Hypocholesterolemic effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rat

Yuanhong Xie Hongxing Zhang Hui Liu Lixia Xiong Xiuzhi Gao Hui Jia Zhengxing Lian Nengsheng Tong Tao Han About the authors

Abstract

To investigate the effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rats, female Wistar rats were fed a high-cholesterol diet (HCD) for 28 d to generate hyperlipidemic models. Hyperlipidemic rats were assigned to four groups, which were individually treated with three different dosages of K. marxianus M3+HCD or physiological saline+HCD via oral gavage for 28 d. The total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels in the serum and liver of the rats were measured using commercially available enzyme kits. In addition, the liver morphology was also examined using hematoxylin and eosin staining and optical microscopy. According to our results, the serum and liver TC, TG, LDL-C levels and atherogenic index (AI) were significantly decreased in rats orally administered K. marxianus M3 (p <0.01), and the HDL-C levels and anti atherogenic index (AAI) were significantly increased (p <0.01) compared to the control group. Moreover, K. marxianus M3 treatment also reduced the build-up of lipid droplets in the liver and exhibited normal hepatocytes, suggesting a protective effect of K. marxianus M3 in hyperlipidemic rats.

Kluyveromyces marxianus ; hypercholesterolemia; high-cholesterol diet


Introduction

Hypercholesterolemia is considered to be a risk factor of cardiovascular disease and is the leading cause of morbidity and mortality in many countries (Law et al., 1994Law MR, Wald NJ, Wu T et al. (1994) Systematic underestimation of association between serum cholesterol concentration and ischaemic heart disease in observational studies: Data from the BUPA study. BMJ 308:363–366.). Elevated serum cholesterol levels are widely recognized as a contributing risk factor for the development of cardiovascular diseases, such as atherosclerosis, coronary heart disease and stroke. It has been reported that a 1% reduction in serum cholesterol could reduce the risk of coronary heart disease by 2 to 3% (Manson et al., 1992Manson JE, Tosteson H, Ridker PM et al. (1992) The primary prevention of myocardial infarction. N Engl J Med 326:1406–1416.). The decrease in cholesterol levels could be achieved by appropriate food intake, such as low-cholesterol, low-fat diets (Lora et al., 2007Lora KR, Morse KL, Gonzalez-Kruger GE et al. (2007) High saturated fat and cholesterol intakes and abnormal plasma lipid concentrations observed in a group of 4- to 8-year-old children of Latino immigrants in rural Nebraska. Nutr Res 27:483–491.), dietary fiber (Jiménez et al., 2008Jiménez JP, Serrano J, Tabernero M et al. (2008) Effects of grape antioxidant dietary fiber in cardiovascular disease risk factors. Nutrition 24:646–53.; Theuwissen et al., 2008Theuwissen E, Mensink RP (2008) Water-soluble dietary fibers and cardiovascular disease. Physiol Behav 94:285–292.), and yogurts containing specific probiotics (Akalin et al., 1997Akalin AS, Gönç S, Düzel S (1997) Influence of yogurt and acidophilus yogurt on serum cholesterol levels in mice. J Dairy Sci 80:2721–2725.; Danielson et al., 1989Danielson AD, Peo ERJ, Shahani KM et al. (1989) Anticholesterolemic property of Lactobacillus acidophilus yoghurt fed to mature boars. J Anim Sci 67:966–974.).

