Screening and Identification of Triglyceride‐Lowering Lactic Acid Bacteria and Preparation of Probiotic Agents

ABSTRACT

To isolate lactic acid bacteria with significant triglyceride‐degrading ability from traditional fermented foods in Guizhou and develop probiotic tablets, providing a basis for the development of functional probiotics. Strains with triglyceride‐degrading ability were screened from 26 isolated strains. Morphological analysis, 16S rRNA gene sequencing, and stress resistance tests were conducted. The lyoprotectant composition for tablet preparation was optimized, and the stability and survival rate of the probiotic tablets were evaluated. Six LAB strains were selected, all exhibiting a triglyceride degradation rate exceeding 60%. Among them, PYC02 (Limosilactobacillus fermentum), SZ02 ( Weissella cibaria ), and PLHB02 ( Weissella cibaria ) showed degradation rates greater than 80%. PYC02 and PLHB02 maintained survival rates above 80% under conditions of pH 3 and 0.3% bile salts. The optimal lyoprotectant composition for maximum probiotic survival was 8% defatted milk powder, 5% sodium alginate, 2% maltodextrin, and 8% trehalose. After 30 days of storage at 4°C and 25°C, the survival rates of the probiotic tablets exceeded 80% and 60%, respectively, with triglyceride degradation rates greater than 60%. The triglyceride‐degrading effects of PYC02 and PLHB02 were significant. These strains exhibited strong stress resistance and high safety, showing potential for the development of functional probiotics, offering new approaches for the prevention and treatment of hypertriglyceridemia.

This study isolated and identified six lactic acid bacteria strains from various fermented foods. Through 16S rRNA sequencing, they were classified as Limosilactobacillus fermentum (PYC02, MHT02, SS01, SC03) and Weissella cibaria (PLHB02, SZ02). Their stress tolerance and safety were evaluated. Results showed that most strains exhibited survival rates exceeding 60% in simulated gastrointestinal acid and bile salt environments, demonstrating excellent tolerance. All six lactic acid bacteria exhibited γ‐hemolysis and sensitivity to four antibiotics. Simultaneously, freeze‐drying protectants for tablets were optimized through single‐factor experiments. Probiotic tablets were formulated using the highly tolerant and safe strains PYC02, PLHB02, SS01, and SZ02, with weight variation, friability, and disintegration time all meeting formulation standards. When investigating the effect of different temperatures on probiotic tablet viability, all four strains maintained high activity after 30 days of storage, with strains PYC02 and PLHB02 exhibiting superior survival rates under both temperature conditions. In summary, strains PYC02 and PLHB02 demonstrated significant triglyceride degradation activity coupled with strong stress resistance and high safety, offering a novel approach for developing functional probiotics. They hold promise for application in the prevention and treatment of hypertriglyceridemia.

1. Introduction

With the global prevalence of obesity and metabolic diseases, hyperlipidemia has become a significant threat to human health (Onwe et al. 2015; Stewart et al. ²⁰²⁰). Among these, triglycerides are a key biomarker of lipid metabolism (Avagimyan et al. 2025; D'Elia et al. ²⁰²⁵), and elevated levels are closely linked to diseases such as atherosclerosis, coronary heart disease, and type 2 diabetes (García‐Poblet et al. ²⁰²⁵; Yamamoto et al. ²⁰²⁴; Zambon et al. ²⁰²⁵). Although drug therapy can help control triglyceride levels to some extent, achieving the goal of treating hyperlipidemia, it struggles to fundamentally regulate lipid metabolism. Moreover, long‐term use may be associated with side effects, including elevated liver enzymes, nephrotoxicity, and myotoxicity (Gong et al. 2020). As a result, the development of safe and effective non‐pharmacological interventions has become a major focus of current research.

Lactic acid bacteria (LAB), as key representatives of probiotics, have been shown to reduce blood lipid levels through multiple mechanisms, including inhibiting fat absorption (Ding et al. 2025), enhancing lipid metabolic activity (Hou et al. 2025), and regulating gut microbiota balance (Mosiej et al. 2025). Furthermore, LAB can further regulate systemic lipid metabolism by lowering blood lipid levels through pathways such as improving intestinal barrier function (Li et al. 2025; Yang et al. ²⁰²⁴) and suppressing inflammatory responses (Liu et al. 2024).

