Effect of some probiotic Lactobacillus spp. on serum lipids of rats

INTRODUCTION

The genus Lactobacillus represent one of the major members of the lactic acid bacteria. Also, during the last three decades Lactobacillus is considered one of most commonly bacteria used as probiotic. However, probiotics are live microorganisms, which when administered in adequate amounts, confer a health benefit on the host (Sanders et al., 2003). Recently, there has been increasing interest in the use of probiotics to prevent, to alleviate or to treat a variety of infectious and inflammatory conditions. These bacteria, may have several therapeutic functions, including antimicrobial activity, ability to assimilate cholesterol, improved lactose utilization and anti-carcinogenic activity (Chou and Weimer, 1999). Therefore, probiotics should meet criteria as identified by FAO/WHO such as safe for consumption and surviving exposure to low pH and important criteria is the ability to confer a health benefit on a host. Therefore, the main target of the present work is to screen three tested strains of Lactobacillus spp. for functional characteristics for probiotics. An additional aim is to evaluate in vivo the impact of feeding rats on fermented milk cultured with different Lactobacillus spp. strains on serum lipids of rats and fecal microflora.

MATERIALS AND METHODS

Materials

Tested strains

Three identified Lactobacillus cultures were used in the present study, namly L. casei AZ1, and L. rhamnosis AZ1 isolated from feces of breast-fed infant, while L. gasseri AZ1 isolated from raw milk.

Bile acid

Bile salt was obtained from Difco Laboratories, Detroit, Michigan, USA.

Pepsin and pancreatin

Pepsin from Procine stomach mucosa and Pancreatin from porcine pancreas were delivered from Sigma Chemical Co. Missouri 63103, USA.

Albino rats

Twenty-five mature male albino rates, obtained from the Osman farm animal experiments, with mean body weights of 130 ± 5 gm, were used in the present study.

Basel diet

The chemical composition of basal or control diet was as follows: raw protein, 23%; raw fat, 6.40%; fibers, 3.60% and starch, 67%.

Buffalo's milk

The chemical composition of buffalo's milk obtained from the herd of Faculty of Agriculture, Al-Azhar Univ. Cairo, was as follows: fat, 6.1 %; lactose, 4.3%; protein, 4.1%; solids not fat, 8.9% and salts, 0.5%.

Blood samples

At the beginning of this experiment and after adaptation period, blood samples were drawn from the retrobulber venous plexus of each rate through a capillary glass tube and left to clot at room temperature to obtain a clear serum. At the end of experiment, the rates were killed by withdrawing blood from the abdominal aorta under light diethyl ether anesthesia.

Faeces samples:

Rate faeces samples were collected every week in sterile Petri dishes transferred to laboratory in ice box and immediately subjected to microbiological analysis.

Media

For propagation and maintenance of Lactobacillus strains MRS broth / medium was used as recommended by De Man Rogosa and Sharp (1960), while for enumeration of coliform count, violet red bile agar (VRBA) medium was adopted as recommended by Klein and Fung (1978). Whereas, the count of staphylococci was determined on Staph 110 media according to APHA, (1992).

Methods

Acid tolerance

Three tested strains were evaluated for their ability to grow in low pH values (1.5, 2 and 3) as described by Pereria and Gibson (2002) with some modifications: MRS broth previously adjusted to pH 1.5, 2 and 3 with HCl, were inoculated with 1 % (v. /v.) of activated tested cultures, the tubes were maintained at 37°C for 3hours. One-milliliter of each samples were taken at various times (0, 60, 120, and 180 min) serially 10-fold diluted and plated into MRS agar, the plates were incubated at 37°C for 48 hours under anaerobic conditions by using double layer technique before enumeration.

Bile tolerance

Bile tolerance was estimated as described by Pereia and Gibson (2002). Overnight tested cultures (1 % (v. /v.) were added into MRS broth with concentrations (0.3, 0.5, and 1 % (w/v.) of bile salts inoculated anaerobically at 37°C for 12h. One-milliliter of each samples were taken at the end of the experiment (12 h), serially diluted, into MRS agar. The plates were incubated at 37°C for 48 hours under anaerobic conditions.

Tolerance of artificial gastric and intestinal juices

Gastric and pancreatic juices were prepared freshly by dissolving pepsin (Sigma) from porcine stomach mucosa (3g/L) and pancreatin (Sigma) from porcin pancreas (1g/L) in sterile saline (5g/L) (Chateris et al. 1998). The pHs of the gastric and pancreatic preparations were adjusted to 2.0 and 8.0 with 5 M/L HCl or 1M/L NaOH, respectively.