Recently, some studies have demonstrated that the hypocholesterolemic effects of probiotics have resulted in an increased interest in this treatment modality, which is less expensive and may be considered a "natural health remedy." Several studies evaluating this effect have found that some lactobacilli or bifidobacteria can exhibit hypocholesterolemic properties in animal models (Fukushima and Nakano 1996Fukushima M, Nakano M (1996) Effects of a mixture of organisms, Lactobacillus acidophilus or Streptococcus faecalis on cholesterol metabolism in rats fed on a fat- and cholesterol-enriched diet. Br J Nutr 76:857–867.; Gilliland et al., 1989; Nguyen et al., 2007Nguyen TD, Kang JH, Lee MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol 113:358–361.; Kumar et al., 2011Kumar R, Grover S, Batish VK (2011) Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague-Dawley rats. Brit J Nutr 105:561–573.) and humans (Agerbaek et al., 1995Agerbaek M, Gerdes LU, Richelsen B (1995) Hypocholesterolaemic effect of a new fermented milk product in healthy middle-aged men. Eur J Clin Nutr 49:346–352.; Anderson and Gilliland 1999Anderson JW, Gilliland SE (1999) Effect of fermented milk (yogurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans. J Am Coll Nutr 18:43–50.; Xiao et al., 2003Xiao JZ, Kondo S, Takahashi N et al. (2003) Effects of milk products fermented by Bifidobacterium longum on blood lipids in rats and healthy adult male volunteers. J Dairy Sci 86:2452–2461.). However, the hypocholesterolemic mechanism of lactic acid bacteria is still no clearly understood, although the bacteria appear to contribute to increased fecal excretion of bile acids and thereby improved overall hepatic cholesterol homeostasis (Jeun et al., 2010Jeun J, Kim S, Cho SY et al. (2010) Hypocholesterolemic effects of Lactobacillus plantarum KCTC3928 by increased bile acid excretion in C57BL/6 mice. Nutrition 26:321–330.). Moreover, some reports have failed to show hypocholesterolemic effects of probiotics (Hatakka et al., 2008Hatakka K, Mutanen M, Holma R et al. (2008) Lactobacillus rhamnosus LC705 together with Propionibacterium freudenreichii ssp shermanii JS administered in capsules is ineffective in lowering serum lipids. J Am Coll Nutr 27:441–447.; Simons et al., 2006Simons LA, Amansec SG, Conway P (2006) Effect of Lactobacillus fermentum on serum lipids in subjects with elevated serum cholesterol. Nutr Metab Cardiovasc Dis 16:531–535.). Thus, additional studies are required to strengthen the proposed hypotheses and to improve our understanding of how bacteria affect cholesterol metabolism, which might result in the more appropriate use of probiotics.

Kefir has been widely recommended in western countries for consumption by healthy people to lower the risk of chronic diseases and has also been provided to some patients for the clinical treatment of a number of gastrointestinal and metabolic diseases, hypertension, and allergy (St-Onge et al., 2002St-Onge MP, Farnworth ER, Savard T et al. (2002) Kefir consumption does not alter plasma lipid levels or cholesterol fractional synthesis rates relative to milk in hyperlipidemic men: a randomized controlled trial. BMC Complement Altern Med 2:1–7.). Yogurt prepared from Tibetan mushrooms and milk has an extraordinary taste and provides excellent nutrition. Tibetan kefir has a granular structure due to the presence of symbiotic microorganisms, such as Lactobacillus and yeast (Simova et al., 2002Simova E, Beshkova D, Angelov A et al. (2002) Lactic acid bacteria and yeasts in kefir grains and kefir made from them. J Ind Microbiol Biotechnol 28:1–6.). In addition, kefir culture was reported to exhibit the ability to assimilate cholesterol in milk (Vujicic et al., 1992Vujicic IF, Vulic M, Konyves T (1992) Assimilation of cholesterol in milk by kefir cultures. Biotechnol Lett 14:847–850.). Furthermore, Liu et al. (2006)Liu JR, Wang SY, Chen MJ et al. (2006) Hypocholesterolaemic effects of milk-kefir and soyamilk-kefir in cholesterol-fed hamsters. Br J Nutr 95:939–946. demonstrated the hypocholesterolemic effect of kefir milk in male hamsters fed with a cholesterol-enriched diet. However, St-Onge et al. (2002)St-Onge MP, Farnworth ER, Savard T et al. (2002) Kefir consumption does not alter plasma lipid levels or cholesterol fractional synthesis rates relative to milk in hyperlipidemic men: a randomized controlled trial. BMC Complement Altern Med 2:1–7. obtained a conflicting result and reported that kefir consumption did not result in the lowering of plasma lipid concentrations, although kefir resulted in increasing fecal isobutyric, isovaleric, and propionic acids as well as the total amount of fecal short chain fatty acids.

