We searched the China National Knowledge Infrastructure, China Biology Medicine, VIP, WanFang, PubMed, EBSCO, Cochrane Library, and Web of Science databases for relevant clinical trials published before January 2024. Taking the PubMed database as an example, the search field was [Title/Abstract], and the search formula was as follows: (Probiotic OR Bifidobacterium OR Bifidobacteria OR Bacillus bifida OR Yeast OR Saccharomyces cerevisiae OR Saccharomyces italicus OR Saccharomyces oviformis OR S cerevisiae OR S. cerevisiae OR Saccharomyces uvarum var. melibiosus OR Candida robusta OR Saccharomyces capensis OR Lactobacillus acidophilus OR Lactobacillus amylovorus OR Lactobacill* OR lactic acid bacteria OR Clostridium butyricum OR Slaysophilus OR Bacillus OR Natto Bacteria OR Streptococcus thermophiles OR Enterococcus) and (Ischemic Stroke OR Ischemic Strokes OR Ischaemic Stroke OR Ischaemic Strokes OR Acute Ischemic Stroke OR Acute Ischemic Strokes OR AIS OR Brain Ischemia OR Middle Cerebral Artery occlusion OR MCA OR Large Vessel Occlusion OR LVO OR Brain Infarction OR Cerebral Infarction). The search terms included components of bifid triple viable and other probiotic strains to ensure the comprehensiveness of the literature search.

The inclusion criteria followed the PICOS principle. (1) Participants: patients diagnosed with AIS by cranial magnetic resonance imaging (Chinese Society of Neurology, Chinese Stroke Society, 2018). (2) Intervention: the experimental group received enteral nutrition and bifid triple viable preparation. (3) Comparison: the control group received enteral nutrition only. (4) Outcomes: the primary outcome measures were nutritional status (total protein [TP], albumin [ALB], prealbumin [PA], hemoglobin [Hb], transferrin [TRF]), and the secondary outcome measures included neurological function (National Institute of Health Stroke Scale [NIHSS], Glasgow Coma Scale [GCS], Neurological Disability Score [NDS]), intestinal barrier function (endotoxin, D-lactic acid, diamine oxidase [DAO], endothelin [ET]), and immune function (immunoglobulin G, immunoglobulin M, and immunoglobulin A [IgG, IgM, IgA]). The safety outcome measures included all adverse events mentioned in the literature that could be meta-analyzed (total adverse events, pulmonary infection, abdominal distension, anorexia, vomiting, reflux, constipation, diarrhea, gastrointestinal bleeding, electrolyte disturbance). (5) Study design: randomized controlled trial.

The exclusion criteria were as follows: (1) literature for which the data was not available would be excluded; (2) duplicate publications would be screened out, retaining only the most comprehensive and recent data; (3) literature not meeting the diagnostic criteria for AIS as defined by cranial magnetic resonance imaging would be excluded; (4) studies failing to meet rigorous quality assessment standards or with significant research design flaws would be excluded; and (5) trials with unclear intervention details or deviations from the specified plans would be excluded.

Two researchers, Kong YM and Deng J, independently screened the literature according to the established inclusion and exclusion criteria using NoteExpress version 3.0 (Aiqinhaiyuezhi Technology Co., Beijing). The duplicates were sequentially excluded, as were literature not related to the study topic and literature lacking complete data, after which the included literature were finalized.

The aforementioned researchers compiled the basic information for each study, including the following: author name(s); country; sources of participants; sample size; male ratio; average age; average ALB; intervention; and treatment duration. These information were then input into a basic information table created by Excel 2010.

Kong YM and Deng J assessed the risk of bias for each study using the Cochrane tool. Assessment items included random generation, allocation concealment, blinding, incomplete outcome data, selective reporting, and other biases. The evaluation ratings were categorized as low, unclear, or high risk.

First, we conducted a meta-analysis using the RevMan 5.3. Mean difference (MD) and 95% confidence interval (CI) were used as the effect sizes for continuous variables, whereas risk ratio (RR) and 95% CI were used as the effect sizes for dichotomous variables. Employing a random effects model for all meta-analyses, irrespective of the presence of low heterogeneity, represents a robust and cautious approach. This method allows for the consideration of potential differences between studies, accounting for both within-study and between-study variations, resulting in more generalizable and conservative estimates of the effect sizes. For the effect models, a p-value <0.05 was considered statistically significant. Second, we conducted a leave-one-out sensitivity analysis to assess the robustness of the results. After sequentially excluding each data point and reanalyzing it, the results were considered robust if the combined effect size did not significantly change. Third, we performed a subgroup analysis to evaluate the impact of different doses and treatment duration on ALB. Fourth, we conducted Egger’s test using Stata 15.0 (StataCorp LLC, United States). A p-value >0.1 in Egger’s test was considered indicative of no publication bias.

