Effects of Probiotics , Prebiotics , and Synbiotics on Hypercholesterolemia : A Review

Cholesterol plays a major role in human health. High cholesterol is a leading risk factor for human cardiovascular disease such as atherosclerosis, coronary heart disease, and stroke. Excess cholesterol in the bloodstream can form plaque in the artery walls. Hypercholesterolemia is a major risk factor for the development of atherosclerosis. A number of pharmacological and nonpharmacological (including dietary) approaches being employed to reduce the cholesterol level. Numerous drugs that lower serum cholesterol have been developed to treat hypercholesterolemic patients, the best example being the statins drugs (Atorvastatin, Simvastatin, Rosuvastatin, and Lovastatin). However, the undesirable side effects of these compounds were observed and have caused concern about their long term therapeutic use. Several studies have been reported that the consumption of fermented dairy products decreases serum cholesterol. Probiotics are live microorganisms that promote health benefits upon consumption, while prebiotics are nondigestible food ingredients that selectively stimulate the growth of beneficial microorganisms in the gastrointestinal tract. Probiotics and prebiotics have been suggested to reduce cholesterol via various mechanisms without any deleterious effect to the human health.This paper may throw some light to prove the ability of these synbiotics as a novel alternative or adjuvant to chemical drugs to help fight the hypercholesterolemic problem.


Introduction
Increased serum cholesterol level can cause several cardiovascular diseases (CVDs) including coronary heart diseases, atherosclerosis, and hypertension. According to the World Health Organization (WHO) survey by 2030, CVD will be one of the major causes of death, and worldwide, 23.6 million people will be affected [1]. Many researchers reported that even a 1% reduction in the serum cholesterol level might reduce the coronary heart disease by 2-3% [2]. During the period of 1999 to 2003, 45% of Western Europe and 35% Central and Eastern Europe populations are affected by heart attacks caused by hypercholesterolemia [3,4]. Elevated serum cholesterol level may increase the heart attack risk by three times compared with people having normal serum cholesterol level. Unhealthy diets such as foods rich in sugar, salt, oil, and fat or food with low fiber content are the major causes of cardiovascular diseases [5].

Good and Bad Cholesterol.
Generally, cholesterol is a waxy, fat-like substance present in the human and animal cells and referred as "good" and "bad" cholesterol. Actually, these descriptions refer to the substances called lipoproteins that carry cholesterol throughout the body in the bloodstream. Lipoproteins are a combination of varying amounts of fats and proteins.
"Good" cholesterol is associated with high-density lipoproteins (HDLs). HDLs are believed to remove excess cholesterol from the body; therefore, higher levels of HDLs are also believed to be associated with lower rates of heart disease. "Bad" cholesterol is associated with low-density lipoproteins (LDLs). LDLs carry cholesterol in the blood to body cells. High levels of LDLs are usually associated with elevated blood cholesterol and an increased risk of heart disease due to cholesterol and fat being deposited in the arteries. ese fatty deposits decrease the interior size of the arteries so the blood supply is reduced, thus increasing the risk of heart disease and stroke.
Several drugs have been developed to treat hypercholesterolemia. Nowadays, numerous nonpharmacological approaches (including dietary ones) have been used to reduce the serum cholesterol. Recently, probiotics and prebiotics are considered as a remedy to reduce the serum cholesterol level.

Probiotics
e word "probiotic" is derived from Greek word "pro bios" which literally means "for life," which is opposite to the meaning of "antibiotics" which is translated to "against life." Greek and Romans began the probiotic history by consuming the fermented foods [6,7]. In 1908, the Nobel laureate Elie Metchnikoff proposed the probiotic bacteria has a beneficial effect on human health. Metchnikoff observed Bulgarian peoples consuming milk which is fermented with rod-shaped Lactobacillus spp. and thus improved their health. And also, he suggested that these probiotic bacteria have positive effects in the gut microflora and exclude the intestinal pathogens in the human intestine [6,8,9].
In 1965, Lilly and Stillwell first used the term "probiotic" to define any substance which kindles the growth of beneficial microorganisms. Later, "probiotic" was also used in different interpretations based on their mechanism and their beneficial effects on human health. Few years later (1974), Parker defined "probiotic" as "substances and microorganisms which contribute to intestinal microbial balance" [10]. In 1989, definition of probiotics was improved by Fuller [11]. Generally, probiotic microorganisms not only present or act in the large intestine but also in other organs, either by modulating immunological parameters, intestinal permeability, bacterial translocation or also by providing bioactive substances or regulatory metabolites. Hence, extensive definitions for probiotics were suggested by many researchers and organizations. Schrezenmeir and de Vrese [12] and the International Life Sciences Institute (ILSI) in Europe defined probiotic as "a viable microbial food supplement which beneficially influences the health of the host" [13]. In normal human intestines, approximately 400 different bacterial species exist, which is a more complex ecosystem, since it includes both facultative anaerobic and anaerobic microorganisms [14]. Generally, the gut microflora is constant but it is affected by numerous factors including age, diet, environment, medication, and stress. Lactobacillus and Bifidobacterium species constitute the majority of probiotic bacteria. Apart from this, several other bacteria and some yeasts are also considered as a probiotic since it has demonstrated some probiotic properties (Table 1). In humans and animals, Gram-positive, lactic acidproducing bacteria such as Lactobacilli and Bifidobacteria are the most common intestinal microflora [15].
Lactobacilli are non-spore-forming bacteria, rod-shape, strictly fermentative, anaerobic, aciduric, or acidophilic bacterial species and require complex nutrition for their growth. Generally, Lactobacilli are found in various habitats including carbohydrate-rich substrates (i.e., fermented milk), plant products, mucosal membranes of both humans and animals, rotten food items, and sewage [15].
Both human and animal intestinal microflora predominantly comprise of several Bifidobacterial species during their lifetime. Normally, the Bifidobacteria populations in the healthy adult colon are about 10 10 -10 11 CFU/g and thus will decrease with age. Most of the Bifidobacterial species are strictly anaerobic, nonmotile, and nonsporulating rods with varying morphological appearance [15].