Moreover, some researchers have found that kefir-fermented milk can decrease plasma cholesterol levels and can promote cancer resistance. Furthermore, it has antioxidant properties, including a role in immune regulation, and can help to protect against pathogenic bacteria and spoilage organisms, as well as assist in the conservation of predominant gastrointestinal probiotic flora (Abd El-Gawad et al., 2005Abd El-Gawad IA, El-Sayed E, Hafez S et al. (2005) The hypocholesterolaemic effect of milk yoghurt and soy-yoghurt containing bifidobacteria in rats fed on a cholesterol-enriched diet. Int Dairy J 15:37–44.; Mathara et al., 2008Mathara JM, Schillinger U, Guigas C et al. (2008) Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya. Int J Food Microbiol 126:57–64.; Nguyen et al., 2007Nguyen TD, Kang JH, Lee MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol 113:358–361.; Akalin et al., 1997Akalin AS, Gönç S, Düzel S (1997) Influence of yogurt and acidophilus yogurt on serum cholesterol levels in mice. J Dairy Sci 80:2721–2725.). The objective of this study was to evaluate the effects of Kluyveromyces marxianus M3 yeast isolated from Tibetan mushrooms on lowering cholesterol in rats.

Materials and Methods

Microbial cultures

K. marxianus M3 was isolated from Tibetan mushrooms and was cultured by a resident of Baicheng, Jilin province, China (Liu et al., 2005Liu H, Dong R, Pan C et al. (2005) Optimization of fermentation conditions of Kluyveromyces marxianus bile salt hydrolase. Food Science 26:97–100 (In Chinese).). M3 strains (1–2%) were inoculated into 10 mL potato lactose liquid medium and grown at 28 °C for 24. The culture was centrifuged and diluted with 0.9% saline water to obtain a preparation of 2.0 × 107 cfu/mL.

Animals, diets and experimental design

Forty female Wistar rats (aged 3 weeks) with a weight of 140 ± 10 g were obtained from the Academy of Military Medical Sciences (Beijing, China). All rats were individually housed at a constant temperature and humidity (18–24 °C, 60%) with a 12 h light/dark cycle. After 1 week of acclimatization, all of the rats were fed a high-cholesterol diet (78.8% basic diet, 1% cholesterol, 10% egg yolk, 10% lard, 0.2% cholate, w/w) for 28 d. In addition, the rats were randomly assigned to four groups (n = 10), respectively. Group (NM): normal rats fed a standard high-cholesterol diet and physiological saline (5 mL/kg); Group α (LD): normal rats fed a standard high-cholesterol diet and K. marxianus M3 (5 mL/kg); Group β (MD): normal rats fed a standard high-cholesterol diet and K. marxianus M3 (10 mL/kg); Group IV (HD): normal rats fed a standard high-cholesterol diet and K. marxianus M3 (20 mL/kg).

The rats were intragastrically administered for 28 d, and food and water consumption and body weight were recorded daily. At the end of the feeding period, all rats were anesthetized by isoflurane and sacrificed by cervical dislocation. The kidney, heart and liver were immediately excised, and the serum was separated from the blood. The liver, heart and kidney were excised, rinsed in ice-cold physiological saline, weighed, and then stored at −20 °C.

Serum lipid analysis

The samples were allowed to stand for 10 min and then centrifuged at 3500 r/min for 15 min, where the sediment was subsequently discarded. The TC (total cholesterol), TG (triglyceride), HDL-C (high density lipoprotein-cholesterol), and LDL-C (low density lipoprotein-cholesterol) levels were analyzed using kits (Bio-technology and Science Incorporation) and a fully automatic biochemical analyzer (Hitachi, Japan). The atherosclerosis index (AI) was calculated as follows: AI = (total cholesterol - HDL cholesterol)/HDL cholesterol.