One milliliter of each activated tested cultures was centrifuged 5000 xg for 10 min at 4°C and washed three time in sterile PBS. Of each washed cell washed cell suspension 0.2 ml was mixed with 1 ml. of gastric or intestinal juice. After brief vortexing the mixtures were incubated at 37°C. When assaying gastric tolerance aliquots of 0.1 ml were removed after 60, 120 and 180 min for determination of total viable count, while for assaying intestinal tolerance, the sampling times were 60, 240 and 360 min.

Feeding Experiment

Rats were randomly and equally divided into five groups, five rats each. The animals were housed in cages at room temperature (25 ± 2°C) and relative humidity (about 55%) for 28 days. These rats were acclimatized on basal diet for one week before starting the experiment.

After an adaptation period for 7 days, the first group was fed on basal diet (80 g.rat.day^-1) and served as control I, while the second group was offered basal diet plus standardized buffalo's milk (40 mL.rat.day^-1) and served as control II. The other groups were fed on: 80 g basal diet plus 40 ml. buffalo's milk and one of the tested Lactobacillus strains for each rat/day. The animals were weighed at the beginning and the end of the experimental period.

Determination of total cholesterol or triglycerides contents

The total cholesterol or triglycerides concentration were determined in the blood serum by using Spectrum diagnostic kits. The total cholesterol concentration was calculated by using the following equation:

Cholesterol (mg/dL) = (A of the tested sample/A of the standard solution) ×200.  

While, triglycerides concentration was calculated using the following formula:

Triglycerides (mg/dL) = (A of the tested sample/A of the standard solution) ×200

where: A= Absorbance at 546 nm.

Determination of High density lipoproteins (HDL) cholesterol content

HDL-cholesterol was determined in blood serum by using Genesis kit, and its concentration calculated by using equation:

Concentration of HDL-cholesterol (mg/dL) = A sample × 570

where: A= Absorbance at 546 nm.

Determination of Low density lipoproteins (LDL) cholesterol content

The LDL-cholesterol was estimated according to the formula of Beena and Prasad (1997) as follows:

LDL-cholesterol (mg/dL) =

Total cholesterol – (HDL + (triglycerides/5)).

Calculation of atherogenic indexes

The atherogenic indexes were calculated as LDL-cholesterol / total cholesterol (index 1) and (total cholesterol – HDL)/HDL (index 2).

Biological Evolution of rat diets

Biological evolution of the different diets was carried out according to Carthew et al. (2001) by using the following equations:

Body weight gain = final weight – initial weight

Food efficiency ratio = Daily growth rate/Daily food intake.

Growth rate, g/day = Body weight gain, g / Experimental period long, day.

Fecal bacterial population

Appropriate dilution of each fecal sample was plated on either violet red bile agar (VRBA) or staph.110media, plated were either incubated at 37°C for 24 or 48h coliform and staphylococcus, respectively (APHA, 1992).

Statistical analysis

Analysis of variance was computed using the General Linear Model procedure of statistical analysis system (SPSS, 2011). Variable means for treatments indicating significant differences in the ANOVA were compared.

RESULTS AND DISCUSSION

Screening of Lactobacillus strains for their probiotic characteristics

Tolerance to acid and bile salts

In the present study, all the tested Lactobacillus spp. were successfully survival in different low pH values (Table 1). They retained varying levels of viability ranged from 100.62 % to 109.04 %. However, pH 1.5 seemed to be more damaging to the tested strains. In this respect, Maffei and Nobrega (1975) stated that the bactericidal effect of acid is evident at pH values below 2.5. Among the tested Lactobacillus strains, L. gasseri AZ1 was the most acidic tolerant, while the least survival was observed for L. rhamnosus AZ1 strain.

                       Table 1. Effect of low pH values on viability of tested Lactobacillus spp. strain.

cfu: colony forming unit.

Fig. 1. Bile tolerance of L. casei AZ1 KY123805 on different bile salt concentrations.

Fig. 2. Bile tolerance of L. gasseri AZ1 KY123806 on different bile salt concentrations.

Fig. 3. Bile tolerance of L. rhamnosus AZ1 KY123789 on different bile salt concentrations.

Tolerance to bile salts is a prerequisite for colonization and metabolic activity of bacteria in small intestine of the host (Havenaar et al., 1992). Once the bacteria reach the intestinal tract, bile entering the duodenal section of small intestine has been found to reduce survival of bacteria. Therefore, probiotics must have an ability to tolerate bile (Kimoto et al., 2000).

In agreement with literate recordings, all tested Lactobacillus strains exhibited excellent bile tolerance (Figures 1, 2 and 3). In this regard, Oh et al., (2000) mentioned that Lactobacillus was capable of surviving in the presence of bile due to its ability to deconjugate bile acids bile acids. Also, it was of interested to notice that all tested Lactobacillus strains showed varying response toward different bile salt concentrations. In this connection, Gopal et al., (1996) reported that the observed differences in tolerance to bile may partly due to the natural differences in growth of individual strains.