Sources of Probiotics.
Fermented milk products are the major sources of probiotics including cultured buttermilk, yoghurt, and cheeses. Buttermilks are prepared by fermenting the milk with lactic acid-producing bacteria which is the reason for its sourness. In cheese industry, probiotic bacteria were used for the maturation of cheese. Kefir is one of the most popular fermented milk products. Generally, yoghurt was fermented by one or two bacterial species, whereas kefir was fermented by more than one probiotic bacterium. Other foods that are either produced by bacterial fermentation are Japanese miso, tempeh, sauerkraut, beer, sour dough, bread, chocolate, kimchi, olives, and pickles [16]. Due to the lower pH of fermented yogurt and milk, they are the major food vehicles for probiotics [17]. However, many studies show probiotics strains are also found in non-dairy-fermented substrates [12]. Some of the non-dairy probiotic products are cereal, legume, cabbage, maize, pearl millet, vegetable, sorghum, etc [18].

Health Benefits of Probiotics.
Probiotic bacteria have several health benefits in various medical conditions such as irritable bowel syndrome, diarrhea, Helicobacter pylori infections, inflammatory bowel disease, gastroenteritis, depressed immune function, infant allergies, cancer, hepatic diseases, hyperlipidemia, and others [19]. e overall health benefits of probiotic microorganism are represented in Figure 1.
e probiotic mechanisms that have been reported include effects on mucin secretion, receptor competition, and immunomodulation of gut-associated lymphoid tissue, decreased proinflammatory mediators, and increased immunosuppression [20]. Probiotic microbes exert beneficial effects through many actions. ese include production of antimicrobial substances, inhibition of adhesion and colonization of intestinal pathogens, promoting the local and peripheral immunity, detoxification, promoting the activity of brush border enzymes, microbial translocation prevention, and stimulating the production of secretory IgA. Generally, normal intestinal microflora has the ability to inhibit the colonization of harmful, enteric pathogens; this phenomenon was referred to as colonization resistance. is colonization resistance ability was attained by the complex interactions among the bacterial community [21]. e inhibitory chemical substance bacteriocin is defined as protein antibiotics of the type of colicin, i.e., substance characterized by lethal biosynthesis, killing activity of predominant intraspecies, and its adsorption to specific receptors on the surface of bacteriocin-sensitive cells [22].

Prebiotics
e term "prebiotic" was first used by Gibson and Roberfroid in 1995 [23]. Prebiotics are defined as "indigestible fermented food substrates that selectively stimulate the growth, composition, and activity of microflora in gastrointestinal tract and thus improve hosts' health and well-being" [24]. Lactulose was used 50 years ago as a prebiotic formula supplement to increase the numbers of Lactobacillus strains in infants' intestines [25].
ese prebiotics are resistance to gastric acidity and hydrolysis by mammalian enzymes. Also human gastrointestinal microflora can digest these prebiotics; hence, it promotes the growth of these probiotic microorganisms. Various new compounds which have gutresistant characteristics and selective fermentability by intestinal microorganisms have been identified and developed as prebiotics and include oligosaccharides (lactosucrose, isomaltooligosaccharides, xylooligosaccharides, and glucooligosaccharides), sugar alcohols, and polysaccharides (starch, modified starch, and resistant starch) [26][27][28].
Fermentation of oligofructose in the colon is due to the presence of intestinal microflora, which confers the beneficial effects to the humans via. increasing the probiotic populations in gastrointestinal microflora, increasing the calcium absorption and fecal weight, shortening the gastrointestinal transit time, and more importantly lowering the serum cholesterol level.