Liver lipid analysis

Isolated livers were weighed after rinsing with phosphate-buffered saline and blotted dry with filter paper. Each liver was homogenized in 20 volumes of extraction solution (chloroform: methanol = 2:1; v/v) and agitated for 60 min at room temperature (Zhao et al., 2012Zhao X, Higashikawa F, Noda M et al. (2012) The obesity and fatty liver are reduced by plant-derived Pediococcus pentosaceus LP28 in high fat diet-induced obese mice. PLoS One 7: e30696.). Liver cholesterol and triacylglycerols were measured using the kits previously described.

Morphology of liver

Fresh livers of rats were fixed with 4% paraformaldehyde for 24 h, gradually dehydrated in a graded series of ethanol, clarified in xylene, and embedded in paraffin wax. The hematoxylin and eosin stained livers were observed using an optical microscope (Wang et al., 2013Wang LX, Liu K, Gao DW et al. (2013) Protective effects of two Lactobacillus plantarum strains in hyperlipidemic mice. World J Gastroenterol 19:3150–3156.).

Statistical analysis

All data were expressed as the mean ± SD. Statistical analysis was performed using SPSS 13.0 software. Differences between the groups were analyzed by One-Way ANOVA followed by Duncan's multiple range tests. Statistical significance was considered at p <0.01.

Results

Effect on plasma lipid profiles

The effects of K. marxianus M3 live yeast supplementation on the serum lipid levels of rats are presented in Table 1. The rats subjected to a high cholesterol diet or high cholesterol diet with K. marxianus M3 had no obvious difference in body weight (BW) during the entire 7 weeks of experiments. And high cholesterol diet dramatically increased the serum TC, TG and LDL-C levels of rats in NM group, which demonstrated the hyperlipidemic model was set up successfully. In addition, oral administration of K. marxianus M3 for 7 weeks significantly decreased (p <0.01) the serum TC, TG and LDL-C levels of rats compared with the NM groups. In contrast, the serum HDL-C levels in the K. marxianus M3 supplemented rats significantly increased (p <0.01, p <0.05) compared to that in NM group after 7 weeks of administration.

Table 1
Effect of Kluyveromyces marxianus M3 from Tibetan Kefir on plasma lipid profiles of rats ( ± s, n = 6).

Moreover, oral administration of various dosages of K. marxianus M3 showed different degrees of changes in serum lipid. The serum TC, TG, and LDL-C levels were the most reduced in the LD group, decreasing by 44.33%, 39.21% and 60.12%, respectively, whereas the serum HDL-C level improved by 44.18% compared to the NM group. In the MD group, the serum TC, TG, and LDL-C levels decreased by 51.01%, 29.41% and 44.3%, respectively, whereas the HDL-C level improved by 16.27%. Moreover, the serum levels of TC, TG and LDL-C in the HD group were decreased by 35.82%, 35.29% and 31.64%, respectively, and the HDL-C level increased by 4.65% (Table 1). As a result, oral administration of K. marxianus M3 in rats significantly decreased (p <0.01) the serum TC, TG, LDL-C levels and atherogenic index (AI), and significantly increased (p <0.01) the serum HDL-C levels and anti-atherogenic index (AAI) compared to the NM group.