In recent years, there has been a significant increase in research on probiotics that can degrade triglycerides. Some researchers have isolated strains from fermented foods to reduce triglyceride levels (Azat et al. 2016; Gao and Li ²⁰¹⁸; Won et al. ²⁰²⁰; Zhao et al. ²⁰²²), while others have investigated the reduction of triglyceride levels and their mechanisms through animal studies (Aminlari et al. 2019; Bo et al. ²⁰²⁰; Kim et al. ²⁰²⁰). In summary, reducing triglyceride levels has become a research hotspot, with its crucial role in metabolic diseases now widely acknowledged. However, reports on the development of triglyceride‐lowering probiotic agents remain limited. This study aims to screen LAB strains with potent triglyceride‐lowering capabilities and develop corresponding probiotic formulations. The research provides scientific rationale and experimental foundations for the development of novel functional probiotic products, which hold significant potential for the prevention and treatment of hyperlipidemia.

2. Materials and Methods

2.1. Materials

The experimental strains were isolated from traditional Guizhou fermented foods, including homemade pickled radish, bamboo shoots, and napa cabbage, as well as commercially available salted vegetables, fermented bean curd, pickled napa cabbage, assorted pickled vegetables, and sour soup. Additionally, strains were obtained from fermented kiwifruit and persimmons, which were purchased from a campus fruit store prior to fermentation and isolation. Triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.2. Methods

2.2.1. Screening of LAB for Triglyceride Degradation

Serially diluted fermentation broths from fermented foods and fruits were inoculated onto MRS agar medium containing calcium carbonate (5 g/L) and incubated at 37°C for 24 h. Twenty‐six LAB strains were obtained through multiple rounds of single‐colony purification. These isolated and purified strains were then inoculated into MRS liquid medium [3% (v/v)] and incubated overnight at 37°C. Following the method described by (Gao and Li 2018), the strains were inoculated into sterile MRS‐triglyceride broth [3% (v/v)] and incubated at 37°C for 48 h. Triglyceride content in the cell‐free supernatant was measured at 500 nm using a triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), with MRS‐triglyceride broth without inoculum serving as a control. The calculation formula was:

$$\text{triglyceride degradation rate}\%=\frac{A_{\text{total}}-A_{\text{residue}}}{A_{\text{total}}}\times 100\%$$

where A _total: the total triglyceride content and A _residue: the residual triglyceride content.

2.2.2. Morphological Observation and Molecular Identification of LAB

1. Morphological Observation of LAB: Six strains selected for their triglyceride‐lowering ability were individually inoculated onto MRS solid and liquid media. After incubation at 37°C for 24 h, individual colonies were examined for size, shape, surface characteristics, edge morphology, color, and transparency. Single colonies were then picked using an inoculating loop, subjected to Gram staining, and examined for color and morphology.

2. 16S rRNA Gene Sequencing of LAB: The 16S rRNA Gene Sequencing of the six triglyceride‐lowering LAB strains was outsourced to Shenzhen Weike Meng Technology Group Co. Ltd. The obtained sequences were analyzed using BLAST in the NCBI database, and a phylogenetic tree was constructed with MEGA 11. Furthermore, the 16S rRNA gene sequences of these six lactic acid bacterial strains were compared against known sequences in the NCBI database. Their corresponding isolation sources, species identification, and GenBank accession numbers are listed in Table 1.

TABLE 1.

Detailed information on 6 strains of lactic acid bacteria.

Strain name	Sample source	Strain types	Gene bank ID
MHT02	Fermented Kiwifruit	Limosilactobacillus fermentum	NR 113335.1
SC03	Kimchi	Limosilactobacillus fermentum	NR 113335.1
SZ02	Fermented Persimmon	Weissella cibaria	NR 036924.1
PYC02	Pickled Vegetables	Limosilactobacillus fermentum	NR 113335.1
PLHB02	Pickled Chinese Cabbage	Weissella cibaria	NR 036924.1
SS01	Fermented Water	Limosilactobacillus fermentum	NR 113335.1

Note: Fermented water comes from the liquid produced during tofu‐maki.

2.2.3. Acid and Bile Salt Tolerance Test

The strain was inoculated at 1% into MRS broth with pH 3.0 and 0.3% bile salt concentration, and incubated at 37°C for 1, 2, and 4 h. After each time point, the fermentation broth was serially diluted to 10⁻⁸. Then, 100 μL of the diluted fermentation broth was spread onto MRS solid medium and incubated at 37°C for 24 h. Plate counts were performed to determine the survival rate. The calculation formula was:

$$\mathrm{SR}\%=\frac{N}{N_0}\times 100\%$$

where N: Live bacteria count measured at 1, 2, and 4 h and N ₀, Live bacteria count measured at 0 h.