A total of 1,376 articles were retrieved from the initial searches. We excluded 419 duplicate articles and 942 unrelated articles that did not meet our inclusion criteria. In total, 15 articles published between 2016 and 2023 were included in the present meta-analysis (Pang, 2016; Zhao, 2017; Yang et al., 2018; Chen et al., 2019; Huang and Jiang, 2019; Yu, 2020; Gao, 2021; Li L. et al., 2021; Wan et al., 2021a,b; Wang et al., 2021; Wang, 2021; Yang, 2021; Li et al., 2023; Zhang et al., 2023). A flow diagram of the study is shown in Figure 1.

A total of 15 clinical trials involving 1,544 Chinese patients were included in the present meta-analysis. Among them, 771 received enteral nutrition and 773 received a bifid triple viable preparation combined with enteral nutrition. All of the studies were from China and the participants were Chinese. The mean male ratio of the participants was 57.6%, the mean age was 66.9 years, and the mean serum ALB level was 33.9 g/L. Additionally, bifid triple viable preparation was administered at doses ranging from 0.84 g/d to 6.00 g/d, with a duration of treatment ranging from 2 weeks to 4 weeks. The basic characteristics of the included studies are presented in Table 1.

Among the included studies, the randomization methods of 2 studies were assessed as having an unclear risk of bias, and the allocation concealment and blinding of participants and personnel in all 15 studies were assessed as having an unclear risk of bias. The risks of bias in the remaining areas were considered low, as shown in Figure 2.

The following subgroups were established based on the therapeutic dose: ≤ 1.0 g/d; 1.1–2.0 g/d; and > 2.0 g/d. The results showed that ≤1.0 g/d (MD, 2.47; 95% CI, 1.99–2.96; p < 0.00001), 1.1–2.0 g/d (MD, 4.08; 95% CI, 2.90–5.25; p < 0.00001), and > 2.0 g/d (MD, 6.48; 95% CI, 4.77–8.19; p < 0.00001) of bifid triple viable preparation significantly increased ALB levels in patients with AIS. The test for subgroup differences showed that p < 0.00001.

Additionally, the subgroups of ‘1–2 weeks’ and ‘3–4 weeks’ were created based on the duration of treatment. The results showed that both 1–2 weeks (MD, 4.40; 95% CI, 2.74–6.06; p < 0.00001) and 3–4 weeks (MD, 3.92; 95% CI, 2.44–5.39; p < 0.00001) of bifid triple viable preparation significantly increased ALB levels in patients with AIS. The test for subgroup differences showed that p = 0.67. As shown in Figure 4.

We evaluated the publication bias of the primary outcome measures using Egger’s test. It showed that there was no publication bias in the results of TP (p = 0.517), PA (p = 0.222), Hb (p = 0.509), and TRF (p = 0.659), while there was publication bias in the results of ALB (p = 0.076), as shown in Figure 5.

In terms of nutritional status, the results of the present study showed that the combined use of bifid triple viable preparations increased the levels of TP, ALB, PA, Hb, and TRF in patients with AIS. The improvements in these indicators not only directly demonstrate the role of bifid triple viable preparation in improving nutritional status, but also indirectly reflect its potential to alleviate nerve damage. Three previous meta-analyses found that probiotics significantly increased the levels of TP, ALB, PA, and Hb in stroke patients (Liu et al., 2021; Zhong et al., 2021; Chen et al., 2022), supporting the findings of the present study. In contrast, the present meta-analysis also identified the benefits of the bifid triple viable preparation on the TRF levels, which further supported the value of the probiotics in improving the nutritional status of patients with AIS.

In terms of neurological function, the results of the present study indicated that the combined use of bifid triple viable preparation and enteral nutrition reduces the NIHSS and NDS scores and increases the GCS scores in patients with AIS. The results of a previous meta-analysis by Liu et al. (2021) are commensurate with our findings on GCS scores, while Chen et al. (2022) did not report the impact of probiotics on neurological function. Of note, the meta-analysis by Zhong et al. (2021) did not show any benefit to the use of bifid triple viable preparation in terms of the NIHSS, which is in stark contrast with the results of the present study. This contradiction may be due to the different probiotic strains studied. Zhong et al. (2021) included various strains, such as Bifidobacterium, Lactobacillus, Enterococcus, Streptococcus thermophilus, and Clostridium butyricum, while the present study only evaluated bifid triple viable preparations. In the present study, we found that the bifid triple viable preparation provided additional benefits in regards to the NIHSS and NDS scores, indicating its potential for the stronger promotion of neurological repair.

In terms of intestinal barrier function, the results of the present study indicated that the combined use of bifid triple viable preparations and enteral nutrition reduced the levels of DAO, D-lactic acid, endotoxin, and endothelin in patients with AIS. The above indicators are commonly used to evaluate the intestinal barrier function, and the benefits observed reflect the advantages of the bifid triple viable preparation in repairing the intestinal barrier in patients with AIS. In previous meta-analyses, Chen et al. (2022) reported the benefits of probiotics on DAO and D-lactic acid for patients with IS, supporting the findings of the present study, while Liu et al. (2021) and Zhong et al. (2021) did not report endpoints related to the intestinal barrier. Additionally, the results of the present study showed that the use of bifid triple viable preparation significantly reduced the levels of endotoxin and endothelin, indicating its potential to alleviate endotoxin damage and improve intestinal barrier repair.