Effect on Liver lipid profiles

After 7 weeks of treatment, the liver lipid levels of rats were also examined. As shown in Table 2, oral administration of K. marxianus M3 for 7 weeks significantly decreased (p <0.01) liver TC, TG, LDL-C levels and the AI compared with the NM groups. In addition, the liver HDL-C levels and AII of the K. marxianus M3 treatment group were significantly higher (p <0.01) compared to the NM group. Moreover, oral administration of K. marxianus M3 at various dosages showed different degrees of decreases in liver TC, TG, LDL-C levels and increases in liver HDL-C levels. After 7 weeks of administration, the liver TG, TC, LDL-C levels in the MD group significantly decreased (p <0.01) by 36.00%, 22.92% and 52.94% compared with the NM group. Furthermore, the liver HDL-C contents in the LD group rats reached 0.31 ± 0.10 mmol/L, which was 72.22% higher than that in the NM group. As a result, K. marxianus M3 treatment in the LD group significantly decreased (p <0.01) the liver AI and increased (p <0.01) the liver AII compared with the NM group.

Table 2
Effect of Kluyveromyces marxianus from Tibetan Kefir on hepatic lipid profiles of rats ( ± s, n = 10).

Effects on viscera organs

As shown in Table 3, a high cholesterol diet increased the heart, liver and kidney weight of the rats. After 7 weeks of administration, the viscera weight (heart, liver and kidney weight) and viscera coefficients in the rats of the LD group were significantly lower (p <0.01) compare to the NM group. The heart weight, heart coefficient, liver weight and liver coefficient in the MD and HD groups were also significantly decreased (p <0.01) compared to the NM group. Moreover, the kidney weight and kidney coefficient in the MD and HD groups were significantly decreased (p <0.05) compared to the NM group.

Table 3
Effect of Kluyveromyces marxianus from Tibetan Kefir on visceral weight and visceral coefficient of rats ( ± s, n = 10).

We further examined the hepatic morphology in rats. As shown in Figure 1a, in the NM group rats, the structure of the hepatic lobule had disappeared, and the liver cell morphology was irregular. There were different degrees of edema, focal necrosis, and fatty degeneration of liver cells. Moreover, the liver cells exhibited massive fatty changes and severe steatosis with cytoplasmic vacuoles, and the infiltration of inflammatory cells were visible. Taken together, these conditions suggested damage due to a high-cholesterol diet on the hepatic cells. In contrast, the size of the lipid droplets in the LD group was remarkably smaller than those in the NM group (Figure 1b), and the hepatic cells exhibited normal histology. In addition, the lipid droplets in the MD and HD groups were also reduced in varying degrees (Figure 1c, d). Taken together, our results indicated that K. marxianus M3 treatment reduced the build-up of lipid droplets and maintained normal hepatocytes.

Figure 1
Histology of liver steatosis in rats. A: high-cholesterol diet group; B: high-cholesterol diet+K. marxianus M3 (5 mL/kg); C: high-cholesterol diet+K. marxianus M3 (10 mL/kg); D: high-cholesterol diet+K. marxianus M3 (20 mL/kg). All the photomicrographs show HE staining (original magnification × 100).