Tolerant to simulated gastric and small intestinal juices:

About 2.5 L. of gastric juice and 0.7 L. of pancreatic juice are secreted each day, these secretions present a pH and enzymatic barrier and act in concert with bile to ensure the survival of ingested microorganisms during digestion. Therefore, surviving gastrointestinal transit was found to be an important functional property of tested probiotic bacteria (Succi et al., 2005 and Vizoso et al., 2006).

As shown from Table 2 gastric juice exerted variable influence on the growth of examined cultures. In general, L. casei AZ1 strain exhibited more gastric juice resistance than other tested cultures. However, variation in the tolerance to gastric juice was previously reported by Mathara et al. (2008) and Kershah (2014). In generally, the three tested Lactobacilluscultures exhibited acceptable levels of survivability and considered intrinsically tolerant to gastric juice, since at least ≥10⁶cfu/ml of each strain survived after 180 min of exposure, as previously concluded by Guerra et al., (2007).

From the foregoing results, it could be pointed out that all tested strains could be successfully transit the human stomach and reaching the intestinal tract and functioning effectively there.

It was obvious, as the data in Figure 4, that all tested cultures retained viability during growth in simulated intestinal juice and considered intrinsically tolerant to intestinal transit. In this respect, Charteris et al., (1998) stated that the majority of probiotic strains were intrinsically resistant to simulated pancreatic juice and showed no reduction in viability up to 4 hours.

According to the obtained results, it might be deduced that L. gasseriAZ1 was markedly with regard to intestinal juice tolerance followed by L. casei AZ1 strain. However, several authors previously concluded that variations and degree of response to intestinal juice may be strain dependent (Sultana et al., 2000; Guerra et al., 2007; Kim et al., 2008). According to the foregoing results, it could be concluded that the three tested lactobacilli strains may be promising candidate strains for use as probiotics.

Feeding Experiment:

An extensive investigation concerning the potential role of probiotic lactobacilli strains was carried out. Data obtained were summarized in Tables 3, 4 and 5.

The effect of different diets on growth parameters of rats are presented in Table 3. At the end of the experiment (28 days), significant differences in final body weights of all rats were detected, but rats received fermented milk products gained higher body weights than those fed only dry diet (cont. I).Anon (1997) concluded that fermentation of milk by lactic acid bacteria was reported to increased its protein availability and its nutritional value. However, our findings are in agreement with those previously reported by Abd El-Gawad et al. (2005), Zommara et al. (2006) and Mohamed (2009). Also, as seen from the same table, rats received milk cultured with L. casie gained the highest final body weight, being 259.3 g.

In addition, the present results revealed that there were no considerable variations between growth rate or food efficiency among different treatments. This statement is consistent with previous finding of Zommara (2002). Moreover, significant differences were detected among dietary groups in food intake (g/ day), while no-significant differences were observed in body weight gain (g). This finding is in contrary to those reported by Abd El-Gawad et al. (2005) and Mohamed (2009).

In order to assess the potential hypocholestrolamic properties of fermented milk made with different Lactobacillus strains albino rats were used as an animal model. Results of determination blood serum lipids of rats were presented in Tables 4 and 5.

The results obtained indicated that serum cholesterol level in control II was significantly higher than in control I, by 24.07 % at the end of the experiment. Compared with control group (cont.I), the total serum cholesterol were lowered by 21.39% and 15.74% in rats fed on L. caseiand  L. rhamnosus, respectively. However, our results are consistent with previous reports of Zommara et al. (2006), Mohamed (2009), Ying et al. (2010), Chuan et al. (2104), Song et al. (2015) and Bobae et al. (2016).

In contrast, L. gasseri achieved an opposite trend, in which their final total cholesterol level was markedly increased by 13.27% as compared with control I.

Also, it could be noticed from obtained results that there were significant differences in HDL-cholesterol level between different treatments at the end of the experiment. However, the levels of serum HDL-cholesterol were increased throughout experiment period and the increases values ranged from 4.02% to 29.44%, as compared with their corresponding initial values. Zilva and Mayne (1991) stated that HDL-cholesterol levels are ''anti-atherogenic'', where their reduced level are associated with increased risk of coronary artery disease.

Since a high blood LDL-cholesterol is associated with increased risk of atherosclerosis and cardiovascular disease, any product that lower this level is of potential value. Therefore, the effect of feeding rats on different lactobacilli strains on LDL-cholesterol level was carried out, results obtained presented in Table (4).

Table 2. Effect of simulated gastric juice on viability of tested Lactobacillus spp. strains.

cfu: colony forming unit.