2.2.4. Safety Evaluation

1. Hemolytic Assay: The hemolytic activity of LAB was determined according to the method described by (Jeong et al. 2014). Strains cultured for three generations were inoculated onto Columbia blood agar plates. The plates were incubated at 37°C for 48 h and examined for hemolysis. If a green, ring‐shaped hemolytic zone appeared around the colony, it was classified as α‐hemolysis; if a clear, transparent hemolytic zone surrounded the colony, it was classified as β‐hemolysis; and if no hemolytic zone was observed, it was categorized as γ‐hemolysis.

2. Antibiotic susceptibility testing: Following the method described by (Nami et al. 2014), the antibiotic susceptibility of LAB strains was tested using four antibiotics: apramycin, ampicillin, trimethoprim, and kanamycin. Susceptibility was determined based on the zone diameter: sensitive (> 20 mm), intermediate resistance (15–19 mm), and resistant (≤ 14 mm) (Humphries et al. 2021).

2.3. Preparation of LAB Tablets

2.3.1. Selection of Lyoprotectants

Using skim milk 5% (w/v) powder as the freeze‐drying protective agent matrix, additional skim milk powder was added at concentrations of 4%, 8%, 12%, and 16%. These mixtures were then blended with fermentation broth at a 1:1 ratio, freeze‐dried, and tested for freeze‐drying protection rates.

Using skim milk 5% (w/v) powder as the freeze‐drying protective agent matrix, sodium alginate was added as the protective agent component to form the freeze‐drying protection mixture. Sodium alginate was incorporated at concentrations of 1%, 3%, 5%, and 7%. These mixtures were then combined with fermentation broth at a 1:1 ratio, freeze‐dried, and the freeze‐drying protection rate was measured.

Using skim milk 5% (w/v) powder as the freeze‐drying protective agent matrix, maltodextrin was added as a freeze‐drying protection factor. The maltodextrin concentrations were set at 1%, 2%, 3%, and 4%, and the mixtures were combined with fermentation broth at a 1:1 ratio. The freeze‐drying survival rate was determined after freeze‐drying.

Mixtures were prepared using skim milk 5% (w/v) powder as the freeze‐drying protective agent matrix and trehalose as the protective agent factor. Trehalose was added at concentrations of 2%, 4%, 6%, and 8%, and the mixtures were combined with fermentation broth at a 1:1 ratio before being freeze‐dried. The freeze‐drying survival rate was determined after freeze‐drying.

The freeze‐drying survival rate (SR) was determined after freeze‐drying:

$$\mathrm{SR}\%=\frac{N_1}{N_2}\times 100\%$$

where N ₁, Live bacteria count before freeze‐drying and N ₂: Live bacteria count after freeze‐drying.

2.3.2. Optimization of Tablet Protectants

To investigate the effects of four factors—skim milk powder, sodium alginate, maltodextrin, and trehalose concentrations—on freeze‐drying protection rates, an orthogonal experiment was conducted for optimization. The goal was to identify the optimal combination of protective agents for freeze‐dried LAB, thereby maximizing the viability of the probiotics. The orthogonal experimental design is shown in Table 2.

TABLE 2.

Orthogonal factor level table.

Levels	Factors
	A (Skim milk) %	B (Sodium alginate) %	C (Maltodextrin) %	D (Trehalose) %
1	8	1	1	4
2	12	3	2	6
3	16	5	3	8

2.3.3. Preparation of Probiotic Tablets

The probiotic tablet formulation consists of active ingredients (40% probiotic bacterial powder), excipients (20% corn starch, 30% arabinose/maltodextrin blend [2:1], 9% mannitol), 1% lubricant, 0.3% food additives, and appropriate amounts of binders and other additives.

Strains stored in a laboratory −80°C freezer were thawed, activated, and plated onto sterilized MRS agar plates. The plates were incubated at 37°C for 24 h. Single colonies were streaked for isolation, and this process was repeated three times. Afterward, a single colony was inoculated into MRS liquid medium and incubated at 37°C for 24 h, with the process repeated three times. The culture was then transferred to a sterile centrifuge tube, centrifuged at 10,000 rpm and 4°C for 10 min to collect the bacterial pellet. The pellet was resuspended in 10 volumes of sterile water, and 10% of its volume was added as cryoprotectant. The sample was pre‐frozen at −80°C for 24 h, then freeze‐dried to obtain bacterial powder. The powder was sieved through a 120‐mesh screen and used to prepare probiotic tablets according to the formulation.