Discussion

Recently, considerable attention has focused on the potential of probiotics in altering lipid metabolism. This interest stems from growing evidence that probiotics reduce the concentration of cholesterol in vivo (Mohan et al., 1995Mohan B, Kadirvel R, Bhaskaran M et al. (1995) Effect of probiotic supplementation on serum/yolk cholesterol and on egg shell thickness in layers. Br Poult Sci 36:799–803.; Abdulrahim et al., 1996Abdulrahim SM, Haddadin SY, Hashlamoun EA et al. (1996) The influence of Lactobacillus acidophilus and bacitracin on layer performance of chickens and cholesterol content of plasma and egg yolk. Br Poult Sci 37:341–346.; Panda et al., 2003Panda AK, Reddy MR, Rama Rao SV et al. (2003) Production performance, serum/yolk cholesterol and immune competence of white leghorn layers as influenced by dietary supplementation with probiotic. Trop Anim Health Prod 35:85–94.; Nguyen et al., 2007Nguyen TD, Kang JH, Lee MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol 113:358–361.; Wang et al., 2009Wang Y, Xu N, Xi A et al. (2009) Effects of Lactobacillus plantarum MA2 isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol diet. Appl Microbiol Biotechnol 84:341–347.; Alkhalf et al., 2010Alkhalf A, Alhaj M, Al-Homidan I (2010) Influence of probiotic supplementation on blood parameters and growth performance in broiler chickens. Saudi J Biol Sci 17:219–25.; Wang et al., 2013Wang LX, Liu K, Gao DW et al. (2013) Protective effects of two Lactobacillus plantarum strains in hyperlipidemic mice. World J Gastroenterol 19:3150–3156.). Generally, a high-cholesterol diet can increase body weight (Xie et al., 2011Xie N, Cui Y, Yin YN et al. (2011) Effects of two Lactobacillus strains on lipid metabolism and intestinal microflora in rats fed a high-cholesterol diet. BMC Complement Altern Med 11:53.). According to our results, addition of K. marxianus M3 live yeast with high-cholesterol diet did not significantly change the body weight of rats. However, K. marxianus M3 treatment for 7 weeks significantly decreased (p <0.01) the serum TC, TG and LDL-C levels in rats. In particular, these effects were more evident in the LD group (TC, TG and LDL-C reduced by 51.01%, 39.22% and 60.13%, respectively) (Table 1). Our results indicated that there was a relationship between the formation and reduction of the metabolism of cholesterol in the serum. Similar results were reported for the cholesterol-reducing activity of yeast (Yalçin et al., 2008Yalçin S, Erol H, Ozsoy B et al. (2008) Effects of the usage of dried brewing yeast in the diets on the performance, egg traits and blood parameters in quails. Animal 2:1780–1785.; Yalçin et al., 2009Yalçin S, Oguz F, Güçlü B et al. (2009) Effects of dietary dried baker's yeast on the performance, egg traits and blood parameters in laying quails. Trop Anim Health Prod 41:5–10.), Lactobacillus (Nielson and Gilliland, 1985Gilliland SE, Nelson CR, Maxwell C (1985) Assimilation of cholesterol by Lactobacillus acidophilus. Appl Environ Microb 49:377–381., Gilliland et al., 1985Gilliland SE, Nelson CR, Maxwell C (1985) Assimilation of cholesterol by Lactobacillus acidophilus. Appl Environ Microb 49:377–381.; Hashimoto et al., 1999Hashimoto H, Yamazaki K, He F et al. (1999) Hypocholesterolemic effects of Lactobacillus casei subsp. casei TMC 0409 strain observed in rats fed cholesterol contained diets. Anim Sci J 72:90–97.; Simons et al., 2006Simons LA, Amansec SG, Conway P (2006) Effect of Lactobacillus fermentum on serum lipids in subjects with elevated serum cholesterol. Nutr Metab Cardiovasc Dis 16:531–535.; Nguyen et al., 2007Nguyen TD, Kang JH, Lee MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol 113:358–361.; Xie et al., 2011Xie N, Cui Y, Yin YN et al. (2011) Effects of two Lactobacillus strains on lipid metabolism and intestinal microflora in rats fed a high-cholesterol diet. BMC Complement Altern Med 11:53.; Wang et al., 2013Wang LX, Liu K, Gao DW et al. (2013) Protective effects of two Lactobacillus plantarum strains in hyperlipidemic mice. World J Gastroenterol 19:3150–3156.) and Bacillus (Fukushima and Nakao, 1995Fukushima M, Nakano M (1995) The effect of a probiotic on faecal and liver lipid classes in rats. Br J Nutr 73:701–710.).

