The Administration of Probiotics against Hypercholesterolemia: A Systematic Review

Featured Application: The current review helps ﬁnd a better probiotic strain or probiotic mixture to develop probiotic-based adjuvant therapy to control hypercholesterolemia. Abstract: Hypercholesterolemia is a key factor in the progression of atherosclerosis and cardiovascular disease (CVD). CVD is a signiﬁcant public health concern with a high death rate. Some of the main factors linked to CVD include genetics and lifestyle. Dyslipidemia has been one of the factors related to the onset of several CVD-related diseases. Several clinicopathological studies have shown a correlation between high cholesterol levels, particularly low-density lipoprotein cholesterol (LDL-c), and CVD development. Probiotics have received a lot of attention for various beneﬁcial effects, especially their ability to reduce blood cholesterol in humans. Probiotics were shown in several investigations to affect hypercholesterolemia by inﬂuencing cholesterol biosynthesis. The current review focuses on the human dietary interventions with probiotics and their effects on CVD risk factors and hypercholesterolemia. The outcomes are debatable and consider various parameters such as probiotic strain, dosing frequency, therapeutic response, dietary changes, and so forth. As a result, probiotics have the propensity to become dietary supplements in moderate/severe hypercholesterolemic patients, which signiﬁcantly reduces the CVD risk. supplementations in hypercholesterolemic subjects.


Introduction
Hypercholesterolemia is a widespread hereditary metabolic disease caused by elevated serum levels of low-density lipoprotein cholesterol (LDL-c), leading to premature coronary artery disease [1]. Hypercholesterolemia is one of the leading causes of CVD, and when treated appropriately, it can reduce the risk of CVD-related morbidity and death. The Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP) recommends LDL-c levels of 100 mg/dL are optimal and >160 is high [2]. It is predominantly caused by the mutation in low-density lipoprotein receptor (LDLR), apolipoprotein B-100 and proprotein convertase subtilisin/Kexin 9 (PCSK9), which leads to decreased LDL-c receptor absorption and evacuation from the circulation [3][4][5]. Due to the emphasis on lifestyle changes, most patients must use medications to achieve a sufficient decline in LDL-c levels. It also implies that any cholesterol-lowering medication should focus on LDL-c as the primary goal [6]. Statins are the most often prescribed treatment for hypercholesterolemia. Studies have shown that statins lower the risk of heart attack and mortality by 30-40% and lower LDL-c levels by 25-40% [7,8]. Statins are also combined with other small molecules such as ezetimibe, proprotein convertase subtilisin-kexin type 9 inhibitors, bempedoic acid, angiopoietin-like 3 protein inhibitors, and nutraceuticals to

Materials and Methods
The literature search was made in PubMed and web of science using a mixture of keywords' probiotics and hypercholesterolemia' and 'hypercholesterolemia and cardiovascular disease.' The scientific reports were collected, which were published till 24 February 2021. The Method PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) was used in this systematic review to set the selection criteria of the collected clinical trials. Clinical studies have been included in the study concerning subjects with high and moderate CVD and hypercholesterolemia without the possibility of CVD.

