the transformation and synthesis of primary and
secondary metabolites, which aligns with the core differential
metabolic pathways identified in this study.
Subsequently, the differential metabolites of the Control and LcZ
groups at various storage time points were analyzed ([107]Fig. 5).
These results indicated that the addition of L. casei Zhang altered the
metabolic profile of fermented camel milk. Notably, several
differential metabolites were observed at the four storage time points,
specifically ADP, oleuropein, Arg-Arg, Ile-Pro, Lys-Lys, and Thr-Try
([108]Fig. 6). Arg-Arg, Ile-Pro, Lys-Lys, and Thr-Try are dipeptides
formed by the linkage of two amino acids through peptide bonds. These
molecules serve as the basic building blocks of proteins and
polypeptide chains and are prevalent, particularly in partially
hydrolyzed or fermented foods, such as fermented dairy products and soy
sauce. These dipeptides have potential applications as therapeutic
agents for the prevention and mitigation of diabetes and its associated
complications ([109]Freund et al., 2018). Furthermore, they are widely
utilized because of their distinctive flavor profiles and nutritional
properties, as exemplified by aspartame. Cakmak et al. hypothesized
that Ile-Pro is associated with the activity of proline enzymes
involved in collagen degradation during airway remodeling ([110]Cakmak
et al., 2009). Concurrently, Ile-Pro possesses antioxidant properties
and enhances bone mineral density. Notably, changes in Ile-Pro
concentrations exhibited contrasting trends between the two groups. The
control group initially increased to its highest value before
subsequently declining, whereas the LcZ group showed an initial
decrease followed by an increase. The underlying mechanism of this
phenomenon was not investigated in the present study and can be
explored in subsequent experiments. The potential function of Thr-Try
remains undetermined and merits investigation by additional researchers
in future studies. Alanine, tyrosine, phenylalanine, and tryptophan
have all been identified as amino acids that occupy terminal positions
in antihypertensive peptide structures ([111]Aguilar-Toalá et al.,
2020). The addition of L. casei Zhang significantly increased Thr-Try
content, which may confer health benefits.
The findings of this study demonstrated that the addition of L. casei
enhanced IGF-1 activity and facilitated the production of beneficial
metabolites. These results suggest that L. casei Zhang holds
significant potential for application in fermented camel milk beverages
and other dairy products, warranting further evaluation of its
performance and adaptability. To achieve this, an in-depth
investigation of the relationship between specific metabolites and
their health effects is essential, necessitating comprehensive animal
and human studies. Our ultimate goal is to develop L. casei Zhang as a
commercially viable starter culture for fermented dairy products.
However, challenges, such as cost management and broad consumer
acceptance, must be addressed. Although this study represents a
significant step forward, ongoing research will be crucial to
overcoming these hurdles and further advance the field.
5. Conclusion
This study evaluated the effect of L. casei Zhang supplementation on
the quality attributes and metabolomic profiles of fermented camel milk
during storage. This study demonstrated that the concentration of L.
casei Zhang in fermented camel milk consistently reached the
recommended standard concentration (10^6 CFU/mL) of probiotics in
functional dairy products. Additionally, supplementation with L. casei
Zhang enhanced the IGF-1 activity retention rate from 54.95% to 59.13%,
suggesting that fermented camel milk could effectively serve as a
carrier for IGF-1. Supplementation with L. casei Zhang also had a
minimal impact on post-acidification and did not negatively affect the
viscosity, water-holding capacity, or texture of the camel milk.
Subsequently, the metabolomics of fermented camel milk was explored.
Metabolomic analysis further revealed significant alterations in the
production of amino acids, peptides, analogs, and carbohydrate
conjugates, indicating modulation of the metabolic profile of fermented
camel milk. Moreover, the application of L. casei Zhang to commercial
starter cultures further verified its potential as an auxiliary starter
for augmenting ADP, Thr-Try, and Iso-Pro levels in fermented camel
milk. In summary, L. casei Zhang has broad application prospects in the
development of fermented camel milk products with enhanced nutritional
and therapeutic properties.
CRediT authorship contribution statement
Dandan Wang: Writing – review & editing, Writing – original draft,
Conceptualization. Wusigale: Visualization, Investigation. Lu Li:
Software, Methodology. Lu Bai: Methodology, Data curation. Yongfu Chen:
Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have influenced the work
reported in this study.
Acknowledgments