High concentrations of TC and LDL-C are strongly associated with an increased risk of coronary heart disease. A reduction in TC and LDL-C in a hypercholesterolemic individual can reduce the incidence of cardiovascular disease (Probstfield and Rifkind 1991Probstfield JL, Rifkind BM (1991) The lipid research clinics coronary primary prevention trial: Design, results, and implications. Eur J Clin Pharmacol 40:S69–S75.). Moreover, elevated levels of oxidized LDL-C are associated with artherosclerotic plaque formation on the artery walls, but increased HDL-C levels may reduce the risk due to the ability of HDL to transport cholesterol back to the liver for excretion or to other tissues of cardiovascular disease (Lewis et al., 2005Lewis GF, Rader DJ (2005) New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ Res 96:1221–1232.). According to our results, K. marxianus M3 supplementation dramatically increased the serum HDL-C level (p <0.01, p <0.05) in rats (Table 1). As a result, the AI of the K. marxianus M3 treatment groups was significantly decreased (p <0.01) compared to the NM group. Thus, we confirmed that K. marxianus M3 exerted a hypolipidemic effect and could alleviate lipid related metabolic syndrome. Similar results were reported by Hashimoto et al. (1999)Hashimoto H, Yamazaki K, He F et al. (1999) Hypocholesterolemic effects of Lactobacillus casei subsp. casei TMC 0409 strain observed in rats fed cholesterol contained diets. Anim Sci J 72:90–97., in which a diet containing L. casei TMC 0409 increased the concentration of HDL-C in blood, which was consistent with other studies (Akalin et al., 1997Akalin AS, Gönç S, Düzel S (1997) Influence of yogurt and acidophilus yogurt on serum cholesterol levels in mice. J Dairy Sci 80:2721–2725.; Danielson et al., 1989Danielson AD, Peo ERJ, Shahani KM et al. (1989) Anticholesterolemic property of Lactobacillus acidophilus yoghurt fed to mature boars. J Anim Sci 67:966–974.; De Smet et al., 1998De Smet I, De Boever P, Versteaete W (1998) Cholesterol lowering in pigs through enhanced bacterial bile salt hydrolase activity. Br J Nutr 79:185–194.). However, conflicting results were reported (Chiu et al., 2006Chiu CH, Lu TY, TsengYY et al. (2006) The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high cholesterol diet. Appl Microbiol Biotechnol 71:238–245.; St-Onge et al., 2002St-Onge MP, Farnworth ER, Savard T et al. (2002) Kefir consumption does not alter plasma lipid levels or cholesterol fractional synthesis rates relative to milk in hyperlipidemic men: a randomized controlled trial. BMC Complement Altern Med 2:1–7.; Keim et al., 1981, Rossouw et al., 1981; De Roos et al., 1998De Roos NM, Schouten G, Katan MB (1998) Yoghurt enriched with Lactobacillus acidophilus does not lower blood lipids in healthy men and women with normal to borderline high serum cholesterol levels. Eur J Clin Nutr 53:277–280.) in humans and animals.

In general, cholesterol is indispensable to the human body, and its levels are subjected to complex regulation. Cholesterol is modified into oxysterols, including 22- and 24-hydroxy cholesterol, when excess cholesterol is deposited in hepatic cells (Satoshi Hirako et al., 2011Satoshi H, Hyoun-Ju K, Saya S et al. (2011) Low-Dose fish oil consumption prevents hepatic lipid accumulation in high cholesterol diet fed mice. J Agric Food Chem 59:13353–13359.). As expected, we demonstrated that the high-cholesterol diet increased hepatic TC, TG and LDL-C levels in rats (Table 2). Rats supplemented with K. marxianus M3 displayed significant reductions in hepatic TC, TG and LDL-C levels. These findings demonstrated that the serum cholesterol and TG levels in K. marxianus M3-treated rats were reduced, rather than merely being redistributed from the blood to the liver. Moreover, our results were consistent with previous reports (Kumar et al., 2011Kumar R, Grover S, Batish VK (2011) Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague-Dawley rats. Brit J Nutr 105:561–573.; Chiu et al., 2006Chiu CH, Lu TY, TsengYY et al. (2006) The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high cholesterol diet. Appl Microbiol Biotechnol 71:238–245.).