2.4. Tablet Performance Evaluation

1. The weight variation shall be tested according to the Weight Variation Test (General Chapter 0101) in the 2020 edition of the Chinese Pharmacopeia.

2. Fragility shall be tested according to the Fragility Test (General Chapter 0923) in the 2020 edition of the Chinese Pharmacopeia.

3. The dissolution time limit shall be tested in accordance with the Dissolution Time Limit Test (General Chapter 0921) in the 2020 edition of the Chinese Pharmacopeia.

2.5. Evaluation of Probiotic Tablet Activity

2.5.1. Probiotic Tablet Triglyceride Degradation Rate

The prepared probiotic tablets were serially diluted with sterile water and spread onto MRS agar plates. After 24 h of inverted incubation, single colonies were picked and subcultured three times. The bacterial suspension was then transferred to MRS‐triglyceride liquid medium, cultured three times, and centrifuged. The bacterial pellet was collected and processed according to the triglyceride assay kit protocol. The optical density (OD) was measured at 500 nm, and the degradation rate was calculated.

2.5.2. Effect of Temperature on the Survival Rate of Probiotic Tablets

The viability of each strain was assessed prior to tablet preparation. The prepared probiotic tablets were sealed into 20 bags under aseptic conditions, with each bag containing 15 tablets. The sealed tablets were then stored at 4°C and 25°C, respectively. On days 10, 20, and 30, two bags were randomly selected from each storage condition to measure their viable bacterial counts. The survival rate (SR) was calculated using the following formula:

$$\mathrm{SR}\%=\frac{N_3}{N_4}\times 100\%$$

where N ₃: Number of viable bacteria measured after freeze‐drying and N ₄: Number of viable bacteria measured before freeze‐drying.

2.6. Data Analysis

Experimental results are presented as the mean ± standard deviation from three independent experiments. Data analysis was performed using IBM SPSS Statistics 26 software, and figures were generated using GraphPad Prism 10.1.2 software. Phylogenetic trees were constructed using MEGA11.

3. Results and Discussion

3.1. Screening of LAB for Triglyceride Degratation

Twenty‐six LAB strains were isolated and purified. Their triglyceride degradation capabilities were assessed by culturing them in MRS broth containing triglycerides. Six strains exhibited triglyceride reduction rates exceeding 60% (p < 0.05), as shown in Figure 1. Strains MHT02, SC03, SZ02, PYC02, PLHB02, and SS01 all demonstrated significant triglyceride degradation capacity (p < 0.05), with degradation rates of 67.69% ± 0.77%, 64.98% ± 0.38%, 80.26% ± 0.44%, 84.87% ± 0.45%, 87.95% ± 0.45%, and 65.64% ± 1.18%, respectively. Among these, SZ02, PYC02, and PLHB02 showed the highest triglyceride degradation capabilities (p < 0.05), with rates of 80.26% ± 0.44%, 84.87% ± 0.45%, and 87.95% ± 0.45%, respectively.

FIGURE 1.

Triacylglycerol‐lowering rates of LAB. Figures with different lowercase letters indicate statistically significant differences (p < 0.05).

In comparison, the reported degradation rates for Lactobacillus rhamnosus R4 and Lactobacillus BH6 were 28.92% and 18.97%, respectively (Azat et al. 2016; Zhao et al. ²⁰²²), suggesting that PYC02 and PLHB02 possess superior lipid‐lowering potential. Furthermore, other probiotics, such as Akkermansia muciniphila , have been shown to prevent fatty liver in obese mice by regulating hepatic triglyceride synthesis and gut homeostasis (Aminlari et al. 2019), while Bacillus coagulans reduces triglyceride levels through intestinal colonization. The triglyceride degradation capacity of these strains may be closely linked to the activity of secreted lipases, as LAB can hydrolyze triglycerides into glycerol and free fatty acids via lipase secretion (Intwala and Barot 2022; Mohammed and Akeel ²⁰²⁴). Additionally, strains may influence triglyceride metabolism indirectly by modulating gut microbiota composition or producing specific metabolites, such as short‐chain fatty acids (Sariyanti et al. 2023).