Sources of Prebiotics.
Generally, the prebiotics are abundant in foods including yacon, soybeans, oats, unrefined wheat, and barley, and inulin was abundant in Jerusalem artichoke, chicory root, and jicama. Apart from this, human breast milk also contains large quantities of oligosaccharides, which promote the health and immune system in infants. Breast-feeding infants contain normal microflora majorly composed of Lactobacilli and Bifidobacteria. ese microflora act as a primary defense layer against the invading pathogens and also promote the immune system [29,30]. e oligosaccharides present in the breast milk nurtured these beneficial microflora and hence are regarded as original and natural prebiotics. Recently, several peptides, proteins, and lipids are also considered as potential prebiotics. However, indigestible carbohydrates, particularly oligosaccharides, gained attraction among the researchers [31].

Synbiotics
Synbiotics are referred to as usage of both probiotics and prebiotics together. e combination of suitable probiotics and prebiotics drastically improves the activity of the probiotic microorganism. e example for synbiotics is a combination of a FOS and a Bifidobacterium strain or lactitol with Lactobacillus strains [23]. Administration of prebiotics and probiotics in combination may provide the synergistic effects and promotes the growth, survival, and adhesion of both probiotic and already existing beneficial bacteria in the colon.

Mechanism of Cholesterol Reduction
Numerous cholesterol lowering mechanisms by Lactobacillus strains have been proposed. e cholesterol-reducing effect of probiotic bacteria might be due to their ability to bind the cholesterol present in the small intestine. One of the most important mechanisms is through the bile salt hydrolase (BSH) enzyme. e BSH enzyme plays a major role in the deconjugation of bile salt in the enterohepatic circulation.
is enzyme hydrolyzes conjugated glycodeoxycholic acid and taurodeoxycholic acid and releases the glyco-and tauro-bile acids. BSH enzyme was identified in several lactic acid bacterial (LAB) species isolated from the gastrointestinal tracts of both humans and animals [32][33][34][35]. Many researchers proposed that BSH activity should be considered as a prerequisite characteristic during the selection of probiotic bacteria along with other cholesterol reducing properties [36]. Over the last few years, the possibility of using bile salt deconjugation by LAB species to treat hypercholesterolemia in humans has gained more attention [37,38]. e major source of cholesterol in human body includes biosynthesis and absorption by the liver and intestines, respectively. ese two attributes determine the overall cholesterol level. Many researchers have found that diverse pathways are involved in the mechanism, which governs the cholesterol-reducing properties of probiotics, but so far these mechanisms are not clearly understood. e overall cholesterol reduction mechanism of probiotic microorganism is represented in Figure 2.

Effects of Probiotics on Cholesterol Reduction.
In 1974, Mann and Spoerry first reported the serum cholesterol reduction by LAB and they witnessed the serum cholesterol level of Masai and Sambura tribes was reduced by 18% and their diet was primarily composed of fermented milk containing Lactobacillus [39] Anderson and Gilliland reported that Lactobacillus acidophilus reduces the blood cholesterol by two mechanisms including breakdown the cholesterol and bile salt deconjugation. ey also reported that the effects of consumption of yoghurt could reduce the serum cholesterol level significantly [40].
Anderson and Gilliland also examined the effect of milk fermented with L. acidophilus L1 on the serum cholesterol level in hypercholesterolemic subjects. Forty-eight volunteers whose serum cholesterol level ranged from 5.40 mmol/ L to 8.32 mmol/L were involved in the randomized, doubleblind, crossover, and placebo-controlled study for about ten weeks. Results showed that the consumption of 200 grams of L. acidophilus L1 containing yogurt after dinner significantly (P < 0.05) reduced the serum cholesterol level when compared to the placebo group [40]. Liong and Shah isolated 11 different Lactobacilli strains and screened for BSH activity and coprecipitation of cholesterol along with deconjugated bile salt. ey reported that Lactobacillus acidophilus strains had higher deconjugation potential compared with the L. casei and the cholesterol coprecipitation with deconjugated bile was increased at lower pH [41].
Similarly, Xiao et al. used thirty-two volunteers to examine the cholesterol-reducing capacity of a low-fat yoghurt comprising 10 8 CFU/g of Bifidobacterium longum BL1 strains. Results showed that the levels of serum total cholesterol, triglycerides, and LDL cholesterol significantly (P < 0.05) decreased after 4 weeks. Interestingly, HDL cholesterol level increased by 14.5% compared to that in the control patients who consumed yoghurt without Bifidobacterium longum BL1 [42]. e probiotic microorganisms isolated from fermented milk reduce the cholesterol in presence of 0.3% bile salt ranging from 28% to 83%. And some strains were able to reduce cholesterol more than 70% [43]. Kim and coworkers demonstrated the cholesterol reduction mechanism of Lactobacillus acidophilus ATCC 43121. ey have demonstrated the cholesterol reduction using the cell-free culture supernatant (CFCS) of L. acidophilus ATCC 43121 [44].
In another study, the cholesterol reduction of Lactobacillus reuteri CRL 1098 was demonstrated using the mice fed with the 10 4 cells/day probiotic cells for 7 days. Results showed the 38% reduction in total cholesterol and among them, 40% were triglycerides. Surprisingly, administration of L. reuteri CRL 1098 increased the high-density lipoprotein by 20% [45].

