Acid-fast staining of
Lactobacillus paracasei HU1 revealed that the organism did not retain carbol fuchsin following acid-alcohol decolorization and was therefore classified as acid-fast negative. This observation is consistent with the established characteristics of lactic acid bacteria, which lack mycolic acid-rich cell walls required for acid-fastness. The acid-fast negative nature of
L.
paracasei HU1 confirms that any survival under acidic or gastrointestinal conditions is attributable to physiological acid tolerance mechanisms rather than structural resistance and supports the appropriateness of evaluating this strain using low-pH survival and gastrointestinal simulation assays rather than classical acid-fast tests.
Bile salt tolerance
The bile salt tolerance of
L.
paracasei HU1 demonstrated a clear concentration- and time-dependent reduction in viability. In the absence of bile salts (control), the viable count increased slightly from 8.20±0.08 log CFU at 0 h to 8.30±0.07 log CFU at 3 h, indicating normal growth and confirming that the assay conditions were favorable for bacterial survival. At the physiological bile salt concentration of 0.3%,
L.
paracasei HU1 exhibited only a moderate decline in viability, decreasing to 7.40±0.12 log CFU after 3 h. This limited reduction suggests good bile tolerance and indicates that the strain is capable of surviving bile concentrations typically encountered in the human small intestine (Table 1).
When the bile salt concentration was increased to 0.5%, a more pronounced decrease in viable counts was observed, with survival declining to 6.60±0.15 log CFU at 3 h. Although inhibitory, this reduction still reflects moderate tolerance, as a substantial proportion of the bacterial population remained viable. At 1.0% bile salts, a marked reduction in viability occurred, with counts decreasing to 4.80±0.18 log CFU after 3 h, indicating strong inhibitory effects under supra-physiological bile stress. Exposure to 2.0% bile salts resulted in severe inhibition, with viable counts dropping sharply to 2.30±0.25 log CFU after 3 h, demonstrating that such elevated bile concentrations exceed the adaptive capacity of
L.
paracasei HU1 and exert near-lethal effects.
Bile salts are known to disrupt bacterial membranes, interfere with nutrient uptake and impair cellular metabolism through their detergent-like action. The ability of
L.
paracasei HU1 to maintain relatively high viability at 0.3% bile salts is therefore of particular significance, as this concentration closely approximates physiological intestinal bile levels. Survival under these conditions is a key criterion for probiotic functionality, as it suggests that the strain can persist during intestinal transit and potentially exert beneficial effects. The progressive decline in viability observed at higher bile concentrations reflects increasing membrane damage and metabolic stress, which is consistent with responses reported for bile-sensitive lactic acid bacteria.
Survival of Lactobacillus paracasei HU1 under simulated gastric and intestinal conditions
The survival of probiotic microorganisms during gastroin-testinal transit is a critical determinant of their functional efficacy. In the present study, exposure of
Lactobacillus paracasei HU1 to simulated gastric juice resulted in a pronounced reduction in viable cell counts, declining from 6.41±0.42 to 3.14±0.43 log CFU/g after 3 h (Table 2), corresponding to an approximate 51% reduction in viability relative to the initial count. This substantial loss highlights the severity of gastric conditions and is consistent with multiple reports demonstrating that acidic gastric environments represent the most severe physiological stress encountered by probiotic bacteria
(Gangakhedkar et al., 2026; Subbalakshmi et al., 2024; Dempsey and Corr, 2022;
Marzotto et al., 2006; Villena et al., 2015).
Previous investigations have shown that exposure to pH values below 3.0 leads to destabilization of bacterial cytoplasmic membranes, disruption of proton gradients and impairment of enzyme activity in
Lactobacillus species
(Gangakhedkar et al., 2026; Subbalakshmi et al., 2024; Mesquita et al., 2021). Marzotto et al., (2006) reported a comparable decline in
L.
paracasei HU1 viability during in vitro gastric digestion, with survival reductions typically exceeding 45-60% for non-encapsulated cells. The magnitude of viability loss observed in the present study therefore falls well within the range reported for unprotected
L.
paracasei HU1 cells suspended in aqueous systems, reinforcing the pronounced sensitivity of this species to gastric acidity.
In contrast, survival under simulated intestinal conditions was comparatively higher, with viable counts decreasing from 6.69±0.51 to 5.02±0.29 log CFU/g over 3 h, corres-ponding to an approximate 25% decline in viability. This comparatively moderate reduction indicates enhanced tolerance under intestinal conditions. Similar patterns have been reported by
Villena et al., (2015) and
Chen et al., (2022), who demonstrated that
L.
paracasei HU1 exhibits greater resilience to bile salts and alkaline pH than to acidic gastric environments
(Villena et al., 2015; Chen et al., 2022). Although bile salts possess antimicrobial properties, including membrane solubilization and protein denaturation,
L.
paracasei HU1 strains have been shown to retain substantial viability at physiologically relevant bile concentrations, particularly over short exposure periods
(Chen et al., 2022).