3.2. Identification of LAB

3.2.1. Morphological Observation of LAB

Six LAB strains with the strongest triglyceride‐lowering capacity were selected. Streak plates were inoculated onto calcium carbonate‐containing agar and incubated at 37°C for 24 h. As shown in Figure 2, all six strains formed calcium‐solubilizing zones, indicating strong acid‐producing capabilities. The colonies were uniformly milky white, with smooth, moist surfaces and central protrusions. Following Gram staining and oil immersion microscopy, all strains appeared purple and rod‐shaped, including both short and long rod forms.

FIGURE 2.

Morphology and shape images of LAB. (a) MHT02 (b) SC03 (c) SZ02 (d) PYC02 (e) PLHB02 (f) SS01.

3.2.2. Identification of LAB 16S rRNA Gene Sequencing

Following 16S rRNA gene sequencing amplification, nucleotide homology comparisons were conducted using NCBI BLAST against target fragment sequences. Results indicated that strains PLHB02 and SZ02 exhibited 99.80% homology with Weissella cibaria and were identified as Weissella cibaria . Meanwhile, PYC02, MHT02, SS01, and SC03 showed over 99.73% homology with Limosilactobacillus fermentum and were thus identified as Limosilactobacillus fermentum. Phylogenetic trees for these six strains were constructed using MEGA, with the results presented in Figure 3.

FIGURE 3.

Phylogenetic tree of six strains of LAB.

3.2.3. Acid and Bile Tolerance Tests

It has been reported that the pH of gastric juice in the human body typically ranges between 2.0 and 3.0, while bile salt concentrations in the intestine range from 0.03% to 0.3% (Das et al. 2016). Therefore, acid and bile salt tolerance are key indicators for evaluating the effectiveness of probiotics. The acid tolerance of six LAB strains at pH 3.0 over time is shown in Figure 4a. The results indicate that, except for MHT02, the survival rates of the remaining strains all exceeded 80%, with a gradual decline over time. Among them, SS01, PYC02, and PLHB02 demonstrated stronger acid tolerance, with significant differences in bile salt survival rates across different time points (p < 0.05). At 1 h, the survival rates were 101.20 ± 0.76%, 101.60 ± 1.20%, and 109.17 ± 1.00%, respectively. After 2 h, the survival rates slightly decreased to 95.70 ± 0.53%, 95.30 ± 1.10%, and 96.67 ± 0.78%, respectively. After 4 h, survival rates remained high at 86.07 ± 0.85%, 88.67 ± 0.67%, and 89.57 ± 0.64%, respectively. These results suggest that SS01, PYC02, and PLHB02 exhibit excellent tolerance to acidic environments at pH 3.0, with minimal changes in survival rates over short periods.

FIGURE 4.

Acid and bile salt tolerance survival rates of six strains of LAB at different time intervals. Figures with different lowercase letters indicate statistically significant differences (p < 0.05). (a) Acid tolerance survival rates of six LAB strains after 1, 2, and 4 h. (b) Bile salt tolerance survival rates of six LAB strains after 1, 2, and 4 h.

Under 0.3% bile salt conditions, the bile salt tolerance of six LAB strains over time is shown in Figure 4b. All strains maintained a survival rate above 73.17% over 4 h. Among them, PYC02, PLHB02, and SZ02 exhibited superior bile salt tolerance, with significant differences (p < 0.05) in survival rates at different time points: the 1‐h survival rates were 100.60 ± 0.92%, 100.37 ± 0.95%, and 100.17 ± 1.55%, respectively. After 2 h, survival rates slightly decreased to 95.30 ± 1.10%, 92.27 ± 0.60%, and 96.67 ± 0.78%, respectively. After 4 h, survival rates remained above 80%. These findings indicate that PYC02, PLHB02, and SZ02 possess strong tolerance to bile salts and maintain stable growth over extended periods.

Literature reports survival rates of certain LAB under similar conditions as 85.25% and 80.79% (Zheng et al. 2020); in contrast, PYC02 and PLHB02 in this study exhibited survival rates of 88.67% ± 0.67% and 89.57% ± 0.64% at pH 3.0, and 81.57% ± 1.57% and 83.30% ± 1.11% under 0.3% bile salt conditions, demonstrating superior adaptability to gastrointestinal environments. The high survival rates of these strains under acidic conditions may be linked to enhanced cell membrane stability, possibly through an increased content of acid‐sensitive lipids (Wang et al. 2018). Furthermore, these strains may mitigate oxidative stress by producing antioxidants, such as superoxide dismutase (Qiu et al. 2021).