Results
From 24 clinical trials, 17 full-text articles were considered after implementing the exclusion criteria and represented in Figure 1. The details of the reported studies on the influence of probiotics on hypercholesterolemia have been summarized in Table 1. The studies revealed the pilot/randomized controlled trial method to investigate the effects of probiotics in hypercholesterolemic patients. Lactobacillus (in nine studies), Bifidobacterium (in eight studies), and Enterococcus (in two studies) were the most widely studied genera in these reports. Saccharomyces and Propionibacterium were used in one study. The studies were planned to assess the effects of probiotics on the lipid profile, considered a crucial causative agent of CVD.
The 33 patients with low and without CVD risk were tested with Bifidobacterium longum BB536 and red yeast rice, which reduced the LDL-c level. The circulating levels of lathosterol in plasma, a marker of cholesterol synthesis, were also significantly reduced (p = 0.0206). It revealed that B. longum BB536 and red yeast rice affect the lathosterol biosynthesis [40]. L. plantarum ECGC 13110402 was administrated with the proper regular diet to 23 people with normal to mildly hypercholesterolemia, which showed a significant difference in the LDL-c concentrations. L. plantarum ECGC 13110402 has high bile salt hydrolase activity, which reduced the LDL-c level and may lower the CVD risk [41]. The isoflavone-supplemented soy product fermented with Enterococcus faecium CRL 183 and L. helveticus 416 was administered to 17 patients. At the end of 42 days, the outcomes were observed that the probiotic intervention significantly reduced the LDL-c level (up to 14.8%) [42].
After eight weeks of daily supplementation to adults with hypercholesterolemia (n = 11) found that Saccharomyces boulardii var. boulardii CNCM I-1079 significantly reduced (about 15.5%) remnant lipoprotein particles. However, there are no significant changes in total cholesterol (TC), LDL-c, HDL-c, triglycerides (TG), TC/HDL-c, and non-HDL-c [43]. L. reuteri NCIMB 30242 with bile salt hydrolase functionality decreased the LDL-c and increased the circulating bile acid. L. reuteri NCIMB 30242 also increases the serum 25-hydroxyvitamin D (25.5%), which significantly reduces the risk of osteoporosis, CVD, diabetes, and some cancers [44,45]. It may reduce CVD risk and other chronic diseases [46]. However, further experiments are needed to warrant the statement.
The probiotic mixture comprised of B. lactis MB 2409, B. bifidum MB 109B, and B. longum BL04 showed a significant decrease in TC, LDL-c, and TG and an increase in HDL-c levels in dyslipidemic children [47]. Fuentes et al. reported that the supplementation of the probiotic mixture contains three strains of L. plantarum which significantly reduced the TC (13.6%), LDL-c, and oxidized LDL-c levels in hypercholesterolemic adults. It significantly reduces the CVD risk in hypercholesterolemic adults [48]. Moroti et al. described that the supplementation of a synbiotic mixture contains L. acidophilus, B. bifidum, and oligofructose effectively increased HDL-c and reduced the fasting glycemia in elderly people with type 2 diabetes mellitus [49]. The oral administration of E. faecium M-74 for one year reduces the LDL-c concentration significantly [50].
Iran Dairy Industries uses Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus for making their regular yogurt. Along with those two bacteria, L. acidophilus and B. lactis were used as a starter to prepare probiotic yogurt. The probiotic yogurt had a cholesterol-lowering effect in healthy hypercholesterolemic subjects. Likewise, the yogurt fermented with L. acidophilus and B. longum did not lower the TC and LDL-c but increased the level of HDL-c significantly, which helped to maintain the LDC-c and HDL-c ratio [51]. The detailed mechanism discussing crucial probiotics used in cholesterol-lowering effects was not elucidated fully. However, the study needs further clarifications using more volunteers and extended study duration [52].     Cross-over study (study of crossover groups of probiotic cohort or prebiotic cohort)