          Fig. 4. Viability of tested Lactobacillus spp. at simulated intestinal juice.

    Table 3. Growth parameters of rats fed on some fermented milk products cultured with Lactobacillus spp.

B.M: Buffalo's milk; n.s: non-significant; *: significant.

Table 4. Levels of serum total cholesterol and HDL-cholesterol in rats fed on some fermented milk

                                                        products cultured with Lactobacillus spp.

B.M: Buffalo's; *: significant; **: high significant; HDL: high-density lipoprotein; (+): increase (-): decrease.

Table 5. Levels of LDL-cholesterol and serum triglycerides in rats fed on some fermented milk products with Lactobacillus spp.

B.M: Buffalo's Milk; n.s: non –significant; **: high significant; LDL: low-density lipoprotein (+): increase; (-): decrease.

It might be gathered from the data obtained that buffalo's milk supplementation to dry diet had markedly increase on level of LDL-cholesterol by 127.3 %. Also, it is noteworthy from the same table the rats fed on by fermented milk cultured by either L. rhamnosusAZ1or L. casei AZ1 possessed the lowest serum LDL-cholesterol concentrations, actually 8.95 mg/dL with the highest reduction level, actually 39.07 %, while the corresponding figures were 11.42 mg/dL and 22.27 %, respectively for L. casei. these results might be considered as promising, because 1 % reduction in cholesterol can reduce the risk of cardiovascular disease for 2 – 3 % (Kourelis et al., 2010). In agreement with our finding, previous studies showed that receiving fermented milk products reduce LDL-cholesterol concentration (Abd El-Gawad et al. 2005; Zommara et al., 2006; Ying Huang et al., 2013 and Song et al., 2015). 

Furthermore, alternation in triglycerides content was studied and data obtained presented in Table 4. From these results it could be noticed that addition of buffalo’s milk to the normal diet (cont. II) markedly increased serum triglycerides by 76.99 % as compared to dry diet (cont. I). In addition, rats received fermented milk cultured by either L. caseior or L. Rhamnosusranked noticeable decreases in serum triglycerides content, actually 16.49 % and 21.99 %, respectively as compared to dry diet (cont. I). Recently, several investigators confirmed the reduction of triglycerides concentrations in rats fed different fermented milk products (Ying Huang et al., 2013; Xu et al., 2013 and Bobae et al., 2016).

As a matter of fact, the atherogenic indexesare indication for the susceptibility for atherosclerosis. Therefore, the atherogenic indices were calculated and results illustrated in Table 5. Buffalo’s milk supplementation to basal diet (cont. II) led to obvious increases in atherogenic indexes 1, 2 and LDL / HDL ratio by mean values of 82.5 %, 51.69 % and 137.91% respectively as compared with control treatment I. The same trend of result was previously reported by Zommara et al. (2006) and Mohamed (2009).

Continuously, the examined Lactobacillus spp.showed different results towards atherogenic indices, were rats received fermented product cultured with L. rhamnosus AZ1, reduced markedly the atherogenic indexes I, 2 and LDL/ HDL ratio by a mean values of 27.5 %, 22.54 % and 32.25 %, respectively, as compared with control treatment I. In contrast, fermented product cultured with L. gasseri showed an opposite trend.

The effect of fermented milk culture products cultured with Laactobacillus spp.on rats' intestinal pathogenic microflora had been studied. Data obtained graphically plotted in Figures 5 and 6. Faeces of rats received dry diet or dry diet plus buffalo’s milk showed increases in the counts of coliform by 4.5 % and 6.65 % respectively, and in staphylococci counts by 7.12 % and 7.29 % respectively.

In contrast, rats fed on fermented products exhibited highly decreases in the populations of either coliform or staphylococci in their faeces. However, the decrease in coliform counts varied from 3.6 % to 30.65 %, while, the reduction values for staphylococci ranged from 7.95 % to 15.88%. This finding may be due to the double effect of developing acidity and the production of antimicrobial agents which suppress the growth of both pathogens (Kebary, 1995 and Badawi & El-Sonbaty, 1997).

Generally, the obtained results strongly suggest that both L. casei and L. rhamnosus are promising candidate as they exhibited pronounced hypolipidemic effect and greater antagonistic effect against either coliforms of staphylococci strains.

Table 6. Levels of atherogenic indices in rats fed on some fermented milk products cultured with Lactobacillus spp.

B.M: Buffalo's; n.s: non-significant; *: significant: atherogenic index 1: (LDL/Total cholesterol); atherogenic index 2: (Total cholesterol-HDL)/HDL.

Fig. 5. Effect of fed on fermented milk products cultured with some Lactobacillus spp.on fecal coliform population.

Fig. 6. Effect of fed on fermented milk products cultured with some Lactobacillus spp.On fecal staphylococci population.