3.3. Safety Evaluation

3.3.1. Hemolytic Test

As shown in Figure 5, no zone of inhibition was observed around any of the strains on the medium, a phenomenon known as gamma‐hemolysis. This absence indicates a lack of hemolytic activity, suggesting that these strains do not produce enzymes or toxins capable of lysing red blood cells on blood agar plates, nor do they cause noticeable changes around their colonies (Elsaadany et al. 2025). This characteristic is important because hemolytic activity is correlated with pathogenicity. Non‐hemolytic strains (γ‐hemolytic) are generally considered less harmful than β‐hemolytic and α‐hemolytic strains. Previous studies have shown that most LAB are typically non‐hemolytic under in vitro conditions (dos Santos Leandro et al. ²⁰²¹; Yang et al. ²⁰²³). The results of this study are consistent with these findings, further supporting the safety of these strains as probiotics. Research indicates that non‐hemolytic manifestations of hemolytic outcomes may be associated with the absence of hemolysin genes (Lad et al. 2022).

FIGURE 5.

Hemolytic test results of six strains of LAB. (a) MHT02 (b) SC03 (c) SZ02 (d) PYC02 (e) PLHB02 (f) SS01.

3.3.2. Antibiotic Susceptibility Testing

Antibiotic susceptibility is commonly used as a criterion to assess the safety of LAB (Wei et al. 2022). Table 3 presents the inhibition zone diameters for four commonly used antibiotics and the antibiotic susceptibility profiles of the six strains. The study found that the inhibition zone diameters of the six strains ranged from 19.4 to 25.7 mm, meeting the sensitivity (S) criterion. The strains PYC02, PLHB02, MHT02, SS01, SC02, and SZ02 demonstrated sensitivity to kanamycin, sulfamethoxazole, apramycin, and ampicillin. These results indicate a low level of antimicrobial resistance and suggest a favorable safety profile for these six lactic acid bacterial strains. Regarding antibiotic susceptibility, the performance of the strains in this study was consistent with most literature reports, and antibiotic susceptibility may be related to resistance mechanisms such as efflux pump systems or cell wall modifications (Baran et al. 2023; Zhang et al. ²⁰²⁴). Additionally, strain susceptibility may be influenced by metabolic pathways or environmental adaptability (Yongqi et al. 2024).

TABLE 3.

Results of strain tolerance.

Antibiotics	Inhibition zone diameter (mm) and antimicrobial susceptibility results
	PYC02	PLHB02	MHT02	SS01	SC02	SZ02
Apramycin	20.5S	20.4S	21.7S	25.7S	20.2S	23.1S
Ampicillin	23.8S	24.5S	23.1S	23.5S	21.6S	22.5S
Trimethoprim	24.1S	23.2S	22.7S	21.9S	22.5S	22.5S
Kanamycin	21.9S	20.5S	20.4S	20.3S	25.7S	23.7S

Note: I, Intermediate; R, Resistance; S, Susceptibility.

3.3.3. Determination of Freeze‐Drying Protective Agents

Single‐factor experiments were conducted on four protective agents: skim milk powder, sodium alginate, maltodextrin, and trehalose. As shown in Figure 6a, the probiotic survival rate peaked at 83.7% when the skim milk powder concentration reached 8%. Figure 6b shows that the LAB survival rate peaked at 81.3% when the sodium alginate concentration was 5%. In Figure 6c, the probiotic survival rate reached a maximum of 89.7% at a maltodextrin concentration of 2%. Figure 6d demonstrates that the probiotic survival rate attained its highest value of 81.4% at a trehalose concentration of 4%.

FIGURE 6.

Survival rate of probiotics under four different protective agents. (a) Survival rate of probiotics when the concentration of skim milk powder is 8%. (b) Survival rate of probiotics when the concentration of sodium alginate is 5%. (c) Survival rate of probiotics when the concentration of maltodextrin is 2%. (d) Survival rate of probiotics when the concentration of trehalose is 4%.