In terms of immune function, the results of the present study indicated that the combination of bifid triple viable preparations and enteral nutrition increased IgG and IgM levels in patients with AIS, with no significant impact on IgA levels. In previous meta-analyses, Chen et al. (2022) reported an increase in IgG and IgA levels in stroke patients treated with probiotics, whereas Liu et al. (2021) and Zhong et al. (2021) did not evaluate the effect of probiotics on immunoglobulins. Interestingly, the results of the present meta-analysis showed benefits for IgM with the use of bifid triple viable preparations, but without the IgA benefits reported by Chen et al. (2022). This difference may be due to variations in the bacterial strains and stroke types evaluated. Nevertheless, the results of both the present study and that of Chen et al. (2022) support the idea that probiotics significantly increase IgG levels, highlighting the role of the bifid triple viable preparation in the regulation of long-term immune function.

The results of the present study showed that the total number of adverse events in the bifid triple viable preparation combination group were comparable to those in the enteral nutrition only group, suggesting that the bifid triple viable preparation has a favorable safety profile and does not increase the risk of additional adverse events. Specifically, pulmonary infection, vomiting, constipation, and diarrhea were significantly reduced in the bifid triple viable preparation combination group compared to the enteral nutrition only group, while abdominal distension, anorexia, reflux, gastrointestinal bleeding, and electrolyte disturbance were comparable. It means that bifid triple viable preparations can reduce the risk of pulmonary infection and some gastrointestinal adverse events in patients with AIS. Two previous meta-analyses reported benefits of probiotics in reducing the risk of pulmonary infection, constipation, diarrhea and vomiting (Zhong et al., 2021; Chen et al., 2022), and another meta-analysis reported benefits of probiotics in reducing gastrointestinal complication (Liu et al., 2021), which supports our findings. However, the study by Chen et al. (2022) also showed that probiotics reduced abdominal distension, while the study by Zhong et al. (2021) noted a reduction in reflux and gastrointestinal bleeding with probiotics, which is different from our results. These differences may be caused with the type of probiotic as well as the type and severity of stroke. Therefore, the effect of bifid triple viable preparation on abdominal distension, reflux and gastrointestinal bleeding needs to be further explored.

The dosage subgroup analysis in the present study showed that bifid triple viable preparation at ≤1.0 g/day, 1.1–2.0 g/day, and > 2.0 g/day all significantly increased the ALB levels, suggesting that all doses of bifid triple viable preparation improved the prognoses of patients with AIS. Previous studies, however, have found that doses of Bifidobacterium at 10⁹ and 10¹⁰ CFU/day significantly reduced colonic tissue damage, whereas low doses did not achieve this benefit (Chen et al., 2020), which indicates that a sufficient dose is a key factor for Bifidobacterium and its combination preparations to achieve beneficial effects. The authors, therefore, recommend that the dosage of the bifid triple viable preparation for patients should be maintained at ≥1.0 g/day.

In terms of treatment duration, the administration of bifid triple viable preparations for 1–2 weeks and 3–4 weeks significantly increased ALB levels, suggesting that both short- and medium-term duration of bifid triple viable preparations achieved additional benefits. However, considering that the standard length of stay for the clinical course of IS is 21–28 days (Office of the National Health and Family Planning Commission, 2016), a treatment course of 3–4 weeks may be more in-line with clinical practice. Based on the current evidence, therefore, we recommend that patients with AIS take bifid triple viable preparation of ≥1.0 g/day and continue for at least 3–4 weeks.

Although the present study has enriched the clinical evidence for the use of bifid triple viable preparations in treating AIS, some limitations need to be acknowledged. First, the literature included in the present study did not report allocation concealment methods, which may pose a potential risk of selection bias. Additionally, the lack of reporting on the use of blinding increases the potential risk of implementation bias. Second, the included studies were all conducted in Chinese, with study subjects of Chinese descent, which reduces the generalizability of the results. Third, the average age of the participants included in the present meta-analyses ranged from 56.9–73.2 years, which does not fully reflect the situation of patients across all age groups. Fourth, although the results of the present study describe the benefits of bifid triple viable preparations in patients with AIS, the optimal dosage and duration of treatment remain unclear.

The authors recommend that future research make efforts to address these limitations: first, strictly control allocation concealment and blinding to provide higher quality evidence for the application of bifid triple viable preparations in patients with AIS patients; second, design multicenter clinical trial protocols to evaluate the effects of bifid triple viable preparations on patients with AIS with a variety of demographic characteristics; third, promote the progress of subgroup clinical studies to reveal the optimal dosage and duration of bifid triple viable preparations for the management of AIS.