​Probiotics for Augmenting Health

The concept of probiotics was introduced in the early 20th century by Elie Metchnikoff (Metchnikoff, 1907). The Food and Agriculture Organization of the United States and World Health Organization, describes them as: ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’.

The probiotic microorganism consists mostly of the strains of the genera Lactobacillus and Bifidobacteria. Bifidobacterial species are common members of the infant gut where they form up to 91 per cent of the total micro flora in breast-fed babies and up to 75 per cent in formula fed infants (Miloud Hadadji et al., 2005). Dairy products containing Bifidobacteria have potential benefits for infants and adults that are generally related to inhibition of pathogens, maintenance and restoration of normal intestinal flora. It has been reported that these organisms are able to exert beneficial effects including improvement of intestinal microflora by preventing colonization of pathogens, amelioration of diarrhoea or constipation, activation of the immune system and increasing protein digestion (Ishibashi and Shimamura, 1993). Owing to these properties, Bifidobacteria are now frequently used to prepare probiotic dietary adjuncts.

Dehydration is a common practice to preserve biological materials so that they are stable in the long run. Among the many cell preservation methods, spray drying is widely used in the food industry because it is economical especially on a large scale. In the current study the spray dried probiotic B. longum was fed to weaned piglets and its probiotic potential was evaluated in terms of weight gain and coliform count in the weaned treated pig.

Bifidobacteria longum (IB10) and (IB12), Bifidobacteria breve (IB39) and Bifidobacteria bifidum  (IB42) were isolated and identified  from breast fed infant faeces by phenotypic and molecular techniques.

Screening of Bifidobacteria for Probiotic properties

Tolerance to bile salt

Tolerance to bile salts was carried out as per the method adopted by Jaya Prasad et al., (1999).

Inhibitory activity assay and Tolerance to acidity

Inhibitory activity assay and tolerance to acidity was carried out as per the method adopted by Zinedine and Faid (2007).

The ideal isolate B. longum (IB10) was incubated for 4 hours in a mixture of 4 per cent whey protein concentrate, 0.4 per cent inulin, 3 per cent honey and 9 percent malted ragi extract and spray dried.

Feeding trials with weaned piglets

Seventeen weaned large white Yorkshire piglets from the University Research Farm, were randomly chosen from 2 litters for the experimental study. The piglets were penned in individual sheds which permitted monitored feeding. The experimental group was administered a daily supplement of 5gms of 107 cfu/g of the probiotic spray dried powder. The piglets were weighed at weekly intervals and the body weight was recorded.

Coliform was enumerated from freshly voided faeces collected from control and experimental group by pour plate method and compared.

The data obtained were analyzed statistically as per the procedure of Snedecor and Cochran (1980).

Screening of bifidobacteria for probiotic properties

Tolerance of bifidobacterial isolates to bile salts

From Table 1, it is seen that the isolated bifidobacterial species had tolerance towards varying levels of bile. The isolate IB10 showed greater tolerance (P≤0.01) to 1 per cent bile when compared to other isolates. All the isolates showed more than 80 per cent survival rate in the presence of 1 per cent w/v bile which was affirmed as probiotic property by Jaya Prasad et al., (1999) who opined that an ideal  probiotic candidate should have a survival threshold of 80 per cent in the presence of 1 per cent w/v. Zinedine and Faid (2007) also reported that various species of Bifidobacterium showed tolerance to bile salts. In the present study bile concentrations of up to 1 per cent w/v were used, though the mean intestinal bile concentration is believed to be 0.3 per cent w/v (Gilliland et al., 1984). Goldin et al., (1992) also reported that a concentration of 0.3 per cent w/v of bile has consequently been used in most studies screening for bile tolerant strains. Interestingly all the four isolates of IB10 and IB12, IB39 and IB42 survived and showed tolerance to bile concentrations of up to 1 per cent w/v. This property may provide these species with an advantage in vivo. Among all the isolates, IB10 showed maximum tolerance.

Table 1: Tolerance of bifidobacterial isolates# to bile salts.

Inhibitory activity of bifidobacterial isolates against pathogenic bacteria

From Table 2 it is evident that the non-neutralized and neutralized cell free supernatants of isolate IB10 showed inhibition against all the four pathogenic species tested. The isolates in the present study showed inhibitory activity against selected pathogenic bacteria. On comparing the inhibitory assay of the four isolates, it was noted that isolate IB10 (B.longum) had an  inhibitory zone  ranging from 3.03 to 2.95 mm against B. cereus, S. aureus, E.coli and P. aeruginosa due to acid and antibacterial compounds which was similar to the work of Zinedine and Faid (2007).

Table 2: Inhibitory activity of bifidobacterial isolates (zone in mm) against pathogenic bacteria.

Tolerance of bifidobacterial isolates to acidity at pH 3

Table 3 shows the tolerance of bifidobacterial isolates to acidity at pH 3 as in most in vitro assays pH 3 has been preferred (Garriga et al., 1998). Isolate IB10 showed a maximum count of 10.375±0.174 (log 10 cfu/ml) at 90 minutes and 9.698±0.254 (log 10 cfu/ml) at 180 minutes post incubation as when compared to the observation of Jaya Prasad et al., (1999) who reported a viable count of 8.1 (log 10 cfu/ml)  at  3 hours incubation at pH 3. This increase in tolerance in the present study may be due to strain variability. In the present study a marginal decrease was noticed in the viable count of Bifidobacteria of all isolates at 90 and 180 minutes. However all the isolates crossed the threshold level of 80 per cent survivability as recommended by Jaya Prasad et al., (1999).

The findings of this study met the recommendation of the FAO / WTO draft guidelines of 2002 and ICMR-DBT guidelines for evaluation of probiotics in food 2011.

Table 3: Tolerance of bifidobacterial isolates# to acidity at pH 3.

Biological trials

Performance (weight) of pigs fed with nutraceutical powder (BMW)

Table 4 presents a comparison of weight of pigs between control and experimental group fed with nutraceutical powder containing B.longum (BMW). The mean values (in kg) of experimental pigs and control on the 70^th day were 55±2.363 and 38.88±0.631 respectively. The inference observed indicated a highly significant difference (P≤0.01) in the assimilation of nutrient leading to enhanced weight gain in BMW fed piglets.

The findings in this study correlated with this theory of Peter Kurti (2000) who reported that probiotics have shown beneficial effects on the intestinal balance and in improving feed utilization and weight gain. He also opined that better digestibility meant better absorption of nutrients and thus better weight gain than probiotic deprived piglets.

Table 4: Performance (weight) of pigs fed with nutraceutical powder (BMW).

Enumeration of faecal coliform (log10 cfu/g) post feeding in control piglets and nutraceutical whey based malt food (BMW) fed piglets

From Table 5 no significant difference (P≤0.05) was observed in the faecal coliform count up to 7 days of feeding of piglets in control and experimental groups. Significant difference (P≤0.05) was noticed from the 14^th to the 21^st day between control (basal diet) and experimental group (nutraceutical whey based malt food with B.longum). A highly significant difference (P≤0.01) was noticed in the reduction of faecal coliform counts between control and experimental group from 28^th day till 70^th day. There was no reduction in the coliform numbers in the control group till the end of the experiment.

The findings of the present study is in accordance to Boomba et al., (2002)  who reported a one log reduction in the E. coli after administration of L.paracasei and maltodextrin. In the present study there was a significant (from 6.80 ± 0.038 to 5.00 ± 0.000) two log reduction in the coliform count in Bifidobacterial fed piglets than the controls.

Table 5: Enumeration of faecal coliform# post feeding in pigs fed control and nutraceutical whey based malt food (BMW).