3.3.4. Optimization of Freeze‐Drying Protective Agents

Orthogonal experiments were conducted based on the orthogonal experimental design to optimize freeze‐drying protective agents, investigating the effects of nine combinations on LAB survival rates. The experimental results indicate that the optimal combination for LAB survival among the nine tested is A1B3C2D3, achieving a survival rate of 84.7% (skim milk powder 8%, sodium alginate 5%, maltodextrin 2%, trehalose 8%). The results of the orthogonal experiment are presented in Table 4.

TABLE 4.

Orthogonal table of the optimal protectant combination.

Numbers	A	B	C	D	SR%
1	1	1	1	1	68.9
2	1	2	3	2	76.5
3	1	3	2	3	84.7
4	2	1	3	3	76.8
5	2	2	2	1	76.4
6	2	3	1	2	76.5
7	3	1	2	2	76.6
8	3	2	1	3	74.7
9	3	3	3	1	75.5
K1	230.1	222.3	220.1	220.8
K2	229.7	227.6	237.7	229.6
K3	226.8	236.7	228.8	236.2
k1	76.7	74.1	73.4	73.6
k2	76.6	75.9	79.2	76.5
k3	75.6	78.9	76.3	78.7
R	1.1	4.8	5.9	5.1

3.4. Tablet Performance Evaluation

1. Weight Variation: Probiotic tablets produced via wet granulation weighed 4.8 g for 20 tablets, with an average tablet weight of 0.24 g. The weights of individual tablets and their variation are detailed in Table 5. The resulting tablets exhibited a snow‐white appearance and uniform texture. No tablets exceeded the weight limits among the 20 samples. The weight variation met the requirements and complied with the relevant provisions of the 2020 edition of the Chinese Pharmacopeia.

2. Fragility Test: The hardness test results for the probiotics are shown in Table 6. The fragility rate was 0.52%, which meets the relevant requirements of the 2020 edition of the Chinese Pharmacopeia.

3. Disintegration Time: The disintegration times of five randomly selected tablets are shown in Table 7. The average disintegration time for the five tablets was 7.68 min, which complies with the relevant provisions of the 2020 edition of the Chinese Pharmacopeia.

TABLE 5.

Limits of weight differences test results.

Piece weight (g)	Average piece weight (g)	Weight difference limit %
0.24	0.24	0.00
0.25	0.24	4.20
0.23	0.24	−4.20
0.23	0.24	−4.20
0.25	0.24	4.20
0.24	0.24	0.00
0.24	0.24	0.00
0.23	0.24	−4.20
0.23	0.24	−4.20
0.25	0.24	4.20
0.23	0.24	−4.20
0.23	0.24	−4.20
0.24	0.24	0.00
0.25	0.24	4.20
0.24	0.24	0.00
0.25	0.24	4.20
0.25	0.24	4.20
0.23	0.24	4.20
0.25	0.24	−4.20
0.24	0.24	0.00

TABLE 6.

Friability test results.

Before weight (g)	After weight (g)	Difference (g)	Friability %
1.92	1.91	0.01	0.52

TABLE 7.

Disintegration time test results.

Numbers	Disintegration time (minutes)
1	6.84
2	5.85
3	9.79
4	7.65
5	8.31

3.5. Probiotic Tablet Triglyceride Degradation Capacity

Probiotic tablets formulated with the four most acid‐ and bile salt‐resistant LAB strains—PYC02, PLHB02, SS01, and SZ02—were tested for triglyceride degradation, as shown in Figure 7a. The triglyceride degradation rates for PYC02, PLHB02, SS01, and SZ02 probiotic tablets were 64.80% ± 2.52%, 66.93% ± 2.01%, 42.77% ± 2.51%, and 34.67% ± 3.18%, respectively. Among these, PYC02 (Limosilactobacillus fermentum) and PLHB02 ( Weissella cibaria ) probiotic tablets exhibited the strongest triglyceride degradation capacity, with degradation rates of 64.80% ± 2.52% and 66.93% ± 2.01%, respectively. These rates were significantly different from those of the other two probiotic tablets (p < 0.05). Potential mechanisms for this observation can be inferred from the literature. It was reported that feeding rats soybean yogurt fermented with lactic acid bacteria downregulated glycerol‐3‐phosphate acyltransferase (involved in triglyceride synthesis) and upregulated enoyl‐CoA isomerase, thereby enhancing fatty acid β‐oxidation (Kitawaki et al. 2009). Supporting this (Noguchi et al. 2022) found that the microbial metabolites 10,13‐dihydroxy‐cis‐6‐octadecenoic acid (γHYD) and 13‐oxo‐cis‐6,cis‐9‐octadecadienoic acid (γKetoD) increased the expression of PPARδ target genes, promoted fatty acid β‐oxidation, and reduced intracellular triglyceride accumulation. Other proposed mechanisms for triglyceride reduction by lactic acid bacteria include the stimulation of lipase activity responsible for triglyceride hydrolysis (Abdullah et al. 2013) and the promotion of hepatic bile acid secretion, which inhibits intestinal absorption of cholesterol and triglycerides, facilitating their fecal excretion (Nasser et al. 2021). Based on these findings and our current results, we plan to conduct animal studies to validate the in vivo triglyceride‐degrading efficacy of strains PYC02 and PLHB02 and to compare functional differences across bacterial genera.