In this study, a high-cholesterol diet promoted the visceral weight (heart, liver and renal) in rats (Table 3). In addition, oral administration of K. marxianus M3 significantly reduced the visceral weights and visceral coefficients, suggesting a protection of K. marxianus M3 to the visceral organs under a high-cholesterol diet. Moreover, the histology of liver steatosis also supported this result. High-cholesterol diet caused different degrees of edema, focal necrosis, and fatty degeneration of liver cells (Figure 1a). In contrast, the K. marxianus M3 treatment could reduce the build-up of lipid droplets and maintained normal hepatocytes (Figure 1b, c). The result of liver tectology proved that the K. marxianus M3 had important potential in alleviating hepatic steatosis attributed to mediation of lipid metabolism and had protective effects on hepatic structure. Similar results have also been reported (Wang et al., 2013Wang LX, Liu K, Gao DW et al. (2013) Protective effects of two Lactobacillus plantarum strains in hyperlipidemic mice. World J Gastroenterol 19:3150–3156.; Xie et al., 2011Xie N, Cui Y, Yin YN et al. (2011) Effects of two Lactobacillus strains on lipid metabolism and intestinal microflora in rats fed a high-cholesterol diet. BMC Complement Altern Med 11:53.).

In recent yeas, several hypotheses have been proposed to explain the hypocholesterolemic effects of the probiotic strains: (1) consumption or absorption of cholesterol by probiotic strains (Pigeon et al., 2002Pigeon RM, Cuesta EP, Gililliand SE (2002) Binding of free bile acids by cells of yogurt starter culture bacteria. J Dairy Sci 85:2705–2710.; Liong and Shah 2005Liong MT, Shah NP (2005) Bile salt deconjugation and BSH activity of five bifidobacterial strains and their cholesterol co-precipitating properties. Food Res Int 38:135–142.); (2) the cholesterol is converted into coprostanol by cholesterol reductase, which is produced by probiotic strains (Lye et al., 2010); (3) some probiotic strains excrete bile salt hydrolase, leading to increased bile excretion in feces (Begley et al., 2010), etc. There are some reports on bile salt hydrolase in different species of Lactobacillus, Enterococcus, Peptostreptococcus, Bifidobacterium, Clostridium, and Bacteroides (Liong and Shah 2005Liong MT, Shah NP (2005) Bile salt deconjugation and BSH activity of five bifidobacterial strains and their cholesterol co-precipitating properties. Food Res Int 38:135–142.; Begley et al., 2010). In our previous research, we have cloned the bile salt hydrolase (bsh) gene in K. marxianus (Genebank Acession: JQ247427.1). So we proposed that the hypocholesterolemic effects of K. marxianus M3 might cased by the activity of bile salt hydrolase. And further research could be conduct in this field.

In conclusion, our results suggested that K. marxianus M3 is a safe probiotic with the potential to reduce serum cholesterol and triglyceride levels. Thus, further studies are required to determine the mechanism underlying the cholesterol-lowering effect. It will also be necessary to test more animals, utilizing varying doses of K. marxianus M3 over longer time periods, to assess the long-term probiotic potential of K. marxianus M3.

Acknowledgments

This work was supported financially by The Comprehensive Reforming Project to promote talents training of BUA, Disease Resistance Transgenic Sheep and Breeding of New Breeds (2013ZX08008-005) and the Key Construction Discipline Program of Beijing Municipal Commission of Education (PXM2014-014207-0000029).

References

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  • Abd El-Gawad IA, El-Sayed E, Hafez S et al. (2005) The hypocholesterolaemic effect of milk yoghurt and soy-yoghurt containing bifidobacteria in rats fed on a cholesterol-enriched diet. Int Dairy J 15:37–44.
  • Abdulrahim SM, Haddadin SY, Hashlamoun EA et al. (1996) The influence of Lactobacillus acidophilus and bacitracin on layer performance of chickens and cholesterol content of plasma and egg yolk. Br Poult Sci 37:341–346.
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Publication Dates

  • Publication in this collection
    Apr-Jun 2015

History

  • Received
    03 Dec 2013
  • Accepted
    14 Sept 2014
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br