These findings indicate thatgastric acidity, rather than bile stress, constitutes the primary bottleneck for probiotic survival, as evidenced by the markedly higher percentage decline observed under gastric conditions compared with intestinal exposure. Consequently, strategies aimed at enhancing probiotic efficacy should primarily focus on improving bacterial protection during the gastric phase of digestion, where the greatest loss of viability occurs.
Influence of food matrices on gastric and intestinal survival
Food matrices are frequently explored as delivery vehicles for probiotics due to their potential to provide physical protection, buffering capacity, or nutritional support; however, their effectiveness is strongly influenced by their physicochemical properties (
Natt and Katyal, 2022;
Meera et al., 2021). In the present study, incorporation of
Lactobacillus paracasei HU1 into apple juice resulted in a rapid and pronounced loss of viability under simulated gastric conditions, with counts decreasing from 1.22±0.18 to 0.11±0.02 log CFU/g within 3 h, corresponding to an approximate 91% decline in viability. This substantial reduction indicates that the apple juice matrix provided minimal protection against gastric stress. These findings are consistent with earlier reports describing limited probiotic survival in acidic fruit-based matrices.
Pimentel et al., (2015) demonstrated that although
L.
paracasei HU1 can remain viable during refrigerated storage in apple juice, its survival during gastrointestinal simulation is substantially compromised due to the low pH of the matrix
(Pimentel et al., 2015). Similarly,
Liang et al., (2022) reported that fermented apple juice formulations supported probiotic viability only when fermentation-induced pH modulation and matrix restructuring occurred, highlighting that unbuffered fruit juices offer minimal protection during gastric digestion
(Liang et al., 2022). The acidic nature of apple juice (pH H≈3.5) likely exacerbates proton influx during gastric exposure, intensifying cellular stress and accelerating bacterial inactivation.
A comparable trend was observed for the mouth freshener matrix, where
L.
paracasei HU1 viability declined from 1.34±0.25 to 0.14±0.03 log CFU/g under gastric conditions, corresponding to an approximate 90% reduction in viability over 3 h. While mouth fresheners may provide a solid or semi-solid physical matrix, their lack of buffering capacity and absence of protective macromolecules likely limit their ability to shield probiotic cells from severe acid stress.
Dixit et al., (2016) emphasized that effective probiotic carriers must possess either buffering components or encapsulating structures to confer meaningful gastric protection, attributes that are typically absent in confectionery-based formulations
(Dixit et al., 2016).
Despite poor survival during gastric exposure,
L.
paracasei HU1 exhibited relatively improved survival in simulated intestinal juice across both food matrices. This pattern further supports the notion that once probiotics successfully transit the gastric phase, intestinal conditions are comparatively permissive. Similar observations have been reported in in vitro digestion studies using non-dairy probiotic carriers, where intestinal survival remained higher provided that a sufficient number of cells survived gastric exposure
(Liang et al., 2022; Pimentel et al., 2015).
The inability of apple juice and mouth freshener matrices to protect
L.
paracasei HU1 during gastric exposure underscores the limitations of relying solely on food carriers for probiotic delivery, particularly when acidic matrices are involved. These findings align with extensive literature demonstrating that substantial improvements in probiotic survival are achieved only when protective formulation strategies, such as encapsulation or buffering systems, are employed, rather than through matrix selection alone.
Microencapsulation approaches using alginate, chitosan, whey proteins, or lipid-based systems have been shown to significantly enhance probiotic survival under simulated gastric conditions by creating a physical barrier that delays acid diffusion (
Matos-Jr et al., 2019;
Ortakci et al., 2012; Yadav et al., 2022; da Conceição et al., 2021). For instance,
Ortakci et al., (2012) reported improved survival of microencapsulated
L.
paracasei during simulated gastric digestion compared to free cells. Similarly,
da Conceição et al. (2021) demonstrated that co-encapsulation with prebiotics such as fructooligosaccharides further enhances probiotic stability by creating a protective microenvironment.
Dairy-based carriers such as milk and yogurt have also been shown to improve probiotic survival due to their buffering capacity, protein content and fat-mediated protection
(Lee et al., 2017; Poon et al., 2020). Lee et al., (2017) and
Poon et al., (2020) reported enhanced gastroin-testinal survival and clinical efficacy of
L.
paracasei when delivered through fermented dairy matrices. These findings suggest that matrix composition plays a decisive role in determining probiotic fate during digestion
(Lee et al., 2017; Poon et al., 2020).