FIGURE 7.

Triglyceride degradation rate and viable bacterial survival rate of probiotic tablets. Figures with different lowercase letters indicate statistically significant differences (p < 0.05). (a) Triglyceride‐lowering rate of probiotic tablets. (b) Survival rate of probiotic tablets under different temperatures for 10 days. (c) Survival rate of probiotic tablets under different temperatures for 20 days. (d) Survival rate of probiotic tablets under different temperatures for 30 days.

3.6. Effects of Temperature on Probiotic Tablets

As shown in Figure 7b–d, the survival rate of viable bacteria in probiotic tablets produced from the four strains decreased after storage at both 4°C and 25°C for 10, 20, and 30 days. Survival rates at 4°C were significantly higher than those at 25°C. After 30 days, survival rates for all four strains exceeded 80%, consistent with the findings reported by (Shu et al. 2018). Survival rates at 25°C gradually decreased over time, with significant differences observed between the 4°C and 25°C conditions (p < 0.05). Notably, PYC02 and PLHB02 maintained higher survival rates across all temperatures: 81.87% ± 0.50% and 81.03% ± 0.60% at 4°C after 30 days, and 60.93% ± 0.25% and 63.10% ± 0.60% at 25°C, respectively, meeting expectations. Thus, the two superior strains, PYC02 and PLHB02, were selected for subsequent studies. This finding is consistent with studies on Lactobacillus plantarum tablets for alleviating lead toxicity, confirming that low‐temperature storage can slow the decline in activity of live bacterial tablets (Heidebach et al. 2010; Liu et al. ²⁰¹⁶). In conclusion, storage at 4°C is more conducive to preserving probiotic tablets, ensuring high survival rates, stability, and efficacy. Although this study provides valuable findings, several limitations should be acknowledged. The functional mechanisms of the lactic acid bacteria, particularly the specific molecular pathways of triglyceride degradation, remain to be fully elucidated. Furthermore, the triglyceride‐lowering effects have not yet been validated in vivo. Therefore, future work will focus on leveraging the promising attributes of strains PYC02 and PLHB02. We plan to employ genomic or metabolomic analyses, combined with animal studies, to further elucidate their underlying mechanisms of action.

4. Conclusion

This study successfully screened and identified six LAB strains with significant triglyceride degradation capabilities. Among them, PYC02 (Limosilactobacillus fermentum) and PLHB02 ( Weissella cibaria ) exhibited strong triglyceride degradation abilities. These strains demonstrated excellent stress resistance and safety profiles. The stability and survival rates of probiotic tablets prepared from these strains were satisfactory, and the tablet performance met the relevant standards of the 2020 edition of the Chinese Pharmacopeia. The findings highlight the significant potential of the six LAB strains in triglyceride reduction, providing scientific evidence and theoretical support for the further development of novel functional foods and dietary supplements.

Author Contributions

Xinglin Ran: conceptualization, writing – original draft, methodology, software, formal analysis. Qian Hu: conceptualization, methodology, writing review and editing, formal analysis. Shengdan Wei: supervision, project administration, funding acquisition. Huizhen Long: investigation, data curation. Xu Luo: investigation, data curation. Boyu Dong: investigation, data curation. Qiuping Yao: investigation, data curation. Dequan Zhu: resources, investigation, data curation.

Funding

This study was supported by the National Natural Science Foundation of China (No. 32260585) and Guizhou Provincial 2026 Central Government Guidance Fund for Local Science and Technology Development Projects (Qiankehe Zhongyindi [2026] No. 034).

Ethics Statement

This study does not involve any human or animal testing.

Consent

Written informed consent was obtained from all study participants.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.