Effects of Prebiotics on Cholesterol Reduction.
Prebiotics or their cofactors act as good alternative for probiotics. ese prebiotics are defined as nondigestible or low-digestible food components which would selectively stimulate the growth and activity of probiotic microorganisms present in the colon of host organism [31,46,47]. Brighenti  Lower the toxigenic/ mutagenic reaction in gut Improve lactose digestion Cholesterol reduction Anticarcinogenic activity healthy persons (male) in which the researchers found that the daily consumption of rice-based ready-to-eat cereal (50 g) comprising 18% inulin significantly (P < 0.05) decreased total cholesterol and triacylglycerol present in the plasma by 7.9% (±5.4) and 21.2% (±7.8), respectively, when compared to control [48]. Fernandez et al. used male Hartley guinea pigs (300-400 g body weight) to study the effect of resistant starch in the plasma cholesterol reduction. is study reported that the administration of 10 g/100 g of resistant starch (obtained from the Meer Corporation) to sixteen male Hartley guinea pigs for about four weeks resulted in the significant reduction (P < 0.001) of plasma cholesterol by 27.4% and LDL cholesterol concentration by 28.0% when compared with the control group [49].
Causey et al. performed a randomized, double-blind, and crossover study using twelve hypercholesterolemic men patients to evaluate the inulin (extracted from chicory roots) effects on blood cholesterol levels of patients. Twelve men were randomly assigned to two different groups, viz., the control group (consumed one pint of vanilla ice cream without inulin) and the inulin group (consumed one pint of vanilla ice cream containing 20 g of inulin). As a result of a 3week study, the researchers found that the daily ingestion of 20 g of inulin significantly (P < 0.05) decreased triglycerides present in the serum [50].
Mortensen et al. reported that when forty male mice were administered with a purified diet containing 10% of long-chained fructan for 16 weeks, the fructan present in the diet significantly decreased the concentration of blood cholesterol by 29.7% (P < 0.001), LDL cholesterol concentration by 25.9% (P < 0.01), VLDL cholesterol concentration by 37.3% (P < 0.05), and the level of IDL cholesterol by 39.4% (P < 0.001) compared to the control group [51].
Favier et al. conducted a 21-day trial study in ten male Wistar rats (body weight of 150 g) to evaluate the hypocholesterolemic effects of β-cyclodextrin in a randomized, placebo-controlled, and parallel design trial. e researchers reported that the daily consumption of β-cyclodextrin (25 g/ kg) significantly (P < 0.05) reduced the level of cholesterol and triacylglycerols present in the plasma by 25.9% and 35.0%, respectively, compared to the control group [52]. Re-entered into liver

Conclusion and Future Prospects
Probiotics, prebiotics, and their combinations have been found to be clinically effective for a large number of disorders which include hypercholesterolemia. e use of any probiotic and prebiotic substances for the enrichment of fermented products provides its delivery into the human gastrointestinal tract and thus stimulates beneficial health effects. Several probiotic-mediated cholesterol removal mechanisms have been suggested, including cholesterol assimilation by growing cells, cholesterol binding to the cell surface, incorporation of cholesterol into the cellular membrane, bile salt deconjugation via bile salt hydrolase, and cholesterol coprecipitation with deconjugated bile. But the exact mechanisms remain unclear and controversial. Much work has already been accomplished to help us to understand probiotics and the manner in which they function. Nevertheless, some concerns like viability and dosage of probiotic strains, safety aspects, and industrial standardization are essential to be studied in detail. In recent times, cholesterol reduction, cancer prevention, and immunology are the major research subjects on probiotics and prebiotics. Genetic engineering and other similar methods are being used to increase the beneficial effects of probiotic strains. Supplementation of promising probiotic strains may indulge better solution to reduce the high cholesterol level issue in human beings. However, broad research is essential for the screening of potent probiotic strains and to evaluate the effective management of good and bad cholesterol present in the body and the sustainability of the desired results obtained by the probiotic strains. e combination of probiotics and prebiotics significantly reduces the serum cholesterol level and that can be used as an alternative remedy for hypercholesterolemic problems without any side effect to the consumers.

Conflicts of Interest
e authors declare that they have no conflicts of interest.