Discussion
The results were correlated with the study of milk products fermented with B. longum, which reduces the serum lipids in healthy moderate hypercholesterolemic male adults [53]. The isoflavone-supplemented soy product fermented with E. faecium CRL 183 and L. helveticus 416 significantly improves the lipid profile in moderately hypercholesterolemic men subjects. The fermented soy product has an anti-inflammatory effect and activates estrogen-receptor complex mechanisms, thereby reducing the LDL-c level. It reduces the serum LDL-c level, and no significant changes were noted in HDL-c and TG levels [43]. The reported results were established previously by Larkin et al. [54] and Greany et al. [55]. However, this study does not include women subjects to estimate the estrogen effects in women. In addition, pickled mustard greens fermented with lactic acid bacteria were beneficial for lowering cholesterol [56]. The possible benefit of a natural health mixture containing B. longum BB536 and RYR (Red Yeast Rice) extract involves two different pathways to facilitate LDL-c and TC reduction. RYR extracts inhibit the liver's cholesterol production, and B. longum BB536, by its high bile salt hydrolase function, reduces intestinal cholesterol absorption [40]. In addition, the expression of LXRa and LXRb/NPC1L1 genes plays a critical role in the absorption of intestinal cholesterol in vitro [57][58][59]. L. plantarum ECGC supports the bile acid function, which reduces the cholesterol in mildly hypercholesterolemic adults without any impact on gastrointestinal and gut microbiota that can be possibly the best natural cholesterol-lowering supplement [42]. However, available data indicate that the study does not target severe hypercholesteremic patients to claim the results are accurate.
S. boulardii var. boulardii CNCM I-1079 supplementation lowered remnant lipoprotein. TG-rich lipoproteins are linked to the incidence and development of coronary artery disease, regardless of LDL-c levels [43]. However, the precise lipoprotein lowering mechanisms of S. boulardii is undisclosed, and the safety and tolerance of the strain were also not evaluated in this study. The safety risk management criteria are required to measure the safety of probiotics. However, due to the wide variety of microorganisms, it is also important to determine the unique hazards connected with each probiotic, including the risk factors associated with the host and the complex interaction between the probiotic, host, and nutrition constituents [60,61].
L. reuteri NCIMB 30242 was found to raise bloodstream bile acid levels involved in several metabolic activities, including energy balance, lipid metabolism, and inflammatory control [62,63]. The administration of Bifidobacterium species claimed to be involved in the control of dyslipidemia in hypercholesterolemic children by improving lipid profile [48]. However, to elucidate the precise molecular mechanisms of action of Bifidobacterium in dyslipidemic children, further studies and clinical examination are needed.
L. reuteri NCIMB lowers cholesterol and reduced sterol absorption. Authors have also analyzed the increased circulation of 25-hydroxyvitamin D in serum during L. reuteri NCIMB administration and found that the level of vitamin D was increased at the end of the study. The increased vitamin D level improves the proper intestinal colonization of probiotics, prevents pathogenic bacterial invasion, reduces chronic inflammation, and maintains enterocyte cell integrity [45,64,65]. Women who consumed yogurt containing L. acidophilus 145 and B. longum 913 regularly for six months had higher HDL-c levels and improved LDL-c/HDL-c ratio [52]. Fukushima and Nakano reported that a probiotic mixture diet containing Bacillus, Lactobacillus, Streptococcus, Clostridium, Saccharomyces, and Candida decreased cholesterol synthesis in rats' liver. The L. acidophilus strains removed cholesterol by binding to the intestine surface area, making it less obtainable for absorption in the intestine [66]. Likewise, L. plantarum KCTC3928 and L. acidophilus ATCC 43121 enhanced the conversion of HMG-CoA and cholesterol to mevalonate and bile acid, respectively, reducing the LDL-c level in hypercholesterolemic patients [67][68][69].
The probiotic mixture of L. plantarum CECT 7527, 7528, and 7529 improves the cholesterol profile and reduces the CVD risk in hypercholesteremic patients [48]. However, the over-growth probiotics mixture may lead to gut dysbiosis, affecting physiology, gut-brain axis function, and other secondary disease outcomes in the host [70,71]. Further studies are needed to warrant the tolerance and safety of long-term consumption of a mixture of L. plantarum strains. The consumption of L. acidophilus, B. bifidum, and oligofructose increases the HDL-c level, reduces CVD risk, and significantly reduces the blood sugar level [49]. Ataie-Jafari et al. stated that probiotic yogurt consumption significantly decreases the total lipid content and reduces the CVD risk [51].
The administration of E. faecium M-74 enriched with selenium reduces the LDL-c level. However, no significant changes were noted in HDL-c and TG levels. Further studies are required to meet the safety and tolerance criteria [50].
The yogurt fermented with B. longum BL1 has the highest efficacy in lowering serum cholesterol in moderately hypercholesterolemic subjects [53]. However, there are no significant changes in HDL-c, TC, and TG levels. Furthermore, studies are needed to warrant the efficacy of B. longum BL1. Therefore, probiotics are the natural replacements that could be beneficial to improve the lipid profile and reduce the CVD risk without causing adverse effects, unlike statin drugs.

Conclusions
Our review concluded that probiotics against hypercholesterolemia could act as an alternative medicine to reduce the CVD risk and other diseases. The obtained clinical trailed data confirm the positive effect of probiotic consumption against hypercholesterolemia. As a result, additional research is needed to understand how probiotics can benefit the cardiovascular system and perhaps other ailments and test out any potential adverse health effects. The present literature review strongly recommends further clinical studies with various mixtures and dosages of probiotics in a greater number of volunteers. Finally, it appears that the potential of probiotics to decrease cholesterol depends on species and strain. L. acidophilus was found to reduce cholesterol levels. Similarly, a fermented milk product containing B. longum 913 and BL1 reduced the LDL-c cholesterol levels in the blood. The L. plantarum and L. reuteri lowers LDL-c levels while increasing vitamin D synthesis. Therefore, the strains of L. acidophilus, B. longum, and L. plantarum used in this study may have hypocholesterolemic properties.

Data Availability Statement:
The data presented in this study are available within the article.